Latitudinal variation in phlorotannin contents from Southwestern Atlantic brown seaweeds

Introduction

Phlorotannins are polymers derived from a simple monomer, phloroglucinol, found exclusively in brown seaweeds (Targett & Arnold, 1998, 2001). These water-soluble secondary metabolites constitute a special class of polyphenols that may exhibit multifunctional ecological roles, acting as a herbivore deterrent (Pereira & Yoneshigue-Valentin, 1999), antifouling agent (Plouguerné et al., 2012), antioxidant (Cruces, Huovinen & Gómez, 2012), UV protector (Henry & Van Alstyne, 2004), and a chelating agent of toxic heavy metal ions (Karez & Pereira, 1995). However, these chemicals may also be classified as primary metabolites when they are structural components of cell walls (Schoenwaelder & Clayton, 1999). In fact, phlorotannins found inside the cells of brown seaweeds are stored in small vesicles called physodes, and these chemicals may exude into the environment due to their water solubility (Jennings & Steinberg, 1994) where they can have several vital ecological roles (Pereira et al., 1990). As cell wall components, where they form a complex with alginic acid, they are insoluble (Schoenwaelder, 2002; Koivikko et al., 2005). Given the smaller amounts of cell-wall-bound phlorotannins compared to soluble phlorotannins, the major function of these chemicals appears to be secondary metabolites (Koivikko et al., 2005).

The concentration of phlorotannins in brown seaweeds is known to be highly variable in several modes and at various scales, supposedly in response to the dynamics of biotic and abiotic environmental conditions (Jormalainen et al., 2003). For example, concentrations may vary in response to environmental factors, either biotic—such as herbivory (Hemmi, Honkanen & Jormalainen, 2004) and epibiosis (Plouguerné et al., 2010)—or abiotic—such as temperature (Cruces, Huovinen & Gómez, 2012), irradiance (Cruces, Huovinen & Gómez, 2013), nitrogen concentrations (Pavia & Toth, 2000), bathymetric variation, and immersion time in the intertidal range (Connan et al., 2004). Phlorotannin content can also vary according to intrinsic aspects of brown seaweeds, such as individual size and age (Pavia et al., 2003), and tissue type (Plouguerné et al., 2012).

Another interesting aspect relating to the distribution, abundance, and function of phlorotannins is the latitudinal differences in content of these chemicals among brown seaweeds living along large temperate-tropical gradients (Steinberg, 1989; Van Alstyne & Paul, 1990). High concentrations of these compounds have been found in species from high latitudes (Ragan & Glombitza, 1986; Steinberg & Paul, 1990; Steinberg & Van Altena, 1992; Hay & Steinberg, 1992; Steinberg, 1992). For example, species of Fucales and Laminariales that are abundant in temperate benthic communities, and Dictyotales found both in temperate and tropical regions, exhibit this biogeographic trend. The most common brown seaweed species in temperate Australasia exhibit more than 10% of total phlorotannins (Steinberg, 1989), whereas there are both phlorotannin-rich and -poor species in some temperate regions of South Africa (Anderson & Velimirov, 1982; Tugwell & Branch, 1989), northwestern Pacific (Katayama, 1951; Estes & Steinberg, 1988), and the European North Atlantic (Ragan & Glombitza, 1986).

Many species of brown seaweeds from North America exhibit low levels of phlorotannins, ranging from 0% to 2% of algal dry weight (DW) (Ragan & Glombitza, 1986). This range is found mainly in kelps dominating both the sublittoral and lower littoral environments (Steinberg, 1992). In contrast, as the most abundant organisms found in littoral and upper sublittoral regions, fucoids commonly contain higher phlorotannin contents (more than 4% DW) (Steinberg, 1985; Van Alstyne, 1988; Denton, Chapman & Markham, 1990; Targett et al., 1992). In general, brown seaweeds from North America exhibit broad variation in phlorotannin contents linked to the bathymetric gradient, with littoral fucoids and subtidal kelps showing high and low levels of these compounds, respectively (Estes & Steinberg, 1988; Steinberg, 1992).

In general, the intensity of selective pressures on organisms increases with decreasing latitude, including higher herbivory and epibiosis (Railkin, 2004; Targett & Arnold, 1998). Consequently, tropical seaweeds are hypothesized to have evolved more effective chemical defenses (Van Alstyne & Paul, 1990; Targett et al., 1992). Contrary to this trend, phlorotannins are sometimes absent or present in very low concentrations in seaweeds from tropical environments (Steinberg, 1989; Van Alstyne & Paul, 1990; Pereira & Yoneshigue-Valentin, 1999). There is only one report of high amounts of these compounds in brown seaweeds from low latitudes (Targett et al., 1995).

However, in almost all studies, the quantification of phlorotannins is based on an analysis of distinct specimens of brown seaweed species extracted together, masking possible variability in amounts of these chemicals in each individual of a population. However, intra-populational variation in seaweed-derived chemicals can be of great magnitude and ecological significance (Oliveira et al., 2013).

Along the Brazilian coast, the few studies on phlorotannin contents in brown seaweeds are united in the fact that they typically reveal low concentrations (Fleury et al., 1994), and that they may be capable of inhibiting grazing when they occur at higher concentrations (Pereira & Yoneshigue-Valentin, 1999). The extensive Brazilian coast covers a broad latitudinal range of the Southwestern Atlantic and harbors numerous species of brown seaweeds. It comprises several environments suitable for exploring chemical defenses via a biogeographic approach. To date, most studies in Brazil have only reported average phlorotannin concentrations, so there is no information concerning the variation within populations or among populations from different latitudes. Thus, more in-depth analysis is needed, as tropical species could have the same mean value as temperate seaweeds, but exhibit greater standard deviation. Here, we hypothesized that contents of brown seaweed phlorotannins would exhibit latitudinal variation along the Brazilian coast. Our aim was to compare the mean phlorotannin concentration, as well as the coefficient of variation, among and within species of brown seaweeds across a broad latitudinal context along the Brazilian coast to evaluate the hypothesis that species from low latitudes exhibit lower amounts of these chemicals relative to those from high latitudes.

Materials and Methods

Study organisms and collection localities

Brown seaweeds were collected from along the Brazilian coast (Instituto Chico Mendes de Conservação da Biodiversidade—Authorization Number 27001-2) in order to best represent various populations of the same species and individuals in each population from the different localities (Fig. 1; Table 1): Giz Beach (6°10′S; 35°05′W) at Tibau do Sul, RN; Itapuama (08°17′S; 34°57′W), Calhetas (08°20′S; 34°56′W), Paraíso (08°21′S; 34°57′W) and Suape beaches (08°22′S; 34°56′W) at Recife, PE; Itapuã Beach (12°57′S; 38°22′W) at Salvador, BA; Pé de Serra Beach (14°28′S; 39°01′W) at Uruçuca, BA; Morro de Pernambuco (14°48′S; 39°01′W) and Back Door beaches (14°56′S; 39°00′W) at Ilhéus, BA; Ponta Beach (16°24′S; 39°02′W) at Porto Seguro, BA; Três Praias (20°38′S; 40°28′W) at Guarapari, ES; Rasa (22°44′S; 41°57′W) and Forno beaches (22°45′S; 41°52′W) at Armação dos Búzios, RJ; and Canasvieiras Beach (27°25′S; 48°28′W) at Florianópolis, SC. We collected individuals of the following species: Canistrocarpus cervicornis (Kützing) De Paula & De Clerck, Colpomenia sinuosa (Mertens ex Roth) Derbés & Solier, Dictyopteris delicatula J.V. Lamouroux, Dictyopteris polypodioides (A.P. De Candolle) J.V. Lamouroux, Dictyota ciliolata Sonder ex Kützing, Dictyota crispata J.V. Lamouroux, Dictyota dichotoma (Hudson) J.V. Lamouroux, Dictyota mertensii (Martius) Kützing, Dictyota pfaffii Schnetter, Lobophora variegata (J.V. Lamouroux) Womersley ex E.C. Oliveira, Padina gymnospora (Kützing) Sonder, Sargassum filipendula C. Agardh, Sargassum stenophyllum Martius, Sargassum ramifolium Kützing, Sargassum vulgare C. Agardh, Sargassum vulgare var. nanum E. De Paula, Sargassum vulgare var. vulgare, Spatoglossum schroederi (C. Agardh) Kützing, and Stypopodium zonale (J.V. Lamouroux) Papenfuss.

Table 1:

Brown seaweeds studied, number of specimens and corresponding collection places (x = The individuals were analyzed together because of the small size/biomass of the specimens; while in the remaining species, the analyzes were performed in each individual).

Seaweed species	Sampling sites
	1. Tibau do Sul—RN	2. Recife—PE	3. Salvador—BA	4. Uruçuca—BA	5. Ilhéus—BA	6. Porto Seguro—BA	7. Guarapari—ES	8. Búzios—RJ	9. Florianópolis—SC
Canistrocarpus cervicornis		7						x
Colpomenia sinuosa						4		x
Dictyopteris delicatula		8	10			x	x
Dictyopteris polypodioides							x
Dictyota ciliolata						11
Dictyota crispata						19
Dictyota dichotoma						x
Dictyota mertensii		7					x	10
Dictyota pfaffii			10
Lobophora variegata		10	x			10
Padina gymnospora	x	10		x	x	7	x	x	8
Sargassum filipendula			9	10
Sargassum ramifolium							7
Sargassum stenophyllum									7
Sargassum vulgare		15		6	10	22		5
Spatoglossum schroederi							10
Stypopodium zonale								32

DOI: 10.7717/peerj.7379/table-1

Results

Variation in phlorotannin contents within populations and across a latitudinal gradient

Intra-populational analyses were carried out for 25 populations (Table 2) of 14 seaweed species (Table 1). For all analyzed populations, we identified a significant difference in the amount of phlorotannins among the individuals that comprised them (t-test, p < 0.01), with CV ranging from 5.2% to 65.3% (Table 2). CV was higher in populations collected from higher latitudes, but the correlation though significant (p < 0.005) was relatively weak (r = 0.55) (Fig. 2).

We assessed phlorotannin contents in brown seaweeds sampled along a broad latitudinal range, from 2° to 22° of southern latitude, representing from Recife to Rio de Janeiro, respectively (Table 2). The highest phlorotannin contents were found in brown seaweeds collected at higher latitudes, but the correlation between amounts and latitude was weak and non-significant (Fig. 3, r = 0.23; p = 0.15).

Discussion

The phlorotannin contents found in the brown seaweeds we investigated were typically very low (<2.0% DW), with only one exception, Spatoglossum schroederi for which we recorded 4.30% DW. These results reinforce a pattern that seems to be typical of tropical areas, including the Brazilian coast, in which low values of phlorotannins have been reported for several brown seaweeds belonging to different orders, ranging from 0.2% to 2.17% DW (Pereira & Yoneshigue-Valentin, 1999; Pereira et al., 1990; Fleury et al., 1994). Low contents of these chemicals, varying from 0.19% to 1.62% DW, were also found in some brown seaweeds from Guam and neighboring areas of the tropical Pacific (Steinberg & Paul, 1990; Van Alstyne & Paul, 1990). Moreover, low levels of phlorotannins (ranging from 0.2% to 1.77% DW) have been found in Sargassum spp. and Turbinaria spp. at two tropical sites, Tahiti and the Great Barrier Reef, Australia, respectively (Steinberg, 1986).

Brown seaweed phlorotannins have been reported as defensive chemicals against herbivores in some studies (Jormalainen & Ramsay, 2009), but only when they occur at concentrations higher than 2.0% DW, that is, levels commonly found in species from temperate regions (Ragan & Glombitza, 1986). However, the evidence for this defensive property of phlorotannins remains disputed, with reports supporting (Van Alstyne & Paul, 1990) and refuting (Steinberg & Paul, 1990) this role. The low levels of phlorotannins in tropical seaweeds may be due to these chemicals having limited impact on tropical fish herbivory, given that fishes from the Great Barrier Reef do not consume more phenolic-poor tropical species than phenolic-rich species (Steinberg & Paul, 1990). However, contradicting this latter finding, phlorotannin-rich seaweeds were not consumed by fishes in Guam (tropical Pacific region), though extracts from phlorotannin-poor species were also not eaten (Van Alstyne & Paul, 1990). Moreover, phlorotannins in amounts higher than those usually found in the Brazilian brown seaweed Sargassum furcatum can inhibit herbivory (Pereira & Yoneshigue-Valentin, 1999). However, according to our results, almost all of the seaweeds we studied probably do not employ this kind of chemical defense to prevent herbivory, since phlorotannin contents were usually lower than 2.0% DW.

The hypothesis of a latitudinal gradient of phlorotannin contents is based on the assumption that herbivory pressure increases with decreasing latitude and that production of seaweed chemical defenses is selected by the action of herbivores. Accordingly, defensive chemicals should be more common and effective in tropical seaweeds. Although chemical defenses are commonly associated with herbivore abundance and pressure, no study has conclusively demonstrated that herbivores impose selective pressures on the production of secondary metabolites (Van Alstyne & Paul, 1990). Moreover, phlorotannins may be present in brown seaweeds for reasons other than herbivore defense, since they have been suggested to exhibit other ecological roles, such as protecting against short-wave UV radiation (Pavia et al., 1997), and as anti-fouling agents (Plouguerné et al., 2010, 2012).

It would be difficult to establish a clear correlation between the latitudinal variability in phlorotannin production by brown seaweeds solely with the different pressures of herbivory along the Brazilian coast, even knowing that this kind of variation exists and that the seaweeds we studied were collected from a broad latitudinal range (ca. 21°). Importantly, it remains controversial if herbivory pressure selects for chemical defense production (Pereira & Da Gama, 2008), even across a global tropical-temperate latitudinal gradient or along the Brazilian coast (Longo, Ferreira & Floeter, 2014). In addition, it is known that concentrations of secondary metabolites may vary according to temperature (Sudatti et al., 2011), nutrient availability (Puglisi & Paul, 1997), light (Pavia et al., 1997), salinity (Kamiya et al., 2010; Sudatti et al., 2011), and herbivory (Weidner et al., 2004). Thus, since the seaweeds we studied are also subjected to unknown variability in all these external conditions, it is perhaps not surprising that we did not establish a direct causal effect between phlorotannin content and latitude.

The extent of genetic control over chemical defense production remains poorly understood. For example, phlorotannin content was demonstrated to be due to genotypic variation in Fucus vesiculosus (Jormalainen et al., 2003; Jormalainen & Honkanen, 2008; Koivikko et al., 2008), as well as for terpenes in the red seaweeds Laurencia nipponica (Masuda et al., 1997; Abe et al., 1999) and Delisea pulchra (Wright et al., 2004). If phlorotannin production is genetically modulated, geographic distance and gene flow would likely contribute to the variation in the content of these phenols in our studied species. In general, seaweeds are considered poor dispersers because their gametes and spores only survive for a few days in the water column (Santelices, 1990; Sosa & Garcia-Reina, 1993). Limited gene flow has been reported for diverse seaweed species (Wright, Zuccarello & Steinberg, 2000; Faugeron et al., 2001, 2004; Zuccarello, Sandercock & West, 2002; Van der Strate et al., 2003), and small-scale dispersal distances are a significant factor in the differentiation of seaweeds (Tatarenkov et al., 2007). Thus, if secondary metabolite production is an inherited character, geographic distance should act as a barrier to gene flow and give rise to quantitative differences in phlorotannin production.

Abiotic differences among collection sites could also support the hypothesis that different field conditions contribute to the between-site variability in phlorotannin concentrations for each of the algal species we studied. Temperature is a determining factor for the survival, geographic distribution, and reproduction of seaweeds (Padilla-Gamiño & Carpenter, 2007), and it is also responsible for many responses of their primary metabolism, such as photosynthesis, growth (Nishihara, Terada & Noro, 2004), nutrient absorption (Tsai et al., 2005), and secondary metabolism (Sudatti et al., 2011). Thus, given the reduced gene flow known for seaweeds (Wright, Zuccarello & Steinberg, 2000) and the different environmental conditions along the Brazilian littoral coast, populations of the same species we studied here could be highly structured, explaining in part the results we obtained. Accordingly, our field data reinforce the idea that genetic heterogeneity contributes to quantitative variation of secondary metabolism and that our sampled populations may represent ecotypes.

The intra-populational variability in the amounts of defensive chemicals we report here corroborates the findings of the few previous studies that investigated this topic in the red seaweeds Portieria hornemannii (Matlock, Ginsburg & Paul, 1999), Delisea pulchra (Wright, Zuccarello & Steinberg, 2000) and Laurencia dendroidea (Sudatti, Rodrigues & Pereira, 2006). However, those studies did not assess as broad a latitudinal context as we did. Our study also reinforces the importance of analysis at the intra-population level (i.e., variation among specimens), since most studies of seaweed chemical ecology overlook this element of chemical variation by examining pooled extracts and/or substances obtained from groups of individuals. Developmental (Bowers & Stamp, 1993), environmental (Agrell, McDonald & Lindroth, 2000), and genetic (Berenbaum & Zangerl, 1992) traits all represent sources of variation that can explain the diversity of plant chemical phenotypes. Moreover, in seaweeds, life-history phases (see Verges, Paul & Steinberg, 2008), ontogenetics (Paul & Van Alstyne, 1988), and chemical races (Abe et al., 1999) may also be included as sources of secondary metabolite variability. In our analysis, the specimens belonged to the sporophytic life-history phase and were approximately of the same size. However, we cannot rule out the possibility that chemical races exist among the individuals of each population we studied.

Conclusion

Overall, our results show that latitude does not explain the variability in total amounts of phlorotannins found in each population of the brown seaweeds we studied along the Brazilian coast, but the significant intra-specific differences in production of these chemicals we report may be important to understanding the ecological drivers of this defensive chemistry in seaweeds. Based on characteristics of the Brazilian coast (Floeter & Soares-Gomes, 1999), the higher phlorotannin levels we recorded in populations from higher latitudes may represent a greater capacity for these seaweeds to respond to seasonal stimuli. Since environments in low latitudes exhibit little seasonal variation, the need for seaweeds in these zones to vary production of these chemicals may be lessened. Thus, brown seaweeds at higher latitudes are more likely to modulate chemical defense production in response to stimuli than those in tropical regions where the environmental conditions are more constant. However, we assert that further studies of intra-populational variability in chemical defense are warranted in the context of marine chemical ecology.

Supplemental Information