Advances in Botanical Research, Volume 43

Defensive and Sensory Chemical Ecology of Brown Algae

CHARLES D. AMSLER AND VICTORIA A. FAIRHEAD

Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294‐1170

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chemical Structure and Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Comparison of Quantification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cellular Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Putative Ecological Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Spatial and Intra/Interspecific Variability . . . . . . . . . . . . . . . . . . . . . . . . . F. Rates and Cost of Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Current Uncertainty and Future Directions . . . . . . . . . . . . . . . . . . . . . . . III. Nonphlorotannin Antiherbivore Defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Dictyotales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Desmarestiales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Activated Defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Testing Chemical Defence Theories with Brown Algae. . . . . . . . . . . . . . . . . A. Optimal Defence Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Induced Defence Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Carbon‐Nutrient Balance and Resource Allocation . . . . . . . . . . . . . . . . D. Tests of Multiple and Other Theories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Nonphlorotannin Defences Against Bacteria, Fouling Organisms, and Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Volatile Halogenated Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Sensory Chemical Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chemoattraction to Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Behaviour and Sensory Capabilities of Spores . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

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0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43001-3

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VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT The ecological interactions of brown algae are important as these macroalgae are common and often keystone members in many benthic marine communities. This review highlights their chemical interactions, particularly with potential herbivores, but also with fouling organisms, with potential pathogens, with each other as gametes, and with their microenvironments when they are spores. Phlorotannins, which are phenolic compounds unique to brown algae, have been studied heavily in many of these respects and are highlighted here. This includes recent controversy about their roles as defences against herbivory, as well as new understanding of their roles in primary cellular functions that may, in many instances, be more important than, and which at least have to be considered in concert with, any possible ecological functions. Brown algae have also been useful models for testing theories about the evolution of and ecological constraints on chemical defence. Furthermore, their microscopic motile gametes and spores have the ability to react to their chemical environments behaviourally.

I. INTRODUCTION Chemical ecology can be described as the study of chemically mediated interactions between organisms or between organisms and their environment, and most such interactions can be grouped into three broad categories. One category is chemical communication between organisms, such as brown algal male gamete attraction to pheromones, which is discussed in Section VII.A. A second is organisms sensing and responding to their chemical environments, and in brown algae this is observed in spores, which are able to sense and respond behaviourally to nutrients as described in Section VII.B. However, most studies of the chemical ecology of brown algae fall into the third category, which is chemical defence. Defences can be mounted against predators, pathogens, biofoulers, or competitors. A majority of the research on chemical defences in brown algae to date, and consequently a majority of this review is focused on defences against predators, but we also discuss defences against pathogens and biofoulers in Sections II. D.3 and V. Outside of speculation about potential allelopathic roles of pheromones, as discussed briefly in Section VII.A, we are aware of little work on brown algae examining chemical interactions between competitors, although the report of Ra˚ berg et al. (2005) is a fascinating recent example. The chemicals mediating defensive interactions are usually secondary metabolites (also called natural products) so termed to distinguish them from

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metabolites with primary metabolic roles within the cells of many taxa. However, as discussed in Section II, brown algal phlorotannins are an important exception to this as they have important primary roles in addition to ‘‘secondary’’ functions as chemical defences. Brown algae have been included in numerous recent reviews that have covered various aspects of algal chemical ecology (Amsler, 2001; Amsler and Iken, 2001; Arnold and Targett, 2002; Cronin, 2001; La Barre et al., 2004; Paul and Puglisi, 2004; Paul et al., 2001; Pohnert, 2004; Pohnert and Boland, 2002; Potin et al., 2002; Steinberg and de Nys, 2002; Steinberg et al., 2001, 2002; Targett and Arnold, 2001; Van Alstyne et al., 2001a). Here we have attempted to take a comprehensive approach in reviewing the current state of brown algal chemical ecology, and we believe that this focus on the group is warranted. Brown algae, which comprise the Class Phaeophyceae, are unique in being very far removed phylogenetically from all other eukaryotic macrophytes (i.e., red macroalgae, green macroalgae, bryophytes, and vascular plants, which clade together in modern phylogenetic trees) with the notable exception of the endosymbiotically derived red lineage chloroplasts of brown algae and their heterokont relatives (Baldauf, 2003; Falkowski et al., 2004; Keeling, 2004). A great deal of the conceptual framework of defensive chemical ecology is based on studies of vascular plants (cf. Section IV), and this phylogenetic distinctiveness of the Phaeophyceae makes them ideal tools to test and extend such ideas in trophically analogous but phylogenetically distinct organisms. Brown algae are also very important members of many marine communities ranging from the tropics to near the poles (Lobban and Harrison, 1994) and often dominate these communities in terms of structure and biomass, particularly in temperate and polar waters (e.g., Dayton, 1985a,b, 1990; Schiel and Foster, 1986; Wiencke and Clayton, 2002). Brown algae do occasionally occur in freshwater (Graham and Wilcox, 2000), but we are aware of no studies of their chemical ecology in freshwater systems. Consequently this review focuses entirely on marine and estuarine environments. With respect to defences against predation, we include only chemical forms of defence even though brown algae can also deter herbivory via morphological or otherwise physical mechanisms (e.g., Lewis et al., 1987; Lowell et al., 1991) or via life history adaptations that expose relatively palatable stages to herbivores over a minimal period of time (Lubchenco and Cubit, 1980). Because of the length of this review, we presume that many readers will consult only some parts at any given time and, consequently, we have chosen to include redundancy in two forms in the text. There are a number of published studies that are referred to, but in diVerent contexts, and often

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with diVerent emphasis, both in Section II on phlorotannins and in Section IV on tests of chemical defence theories. Also, particularly because this review is focused on brown algae as a specific taxonomic group, we feel that it is important throughout to be explicit about the ordinal‐level taxonomic relationships between the species discussed. Both similarities and diVerences in the chemical ecology of diVerent brown algal species, as described later, should be considered within a phylogenetic context. To provide such context, we have attempted to exhaustively identify the ordinal‐level classification of each species every time it is discussed, although sometimes using terms such as ‘‘kelp’’ for members of the Laminariales, ‘‘fucoid’’ for members of the Fucales, or ‘‘dictyotalean’’ for members of the Dictyotales. It is hoped that a reader of the entire review or of multiple parts thereof will understand the reasons for this redundancy.

II. PHLOROTANNINS Phlorotannins are polyphenolic polar metabolites with both primary and secondary roles. They occur only in the Phaeophyceae and account for over 10% dry weight in many species or up to 20% in others (Ragan and Glombitza, 1986). Their roles and functions have been the subject of many studies over the last few decades, particularly those roles that relate to antiherbivory and antifouling. Recent reviews have covered several aspects of our current knowledge in relation to phlorotannins and this work will not specifically focus on these areas, but it will still briefly address important areas such as chemical structure and roles at the cellular level. The comprehensive review by Ragan and Glombitza (1986) covered work up to that time, and recent reviews have concentrated on more specific aspects, such as the mediating role phlorotannins play in the interaction between algae and herbivores (Targett and Arnold, 1998, 2001) and on the putative roles of phlorotannins at the cellular level (Schoenwaelder, 2002). Arnold and Targett (2002) provide an overview of marine tannins in general, reviewing work on both vascular and nonvascular taxa, and Paul and Puglisi (2004) include a discussion of phlorotannins in their wide‐ranging review of chemical interactions between marine organisms. A. CHEMICAL STRUCTURE AND SYNTHESIS

1. Structure Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene; Fig. 1) and are classified into six groups on the basis of the chemical structure of the polymer (fucols, phlorethols, fucophlorethols, fuhalols,

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Fig. 1. Chemical structures of phloroglucinol and subunits of the six diVerent structural classes of phlorotannins.

isofuhalos, and eckols; Fig. 1) (Ragan and Glombitza, 1986). They have several properties in common with some vascular plant tannins, although they remain chemically quite diVerent. Properties in common with the condensed tannins of vascular plants include the ability to bind to metal ions and to precipitate protein and carbohydrate from solution (Ragan and

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Glombitza, 1986). The molecular masses of phlorotannins vary between 126 Da and 650 kDa (Targett and Arnold, 2001), but are found most commonly in the 10 to 100 kDa range (Boettcher and Targett, 1993). 2. Location in the cell: soluble and cell wall bound forms The highest concentration of phlorotannins in the cell is found in physodes, which are membrane‐bound vesicles appearing as light‐refractive bodies in the cytoplasm (for reviews, see Ragan and Glombitza, 1986; Schoenwaelder, 2002). Phlorotannins in this form are soluble (Ragan and Glombitza, 1986). Physodes may occur in most tissues of the thallus but commonly occur in the outermost layers (Ragan and Glombitza, 1986; Tugwell and Branch, 1989). Lu¨ der and Clayton (2004) suggest that as much as 90% of the total phlorotannin content of Ecklonia radiata (Order Laminariales) can be found in the epidermal layer. Shibata et al. (2004) reported that the phlorotannin distribution in tissues of three other Ecklonia spp. is similarly concentrated in epidermal layers. Phlorotannins are also found as a constituent of cell walls (Schoenwaelder and Clayton, 1999a), where they are incorporated after release from the physodes (see Section II.C.1). Physodes are able to move to areas of active cell wall formation via interactions with the cytoskeleton (Schoenwaelder and Clayton, 1999b). The cell wall bound phlorotannins are thought to occur in levels an order of magnitude lower than levels of soluble phlorotannins in the cell (Koivikko et al., 2005). 3. Synthesis and metabolic turnover Phlorotannins are generally thought to be synthesised via the acetate–malonate pathway (Herbert, 1989) in a process that may involve a polyketide synthase (PKS) type enzyme complex (Arnold and Targett, 2002). However, Chen et al. (1997) have proposed an alternative hypothesis that they are produced by the shikimic acid pathway, in a manner analogous to vascular plant tannins. Identification of the phlorotannin synthetic pathway is an important goal for future research. This is particularly so if it leads to methodologies to monitor phlorotannin synthesis at the genetic or enzymatic levels, which could potentially help resolve some of the uncertainties in studies of phlorotannins as described later. Arnold and Targett (1998) developed a method using stable isotope (13C) techniques to determine rates of phlorotannin synthesis. In Lobophora variegata (Order Dictyotales) and in Sargassum pteropleuron and Fucus distichus (Order Fucales), phlorotannin synthesis costs represented
1 % of total assimilated carbon when calculated over a range of carbon assimilation rates. Arnold and Targett (1998) emphasised that these investment rates do not include other costs, such as maintaining the metabolic pathways and

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storage structures, which may diVer between species and for diVerent metabolites. They also investigated polymerisation (‘‘aging’’) in Sargassum pteropleuron, observing that
30 kDa polymers formed rapidly in a 5‐h period following the labelling incubation, and that this size class was gradually replaced with 30 kDa polymers from that time on. In a later study these authors used the same methods in field and laboratory experiments that provided evidence for relatively high rates of metabolic turnover in two tropical species (L. variegata and Sargassum hystrix var buxifolium), although the rate for L. variegata (2 days for complete turnover) was considered to be related to exudation under the stressful experimental conditions (Arnold and Targett, 2000). The turnover rate for S. hystrix var. buxifolium was slower at 17 days for a complete removal of 13C from the measurable phlorotannin pool, which was recorded in field experiments considered to involve very low rates of exudation. B. COMPARISON OF QUANTIFICATION METHODS

Several methods provide measures of phlorotannins, either by quantifying the total level of polyphenolics or by specifically measuring levels of tannins (for reviews, see Ragan and Glombitza, 1986; Targett and Arnold, 1998). The chemical behaviour of phlorotannins (as reactive, large, structurally similar, polar metabolites) has led to colorimetric methods becoming accepted as the preferred technique to eVectively quantify the levels of soluble, physode‐bound phlorotannins (Ragan and Glombitza, 1986; Targett and Arnold, 1998). These colorimetric methods, of which there are several, measure the total level of phenolics in the sample, which does not allow diVerentiation of size classes (Stern et al., 1996b), considered an important factor in determining the bioactivity of phlorotannins (see Section II.D.2). Results gained from each procedure will be variously aVected by the exact chemical structure of the phlorotannins in the sample (including size and bond types) and by the choice of reference compound (Ragan and Glombitza, 1986; Stern et al., 1996b; Van Alstyne, 1995). The proliferation of methods means it is sometimes diYcult to make comparisons between studies, but as each method has its advantages in particular instances, it is hard to advocate a single preferred method, and relative comparisons within studies (when results are gained using the same procedure) are still valid. The strong hydrogen‐bonding capacity of phlorotannins means they can be eVectively removed from a sample by treatment with the resin polyvinylpolypyrrolidone (PVPP) (reviewed by Toth and Pavia, 2001). This allows quantification methods to be assessed (e.g., Targett et al., 1995) or to be modified by including PVPP‐treated samples (e.g., Cronin and Hay, 1996a; Peckol

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et al., 1996; Yates and Peckol, 1993) and is useful for creating appropriate controls in experimental design (e.g., Wilkstro¨ m and Pavia, 2004). Extraction of phlorotannins prior to quantification is problematic no matter what assay is preferred. Polyphenolics are prone to oxidation and thus an inert atmosphere and dark, cold conditions are required for any procedure. The ability to oxidise rapidly, in combination with their tendency to precipitate proteins, is likely to result in underestimates of concentrations unless these factors are controlled for (Ragan and Glombitza, 1986). Extraction of phenolics is normally by aqueous alcohol (usually methanol) or aqueous acetone (Ragan and Glombitza, 1986), which minimises extraction of nonphenolic substances (e.g., proteins) that may be inadvertently measured in the quantification assay (Van Alstyne, 1995). Koivikko et al. (2005) compared the eYciency of various solvents for extracting phlorotannins from Fucus vesiculosus, indicating that 70% aqueous acetone was the most eYcient in that case. 1. Folin–Denis A modification of the Folin–Denis procedure (Folin and Denis, 1915) is perhaps the most widely used method of quantifying phlorotannins. During the Folin–Denis assay, polyphenolics are oxidised in reactions linked to the production of stable blue‐coloured molecules (via the reduction of phosphomolybdic and phosphotungstic acids), which allows measurement through spectrophotometry and quantification using a commercially available standard (Ragan and Glombitza, 1986). Although several compounds are known to interfere with the reactions (Ragan and Glombitza, 1986) and the reagent is also reactive with certain nonpolyphenolic compounds, these factors are not considered to aVect results beyond normal error limits (Targett and Arnold, 1998; Targett et al., 1995; but see Appel et al., 2001). 2. Folin–Ciocalteu The Folin–Ciocalteu assay, using a modified reagent (Folin and Ciocalteu, 1927; Waterman and Mole, 1994), represents an improvement of the Folin– Denis assay by reducing levels of precipitates (Van Alstyne, 1995), which allows use with small sample volumes (Stern et al., 1996b). In common with the Folin–Denis assay, the reagent is reactive with compounds other than phenolics, but this represents only a small percentage of the overall measurement (Van Alstyne, 1995). 3. 2,4‐Dimethyloxybenzaldehyde The DMBA assay, another colorimetric assay, depends on the ability of 2,4‐ dimethyloxybenzaldehyde (DMBA) to react specifically with phlorotannins (1,3‐ and 1,3,5‐substituted phenols), but not other phenolics, producing a

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pink‐coloured chromophore (Stern et al., 1996b). This assay has the disadvantage of requiring a standard to be produced for each species (by purification of phlorotannins from it or a very closely related species) in contrast to those aforementioned assays that utilise commercially available phloroglucinol. This is due to the larger variability in reactivity depending on the specific nature of the phlorotannins (Stern et al., 1996b). However, the DMBA assay is less sensitive to interferences from nonphenolic substances (Stern et al., 1996b), and once a standard has been prepared, the simplicity of the assay procedure allows more easily for a sample design with much larger numbers of samples (as long as the samples come from the same, or closely related, species). 4. Cell wall bound Koivikko et al. (2005) published a method for measuring cell wall bound phlorotannins. The method was adapted from vascular plant studies and involves alkaline degradation of the bonds between phenolics and alginic acid in the cell walls. C. CELLULAR ROLES

1. Cell wall structure Phlorotannins have recently been confirmed as being an important component of the brown algal cell wall (Schoenwaelder and Clayton, 1999a) and as being vital for the process of cytokinesis (for a review, see Schoenwaelder, 2002). In zygotes of Acrocarpia paniculata and Hormosira bankisii (Order Fucales), phlorotannins are secreted into cell walls following the fusion and breakup of physode membranes (Schoenwaelder and Clayton, 1998a,b) where they are then thought to complex with alginic acid (Schoenwaelder and Clayton, 1999a). Investigations in the same species also revealed that prior to cell division the physodes form a distinct line across the centre of cell, which occurs before any other cell wall constituents accumulate (Schoenwaelder and Clayton, 1998b). 2. Adhesion to the substrate A secretion of phenolics coincides with the adhesion of zygotes in Acrocarpia paniculata and Fucus gardneri (Order Fucales) where secretion is localised at the point of adhesion (Schoenwaelder and Clayton, 1998a; Vreeland et al., 1998). Secretion of polyphenolics also occurs after fertilisation in Durvillaea potatorum (Order Durvillales; Clayton and Ashburner, 1994). Adhesion of these newly formed zygotes is thought to be via an extracellular glue composed, in part, by phenolics (Vreeland et al., 1998; reviewed in Schoenwaelder, 2002).

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3. Spermatozoid inhibitors During the period in which newly fertilised zygotes remain unprotected from polyspermy (which is potentially deadly), secreted phlorotannins are thought to protect the zygote by impairing sperm motility (Schoenwaelder and Clayton, 1998a; reviewed in Schoenwaelder, 2002). 4. Wound healing That phlorotannins play a role in wound healing has been recognised for over three decades (Fagerberg and Dawes, 1976; Fulcher and McCully, 1971), and recent work has further clarified this role (Lu¨der and Clayton, 2004). Lu¨ der and Clayton (2004) created small wounds with a cork borer in sections of Ecklonia radiata (Order Laminariales) and then followed the healing process for 9 days using microscopy techniques (light, fluorescence, and transmission electron). They found clear evidence of an accumulation of phlorotannins around wound sites, which was evident 1 day after wounding. This became prominent in the nearby medulla after only 3 days and by 9 days dense accumulations were found throughout the medulla of the entire algal section. They observed that new medullary cells produced at the wound site were structurally diVerent and contained several physodes, which increased in number and size over the next few days. Cortical cells were also observed to accumulate physodes. By day 5 the wound surface was composed entirely of cells dense with physodes, which diVerentiated into epidermal cells that remained rich in physodes. They concluded that phlorotannins function in both wound‐sealing (which is consistent with the clotting proposal of Fagerberg and Dawes, 1976) and structural wound‐healing roles (consistent with their role in cell wall formation; see Section II.C.1). In addition, the general accumulation of phlorotannins throughout the medulla was considered to be an antiherbivore response (see Section II.D.1), which may reduce infection (see Section II.D.3). D. PUTATIVE ECOLOGICAL ROLES

1. Antiherbivory: evidence for and against deterrence A multitude of studies have focused on the role of phlorotannins in herbivore defence. These studies have generally utilised feeding experiments that give herbivores the choice between tissues with diVerent phlorotannin contents and artificial foods containing algal extracts or pure compounds, with only the latter method being a test of phlorotannin bioactivity. The concentration and dose of phlorotannins in the oVered food influence greatly the outcome of the algal–herbivore interaction (Pereira and Yoneshigue‐ Valentin, 1999; Targett and Arnold, 1998; Van Alstyne et al., 1999c), and the

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type and nutritional quality of the bioassay food may also be important (Deal et al., 2003; Kubanek et al., 2004). A positive deterrent eVect has not been found consistently and seems to be highly dependent not only on the selection of algae but also on the herbivore species. Even when the same herbivore species is used, the interactions mediated by phlorotannins do not always yield the same results. The numerous studies conducted using Fucus vesiculosus (Order Fucales) demonstrate these points. The gastropod Littorina littorea preferred to graze on F. vesiculosus with a lower phenolic content when oVered the choice between this and higher phenolic content foods (Geiselman and McConnell, 1981; Yates and Peckol, 1993). In contrast, Jormalainen et al. (2004) found a preference of Idotea baltica for phlorotannin (extracted from F. vesiculosus) containing food, an eVect that increased with higher concentrations (10% compared to 5%), which they suggest to be related to host recognition. Also, Hemmi et al. (2004) established that while the I. baltica preferred damaged F. vesiculosus for at least 10 days after clipping, this was not associated with diVerences in phlorotannin levels, as clipping did not induce the production of phlorotannins. Furthermore, crude polar extracts from F. vesiculosus deterred feeding by sea urchins (Arbacia punctulata), but bioassay‐guided fractionation of this extract revealed that deterrence was not due to phlorotannins (Deal et al., 2003). Feeding assays showed that even at concentrations 400% of the natural level (1% dwt), isolated phlorotannins were not an eVective deterrent to feeding by A. punctulata (Deal et al., 2003). Kubanek et al. (2004) also used bioassay‐guided fractionation techniques and found that the deterrent eVect of crude F. vesiculosus extracts was due to defensive compounds other than phlorotannins, but they were not able to isolate the bioactive compounds and did not detect the galactolipid reported by Deal et al. (2003). They tested the eVect of purified phlorotannins (at 3, 6, and 12 natural yield) on the feeding of amphipods (Ampithoe longimana) and sea urchins (A. punctilata), which normally avoid F. vesiculosus, and found no deterrent eVects. The amphipod Ampithoe valida, which is commonly known to eat F. vesiculosus in the field, was deterred only at the 12 concentration (Kubanek et al., 2004). Investigations of other taxa have also produced mixed results. Within the Fucales, Van Alstyne (1988) found that snails (Littorina sitkana) moved away from areas of wounded Fucus gardneri (as F. distichus), which showed phlorotannin accumulation, and preferred to feed on undamaged algae with lower phlorotannin contents. The isopod Idotea granulosa preferred the high phlorotannin content tissue of Ascophyllum nodosum, which may have been due to the higher nitrogen content of that tissue (Pavia et al., 1997). Feeding by the snail Littorina obtusata on Ascophyllum nodosum tissue with a high

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phlorotannin content was decreased (Pavia and Toth, 2000a; Toth and Pavia, 2000b), but feeding by I. granulosa was not aVected by phlorotannin content (Pavia and Toth, 2000a). In a subsequent study the basal stipes of A. nodosum were found to have the highest phlorotannin contents and were consumed less by L. obtusata than other tissue types (Pavia et al., 2002). Phenolics extracted from Sargassum furcatum were deterrent at 2 and 5% of food content to amphipods (Parhyale hawaiensis), but the natural concentration (0.5%) did not deter feeding, consistent with the observed palatability of this species to P. hawaiensis (Pereira and Yoneshigue‐Valentin, 1999). In contrast (Cronin and Hay, 1996a) reported that feeding by amphipods was not influenced by the phlorotannin concentration of Sargassum filipendula. In members of the Laminariales (kelps), Johnson and Mann (1986) reported that the avoidance of the intercalary meristem region of Laminaria longicruris by grazing snails (Lacuna vincta) correlated with a high phenolic content. Abalone (Haliotis rufescens) preferred to feed on phenolic poor species, and phenolics extracted from Dictyoneurum californicum deterred feeding by 90% (Winter and Estes, 1992). However, Wakefield and Murray (1998) concluded that the feeding preferences of Norrisia norrisi (herbivorous snail) were determined more by factors such as habitat and refuge provision or toughness than by phlorotannin content. Feeding preference assays revealed that the snail consistently preferred the high phlorotannin content kelps over other algal species, although comparisons within the kelps did show the least preferred species (Egregia menziesii) was that with the highest phlorotannin content, but this was also the toughest kelp species. In a study of Zonaria angusta (Order Dictyotales), amphipods (Tethygeneia sp. and Hyale rubra) preferred to consume young tissue with a low density of physodes (Poore, 1994). The amphipods preferentially consumed the area directly behind the apical cell row, which has a low density of physodes, in preference to both the single layer of physode‐dense apical meristematic cells and older tissue with moderate densities of physodes (Figs. 2 and 3). A number of studies comparing palatability across orders have also been reported. Steinberg and van Altena (1992) found that some of the Australasian herbivores they tested were deterred from feeding by extracts from some algal species, but in general they found that Australasian marine herbivores were not deterred by phlorotannins. This contrasts to North American herbivores tested (e.g., Tegula funebralis and Tegula brunnea), which were strongly deterred by the presence of phlorotannins (Steinberg, 1984, 1985, 1988; Steinberg and van Altena, 1992). Steinberg et al. (1991) showed that the concentration of phlorotannins (from tropical and temperate species) in foods could not be related to feeding deterrence of tropical herbivorous fish. Also, the herbivorous fish Cebidichthys violaceus was

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Fig. 2. Apical region of Zonaria angustata branches: intact (left) and displaying damage from amphipod feeding (center and right). On the right is a branch in which the apical cell row has been breached by amphipods with the area behind it having been consumed. The center shows a branch in which the dangling apical cell row has broken away. From Poore (1994).

Fig. 3. Distribution of physodes in a longitudinal section of the apical tissues of Zonaria angustata. Physode distribution was compiled from photomicrographs. Physodes are visible as dark‐staining subcellular bodies with high densities in the apical meristem (left) and old growth (right) and in low densities adjacent to the meristem (center). From Poore (1994).

deterred from feeding by the presence of polar extracts of the fucoid Fucus gardneri in its diet, but not by polar extracts from the kelp Macrocystis integrifolia (Ireland and Horn, 1991). Van Alstyne et al. (2001b) examined the feeding preferences of four herbivores (sea urchins and snails) towards tissue that diVered in phlorotannin concentrations (juvenile and adult tissue from six members of the Laminariales and two members of the Fucales). Although they found much variation amongst species and life stages in terms of deterrence level, phlorotannins were apparently not responsible.

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The lack of a consistent feeding response to phlorotannins has led some authors to question whether they can be considered a defensive metabolite (Jormalainen and Honkanen, 2004; Jormalainen et al., 2003) and also highlights the complex role other metabolites play in defence, a fact that is often overlooked but which can confound experimental designs (Deal et al., 2003; Kubanek et al., 2004). 2. EVects on digestion and reproduction Studies of eVects of phlorotannins on the assimilation eYciencies of herbivores, as with those on feeding deterrence, have produced results that are often ambiguous. The specific structure of the phlorotannins occurring in an algal species, in combination with diVerences in the gut environment of herbivores, is thought to be partly responsible for this confusion (see review in Targett and Arnold, 2001). Several features of the gut are important in determining the activity of ingested phlorotannins (such as morphology, pH, enzyme composition, microbial activity) and these factors diVer widely among diVerent herbivores (Targett and Arnold, 2001). The acidity of the gut may be most important in fish, with basic guts correlated with higher assimilation eYciencies (Targett and Arnold, 2001). The presence of surfactants in herbivore gut fluid has also been shown to be involved in ameliorating the eVects of phlorotannins (Tugwell and Branch, 1992). Nevertheless, phlorotannins are considered to aVect food value via their ability to complex with proteins and other macromolecules in the gut, which can aVect assimilation eYciency (Stern et al., 1996a; see review in Targett and Arnold, 2001). Polar extracts (that should include most phlorotannins) of Fucus gardneri (Order Fucales), but not of Macrocystis integrifolia (Order Laminariales), significantly reduced the ability of the fish Cebidichthys violaceus to digest a palatable green alga (Ulva lobata), in particular reducing the assimilation eYciency of nitrogen (Ireland and Horn, 1991). Shell growth of abalone (Haliotis rufescens) was inhibited by the addition of polyphenolics from Dictyoneurum californicum (Order Laminariales) into its diet (Winter and Estes, 1992). In addition, the total carbon and nitrogen assimilations of the herbivorous isopod Idotea baltica decreased with increasing phlorotannin content in an artificial food diet, although as mentioned earlier this herbivore was not deterred from feeding by higher phlorotannin content (Jormalainen and Honkanen, 2004). However, Hemmi and Jormalainen (2004) reported that the body size of female I. baltica was significantly correlated with the phlorotannin content of Fucus vesiculosus (Order Fucales) in their habitat. Also, Toth et al. (2005) found that an increased level of phlorotannins in the diet of Littorina obtusata (fed on Ascophyllum nodosum, Order Fucales) did

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not have an eVect on growth rates, but did reduce significantly the number of viable eggs that the gastropod produced. A high dietary phlorotannin content did not aVect the assimilation eYciencies of three tropical herbivores (fish Sparisoma radians and Sparisoma chrysopterum, which have a basic gut environment, or the crab Mithrax sculptus) (Targett and Arnold, 2001; Targett et al., 1995). Similarly, the growth and conversion eYciencies of the Australasian herbivores Tripneustes gratilla (sea urchin) and Turbo undulata (snail) were not aVected by feeding on a diet high in phlorotannins for several months (Steinberg and van Altena, 1992). Also, a phlorotannin‐rich diet (containing 3 natural concentration of phlorotannins from F. vesiculosus) was correlated with enhanced growth and survivorship of the amphipod Ampithoe valida (Kubanek et al., 2004). In common with vascular plant tannins, molecular size is an important determinant of the eVects of phlorotannins on herbivores. Boettcher and Targett (1993) studied the eVect of diVerent molecular size fractions on the assimilation eYciency of the fish Xiphister mucosus (which has an acidic gut environment; Targett and Arnold, 2001). They utilised force‐feeding bioassays with extracts from various temperate and tropical browns with which X. mucosus cooccurs but is not observed to eat. The high molecular size (>10 kDa) phlorotannins had a significant eVect in reducing the assimilation eYciency (total, organic and protein) of the herbivore. This is the same size class (10–100 kDa) of phlorotannins that was found in the highest concentrations in all but one of the surveyed species. Boettcher and Targett (1993) suggested that diVerences in the molecular size distribution of phlorotannins in diVerent species could be part of the explanation for such wide‐ranging results in deterrence studies. Phlorotannins do, in some cases, aVect the nutritional value of foods, but animals (including marine herbivores) can engage in compensatory feeding to overcome this eVect (Cruz‐Rivera and Hay, 2001). This may mean that some herbivores simply eat more of a particular (phlorotannin containing) species in order to meet nutritional requirements (but see Honkanen et al., 2002). In addition, some herbivores preferred phlorotannin‐containing foods (Jormalainen and Honkanen, 2004; Pavia et al., 1997). These outcomes are not consistent with the fitness benefit that should be associated with a putative defensive metabolite. 3. Antibacterial and antifouling activity The potential antifouling and antibacterial roles of phlorotannins were first proposed in the 1940s and 1960s, respectively, and many subsequent studies have investigated the validity of these roles (reviewed in Ragan and Glombitza, 1986), with results to date also being inconclusive, at least for the

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antifouling role. In addition, note the caveats about the ecological relevance of antifouling or antiepiphytic bacteria bioassays noted in Section V. Fletcher (1975) reported that phlorotannins in the crustose alga Ralfsia spongiocarpa (Order Ectocarpales) were eVective in protecting the thallus from epiphytic fouling. In studies of members of the Fucales, Sieburth and Conover (1965) found that extracts containing Sargassum natans polyphenolics acted as an antibiotic. Phlorotannins extracted from Sargassum tenerrimum inhibit bacterial growth as well as larval settlement of polychaetes (Lau and Qian, 1997) and of barnacles (Lau and Qian, 2000) at ecologically relevant concentrations. Phlorotannins from Fucus spiralis and Ascophyluum nodosum aVected the survival and therefore the habitat choice of the ciliate Voticella marina (Langlois, 1975). A study by Wilkstro¨ m and Pavia (2004) showed that phlorotannins from Fucus vesiculosus inhibit settlement of the fouling barnacle Balanus improvisus at ecologically relevant concentrations (1 mg liter1). The second species they studied was Fucus evanescens, an invasive species, which had quantitatively much lower natural levels of fouling that the native F. vesiculosus. However, in settlement bioassays, Balanus improvisus settled on F. evanescens at higher rates when given a choice between the two species. The authors found that whilst the phlorotannin concentration of the thallus did not diVer between the two species (about 10% dwt in both), the inhibitory eVect of phlorotannins from F. vesiculosus was greater and eVective at lower concentrations than those extracted from F. evanescens. Contradictory results from field experiments (i.e., lower fouling on F. evanescens) and laboratory work (i.e., phlorotannins from F. vesiculosus more eVective antifouling) indicated that the higher eVectiveness of antifouling mechanisms in F. evanescens was not due to settlement inhibition by phlorotannins, but was due to a higher postsettlement mortality (Wilkstro¨ m and Pavia, 2004). The high level of fouling on F. vesiculosus indicates that while phlorotannins from this species can deter larval settlement, other factors are responsible for juvenile mortality. Furthermore, this demonstrates that the settlement inhibition eVect of phlorotannins is not constant even among congeneric species. In another study of F. vesiculosus, Honkanen and Jormalainen (2005) found that the variation in fouling biomass levels, which they observed on diVerent genotypes, was not correlated with tissue phlorotannin content. Lu¨ der and Clayton (2004) suggested that the presence of increased numbers of physodes at wound surfaces in Ecklonia radiata (Order Laminariales) may serve to reduce microbe infection. Molecular size may be an important factor in determining the bioactivity of phlorotannins, including antibiotic roles (Ragan and Glombitza, 1986). By contrast, Jennings and Steinberg (1997) concluded that natural concentrations of phlorotannins at the thallus

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surface of Ecklonia radiata were unlikely to be able to eVectively reduce epiphytism by the green alga Ulva lactuca and found no correlation between epiphyte load and tissue phlorotannin content. Their study of phlorotannin exudation rates in E. radiata found these to be much lower than rates reported in other studies, which mainly utilised stressed algae (Jennings and Steinberg, 1994, 1997). An inhibitory eVect of phlorotannins from E. radiata and Sargassum vestitum (Order Fucales) on the germination of U. lactuca was found, but only at unnaturally high concentrations (>10 mg liter1) (Jennings and Steinberg, 1997). The authors highlighted the importance in these types of studies of measuring or estimating concentrations of phlorotannins where the encounter occurs (i.e. at the boundary layer) and which the larvae or epiphytes are actually likely to encounter so that measuring tissue content is probably not appropriate for studies of epiphytism (Jennings and Steinberg, 1997). 4. Sunscreen Depletion of the ozone layer over the past few decades has led to higher levels of damaging UV‐B radiation reaching shallow water benthic communities (Frederick et al., 1998), which has led to concern over the eVects of UV‐B on benthic organisms (Karentz, 2001; Karentz and Bosch, 2001). Phlorotannins absorb in the UV‐B range of the spectrum (Pavia et al., 1997) and are considered to be one way in which the algal thallus can protect itself from photodestruction caused by UV radiation (reviewed in Schoenwaelder, 2002). A 2‐week exposure to increased UV‐B radiation led to a significant increase in the phlorotannin content of Ascophyllum nodosum (Order Fucales) (Pavia et al., 1997). In a subsequent experiment, Pavia and Brock (2000) found that A. nodosum exposed to ambient UV‐B increase phenolic content after 7 weeks (but not after 2 weeks) in comparison to individuals that had had UV‐B removed from the available spectrum. However, in a field study involving a smaller increase in UV‐B radiation, no increase was found in phlorotannin content in Desmarestia anceps or in Desmarestia menziesii (Order Desmarestiales; V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result). Swanson and Druehl (2002) reported that tissue phlorotannin content increased in Macrocystis integrifolia (Order Laminariales) after exposure to UV‐B radiation (but also UV‐A, which phlorotannins do not absorb well). Phlorotannin exudate from M. integrifolia reduced transmission of UV‐B radiation and also reduced the harmful eVects of UV‐B on developing kelp meiospores (germination rates were higher when water contained exudate) (Swanson and Druehl, 2002). The authors suggested that in regions with high kelp densities the concentration of phlorotannins in the water body

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C. D. AMSLER AND V. A. FAIRHEAD

could alter the spectral characteristics and shield marine organisms from biologically harmful UV‐B (Swanson and Druehl, 2002). However, UV-B did not affect phlorotannins in Fucus gardneri germlings (Order Fucales; Henry and Van Alstyne, 2004). 5. Heavy metal chelation The capacity of phlorotannins to bind to metal ions is well established, which has led to the suggestion that phlorotannins may be able to reduce the toxicity of certain heavy metal pollutants (reviewed in Ragan and Glombitza, 1986; Toth and Pavia, 2000a), but very little work has been done in this area. Ragan et al. (1979) found that phlorotannins are particularly good at chelating divalent metal ions such as copper and lead. The toxicity and anthropogenic release of heavy metals into the oceans means that an ability to detoxify both the cell (e.g., Skipnes et al., 1975; Smith et al., 1986) and the surrounding environment (e.g., Ragan et al., 1980) would be advantageous for the marine system. However, Toth and Pavia (2000a) concluded that probably substances other than phlorotannins (e.g., polysaccharides and/or phytochelatins) are important for the detoxification and resistance to copper accumulation in Ascophyllum nodosum (Order Fucales). Increasing the copper concentration in the surrounding water resulted in accumulation of copper in the algal tissues (without aVecting growth rates), but not in an increase in phlorotannin content. Karez and Periera (1995) reported that a majority of the copper, lead, cadmium, zinc, and chromium in Padina gymnospora (Order Dictyotales) coextracted with phlorotannins and hypothesised that this was because they were chelated by the phenolics. E. SPATIAL AND INTRA/INTERSPECIFIC VARIABILITY

The concentration of phlorotannins has been found to vary from undetectable levels to up to 20% of thallus dry weight, varying within and between species, as well as showing spatial variation on just about every scale (for reviews, see Ragan and Glombitza, 1986; Van Alstyne et al., 2001a). Phlorotannin content has been found to vary within the thallus of an individual (Fairhead et al., 2005a; Pavia et al., 2003; Pfister, 1992; Poore, 1994; Steinberg, 1984; Toth and Pavia, 2002b; Tugwell and Branch, 1989; Tuomi et al., 1989; Van Alstyne et al., 1999a), between individuals in the same population (Fairhead et al., 2005a; Pavia et al., 2003), between diVerent sites ˚ berg, 1996; Pavia et al., 2003; Steinberg, 1989; and populations (Pavia and A Stiger et al., 2004; Targett et al., 1992; Toth and Pavia, 2000a; Van Alstyne et al., 1999a,b), across depths (Connan et al., 2004; Fairhead et al., 2005a; Martinez, 1996), between diVerent species (Fairhead et al., 2005a; Ragan

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and Glombitza, 1986; Steinberg, 1989; Steinberg and van Altena, 1992; Tugwell and Branch, 1989; Van Alstyne et al., 1999a,b; Stiger et al., 2004), between diVerent aged tissue (Pavia et al., 2003; Toth and Pavia, 2002b), across seasons (Connan et al., 2004; Stiger et al., 2004), and across large geographical areas (Steinberg, 1989; Targett et al., 1992; Van Alstyne and Paul, 1990; Van Alstyne et al., 1999b). Because the bioactivity of phlorotannins is often concentration dependant, this high degree of variation in phlorotannin content has important consequences for mediating the interactions between herbivores and brown macroalgae (Targett and Arnold, 2001; Van Alstyne et al., 2001a). Biogeographical patterns in phlorotannin content exist, with Australasian brown algae tending to have, on average, concentrations over five times higher than those from the northeast Pacific (Steinberg, 1989; Steinberg et al., 1995). The ecological and evolutionary consequences of this variation for algal–herbivore interactions are reviewed in Van Alstyne et al. (2001a). There is also evidence for a tropical‐temperate pattern, with tropical brown algae often containing low levels of phlorotannins (14%) and that all the species surveyed had concentrations >2% dwt. A later study broadened the geographical range of high phlorotannin concentration phaeophytes to the western tropical Atlantic (Targett et al., 1995). Other studies of members of the Fucales and Laminariales have found within‐species diVerences in the concentration of phenolics across diVerent regions separated by tens to hundreds of kilometres, such as the southern and eastern coasts of Australia and northern New Zealand (Cystophora moniliformis and Ecklonia radiata; Steinberg, 1989), the eastern coast of North America (Fucus vesiculosus; Target et al., 1992) and the western U.S. coastline (a variety of kelps and fucoids, Van Alstyne et al., 1999b). For other species, smaller scales (