MARINE ECOLOGY PROGRESS SERIES I Published February 28 Mar Ecol Prog Ser
Extrinsic factors influencing phlorotannin production in the brown alga Ascophyllum nodosum
Henrik Pavia*, Elisabet Brock
Department of Marine Botany, Goteborg University, Box 461,40530 Goteborg, Sweden
ABSTRACT: Many brown seaweeds contain high concentrations of phlorotannins (polyphenolics) that show extensive phenotypic variation, which can be generated by variation in a number of biotic and abiotic extrinsic factors. Extrinsically induced changes in the phlorotannin content of brown macro- algae can have significant consequences for secondary production in nearshore marine communities since they will affect the palatability and degradability of a dominating type of macroalgal tissue. In the present study the separate and interactive effects of natural herbivory and 3 abiotic factors on phlorotannin production in the brown alga Ascophyllum nodosum were tested in 2 different induction expenments. In the first experiment the hypothesis that grazing by the gastropod Littorina obtusata, nltrogen enrichm.ent, and enhanced or ambient UV-B radiation (UVBR) can affect the phlorotann~n content of A. nodosum was tested, and in the second experiment the effects of ambient UVBR and a variable water level (desiccation)were examined. In the first experiment it was found that 3 wk of graz- ing by L. obtusata caused a significant increase in the phlorotannin content of the algae and this effect was independent of the nitrogen and UVBR treatments which had no effects on phlorotannin levels. This is one of few demonstrated examples of induced chemical responses in seaweeds caused by nat- ural herbivory. Measurements of biomass changes and tissue nitrogen content of the experimental algae showed that the nitrogen uptake of the algae was unaffected by herbivory and UVBR, and that the growth of A. nodosum plants was nitrogen-limited but not affected by UVBR. In the second exper- iment there was a significantly higher phlorotannin content in algae exposed to natural UVBR, com- pared to no UVBR, after 7 wk, but not after 2 or 4 wk. Furthermore, there was a significant interaction effect between the UVBR and the water level treatment. The phlorotannin content was higher in algae exposed to a variable water regime compared to continuously submerged algae when UVBR was fil- tered out, but not under ambient UVBR. In summary, the results of this study support the induced defence model (IDM),but not the carbon-nutrient balance model (CNBM),in explaining intraspecific variation In the phlorotannin content of A nodosum. The results also support the previously proposed hypothesis that phlorotannins play a role as inducible screens aga~nstharmful UV radiation.
KEY WORDS: Brown algae - Ascophyllum . Herbivory . Inducible chemical defence . Liltorina obtusata - Nitrogen . Phlorotannins . Polyphenolics . UV-B radiation
INTRODUCTION intensity of these extrinsic factors most likely causes much of the observed intraspecific phenotypic varia- Production of secondary metaboli.tes in plants is gen- tion in secondary metabolite content in natural pop- erally a plastic trait that can be influenced by a number ulation~of both terrestrial plants (Bazzaz et al. 1987, of biotic and abiotic extrinsic factors (Waterman & Herms & Mattson 1992, Waterman & Mole 1994, Kar- Mole 1989). Spatial and temporal variation in the ban & Baldwin 1997) and marine macroalgae (Hay & Fenical 1992, Targett et al. 1992). Many brown macro- algae contain substantial concentrations of polyphe- 'E-mail: henrik.pavia@marbot,gu.se nolic secondary metabolites known as phlorotannins
O Inter-Research 2000 Resale of fullarticle not permitted Mar Ecol Prog Ser 193: 285-294, 2000
(polypl~loroglucinolphenolics). The phlorotannin con- sis) and essential nutrients, e.g. nitrogen. Although the tent of brown seaweeds can show extensive intra- IDM and the CNBM differ in some of their basic specific variation (see reviews by Ragan & Glombitza assumptions and give alternative explanations for phe- 1986, Steinberg 1992, Targett & Arnold 1998) which notypic variation in secondary metabolites of plants, may be generated by variation in extrinsic factors such they are not mutually exclusive models (Berenbaum as nitrogen availability (Yates & Peckol 1993, Arnold et 1995). Evaluations of their relative importance require al. 1995, Peckol et al. 1996), solar radiation (H. Pavia factorial experiments where both herbivory intensity unpubl.), ultraviolet-B radiation (UVBR; 290 to 320 nm) and resource availabihty are manipulated simultane- (Pavia et al. 1997), and physical damage (Van Alstyne ously (see Peckol et al. 1996). 1988, Yates & Peckol 1993, Hammerstrom et al. 1998). In the present study we tested for the separate and Phlorotannins have been shown to deter herbivory by interactive effects of extrinsic factors on phlorotannin fishes, urchins and mesoherbivores, although their production in the brown alga Ascophylluni nodosum effectiveness as herbivore deterrents is highly variable (L.)Le Jol. on the Swedish west coast. Previous studies (see Hay & Steinberg 1992, Targett & Arnold 1998 for in the study area have shown that A. nodosum contains reviews). Changes in phlorotannin production caused high concentrations (4 to 15% of dry weight [DW]) of by extrinsic factors may consequently affect marine phlorotannins with extensive variation among individ- plant-animal interactions by changing the susceptibil- ual plants at different spatial scales and among years ity of brown seaweeds to herbivory (e.g. Cronin & Hay (Pavia & Aberg 1996, Pavia et al. 1999b). Tlt7o factorial 1996). Extrinsically induced alterations of phlorotan- experiments were done to examine the effects of 4 dif- nin levels may also have significant consequences for ferent extrinsic factors on the phlorotannin content of marine communities in coastal waters, since brown A. nodosum. In the f~rstexperiment we tested the macroalgae are important sources of detritus in these hypothesis that natural grazing by the gastropod Wtto- habitats and changes in the phlorotannin content can rind obtusata (L.),nitrogen availability and 1!VBR can, strongly affect the quality of the algal detritus as a food separately or interactively, induce changes in the source for detritus feeders (Duggins & Eckman 1997). phlorotannin content of A. nodosurn. We used natural In natural populations of brown algae, phlorotannin grazers ]rather than herbivory simulat~ons,since clip- production is likely to be affected by complex interac- ping damages have previously failed to induce phloro- tions between several biotic and abiotic factors. Exper- tannin responses in A. nodosum (Lowell et al. 1991, imental tests of the occurrence and magnitude of such Pavia et al. 1997). Moreover, studies on vascular plants interactions require that treatments be applied simul- have sometinles shown that lllechanical sinlulations of taneously in factorial combinations, something which herbivory through clipping do not adequately mimic has only rarely been done in studies on phlorotannin natural herbivory, with regard to induced changes in production (see Cronin & Hay 1996, Peckol et al. 1996, secondary metabo1is.m (see reviews by Baldwin 1990, Pavia et al. 1997). In the only example, as far as k7e Karban & Baldwin 1997). Nitrogen availability was know, demonstrating interactive effects of extrinsic chosen as a potentially interesting extrinsic factor, factors on phlorotannin production, Fucus vesiculosus since nitrogen is commonly the most limiting nutrient pldnts were exposed simultaneously to simulated her- for the growth of marine macroalgae (Chapman & bivory (clipping) and manipulations of nitrogen avail- Craigie 1977, Hanisak 1983, Lobban & Harrison 1994), ability (Peckol et al. 1996). The results showed that and the CNBM would thus pred~ctthat the concentra- both clipping and nitrogen availability affected phloro- tion of phlorotannins would decrease with increasing tannin production and that the effect of clipping was, nitrogen availability. Furthermore, nitrogen enrichment at times, dependent on nitrogen availability, in support experiments with other brown algae have revealed of both the induced defence model (IDM)(e.g. Karban that nitrogen availability can affect phlorotannin pro- & Myers 1989, Harvell 1990) and the carbon-nutrient duction (Yates & Peckol 1993, Arnold et a1 1995, balance model (CNBM) (Bryant et al. 1983, 1989), 2 of Peckol et al. 1996, but see Cronin & Hay 1996). The the dominant models for explaining ~ntraspecificphe- UVBR treatment was included because addition of notypic variation in secondary metabolites of plants. UVBR has previously been shown to induce increased The IDM is based on the assumption that defences are phlorotannin levels in A. nodosurn (Pavia et al. 1997). costly, and predicts that plants should allocate more In the second experiment we tested the hypothesis that resources to the production of costly defences when the 2 factors. UVBR and water level (desiccation), can, herbivores are present and active. The CNBM focuses separately or interactively, induce changes in the on resource availability rather than herbivore activity, phlorotannin content of A. nodosum. Desiccation stress and predicts that the production of carbon-based sec- has been shown to influence production of other ondary metaboliles such as tannins will be governed defence chemicals in 1)rown seaweeds (Renaud et al. by the relative supply of carbon (through photosynthe- 1990, Cronin & Hay ISStil, but the effect of desiccation, Pavia & Brock: Phlorotannin prc ducti ion in AscophyUum nodosum 287
or its interactive effects with ambient UV light, on the cooling basin (6 X 2.5 X 0.5 m) and filled with filtered production of phlorotannins has not been previously (cotton cartridge filters, 100 and 5 pm) surface sea- explicitly tested. We hypothesized that UV light and water which was exchanged every morning. After desiccation would be likely to affect phlorotannin pro- weighing, the A. nodosum plants were randomly and duction of the algae interactively because algae that separately placed in the aquaria and anchored at the are periodically air-exposed, and not continuously bottom to wooden pegs fastened in cement weights. submerged in UV-absorbing seawater, are exposed to Each alga was exposed to one of 12 factorial combina- higher levels of UV radiation. tions of treatments (includng controls), i.e. there were 4 replicate aquana for each treatment combination. For the herbivory treatment 20 Littorina obtusata snails MATERIAL AND METHODS were placed in each of 24 aquaria. The UVBR treat- ment included both addition and filtering of UVBR, as Study site and organisms. The experiments were well as ambient UVBR from solar radiation. To screen carried out in 1996 and 1997 at Tjarno Marine Biologi- out UVBR, 16 of the aquana were covered wth Mylar cal Laboratory on the Swedish west coast. Field collec- film (Dupont, Mylar D). The Mylar film has a sharp tions of algae and herbivores were done in Ascophyl- wavelength cut-off with 50 % transmittance at 320 nm lum nodosum populations on several small islands in and =g0 % transmittance in the photosynthetically the archipelago around the field station. A. nodosum is active radiation (PAR) range (Wangberg et al. 1996). a long-lived seaweed that grows on sheltered rocky Each of the 16 aquaria with enhanced UVBR were shores in the intertidal zone on the coasts of the North- equipped with a battery-powered UVBR fluorescent ern Atlantic. At shores with large tidal amplitudes, A. tube (Philips TL 4W/12). To filter out wavelengths nodosum plants can form large, almost monospecific <295 nm, the fluorescent tubes were covered with cel- stands. Because of the small tidal range ($0.3 m) at the lulose diacetate f~lrns(Erik S. Ekman AB, Stockholm) Swedish west coast, the vertical distribution of A. whlch were exchanged every 5 d. The tubes were nodosum is limited and the width of the A. nodosum mounted at a distance of 10 cm from the water surface zone is 3 to 4 m max. and usually
gen or UVBR), aquaria with enhanced nitrogen (with- 18 d, with no single period of desiccation lasting more out herbivores or UVBR manipulations) and aquaria than 48 h. The weather was clear and sunny on 26 out with herbivores (without manipulations of nitrogen or of 49 d. To screen out ambient UVBR, 18 of the aquaria UVBR). Samples were taken from the aquaria with L. were covered with Mylar films which were exchanged obtusata to test for possible effects on nitrogen levels every 5 d. Samples for phlorotannin analyses were col- caused by the newly produced faeces of the gastropods lected after 2, 4 and 7 wk, with one-third of the aquaria (faeces were rinsed out every morning when water being sampled each time to obtain independent data was exchanged). Ammonium and nitrate concentra- for each sampling time. The tissue samples (apical tions were analyzed on the day of sampling according shoots) were freeze-dried, ground to a fine powder in to the method described in Carlberg (1972). a mortar, extracted and analyzed using the Folin- The experiment was run for 21 d, after which the Ciocalteus method as described above. WW changes algae were collected and their WW changes were were determined for the 12 algae remaining on the last recorded. Tissue samples (apical shoots) for phlorotan- sampling date. nin analyses were immediately frozen at -80°C, while Statistical analyses. Data were analyzed using dif- samples for nitrogen analyses were weighed, dried for ferent models of analysis of variance (ANOVA). Prior 48 h at 60°C, and weighed again to determine individ- to the statistical analyses, data were tested for homo- ual DW to W ratios. For phlorotannin analyses the geneity of variances with Cochran's C-test (Winer et al. frozen samples were ground to a fine powder with liq- 1991, Underwood 1997). Multiple mean comparisons uid nitrogen in a mortar, placed in aqueous acetone were made with the Student-Newman-Keuls (SNK) (60 %) and shaken under nitrogen in the dark at 4°C for procedure (Winer et al. 1991, Underwood 1997). In 24 h. After extraction the samples were centrifuged, the first experiment, data on algal WW changes and evaporated in vacuo at less than 40°C to a small aque- phlorotannin and tissue nitrogen content were ana- ous volume, filtered to remove precipitated lipophilic lyzed using 3-way ANOVAs with herbivory, UVBR and material, and diluted to a known volume. Phloro- nitrogen as fixed, orthogonal factors. In the second tannins were quantified colourimetrically using the experiment, data on phlorotannin content were ana- Folin-Ciocalteus method (Waterman & Mole 1994, Van lyzed using a 3-way ANOVA with UVBR, water level Alstyne 1995). Phloroglucinol (1,3,5-trihydroxyben- and time as fixed orthogonal factors. Data on WW zene, Merck art. 7069) was used as a standard. Sam- changes at the last sampling time of the second exper- ples for tissue nitrogen content were ground with a iment were analyzed by a 2-way ANOVA with UVBR ball mill and analyzed in an elemental analyzer and water level as fixed, orthogonal factors. (FISONS Instruments NA 1500 NC). Expt 2. For the second induction experiment 36 healthy Ascophyllum nodosum plants of similar size RESULTS (-50 g W) were collected in May 1997 and handled the same as the algae in the first induction experiment. Expt l However, instead of daily exchange of seawater, the 36 Plexiglas aquaria were continuously supplied with the Nitrogen analyses of seawater in the aquaria showed filtered surface seawater through a tube system. The low ambient nitrogen concentrations, with concentra- algae were continuously submerged in 18 of the tions of nitrate c0.4 pM and of ammonium c1.6 pM. aquaria, while the water level was adjusted 3 times d-' There was no effect of the presence of Littorina in the other 18 aquaria, in order to correspond to the obtusata on nitrogen levels in the aquaria (Fig. 1).The water level in the surrounding sea. The small tidal nitrate and ammonium concentrations in the aquaria range (10.3m) on the Swedish west coast results in sto- where nitrogen had been added were still high chastic, weather-induced changes in the water level, (210 PM) 6 h after nitrogen addition, with a somewhat which affect all organisms in the upper littoral zone steeper reduction in ammonium concentrations over (Johannesson 1989). A. nodosum and other fucoids can time (Fig. 1). be In or out of the water for several consecutive days, At the end of the experiment, there was a distinct depending on the prevailing barometric conditions. difference in colour between plants with and with- During high pressure m the summer months the fu- out nitrogen addition. Algae from nitrogen-enriched coids could thus experience exposure to solar radiation aquaria had a more brownish colour compared to the and extreme desiccation during periods of several brighter untreated algae. Tissue nitrogen analyses days, which can lead to severe tissue damages on the revealed that the tissue nitrogen concentration was algal thalli (H. Pavia pers. obs.). AIgae exposed to a significantly (F,,36= 228.9, p < 0.0001) higher in nitro- naturally varying water level during the 7 wk experi- gen-enriched Ascophyllum nodosum plants, with lev- ment were submerged 31 d and in the air for a total of els about twice as high in plants given extra nitrogen Pavia & Brock: Phlorotannin production in Ascophyllum nodosum 289
0 Control 1 Lirrorina obtusara
Ambient nitrogen Nitrogen ennched
Fig. 2. Tissue nitrogen content of Ascophyllum nodosum plants exposed to enriched and ambient nltrogen cond~t~ons. Error bars are +SE (n = 24)
The ANOVA for data on phlorotannin content showed that Ascophyllum nodosum plants which had been grazed by Littorina obtusata had significantly = 29.0, p < 0.001) higher phlorotannin concentra- 0 2 6 tions (Fig. 4). This effect was independent of the nitro- Time (h) gen and UVBR treatments, which had no significant
Fig. 1. Concentrations of (a) nitrate and (b) ammonium in the separate or interactive effects on phlorotannin concen- filtered seawater of experimental aquaria from 3 different trations. treatment combinations: control aquaria (without herbivores or manipulations of nitrogen or UVBR), aquaria with en- hanced nitrogen levels (without herbivores or UVBRmanipu- Expt 2 lations) and aquaria with herbivores (without manipulations of nitrogen or UVBR). Samples from t = 0 were taken before the addition of amrnoniumnitrate at a concentration of 20 pM. At the end of the 7 wk experiment the Ascophyllum Error bars are *SE (n = 3) nodosum plants had increased their WW by an aver- age of 39 % of the initial W, with no significant sepa- rate or interactive effects of the UVBR or water level (Fig. 2). There were no significant separate or inter- treatments on WW changes of the algae. active effects of herbivory or UVBR treatments on The 3-way ANOVA for phlorotannin concentration tissue nitrogen content, indicating that the nitrogen showed that there were significant interactive effects uptake of A. nodosum was not affected by these treat- between the UVBR treatment and the factor tune, and ments. between the UVBR and the water level treatment Ascophyllum nodosum plants from aquaria with Lit- (Table 1). SNK tests revealed that there were signifi- torina obtusata snails showed extensive damage, and cantly (p < 0.05) higher phlorotannin levels in the on some plants the snails had eaten through the basal parts of the primary shoots, resulting in detachment Nitrogen ennched of algal pieces of various sizes. The detached pieces T were included in the WW determination of the algae Ambient nitrogen at the end of the experiment in order to analyze WW T changes. The ANOVA showed that there was a signif- icant interactive effect between the nitrogen and her- T T bivory treatment on algal WW changes = 4.84, p = 0.034), while the UVBR treatment caused no sig- nificant separate or interactive effects. The SNK test revealed that nitrogen addition had a significantly (p < 0.05) positive effect on algal growth in the absence of No grazer Littorina obtusara grazers. Furthermore, A. nodosum plants exposed to Fig. 3. Relative change in the wet weight of Ascophyllum grazing had a significantly smaller increase in WW nodosum plants exposed to enriched and ambient nitrogen than algae without grazers, and this difference was conditions and to grazing by the gastropod Littorina obtusata. significantly larger for nitrogen-enriched algae (Fig. 3). Error bars are +SE (n = 12) 290 Mar Ecol Prog Ser 193: 285-294, 2000
10 - Variable water level 1 High water level S- r l a 8- E 5 6- c 0U .-c 4- dS 2 2- -0 go No grazer Lifforina oblusata Natural WBR No WBR Fig. 4. Phlorotannin content of Ascophyllum nodosum plants following grazing by the gastropod Littorina obtusata. Error Fig. 6. Phlorotannin content of Ascophyllum nodosum plants bars are +SE (n = 24) exposed to ambient and no (Mylar-f~lter)W-B radiation (UVBR) and to a variable and a continuously high water level. Error bars are +SE (n = 9)
DISCUSSION
0 Ambient UVBR The results of this study show that both biotic (nat- No UVBR ural herbivory) and abiotic (UVBR and variable water level) extrinsic factors can affect the phlorotannin con- tent of Ascophyllum nodosum. Three wk of grazing by Littorina obtusata resulted in a mean phlorotannin 2 4 7 concentration of 9.1% DW in grazed A. nodosum Time (weeks) plants, compared to 7.0% DW for ungrazed algae, Fig. 5. Phlorotannin content of Ascophyllum nodosum plants indicating that the IDM can be relevant for explaining following 2,4 and 7 wk of exposure to ambient and no (Mylar- intraspecific variation in the phlorotannin content of A. filter) UV-B radiation (UVBR). Error bars are *SE (n = 6) nodosum. This is 1 of few demonstrated examples of herbivory-induced production of secondary metabo- lites in marine algae (see also Van Alstyne 1988, Ascophyllum nodosum plants exposed to ambient Cronin & Hay 1996). The shortage of evidence for UVBR after 7 wk, but not after 2 or 4 wk (Fig. 5). Fur- inducible chemical responses in marine algae com- thermore, there was significantly lower phlorotannin pared to vascular plants (see Karban & Baldwin 1997) concentration in algae exposed to variable water level has been attributed to differences in herbivory, e.g. in when UVBR was screened out, but not under ambient the relative frequency of specialist and generalist her- UVBR (Fig. 6). bivores, between marine and terrestrial habitats, and to structural and functional differences between sea- weeds and vascular plants (see Hay & Steinberg 1992, Table 1. ANOVA results for the phlorotannin concentration In Steinberg 1994, Cronin & Hay 1996 for further discus- Ascophyllum nodosum plants following 2,4 and 7 wk of treat- sion). However, there are only a few studies that have ments with UV-B radiation [UVBR) filterdambient UVBR and evaluated inducible production of defence chemicals variable water IeveVcontinuously high water level in marine macroalgae for a limited selection of taxa, and most of these studies have looked for effects of Factor df MS F p artificial clipping or grazing by large and mobile herbi- UVBR 1 7.60 45.24 ~0.01 vores, i.e. fishes and large urchins. Hay (1996) sug- qested that it is most likely to be grazing by smaller, less mobile mesoherbivores (sensu Brawley & Fei UVBRX Water level 5.60 <0.05 1987) that cue for chemical induction in marine algae. UVBR X Time 6.59 <0.01 These herbivores are active over spatial and temporal Water level X Time 0.20 1.17 >0.25 UVBR X Water level X Time 0.02 0.12 >0.75 scales that would allow changes in defence chemicals Residual 26 0.17 to become effective in reducing grazing damages, even if the induction would take days or weeks to be Pavia & Brock: Phlorotannin p1 .eduction in Ascophyllum nodosum 291
manifested. The hypothesis of Hay (1996) is supported content with time in algae exposed to ambient UVBR by results from experiments on the interactions be- was more similar to natural changes than the lack of tween Fucus distichus and the gastropod Littorina significant change found in algae from aquaria with sitkana (Van Alstyne 1988), between Dictyota men- UVBR filters. It is notable that the time required (4 to strualis and the amphipod Amphitoe longimana 7 wk) for a detectable difference in phlorotannin levels (Cronin & Hay 1996, and between A. nodosum and L. to be manifested when screening out 100% of UVBR obtusata (present study, Pavia & Toth in press) was much longer than the time required (52 wk) to In the first induction experiment we found no sig- induce increased phlorotannin levels under enhanced nificant separate or interactive effects of enhanced or (-50%) UVBR (Pavia et al. 1997). This relatively slow ambient UVBR on phlorotannin concentrations in response may also explain the lack of effect on phloro- Ascophyllum nodosum. In contrast with these results, tannin production in the UVBR-screening treatment of Pavia et al. (1997) found a significant increase in the the first induction experiment, which only lasted for phlorotannin content of A, nodosum after 2 wk of expo- 3 wk. sure to increased UVBR. There are several factors that In the second induction experiment there was a sig- could possibly explain the difference between these 2 nificant interactive effect between UVBR and water experiments in the chemical response of A. nodosum to level treatments on the phlorotannin content of Asco- WBR treatments. The addition of UVBR irradiance in phyllum nodosum, but the effect was different from Pavia et al. (1997) was more than twice as intensive, what we had expected. We hypothesized that the and the daily exposure time about 3 times as long (6 vs effect of ambient UVBR on phlorotannin production 2 h), than in the present study. Furthermore, the exper- should be more pronounced when the water level was imental algae in Pavia et al. (1997) were placed in the variable and the algae were periodically out of UV- sea with natural variation in water level and the ex- absorbing seawater. The results, however, showed that periment was conducted during another season (early under ambient WBR there was no difference in the spring instead of summer), with generally lower phloro- phlorotannin content between algae exposed to a vari- tannin concentrations in the experimental algae (4.8 vs able water level and algae which were continuously 7.0% DW). The first induction experiment of the pre- submerged in seawater. In contrast, there was a statis- sent study was preceded by a period of several weeks tically significant, although relatively modest (4.6 vs with clear weather and many hours of sunlight during 3.9% DW) difference when WBR was filtered out, most of the days, and any light-induced production of with higher phlorotannin content in A, nodosum plants phlorotannins in the algae could have been already exposed to a variable water level. We can only specu- activated before the start of the experiment. The sec- late about the causes of this difference, e.g. that algae ond experiment of the present study started in early under a variable water level regime had a slightly May and the algae had relatively low initial phlorotan- higher carbon:nutrient balance, due to reduced nutri- nin levels (-4.5% DW). After 2 and 4 wk the phlorotan- ent uptake, which could have resulted in a somewhat nin levels in A, nodosum plants exposed to ambient higher production of phlorotannins, in accordance with UVBR were only slightly and not significantly higher the CNBM. In algae exposed to UVBR, any such rela- than in algae from aquaria with UVBR filters, but after tively subtle effect may have been masked by the need 7 wk the difference was substantial (5.9 vs 4.5 % DW) to produce UV-absorbing metabolites. and statistically significant. This result gives further The herbivory-induced increase of phlorotannin lev- support to the hypothesis of Pavia et al. (1997) (see also els in Ascophyllum nodosum was independent of nitro- Berthold 1882) that phlorotannins may function as gen availability. In the absence of grazers there was inducible screens against UV radiation, even though also a lack of effect of nitrogen enrichment on phloro- their absorption maxima are at wavelengths (260 to tannin production in A. nodosurn, in contrast with the 280 nm) somewhat shorter than incident UV radiation. predictions of the CNBM and with the results of previ- Unfortunately, changes in phlorotannin levels in nat- ous studies on other brown algal species showing that ural populations of A. nodosurn were not monitored nitrogen availability can affect phlorotannin levels during the course of the second experiment, but data (Ilvessalo & Tuorni 1989, Yates & Peckol 1993, Arnold from other years in the study area imply that phloro- et al. 1995, Peckol et al. 1996, but see Cronin & Hay tannin concentrations in A. nodosum are generally low 1996). The results of the present study alone cannot (-4 to 5 % DW) in April-May, and increase up to levels justify the conclusion that nitrogen availability or the >8% in July-September, although with substantial CNBM are irrelevant for explaining intraspecific vari- small-scale spatial variation and moderate among-year ation in the phlorotannin content of A. nodosum, since variation (H. Pavia unpubl., see also Ragan & Jensen the effects of nutrient enrichment on secondary metab- 1978 for data on seasonal variation in an adjacent olism could be dependent on other environmental fac- area). This implies that the increased phlorotannin tors not tested in the present study, and also on the 292 Mar Ecol Prog Ser 193: 285-294, 2000
developmental stage of the plant (Yates & Peckol 1993, defence chemicals without affecting growth processes Peckol et al. 1996). Given the pronounced seasonality is smaller or lacking (Herms & Mattson 1992). The of environmental conditions in the study area, the results of the first induction experiment fit these theo- result might have been different if the induction exper- retical predictions. Assuming that the amount of algal iment would have been conducted in another season tissue consumed by L. obtusata was relatively small, with different light, temperature and ambient nutrient the interactive effect between the herbivory and nitro- conditions. The lack of any effect of nitrogen availabil- gen treatments could reflect a higher cost, in terms of ity on phlorotannin production in A, nodosum is, how- growth, for the herbivory-induced phlorotannin pro- ever, also supported by the absence of a relationship duction in nitrogen-enriched algae, compared to algae between the concentrations of tissue nitrogen and under low nitrogen levels. Unfortunately, there is no phlorotannins in A. nodosum plants from the Tjarno rigorous way, a posteriori, of separating the relative area in spring (Pavia et al. 1997). Furthermore, in a 2 yr importance of actual algal tissue consumption by the correlative field study comprising chemical analyses of herbivores, and any trade-off between phlorotannin 300 A. nodosum plants from the Tjarno area and from production and growth, for the observed pattern of the Irish Sea, it was found that the relationship be- algal biomass changes. This would require a measure tween tissue nitrogen and phlorotannins was weak of herbivore consumption that is independent of algal and variable (Pavia et al. 1999b). Together, these biomass changes in the experiment, e.g. the quantity results suggest that nitrogen availability is not a good of faeces produced or the amount of tissue damages on predictor of the phlorotannin content of A. nodosum. the algae. We made no rigorous estimations of the The measurements of algal WW changes during the amount of grazing damages on the algae in the present experiments clearly showed that the growth of the experiment, but there was no apparent difference Ascophyllum nodosum plants in the aquaria was nitro- between the 2 nitrogen treatments. This visual obser- gen-limited, whle there was no apparent effect of the vation is supported by the almost equal mean amounts UVBR or water level treatments on algal growth. The of detached algal pieces from the grazed A. nodosum statistical analysis of data on WW changes in the first plants in aquaria with and without added nitrogen (8.8 experiment also revealed a significant interaction vs 8.7 g WW per aquaria). A significantly larger pro- effect between the herbivory and nitrogen treatments. portion of detached algal parts would have been The post hoc comparisons of mean values showed that expected in the aquaria with enhanced nitrogen levels, in the absence of grazers, nitrogen-enriched A. no- if these algae were exposed to more intense grazing dosum plants grow substantially more than algae than algae which were not given additional nitrogen. under ambient nitrogen levels, but in the presence of These findings could thus imply that the interactive Littorina obtusata snails there was no difference in effect of herbivory and nitrogen treatments on algal WW changes between different nitrogen treatments. WW changes is, at least partly, an indirect effect of the This effect could reflect the fact that the feeding rate of induced increase in phlorotannin production. L. obtusata snails were positively affected by increased In summary, the results of the present study show tissue nitrogen content of A. nodosum. If so, it would that both biotic and abiotic factors can affect phloro- imply that when herbivores are abundant, the effect of tannin production in Ascophyllum nodosum, and they increased nutrient availability may not have a positive provide support to the IDM, but not to the CNBM, as a net effect for the growth of A. nodosum plants. There relevant model for explaining intraspecific variation in is, however, an alternative model to explain the ob- phlorotannin content in natural populations of A. served interaction effect between the nitrogen and nodosum. The results also give further support to the herbivory treatments on the WW changes of the A. hypothesis of Pavia et al. (1997),that UV radiation can nodosum plants. The IDM and other optimalitv models be an important factor in explaining phenotypic varia- for the evolution of defensive characters are based on tion in phlorotannin content of natural populations of the assumption that these characters are costly to pro- intertidal brown seaweeds. The observed differences duce (e.g. Rhoades 1979, Fagerstrom et al. 1987, in phlorotannin levels caused by gastropod grazing as Simms & Rausher 1987). A few correlative studies pro- well as UVBR are probably large enough to affect the vide support for the assumption that phlorotannin pro- feeding behavior of Littorina snails. Van Alstyne (1988) duction is costly, in terms of individual qrowth, for found that a -20% increase in the phlorotannin con- brown algae (Yates & Peckol 1993, Steinberg 1995), tent of Fucus djstichus reduced grazing by L. sitkana including A. nodosum (Pavia et al. 1999b). Theory by -50%, and experiments with A. nodosum and L. predicts that the cost of production of carbon-based obtusata showed that a 20 to 40% difference in defence chemicals, such as phlorotannins, will be rela- phlorotannin concentrations of algal shoots signifi- tively higher in nutrient-rich environments, since the cantly affected the feeding preferences of the gastro- excess of carbon that can be used for production of pods (Pavia & Toth in press). From studies in terrestrial Pavia & Brock Phlorotannin pr oduction in Ascophyllum nodosum 293
communities, there is substantial evidence that ex- pling and analyses of physical, chemical and biological trinsic factors can influence production of secondary parameters. ICES Coop Res Rept Ser A V0129 metabolites in vascular plants and that these changes Chapman ARO, Craigie JS (1977) Seasonal growth in Lami- naria longicruris: relations with dissolved inorganic nutri- can have important secondary consequences by af- ents and internal reserves of nitrogen. Mar Biol 40: fecting the palatability and degradability of plant 197-205 tissue (see reviews by Waterman & Mole 1989, Rozema Cronin G, Hay M (1996) Induction of seaweed chemical et al. 1997). In marine communities much less is known defenses by amphipod grazing. Ecology ?7:2287-2301 Duggins DO, Eckman JE (1997) Is kelp detritus a good food about the effect of extrinsic factors on secondary for suspension feeders? Effects of kelp species, age and metabolism of seaweeds and its importance for the secondary metabolites. Mar Biol 128:489-495 nearshore foodweb (Hay 1996). Brown algae, espe- Fagerstrom T, Larsson S, Tenow 0 (1987) On optimal defence cially fucoids, are good candidates for more studies on in plants. Funct Ecol 1:73-81 these issues since they are important components of Harnmerstrom K, Dethier MN, Duggins DO (1998) Rapid phlorotannin induction and relaxation in five Washington coastal marine communities and they contain sub- kelps. Mar Ecol Prog Ser 165:293-305 stantial and highly variable concentrations of phloro- Hanisak MD (1983) The nitrogen relationships of marine tannins. macroalgae. In: Carpenter EJ, Capone DC (eds) Nitrogen in the marine environment. Academic Press, New York, p 699-730 Acknowledgements. We thank Inger Wallentinus. GuniUa Harvell CD (1990) The ecology and evolution of inducible Toth and Per Aberg for comments that improved the manu- defenses. 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Editonal responsibility: Otto finne (Editor), Submitted: March 12, 1999; Accepted: September 6, 1999 Oldendorf/Luhe, Germany Proofs received from author(s): January 28, 2000