Mar Biol (2008) 15551ndash62

DOI 101007s00227-008-1006-z

ORIGINAL PAPER

Species-speciWc defense strategies of vegetative versus reproductive blades of the PaciWc kelps Lessonia nigrescens and Macrocystis integrifolia

Christian Pansch middot Ivan Goacutemez middot Eva Rothaumlusler middot Karina Veliz middot Martin Thiel

Received 12 November 2007 Accepted 16 May 2008 Published online 3 June 2008copy Springer-Verlag 2008

Abstract Chemical defense is assumed to be costly andtherefore algae should allocate defense investments in away to reduce costs and optimize their overall Wtness Thuslifetime expectation of particular tissues and their contribu-tion to the Wtness of the alga may aVect defense allocationTwo brown algae common to the SE PaciWc coasts Lesso-nia nigrescens Bory and Macrocystis integrifolia Bory fea-ture important ontogenetic diVerences in the developmentof reproductive structures in L nigrescens blade tissuespass from a vegetative stage to a reproductive stage whilein M integrifolia reproductive and vegetative functions arespatially separated on diVerent blades We hypothesizedthat vegetative blades of L nigrescens with importantfuture functions are more (or equally) defended than repro-ductive blades whereas in M integrifolia defense shouldbe mainly allocated to reproductive blades (sporophylls)

which are considered to make a higher contribution toWtness Herein within-plant variation in susceptibility ofreproductive and vegetative tissues to herbivory and in allo-cation of phlorotannins (phenolics) and N-compounds wascompared The results show that phlorotannin and N-con-centrations were higher in reproductive blade tissues forboth investigated algae However preferences by amphi-pod grazers (Parhyalella penai) for either tissue typediVered between the two algal species Fresh reproductivetissue of L nigrescens was more consumed than vegetativetissue while the reverse was found in M integrifolia thusconWrming the original hypothesis This suggests thatfuture Wtness function might indeed be a useful predictor ofanti-herbivore defense in large perennial kelps Resultsfrom feeding assays with artiWcial pellets that were madewith air-dried material and extract-treated Ulva powderindicated that defenses in live algae are probably not basedon chemicals that can be extracted or remain intact after air-drying and grinding up algal tissues Instead anti-herbivoredefense against amphipod mesograzers seems to depend onstructural traits of living algae

Introduction

Herbivores can inXuence the structure of benthic algal com-munities through the consumption of large amounts of algalbiomass (Lubchenco and Gaines 1981 Carpenter 1986Vinueza et al 2006 Jormalainen and Honkanen 2008) Inresponse to this herbivore pressure macroalgae have devel-oped diVerent strategies one of which is the defense of tis-sues in ways that makes them less palatable for potentialconsumers (reviewed in DuVy and Hay 1990 Cronin2001) To reduce tissue palatability algae use defense strat-egies known as (1) structural or morphological defense

Communicated by P Kraufvelin

C Pansch middot E RothaumluslerInstitut fuumlr Biowissenschaften Lehrstuhl fuumlr Meeresbiologie Universitaumlt Rostock Albert-Einstein-Str3 18057 Rostock Germany

I Goacutemez middot K VelizInstituto de Biologiacutea Marina Facultad de Ciencias Universidad Austral de Chile Casilla 567 Valdivia Chile

M Thiel (amp)Departamento de Biologiacutea Marina Facultad de Ciencias del Mar Universidad Catoacutelica del Norte Larrondo 1281 Coquimbo Chilee-mail thielucncl

M ThielCentro de Estudios Avancados en Zonas Aridas CEAZA Coquimbo Chile

123

52 Mar Biol (2008) 15551ndash62

eg the calciWcation of tissue (Littler and Littler 1980) (2)chemical defense eg synthesis and accumulation ofunpalatable compounds (Amsler and Fairhead 2006) and(3) nutritional defense eg algae are less palatable due totheir low nutritional quality (Lubchenco and Gaines 1981DuVy and Paul 1992)

Many studies have been conducted on algal chemicaldefense Deterrent compounds are usually produced via thesecondary metabolic pathway (Maschek and Baker 2008)and numerous examples have conWrmed that a variety ofsubstances can eYciently deter diVerent grazers (reviewedin Cronin 2001 Amsler and Fairhead 2006) For examplediterpenes and phlorotannins the most investigated groupsof metabolites isolated from brown algae (Maschek andBaker 2008) were identiWed to act both as anti-herbivoryand anti-fouling substances (summarized in Amsler andFairhead 2006) Phlorotannins have also been invoked toserve or contribute to other vital functions such as protec-tion against UV-radiation cell-wall formation and cytoki-nesis (Pavia et al 1997 Schoenwaelder and Clayton 1999Schoenwaelder 2002) On the other hand herbivores areconsidered to be mainly N-limited and therefore selectivein foraging for N-rich algal tissue (Mattson 1980 DuVy andPaul 1992 Cruz-Rivera and Hay 2000) Consequentlychemical defense as well as nutritional status of algae willinXuence the feeding preferences of herbivores (Cruz-Rivera and Hay 2003)

It is widely assumed that the production maintenanceand translocation of deterring metabolites are associatedwith metabolic costs because defenses use resources thatcould have been allocated to growth or reproduction (Hayand Fenical 1988 Fagerstroumlm 1989) Many hypotheses onchemical defenses (summarized in Cronin 2001 Pavia andToth 2008) seek to explain the allocation of overallresources and defense metabolites in macroalgae Thegrowth-diVerentiation balance hypothesis (GDBH) predictsthat actively growing and reproductive tissues are lessdefended because of lacking cell diVerentiation when com-pared to diVerentiated vegetative tissue (Herms and Matt-son 1992) which has been discussed in former studies(Cronin and Hay 1996 Van Alstyne et al 1999) Neverthe-less highly diVerentiated large brown algae (eg Laminar-iales Fucales) may translocate low molecular weightcompounds (eg precursors of phlorotannins) among func-tionally diVerent tissues (Raven 2003) which makes pre-dictions from the GDBH diYcult to test (see discussion inCronin and Hay 1996) The most widely accepted hypothe-sis the optimal defense theory (ODT) predicts that chemi-cal compounds for defense are allocated within the algae ina way that optimizes the overall Wtness of the organism(Cronin 2001 Pavia and Toth 2008) Thus algal parts withhigh Wtness values that are susceptible to grazers should bemost intensely defended resulting in a within-plant varia-

tion in defense allocation (reviewed in Jormalainen andHonkanen 2008)

For example meristematic and reproductive blade por-tions should be proportionally more defended than non-meristematic vegetative blades which are less importantfor the plant Wtness (Steinberg 1984 Tugwell and Branch1989 Tuomi et al 1989 Van Alstyne et al 1999 Toth andPavia 2002b) However these predictions are highly depen-dent on speciWc Wtness values that are previously assignedfor the tested tissues (see discussion in Pavia et al 2002)This pattern might also vary between taxonomic groups(Tuomi et al 1989 Van Alstyne et al 1999 Pavia et al2002) or between particular algae that show distinct repro-ductive morphologies For example in kelps (Laminari-ales) higher chemical defenses (eg phlorotanninconcentrations) were found in reproductive than in vegeta-tive blades (Steinberg 1984 Paul and Fenical 1986 Tug-well and Branch 1989 Van Alstyne et al 1999) while inthe rockweeds (Fucales) the defense allocation showed theopposite pattern (Tuomi et al 1989 Van Alstyne et al1999 Pavia et al 2002)

The goal of the present study was to examine whethertwo kelp species from the northern-central coast of ChileLessonia nigrescens Bory 1826 and Macrocystis integrifo-lia Bory 1826 (Laminariales) display diVerent defensestrategies in response to herbivore (amphipod) attacks onblades It is well known that stipes and holdfasts of peren-nial algae are more defended than non-meristematic bladeportions (Tugwell and Branch 1989 Macaya et al 2005see also discussion in Pavia et al 2002) However bladesof these large kelps fulWll diVerent functions and defensesmight vary depending on the function of a blade In particu-lar both kelp species show important diVerences in theirreproductive phenology blades of L nigrescens changeduring ontogeny from a vegetative to a reproductive stagethat is characterized by the maturation of sori (Santeliceset al 1980 Edding et al 1994 HoVmann and Santelices1997) while in M integrifolia the sori are developed onspecialized reproductive blades called sporophylls (Neus-hul 1963) Based on this important diVerence of individualblade structures we expected diVerent defense strategies inthese two algal species SpeciWcally we hypothesized thatin L nigrescens the vegetative blades which are mainlyphotosynthetic structures are more (or equally) protectedthan reproductive tissue parts because ontogeneticallythey will develop sporangia and contribute to the Wtness ofthe alga Thus vegetative blades of L nigrescens not onlyparticipate in photosynthesis but also contribute to thefuture production of reproductive tissues later Howeverafter having fulWlled their vegetative function and once thesporulation took place the need in defending the reproduc-tive structures should diminish On the other hand Mintegrifolia is expected to protect the reproductive blades

123

Mar Biol (2008) 15551ndash62 53

more than the vegetative ones because the alga could dowithout (part of) the latter while the loss of reproductiveblades means a signiWcant loss for the plant with regard toits Wtness Herein we measured the consumption rates ofmesograzers in three distinct feeding assays (fresh materialalgal pellet and extract pellet) to gain insights into struc-tural chemical (phlorotannin content) and nutritional(N-content) defense mechanisms of the two kelp species inthe context of the ODT

Materials and methods

Study site and organisms

The study was conducted at the end of the austral summer(March) 2007 in the Laboratorio de Botanica Marina at theUniversidad Catoacutelica del Norte Coquimbo Chile Vegeta-tive and reproductive (bearing sori) blades of Lessonianigrescens were collected in the exposed rocky intertidalzone at La Pampilla Coquimbo (29deg57S 71deg20W) In thecase of Macrocystis integrifolia blades and sporophyllswere collected in the subtidal zone oV Punta de Choros(29deg14S 71deg28W) The amphipod Parhyalella penaiPeacuterez-Schultheiss and Crespo 2008 which is a generalistmesograzer that feeds on a variety of diVerent macroalgaeincluding L nigrescens and M integrifolia (Macaya et al2005 Rothaumlusler et al 2005) was used for testing algalpalatability in the diVerent feeding assays This littoralamphipod species was called P ruVoi Lazo-Wasem andGable 2001 in earlier publications but careful examinationrevealed that it is a new species which led to the recentspecies description under a new name (Peacuterez-Schultheissand Crespo 2008) This grazer can be found in accumula-tions of drift algae (diverse species) in the shallow subtidalzone of sheltered beaches from northern-central ChileAmphipods for this study were collected from Playa Chicaof Bahiacutea La Herradura Coquimbo by collecting accumula-tions of drift algae The amphipods were separated from thealgae by gently shaking them over a large tray Wlled withseawater

Design of feeding assays

For each kelp species both vegetative and reproductiveblades were sampled from ten sporophytes and conse-quently the reproductive and vegetative tissues weredependent on each other For both species and blade typesnon-meristematic sections of raquo15 cm length from middleparts of the blades were cut for the assays (ensuring thatsori-bearing tissues from the reproductive blades wereobtained) The large number of assays and analysesrequired subdivision of the materials but we had suYcient

materials for at least seven replicates in all feeding assaysor tissue analyses with exception of the assays with algalpellets of Macrocystis integrifolia Problems in preparationof the pellets caused additional loss of replicates but wewere able to recover at least three replicates in each of thesetwo assays (choice and no-choice assays)

The palatability of the diVerent blades to the mesograzerParhyalella penai was tested in feeding assays with (1)fresh material (2) agar-based food from air-dried and pow-dered algae (algal pellets) and (3) agar-based food madewith crude extract of the algae dropped onto powder of thepalatable green alga Ulva lactuca L 1753 (extract pellets)Following logistic restrictions (availability of tissue) forfresh-algal material (ie natural food) we only conductedno-choice assays with fresh-algal material accounting forautogenic changes of the living algal tissues by growth con-trols (Cronin and Hay 1996 Taylor et al 2002 Toth andPavia 2002a) It had been discussed by Peterson andRenaud (1989) that results from no-choice assays can reX-ect diVerences in attractiveness or palatability of variouspotential foods Supporting this assumption several recentstudies had shown no-choice assays producing a similaroutcome as choice assays (Taylor et al 2002 Macaya et al2005 Macaya and Thiel 2008) as was also veriWed in arecent meta-analysis by Toth and Pavia (2007) In the caseof the agar-based pellets (ie artiWcial food) we conductedboth no-choice and choice assays

Furthermore the concentration of phlorotannins wasmeasured since these secondary metabolites have beenshown to serve as defense substances in brown algae (Ams-ler and Fairhead 2006) Additionally the concentration ofnitrogen is commonly used as a proxy for food quality traitsin algal tissues (reviewed in Mattson 1980 see alsoCruz-Rivera and Hay 2000 2003) In the present study theN-concentration in algal tissues was measured in order tocompare them with consumption rates of Parhyalella penaion the diVerent algae tissues

Preparation and evaluation of feeding assays

In the no-choice assays one piece of fresh-algal material orone pellet was oVered to eight individuals of the mesograzerParhyalella penai (adult specimens body length raquo4ndash6 mm)in one Petri dish (9 cm diameter Wlled with raquo80 ml seawa-ter) In the choice assays the reproductive and the vegeta-tive materials from the same algal individual were oVeredsimultaneously All feeding assays were conducted in a con-stant temperature room (15 sect 1degC) with a 12 h light cycle atan irradiance of 40 sect 10 mol miexcl2 siexcl1 (Xuorescent lamp40 W Phillips) A maximum consumption period of 72 hwas used during which we exchanged the water andreplaced dead amphipods daily (mortality rates were gener-ally very low in all assay combinations with an absolute

123

54 Mar Biol (2008) 15551ndash62

maximum of two dead amphipods per Petri dish in 1 daybut this only occurred in very few replicates) If necessaryassays were stopped earlier to avoid a total consumption offresh material or pellets The data from the feeding assayswere converted to consumption rates as mg (fresh weight) orpercent of the total of 200 squares consumed by one individ-ual of the amphipod P penai within 24 h

Feeding assays with fresh-algal material

After blotting the algal pieces (raquo03 g) with absorbent tis-sue paper these were weighed to the nearest mg using ananalytical balance (sect02 mg) Following exposure to theamphipods for a maximum period of 72 h the pieces werere-weighed Another algal piece was kept under the sameconditions without grazers as a growth control The totalconsumption by the herbivores was then calculated usingthe formula described in Cronin and Hay (1996) asCreal = Ti pound (Cf Ci) iexcl Tf where Ti and Tf are the initial andWnal wet weight of the algal material that was subject tograzing and Ci and Cf the initial and Wnal wet weight of thegrowth control

Feeding assays with algal pellets

The algal material was dried at room temperature in a darkpaper box to avoid photolysis of light-sensitive compoundsand then ground in an ultra-centrifugal mill The pelletswere prepared with 05 g of the algal powder and thenmixed with 4 ml of distilled water A speciWc amount ofagar (036 g) was added to 6 ml distilled water and boiledthree times in a microwave until a clear solution was visi-ble Once the agar cooled down to at least 40degC the algalpowder was added and mixed This mixture was immedi-ately poured onto pieces of a gauze mesh (mesh size1 mm2) consisting of 200 squares and pressed between twoglass plates After hardening the pieces were oVered to thegrazer as agar-based food (pellet) in choice and no-choicefeeding assays Consumption rates of agar-based food weredetermined by counting the total mesh squares (1 mmsup2 sur-face area) consumed after the feeding period

Feeding assays with extract pellets

To examine whether diVerences in algal palatability arecaused by chemical compounds extracts from fresh-algalmaterial were prepared mixed with Ulva powder incorpo-rated into an agar-matrix and oVered to grazers as agar-based food For the extraction fresh material of the algawere shortly dried with tissue paper and cut into smallpieces to facilitate the extraction procedure (Rothaumlusleret al 2005 Fairhead et al 2005a b Medeiros et al 2007)Pieces of 3 g wet weight were weighed with an analytical

balance and added to glass Xasks (100 ml) which were thenWlled with 50 ml of a 11 hexanendashmethanol mixture (toextract most secondary metabolites from polar to non-polar) The extraction lasted 48 h and the mixture of solventand algal material was then Wltered (coVee Wlters) into asmall vial to separate the algae pieces from the extractAfter evaporation raquo05 g dry Ulva powder was mixed withthe crude extract obtained from 3 g wet weight (raquo05 g dryweight dw) To achieve natural concentrations of extractedcompounds in the agar-based food we used the followingrelationship for the pellet preparation 05 g dw of theextracted algal material t05 g dw of the Ulva powder forthe pellet Subsequently these extract pellets were preparedas described above

Chemical composition of algal tissues

The phlorotannin content of the diVerent tissue parts wasmeasured at the Universidad Austral de Chile in ValdiviaAlgal material was air-dried in darkness and room tempera-ture and stored in silica gel To determine the concentrationof soluble phlorotannins we used the Folin-Ciocalteu assay(Van Alstyne 1995) and compared the values with a phloro-glucinol standard from a calibration curve Algal samplesof raquo01 g dw were incubated in 10 ml of 70 acetone for12 h at 4degC in total darkness following the extraction-method described in detail by Koivikko et al (2005) Fol-lowing multiple extractions 1 ml of the Folin-Ciocalteureagent was added to the phlorotannin extract which waskept for 5 min before adding 2 ml of a sodium carbonatesolution (02 g mliexcl1) After 1 h the absorbance at 730 nmwas read on a SUV-2120 spectrophotometer (SCINCOKorea)

Tissues were analyzed for their N-concentrations at theUniversity of Rostock Germany Air-dried material wasground using a mortar and samples of 1ndash3 mg were loadedinto small tin boats (6 pound 6 pound 12 mm) and packaged Thesepackages were burned (900degC) and total concentrations ofnitrogen were measured automatically using acetanilide asan internal standard (Elementar Vario EL III Germany)

Statistical analyses

All data were tested for normality with the Shapiro-WilkrsquosW test and if non-normal square-root transformed beforebeing used in parametric statistical tests An arcsine trans-formation was used for percentage data To analyze thediVerences between means of the treatments a t test fordependent samples was used since reproductive and vegeta-tive tissues came from the same individual plant This wasdone for all assays and only in the case of feeding assayswith algal pellets of Macrocystis integrifolia a t test forindependent samples was applied (because there were

123

Mar Biol (2008) 15551ndash62 55

insuYcient dependent replicates) In case the data were notnormally distributed after transformation a non-parametricWilcoxon matched pairs test was used to analyze diVer-ences between means of dependent data and a non-parametricMannndashWhitney U test was used for analyses of non-normaldata from independent samples Homogeneity of varianceswas checked using the Levenersquos test All statistical testswere performed using the software STATISTICA 60 (Stat-Soft Inc USA)

Results

Palatability of vegetative and reproductive algal tissues

In the feeding assay with fresh-algal material of Lessonianigrescens the amphipods consumed signiWcantly morereproductive than vegetative material (Table 1 Fig 1)When food was oVered to amphipods in form of agar-basedfood pellets made from air-dried algal material of L nigres-cens no signiWcant diVerences were found in consumption

rates even though a trend was observed that the amphipodspreferred the vegetative material in the choice feedingassay (Table 1 Fig 2 algal pellets) Some problemsappeared in no-choice assays with algal pellets whereamphipods consumed the total amount of squares in fewpellets over night before the assay could be stopped In thecase of agar-based food made from Ulva powder containingalgal crude extract the amphipods consumed signiWcantlymore from the vegetative than from the reproductive mate-rial both in choice and no-choice feeding assays (Table 1Fig 2 extract pellets)

When fresh-algal material of Macrocystis integrifoliawas oVered the amphipods consumed signiWcantly morevegetative than reproductive material (Table 1 Fig 1)Although problems appeared in the preparation of algal pel-lets (slime production of the powdered algae when gettingin contact with water leading to the loss of several repli-cates) consumption rates diVered signiWcantly a signiW-cant preference for reproductive material was found in bothchoice and no-choice assays (Table 1 Fig 2 algal pellets)However as mentioned for Lessonia nigrescens in some

Table 1 Results from statistical analysis of the consumption rates onalgal material [containing mechanical (m) nutritional (n) or chemical(c) traits] of the phlorotannin contents and the N-concentrations

between reproductive (R) and vegetative (V) blade parts of Lessonianigrescens and Macrocystis integrifolia in diVerent assays using t testsfor dependent samples

Lessonia nigrescens Macrocystis integrifolia

df t P df t P

Fresh material (mg) m n c 8 3714 0006 V lt R 6 iexcl4557 0004 V gt R

Algal pellets no-choice ( squares) n c 0735a V = R 7b 2390b 0048b V lt R

Algal pellets choice ( squares) n c 8 iexcl1564 0156 V = R 0034c V lt R

Extract pellets no-choice ( squares) c 8 iexcl2359 0046 V gt R 8 1855 0101 V = R

Extract pellets choice ( squares) c 8 0019 0006 V gt R 9 3616 0006 V lt R

Content of soluble phlorotannins ( dw) 6 3078 0022 V lt R 6 8970 lt0001 V lt R

Nitrogen content ( dw) 6 4823 0003 V lt R 7 3355 0012 V lt R

a Non-parametric Wilcoxon matched pairs test for analyses of non-normal data from dependent samplesb t test for independent samples that we used because of insuYcient dependent replicationc Non-parametric MannndashWhitney U test for analyses of non-normal data from independent samples

Fig 1 Consumption of fresh material (mg) of Lessonia nigrescens and Macrocystis integrifolia by one individual of Parhyalella penai within 24 h White boxes indicate reproduc-tive and grey boxes vegetative material (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates) All assays lasted a maximum time period of 72 h

0

2

4

6

8

0

2

4

6

8

amo

un

t co

nsu

med

[m

g]

reproductive

Lessonia nigrescens

N=9

Macrocystis integrifolia

N=9 N=7 N=7

reproductive vegetative vegetative

123

56 Mar Biol (2008) 15551ndash62

replicates the amphipods consumed all available food inno-choice assays with algal pellets before the feeding assaywas stopped Similar as for the algal pellets in the assayswith extract pellets amphipods preferred material of repro-ductive blades albeit this was only signiWcant in the choiceassay (Table 1 Fig 2 extract pellets)

Mean diVerences in consumption rates between freshreproductive and vegetative material were much stronger inMacrocystis integrifolia (mean diVerence 3316 mg) thanin Lessonia nigrescens (mean diVerence 433 mg) (t testP lt 0001)

Chemical composition of vegetative and reproductive tissues

The content of soluble phlorotannins diVered signiWcantlybetween reproductive and vegetative material in both algalspecies (Table 1 Fig 3) Phlorotannin concentrations inreproductive blade parts of Lessonia nigrescens were close to49 dw while vegetative blades contained less than 35dw In Macrocystis integrifolia the pattern was similarshowing phlorotannin concentrations close to 46 dw inreproductive and about 21 dw in vegetative tissue In bothL nigrescens and M integrifolia the nitrogen content variedbetween 13 and 23 dw with values signiWcantly higher inreproductive than in vegetative tissues (Table 1 Fig 3)

Discussion

The results of this study show that the interactionbetween amphipod grazers and algae proceeded as previ-ously hypothesized when fresh-algal material wasoVered The amphipods preferred the reproductive tis-sues of Lessonia nigrescens which suggests that theseparts are less defended or simply tastier for the amphi-pods In Macrocystis integrifolia vegetative bladeswere strongly preferred over reproductive blades indi-cating a low defense level in vegetative tissues EVectsizes (ie diVerences in consumption rates between veg-etative and reproductive tissues) have in this case beenmuch higher in assays using fresh material of M integri-folia (Fig 1) However in both kelp species grazers thatwere oVered fresh material consumed more of the tissueswith lower assigned Wtness values which provides sup-port for the ODT (valuable tissues are more defended)Surprisingly feeding assays with pellets based on air-dried algal material or algal extracts (ie after destroy-ing structural characteristics of the algae) showed theopposite pattern in grazer preferences for both algal spe-cies This suggests that structural or mechanical traits offresh-algal tissues seem to be more eYcient in deterringamphipod grazers than chemical compounds present inagar-based pellets

Fig 2 Consumption of agar-based food ( of total squares) of Lessonia nigrescens and Macrocystis integrifolia by one individual of Parhyalella penai within 24 h on algal pellets (above) and extract pellets (below) White boxes indicate reproductive and grey boxes vegetative material (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates) All assays lasted a maximum time period of 72 h

0

5

10

15

0

5

10

15

0

5

10

15

0

5

10

15

sq

uar

es c

on

sum

ed [

]

no choice

Lessonia nigrescens

reproductive

vegetative

N=9

Macrocystis integrifolia

N=9 N=9 N=9 N=3 N=6 N=3 N=4

N=9 N=9 N=9 N=9 N=9 N=9 N=10 N=10

choice no choice choice

123

Mar Biol (2008) 15551ndash62 57

Palatability of fresh tissue and nutritional qualities

Both algae had higher N-concentrations ( dw) in repro-ductive than in vegetative tissues (as also found for Alariamarginata by Steinberg 1984) which might result fromactive spore production within the reproductive tissues(Reed et al 1996) Following general assumptions (Matt-son 1980 DuVy and Paul 1992 Cruz-Rivera and Hay2003) fresh reproductive material of these two algaeshould thus be more valuable for amphipods (and moreconsumed) because of its higher nutritional quality How-ever this was not supported by our results since prefer-ences in food choice by amphipods were not alwaysconsistent with higher N-concentrations within the pre-ferred food Amphipods consumed signiWcantly morereproductive than vegetative fresh tissue from Lessonianigrescens but preferred vegetative blades from Macrocys-tis integrifolia The reverse pattern was found in feedingassays with agar-based food (algal and extract pellets) inboth algae which led us to assume that feeding preferencesin fresh-algal tissue are based on structural or mechanicaltissue characteristics rather than on nutritional or chemicaltraits This had also been suggested by Steinberg (1985) forchemically weakly defended (or undefended) algae Mor-phological characteristics of algal tissue had previouslybeen emphasized to aVect feeding preferences of grazers(Littler and Littler 1980 Steneck and Watling 1982) Thepattern observed herein for small amphipods might have

been diVerent with other grazers that are less aVected by tis-sue hardness (eg sea urchins or gastropods) (Rothaumlusleret al 2005)

In Macrocystis integrifolia we found vegetative bladesto be much softer and thinner than reproductive blades (per-sonal observation) which might explain the extremely highconsumption rates of vegetative fresh material Similarresults were found by Steinberg (1984) for Alaria margin-ata (Laminariales) which also has distinctive reproductiveand vegetative tissues the herbivorous snail Tegula funeb-ralis consumed much more fresh material from vegetativeblade portions than from the reproductive portionsSteinberg (1984) also measured the tissue toughness inA marginata with a ldquopenetrometerrdquo and he showed thatreproductive tissues are tougher than vegetative tissuessupporting our suggestions for M integrifolia Further-more it is important to consider that M integrifolia is pro-ducing large amounts of mucus which might have beenmore intense in reproductive tissues and could haveaVected amphipod preferences in fresh feeding assays Theproduction of mucus by the alga Carpoglossum conXuens isthought to reduce the level of competition from other algaeand to deter animals from being on or around the alga(Edgar 2000) Wotton (2004) discusses further roles ofmucus (exopolymers) in aquatic systems As an examplemucus might prevent damage by abrasion forming a slip-pery layer on macroalgal fronds It was also observed thatsome algal species (Fucales) release spores with large

Fig 3 Content of soluble phlorotannins and nutrients ( dry weight) in reproductive (white boxes) and vegetative (grey boxes) tissues of Lessonia nigrescens and Macrocystis integrifolia (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates)

reproductive

Lessonia nigrescens

ph

loro

tan

nin

s [

dw

]

N

[

dw

]

0

1

2

3

0

1

2

3

0

2

4

6

0

2

4

6

N=7

Macrocystis integrifolia

vegetative reproductive vegetativeN=7 N=8 N=8

N=7 N=7 N=7 N=7

123

56 Mar Biol (2008) 15551ndash62

replicates the amphipods consumed all available food inno-choice assays with algal pellets before the feeding assaywas stopped Similar as for the algal pellets in the assayswith extract pellets amphipods preferred material of repro-ductive blades albeit this was only signiWcant in the choiceassay (Table 1 Fig 2 extract pellets)

Mean diVerences in consumption rates between freshreproductive and vegetative material were much stronger inMacrocystis integrifolia (mean diVerence 3316 mg) thanin Lessonia nigrescens (mean diVerence 433 mg) (t testP lt 0001)

Chemical composition of vegetative and reproductive tissues

The content of soluble phlorotannins diVered signiWcantlybetween reproductive and vegetative material in both algalspecies (Table 1 Fig 3) Phlorotannin concentrations inreproductive blade parts of Lessonia nigrescens were close to49 dw while vegetative blades contained less than 35dw In Macrocystis integrifolia the pattern was similarshowing phlorotannin concentrations close to 46 dw inreproductive and about 21 dw in vegetative tissue In bothL nigrescens and M integrifolia the nitrogen content variedbetween 13 and 23 dw with values signiWcantly higher inreproductive than in vegetative tissues (Table 1 Fig 3)

Discussion

The results of this study show that the interactionbetween amphipod grazers and algae proceeded as previ-ously hypothesized when fresh-algal material wasoVered The amphipods preferred the reproductive tis-sues of Lessonia nigrescens which suggests that theseparts are less defended or simply tastier for the amphi-pods In Macrocystis integrifolia vegetative bladeswere strongly preferred over reproductive blades indi-cating a low defense level in vegetative tissues EVectsizes (ie diVerences in consumption rates between veg-etative and reproductive tissues) have in this case beenmuch higher in assays using fresh material of M integri-folia (Fig 1) However in both kelp species grazers thatwere oVered fresh material consumed more of the tissueswith lower assigned Wtness values which provides sup-port for the ODT (valuable tissues are more defended)Surprisingly feeding assays with pellets based on air-dried algal material or algal extracts (ie after destroy-ing structural characteristics of the algae) showed theopposite pattern in grazer preferences for both algal spe-cies This suggests that structural or mechanical traits offresh-algal tissues seem to be more eYcient in deterringamphipod grazers than chemical compounds present inagar-based pellets

Fig 2 Consumption of agar-based food ( of total squares) of Lessonia nigrescens and Macrocystis integrifolia by one individual of Parhyalella penai within 24 h on algal pellets (above) and extract pellets (below) White boxes indicate reproductive and grey boxes vegetative material (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates) All assays lasted a maximum time period of 72 h

0

5

10

15

0

5

10

15

0

5

10

15

0

5

10

15

sq

uar

es c

on

sum

ed [

]

no choice

Lessonia nigrescens

reproductive

vegetative

N=9

Macrocystis integrifolia

N=9 N=9 N=9 N=3 N=6 N=3 N=4

N=9 N=9 N=9 N=9 N=9 N=9 N=10 N=10

choice no choice choice

123

Mar Biol (2008) 15551ndash62 57

Palatability of fresh tissue and nutritional qualities

Both algae had higher N-concentrations ( dw) in repro-ductive than in vegetative tissues (as also found for Alariamarginata by Steinberg 1984) which might result fromactive spore production within the reproductive tissues(Reed et al 1996) Following general assumptions (Matt-son 1980 DuVy and Paul 1992 Cruz-Rivera and Hay2003) fresh reproductive material of these two algaeshould thus be more valuable for amphipods (and moreconsumed) because of its higher nutritional quality How-ever this was not supported by our results since prefer-ences in food choice by amphipods were not alwaysconsistent with higher N-concentrations within the pre-ferred food Amphipods consumed signiWcantly morereproductive than vegetative fresh tissue from Lessonianigrescens but preferred vegetative blades from Macrocys-tis integrifolia The reverse pattern was found in feedingassays with agar-based food (algal and extract pellets) inboth algae which led us to assume that feeding preferencesin fresh-algal tissue are based on structural or mechanicaltissue characteristics rather than on nutritional or chemicaltraits This had also been suggested by Steinberg (1985) forchemically weakly defended (or undefended) algae Mor-phological characteristics of algal tissue had previouslybeen emphasized to aVect feeding preferences of grazers(Littler and Littler 1980 Steneck and Watling 1982) Thepattern observed herein for small amphipods might have

been diVerent with other grazers that are less aVected by tis-sue hardness (eg sea urchins or gastropods) (Rothaumlusleret al 2005)

In Macrocystis integrifolia we found vegetative bladesto be much softer and thinner than reproductive blades (per-sonal observation) which might explain the extremely highconsumption rates of vegetative fresh material Similarresults were found by Steinberg (1984) for Alaria margin-ata (Laminariales) which also has distinctive reproductiveand vegetative tissues the herbivorous snail Tegula funeb-ralis consumed much more fresh material from vegetativeblade portions than from the reproductive portionsSteinberg (1984) also measured the tissue toughness inA marginata with a ldquopenetrometerrdquo and he showed thatreproductive tissues are tougher than vegetative tissuessupporting our suggestions for M integrifolia Further-more it is important to consider that M integrifolia is pro-ducing large amounts of mucus which might have beenmore intense in reproductive tissues and could haveaVected amphipod preferences in fresh feeding assays Theproduction of mucus by the alga Carpoglossum conXuens isthought to reduce the level of competition from other algaeand to deter animals from being on or around the alga(Edgar 2000) Wotton (2004) discusses further roles ofmucus (exopolymers) in aquatic systems As an examplemucus might prevent damage by abrasion forming a slip-pery layer on macroalgal fronds It was also observed thatsome algal species (Fucales) release spores with large

Fig 3 Content of soluble phlorotannins and nutrients ( dry weight) in reproductive (white boxes) and vegetative (grey boxes) tissues of Lessonia nigrescens and Macrocystis integrifolia (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates)

reproductive

Lessonia nigrescens

ph

loro

tan

nin

s [

dw

]

N

[

dw

]

0

1

2

3

0

1

2

3

0

2

4

6

0

2

4

6

N=7

Macrocystis integrifolia

vegetative reproductive vegetativeN=7 N=8 N=8

N=7 N=7 N=7 N=7

123

Mar Biol (2008) 15551ndash62 57

Palatability of fresh tissue and nutritional qualities

Both algae had higher N-concentrations ( dw) in repro-ductive than in vegetative tissues (as also found for Alariamarginata by Steinberg 1984) which might result fromactive spore production within the reproductive tissues(Reed et al 1996) Following general assumptions (Matt-son 1980 DuVy and Paul 1992 Cruz-Rivera and Hay2003) fresh reproductive material of these two algaeshould thus be more valuable for amphipods (and moreconsumed) because of its higher nutritional quality How-ever this was not supported by our results since prefer-ences in food choice by amphipods were not alwaysconsistent with higher N-concentrations within the pre-ferred food Amphipods consumed signiWcantly morereproductive than vegetative fresh tissue from Lessonianigrescens but preferred vegetative blades from Macrocys-tis integrifolia The reverse pattern was found in feedingassays with agar-based food (algal and extract pellets) inboth algae which led us to assume that feeding preferencesin fresh-algal tissue are based on structural or mechanicaltissue characteristics rather than on nutritional or chemicaltraits This had also been suggested by Steinberg (1985) forchemically weakly defended (or undefended) algae Mor-phological characteristics of algal tissue had previouslybeen emphasized to aVect feeding preferences of grazers(Littler and Littler 1980 Steneck and Watling 1982) Thepattern observed herein for small amphipods might have

been diVerent with other grazers that are less aVected by tis-sue hardness (eg sea urchins or gastropods) (Rothaumlusleret al 2005)

In Macrocystis integrifolia we found vegetative bladesto be much softer and thinner than reproductive blades (per-sonal observation) which might explain the extremely highconsumption rates of vegetative fresh material Similarresults were found by Steinberg (1984) for Alaria margin-ata (Laminariales) which also has distinctive reproductiveand vegetative tissues the herbivorous snail Tegula funeb-ralis consumed much more fresh material from vegetativeblade portions than from the reproductive portionsSteinberg (1984) also measured the tissue toughness inA marginata with a ldquopenetrometerrdquo and he showed thatreproductive tissues are tougher than vegetative tissuessupporting our suggestions for M integrifolia Further-more it is important to consider that M integrifolia is pro-ducing large amounts of mucus which might have beenmore intense in reproductive tissues and could haveaVected amphipod preferences in fresh feeding assays Theproduction of mucus by the alga Carpoglossum conXuens isthought to reduce the level of competition from other algaeand to deter animals from being on or around the alga(Edgar 2000) Wotton (2004) discusses further roles ofmucus (exopolymers) in aquatic systems As an examplemucus might prevent damage by abrasion forming a slip-pery layer on macroalgal fronds It was also observed thatsome algal species (Fucales) release spores with large

Fig 3 Content of soluble phlorotannins and nutrients ( dry weight) in reproductive (white boxes) and vegetative (grey boxes) tissues of Lessonia nigrescens and Macrocystis integrifolia (each box mean sect SE P lt 005 P lt 001 P lt 0001 N number of replicates)

reproductive

Lessonia nigrescens

ph

loro

tan

nin

s [

dw

]

N

[

dw

]

0

1

2

3

0

1

2

3

0

2

4

6

0

2

4

6

N=7

Macrocystis integrifolia

vegetative reproductive vegetativeN=7 N=8 N=8

N=7 N=7 N=7 N=7

123

58 Mar Biol (2008) 15551ndash62

amounts of viscous mucus that might reduce the dispersalof spores (Brawley and Johnson 1992 Brawley et al 1999)Unfortunately to date the eVect of mucus on algae grazerinteractions has not been experimentally tested

In Lessonia nigrescens our Wndings suggest that feedingrates might also be inXuenced by structural or mechanicaltraits Here the higher consumption of fresh reproductivematerial might be explained by constraints due to physio-logical changes that must occur when the tissue transformsfrom vegetative to reproductive Furthermore the repro-ductive structures are known to decay and be shed oV afterreleasing spores (F Tala personal communication) whichmight expose the undefended inner parts (Tugwell andBranch 1989 Shibata et al 2004) of the blade (medulla) toamphipod grazers Possibly the spores themselves mightbe easily consumed and digested by crustacean mesogra-zers a situation observed in interactions between red algaeand micro-grazers (Buschmann and Santelices 1987) Addi-tionally in L nigrescens blades accumulate products ofphotosynthesis during maturation (eg polysaccharides)which might be particularly concentrated in older reproduc-tive blades (Goacutemez et al 2007) The high N-concentrationsthat we found in mature reproductive tissues of L nigres-cens might be a reXection of these processes and mightdrive the feeding preferences of the amphipods

Palatability of pellets and the role of phlorotannins

Phlorotannin concentrations ( dw) were higher in repro-ductive than in vegetative tissues in both algal species aspreviously suggested by Van Alstyne et al (1999) for algaebelonging to the order Laminariales (see also Steinberg1984 Tugwell and Branch 1989) Phlorotannins have alsobeen shown to occur in very low concentrations of raquo1 dwin vegetative blades of the congener Macrocystis pyrifera(Steinberg 1985 Winter and Estes 1992) Following gen-eral assumptions one could expect that based on thesediVerences in phlorotannin concentrations reproductive tis-sues are more defended than vegetative tissues Surpris-ingly we found no consistent evidence for this assumptionin feeding assays with fresh material (in Lessonia nigres-cens) or in feeding assays with agar-based food (in Macro-cystis integrifolia) This led us to assume that the grazerParhyalella penai does not respond to extracted phlorotan-nins at least not at the concentrations found in the twostudied algae There might be long-term eVects of phloro-tannin consumption eg on reproductive or food-assimila-tion rates of the amphipod grazers (Cruz-Rivera and Hay2000 Targett and Arnold 2001) but this was not examinedherein It also should be considered that the highly water-soluble phlorotannins might have leached out of the pre-pared food pellets during the assays (Jormalainen et al2005) possibly reducing the deterrence eVect on P penai

Martinez (1996) demonstrated that individuals of Lesso-nia nigrescens with higher contents in phlorotannins(raquo5 mg giexcl1 dw) were less palatable to herbivorous snailsand Wsh than individuals with lower phlorotannin contents(raquo1 mg giexcl1 dw) These values however seem to beextremely low (raquo01ndash05 dw) when compared to ourresults and concentrations cited for other brown algae Nev-ertheless there appear to be eVects of phlorotannins onlarger grazers (Martinez 1996) but no or only minor eVectson small crustacean mesograzers (eg Parhyalella penai)as seen in our study There might be other reasons why thephlorotannin content is higher in reproductive than invegetative blades since phlorotannins are also involved inprimary functions such as eg structuring cell walls(Schoenwaelder and Clayton 1999 Arnold and Targett2003) It must be emphasized that synthesis and allocationof phlorotannins in brown algae are complex and requirefurther examination Since the production of solublephlorotannins is almost exclusively a function of cortexcells (Shibata et al 2004) and thus a large proportion of theblade tissues do not contain phlorotannins some micro-and mesograzers might be able to distinguish the diVerenttissue types feeding mainly on the phlorotannin-free zonesin eg the reproductive tissues of L nigrescens

We observed a repelling eVect in agar-based food fromreproductive material of Lessonia nigrescens to the grazerParhyalella penai Although this pattern was weak inassays with algal pellets it was highly signiWcant in assayswith extract pellets ie where we excluded the simulta-neous eVect of nutrients from L nigrescens However justthe opposite pattern was found in Macrocystis integrifoliaConsidering that we used hexane and methanol to extractcompounds from the algal tissue we can expect a widespectrum of polar as well as non-polar compounds beingpresent in the crude extracts (Amsler and Fairhead 2006)Since methanol does not extract phlorotannins veryeYciently (Koivikko et al 2005) phlorotannins might evenbe under-represented in the extract pellets when comparedto non-polar compounds Consequently our suggestedexplanation cannot only be based on phlorotannins butneeds to include possible eVects of a wide variety of addi-tional extracted metabolites Although a suite of non-phlorotannin secondary metabolites like galactolipids orhydrophilic non-phenolic compounds is known (Harperet al 2001) relatively few studies have assayed their deter-ring roles (Amsler and Fairhead 2006 Maschek and Baker2008) Since phlorotannin-rich tissues did not show consis-tent deterring eVects on the grazer in this study we mightexpect untested deterring secondary metabolites beingresponsible for diVering consumption rates in assays withagar-based food As a support for this assumption Rothaumlus-ler and Thiel (2006) found slight chemically mediateddefense in non-polar extracts (ie not containing polar

123

Mar Biol (2008) 15551ndash62 59

phlorotannins) of L nigrescens On the other hand theremight be other substances in crude extracts (polar and non-polar) from eg reproductive M integrifolia that attractthese amphipods (Pansch et al unpublished data) VanAlstyne et al (2001) and Van Alstyne and Houser (2003)showed simultaneously deterring and attracting (dependenton the metabolite concentrations) functions of activatedsecondary metabolites in macroalgae responding to seaurchins

Finally not all herbivores are deterred by phlorotannins(Jormalainen et al 2003) indicating that some species haveadapted to tolerate or utilize these algal compounds (Targ-ett and Arnold 1998 Pavia et al 1997 Jormalainen et al2003) Variations in eVectiveness are even found at a bio-geographic scale the comparatively low polyphenol con-tents found in North American Phaeophycean have been

found to deter a range of herbivores but Australasian inver-tebrate herbivores are unaVected by high levels of phloro-tannins (Steinberg and Van Altena 1992) which underlinesthe suggestion that phlorotannins have roles other thandefense in these species A similar situation occurs wheninduction of defensive responses of phlorotannins to graz-ing have been examined in some cases there is evidence ofinduction (Peckol et al 1996 Luumlder and Clayton 2004)whereas some authors have failed to demonstrate inducedincreases of phlorotannins in various species of Laminari-ales (Yates and Peckol 1993 Martinez 1996 Pavia et al1997 Toth and Pavia 2002b) Overall the role of phloro-tannins as herbivore deterrents remains ambiguous mainlybecause the anti-herbivore eVectiveness is also a functionof herbivore-speciWc factors (eg characteristics of gutenzymatic adaptations etc)

Table 2 Concentrations of phlorotannins and relationships between diVerent tissues in chemical deterrents and nitrogen contents as well as ingrazer consumption of fresh tissues and artiWcial food for several species of macroalgae

If food preferences reXect expectations based on chemical deterrents they were italicized if food preferences correspond with nitrogen contentsthey were highlighted with bold letters

V vegetative blades R reproductive tissue S stipes H holdfasts M meristems y young o old b basal a apicala Approximated mean phlorotannin values in the diVerent thallus parts corresponding to the comparisons of the ldquocontents of chemical deterrentsrdquominndashmaxb Present study c Tugwell and Branch 1989 d Toth and Pavia 2002a e Toth and Pavia 2002b f Steinberg 1984 g Van Alstyne et al 1999 h Tuomiet al 1989 i Honkanen et al 2002 j Pavia et al 2002 k Toth et al 2005 l Taylor et al 2002 m Cronin amp Hay 1996 n Fairhead et al 2005ao Fairhead et al 2005b

Phlorotannins raquo[ dw]a

Content of chemical deterrents

Nitrogen content

Consumption fresh tissue

ArtiWcial food

Laminariales

Lessonia nigrescensb 35ndash49 V lt R V middot R V middot R V gt R

Macrocystis integrifoliab 21ndash46 V lt R V lt R V gt R V lt R

Macrocystis angustifoliac V lt R H S

Ecklonia maximac V lt R H S lt M

Laminaria pallidac V R lt H S M

Laminaria hyperboread 12ndash16 yV = oV yV = oV yV gt oV

Laminaria hyperboreae 18ndash32 yV oV lt yM lt oM

Alaria marginataf 10ndash50 V lt R V = R V gt R

Alaria marginatag 10ndash21 V M lt R

Alaria nanag 20ndash31 V lt R M

Fucales

Pelvetia compressag 28ndash57 V M gt R

Fucus gardnerig 32ndash49 M gt V gt R

Fucus vesiculosush 16ndash54 oV yV oR gt yR oR lt yR

Fucus vesiculosusi 90ndash105 aV lt bV aV cedil bV aV cedil bV

Ascophyllum nodosumj 35ndash75 S gt V gt R S lt V lt R

Ascophyllum nodosumk 38ndash63 aV lt bV aV gt bV

Sargassum Wlipendulal aV aS gt bV gt Bs

Dictyota ciliolatam yV lt oV yV gt oV yV gt oV

Desmarestia ancepsno 100ndash120 V gt S V H gt S

Desmarestia menziesiino 50ndash53 V = S V = H = S

123

60 Mar Biol (2008) 15551ndash62

Conclusions and outlook

The brown alga Lessonia nigrescens seems to follow astrategy in which fresh vegetative tissues are eYcientlydefended against amphipod grazing (Table 2) These pho-tosynthetically active tissues have the potential to producesori and to contribute to reproduction of the alga therebyexhibiting an important role for future Wtness Reproduc-tive structures seem to be little defended or somehowattractive to amphipod mesograzers In fact in some algalspecies grazing on reproductive structures might improvespore release and enhance dispersal (Porter 1976 Busch-mann and Santelices 1987 Santelices and Ugarte 1987)thus improving algal Wtness Algae with distinct reproduc-tive blades such as the giant kelp Macrocystis integrifoliaseem to follow a diVerent strategy Reproductive bladesare presumably of higher value for these kelp species andthus much stronger defended than vegetative blades(Table 2) Particularly in M integrifolia this also coin-cides with the basal position of reproductive blades andtheir exposure to benthic grazers such as sea urchins andsnails On the other hand vegetative blades seem to behighly susceptible to amphipod grazing This alga hadbeen shown to coincidentally express remarkable growthrates in vegetative plant portions (Stekoll and Else 1990)and might therefore compensate grazer-induced losses ofvegetative blades by growth as suggested by Carpenter(1986)

Herein it was shown that defense traits for both algae(Lessonia nigrescens and Macrocystis integrifolia) canbe explained by the ODT Although the Wtness valuesassigned to reproductive and vegetative tissues are ratherqualitative estimates they seem to correspond with thedefense strategies of the two studied algae Results fromformer studies (Table 2) indicate that phlorotannin valuesdiVer between diVerent tissues over small spatial scales(as also seen in the present study) However in somecases the grazer preferences seem to vary independentfrom chemical deterrents and possibly responding to foodquality of the studied tissues or other factors Our resultssuggest a trade-oV in amphipod preferences that dependsmainly on (i) tissue toughness but also on (ii) nutritionalquality and (iii) untested secondary metabolites of thefood How these diVerent traits interact in driving thefeeding preference of mesograzers needs to be examinedin future studies

Acknowledgments We are thankful to the leaders of the GAMEproject Mark Lenz and Martin Wahl Fadia Tala made many helpfulsuggestions on a Wrst draft of the manuscript We would also like tothank Prof Dr Ulf Karsten and coworkers for the use of the laboratoryat the University of Rostock Germany This project was Wnanced bythe Mercator Stiftung GmbH and FONDECYT grants 1060127 (MTIG) and 1060503 (IG)

References

Amsler CD Fairhead VA (2006) Defensive and sensory chemicalecology of brown algae Adv Bot Res 431ndash91 doi101016S0065-2296(05) 43001-3

Arnold TM Targett NM (2003) To grow and defend lack of tradeoVsfor brown algal phlorotannins Oikos 100406ndash408 doi101034j1600-0706200311680x

Brawley SH Johnson LE (1992) Gametogenesis gametes and zygotesan ecological perspective on sexual reproduction in the algae BrPhycol J 27233ndash252 doi10108000071619200650241

Brawley SH Johnson LE Pearson GA (1999) Gamete release at lowtide in fucoid algae maladaptive or advantageous Am Zool39218ndash229

Buschmann A Santelices B (1987) Micrograzers and spore release inIridaea laminarioides Bory (Rhodophyta Gigartinales) J ExpMar Biol Ecol 108171ndash179 doi101016S0022-0981(87)80021-7

Carpenter RC (1986) Partitioning herbivory and its eVects on coral reefalgal communities Ecol Monogr 56345ndash363 doi1023071942551

Cronin G (2001) Resource allocation in seaweed and marine inverte-brates chemical defense patterns in relation to defense theoriesIn McClintock JB Baker BJ (eds) Marine chemical ecologyCRC Press Boca Raton pp 325ndash353

Cronin G Hay ME (1996) Within-plant variation in seaweed palatabil-ity and chemical defenses optimal defense theory versus thegrowth-diVerentiation balance hypothesis Oecologia 105361ndash368 doi101007BF00328739

Cruz-Rivera E Hay ME (2000) Can quantity replace quality Foodchoice compensatory feeding and Wtness of marine mesograzersEcology 81201ndash219

Cruz-Rivera E Hay ME (2003) Prey nutritional quality interacts withchemical defenses to aVect consumer feeding and Wtness EcolMonogr 73483ndash506 doi1018900012-9615(2003) 073[0483PNQIWC]20CO2

DuVy JE Hay ME (1990) Seaweed adaptation to herbivory Biosci-ence 40368ndash375 doi1023071311214

DuVy JE Paul VJ (1992) Prey nutritional quality and eVectiveness ofchemical defenses against tropical reef Wshes Oecologia 90333ndash339 doi101007BF00317689

Edding M Fonck E Macchiavello J (1994) Lessonia In Akatsuka I(ed) Biology of economic algae Academic Publishing TheHague Netherlands pp 407ndash446

Edgar GJ (ed) (2000) Australian marine life the plants and animals oftemperate waters Reed New Holland Sydney

Fagerstroumlm T (1989) Anti-herbivore chemical defense in plants a note onthe concept of cost Am Nat 133281ndash287 doi101086284918

Fairhead VA Amsler CD McClintock JB Baker BJ (2005a) Within-thallus variation in chemical and physical defenses in two speciesof ecologically dominant brown macroalgae from the AntarcticPeninsula J Exp Mar Biol Ecol 3221ndash12 doi101016jjembe200501010

Fairhead VA Amsler CD McClintock JB Baker BJ (2005b) Variationin phlorotannin content within two species of brown macroalgae(Desmarestia anceps and D menziesii) from the Western Antarc-tic Peninsula Polar Biol 28680ndash686 doi101007s00300-005-0735-4

Goacutemez I Orostegui M Huovinen P (2007) Morpho-functional patternsof photosynthesis in the south PaciWc kelp Lessonia nigrescensEVects of UV radiation on 14C Wxation and primary photochem-ical reactions J Phycol 4355ndash64 doi101111j1529-8817200600301x

Harper MK Bubni TS Copp BR James RD Lindsay BS RichardsonAD et al (2001) Introduction to the chemical ecology of marine

123

Mar Biol (2008) 15551ndash62 61

natural products In McClintock JB Baker BJ (eds) Marinechemical ecology CRC Press Boca Raton pp 3ndash69

Hay ME Fenical W (1988) Marine plantndashherbivore interactions theecology of chemical defense Annu Rev Ecol Syst 19111ndash145doi101146annureves19110188000551

Herms DA Mattson WJ (1992) The dilemma of plants to grow ordefend Q Rev Biol 67285ndash335 doi101086417659

HoVmann A Santelices B (eds) (1997) Flora marina de Chile centralmarine Xora at central Chile Universidad Catoacutelica de ChileSantiago

Honkanen T Jormalainen V Hemmi A Maumlkinen A Heikkilauml N(2002) Feeding and growth of the isopod Idotea baltica on thebrown alga Fucus vesiculosus roles of inter-population and with-in-plant variation in plant quality Ecoscience 9332ndash338

Jormalainen V Honkanen T (2008) Macroalgal chemical defenses andtheir roles in structuring temperate marine communities InAmsler CD (ed) Algal chemical ecology Springer Heidelbergpp 57ndash89

Jormalainen V Honkanen T Koivikko R Eraumlnen J (2003) Inductionof phlorotannin production in a brown alga defense or resourcedynamics Oikos 103640ndash650 doi101034j1600-0706200312635x

Jormalainen V Honkanen T Vesakoski O Koivikko R (2005) Polarextracts of the brown alga Fucus vesiculosus (L) reduce assimi-lation eYciency but do not deter the herbivorous isopod Idoteabaltica (Pallas) J Exp Mar Biol Ecol 317143ndash157 doi101016jjembe200411021

Koivikko R Loponen J Honkanen T Jormalainen V (2005) Contentsof soluble cell-wall-bound and exuded phlorotannins in thebrown alga Fucus vesiculosus with implications on their ecolog-ical functions J Chem Ecol 31195ndash212 doi101007s10886-005-0984-2

Littler MM Littler DS (1980) The evolution of thallus form and sur-vival strategies in benthic marine macroalgae Weld and laboratorytests of a functional form model Am Nat 11625ndash44 doi101086283610

Lubchenco J Gaines SD (1981) A uniWed approach to marine plant-her-bivore interactions I Populations and communities Annu RevEcol Syst 12405ndash437 doi101146annureves12110181002201

Luumlder UH Clayton MN (2004) Induction of phlorotannins in thebrown macroalga Ecklonia radiata (Laminariales Phaeophyta) inresponse to simulated herbivorymdashthe Wrst microscopic studyPlanta 218928ndash937 doi101007s00425-003-1176-3

Macaya EC Thiel M (2008) In situ tests on inducible defenses in Dict-yota kunthii and Macrocystis integrifolia (Phaeophyceae) fromthe Chilean coast J Exp Mar Biol Ecol 35428ndash38 doi101016jjembe200710005

Macaya EC Rothaumlusler E Thiel M Molis M Wahl M (2005) Induc-tion of defenses and within-alga variation of palatability in twobrown algae from the northern-central coast of Chile eVects ofmesograzers and UV radiation J Exp Mar Biol Ecol 325214ndash227 doi101016jjembe200505004

Martinez EA (1996) Micropopulation diVerentiation in phenol contentand susceptibility to herbivory in the Chilean kelp Lessonianigrescens (Phaeophyta Laminariales) Hydrobiologia 326327205ndash211 doi101007BF00047808

Maschek JA Baker BJ (2008) The chemistry of algal secondarymetabolism In Amsler CD (ed) Algal chemical ecology Spring-er Heidelberg pp 1ndash24

Mattson WJ (1980) Herbivory in relation to plant nitrogen contentAnnu Rev Ecol Syst 11119ndash161 doi101146annureves11110180001003

Medeiros HE da Gama BAP Gallerani G (2007) Antifouling activityof seaweed extracts from Guarujaacute Sao Paulo Brazil Braz J Oce-anogr 55257ndash264 doi101590S1679-87592007000400003

Neushul M (1963) Studies on the giant kelp Macrocystis II Repro-duction Am J Bot 50354ndash359 doi1023072440152

Paul VJ Fenical W (1986) Chemical defense in tropical green algaeorder Caulerpales Mar Ecol Prog Ser 34157ndash169 doi103354meps034157

Pavia H Toth GB (2008) Macroalgal models in testing and extendingdefense theories In Amsler CD (ed) Algal chemical ecologySpringer Heidelberg pp 147ndash172

Pavia H Cervin G Lindgren A Aberg P (1997) EVects of UV-B radi-ation and simulated herbivory on phlorotannins in the brown algaAscophyllum nodosum Mar Ecol Prog Ser 157139ndash146doi103354meps157139

Pavia H Toth GB Aberg P (2002) Optimal defense theory elasticityanalysis as a tool to predict intraplant variation in defenses Ecol-ogy 83891ndash897

Peckol P Krane JM Yates JL (1996) Interactive eVects of inducibledefense and resource availability on phlorotannins in the NorthAtlantic brown alga Fucus vesiculosus Mar Ecol Prog Ser138209ndash217 doi103354meps138209

Peacuterez-Schultheiss J Crespo JE (2008) A new species of ParhyalellaKunkel 1910 (Amphipoda Talitroidea Dogielinotidae) from thecoast of Chile Zootaxa 172461ndash68

Peterson CH Renaud PE (1989) Analysis of feeding preference exper-iments Oecologia 8082ndash86 doi101007BF00789935

Porter KG (1976) Enhancement of algal growth and productivity bygrazing zooplankton Science 1921332ndash1334 doi101126science19242461332

Raven JA (2003) Long-distance transport in non-vascular plants PlantCell Environ 2673ndash85 doi101046j1365-3040200300920x

Reed D Ebeling A Anderson T Anghera M (1996) DiVerentialreproductive responses to Xuctuating resources in two seaweedswith diVerent reproductive strategies Ecology 77300ndash316doi1023072265679

Rothaumlusler E Thiel M (2006) EVect of detachment on the palatabilityof two kelp species J Appl Phycol 18423ndash435 doi101007s10811-006-9053-7

Rothaumlusler E Macaya EC Molis M Wahl M Thiel M (2005) Labora-tory experiments examining inducible defense show variable re-sponses of temperate brown and red macroalgae Rev Chil HistNat 78603ndash614

Santelices B Ugarte R (1987) Algal life-history strategies and resis-tance to digestion Mar Ecol Prog Ser 35267ndash275 doi103354meps035267

Santelices B Castilla JC Cancino J Schmiede P (1980) Comparativeecology of Lessonia nigrescens and Durvillaea antarctica(Phaeophyta) in Central Chile Mar Biol (Berl) 59119ndash132doi101007BF00405461

Schoenwaelder MEA (2002) The occurrence and cellular signiWcanceof physodes in brown algae Phycologia 41125ndash139

Schoenwaelder MEA Clayton MN (1999) The presence of phenoliccompounds in isolated cell walls of brown algae Phycologia38161ndash166

Shibata T Kawaguchi S Hama Y Inagaki M Yamaguchi K Nakam-ura T (2004) Local and chemical distribution of phlorotannins inbrown algae J Appl Phycol 16291ndash296 doi101023BJAPH0000047781249930a

Steinberg PD (1984) Algal chemical defense against herbivoresallocation of phenolic compounds in the kelp Alaria marginataScience 223405ndash407 doi101126science2234634405

Steinberg PD (1985) Feeding preferences of Tegula funebralis andchemical defenses of marine brown algae Ecol Monogr 53333ndash349 doi1023071942581

Steinberg PD Van Altena IA (1992) Tolerance of marine invertebrateherbivores to brown algal phlorotannins in temperate AustralasiaEcol Monogr 62189ndash222 doi1023072937093

123

62 Mar Biol (2008) 15551ndash62

Stekoll MS Else PV (1990) Cultivation of Macrocystis integrifolia(Laminariales Phaeophyta) in southeastern Alaskan waters Hyd-robiologia 204205445ndash451 doi101007BF00040269

Steneck RS Watling LE (1982) Feeding capabilities and limitations ofherbivorous molluscs a functional group approach Mar Biol(Berl) 68299ndash319 doi101007BF00409596

Targett NM Arnold TM (1998) Predicting the eVects of brown algalphlorotannins on marine herbivores in tropical and temperateoceans J Phycol 34195ndash205 doi101046j1529-88171998340195x

Targett NM Arnold TM (2001) EVects of secondary metabolites ondigestion in marine herbivores In McClintock JB Baker BJ(eds) Marine chemical ecology CRC Press Boca Raton pp 391ndash411

Taylor RB Sotka E Hay ME (2002) Tissue-speciWc induction ofherbivore resistance seaweed response to amphipod grazingOecologia 13268ndash76 doi101007s00442-002-0944-2

Toth GB Pavia H (2002a) Intraplant habitat and feeding preferenceof two gastropod herbivores inhabiting the kelp Laminariahyperborea J Mar Biol Assoc UK 821ndash5 doi101017S0025315402005416

Toth GB Pavia H (2002b) Lack of phlorotannin induction in the kelpLaminaria hyperborea in response to grazing by two gastropodherbivores Mar Biol (Berl) 140403ndash409 doi101007s002270100707

Toth GB Pavia H (2007) Induced herbivore resistance in seaweeds ameta-analysis J Ecol 95425ndash434 doi101111j1365-2745200701224x

Toth GB Langhamer O Pavia H (2005) Inducible and constitutivedefences of valuable seaweed tissues consequences for herbivoreWtness Ecology 86612ndash618 doi10189004-0484

Tugwell S Branch GM (1989) DiVerential polyphenolic distributionamong tissues in the kelps Ecklonia maxima Laminaria pallidaand Macrocystis angustifolia in relation to plant-defense theory

J Exp Mar Biol Ecol 129219ndash230 doi1010160022-0981(89)90104-4

Tuomi J Ilvessalo H Niemelauml P Sireacuten S Jormalainen V (1989) With-in-plant variation in phenolic content and toughness of the brownalga Fucus vesiculosus L Bot Mar 32505ndash509

Van Alstyne KL (1995) Comparison of three methods for quantifyingbrown algal polyphenolic compounds J Chem Ecol 2145ndash58doi101007BF02033661

Van Alstyne KL Houser LT (2003) DimethylsulWde release duringmacroinvertebrate grazing and its role as an activated chemicaldefense Mar Ecol Prog Ser 250175ndash181 doi103354meps250175

Van Alstyne KL McCarthy JJ Hustead CL Kearns LJ (1999) Phloro-tannin allocation among tissues of northeastern paciWc kelps androckweeds J Phycol 35483ndash492 doi101046j1529-881719993530483x

Van Alstyne KL Wolfe GV Freidenburg TL Neill A Hicken C(2001) Activated defense systems in marine macroalgae evi-dence for an ecological role for DMSP cleavage Mar Ecol ProgSer 21353ndash65 doi103354meps213053

Vinueza LR Branch GM Branch LM Bustamente RH (2006) Top-down herbivory and bottom-up El Nino eVects on Galapagosrocky-shore communities Ecol Monogr 76111ndash131 doi10189004-1957

Winter FC Estes JA (1992) Experimental evidence for the eVects ofpolyphenolic compounds from Dictyoneurum californicum (Pha-eophyta Laminariales) on feeding rate and growth in the red aba-lone (Haliotus rufescens) J Exp Mar Biol Ecol 155263ndash277doi1010160022-0981(92) 90067-K

Wotton RS (2004) The ubiquity and many roles of exopolymers (EPS)in aquatic systems Sci Mar 6813ndash21

Yates JL Peckol P (1993) EVects of nutrient availability and herbivoryon polyphenolics in the seaweed Fucus vesiculosus Ecology741757ndash1766 doi1023071939934

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