Top-down and bottom-up controls on sediment organic matter composition in an

experimental seagrass ecosystem

Amanda C. Spivak1, Elizabeth A. Canuel, J. Emmett Duffy, and J. Paul RichardsonVirginia Institute of Marine Science, P.O. Box 1346, Gloucester Point, Virginia 23062

Abstract

We tested the singular and interactive effects of resource availability (light) and community composition (foodchain length and herbivore species richness) on eelgrass (Zostera marina) ecosystem properties and functioningwith an experimental mesocosm system. Food chain length was manipulated through the presence or absence ofblue crab (Callinectes sapidus) predators, whereas grazer species richness varied across three levels (zero, two, orfour crustacean species). We found important and interacting effects of bottom-up and top-down forcings onsediment organic matter (SOM) composition. Light increased eelgrass and algal biomass and sediment organiccarbon and nitrogen content. Increasing grazer diversity generally decreased algal biomass and ecosystemproduction but interacted with food chain length (i.e., presence of predatory crabs) and light. Predators generallyincreased algal biomass and ecosystem production through a trophic cascade, which was stronger at high grazerdiversity and under ambient light. SOM composition, determined with fatty acid (FA) biomarkers, was sensitiveto all manipulated variables. Increasing grazer species richness often decreased the contributions of FAs derivedfrom plant and algal sources, whereas increasing light had the opposite effect. Food chain length was generallya less important determinant of SOM composition than light, although predators did increase FAs representativeof heterotrophic bacteria. Overall, resource availability and epibenthic community composition stronglyinfluenced organic matter cycling, SOM composition, and the bacterial community in seagrass-bed sediments.

Coastal ecosystems are often affected by multipledisturbances that alter both resource availability andcommunity composition simultaneously. In the Chesa-peake Bay, for example, seagrass beds are affected bycommercial harvesting of the blue crab, Callinectes sapidus(Stephan et al. 2000), and by suspended sediment andnutrient loading that can lead to reduced light availability(Kemp et al. 2004). Changes in abundance of importantpredators, such as the striped bass or the blue crab, mayprecipitate changes in the biomass of lower trophic levels(Hairston et al. 1960; Strong 1992; Pace et al. 1999). Theseshifting trophic interactions, along with reduced lightavailability, can affect primary producer abundance andproductivity (Heck et al. 2000; Hughes et al. 2004; Borer et

al. 2006) and, in turn, sediment organic matter (SOM)content (Canuel et al. 2007). Consequently, cascadingchanges in animal and plant biomass may alter the ratesand pathways by which organic matter (OM) is cycled in anecosystem (Schindler et al. 1997; Dangles and Malmqvist2004).

Predicting how changing trophic structure affects OMcycling is complicated by the fact that predators induceshifts not only in prey biomass but also in prey communitystructure. In seagrass systems, for example, grazinginvertebrates can consume epiphytic algae, macroalgae,benthic microalgae, and vascular plants (Valentine andDuffy 2006). Thus, shifts in grazer community compositionmay affect the abundance of different primary producers.Because seagrasses, macroalgae, and epiphytes differ intheir biochemical composition and proportion of structuralcomponents, the food preferences of grazing invertebratesmay, affect the quantity and lability of organic carbondelivered to the sediments and thus, the quantity andquality of sediment organic carbon (Canuel et al. 2007).Such compositional changes need not be dramatic to affectecosystem properties: small shifts in grazer richness andspecies composition can significantly affect plant and algalbiomass and influence total sediment organic carbon (e.g.,Duffy et al. 2003; Canuel et al. 2007).

Because sediment microbial communities are importantmediators of carbon and other elemental cycles in coastalenvironments (Boschker et al. 1999; Holmer et al. 2001,2004), changes in aboveground trophic structure anddiversity that alter OM delivery to seagrass sedimentsmay have important consequences for carbon cycling andstorage. In terrestrial soils, by analogy, microbial commu-nity composition and activity are sensitive to changes inaboveground community structure (Setälä et al. 1998;Wardle et al. 2005). Although there are fewer studies from

1 Corresponding author (amanda@vims.edu).

AcknowledgmentsWe thank J. G. Douglass, K. E. France, S. Grill, and M. A.

Wunderly for assistance in setting up, maintaining, and samplingthe experiments. We also thank M. A. Cochran, A. K. Hardison,and E. J. Waterson-Lerberg for help with laboratory analyses.This study was improved by input from I. C. Anderson, D. Bronk,and J. J. Stachowicz. We also thank two anonymous reviewersand B. Thamdrup for constructive comments that improved themanuscript.

This research was supported by NSF grants OCE 00-99226 andOCE 03-52343 to J.E.D., NSF grants DEB 0542645 and DEB0454736 to E.A.C., and an NCER, STAR program, EPA grantFP-91656901 to A.C.S. Although the research described in thearticle has been funded in part by the U.S. EPA’s STAR program,it has not been subjected to any EPA review and therefore doesnot necessarily reflect the views of this agency, and no officialendorsement should be inferred. Additional support was providedby the Virginia Institute of Marine Science.

This article is contribution 2834 of the Virginia Institute ofMarine Science, The College of William and Mary.

Limnol. Oceanogr., 52(6), 2007, 2595–2607

E 2007, by the American Society of Limnology and Oceanography, Inc.

2595

marine habitats, microbial activity in sediments is stronglyrelated to OM deposition (Canuel and Martens 1993;Boschker and Cappenberg 1998; Boschker et al. 2000). Thepotential cascade from consumer control of abovegroundproduction to delivery and accumulation of belowgroundOM may thus be important to carbon remineralization,recycling, and sequestration in the sediments.

Effective conservation and management of seagrassecosystems requires a clear understanding of relationshipsbetween community ecology and biogeochemical cycling. Avariety of studies have investigated coastal eutrophication(Cloern 2001; Duarte 2002 and references therein), trophicinteractions in seagrass beds (Heck and Valentine 2006 andreferences therein; Valentine and Duffy 2006), and inter-actions between nutrient enrichment and food-web ecology(McClelland and Valiela 1998; Deegan et al. 2002; Tewfiket al. 2005). Others have examined sediment nutrient andbacterial processes in seagrass beds (Holmer et al. 2001,2004). Yet few studies have examined the relationships andfeedbacks between aboveground ecology and belowgroundgeochemical cycling. Geochemical tools provide a way todetect and quantify such linkages between communitystructure and OM cycling. Specifically, lipid biomarkers arecompounds reliably produced by a specific group oforganisms that are sufficiently resistant to degradation tobe preserved in sediments (Killops and Killops 1993).Diagnostic biomarkers often have site-specific methylgroups, double bonds, or cyclic side chains useful in tracingthe sources of OM (Killops and Killops 1993). Bacteria, forexample, synthesize iso- and anteiso-branched fatty acids(BrFAs), while microalgae contain highly unsaturatedlong-chain fatty acids (or alkanoic acids) (Volkman et al.1998). One class of lipids, the FAs, is particularly useful,because they have high source fidelity and exhibit a rangeof chemical reactivity (Canuel et al. 1995; Canuel andMartens 1996). Additionally, a subclass of the FAs, thephospholipid-linked fatty acids (PLFAs), are good indica-tors of recently viable cells because they are mainly derivedfrom membrane lipids, which are rapidly hydrolyzed aftercell death (White et al. 1979; Killops and Killops 1993). Byquantifying both the total FAs and the PLFAs, it ispossible to compare OM contributions from detrital andviable or recently viable sources. Thus, lipid biomarkers,and FAs, in particular, provide a quantifiable link betweenthe aboveground community and sediment geochemistry.

To assess the effects of changing community structure oncarbon fate and storage in seagrass beds, we conducted anexperimental manipulation of bottom-up forcing (lightavailability), community composition (grazer diversity),and food chain length (predator presence) and measuredtheir interacting effects on ecosystem productivity, SOMquality, and sediment microbial activity. Specifically, webuilt on previous studies examining top-down effects on theaboveground community (Duffy et al. 2003) and on SOM(Canuel et al. 2007) to test several hypotheses. First, higherdiversity of epibenthic grazers will reduce algal biomass butincrease seagrass and benthic algal biomass, leading tochanges in the composition and quality of algal materialincorporated in sediments. Second, predators will increaseaccumulation of algal biomass through a trophic cascade,

thereby increasing SOM quantity, quality, and sedimentmicrobial activity. Finally, high light availability willincrease biomass accumulation of aboveground andbenthic algae, SOM lability, and sediment microbialactivity.

Methods

Experimental design—We conducted a mesocosm exper-iment to examine the main and interactive effects of grazerspecies richness, food chain length, and light intensity onecosystem properties, including production, algal biomassaccumulation, and SOM content and composition. Weestablished three grazer richness treatments containing nograzer species, random combinations of two grazer species,or four grazer species. Grazers were chosen from a pool ofsix species, including three amphipod crustacean species(Ampithoe longimana, Gammarus mucronatus, and Caprellapenantis), two isopods (Idotea baltica and Erichsonellaattenuata), and a gastropod (Bittium varium). Theseinvertebrate grazers are common in the York River estuaryduring the spring and summer (Duffy et al. 2001, 2003).Food chain length was manipulated by exposing a parallelset of grazer treatments to a generalist predator common inthe Chesapeake Bay, the blue crab, C. sapidus. Lightintensity was manipulated by covering half of the tankswith shade cloths (69% attenuation). There were a total of12 treatments, each replicated five times. Because ofextinctions and contaminations, however, six replicateswere removed from the final analyses. Consequently, 54replicates were used in statistical analyses; the zero-grazertreatments had five replicates in ambient light and fourreplicates in low light, the two grazer treatments had fourreplicates, and the four grazer treatments had fivereplicates.

Outdoor mesocosm experiments were conducted for6 weeks during the summer of 2003 in an array of 113-liter,translucent fiberglass tanks that were continuously sup-plied with flowing estuarine water from the York River,Virginia (Duffy et al. 2003). Water passed first througha sand filter and then through a 150-mm mesh. Thiseliminated larger invertebrates and minimized invasion bynontarget animals while permitting passage of invertebratelarvae and algal spores, which often colonized the tanks.Water was supplied through ‘‘dump buckets,’’ whichregularly spilled the filtered water into the tanks, providingboth turbulence and aeration. Tanks were stocked withclean sand to a depth of 10 cm; the percent total organiccarbon (%TOC) was below detection. Low OM contentsand was used as a substrate to reduce initial heterogeneitybetween the tanks and to increase our ability to detectnewly deposited SOM (Canuel et al. 2007). Seventy-fivepreweighed eelgrass (Zostera marina) shoots, cleaned ofgrazers and epiphytes, were planted in the sand in eachtank. This eelgrass density is within the range found in theYork River estuary system (Orth and Moore 1986). Oneweek after the grass was planted, invertebrate grazers wereadded to each grazer mesocosm (45 each for two-speciestreatments, 15 of each for four-species treatments); thesedensities were near the low end of those found in the York

2596 Spivak et al.

River. Blue crabs (C. sapidus) were added 2 d after thegrazers had acclimated. The 6-week experimental incuba-tion time was chosen to minimize the risks of invasion bynontarget grazer species and of complete consumption ofthe eelgrass, which increase at longer time intervals. Thistime period permits major changes in animal (one to twograzer generations) and plant community development andin surface sediment characteristics (see Duffy et al. 2003,2005; Canuel et al. 2007). Despite limitations, thisexperimental infrastructure simulates several aspects ofthe biotic and abiotic field situation well (Duffy et al. 2001).Results of the experiment for aboveground biomass andcomposition of seagrass and the associated community arereported elsewhere (Duffy et al. unpubl. data). Here, wefocus on patterns of SOM accumulation and composition.

Gross ecosystem production—As an estimate of whole-ecosystem metabolism, we measured gross ecosystem pro-duction (GEP; mmol L21 O2 d21 m22) 1 week before theexperiment was terminated. Because of time constraintsand instrument availability, these measurements wereconducted only in ambient light treatments. Clear plasticwrap was placed on the water’s surface of each tank tominimize oxygen exchange with the atmosphere, and thewater supply was shut off. Dissolved oxygen (DO)measurements were taken three to four times during eachof two 4-h incubations (10:00–14:00 h and 22:00–02:00 h)with a YSI Data Sonde to capture net daytime productionand total respiration, respectively (assuming that little-to-no production occurs at night). Tank water was stirredprior to each reading to disrupt any temperature or DOstratification that may have formed while maintaininga closed system. If DO fell to hypoxic levels (2 mg L21),measurements ceased on that tank, and the plastic coverwas removed. We calculated the slope of changes in DOconcentration versus the time elapsed and divided this bythe area of the tank to obtain flux in O2 mmol L21 d21

m22. Hourly light and dark rates were scaled to 14 h ofdaylight and 10 h of darkness to estimate net dailysummertime GEP.

Bulk SOM—At the end of the experiment, threesediment cores (2.6 cm in diameter) were collected fromeach mesocosm, and the upper 1 cm from each core wasremoved. Subsamples from each core were combined intoa composite sample in a precombusted (450uC) jar. Thesediment sample was homogenized, and aliquots wereremoved to precombusted glass scintillation vials foranalyses of benthic chlorophyll a (Chl a; a measure ofmicroalgal biomass) and sediment TOC and total nitrogen(TN). All samples were stored at 220uC until analysis.Samples of benthic Chl a were analyzed within 6 weeks ofcollection according to Neubauer et al. (2000). Concentra-tions of TOC and TN were analyzed by standard methodswith a Fisons CHN analyzer (model EA1108) afterremoving inorganic carbon (Hedges and Stern 1984);acetanilide was used as the standard.

Lipid biomarker analyses—Lipid biomarker compoundswere analyzed by a modified Bligh and Dyer (1959) method

(Canuel and Martens 1993; Canuel et al. 2007). Briefly,sediment samples were extracted with methylene chlo-ride : methanol (2 : 1, v : v) with an accelerated solventextraction system (Dionex ASE 200). Following extraction,the samples were partitioned, and the organic phase wasremoved. Hexane was added to the aqueous phase, and thesamples were partitioned a second time, after which thehexane layer was added to the original organic phase. Thecombined organic phases sat over anhydrous Na2SO4overnight to remove traces of water and were concentratedto 1 mL (Zymark Turbo Vap 500). The total lipid extractswere separated into nonpolar (F1/2) and polar (F3)fractions by eluting solvents of increasing polarity throughsilica gel columns (Guckert et al. 1985). F1/2 (neutral andglycolipids) and F3 (phospholipids) were each saponifiedby procedures described in Canuel et al. (2007). Followingsaponification, the residue was extracted under basic(saponified-neutral; SAP-N) and acidic pH (saponified-acids; SAP-A). The SAP-A fractions were methylated withBF3-CH3OH and purified by silica gel chromatography.Just before gas chromatographic (GC) injection, sampleswere evaporated to dryness under N2, and a small volumeof hexane (30 mL for the polar fraction and 100 mL for thenonpolar) was added. The FAs (as methyl esters) wereanalyzed by gas chromatography following previouslypublished procedures (Canuel et al. 2007 and referencestherein). Peaks were quantified relative to an internalstandard, methyl heneicosanoate, added just prior to GCanalysis. Peak identities were verified by reference stan-dards and by combined gas chromatography–mass spec-trometry (GC-MS) with a Hewlett-Packard 6890 GCinterfaced with a mass selective detector operated inelectron impact mode. FAs are designated A:BvC, whereA is the total number of carbon atoms, B is the number ofdouble bonds, and C is the position of the first double bondfrom the aliphatic ‘‘v’’ end of the molecule. The prefixes‘‘i’’ and ‘‘a’’ refer to iso and anteiso methyl BrFAs (seeCanuel et al. 1995 and references therein). Results for twoclasses of FAs are presented: PLFAs, which representviable or recently viable biomass, and total FAs, whichrepresent neutral, glycolipids, and phospholipids andinclude the sum of the viable and detrital contributions.

Statistical analyses—The experiment was analyzed asa fully factorial three-way analysis of variance, with grazertreatment (df 5 2), food chain length (i.e., predatorpresence or absence, df 5 1), and light availability (df 51) as fixed variables, by SAS version 9.0 for Windows.Analyses of FA data were conducted on percent abun-dance. GEP data were subjected to a two-way analysis ofvariance, because data were only available for ambient lighttreatments. From the analyses of variance, we calculatedthe magnitude of main and interactive effects (v2,percentage of the variance explained). Because of contam-inations and extinctions, two control and four 2-speciesmesocosms were removed from all statistical analyses;results presented here use the type III sum of squares (SS)from the analysis of variance model. Included in thestatistical analyses were five replicates in ambient light andfour in low light of the zero-grazer treatments, four

Biodiversity and sediment organic matter 2597

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2598 Spivak et al.

replicates of the two grazer treatments, and five replicatesof the four grazer treatments. There were two criteria forelimination: (1) grazer contamination totaled more than500 mg of ash-free dry matter and (2) failure of two grazerspecies (Caprella and Bittium) to establish necessitatedelimination of mesocosms where this pair of species wasinitially stocked. To separate effects of grazer presenceversus grazer species richness, we conducted a prioricontrasts that partitioned the grazer SS from the analysisof variance into two orthogonal components (see Duffy etal. 2005). The first contrast compared the two- and four-species treatments against the zero-species treatment(species presence contrast), and the second compared thetwo- versus four-species treatments (species richnesscontrast).

To aid in interpreting the FA data, we performedmultiple regression analyses modeling the FA groups asa function of biomass of the major primary producers,eelgrass, total algae, and benthic Chl a. The partial r2 wascalculated by dividing the type III SS for each responsevariable by the total SS. The analyses were performed on%TOC, individual FAs, and groups of FAs normalized tothe sum of all FAs (%total FA or %PLFA). Additionally,we conducted principal components analysis (PCA; byMinitab 14) to better elucidate relationships betweenmanipulated and response variables. We only performedPCA on SOM variables, as these responded to primaryproducer abundance determined by grazers and crabpredators. PCA loadings describe the relationships betweenthe SOM response variables and the dominant principalcomponents. PCA scores illustrate relationships betweenthe observations and the dominant principal components.PCA loadings were also regressed against the majorprimary producer groups (Z. marina, total algal biomass,and benthic Chl a) to help interpret the nondimensionalresults.

Results

Primary producer biomass and GEP—In general, primaryproducer biomass was enhanced by light and predatorpresence and decreased by grazers. Aboveground, lightincreased the biomass of both Z. marina and algae(Table 1; Fig. 1A,B). Cascading predator effects resultedin grazers reducing primary producer biomass only in theabsence of predators (grazer 3 predator interaction,Table 1). For example, grazer presence and richnessdecreased Z. marina biomass in the absence of predators(p 5 0.002, v2 5 0.28). Further, total algal biomass wasreduced by grazer presence and richness but increased whenpredators were present. In the sediments, benthic Chl a wasincreased by ambient light (p 5 0.023, v2 5 0.06, Fig. 1C),decreased by grazer presence (p 5 0.004, v2 5 0.01), andunaffected by crab predators.

GEP in the ambient light mesocosms was influenced bythe interaction of predators and grazers (p 5 0.002, v2 50.19, Table 1; Fig. 2). Overall, blue crab predators in-creased GEP (p , 0.001, v2 5 0.29) but only in thepresence of grazers, reflecting a trophic cascade. Increasinggrazer species richness reduced GEP but only in the

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Biodiversity and sediment organic matter 2599

absence of predators (p 5 0.001, v2 5 0.39). Thus, grazerpresence, richness, and predator presence are all important,interacting determinants of GEP (Table 1).

Bulk SOM—During the 6-week experiment, measurablelevels of TOC and TN accumulated in surface sediments(Table 1; Fig. 3). Sediment %TOC and %TN contentwere higher in ambient light than in shaded treatments (p5 0.004, v2 5 0.12 and p , 0.001, v2 5 0.18,respectively). Neither grazers nor predators significantlyaffected %TOC or %TN. Thus, bottom-up forcing hada stronger effect on TOC and TN accumulation than top-down processes.

Total FAs—While bulk indicators of SOM were sensitiveonly to light availability, FA composition was stronglyinfluenced by both bottom-up and top-down forcing. Onaverage, total FA abundance normalized to TOC(mg mg21) was significantly reduced by light but wasunaffected by predators and grazers (Fig. 4A). For furtheranalysis, both the total FAs and PLFAs were categorizedinto subclasses on the basis of chain length, number ofdouble bonds, and carbon branching patterns representingdifferent sources of OM (Fig. 5).

Total FA composition was dominated (29–47% totalFA) by even-numbered saturated compounds (C12:0–C18:0), representing algal and bacterial sources. Therelative abundance of short-chain FAs (SCFAs; %(C12:0+ C14:0)) was highest in ambient light in the presence ofpredators (Table 1; Fig. 5A). Grazer presence, however,decreased SCFAs. SCFAs were also positively related tobenthic Chl a (Table 2). The contributions of %C16:0 and%C18:0 FAs were unaffected by any of the treatments andwere unrelated to either eelgrass or benthic Chl a abun-dance (Table 1). The long-chain FA C24:0, composing 3–16% of the total FAs, was increased by ambient light onaverage (Table 1; Fig. 5B) and decreased by grazers butmore so in ambient light and predator treatments. C24:0was also positively related to benthic Chl a (Table 2).Overall, light increased FAs that were positively associat-ed with benthic microalgae (Chl a), whereas grazers, thedominant top-down control, generally had the oppositeeffect.

Relative abundance of polyunsaturated FAs (C18:4,C20:4, C20:5, C22:5, and C22:6; grouped as polyunsaturatedFAs [PUFAs]) was reduced by predators but only inshaded treatments, reflecting an interaction between pred-ators and light (Table 1; Fig. 5C). %PUFA abundance wasnot related to either eelgrass biomass or benthic Chla (Table 2). Linoleic (C18:2v6) and linolenic (C18:3v3) acidswere decreased when grazers were present (p 5 0.035, v2 50.07; Table 1; Fig. 5D) but only in ambient light treatments(grazer 3 light interaction, p 5 0.007, v2 5 0.12). Z. marinawas positively related to %(C18:2v6 + %C18:3v3) (Table 2).Overall, top-down controls were important determinants ofPUFA abundance, with predators decreasing %PUFA andgrazers decreasing linoleic and linolenic acids.

BrFAs (iso- and anteiso-C13:0, C15:0, C17:0, and C19:0),representative of sediment heterotrophic bacteria, weresensitive to all three manipulated variables (Fig. 5E). Light

Fig. 1. Effects of grazers, predators, and light availability onaboveground primary producers (Z. marina and total algae) andbenthic Chl a. Light, generally, increased primary producerbiomass. Grazers decreased Z. marina biomass (in the absenceof predators) and total algal biomass. Predators decreased both Z.marina and total algal biomass, but the magnitude of this effectvaried with grazer richness, resulting in grazer 3 predatorinteractive effects. Error bars represent standard error. Therewere four replicates of each zero-grazer treatment in low light andfive in ambient light: four replicates of each two grazer treatmentand five replicates of each four grazer treatment. Statistical resultsare reported in Table 1.

2600 Spivak et al.

generally decreased the relative abundance of BrFAs,though this effect was driven mainly by the two-grazerspecies treatment and translated into a grazer 3 lightinteraction effect (Table 1). Relative abundance of BrFAswas consistently higher in predator treatments (p 5 0.013,v2 5 0.07). BrFAs were positively related to benthic Chla (Table 2). These results suggest that sediment heterotro-phic bacteria are sensitive to both bottom-up and top-downcontrols.

PCA provided a summary of these changes in SOM withmanipulation of light and epibenthic community composi-tion. Principal components 1 (PC1) and 2 (PC2) explained31.7% and 25.9% of the variance in total FA composition,respectively (Fig. 6A,B). %TOC, %C24:0, and %(C12:0 +C14:0) had the most positive loadings on PC1 (Table 3) andalso responded positively to ambient light (Figs. 3, 5). Theassociation between PC1 and light is also supported by thepositive relationship between benthic Chl a and PC1loadings (r2 5 0.33; p , 0.001). In contrast, PC2 separatedSOM variables according to crab predator or grazer effects.Variables with negative PC2 loadings (%PUFA and%BrFA) were affected by crab predators, albeit in oppositedirections, whereas those with positive PC2 loadings(%(C12:0 + C14:0), %(C18:2 + C18:3), and %C24:0 (ambientlight only)) were decreased by grazers (Table 3). Bottom-upforcing interacted with top-down forcing of SOM compo-sition, as PC scores were influenced by crab predators andgrazers differently, depending on light availability(Fig. 6A,B). In ambient light (Fig. 6A), grazer-free treat-ments had positive PC1 and PC2 scores, whereas the two-and four-grazer treatments were near zero or negative onPC2. In contrast, under low light (Fig. 6B), treatments withcrabs had more positive PC2 scores, whereas no-crab

treatments were negative. Under both light regimes, thepattern is most evident for the zero- and four-grazertreatments (Fig. 6A,B). Thus, PCA results suggested thatthe dominant top-down control (grazers vs. crab predators)influenced total FA composition differently with lightavailability.

Phospholipid-linked FAs—Like total FAs, PLFAs (mgPLFA mg21 TOC; Fig. 4B), indicative of viable or recentlyviable OM sources, were sensitive to top-down and bottom-up influences. None of the manipulated treatments affectedtotal PLFAs, %(C12:0 + C14:0), %C16:0, or %C18:0 PLFAs(Table 1). The relative abundance of C24:0 PLFAs andlinoleic and linolenic PLFAs (%C18:2v6 and %C18:3v3) washigher under ambient light but only in the absence ofgrazers, which reduced linoleic and linolenic acid contribu-tions (Fig. 5G,I). Predators increased linoleic and linolenic(C18:2v6 and C18:3v3) PLFAs only under ambient light; thistranslated into a predator 3 light interaction (Table 1).BrFAs were lower in ambient light treatments (p 5 0.006,v2 5 0.08). In addition to main effects, there were a variety

Fig. 2. Effects of grazers and predatory crabs on summergross ecosystem production, measured as dissolved oxygen (DO)flux. Predators (crabs) mediated a negative grazer effect on grossecosystem production through a trophic mechanism. The magni-tude of the predator effect increases with grazer richness. Data areonly from ambient light treatments. Error bars represent standarderror. Statistical results are reported in Table 1.

Fig. 3. Effects of light, grazers, and predators on sedimentnitrogen and carbon. (A) Light increased sediment total nitrogen(%TN) and (B) total organic carbon content (%TOC). Neithergrazer richness nor food chain length affected %TN or %TOC.Error bars represent standard error. Statistical results are reportedin Table 1.

Biodiversity and sediment organic matter 2601

of interactive effects on PLFA composition and abundance(Table 1). Overall, the PLFA results echo those for totalFAs, showing that community structure and light avail-ability alter SOM deposition and probably sedimentmicrobial response.

PC1 and PC2 explained 25.3% and 19.5% of thevariance, respectively, in PLFA composition (Fig. 6C,D).Similar to the results for total FAs, PC1 separated PLFAvariables according to light availability. %TOC, %C24:0,which were increased by light (Figs. 3, 5) %(C18:2 + C18:3),which was increased by light and correlated with benthicChl a (Fig. 5; Table 2), had more positive PC1 loadings(Table 3). PC1 was also positively related to total algalbiomass, which increased in ambient light (r2 5 0.10; p 50.018). In ambient light, PC2 separated response treatmentsby grazer presence (near zero) and absence (more negative)(Fig. 6C). The association of PC2 with grazers is supportedby the negative relationship between PC2 and benthic Chla (r2 5 0.10; p 5 0.017). In shaded treatments, neither PC1nor PC2 clearly separated grazer and crab treatments(Fig. 6D).

Discussion

A realistic assessment of ecosystem functioning underchanging conditions requires simultaneous consideration of

Fig. 4. (A) Abundance of total fatty acids (total FAs) and(B) phospholipid-linked fatty acids (PLFAs) normalized to totalsediment organic carbon content (mg mg {1TOC). Light decreasedtotal FA (mg mg {1TOC (A)) but had no effect on PLFA (mg mg

{1TOC

(B)). Error bars represent standard error. Statistical results arereported in Table 1.

Fig. 5. (A–J) Effects of light, grazers, and predators on totalfatty acids (total FAs) and phospholipid-linked fatty acids(PLFAs) subclasses. Light, predators, and grazers had strongsingular and interactive effects on total FAs and PLFAs. Thepolyunsaturated fatty acid (%PUFA) subclass, representing freshalgal material, is composed of C18:4, C20:4, C20:5, C22:5, and C22:6.The branched fatty acid (%BrFA) subclass, representing hetero-trophic bacteria, includes iso- and anteiso-C13:0, C15:0, C17:0, andC19:0. Error bars represent standard error. See text for biomarkersources and Table 1 for statistical results.

2602 Spivak et al.

top-down and bottom-up effects (Strong 1992; Hughes etal. 2004; Borer et al. 2006). In benthic, sedimentarysystems, this should include effects on biomass andcomposition of the aboveground primary producers andanimals (Heck et al. 2000; Hughes et al. 2004; Borer et al.2006), the belowground community (Wardle et al. 2005),and the OM composition in sediments (Holmer et al. 2004;Canuel et al. 2007). In this study, we showed experimentallythat epibenthic food-web structure and resource (light)availability strongly influenced the abundance and compo-

sition of SOM. Specifically, light increased and grazersdecreased most measures of primary producer biomass andSOM. Grazer effects on primary producers and SOMcomposition were generally stronger in ambient lighttreatments, showing that animal communities and resourceavailability together shaped properties of this seagrassecosystem. Perhaps surprisingly, given the strong effects ofpredators on aboveground algal biomass in this system(Duffy et al. 2005), effects of predators (food chain length)on SOM were less pervasive than those of light availability

Fig. 6. Score plots from principal component analysis for total fatty acids (total FAs) andphospholipid-linked fatty acids (PLFAs) in ambient light and shaded treatments. Error barsrepresent standard error. G, grazers; C, crab predators.

Table 2. Regression analyses of Z. marina biomass (ash-free dry weight, g) and benthic Chl a (mg cm22) against the major fatty acidgroups. Significant relationships (p , 0.05) are noted in bold. TFA, total fatty acids; PLFA, phospholipid-linked fatty acids; i,a, iso-,anteiso-.

Response

Z. marina Benthic Chl a Total

Coefficient Partial r2* p Coefficient Partial r2* p Model r2

TFA

%SCFA (C12:0+C14:0) of TFA 0.09 0.03 0.171 0.12 0.22 ,0.001 0.25%C24:0 of TFA 0.00 0.00 0.968 0.07 0.22 ,0.001 0.22%PUFA of TFA 20.13 0.01 0.428 0.03 0.00 0.718 —%(C18:2+C18:3) of TFA 0.26 0.08 0.041 0.05 0.01 0.439 0.09%BrFA (i,a C13–C19) of TFA 20.20 0.06 0.066 0.11 0.08 0.034 0.14

PLFA

%SCFA (C12:0+C14:0) of PLFA 0.06 0.02 0.285 0.00 0.00 0.897 —%C24:0 of PLFA 0.05 0.07 0.052 0.00 0.00 0.813 —%PUFA of PLFA 20.07 0.01 0.588 20.05 0.01 0.449 —%(C18:2+C18:3) of PLFA 0.32 0.04 0.121 0.20 0.08 0.037 0.12%BrFA (i,a C13–C19) of PLFA 20.16 0.04 0.137 20.03 0.00 0.509 —

* Partial r2 values were calculated by dividing the type III SS by the total SS.

Biodiversity and sediment organic matter 2603

or grazers. Nevertheless, predators increased OM contribu-tions from microbial sources generally (%SCFAs, totalFAs), and from sediment heterotrophic bacteria specifically(%BrFA, total FA). This suggests that the previouslydemonstrated cascading effects of crab predators onprimary producer biomass (Duffy et al. 2005; Canuel etal. 2007) also affect the accumulation of labile OM, elicitinga bacterial community response.

Bottom-up forcing—Many seagrass ecosystems sufferfrom suspended sediment and nutrient loading, both ofwhich can reduce light availability (Duarte 2002; Kemp etal. 2004). Decreased water clarity negatively affectsseagrass performance and has cascading effects on associ-ated fauna, water quality, and sediment erosion (Orth andMoore 1983; Duarte 2002). With such wide-ranging effects,it is likely that decreased water transparency would alsoaffect SOM accumulation and biogeochemical processes inseagrass sediments (McGlathery et al. 1998; Holmer et al.2004). Thus, a primary goal of our study was to elucidatehow light availability, alone and in concert with changingfood-web structure, influences OM composition.

In our experimental system, light strongly increasedaboveground plant and algal biomass (Table 1; Fig. 1),confirming that the level of shading we used limitedprimary production and accumulation of producer bio-mass. In the sediments, light increased benthic microalgalbiomass and, presumably as a result, TN and TOC. Theselight effects translated into changing SOM composition byincreasing the abundance of algal and microbial FAs(%(C12:0 + C14:0); total FA), %C24:0 (total FAs andPLFAs), and linoleic and linolenic acids (PLFAs) and bydecreasing heterotrophic bacterial fatty acids (%BrFA;total FAs, and PLFAs) (Fig. 5). Linoleic and linolenicacids (total FA) were positively correlated with eelgrassbiomass, while %(C12:0 + C14:0) (total FAs), %C24:0 (totalFAs), and BrFAs (total FAs) were positively correlatedwith benthic microalgal biomass (Chl a). The positiverelationship between benthic Chl a and heterotrophicbacterial FAs suggests that, in our system, microalgaeserved as a primary OM source for sediment bacteria. Thisis consistent with recent studies showing that microalgae

are often a major source of SOM and drive microbialdegradation processes in seagrass beds (Boschker et al.2000; Bouillon and Boschker 2006).

Although it is generally accepted that C12:0 + C14:0 derivesfrom aquatic algal and microbial sources, the origin of C24:0is less clear. Vascular plants are typically considered thesource of long-chain FAs; however, diatoms have beenreported to contribute as much as 30% of C24:0 in somesediments (Volkman et al. 1980). Other studies have reportedC24:0 FAs in cyanobacterial mats (Edmunds and Eglinton1984), diatoms (Viso and Marty 1993), and microalgae(Volkman et al. 1998 and references therein). These organ-isms are often associated with the community of organismscomposing the microphytobenthos. In addition, %(C12:0 +C14:0) (TFA; aquatic algal and microbial OM) and %C24:0(TFA) had similar PC1 and PC2 scores (Table 3). These FAclasses responded similarly to light and food-web treatments,suggesting that they share an OM source in our system.

Overall, light availability increased the abundance ofaboveground primary producers, sediment TN and TOCcontent, and the relative contributions of FA typicallythought to derive from aquatic sources, such as algae andmicrobes. These results demonstrate that resource avail-ability affects belowground OM storage and cycling in thisseagrass system in addition to the more obvious accumu-lation of plant biomass aboveground. Consequently,changes in water quality that result in reduced lightavailability may alter carbon cycling and storage inseagrass ecosystem sediments.

Community structure and top-down forcing—The com-munity structure of seagrass ecosystems is rapidly changingas a result of reduced water quality, fishing pressure, andother human influences (Duarte 2002; Orth et al. 2006).The resulting shifts in community composition at multipletrophic levels may precipitate changes in ecosystemfunctioning (Heck et al. 2000; Duffy 2002). For example,loss of a top predator can indirectly reduce primaryproducer biomass via a trophic cascade (Hairston et al.1960; Pace et al. 1999; Shurin et al. 2002). In seagrasssystems specifically, shifts in species composition at in-termediate trophic levels may also alter ecosystem proper-ties and OM accumulation (Duffy et al. 2003; Canuel et al.2007). A goal of this experiment was to determine howsimultaneous changes in food-web composition and re-source availability influence ecosystem properties andfunctioning.

Food chain length (predator presence or absence)strongly influenced GEP, total algal biomass, and SOMcomposition (Table 1). This effect of crab predators wasevidently mediated indirectly; as crabs inhibited or con-sumed grazing invertebrates, increasing algal biomass and,consequently, GEP. In the sediments, predators increasedalgal and microbial OM (%(C12:0 + C14:0) total FA;Fig. 5A), presumably through the same trophic cascademechanism. Interestingly, predators decreased the relativecontribution of even-numbered PUFAs (%PUFA totalFAs; Fig. 5C), which are considered proxies for ‘‘fresh’’algal material (Canuel and Martens 1993). This effect wasstrongest in shaded treatments where primary producer

Table 3. Loadings from principal components analysis ofsediment organic matter composition and content for total fattyacids (total FA) and phospholipid-linked fatty acids (PLFA).Polyunsaturated fatty acids (PUFA) are composed of C18:4, C20:4,C20:5, C22:5, and C22:6. i,a, iso-, anteiso-.

Variable

Total FA PLFA

PC1 PC2 PC1 PC2

TOC (mg g21) 0.494 20.221 0.324 20.148%(C12:0+C14:0) 0.512 0.169 0.526 0.140%C16:0 20.229 0.527 20.536 20.168%(C18:2+C18:3) 0.222 0.453 0.137 20.662%C24:0 0.583 0.118 0.216 20.557%PUFA 20.018 20.632 0.492 0.290%BrFA (i,a C13:0–C19:0) 0.228 20.160 20.152 0.312

2604 Spivak et al.

biomass was lower. Importantly, predators also increasedOM contributions from sediment heterotrophic bacteria(%BrFA, total FA; Fig. 5E), suggesting that trophiccascades can extend beyond animals and plants to OMand biogeochemical cycling. Consequently, the removal oftop predators may alter not only biomass and productionof herbivores and plants, but also ecosystem processesmediated by sediment or soil communities (Setälä et al.1998; Wardle et al. 2005). This has implications for seagrassecosystems in Chesapeake Bay and elsewhere where bluecrabs and predatory fishes are commercially harvested.

Overall, grazers strongly decreased ecosystem produc-tion, plant and algal biomass, and the contributions to thesediments of FA deriving from these sources (Table 1;Figs. 1, 2, 5). Aboveground, grazer presence decreasedtotal algal and Z. marina biomass, resulting in reducedGEP, but only in the absence of predators, reflecting thestrong trophic cascade demonstrated previously in theaboveground portion of this system (Duffy et al. 2005).Both grazer presence and richness were strong determi-nants of GEP, confirming that invertebrate speciescomposition and diversity can influence ecosystem-levelrate processes (Jonsson and Malmqvist 2003; Dangles andMalmqvist 2004). In the sediments, grazer presence de-creased benthic microalgal biomass (Chl a), microbial FAs(%(C12:0 + C14:0) total FAs), linoleic and linolenic acids(total FAs and PLFAs), and %C24 (ambient light and withpredators, total FAs) (Fig. 5). Thus, grazing reduced thecontribution of FAs characteristic of eelgrass and algae toSOM. Grazer richness influenced only heterotrophicbacterial FA abundance (%BrFA, total FA and PLFA;Fig. 5E), though this effect was mainly driven by the two-species treatment. Overall, our results indicate that thepresence of grazers is more important than the number ofspecies in determining SOM composition and quality.

Overall, food chain length and grazers strongly affectedGEP, primary producer biomass, and SOM composition.Predators mediated carbon flow and accumulation betweenlower trophic levels while grazers altered the compositionof OM delivered to the sediment. Further, our resultssuggest that aboveground communities may influencesediment heterotrophic bacteria. Consequently, human-induced shifts in the abundance or composition of above-ground communities can indirectly affect sediment bio-geochemistry by influencing the pathways (invertebrategrazers vs. bacteria) through which OM is cycled.

Interactions between bottom-up and top-down forcings—Because seagrass habitats are perturbed by multiplestressors, developing a comprehensive understanding ofecosystem responses is imperative for conservation andrestoration (Duarte 2002; Orth et al. 2006). However, moststudies have investigated the effects of human stressors,such as eutrophication (see Cloern 2001) or changingbiodiversity (see Duffy 2006), on seagrass systems singu-larly (but see Heck et al. 2000; Hughes et al. 2004). Thus,a major goal of this study was to investigate howinteractions between decreased resource (light) availabilityand altered food-web structure (grazer community andpredator presence) affect ecosystem properties. Interactions

between the three manipulated variables had pervasiveeffects on the abundances of aboveground eelgrass andalgal biomass and SOM composition. Although themajority of interactions were between grazers and light orpredators, there were also several three-way interactions.

Overall, most interactive effects of the treatments onSOM largely stemmed from light or predators mediatinggrazer effects on primary producer biomass and OM.Generally, grazer effects were stronger in ambient light,while predator controls were more prevalent in shadedtreatments (Table 1), suggesting that the strength oftrophic cascades depends on the availability of light orother resources, as in some freshwater systems (Chase2003). The results of the PCA analyses best summarize theinteractive effects of light, grazers, and predators on SOM(Table 3; Fig. 6), suggesting that grazers can stronglydetermine SOM composition, that their effects are dampedby predators, and that changing light intensity affects therelative strength of this trophic cascade.

Our results largely confirm our original hypotheses.Grazers decreased total algal biomass and altered SOMcomposition. Predator inclusion resulted in a trophiccascade whereby total algal biomass, algal and microbialOM (%(C12:0 + C14:0) total FA), and bacterial FAabundance (%BrFA, total FA) in the sediments wereincreased. Ambient light increased aboveground andsediment primary producer abundance, sediment TN andorganic carbon, and algal and microbial OM (%(C12:0 +C14:0) total FA, %C24:0 total FA and PLFA). Unlike ourpredictions, grazers decreased Z. marina biomass, benthicChl a, and FAs derived from algal and microbial OM(%(C12:0 + C14:0) total FA), while reduced light availabilityincreased bacterial OM (%BrFA, total FA). This latterresult was largely driven by the treatment with two grazerspecies. The complex interactive effects among resources,predators, and grazers suggest that aboveground andsediment properties are unlikely to respond in simple,predictive ways to multiple disturbances. Further, ourresults demonstrated that resource availability and food-web structure strongly influence ecosystem properties andthat synergism between bottom-up and top-down controlsmay affect sediment carbon composition and storage innatural seagrass beds. This underscores the need foradditional multifactorial experimental and field approachesto understanding the cycling of OM in estuarine systems.Realistic mesocosm experiments are initially helpful inidentifying subtle changes in SOM and focusing researchquestions and methods. However, field experiments willclearly be necessary to explore how linkages betweenaboveground processes and SOM are related in the morecomplex natural environment. Combined, results fromboth approaches should be useful in designing moreeffective management strategies for the preservation ofproductive seagrass ecosystems.

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Received: 17 January 2007Accepted: 2 July 2007Amended: 5 June 2007

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