### Responses of Seagrass Communities to Fertilization along a Gradient of Relative Availability

of Nitrogen and Phosphorus in a Carbonate Environment Author(s): Meredith Ferdie and James W. Fourqurean Source: Limnology and Oceanography, Vol. 49, No. 6 (Nov., 2004), pp. 2082-2094 Published by: American Society of Limnology and Oceanography Stable URL: http://www.jstor.org/stable/3597513 Accessed: 15/12/2009 12:47

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http://www.jstor.orgLimnol. Oceanogr., 49(6), 2004, 2082-2094

? 2004, by the AmericanSociety of Limnology and Oceanography, Inc. 

## Responses of seagrass communities to fertilization along a gradientof relative availability of nitrogen and phosphorusin a carbonateenvironment

MeredithFerdiel and James W. Fourqurean2 Departmentof Biological Sciences and Southeast EnvironmentalResearch Center,Florida International University, Miami, Florida 33199

Abstract Patterns of relative

nutrient availability

in south Florida suggestspatial

differences

regarding theimportance

of nitrogen (N) andphosphorus

(P) to benthic

primary producers.

Wedida 14-month

in situfertilization

experiment to testpredictions

of N andP limitation

in thesubtropical

nearshore marine waters of theupper

Florida Keys.Six sitesweredivided

intotwo groups

(nearshore,

offshore) representing

theendpoints

of anN:P stoichiometric

gradient. Twenty-four

plotswere

established

ateachsitewithsixreplicates ofeach treatment (+N, +P, +N+P,control), for a totalof 144experimental plots.Theresponses

of benthic

communities

to N andP enrichment

variedappreciablybetweennearshore andoffshore

habitats. Offshore seagrass beds were strongly

limitedby nitrogen,

and nearshore bedswereaffected

by nitrogen

andphosphorus.

Nutrient addition at offshore

sitesincreased

thelength andaboveground

standing cropof thetwo seagrasses, Thalassia testudinum

andSyringodium

filiforme, andgrowth rates of T.testudinum.

Nutrient addition atnearshore

sitesincreased

therelative

abundance

ofmacroalgae,

epiphytes, andsediment

microalgae.

N limitation

of seagrass

in this carbonate

systemwas clearlydemonstrated. However, added phosphorus

was retained

in the systemmoreeffectivelythanN, suggesting thatphosphorus

mighthave important

long-term

effects on these benthiccommunities. The observedspecies-specific responses to nutrient enrichment

underscores

theneedto monitor

allprimary

producers whenaddressing

questions of nutrient

limitation andeutrophication

in seagrass

communities.

The initial response of a coastal ecosystem to eutrophication will be influenced by the status of nutrientlimitation at the time of enrichment.In coastal marine systems, nitrogen (N) limitation is common in temperateecosystems with siliciclastic sediments, whereas phosphorus (P) limitation is often assumed to be restricted to tropical ecosystems with carbonate sediments (reviewed by Short 1987; Howarth 1988). This generalization is largely attributedto biogeochemical sorption processes: strong P affinity to carbonate sediments results in a loss of phosphorusavailable for plant uptake. However, not all tropical carbonatebenthic ecosystems are P-limited (e.g., Erftemeijer 1994; Udy et al. 1999), so there must be other determinantsof the limiting nutrient in coastal marine ecosystems. Confirming the quality (N vs. P or some other element)

1 Present address: Department

of Environmental

Sciences,University of Virginia,

Charlottesville,

Virginia 22903. 2 Corresponding

author (jim.fourqurean@fiu.edu). Acknowledgments Theauthors

thank Pursell Technologies

andIMC Phosphates

for donating fertilizer usedin thisstudy.

JoeBoyerand

JenniferRichards served onM.F.'s thesis committee

and provided valuableguidanceforthisproject.

Tom Frankovich,

CeciliaGordon,

AimeeRotaru, DottieByron,

andKevinCunniff

all contributed

valuable lab andfield assistance.

TomPhilippi

provided valuable guidance on the statistical

analyses. Twoanonymous

reviewers providedcomments that helped strengthen

thepaper. Thisstudy

wasfunded

by grant X994620-94-5

awarded toJ.W.F from theU.S.Environmental

Protection

Agencyaspart

oftheFloridaKeysNational

Marine Sanctuary Water Quality Protection

Program andby a Florida

International

University

(FIU)Tropical

BiologyProgram

research grant awarded to M.F This is contribution

221oftheSoutheast

Environmental

Research Center and contribution

74 oftheTropical

BiologyProgram

atFIU. of nutrient limitation in an ecosystem can be achieved by

nutrientaddition assays, but these assays require relatively involved experimentaldesigns, and theirinterpretation is not always clear-cut. In order to generate hypotheses about the nature of nutrientlimitation, it is possible to examine the elemental content of organic matter from ecosystems. The elemental content of plant tissue and the ratios of elements (i.e., C:N:P) might reflect relative availability and nutrient limitation status. Stoichiometry has been used to infer nutrientlimitation of phytoplankton(e.g., Redfield 1958), wetland macrophytes (e.g., Bedford et al. 1999), sediment microalgae (e.g., Hillebrand and Sommer 1999), macroalgae (e.g., Lyngby et al. 1999), and seagrass (e.g., Atkinson and Smith 1983). The benthic ecosystem of the Florida Keys are an interesting locale to study nutrientlimitation of coastal systems. The dominant primary producers in this environment are seagrasses and macroalgae, and the water column is transparent and unproductive. Spatial patternsin elemental stoichiometricratios suggest varying importanceof nitrogenand phosphorus.Elemental analysis of seagrass (Fourqurean and Zieman 2002), water quality (Jones and Boyer 2001), and sediment (Szmant and Forrester 1996) display a patternof relatively high N: P nearshore and relatively low N: P offshore toward the Florida Keys BarrierReef (-10 km from the shoreline). On the basis of these stoichiometricpatterns, it has been hypothesized thatthe benthic communities in the nearshorewaters of the Florida Keys are P-limited and offshore waters are N-limited, despite the fact that the entire region is underlain with carbonate sediments (Fourqurean and Zieman 2002). This implies thatbenthic communityresponses to localized nutrientenrichmentmight differ significantly on the spatial scale of a few kilometers. However,
2082Fertilization of seagrass communities

nutrient limitation hypotheses derived from stoichiometric evidence should be confirmed with nutrientaddition assays. The classic response of benthic communities to coastal marine eutrophicationis a shift in vegetation structure from slow-growing seagrass and macroalgae to fast-growing microalgae and phytoplankton (e.g., Valiela et al. 1992). This sequence is a consequence of interspecific competition and changing resource availability. The relative availability of
resources (e.g., nutrientand light) at a given time will favor those species with greater competitive ability for the most limiting resource (Tilman 1982). In low-nutrient environments, seagrasses and slow-growing macroalgae are competitive dominantsbecause they have relatively low nutrient requirements,

can access nutrientsin the sedimentporewater, and possess efficient internal nutrientrecycling (see review by Duarte 1995). An increase in environmental nutrient availability is concomitant with a decrease in light availability to the benthos. Seagrasses have higher light requirements comparedwith algae because they support metabolism for greater belowground biomass. As nutrient availability continues to increase, epiphytes and fast-growing macroalgae dominatebecause they takeup nutrients more effectively and quickly than seagrass and have relatively lower light requirements to sustain growth (e.g., Harlin and ThorneMiller 1981; Twilley et al. 1985). Seagrass communities are ideal candidates for nutrient limitation studies because they tend to proliferatein shallow oligotrophic waters. The seagrassplantsthemselves aregood indicators of changes in nutrient regimes because they are rooted plants with relatively long lives, they are sensitive to changes in water quality, and tissue nutrientcontent reflects the relative availability of nutrients in a system (Atkinson and Smith 1983; Duarte 1990). Knowledge of species-specific life history traitsand shifts in species dominancemight also provide insight on changes in nutrient regimes. The threemost common seagrass species in the Caribbean region are Thalassia testudinum,Syringodium filiforme, and Halodule wrightii. From a life history perspective, T. testudinum is often dominant in areas of high light and low nutrients. S. filiforme and H. wrightii possess faster colonization rates and arerelatively more efficient at nutrient uptakecompared with T. testudinum(Williams 1990). We could then predict that the relatively slow-growing T. testudinumwould be replaced by faster-growing species if nutrientloads were increased. Changes in the distributionand abundanceof seagrass, relative to other seagrass species or primary producers, could therefore be used to infer changes in the nutrientstatus of a system. In this study, we sought to test the hypothesis that seagrass-dominated benthic communities close to shore with high N: P ratios areP-limited, whereasthose further offshore with low N:P are N-limited. We expected that these ecosystems would respond to nutrientenrichmentwith increasing biomass and productivity.We furtherexpected that additions of nitrogen, phosphorus, or both would alter the N: P of the primary producers and that we would see a shift from dominanceby slow-growing T. testudinum to morerapidly growing primaryproducersas a consequence of nutrient addition. Materials and methods
Study site-The Florida Keys (ca. 250N, 80?S) are a 250- km-long archipelago of low-lying islands stretching from Key Largo to the Dry Tortugas (Fig. 1). The Keys are flanked by the Straits of Florida and Atlantic Ocean to the south and east and the Gulf of Mexico and Florida Bay to the north and west. South Florida contains the largest continuous seagrass beds in the continental United States and
some of the most expansive documented seagrass communities in the world (Fourqurean et al. 2002). T. testudinum is a common component of the oceanside benthos in the Florida Keys. S. filiforme distributionis patchy in the nearshore oceanside areasbut increases in density in an offshore direction. T. testudinumwas selected as a primaryresponse species because it is the dominant seagrass species in the region and has been identified as nutrientlimited in adjacent Florida Bay (Powell et al. 1989).
Experimentaldesign and statistical analyses-A factorial nutrientloading experiment was conducted at six sites, divided into two groups that represent the endpoints of the stoichiometric gradient:nearshore (<1 km from the shoreline) and offshore (<1 km inside the reef tract).The number of replicates for each treatmentwas determinedby a preliminary power analysis. At each site, 24 plots were established in a grid (50 X 50 m) with six replicates of each treatment (nitrogen addition, +N; phosphorus addition, +P; nitrogen andphosphorusaddition; +N+P; and control, C). Treatment plots (0.5 m2) were evenly spaced within the grid with 10
m of separationbetween treatments. This experimentaldesign was of the form of a split plot. Sites (n = 6) were the (random)whole plots; onshore versus offshore were fixed whole-plot "treatments."Quadratswithin plots were the subplots (random); nutrient treatments

were fixed subplot treatments.We treated N and P additions as separatefactors, with two levels of nitrogen (control, +N) and two levels of phosphorus (control, +P). Response variables that were only measured at the termination of the experiment were tested with mixed-model analyses of variance (PROC MIXED, SAS v9.0), with denominatordegrees of freedom calculatedvia the containment method. Nearshore-offshore was tested against sites (1 and 4 degrees of freedom); nutrient treatmentsand all interactions between nutrient treatments and nearshore-offshore
were tested against quadrats (1 and 132 degrees of freedom). For response variables sampled repeatedly throughout the experiment, sampling dates as categoricalrepeatedmeasures with quadrats within sites as subjects were added to the above split plot design. Sampling date was treated as categorical rather

than ordered,and errorcovariances were treated as unstructured

ratherthan autoregressivebecause of the underlying seasonality in many of the measured response variables. In order to evaluate differences in environmental conditions between onshore andoffshore sites, t-tests assuming equal varianceswere performedon physicochemical and sediment parameters.

Unless otherwise stated, we deemed a comparison statistically significant atp - 0.05. 2083Ferdie and Fourqurean

Fig. 1. Mapof study

sitesin theupper

Florida Keys.

Nutrient loading rates-Loading rates used in nutrient enrichmentexperiments ideally must satisfy the following two requirements:they must be ecologically relevant, and they must be sufficient to amelioratenutrientlimitation. Loading rates used in previously published experiments in seagrass ecosystems vary widely, and these rates rarely have been justified. The use of excessive loading rates in experiments is problematicwhen the goal is to apply experimentalresults to ecological and coastal management applications. To ensure the ecological relevance of this experiment, nutrient loading rates were based on regulatoryestimates of potential maximum wastewater and stormwater discharge (MCSM 2001), which are the dominantsources of anthropogenicnutrient inputs to the nearshore marine waters of the Florida Keys and have been identified as severe threatsto regional water quality (EPA 1999). Adjustmentswere made for geochemical (e.g., sorption) and biological (e.g., denitrification) processes. A final loading rate of 0.77 g N m-2 d-l and 0.12 g P m-2 d-l representeda N: P (mole: mole) ratioof 14. The rate used in this experiment exceeded the N and P demand of T. testudinumduring peak growth as assessed by multiplying peak growth rates by nutrientcontent (Fourqurean et al. 1992). Fertilizer was added monthly to the sediment. Nitrogen was added in the form of urea-coatedslow-release N fertilizer (Polyon, Pursell Technologies; 38-0-0, 94% nitrogen as urea and phosphorus as deflourinated granular phosphate rock [Multifos, IMC Phosphates] Ca3(PO4)2, 18% P). Urea is rapidly converted via microbial activity to ammonium (Fenchel and Blackburn 1979), which is the dominantform of inorganic nitrogen in the coastal waters of the Florida Keys (Boyer and Jones 2002). Fertilizerwas applied evenly to each 0.5-m2 plot and was gently mixed by hand into the surface 5 cm of sediment. The sediment surface of control plots was also mixed in a similar manner.Fertilizer application nearthe sediment-water column interfaceallowed for nutrients to diffuse throughthe sedimentporewaterandfrom the sediment into the water column, thus exposing both above- and belowground macrophyte biomass to nutrient manipulations. Sampling-The experimentwas initiatedin May 2001 and was terminatedin July 2002. Sediment fertilizationand collection of abiotic data (light availability,salinity, surfacewater temperature,turbidity) occurred monthly at each site. Light attenuationby the water column was assessed with a 47r photosynthetically active radiation (PAR) sensor (LICOR, Lincoln, Nebraska). Biotic response variables were measured during four major sampling events that occurred over the course of 14 months (May 2001, September2001, March 2002, July 2002), in which the first represented a prefertilizationsampling event. Biomass and nutrient sampling was conducted on those primaryproducersconsidered most sensitive to changes of nutrientavailability in coastal marine systems: seagrass, macroalgae, epiphytes, and sediment microalgae. The following response variables were measuredat all sampling events: T. testudinumleaf productivity, morphology, standing crop, and leaf elemental con2084Fertilization of seagrass communities

tent. At the end of the experiment, we measured sediment porosity, bulk density, and organic content, as well as T. testudinumrhizome and S. filiforme leaf elemental content; S.filiforme leaf length and standingcrop; T. testudinum total epiphyte load and epiphyte chlorophyll a (Chl a) content; sedimentmicroalgae abundance; sedimenttotalN andP content; and nonepiphytic macroalgae abundance. At each sampling event, a quadrat (10 X 10 cm) was placed inside each treatment plot. T. testudinum

growthrates
were measuredon plants within this quadrat by the modified hole-punch methodology (Fourqurean et al. 2001). Morphological measurementsof all T. testudinum leaves (length and width) were recorded, and seagrass standing crop (g m-2), areal productivity (g m-2 d-l), and leaf emergence rates of marked short shoots (SS) (leaves SS-1 d-1) were calculated. The number of S. filiforme short shoots, leaves, and leaf length found within the productivityquadrats were recorded. Epiphytic material,including inorganic carbonates,adhering to T. testudinum

leaves was removed by gentle scrapingwith
a razorblade. Epiphytes were placed into preweighed 20-ml glass scintillation vials and stored at -20?C. Seagrass leaf materialwithin each productivityquadrat was pooled by species, dried at 70?C, and groundto a fine powder.Carbonand nitrogencontents were determinedwith a CHN analyzer(Fisons NA1500). Phosphorus content was determined by a dry-oxidation, acid hydrolysis extraction followed by colorimetricanalysis of phosphateconcentration(Fourqurean et al. 1992). Elemental content and ratios were calculated on a dry weight and mole: mole basis, respectively. Epiphytes were freeze-dried to obtain dry weight, and total epiphyte load (g dry weight of epiphytes g-l dry weight of seagrass leaves) was determined. Twenty milliliters of 90% acetone was added to each vial. Vials were shaken and stored at -20?C for a minimum of 72 h. Chl a content of the acetone extracts was measured fluorometrically(Strickland and Parsons 1972) on a Gilford Fluoro IV spectrofluorometer (excitation = 435 nm, emission = 667 nm). Epiphyte autotrophic content (/,g Chl a mg-' epiphyte) was used to estimate the relative contribution of algal epiphytism (Frankovichand Fourqurean1997). Abundance of macrophytes (seagrasses and macroalgae) was assessed by the Braun-Blanquetprocedure(Fourqurean et al. 2001). This technique results in a score of relative cover, in which cover is defined as the fractionof the bottom that is obscured by specific benthic taxa when viewed by a diver from directly above. Score values were as follows: 0, absent; 1, <5% cover; 2, 5-25% cover; 3, 25-50% cover; 4, 50-75% cover; 5, 75-100% cover. Two sediment cores (1.15 cm diameter, 2.61 cm depth) were taken from each treatmentplot. One core was used to estimate sediment microalgal abundance via determination of Chl a content of the sediment (mg Chl a m-2). Sediment samples were transferred into 20-ml glass scintillation vials and placed on ice in the dark. In the lab, 10 ml of 100% acetone was added to the wet sample to extract Chl a, and furtheranalysis followed the method used for epiphyte Chl a content. The second core was used to analyze sediment porosity, bulk density, organic content, and elemental content (C, N, P). Sediment was transferred into preweighed20- The sample was dried at 70?C for 48 h to obtain a dry weight. Dry samples were groundwith a ceramic mortar and pestle. Sediment total nutrientswere analyzed by the same methods as seagrass nutrients. Sediments were ashed at 500?C for 5 h, and percent organic content was calculated
as loss on ignition. T. testudinum rhizomes for nutrient analysis (C:N:P) were randomly collected from each quadrat,rinsed to remove carbonate sediments, dried at 70?C, ground to a fine powder, and analyzed in the method described above. Nonepiphytic macroalgal abundance was calculated for each treatmentplot with a modification of the Braun-Blanquet quadratmethod (Fourqurean et al. 2001).
Retention efficiencycalculations-Changes in community structureoften occur on timescales greaterthan this experiment. In orderto gain insight into the fate of addednutrients and the relative long-term importanceof N and P additions, we estimated the retention efficiency of nitrogen and phosphorusin both nearshoreand offshore seagrass systems. Absolute quantitiesof nitrogen andphosphoruswere calculated for compartmentswithin a 0.25-m3 (25 X 100 X 100 cm) seagrass habitat containing 200 liters of water column and 50 liters of sediment. The following compartmentswere included: water column, T. testudinumleaves, T. testudinum rhizomes, S. filiforme leaves, nonepiphytic macroalgae, T. testudinum

epiphytes, and sediment.Waterquality datawere obtainedfrom the SoutheastEnvironmental ResearchCenter Water Quality Monitoring Network at Florida International University. Absolute quantities(g) of nitrogen andphosphorus in each component were calculated by multiplying nutrient content (percent dry weight) and total mass (g m-2). Estimates for macroalgal biomass (113 g dry weight m-2; Davis and Fourqurean2001) and epiphyte nutrientcontent (N = 1.48% of ash free dry weight (AFDW), P = 0.22% of AFDW; Lin et al. 1996) were obtained from the literature. T. testudinumbelowground biomass was estimated as 85% of total T.testudinum

mass. The remainingdatawere derived directlyfrom this experiment.Mass balances were calculated for control and +N+P treatmentsat both nearshoreand offshore sites. Net system retention ([total system nutrient(g), +N+P] - [total system nutrient(g), control]) and retention efficiency ([net system retention (g)/mass applied (g)] X 100%) were calculated.

Results
Nearshoreandoffshore waters exhibited similarmean water temperature

and salinity (Table 1). There was an inverse relationshipbetween depth and light attenuation: the deeper offshore sites hadhigherwaterclarity,as evidenced by lower light attenuationand lower turbidity, compared with nearshore sites. Despite the difference in water clarity, the amount of light reaching the benthos (% lo) was not very differentbetween nearshoreand offshore locations: between 14.2% and 27.8% of the incoming solar radiation reached the seagrass canopy. Sediments at nearshoresites had higher porosity, lower bulk density, and higher organic content compared with offshore sites, which consisted of coarser ml glass scintillation vials, and wet weight was calculated. 2085

sand and a low contributionof organic material.NearshoreFerdie and Fourqurean

Table1. Summary

of site descriptions

andenvironmental

parameters.

Temperature

andsalinity

areannual

means(n = 10-18). The diffuse attenuation

coefficient (kd)and the amountof light reaching the benthos (% I,) are means of quarterly measurementsfrom 1995 to 2002 (n = 18-27). Turbiditywas measuredin Nephelometric TurbidityUnits (NTU). Sediment characteristicsand abundancevalues are site means (n = 24) from the beginning of the experiment.Differences in means between onshore and offshore sites were evaluatedby ttests.Parameters

withdifferent

means(p - 0.05) arebold.

Nearshore Offshore t t-test I-1 1-2 I-3 O-1 0-2 0-3 p value Water Depth(m) 5 5 4 8 8 6 0.023 Salinity 35.9 36.0 36.1 36.2 36.2 36.1 0.067 Temperature

(?C) 24.6 26.2 26.5 26.7 26.6 26.9 0.180 Turbidity (NTU) 0.64 0.39 0.59 0.30 0.26 0.25 0.026 Lightattenuation,

kd (m-1) 0.32 0.39 0.33 0.23 0.16 0.29 0.051 Io(%) 20.2 14.2 26.7 15.9 27.8 17.6 0.990 Sediment Porosity (g cm-3) 0.71 0.69 0.77 0.54 0.52 0.60 0.007 Bulkdensity

(g cm-3) 0.77 0.79 0.93 1.20 1.21 1.23 0.002 Organiccontent (%) 4.52 3.78 3.47 2.25 2.07 1.91 0.005 Macrophyte

abundance Thalassia 3.1 4.0 3.9 3.9 3.7 3.6 0.834 Syringodium 0.0 0.1 0.3 0.6 1.1 2.5 0.092 Nonepiphytic

macroalgae 1.5 1.0 1.0 0.1 0.8 1.4 0.386

seagrassbeds were dominatedby T. testudinum interspersed with abundantmounds of fine mud deposited by infauna. Offshore seagrass beds were more homogeneous, and S.filiforme was more abundantin offshore comparedwith nearshore sites. T. testudinumrhizomes were primarilylocated in the top 10 cm of sediment at nearshore sites, whereas offshore rhizomes were found at greaterdepth (-5-20 cm). Nonepiphytic macroalgalcomposition consisted primarilyof calcareous green algae species (Penicillus spp., Halimeda spp., Udotea spp., Rhipocephalus spp.). Also present in sparse amounts were green algae (e.g., Avrainvillea spp., Codium spp., Dasycladus spp., Batophora spp., Caulerpa spp.), red algae (e.g., Laurencia spp., Gracilaria spp.), and brown algae (e.g., Dictyota spp.). Seagrass morphology,standingcrop, and growth responses Sediment fertilization increased sediment nutrientcontent and increasedplant nutrientcontent,providingevidence that fertilization increased availability of both nitrogen and phosphorus.Plant nutrientenrichmentresultedin significant changes in T. testudinumleaf nutrientcontent (Fig. 2) and elemental ratios (Fig. 3). Nitrogen addition led to rapiduptake of nitrogen by seagrass at both nearshoreand offshore sites, as evidenced by increases in leaf N content and decreases in C:N. The response to P addition was delayed comparedwith N addition.Whereasthe effects of N addition
were evident at both nearshoreand offshore sites in September 2001 (4 months of fertilization), elevated leaf P content
was not observed at nearshore sites until March 2002 (10 months) and at offshore sites until July 2002 (14 months). The nutrient

contents of S.filiforme leaves and T. testudinum rhizomes were significantly affected by nutrient addition (Fig. 4). Nitrogen additiondecreased S. filiforme leaf and T. testudinumrhizome C: N. Phosphorusadditiondecreased S. filiforme leaf C: P. Significant changes in rhizome P content and C: P values with P additionwere observed at nearshore, but not offshore, sites. Nutrient addition significantly affected T. testudinum length, standingcrop, and growthrates (Fig. 5). Leaf length, standing crop, and areal productivity increased with N addition at offshore sites, but not at nearshoresites. Therewere no significanteffects of P additionon T. testudinumproductivity. Nitrogen addition significantly increased S. filiforme length and standingcrop at offshore, but not nearshore,sites (Fig. 6). There were no significant effects of nutrientadditions on T. testudinumleaf emergence rates. Algal responses to nutrientenrichmentoccurredprimarily at nearshore sites (Fig. 7). Nonepiphytic macroalgal abundance increased with N additionat nearshoresites only (significant Location X N interaction).Both N and P additions affected epiphyteloads, but in a complex manner(significant main effects of N and P additions on epiphyte load). Nearshore, P additionincreasedepiphyteloads, whereasoffshore, N additions decreased loads on the seagrass blades. Additions of both nitrogenandphosphorussignificantlyincreased the epiphyte Chl a content at inshore sites, but at offshore sites, only N addition increased the autotrophicindex. Sediment microalgae abundance increased only with +N+P enrichment at nearshoresites.
Retentionefficiencyof nitrogen andphosphorus-The relative allocation of nitrogen and phosphorus to various seagrass system compartmentswas similar between nearshore and offshore sites. However, the net retention of added nutrients varied considerably on the basis of nutrientand location (Table 2). The majority of nitrogen and phosphorus was located in the sediment, and the water column contribution was negligible (<0.1%). Nutrient enrichmentnearly doubled both nearshore and offshore total nitrogen in the system, and greatly elevated total nearshorephosphorusand 2086Fertilization of seagrass communities

0.2 - 0.15 - ^ T L E--- Nearshore
E * | X

'====1 Offshore

q.a 2 0.10-

0

. I IT TI T ??-TI

= 0.05-

0.0 0.00

C +N +P +N+P C +N +P +N+P g 0.20 2.5 20 o 0.15- 2.0'

....-~~~~~~~ .S 0.10-

1.0 C +N +P +N+P C +N +P +N+P Fig. 2. Phosphorus and nitrogen content of sediments and leaves of Thalassia testudinumat nearshoreand offshore sites following 14 months of fertilization, expressed as percentage of dry weight. Values are treatmentmeans (n = 18), and errorbars are ? 1 SE.

offshore phosphorus. Net system retention for both nitrogen Discussion and phosphorus were higher at nearshore sites compared with offshore sites. Retention efficiency was large for phos- This experiment demonstrated that nearshore benthic phorus (nearshore = 82%, offshore = 49%) and relatively communities in south Florida are indeed nutrient limited, but small for nitrogen (nearshore = 9%, offshore = 4%). the nature of that limitation differs for nearshore and off35 - 35 Nearshore Offshore

30- 30-

25 - 25

| 20 - +N 20-

-f- +N+P

15
. . 1
. 5 May 01 Sep 01 Mar 02 July 02 May 01 Sep 01 Mar 02 July 02

Nearshore Offshore 1600 1600 -

1200 1200 -

800 800-

400 . . 400 May 01 Sep 01 Mar 02 July 02 May 01 Sep 01 Mar 02 July 02 Fig. 3. Thalassia testudinumleaf nutrientratios during the course of the experiments. Values
are treatmentmeans (n = 18), and errorbars are 1 SE. 2087Ferdie and Fourqurean

C +N +P +N+P :. 1500 U

0

*I 1000

500
3500

Co U E

o '= 2000
t_

500 

## I

IL 

# I .I

L ; +N +P +N+P C

C +N +P +N+P C +N +P +N+P Fig. 4. Thalassia testudinum rhizome and Syringodium filiforme leaf nutrient

content responses at nearshoreand offshore sites following 14 months of fertilization,expressed as percentageof dry weight. Values are treatment means (n = 18), and errorbars are + 1 SE. Syringodium filiforme was not present in any +N treatmentsat nearshoresites.

shore sites and for seagrass and algae (Table 3). The original hypothesis that seagrass communities are P-limited nearshore and N-limited offshore was partially supported by the responses of primary producers to nutrient enrichment. There was an increase in T. testudinunz leaf length, standing crop, and growth rates (Fig. 5) in response to N, but not P, addition at offshore sites. Offshore S. filiifrme leaf length and standing crop also increased with N addition. This implies that seagrass in offshore habitats in the upper Florida Keys are N-limited. Nutrient addition at nearshore sites had
no significant effect on T. testudinum or S. filiforme length or standing crop. T. testudinum areal productivity at nearshore sites increased with +N and +N+P treatments, suggesting that nearshore T. testudinum might be nutrient limited, but this limitation is not as severe as offshore seagrass. Contrary to our a priori predictions based on stoichiometric evidence, there was no clear increase in biomass or primary productivity of seagrasses at nearshore sites in response to P addition. Significant increases in the relative abundance of nonseagrass primary producers were found almost exclusively at nearshore sites (Fig. 7) and encountered with all nutrient treatments (+N, +P, +N+P). Macroalgal abundance, total epiphyte load, epiphyte autotrophic content, and sediment microalgae abundance increased with nutrient addition at nearshore sites. A decrease in total epiphyte loads at offshore sites with +N and +N+P addition might have been caused by higher leaf production and leaf mass associated with offshore N addition. The only significant effect of nutrients on an offshore algal response variable was an increase in epiphyte Chl a content with N addition (+N, +N+P), although the increase was not as great in magnitude as the nearshore response. This suggests that the autotrophic component of the offshore epiphyte complex might be N-limited, as was observed for the offshore seagrass. The strong epiphyte responses at nearshore sites might be
a function of benthic microalgal mats. Qualitative observations by divers of microalgal blooms were identified exclusively in nutrient treatments (+N, +P, +N +P) at nearshore, but not offshore, sites. The absence of benthic algae increases at offshore sites might be a result of higher water energy and strong water mixing at offshore sites, which might have physically inhibited the development of benthic microalgal mats, or of observation times that do not coincide with mat formation, or of microalgal mats that are not nutrient limited at offshore sites. The dynamics of benthic microalgae in response to nutrient enrichment deserve more attention in future research. The competitive outcome following changes in nutrient availability plays a critical role in determining the community response to eutrophication. Although the time frame of this experiment was not long enough to observe community structure shifts, changes in the relative abundance of primary producers allowed us to predict the future trajectory of community composition. Increases in S. filiforme, macroalgae, and epiphyte abundance observed in this study might lead to direct competition with T. testudinum for resources. Mean leaf length for S. filiforme in offshore +N+P treatments (198 + 48 mm) were higher than T. testudinum (176 + 39 mm), suggesting that shading effects on T. testudinum by S. filiforme can occur with fertilization. These shading effects might be exacerbated by a higher T. testudinum minimum light requirement than S. filiforme (Wiginton and McMillan 1979). Macroalgal species, especially rhizophytic calcareous 35

25

15
100 4

E 0 -N

L. 60

20 11 

# l 2088Fertilization of seagrass communities

Nearshore 180 - 160 - 140 - 120 - 100 - 80 May01 Sep01 Mar 02 July 02

Nearshore

Sep01 Mar02 July02
Nearshore

* C

---- +N

- +N+P *- +P 180 160 140 120 100 80 60 40

4-

3- Offshore

May01 Sep01 Mar 02 July 02

Sep01 Mar 02 July 02

Offshore I

1

Sep01 Mar 02 July 02 Sep01 Mar02 July02 Fig. 5. Morphology and growth measurementsof Thalassia testudinumover the course of the experiments at nearshoreand offshore sites. Values are treatmentmeans (n = 18), and errorbars
are 1 SE.

225- E

150'

75 .iz
C2 75- 

# 1

- Nearshore r 'lOffshore 

## I

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._

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# I



# I

+N+P Fig. 6. Syringodium

filiforme mean leaf length and standingcrop at nearshore and offshore sites following 14 months of fertilization. Values are treatmentmeans (n = 18), and errorbars are ?_1 SE. 180

E 160
140

, 120

; 100
80

180
' 160

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I 80 60 40

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Fig. 7. Epiphyte load and autotrophiccontent and nonepiphytic macroalgal and sediment microalgal responses at nearshoreand offshore sites following 14 months of fertilization.Values are treatment means (n = 18), and error bars are ? 1 SE.

greens, are conspicuous in south Florida seagrass communities and might compete with seagrasses for light, space, and nutrients (Davis and Fourqurean 2001). Seagrasses are especially prone to the negative shading effects of epiphytes and benthic algal blooms (e.g., Twilley et al. 1985). Nutrient enrichment in this experiment resulted in an increase in seagrass biomass at offshore sites and an increase in algal biomass at nearshore sites. This demonstrates that both regions are nutrient limited but that taxon-specific responses to nutrient enrichment are different. Continuous nutrient enrichment could lead to a shift from T. testudinum to S. filiforme dominance at offshore sites and to algae dominance at nearshore sites. The time course of seagrass community responses to changes in nutrient availability has been shown to occur on the scale of years and decades. In Florida Bay, a monoculture T. testudinum bed was replaced by H. wrightii following 8 yr of fertilization (Fourqurean et al. 1995). Changes have also been shown to occur following the cessation of nutrient addition. In the Mediterranean, a Posidonia oceanica meadow displayed persistent seagrass declines 3 yr following the closure of a fish farm (Delgado et al. 1999). These long-term community changes suggest that nutrients were retained and recycled in the system. Benthic communities in this experiment differed dramatically in their retention of nitrogen and phosphorus (Table 2). Although total system nitrogen douTable 2. Absolute quantities (g) and retentionefficiency (%) of nitrogen and phosphorusfor compartmentswithin 0.25 ml of seagrass habitat containing 200 liters of water column and 50 liters of sediment. Measurementsare listed for control (C) and nutrient(+N+P) treatmentsat nearshoreand offshore sites. Nitrogen Phosphorus

Compo t Nearshore Offshore Nearshore Offshore Component [g (0.25 m-3)-'I C +N+P C +N+P C +N+P C +N+P Sediment 43.5 67.1 21.4 24.5 3.4 44.3 10.2 33.7 Thalassia aboveground 1.7 2.2 1.2 2.6 0.1 0.1 0.1 0.2 Thalassia belowground 3.4 7.8 2.4 10.6 0.2 0.4 0.4 1.0 Syringodiumleaves 0.1 0.4 0.1 0.9 0.003 0.03 0.01 0.05 Macroalgae 0.3 0.3 0.3 0.3 0.04 0.04 0.04 0.04 Epiphytes 0.4 0.6 0.6 0.7 0.07 0.1 0.1 0.1 Watercolumn 0.04 0.04 0.04 0.04 0.001 0.001 0.001 0.001 Total system (g) 49.4 78.5 26.1 39.7 3.8 45.1 10.8 35.2 Net retention (g) 29.1 13.6 41.2 24.4 Mass applied (g) 323 323 50 50 Retention efficiency (%) 9.0 4.2 82.5 48.7 C +N +P +N+P



# iC
+

C +N +P +N+P

l 2090Fertilization of seagrass communities

Table 3. A summaryof statistically significant (ANOVA main effects, p - 0.05) responses after 14 months of fertilization.A plus (+) indicates that the variable was greaterthan the controls, a minus (-) indicates a decrease comparedwith controls. ND indicates there was no comparisonpossible, because Syringodium filiforme was absent from all +N treatmentsat the inshore locations.

Nearshore Offshore Response variable +N +P +N+P +N +P +N+P Thalassia testudinum Areal productivity Specific productivity Productivityper short shoot Standingcrop Leaf length Syringodium

filiforme Standingcrop Short shoot density Leaf length Macroalgae Abundance Epiphytes Load Chl a load Autotrophicindex Sediment microalgae Abundance Nutrientcontent of seagrasses Thalassia Leaf N content Leaf P content Leaf C N Leaf C P Leaf N:P Rhizome N content Rhizome P content Rhizome C N Rhizome C: P Rhizome N: P Syringodium Leaf N content Leaf P content Leaf C: N Leaf C: P Leaf N:P + + + + ?
+

ND ND ND

+

+ + +

+- + +

+ + +

- ?

+ +

+- 4- +

+

+ + + + +

+

+

+ +

+

+ +

ND ND ND ND ND + + +

+ + + + + +

+ + + + +

+

+ + + + + +

+ +

bled with N addition, <10% of the added nitrogen was retained. In contrast, these sites were extremely efficient at P retention: 82% and 49% of added phosphorus was retained at nearshore and offshore sites, respectively. These differences suggests that long-term P enrichment, unlike N enrichment,has the potentialto be an important driverof longterm community changes because most of the phosphorus was retained within the system, whereas much of the nitrogen was lost from the system. Nutrients can be lost from a system via several pathways, including mineralizationprocesses, sedimentation,diffusion from the sediment, physical removal, and herbivory.The sediment served as a large sink for phosphorusin this study. The high loss rates of nitrogen was likely a function of microbial transformations (e.g., denitrification).Because of the high affinity of carbonatesediments for phosphate,it is also likely thatinorganicnitrogen is more mobile and thereforemore easily lost from the system. Seagrass leaf nutrient content increased at all sites with the additionof nutrients, but the magnitudeof these responses varied by time and location. Increases in T. testudinum leaf N content were apparent quickly (4 months of fertilization), whereas significant increases in P content were not observed at nearshoresites until 10 months and at offshore sites until 14 months (Fig. 3). The overall delayed response of T. testudinumleaf P content with P addition might have been caused by sorption of added phosphorus on carbonate sediments (Short et al. 1990). However, this geochemical process does not explain the difference between nearshore and offshore T. testudinumP content responses to +P ad2091Ferdie and Fourqurean

dition. Sediment grain size has been shown to possess an inverse relationshipto sorptionpotential:smaller grain sizes provide more surface areafor P adsorption,resulting in less phosphorusavailablefor plantuptake(Erftemeijer1994). On the basis of this phenomenon, we could predict that nearshore sites, with smaller grain size, would display delayed P uptakeby seagrass comparedwith offshore sites. The opposite patternwas observed. Dissolution andprecipitation of carbonates are also influenced by organic matter (Morse et al. 1985) and sediment pH (Jensen et al. 1998), where CaCO3 dissolution is relatedto higher organic content.Nearshore sediments have relatively higher organic content (Table 1), so this factor might affect P availability. It is also possible that the greater depth of the root zone at offshore sites might account for the delay in seagrass P uptake. An alternateexplanation is that P uptake by T. testudinumdid not occur at offshore sites because phosphoruswas not limiting. The increase of rhizome P content with +P at nearshore, but not offshore, sites is furtherevidence that phosphorus did not limit seagrass growth or metabolic processes at offshore sites (Fig. 4). The additionof nitrogennot only increased T. testudinum N content, but it also affected the metabolism and uptakeof phosphorus.A significant decrease in leaf P content with N addition was observed at both nearshoreand offshore sites (Figs. 2, 3). A probable explanation is that the addition of nitrogen increased N assimilation and led to higher P demand, a phenomenaobserved with nutrientenrichmentin an Australianseagrass bed (Udy and Dennison 1997). This net decrease was not seen with +N+P, presumablybecause the increased P demand was met by fertilization. The addition of phosphorus alone did not have a significant negative effect on N content, emphasizing the difference in metabolic characteristicsof specific nutrients. Growth and biomass responses in this experimentdid not consistently follow the nutrientcontent responses. Although N additionincreased leaf N content at all sites, increases in leaf length and standing crop were found only at offshore sites. The addition of phosphorus alone to nearshore and offshore sites increased leaf P concentrations but did not affect length, standing crop, or growth rates. Increases in T. testudinum

leaf nutrientcontent in the absence of a positive standingcrop or growthresponse implies thatnutrientavailability in the study area was either insufficient to satisfy all plant metabolic demands for nitrogen, phosphorus,or both, or that T. testudinumis capable of storing excess nutrients when demandis less than supply, also known as luxury consumption.Luxury consumptionof nutrients by algae is common (e.g., Campbell 2001) and has also been identified in seagrass (e.g., Murray et al. 1992; Van Lent et al. 1995). Nutrient storage differs between T. testudinum photosynthetic (leaves) and structural(rhizomes) tissues; the photosynthetic short shoots contain the largest amountof nitrogen and phosphorus, whereas rhizomes serve as importantcarbohydratestorage organs. The only instance in this study in which the addition of a solitary nutrient (N or P) had no effect on plant nutrient content for that particularnutrient
was for rhizome C: P and P content (Fig. 4) at offshore sites. The absence of a rhizome P increase might be a function of accessibility, in which the addedphosphorusdid not migrate downward to the relatively deep rhizome-root complex because of sorption activity, relatively lower mobility, or both compared with nitrogen. It is also possible that the added phosphoruswas not integratedinto offshore rhizome tissue because the element is in excess at offshore sites and not required, which is likely because offshore P availability is relatively high and offshore growth is strongly N-limited.
Ecological applications of this study-Phosphorus limitation of coastal primaryproductionis frequentlyassociated with tropical marine carbonatesystems in which the strong sorptionaffinity of phosphorusto calcium carbonate mineral surfaces reduces the amount of phosphorus available for plant uptake (Morse et al. 1985). Biological availability of phosphorus is furtherregulated by geochemical processes, such as sediment grain size (Erftemeijer1994), sorption-site saturation(McGlathery et al. 1994), and carbonatedissolution dynamics (e.g., Jensen et al. 1998; Burdige and Zimmerman 2002). There is building evidence that seagrass communities found in carbonate-rich systems can be limited by nitrogen ratherthan phosphorus.A review of in situ seagrass fertilization experiments that included separateN and P enrichment found only one out of seven studies which clearly identified P limitation of seagrass growth in a carbonate system (Table 4). In this frequently cited investigation, Short et al. (1990) found significant effects of P addition on S. filiforme growth only when porewater P concentrationswere increasedto 2,000 times ambientlevels, a concentration

thatmight be excessive in real-life scenarios. Other fertilization experiments that used only +N+P combined addition have inferred P limitation with changes in leaf nutrientcontent, biomass measurements,or both (Agawin et al. 1996; Powell et al. 1989), but atthis time, adequate experimental data is lacking to justify a broad assumption of P limitation in carbonatesystems. In temperatesystems, three out of six fertilization studies identified seagrass growth with P addition (Harlin and Thorne-Miller 1981; Perez et al. 1991; Murray et al. 1992), suggesting that P limitation is more common on a global scale and that parameterssuch as locale (tropicalvs. temperate)and sediment composition (carbonatecontribution)are not effective predictorsof seagrassnutrient limitation.This experimentclearly demonstrated

N limitation of T. testudinumand S.filiforme in the offshore waters of the upperFloridaKeys, despite predictions of P limitation in carbonatesystems. This study partially supports our original hypotheses of nutrientlimitation on the basis of patternsof nutrientavailability: nearshorealgae were affected by nitrogen and phosphorus, and offshore seagrasses were affected by nitrogen. Predictions of nearshoreP limitation were not strongly supported by our experimental results. It is possible that nearshore habitatsare P-limited, but the signal was not observed because of factors such as the relatively short experimental time period, high nutrientconservation, and recycling of T. testudinumas an adaptationto low nutrient environments. These problems are associated with P-loading, sediment sorption, or the indirect effects of microalgal mats, which can cause direct shading of seagrassphotosynthetictissue or affect sediment processes such as oxygen demand. Predicting the response of a system to eutrophication is confounded 2092Fertilization of seagrass communities

Table 4. Responses of seagrass to in situ nutrientenrichmentexperimentsinvolving separatenitrogen (N) and phosphorus(P) addition. Entries are groupedby climate (temperatevs. tropical/subtropical) and by sediment regime (carbonatevs. siliciclastic).

Response Location Species Tissue Growth Source Tropical/subtropical

carbonate Indonesia (coral islands) Thalassia hemprichii +N None Erftemeijeret al. (1994) Bahamas Syringodium filiforme +N+P +P Short et al. (1990) NE Australia Halodule uninervis +N +N Udy et al. (1999) NE Australia Syringodiumisoetifolium +N +N Udy et al. (1999) Florida Keys Thalassia testudinum +N+P +N This investigation, offshore Florida Keys T. testudinum +N+P +N+P This investigation, nearshore Tropical/subtropical

siliciclastic Indonesia (coastal site) T. hemprichii None None Erftemeijeret al. (1994) E Australia H. univervis +N +N Udy and Dennison (1997) E Australia Zostera capricorni +N+P +N+P Udy and Dennison (1997) E Australia Cymodoceaserrulata +N None Udy and Dennison (1997) Temperatecarbonate NE Spain Cymodoceanodosa +P +P Perez et al. (1991) Temperatesiliciclastic SE Australia Heterozostera tasmanica +N+P +N Bulthuis et al. (1992) Italy C. nodosa None None Ceccherelli and Cinelli (1999) Rhode Island Zostera marina +N +N+P Harlinand Thorne-Miller(1981) ChesapeakeBay Z. marina +P +P Murrayet al. (1992)

by the fact that primary producers in coastal systems have species-specific nutrient requirements and can exhibit dissimilar forms of limitation. The limiting nutrient has been shown to vary among trophic levels such as plants and bacteria in coastal ecosystems (Sundareshwar et al. 2003), but this study clearly demonstrates that members within a trophic level might also respond differently to N and P enrichment. The response of seagrass communities in the upper Florida Keys to N and P enrichment varies appreciably between the nearshore and offshore environment, and the responses of all primary producers should be considered before categorizing a natural system with a specific nutrient limitation.

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