spatial and seasonal variability in elemental content, c,...

447Q 2005 Estuarine Research Federation

Estuaries Vol. 28, No. 3, p. 447–461 June 2005

Spatial and Seasonal Variability in Elemental Content, d13C, and

d15N of Thalassia testudinum from South Florida and Its

Implications for Ecosystem Studies

JAMES W. FOURQUREAN1,*, SUSIE P. ESCORCIA1, WILLIAM T. ANDERSON2, and JOSEPH C.ZIEMAN3

1 Department of Biological Sciences and Southeast Environmental Research Center, FloridaInternational University, Miami, Florida 33199

2 Department of Earth Sciences and Southeast Environmental Research Center, FloridaInternational University, Miami, Florida 33199

3 Department of Environmental Sciences, University of Virginia, Charlottesville, Virginia 22903

ABSTRACT: Elemental and isotopic composition of leaves of the seagrass Thalassia testudinum was highly variable acrossthe 10,000 km2 and 8 years of this study. The data reported herein expand the reported range in carbon:nitrogen (C:N)and carbon:phosphorus (C:P) ratios and d13C and d15N values reported for this species worldwide; 13.2–38.6 for C:Nand 411–2,041 for C:P. The 981 determinations in this study generated a range of 213.5‰ to 25.2‰ for d13C and24.3‰ to 9.4‰ for d15N. The elemental and isotope ratios displayed marked seasonality, and the seasonal patternscould be described with a simple sine wave model. C:N, C:P, d13C, and d15N values all had maxima in the summer andminima in the winter. Spatial patterns in the summer maxima of these quantities suggest there are large differences inthe relative availability of N and P across the study area and that there are differences in the processing and the isotopiccomposition of C and N. This work calls into question the interpretation of studies about nutrient cycling and food websin estuaries based on few samples collected at one time, since we document natural variability greater than the signaloften used to imply changes in the structure or function of ecosystems. The data and patterns presented in this papermake it clear that there is no threshold d15N value for marine plants that can be used as an unambiguous indicator ofhuman sewage pollution without a thorough understanding of local temporal and spatial variability.

IntroductionElemental and isotopic composition of macro-

phytes are increasingly being used as indicators ofelemental supply and processing in marine ecosys-tems (McClelland and Valiela 1998b; Miller andSluka 1999; Fourqurean and Zieman 2002). Mac-rophytes are fixed in place and continuously sam-ple the environment around them; their chemicalcomposition is to a large part determined by en-vironmental conditions. The ratios of elements inmacrophyte tissues can be used much like the Red-field ratio for particulate organic matter, with de-partures from taxon-specific means as indicators ofnutrient limitation (Atkinson and Smith 1983;Duarte 1990). Spatial patterns in elemental stoi-chiometry can be used to identify sources andsinks of nutrients in aquatic ecosystems (Fourqur-ean et al. 1992, 1997; Miller and Sluka 1999; Four-qurean and Zieman 2002). Paleoecological recordsof elemental ratios of organic matter in sedimentcores have been used to infer temporal change in

* Corresponding author; tele: 305/348-4084; fax: 305/348-4076; e-mail: [email protected]

nutrient sources in coastal and lake ecosystems(e.g., Meyers 1997; Orem et al. 1999; Teranes andBernasconi 2000).

In addition to elemental stoichiometry, ratios ofthe stable isotopes of carbon (C) and nitrogen (N)have proven useful indicators of supply and pro-cessing of nutrients. Stable isotope ratios in mac-rophytes and consumers have proven valuable intracing the flow of energy in marine food webs (Pe-terson et al. 1985; Peterson 1999). Stable isotoperatios can also be used to identify nutrient sourcesand processing in ecosystems. 13C:12C ratios in mac-rophytes have been used to identify the impor-tance of allochthonous C to marine ecosystems(Zieman et al. 1984; Lin et al. 1991; Hemminga etal. 1994). Bacterially-mediated processing of N canstrongly influence stable N isotope ratios, and as aconsequence, spatial pattern in 15N:14N ratios inmacrophytes can be used to infer ecosystem-scaleprocessing of organic matter (Fourqurean et al.1997). C and N isotopes have been used in bothpaleoceanography and paleolimnology to inferchanges in water column nutrient cycles (Schelskeand Hodell 1991; Haug et al. 1998; Teranes and

448 J. W. Fourqurean et al.

Fig. 1. Distribution of the 30 seasonal monitoring stationsin the Florida Keys National Marine Sanctuary (heavy crossesdenote monitoring sites). Isopleths of mean N:P of Thalassiatestudinum leaves for quarterly samples collected during the pe-riod 1995–2001 are shown.

Bernasconi 2000). Owing to the isotopically heavyN associated with many anthropogenic nutrientsources, stable isotopes of N in macrophytes arepotentially invaluable tools for gauging the effectof man on coastal water bodies (McClelland et al.1997; McClelland and Valiela 1998b). This tool ispotentially of primary importance because of themagnitude of the effect man is having on coastalwater bodies through anthropogenically-increasedN loading (Paerl 1997; Vitousek et al. 1997; Tilmanet al. 2001).

Despite the promise of these stoichiometric andisotopic tools for understanding elemental cyclesin the coastal ocean, there are still many problemswith their routine application. Chief among theseproblems is that unambiguous application of thesetools requires an understanding of the natural var-iability in elemental and isotopic composition oforganisms in both time and space. Relatively small-scale surveys of marine macrophytes have docu-mented spatial variability in elemental and isotopiccomposition (Stephenson et al. 1984; Fourqureanet al. 1992; Boon and Bunn 1994; Boyce et al.2001), and meta-analyses have brought to light dif-ferences between macrophyte species (Atkinsonand Smith 1983; Hemminga and Mateo 1996).Less attention has been given to analysis of tem-poral patterns in elemental and isotopic composi-tion of macrophytes, which could be important ininterpreting food web structure from isotopic evi-dence, since different consumers integrate isotopicsignals of their food sources over different timescales (O’Reilly et al. 2002). It has recently beenshown that the stable isotopic composition of themarine macrophytes that form the base of manycoastal food webs can exhibit marked seasonality(Cloern et al. 2002; Anderson and Fourqurean2003). There have been few, if any, detailed inves-tigations of the interrelationships between elemen-tal and isotopic composition of marine macro-phytes despite evidence that elemental composi-tion and isotopic composition can be related in saltmarsh plants (Cloern et al. 2002) and terrestrialold-field grasses and herbs (Handley and Scrim-geour 1997).

Before the variability in elemental and isotopiccontent of primary producers can be used to ex-plore the nature of elemental cycling in coastalecosystems, the nature and causes of the variabilitymust be fully understood. A large monitoring pro-gram designed to assess the status and trends ofseagrass beds in south Florida allowed for us todetermine both spatial and temporal patterns inthe elemental and isotopic composition of Thalas-sia testudinum, the dominant marine macrophyte inthis region (Fourqurean et al. 2001; Fourqureanand Rutten 2003). Coupled with knowledge of wa-

ter chemistry and primary productivity of the re-gion, this data set allows us to explore the relation-ships between environment, nutrient concentra-tions, nutrient demand, time of year, and the ele-mental and isotopic composition of thiswidespread marine macrophyte.

MethodsSITE SELECTION

The seagrass Thalassia testudinum Banks ex Kon-ig is widespread in tropical and subtropical coastalmarine areas of the Atlantic and Caribbean, and itis the dominant species in the seagrass beds thatare common in south Florida. In order to assessseasonality and interannual patterns in elementaland isotopic composition of T. testudinum, datawere gathered from the network of 30 permanentseagrass monitoring sites in the Florida Keys Na-tional Marine Sanctuary, a subtropical-tropical,shallow-water, marine ecosystem of seagrass beds,coral reefs, and mangroves (Fig. 1, see Fourqureanet al. 2001; Fourqurean and Rutten 2003 for a de-scription of the monitoring program). Extensivewater quality monitoring is done at these sites; datafrom that program (Boyer and Jones 2002) wereused to compare elemental composition with nu-trients in the overlying water column. These siteswere originally located using a stratified-randomapproach, with 2 random locations being chosenwithin three strata (inshore, offshore, and inter-mediate) in each of the defined segments of theSanctuary (Klein and Orlando 1994). Data on el-emental and isotopic composition of seagrasses atthe permanent sites were collected 4 times per yearduring the period December 1995–March 2003. Apreliminary examination of 2 yr of seasonal pat-

Seagrass Elemental and Isotopic Variability 449

tern in stable isotopic composition from 4 sites waspublished previously (Anderson and Fourqurean2003); those data are analyzed in a larger contextin this paper.

To describe the spatial patterns in elemental andisotopic composition of T. testudinum, data werecollected from an additional 425 randomly select-ed sites in the Sanctuary. These stations were sam-pled during the summer months of 1996–2000;during each year approximately 100 samples werecollected across the spatial extent of the Sanctuary.Data from all years were pooled to produce a syn-optic view of the spatial pattern. Owing to the costof stable isotopic analyses, d13C and d15N data weredetermined only for data from sites sampled in1999. A limited set of the elemental compositiondata has been published previously (about 50 ofthe present data points were included in the anal-yses presented in Fourqurean and Zieman [2002]).In order to more fully describe the regional vari-ation in elemental and isotopic composition of T.testudinum, literature values on the range and dis-tribution of C, N, and phosphorus (P) contentfrom Florida Bay (Fourqurean et al. 1992; Fran-kovich and Fourqurean 1997) were incorporatedinto our analyses for this paper. Some previously-published d15N data from Florida Bay (15 pointsfrom Corbett et al. [1999]) and previously unpub-lished d13C and d15N data from Florida Bay collect-ed in 1994 were also incorporated in maps andsummary figures.

At each sampling site, 5 intact short shoots of T.testudinum were haphazardly collected from a 10-m2 area. These short shoots were returned to thelab, where all attached green leaves were cut fromthe short shoots and cleaned of adhering epiphytesby gently scraping with a razor blade. Samples werenot acid washed to remove the epiphytes becauseof the known artifacts in stable isotope determi-nations of acid washed samples (Bunn et al. 1995);we have found thorough scraping of fresh bladesto be efficient at removing adhering carbonate ma-terial. All leaves from a site were pooled and driedat 808C. Dried leaves were ground to a fine powderusing a ceramic mortar and pestle. Powdered sam-ples were analyzed in duplicate for C and N con-tent using a CHN analyzer (Fisons NA1500). P con-tent was determined by a dry-oxidation, acid hy-drolysis extraction followed by a colorimetric anal-ysis of phosphate concentration of the extract(Fourqurean et al. 1992). Elemental content wascalculated on a dry weight basis (i.e., mass of ele-ment/dry weight of sample 3 100%); elementalratios were calculated on a mole:mole basis.

All isotopic analyses were measured at the South-east Environmental Research Center (SERC) Sta-ble Isotope Laboratory using standard elemental

analyzer isotope ratio mass spectrometer (EA-IRMS) procedures. The EA was used to combustthe organic material and to reduce the formed gas-es into N2 and CO2, which were measured on aFinnigan MAT Delta C IRMS in a continuous flowmode. The samples’ isotopic ratios (R) are report-ed in the standard delta notation (‰): d (‰) 5[(Rsample/Rstandard) 2 1] 3 1000. These results arepresented with respect to the international stan-dards of atmospheric N (AIR, N2) and Vienna PeeDee belemnite (V-PDB) for C. Analytical reproduc-ibility of the reported d values, based on samplereplicates, was better than 6 0.2‰ for d15N and 60.08‰ for d13C.

Net aboveground productivity of T. testudinumwas measured on a quarterly basis at each perma-nent site using a modified leaf marking technique(Fourqurean et al. 2001). Six 10 3 20 cm quadratswere haphazardly distributed within 10 m of a per-manent steel rod that marked the site. Within eachquadrat, all short shoots of the seagrass T. testudin-um were marked near the base of the leaves bydriving an 18-gauge hypodermic needle throughall of the leaves on a short shoot. Care was takennot to disturb other plant and animal taxa in thequadrats. The marked short shoots were allowedto grow for 10–14 d, after which all abovegroundseagrass material in the quadrats was harvested.The number of short shoots was counted, andleaves were separated into newly produced (belowthe marks) and older leaf material.

Data were analyzed using a variety of parametricand nonparametric methods. Pearson’s correlationcoefficients and standard linear regression wereused to assess the strength of relationships betweennormally distributed variables. The nonparametricSpearman’s r was used to assess correlation whereparametric tests were not appropriate. To describethe seasonal pattern in elemental and isotopic con-tent, we fit a sine model of the form y 5 mean 1amp 3 sin(time 1 F), where amp is the amplitudeof a sine wave and F is a phase angle in radians(2p radians 5 365 d), to time series of data usingan iterative least-squares nonlinear curve fittingroutine. Analysis of covariance (ANCOVA) wasused to test for relationships between variables atthe seasonal monitoring sites, e.g., the effect of sea-grass growth rate on stable isotopic compositionwas assessed using ANCOVA with sites as a maineffect and seagrass growth rate as a covariate withinsites. To aid in the visualization of spatial patternin the elemental and isotopic data, a kriging al-gorithm (point kriging using a linear variogramand no nugget) was used to interpolate betweendata points and contour maps were generatedfrom these interpolated fields.


Fig. 2. Frequency distribution of the C:N, C:P, and N:P ratiosof green leaves of Thalassia testudinum from south Florida. Datafrom Fourqurean et al. (1992) and Frankovich and Fourqurean(1997) have been added to the 1,015 data points collected forthis paper to give a broader view of the distribution of theseratios in the region.

Fig. 3. Relationship between the mean N:P of Thalassia tes-tudinum leaves measured over the period 1995–2001 and themedian total Nitrogen: total Phosphorus (N:P) in the water col-umn at the 30 seasonal monitoring. Water column data are themedians of quarterly observations from these sites over the sameperiod, the data were provided by J. N. Boyer (unpublisheddata), Florida International University. Standard parametric lin-ear regression statistics are shown. 95% confidence interval ofthe regression is indicated.

ResultsELEMENTAL CONTENT OF T. TESTUDINUM IN

SOUTH FLORIDA

There was a broad range in the N and P contentof T. testudinum collected across south Florida. TheN content ranged from 1.15% to 3.15% and P con-tent ranged from 0.049% to 0.241%, while C con-tent was much less variable in the 1,065 samples.For the samples collected from the seasonal mon-itoring stations over the period 1995–2003, the

mean C:N was 22.6, compared to a slightly highermean of 24.4 for the synoptic mapping sites. C:Nwas normally distributed about the mean (Fig. 2).The C:P distribution was skewed, with a substantialnumber of high values; the median C:P was similarbetween the synoptic mapping samples and theseasonal monitoring samples (780.1 versus 780.7).The distribution of N:P was also skewed, with a tailtowards the higher values. The median N:P wassimilar between the two sets of samples (35.4 forthe seasonal monitoring stations compared to 33.5for the synoptic monitoring sites).

NUTRIENT RATIOS AT THE SEASONALMONITORING STATIONS

There was a spatial pattern in the relative N andP content of T. testudinum leaves across the 30 sea-sonal monitoring stations in the Florida Keys Na-tional Marine Sanctuary, with generally lowermean N:P offshore of the Florida Keys on the At-lantic Ocean side of the island chain, and higherN:P close to shore and on the Gulf of Mexico sideof the island chain (Fig. 1). The N:P of seagrassleaves was significantly correlated with the medianratio of total N to total P of the overlying watercolumn (Fig. 3), but the slope of the relationshipis less than 1, suggesting that processes act in thebenthos to either remove N or store P relative tothe water column.

There were repeatable, seasonal patterns in thenutrient content of seagrass. C:N and C:P were lowin winter months and high in summer months, but


Fig. 4. Seasonal pattern in C:N, C:P, and N:P of green leavesof Thalassia testudinum in south Florida. Each point is the meanof the observations from the 30 seasonal monitoring sites shownin Fig. 2, error bars indicate 6 1 standard error. The best fitsine model of the form y 5 mean 1 amp 3 sin(time 1 f),where amp is the amplitude of a sine wave and f is a phaseangle in radians (2p radians 5 365 d), is shown for C:N and C:P; estimates of the model parameters for the N:P data were notsignificantly different than 0.

the degree of seasonality was not constant amongstations. The sine model was fit to the mean C:Nand C:P ratios for all 30 sites at each samplingevent, the model explained 61% of the variance inC:N (Fig. 4). A F 5 4.6 6 0.2 indicated that thepeak in C:N occurred on average on the 185th dayof the year ( July 4), 6 9 d. The sine model de-scribed 35% of the variance in C:P, with a maxi-mum on July 14 6 15 d. The timing of the peakswere not significantly different for C:N and C:P.Note that the average annual excursion in the datais 2 3 amp. The sine model was not appropriateto describe the time series of N:P, as the estimatesof neither amp or F were significantly differentfrom 0.

The sine model was fit to the time series of C:Nand C:P from each of the 30 seasonal monitoringstations. The amplitude of the seasonal variation inelemental content at a station varied as a function

of the mean annual nutrient content. Sites withlow mean C:N or C:P had low amplitude, whilesites with high ratios had high amplitudes (Fig. 5).Seagrass growth rates at these sites have also beenshown to follow the sine model (Fourqurean et al.2001). Within sites, there were strong relationshipsbetween seagrass growth rate and elemental con-tent; as growth rate increased, so did the C:N andC:P. Across all sites, there were significant relation-ships between growth rate and N and P content ofT. testudinum. The mean slope of the relationshipfor C:N was 2.4 6 0.3 (g m22 d21)21 (ANCOVA, F5 72.0, p , 0.001), while for C:P the mean slopewas 88.6 6 18.2 (g m22 d21)21 (ANCOVA, F 5 23.8,p , 0.001).

C AND N STABLE ISOTOPE RATIOS OF T. TESTUDINUMIN SOUTH FLORIDA

In addition to the broad range in the elementalcontent of leaves of T. testudinum, there was also abroad range in the d13C and d15N values. d13Cranged from a minimum of 213.54‰ to a maxi-mum of 25.23‰, and d15N ranged from 24.31‰to 9.40‰ in the 981 samples. The mean values ofd13C were similar for samples collected at the sea-sonal monitoring stations and the synoptic map-ping stations (28.68‰ compared to 28.75‰),but the values from the seasonal monitoring sta-tions had a more peaked distribution than the datafrom the synoptic mapping stations, which hadheavier tails (Fig. 6). The mean values of d15N werealso similar between the two sets of samples(2.28‰ versus 2.34‰). Both the seasonal moni-toring data and synoptic station data had long left-hand tails to their distributions, but the synopticdata also had a long right-hand tail, with relativelyinfrequent heavy values of d15N.

C AND N STABLE ISOTOPE RATIOS AT THE SEASONALMONITORING STATIONS

The average d13C and d15N values of T. testudinumleaves at the seasonal monitoring sites were not sig-nificantly correlated (r 5 20.07, p 5 0.72), but theaverage isotopic values were related to the elemen-tal content. Sites with relatively low N:P had moredepleted d13C signatures, while sites with high N:Pwere more enriched (Fig. 7). Average d15N in-creased as the mean C:N at a site increased.

There was a marked seasonal variation in bothd13C and d15N values of T. testudinum at the seasonalmonitoring stations, but the magnitude of the sea-sonal change varied among stations and timing ofthe peaks varied somewhat between years and sta-tions. In general, values for both d13C and d15Nwere heavier in summer months and lighter in win-ter months. To describe the average seasonal pat-tern in the isotope ratios, we fit the sine model to


Fig. 5. The seasonal amplitude in the C:N and C:P of Thal-assia testudinum leaves increased as a function of the mean C:Nor C:P in south Florida. Amplitude and mean were determinedfor each of the 30 seasonal monitoring sites by fitting the modely 5 mean 1 amp 3 sin(time 1 f), where amp is the amplitudeof a sine wave and f is a phase angle in radians (2p radians 5365 d). Standard parametric linear regression statistics areshown. 95% confidence interval of the regressions are indicat-ed.

Fig. 6. Frequency distribution of the d13C and d15N of greenleaves of Thalassia testudinum from south Florida. Data from Cor-bett et al. (1999), as well as unpublished data from Florida Bay,have been added to the 744 data points collected for this paperto give a broader view of the regional distribution.

the mean values for all 30 sites at each samplingevent (Fig. 8). The sine model described 63% ofthe variance in the d13C data, with a mean of28.63‰ 6 0.09, and an amp of 0.70‰ 6 0.13with a peak on August 11 6 12 d. The sine modeldescribed 40% of the variance in the d15N pattern,with a mean of 2.3‰ 6 0.1 and an amp of 0.4‰6 0.1. The yearly peak occurred on July 26 6 16d, statistically the same as the peak in the d13C data.Averaging across all sites and years, the average sea-sonal range (2 3 amp) was 1.40‰ in d13C and0.8‰ in d15N. Calculated in this fashion, the ampparameter underestimates the magnitude of thetrue average seasonal variation in the isotope val-ues because the stations are slightly out of phasewith one another. Calculating the range of valuesencountered within each year between 1996–2003

at each site, and then calculating the mean of theseyearly ranges at each site, gives a better estimate ofthe average annual variation in the isotope valuesat a site. Average annual variation in d13C was1.90‰ 6 0.11 for all sites, with minimum of1.14‰ and a maximum of 3.58‰. Average annualvariation in d15N was 1.7‰ 6 0.1, with a minimumof 0.8‰ and a maximum 3.4‰.

Seagrass growth rate (Fourqurean et al. 2001),C:N and C:P ratios (Fig. 4), and C and N stableisotope ratios (Fig. 8) all showed peaks during thesummer months. Across sites, there was a signifi-cant relationship between the seagrass growth rateat a site and d13C values; d13C increased by 0.30‰6 0.09 for every 1 g m22 d21 of productivity in-crease (ANCOVA, F 5 10.7, p 5 0.001). There wasno significant relationship between growth rateand d15N across all sites (slope of relationship in


Fig. 7. Relationships between stable isotopic compositionand the elemental ratios of green leaves of Thalassia testudinum.Each point represents the mean of all values collected between1995–2001 at each of the 30 seasonal monitoring stations.

Fig. 8. Seasonal pattern in d13C and d15N of green leaves ofThalassia testudinum in south Florida. Each point is the mean ofthe observations from the 30 seasonal monitoring sites shownin Fig. 2, error bars indicate 6 1 standard error. The best fitsine model of the form y 5 mean 1 amp 3 sin(time 1 f),where amp is the amplitude of a sine wave and f is a phaseangle in radians (2p radians 5 365 d), is shown.

ANCOVA not significantly different from 0). Therewas a significant relationship between d15N and C:N, with an increase in d15N by 0.03‰ 6 0.01 foreach unit increase in C:N (ANCOVA, F 5 4.8, p 50.03).

SPATIAL PATTERN IN ELEMENTAL RATIOS

Samples collected across the study area duringthe summer months (to coincide with peaks in C:N and C:P ratios, Fig. 4) indicated there was astrong spatial pattern to the nutrient content ofleaves of T. testudinum (Fig. 9). C:N was lowest inthe interior of Florida Bay, with a general trend ofincreasing C:N as distance from the Florida Keysincreased both towards the Atlantic and Gulf ofMexico. C:P ratios were highest in the interior ofFlorida Bay, with a general decrease in C:P withdistance offshore of the Florida Keys. These twopatterns together lead to a clear pattern in N:P,with maximum N:P in the interior of Florida Bay,with decreases in N:P with distance offshore of theFlorida Keys. Using an N:P of 30 as a rough indi-

cation of the optimum relative availability of N andP for T. testudinum (Atkinson and Smith 1983;Duarte 1990; Fourqurean and Zieman 2002), thistrend in N:P indicated that, for the most part, theseagrasses of south Florida were P limited in Flor-ida Bay and near shore in the Florida Keys, with atendency towards N limitation in the more off-shore seagrass beds.

SPATIAL PATTERN IN ISOTOPE RATIOS

As with the elemental ratios, there were strongspatial patterns in the C and N stable isotopic com-position of T. testudinum (Fig. 10). C isotope ratioswere more depleted in the northwest corner ofFlorida Bay (212‰ to 211‰), and more en-riched (28‰ to 26‰) in areas close to the Flor-ida Keys. A much different spatial pattern was ev-ident for 15N:14N ratios. The heaviest d15N values(. 8‰) were found in Florida Bay, while the light-est d15N values (, 2‰) were found in the centralregion of our study area. In contrast to the ele-mental ratios (Fig. 9) and d13C, there was no pat-


Fig. 9. Spatial pattern of the C:N, C:P, and N:P of green leaves of Thalassia testudinum in south Florida. All data was collected inthe summer months to control for the seasonal pattern. Bottom panel: an N:P ratio of 30 was used to delimit N-limited and P-limitedareas.

tern in d15N attributable to the distance offshorefrom the Florida Keys.

RELATIONSHIPS BETWEEN ELEMENTAL RATIOS ANDISOTOPE RATIOS AT THE SYNOPTIC SCALE

The spatial patterns in elemental composition ofT. testudinum (Fig. 9) were based on 836 samples

for which C, N, and P content were known. TheC:N and C:P ratios of these samples were both cor-related with N:P, but C:N and C:P were not cor-related with each other (Table 1), indicating thatN and P content of T. testudinum were independentof each other on this spatial scale. For 350 of thesesamples, the C and N stable isotopic composition


Fig. 10. Spatial pattern of the d13C and d15N of green leaves of Thalassia testudinum in south Florida. All data was collected in thesummer months to control for the seasonal pattern.

was also known. Both the C:P and N:P of T. testu-dinum leaves were positively correlated with d13C(Table 1). This relationship of decreasing d13C withincreasing P content at the synoptic scale mirroredthe relationship seen at the seasonal monitoringstations (Fig. 7). The relationship of increasingd15N with decreasing N content that was observedat the seasonal monitoring stations (Fig. 7) was alsoseen at the synoptic scale (Table 1). There was nosignificant relationship between the d15N and d13Cof T. testudinum leaves from the 350 synoptic map-ping stations (Table 1).

DiscussionElemental and isotopic composition of leaves of

the seagrass T. testudinum was highly variable acrossthe 10,000 km2 and 8 yr of this study, and the datapresented herein generally expand the reportedranges of elemental and isotopic composition forthis species (Table 2). The striking spatial and tem-poral patterns in the variability of elemental andisotopic content suggest that the measured varia-tion is not merely random noise, but instead that

the variation contains useful information about theeffects of the environment on the nutrient contentand physiological status of seagrasses. Our workcalls into question the interpretation of studiesabout nutrient cycling and food webs in estuariesbased on few samples collected at one time, sincewe document natural variability of elemental andisotopic content greater than the signals often usedto imply changes in the structure or function ofecosystems.

Despite the tropical nature of the study site,there is substantial seasonal variability in thegrowth rate and abundance of the perennial T. tes-tudinum, with enhanced summertime growth ratesleading to 64% higher abundance in summer com-pared to winter (Fourqurean et al. 2001). Thesedifferences are correlated with seasonal patterns inboth light availability and water temperature. It hasbeen shown experimentally that decreases in lightavailability lead to lighter d13C values for seagrasses(Grice et al. 1996). Photosynthesis of T. testudinumin south Florida is rarely photosaturated (Fourqur-ean and Zieman 1991), so increases in light lead


TABLE 1. Correlations (nonparametric Spearman’s r) between elemental ratios and stable isotopic composition of Thalassia testu-dinum leaves from the synoptic sampling. n 5 836 for elemental ratios; n 5 350 for stable isotope ratios. Values of Spearman’s r areabove the diagonal, p values are below the diagonal. Bold type indicates significant correlations.

C : N C : P N : P d13C d15N

C : NC : PN : Pd13Cd15N

—0.011

,0.0010.7870.019

0.088—

,0.001,0.001

0.998

20.3880.860

—,0.001

0.066

20.0140.2940.308

—0.371

0.12620.00020.09920.045

—

TABLE 2. Reported values of d15N and d13C of green leaves of Thalassia testudinum.

d15N d13C Location Reference

This study24.3 to 9.4 213.5 to 25.2 South Florida

South Florida

1.9 to 3.2

212.5216.3 to 27.3214 to 27.0215.3 to 28.7

Florida KeysSouth FloridaSouth FloridaSouth Florida

McMillan et al. 1980Lin et al. 1991Zieman et al. 1984Fleming et al. 1990

3.421.0 to 9.4

5.3 to 5.921.2 to 3.7

213.6

29.1 to 28.5212.3 to 26.1

Florida BayFlorida BayFlorida BaySouth Florida

Harrigan et al. 1989Corbett et al. 1999Orem et al. 1999Anderson and Fourqurean 2003

5.521.2 to 9.4

210.5216.3 to 26.1

Florida Keys Schwamborn and Criales 2000Range of literature values, south Florida

Other locations3.53.94.32.9 to 5.7

213.228.9

211.1

211.0 to 28.3210.0 to 29.9

NicaraguaTexasJamaicaTexasTexasSt. Croix, U.S.Virgin Islands

Macko 1981Macko 1981Macko 1981Fry et al. 1987McMillan et al. 1980McMillan et al. 1980

210.929.04

211.4212.3 to 211.128.929.6 to 211.0

Veracruz, MexicoTexasTexasBelizeTexasTampa Bay

McMillan et al. 1980Benedict and Scott 1976Benedict et al. 1980Ambler et al. 1994Fry and Parker 1979Durako and Hall 1992

21.2 to 9.4 216.3 to 26.1 Range of literature values

to increases in photosynthetic C fixation, whichshould lead to decreased isotopic discriminationduring photosynthesis and heavier values of d13Cin plant tissues, if the increased rates cause a draw-down in the availability of CO2 (Farquhar et al.1989). We recorded a highly seasonal and repeat-able change in the d13C of T. testudinum, with anaverage yearly increase of 1.4‰ from winter min-ima to summer maxima (Fig. 8). The seasonal in-crease in d13C values is a potential sign that seagrassphotosynthesis may begin to be C limited in sum-mer months in south Florida. A similar seasonalityin stable C isotope ratios in phytoplankton hasbeen used to infer a drawdown of CO2 pools dur-ing productivity blooms (Laws et al. 1995; Ostromet al. 1997). Previous studies of more limited du-ration have also documented some seasonal pat-

terns in d13C of seagrasses, and in more temperatelocations with a greater seasonal difference in lightand temperature, the seasonal difference in d13Ccan be more pronounced than we measured in thistropical ecosystem (Stephenson et al. 1984; Four-qurean et al. 1997).

It is also possible that factors other than seasonalchanges in photosynthetic rates may be responsiblefor the seasonal pattern in d13C of seagrass leaves.A decrease in solubility of CO2 with increasing tem-peratures can also lead to a smaller CO2 pool andless isotopic discrimination and heavier d13C valuesof marine plants in summer months (Francois etal. 1993). It is also possible that the d13C of thedissolved inorganic carbon (DIC) in seawater maychange as a result of seasonal changes in inputs ofterrestrially-derived organic matter and the bal-


ance between ecosystem production and respira-tion. Swart et al. (2001) documents substantial var-iability in the d13C of the DIC in Florida Bay, butthere were no clear seasonal patterns in this vari-ability. Surge and Lohmann (2002) found that d13Cof DIC was enriched by about 5‰ in summer com-pared to winter in mangrove-lined estuaries 200km north of our study area.

Seasonal patterns in root-zone processes couldinfluence the d13C of seagrass tissues. About 90%of the biomass of T. testudinum is in belowgroundstructures (Fourqurean and Zieman 1991), andthe lacunal system of seagrasses allows for diffusionof gases from the root zone to the leaves wherephotosynthesis occurs. Respiratory processes candissolve carbonate sediments ( Jensen et al. 1998;Burdige and Zimmerman 2002), and carbonatesediments are enriched in 13C relative to seagrasstissues, providing for the possibility that our ob-served increases in d13C during the warm summermonths could be a result of increased dissolutionof carbonates in response to increased respirationin the sediments. Such an effect could also helpexplain the enrichment of the d13C of the DIC ofsurface waters observed in the region (Surge andLohmann 2002).

Increased photosynthetic rates can result inhigher growth rates for plants, but elements otherthan C are needed to synthesize new plant tissues.Given that the seagrass-specific Redfield ratio is ap-proximately 550:25:1 for C:N:P (Atkinson andSmith 1983; Duarte 1990), on average 1 mol of Nis required for every 22 mol of C and 1 mol of Pfor every 550 mol of C allocated to growth. Thelikely mechanism underlying the repeatable sea-sonal pattern in C:N and C:P ratios (Fig. 4) is thatN and P can not be supplied from the environ-ment in sufficient quantities to satisfy the growthdemands of the seagrasses when photosyntheticrates, and the potential for growth, are high duringthe summer months. Additional evidence for sea-sonal nutrient limitation as a function of potentialgrowth rate comes from the relationship betweenthe mean C:N and C:P ratios and the amplitude ofthe seasonal signal of those ratios (Fig. 5). Siteswith ample N to meet their growth demands, i.e.,those with low average C:N ratios, have little sea-sonal variability compared to sites at which thehigh mean C:N ratios indicate N deficits. The sameholds for the relationship between mean C:P ratiosand the amplitude of the seasonal pattern in C:Pratios.

Sites with evidence of higher average photosyn-thetic rates are the ones that show deviation fromseagrass Redfield N:P stoichiometry; sites with highN:P ratios, indicating P limitation, had the highestaverage d13C values (Fig. 7). This was likely a result

of relative light availability. Low light would be ex-pected to produce low photosynthetic rates andisotopically depleted d13C in plant tissues. Such aneffect can be seen along natural gradients in lightavailability (Cooper and DeNiro 1989), and exper-imental reductions in light availability lead to de-creases in d13C of seagrasses (Abal et al. 1994; Griceet al. 1996). Low light levels also result in seagrassRedfield-like N:P ratios (Fourqurean and Rutten2003); only at relatively high light levels (relativeto nutrient supply) would one expect deviationfrom an N and P balance.

The cause of the seasonal cycle in d15N (Fig. 8)needs to be determined by careful experimenta-tion. The two most likely causes are a seasonal var-iability in the d15N of the source dissolve inorganicnitrogen (DIN) or seasonal differences in the frac-tionation of the DIN pool during uptake. We feelthat the most probable cause is a decreased frac-tionation in summer when plant growth demandsoutstrip the N supply and draw down the pool ofavailable N, in a manner similar to the decrease infractionation of the DIC pool in summer whenphotosynthetic rates are high. On average, therewas a 0.8‰ repeatable seasonal pattern of isoto-pically light values in winter and heavy values insummer as determined by fitting the sine modelto the station mean data. This degree of seasonalpattern was less than the mean annual range at allsites of 1.7‰ because of phasing differencesamong sites. At some sites, there was as much as a3.4‰ average annual range, which is about thesame range that had been previously documentedfor T. testudinum from all locations in the species’range outside of south Florida (Table 2). Evidenceto support the hypothesized mechanism behindthe increase in d15N values during summer monthscan also be seen in the relationship between C:Nand d15N (Fig. 7); as the C:N increases indicatingdecreasing N availability, the d15N values also in-creased in a manner consistent with decreasedfractionation of a DIN pool of constant d15N as theavailability of N decreased. It is also possible thatthere are seasonal patterns in the d15N of the DINsource for seagrass growth, especially in the sedi-ments underlying the seagrass beds. The d15N ofsoil DIN is a complex function of supply (e.g., de-position, nitrogen fixation, and remineralization),utilization (e.g., plant uptake and denitrification),and losses both advective and diffusive. Many ofthese processes strongly fractionate the DIN pool(see Handley and Scrimgeour 1997 for a review ofthis topic).

At the spatial resolution of the 30 monitoringsites, T. testudinum elemental content was a reflec-tion of the relative availability of N and P in thewater column (Fig. 3), suggesting that seagrass stoi-


chiometry does reflect the relative availability ofnutrients in the environment. The long lifespan ofseagrass shoots (often over 10 yr in south Florida,Peterson and Fourqurean 2001) results in seagrass-es being long-term integrators of nutrient avail-ability; we have found the spatial patterns in sea-grass N:P to be much less variable in time than N:P in the water column. The slope of the relation-ship between water column and seagrass leaf N:Pis 0.5, indicating that P is more available relativeto N for benthic primary producers than in thewater column. This is likely a result of the loss ofN from the sediments through diffusion and de-nitrification, while P is retained relatively efficient-ly in the sediments as a result of the kinetics ofphosphate sorption onto carbonate sediments (deKanel and Morse 1978; Short et al. 1990).

The samples collected to describe fine-scale spa-tial variability in elemental and isotopic content ofT. testudinum leaves were all collected in the sum-mer months in order to control for the markedseasonality. Summer sampling also is the only ap-propriate time to collect elemental content datawhen assaying for regional pattern in elementalcontent, since the nutrient limitation signal is onlyexpressed during periods of rapid growth. Oursummer samples contained as much variation inelemental and isotopic content as the seasonalsamples (Figs. 2 and 6), indicating spatial differ-ences in environmental conditions were as impor-tant in determining elemental and isotopic con-tent of the seagrass leaves as seasonal patterns inphotosynthesis and growth. Across the landscape,there was no significant correlation between C:Nand C:P, indicating that N and P availability wereindependently varying across the study area. If re-gional patterns in light availability were causing thepatterns in C:N and C:P, then these quantitieswould be correlated. The general pattern in rela-tive availability of N and P to seagrasses across the10,000 km2 of the study area was low N:P on theoffshore areas on the Atlantic Ocean side of theFlorida Keys, high N:P in the enclosed Florida Bayestuary, and relatively low N:P along the northernboundary of the study area in the Gulf of Mexico(Fig. 9). The spatial patterns in elemental ratioshave been previously reported for Florida Bay(Fourqurean et al. 1992; Frankovich and Fourqur-ean 1997) and the back reef environment in theeastern third of the study area (Fourqurean andZieman 2002), but this is the first documentationof the trend in the western Florida Keys. The pre-vailing view of ubiquitous P limitation of seagrassbeds underlain by carbonate sediments (Short etal. 1990) is brought into question with these data,since broad areas offshore on the Atlantic Oceanside of the Florida Keys and west of the island of

Key West have N:P values less than 30, indicatingpotential N limitation. Experimental evidence sup-ports the hypothesis of N limitation in the regionsof low N:P in the eastern part of our study area(Ferdie and Fourqurean 2004), but this experi-mental evidence also indicates that, at intermedi-ate N:P ratios, that seagrass Redfield ratios are im-perfect predictors of the response of seagrass eco-systems to nutrient addition.

Areas closest to the islands of the Florida Keyshad the heaviest d13C values for T. testudinum leaves(Fig. 10). These areas tend to be shallow (ca. 1–2m) compared to areas farther from the islands onthe Atlantic Ocean side of the Florida Keys, whered13C was lower by 2–3‰. This was a pattern to beexpected if less light reached the seagrasses in thedeeper offshore areas. The enclosed regions ofFlorida Bay also had isotopically light d13C valuesfor T. testudinum compared to the shallow areas ad-jacent to the islands, despite the very shallow na-ture of Florida Bay (ca. 1 m average depth). It islikely that the isotopically light seagrass leaf carbonis a result of the influence of a DIC pool that hasbeen influenced by CO 2 produced by the oxida-tion of organic matter derived from mangrovesand other emergent and terrestrial plants (Lin etal. 1991; Swart et al. 2001). Seagrass stable C iso-tope values were positively correlated with both theN:P and C:P ratios (Table 1), indicating that areaswith lower relative P availability were characterizedby relatively heavy d13C values across the spatial ex-tent of this study. This relationship mirrors thatseen at the individual monitoring sites, where C:Pand d13C values both increased during times ofhigh productivity, as well as the relationship be-tween the yearly mean N:P and yearly mean d13Cat the 30 monitoring sites.

The broad spatial patterns in the d15N value ofleaves of T. testudinum (Fig. 10) may have beencaused by either regional patterns in the isotopicsignature of the DIN available to the seagrasses, bydifferent amount of fractionation of the availableDIN pool during uptake, or a combination of thesefactors. The very high d15N values in north-centralFlorida Bay have been attributed to progressive de-nitrification of DIN advected into Florida Bay fromthe Gulf of Mexico and to seepage of 15N-richgroundwater into the surface waters (Corbett et al.1999). It is interesting to note that there was nosignal of heavy d15N values close to the heavily pop-ulated Florida Keys, despite the fact that ground-water under the islands is human sewage-influ-enced (Lapointe et al. 1990) and likely enrichedwith 15N. The very low d15N values (, 2‰) typicalof the middle of our study area potentially indicatelow average growth rates and/or high DIN avail-ability, resulting in large fractionation of the DIN


pool. The low d15N values are largely coincidentwith an area known as the Sluiceway that allowsterrestrially-influenced Gulf of Mexico water topass through the Florida Keys to the FloridaStraights (Schomer and Drew 1982). Another pos-sible explanation of these low values is a DIN poolheavily influenced by fertilizer-laden runoff fromthe agricultural fields of Florida, because artificialfertilizers have a d15N of near 0‰, but as yet wehave no data on the d15N of DIN from the surfacewater or sediment porewaters of this area. Large Ninputs from the agricultural areas are not likely,since most of the DIN that enters the Evergladesecosystems that run off into this region is lost touptake and denitrification before it reaches theGulf of Mexico (Rudnick et al. 1999), but the po-tential for this low d15N signal to be an indicatorof anthropogenic N merits further study.

Anthropogenically-increased N loading is one ofthe largest threats to the coastal ocean worldwide(Paerl 1997; Vitousek et al. 1997; Tilman et al.2001). Given the increasing use of N isotopes invegetation as an indicator of anthropogenic alter-ations of coastal N cycling, it is very important thatwe understand natural causes of spatial and tem-poral variability in 15N:14N. The data and patternspresented in this paper make it clear that there isno threshold d15N value for marine plants that canbe used as an unambiguous indicator of humansewage pollution without a thorough understand-ing of local temporal and spatial variability. Itwould be possible to misinterpret a seasonal in-crease in d15N values associated with seasonal in-creases in growth rates as indication of an increasein the influence of human wastes, which generallyhave heavy d15N signatures. Likewise, sampling anarea without synchronizing the season of samplingacross the area could lead to spurious spatial pat-terns in N and C isotope ratios.

Enriched d15N values in benthic vegetation havebeen linked to anthropogenic N inputs that resultin deleterious effects in N-limited marine ecosys-tems (McClelland et al. 1997; McClelland and Val-iela 1998a). We found the opposite relationship tobe true; there was an inverse relationship betweenplant-assessed N availability (C:N ratio) and thed15N values of seagrass tissues (Fig. 7 and Table 1).Apparently, there are many ways to induce a heavyd15N signature in a marine plant. Anthropogenicloading of 15N-enriched sewage-derived DIN, pro-gressive denitrification of an N source and the re-sulting isotopically-enriched remaining DIN (Four-qurean et al. 1997), and N limitation during peri-ods of rapid growth can all lead to isotopicallyheavy d15N signatures.

Our unpublished data on other marine plantsfrom south Florida and other seagrasses from oth-

er places in the world suggest that the variabilitywe document herein is not peculiar to T. testudin-um. The existence of a substantial amount of non-random variability in the elemental and isotopiccomposition of T. testudinum in south Florida au-gurs well for the utility of using elemental and sta-ble isotopic content as indicators of ecosystem pro-cesses and for tracing elements in ecosystems, butit is important that the intra-annual and small-scalespatial variability of these plants is understood be-fore interpreting the consequences of differencesin samples.

ACKNOWLEDGMENTS

This research was funded by the U.S. Environmental Protec-tion Agency as part of the Florida Keys National Marine Sanc-tuary Water Quality Protection Program (X994620-94) and bythe U.S. National Science Foundation as part of the FCE-LTERprogram (NSF/OCE-9910514). Salary support to J. W. Fourqur-ean for data analysis and document preparation was providedby the Secretaria de Estado de Educacion y Universidades, Min-isterio de Educacion, Cultura y Deporte, Spain; Carlos Duartekindly provided office space. Water quality data provided by JoeBoyer and Ron Jones of Florida International University (FIU).Brian Fry analyzed some early stable isotope samples from Flor-ida Bay, and provided invaluable guidance early in this study.Alan Willsie, Craig Rose, Leanne Rutten, and Meredith Ferdiecollected and analyzed the elemental content of most of thesamples. The services of Captain Dave Ward (R/V Magic) andCaptain Mick O’Connor (R/V Expedition II) were vital to thecompletion of this project. This is contribution no. 260 fromthe Southeast Environmental Research Center at FIU.

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BOYER, J. Unpublished data. Southeast Environmental ResearchCenter, Florida International University, Miami, Florida33199.

Received, June 23, 2004Revised, February 1, 2005Accepted, March 2, 2005

spatial and seasonal variability in elemental content, c,...

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