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BULLETIN OF MARINE SCIENCE. 88(1):000–000. 2012http://dx.doi.org/10.5343/bms.2010.1105

1Bulletin of Marine Science© 2012 Rosenstiel School of Marine and Atmospheric Science of the University of Miami

NitrogeN aNd CarboN isotopiC systematiCs of the florida reef traCt

K Lamb, PK Swart, and MA Altabet

abstraCt

The nitrogen isotopic composition of a variety of organisms in coral reefs has been used extensively, not only to study nitrogen dynamics, but also as a tracer for the input of anthropogenic nitrogen. however, the interplay between isotopic fractionation associated with the internal nitrogen cycling and variations in the absolute values supplied by external source signatures is poorly understood. here we report the δ15N and δ13C of algae, sponges, and fishes, the δ15N and δ18o of dissolved No3

−, and the δ15N of dissolved organic nitrogen in samples collected from the florida reef tract over a 2.5-yr period (2003–2005). our data are synthesized with results from previous studies of the δ15N of particulate organic material and coral tissue and zooxanthellae from the same area to provide a more detailed understanding of factors controlling coral reef δ15N and variation among biogenic components. These data show that during the study period there were (1) no clear spatial patterns in the δ15N of biogenic components related to proximity to the florida reef tract, and (2) no temporal patterns related to the wet or dry seasons. The range of δ15N and δ13C in the particulate organic material could be best explained as a mixture of material derived from seagrass, algae, mangroves, and fishes. The δ15N and δ18o of No3

− support a model in which variations in nitrogen isotopic composition are derived mainly from the isotopic effects associated with the nitrification of Nh4

+ to No3

− and subsequent assimilation by primary producers rather than through the input of isotopically distinct No3

− from external sources.

The overall health of coral reef ecosystems is of concern worldwide. in many areas, there is a perceived and real decline in the health of these communities as a result of changes in climate, population pressure, overfishing, pollution, and an excess supply of nutrients. in the case of the florida Keys (fK), there has been a phase shift from coral dominated communities to ones largely composed of macroalgae and soft cor-als (porter 1992). This adjustment is not unique to south florida and many reefs have experienced the same phenomenon, a change many workers have attributed to the increasing input of anthropogenic nitrogen both in the form of sewage (lapointe et al. 2005a) and from artificial fertilizers (marion et al. 2005). some of these studies have attempted to identify anthropogenic sources of nitrogen using the δ15N value of organic material derived from plants and animals. more positive values are thought to be indicative of anthropogenic nitrogen input, while values closer to zero are con-sidered typical of more pristine systems. for example, heikoop et al. (2000) reported that the bulk δ15N values of coral tissues (coral and zooxanthellae combined) from putatively contaminated sites were more positive than those from pristine areas, but did not offer any other evidence that there were real differences in nitrogen input or isotopic signature between them. other investigations have similarly used elevated δ15N values for algae and other organisms as evidence of anthropogenic influence (risk and erdmann 2000, Costanzo et al. 2001, barile and lapointe 2005, lapointe et al. 2005a). however, it is well known that δ15N values are influenced by common

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BULLETIN OF MARINE SCIENCE. VOL 88, NO 1. 20122

nitrogen cycling processes (e.g., nitrification, assimilation, and denitrification). in particular, processes removing N from the environment often leave the remainder enriched in 15N, leading to elevated δ15N for organisms subsequently utilizing this nitrogen. Therefore, it would be incorrect to ascribe all observations of “heavy” δ15N values to anthropogenic processes without eliminating the effects of these other processes.

in the fK, δ15N values as low as +4.5‰ have been cited as evidence for contami-nation by anthropogenic nitrogen (lapointe et al. 2004). other studies have shown elevated δ15N values in sponges in the fK relative to other areas and increasing trends in gorgonian tissue δ15N (Ward-paige et al. 2005b) over the past 50 yrs. These studies asserted that the fK have been subject to an increased input of anthropogenically-derived nitrogen. however, other recent studies have indicated that the situation in the fK is not so clear. for example, the δ15N of coral tissues and zooxanthellae from a number of locations in the upper fK matched those of reefs more clearly unaf-fected by pollution (swart et al. 2005). in addition, studies of the δ15N of particulate organic material (pom) have shown no evidence of an anthropogenic source (lamb and swart 2008), while the δ15N of the No3

− upwelled from deeper waters have val-ues similar to algae (leichter et al. 2007), previously reported as indicating pollution from anthropogenic sources (lapointe et al. 2005b).

in response to continuing uncertainty regarding the interpretation of variation in the δ15N of marine organisms in the fK and other areas, a study was initiated to examine the isotopic influence of nitrogen cycling within this coral reef ecosystem. here we report on the δ15N and δ13C of macroalgae, sponges, mangroves, seagrasses, the δ15N and the δ18o of No3

−, and the δ15N of dissolved organic nitrogen (combined with Nh4

+) from the fK reef tract over a 2.5-yr period (2003–2005). in addition, we examined samples of fishes that had been collected on six occasions between 2000 and 2005. These data are combined with previously published δ15N values for corals (swart et al. 2005), pom (lamb and swart 2008), and dissolved inorganic nitrogen (diN, leichter et al. 2007) from the same area.

methods

field samplessamples of benthic algae (Dictyota pinnatifida Kützing and Halimeda sp.), seagrass

(Thalassia testudinum banks & sol. ex K.d. Koenig), red mangrove (Rhizophora mangle linnaeus), and turf algae were collected approximately monthly between July 2003 and december 2005 from within the boundaries of the florida Keys National marine sanctuary (fKNms) and John pennekamp Coral reef state park (fig. 1). The two transects delineated in the study area include three stations per transect with a mangrove island site, an inshore reef site, and an offshore reef site. The mangrove sites were adjacent to the mangrove fringe of Key largo and adjacent to rodriguez Key. Water depth was < 1 m and the bottom sediment was principally composed of carbonate mud, seagrasses, and fragments of the coral Porites furcata lamarck, 1816. as a result of the paucity of suitable fishes at the mangrove sites, they were not collected here. The inner reef sites (triangles and White banks) are small patch reefs surrounded by sandy to muddy areas containing seagrasses. The outer reef sites are situated on or near the shelf break and are exposed to higher energy than the other sites. fish samples were taken may, June, and september 2000, June and July 2003, february 2004, and June and July 2005. additional samples of fishes were collected from algal reef and little grecian. These sites are situated slightly landward of the shelf margin and are classified as being intermediary between the inner and outer reef sites. The location and water depths of

LAMB ET AL.: NITROgEN ANd CARBON ISOTOpE SySTEMATICS OF FLORIdA REEFS 3

the sites are given in table 1 and figure 1. in addition, some of the diN samples were collected from the interior canals of Key largo and from mangrove areas. Where possible, samples were identified to the genus and species level using a taxonomic key. We collected samples of the following taxa: (1) macroalgae (D. pinnatifida and Halimeda sp.), (2) mangove (R. mangle), (3) sponges, (4) seagrass (T. testudinum), and (5) turf algae. a range of fish species was collected from all sites [Acanthurus bahianus Castelnau, 1855, Kyphosus sectatrix linnaeus, 1758, Scarus croicensis bloch, 1790, Sparisoma atomarium (poey, 1861), Sparisoma aurofrenatum (Valenciennes in Cuvier and Valenciennes, 1840), Sparisoma chrysopterum (bloch and schneider, 1801), Sparisoma viride (bonnaterre, 1788), Stegastes dorsopunicans (poey, 1868)]. samples of the coral Montastraea faveolata (ellis and solander, 1786) were collected between 1995 and 1997 in the same general area (swart et al. 2005). in addition, pom samples were collected on 13 cruises between 2000 and 2002 (lamb and swart 2008). These data have been compared to the data produced in this paper.

analytical methodsMacroalgae, Sponges, and Seagrass Samples.—samples were collected by snorkelers and

transported chilled to the laboratory. in the laboratory, they were rinsed in deionized water, and dried in a low temperature (40 °C) oven until a constant dry weight was achieved (usually 24–48 hrs). only the seagrasses typically have epiphytes, but these were not extensive and

Figure 1. Study location in the upper Florida Keys. Canals are not shown. The two inshore to offshore transects are represent by Pennekamp–White Banks Reef–Sand Island and Rodriguez–Triangles–Pickles. In addition, only fishes were sampled from Little Grecian and Algal Reef. Coral samples analyzed for δ15N were previously collected from Triangles and Pickles (Swart et al. 2005). Cruise transects made in the analysis of particulate organic material are shown as lines. Along each transect, four stations were sampled. These data are reported in Lamb and Swart (2008).


no attempt was made to physically remove them. The dried materials were milled (Thomas scientific Wylie mill) and treated with ~25 ml of 10% hCl to remove carbonate. after rins-ing and drying, the samples were prepared in triplicate for later δ13C and δ15N analysis (~1 mg for D. pinnatifida and Halimeda sp. samples; ~3 mg for sponges, mangrove, and seagrass samples). to test for possible isotopic effects resulting from the acidification, nine samples of plant material with known C and N isotopic compositions were processed in the same man-ner. Theses analyses revealed there to be no significant differences between the δ15N, δ13C, and C:N of the treated and untreated samples.

Fish Samples—fishes were collected by spear and handnet (paddack et al. 2009) and stored in a freezer until dissection. The approximate amount of muscle tissue removed varied based on the size of the fish and ranged up to 5 g (wet weight, up to 3 g dry weight). in addition, ~3–10 individual scales were plucked from a subset of S. viride and A. bahianus for separate isotopic analysis of δ15N and δ13C. The scales were removed from the dorso-lateral side of the fish, typically where the incision was made for muscle tissue removal. samples were dried and powdered. scales were cut into lengths of appropriate weights (~ 3 mg). of the sample set, 29 juvenile fishes were too small for dissection. These were dried whole and completely pulver-ized. samples were prepared in triplicate.

Water Samples.—Water samples were collected at the sea surface using pre-cleaned wide-mouthed bottles and stored frozen until analysis. at the time of sample processing, samples were first thawed then vacuum filtered through a pre-combusted 47-mm Whatman gf/C (1.2-µm pore size) glass fiber filter. samples were colorimetrically measured for No2

− and Nh4

+ concentration according to strickland and parsons (1972). The concentration of [No2−]

and [Nh4+] averaged 0.07 µm (± 0.08, n = 87) and 2.05 µm (± 2.15, n = 95), respectively. given

these low concentrations, no additional steps were needed for No2− removal prior to No3

− isotope analysis or for separate Nh4

+ isotopic analysis. analysis of the δ15N of the dissolved nitrogen species followed mcilvin and altabet (2005), and entailed the chemical stepwise reduction of nitrate (No3

−) to nitrite (No2−) using cadmium (Cd), and No2

− to gaseous ni-trous oxide (N2o) through reaction with sodium azide (NaN3). The principal difference in the analytical technique employed was that commercially available powdered Cd was used following activation by dilute hCl rather than making spongy Cd in-house. dissolved organic nitrogen (doN) was analyzed in a similar manner, but was first oxidized to No3

− using basic persulfate digestion (Nydahl 1978).

it is possible that freezing samples prior to filtration can lyse cells and introduce intracel-lular materials into the water samples. Therefore, we tested the procedure by analyzing four seawater samples with a range of No3

− and Nh4+ concentrations to assess possible artifacts

from the freezing procedure. These analyses showed no significant differences in either the concentrations of No3

− and doN or the δ15N and δ18o isotopic composition between the frozen samples and those preserved by filtering and acidification.

Table 1. Location of sample sites in the upper Florida Keys. Algal Reef and Little Grecian were only sampled for fish. NA = not applicable.

Station Latitude Longitude Depth (m) Transect Station positionPennekamp 25.1022 80.4003 0.0–1.0 North Mangrove White Banks 25.0383 80.3693 0.5–1.0 North Inshore reefSand Island 25.0189 80.3665 1.0–1.5 North Offshore reefRodriguez 25.0554 80.4492 0.0–1.0 South Mangrove Triangles 25.0183 80.4333 0.0–1.0 South Inshore reefPickles 24.9877 80.4100 0.5–2.0 South Offshore reefAlgal Reef 25.1023 80.3031 0.5–2.0 NA Mid-offshoreLittle Grecian 25.1152 80.3033 0.2–2.0 NA Mid-offshore


Mass Spectrometry.—The δ15N and δ13C of the samples were determined using a continu-ous-flow isotope-ratio mass spectrometer (Cfirms, europa scientific) at the University of miami. data were reported relative to atmospheric N2 and V-pdb (Vienna pee dee belemnite) for nitrogen and carbon, respectively. isotopic data produced from each run were scrutinized for standard precision and overall drift throughout individual runs. all data were corrected for such variations by comparing the run-generated δ15N and δ13C values of the standards to in-house standards. typical precisions of in-house organic standards are ± 0.11‰ for N and ± 0.07‰ for C. reported molar C:N ratios for the samples were calculated from the standard weight and corresponding beam intensities of standards and samples. as samples were run in triplicate, the mean of the three samples was treated as the mean value for the sample processed.

The δ15N and δ18o composition of N2o generated from dissolved No3− and doN were

determined on a gV isoprime isotope ratio mass spectrometer (irms) with an external automated purge-and-trap system at the University of massachusetts, dartmouth, smast campus. data are reported relative to atmospheric N2 and VsmoW (Vienna standard mean ocean Water) for nitrogen and oxygen, respectively. analytical precision measured from multiple determinations on standards was approximately ± 0.2‰ for δ15N and ± 0.7‰ for δ18o. isotopic data produced from each run were scrutinized for standard precision through-out individual runs. data were calibrated against standards (Usgs 34, 35, and one laboratory standard) as described in mcilvin and altabet (2005). The results for doN isotopic measure-ments were corrected for preexisting No3

− (little or no No2− or Nh4

+ initially present):

TN NOTN NO

DON3

TN 3 NO3) )d

d d=

-

--

--

^

^ ^

h

h h

6 66 6

@ @@ @

eq. 1

where [tN] is the measured concentration of the No3− + doN, δtN is its measured isotopic

composition, [No3−] is the measured No3

− concentration, and δNo3− is its measured isotopic

composition.

Statistical Analysis.—all comparisons of the differences in δ15N, δ13C, and C:N among loca-tions was performed using a mann-Whitney U test. all reported statistical significances in the present study are at the 95% confidence interval unless specified otherwise. Where ap-plicable, correlation between variables was determined using a spearman’s rank correlation coefficient.

results

algae, mangroves, and seagrassesGenus and Species Variations.—The mean δ15N and δ13C values and C:N ratio of all

the samples (D. pinnatifida, Halimeda sp., R. mangle, T. testudinum, and turf algae) are shown in tables 2 and 3, and figure 2. raw data can be accessed online (table s1). The δ15N and δ13C values, regardless of location, were tested for significant dif-ferences and with few exceptions, most of the samples differed significantly in both δ15N and δ13C (table 4). There were fewer significant differences in mean C:N values among genera (table 2).

Temporal Variations.—first-order patterns of δ15N, δ13C, and C:N ratios through time were also investigated (table 5). samples were grouped according to collection season; in this case, wet season (June–November) or dry season (december–may). This grouping revealed no significant differences based on season for any of the pa-rameters. over the course of the 2.5-yr study, a slight increasing trend in the δ15N and δ13C values was perceptible (fig. 3) in some of the groups. The trend in δ15N


was only significant in the case of D. pinnatifida and the sponges (spearman’s rank: p < 0.05). sponges and mangroves showed a significant increase in δ13C over time (spearman’s rank: p < 0.05).

Spatial Variations.—groups of organisms found at the majority of the sites (not including mangroves) were tested to ascertain whether there were any spatial pat-terns in δ15N and δ13C. The significance of these comparisons is shown in tables 6–9. The overall pattern is that there were generally no differences between various sites for δ15N. for example, of the 61 possible comparisons, only eight (13%) were sig-nificantly different. in all cases in which there were significant differences, the inner sites possessed more negative δ15N values than the more seaward sites. in contrast, the δ13C values differed in 55% of the comparisons (tables 6–9). once again, these differences were almost always manifest when comparing the inner to other sites, with the inner sites being more depleted than the outer sites.

spongessponges had δ15N values that were significantly more positive (+4.2‰ ± 1.8) than

all of the groups with the exception of the turf algae and fishes. sponge δ13C values (−17.2‰ ± 1.6) were statistically similar to the turf algae, Halimeda sp., and D. pinnatifida, but more positive than the mangroves and seagrass (tables 2, 10, fig. 2). The sponge C:N ratio (5.6 ± 5.72) was significantly different from all of the primary producers (mann-Whitney U test for all comparisons: p < 0.05). generally, no significant spatial differences were evident in either the δ13C or δ15N among the sites (table 10). The exception was the δ13C at the pennekamp site, which was significantly more negative than at any of the other sites; and the δ15N at rodriguez site, which was significantly more positive than at triangles, White banks, and pennekamp (table 10). raw data can be accessed online (table s1).

fishesSpecies.—mean δ15N, δ13C, and C:N values of the fishes collected during the study

are shown in table 11 and figure 2. There were significant inter-species differences in δ13C and δ15N in 70% of the comparisons (table 12). Stegastes dorsopunicans had the most enriched mean δ15N signatures (+8.5‰ ± 0.2) and S. viride the most de-pleted mean δ15N (+4.9‰ ± 1.0; table 11, fig. 2). Sparisoma viride also had the most

Table 2. δ13C, δ15N, and C:N ratios of samples measured in this study together with data on corals and POM from a previous study. Data in brackets represent one standard deviation. nm = not measured.

Sample analyzed δ15N (‰) δ13C (‰) C:N nDictyota pinnatifida +2.4 (± 1.3) −16.5 (± 1.4) 16.0 (± 6.0) 87Halimeda sp. +1.6 (± 1.3) −17.0 (± 2.5) 10.8 (± 4.5) 138Rhizophora mangle +1.4 (± 1.0) −28.0 (± 5.6) 69.2 (± 38.1) 52 Thalassia testudinum +1.9 (± 1.1) −10.5 (± 2.1) 23.8 (± 11.4) 57 Sponges +4.2 (± 1.8) −17.2 (± 1.6) 5.6 (± 5.7) 155Turf algae +2.7 (± 0.9) −16.6 (± 3.1) 10.4 (± 3.8) 20POM1 +3.7 (± 3.1) −20.0 (± 2.0) 8.6 (± 2.6) 274Montastraea faveolata (coral tissue)2 +6.6 (± 0.6) −13.3 (± 0.5) nm 60Montastraea faveolata (zooxanthellae)2 +4.7 (± 1.1) −12.2 (± 1.0) nm 601 Lamb and Swart 20082 Swart et al. 2005


Table 3. Summary of δ15N, δ13C, and C:N for Dictyota pinnatifida (D), Halimeda sp. (H), Thalassia testudinum (Th), turf algae (T), Rhizophora mangle (R), and sponges (S) from stations shown in Figure 1. Data in brackets represent one standard deviation. nc = samples not collected.

Station δ15N (‰) δ13C (‰) C:N nTriangles D 2.7 (± 1.8) −15.3 (± 1.0) 14.9 (± 7.5) 21White Bank D 2.2 (± 1.3) −16.5 (± 1.1) 16.4 (± 4.6) 13Sand Island D 2.6 (± 1.8) −16.5 (± 1.1) 15.7 (± 6.0) 22Pennekamp D 1.8 (± 1.3) −19.2 (± 0.9) 15.2 (± 13.6) 3Rodriguez D 1.8 (± 1.6) −19.3 (± 1.4) 16.7 (± 3.4) 5Pickles D 2.4 (± 0.9) −16.2 (± 1.0) 16.3 (± 5.9) 23

Triangles H 1.8 (± 1.1) −16.1 (± 1.5) 11.0 (± 3.8) 17White Bank H 1.8 (± 1.0) −16.9 (± 1.8) 10.8 (± 4.3) 29Sand Island H 1.7 (± 0.9) −18.2 (± 2.0) 9.7 (± 3.1) 31Pennekamp H 1.1 (± 1.4) −19.5 (± 2.7) 10.6 (± 2.5) 20Rodriguez H 1.0 (± 1.5) −19.6 (± 3.1) 14.3 (± 8.6) 16Pickles H 2.2 (± 0.8) −17.2 (± 2.2) 9.5 (± 2.9) 25

Triangles Th 2.2 (± 0.9) −8.7 (± 0.7) 23.4 (± 10.9) 16White Bank Th 2.2 (± 1.3) −10.2 (± 0.9) 24.8 (± 12.2) 7Sand Island Th nc nc nc ncPennekamp Th 1.4 (± 0.8) −13.0 (± 1.8) 21.7 (± 11.5) 16Rodriguez Th 1.9 (± 1.5) −10.5 (± 2.2) 24.2 (± 11.7) 16Pickles Th 1.5 (± 0.3) −9.0 (± 0.8) 28.2 (± 15.2) 2

Triangles T 2.2 (± 0.8) −14.4 (± 1.7) 10.5 (± 3.5) 5White Bank T 2.5 (± 0.6) −16.1 (± 0.9) 12.1 (± 6.6) 4Sand Island T 2.7 (± 1.0) −16.8 (± 1.3) 9.9 (± 1.7) 6Pennekamp T 2.3 (± 0.9) −18.7 (± 1.0) 6.9 (± 5.9) 2Rodriguez T nc nc nc ncPickles T 3.7 (± 0.1) −16.5 (± 1.8) 11.4 (± 2.9) 3

Pennekamp R 1.4 (± 1.0) −27.7 (± 2.1) 64.3 (± 42.1) 27Rodriguez R 1.4 (± 1.3) −28.4 (± 1.5) 74.2 (± 39.7) 25

Triangles S 3.8 (± 1.8) −16.6 (± 1.5) 7.3 (± 12.0) 30White Bank S 4.3 (± 1.3) −17.1 (± 1.7) 4.8 (± 2.3) 39Sand Island S 4.5 (± 1.0) −17.1 (± 1.1) 4.1 (± 1.5) 27Pennekamp S 3.2 (± 2.5) −18.6 (± 1.9) 6.4 (± 3.0) 23Rodriguez S 4.9 (± 2.6) −16.9 (± 1.3) 7.0 (± 3.0) 19Pickles S 4.5 (± 1.1) −17.2 (± 1.3) 4.5 (± 2.8) 17

enriched mean δ13C signatures (−13.4‰ ± 1.6), while K. sectatrix had the most de-pleted mean δ13C signatures (−15.9‰ ± 0.8). molar ratios of C:N were greatest for K. sectatrix (5.0 ± 0.1) and smallest for S. dorsopunicans (3.6 ± 0.7).

Tissue-Type Variations.—a subset of S. viride and A. bahianus muscle tissue and scales was examined, revealing that the δ13C values and C:N ratios of samples differed significantly between tissue types, while δ15N composition did not. for A. bahianus, the two tissue types differed significantly in δ13C, while in S. viride the two tissue types differed significantly in both δ13C and C:N values (p < 0.05). in addition,


there was a clear correlation between tissue δ15N of scales and δ15N of muscle tissue for both species, with a best fit regression line slope of 0.86 (r2 = 0.91), very close to the 1:1 line. The δ13C of the scale and muscle tissue were also strongly correlated (spearman’s rank: p < 0.05).

Spatial Variations.—data from S. viride and A. bahianus were compared among sites to assess spatial differences. for both species, there was generally no significant differences in the δ15N values between any of the stations (tables 13, 14; the only ex-ception being between pickles and algal reef for S. viride). The δ13C data exhibited a larger number of significant differences. in particular, little grecian and algal reef had significantly lighter δ13C values than the other stations (tables 13, 14).

Figure 2. Mean δ15N and δ13C of algae, mangroves, seagrass, sponges, and fishes from the upper Florida Keys. Error bars = 1 standard deviation. Fishes are shown in the solid circles with num-bers referring to the various species; (1) Acanthurus bahianus (ocean surgeonfish), (2) Kyphosus sectatrix (chub), (3) Scarus croicensis (striped parrotfish), (4) Sparisoma atomarium (greenblotch parrotfish), (5) Sparisoma aurofrenatum (redband parrotfish), (6) Sparisoma chrysopterum (red-tail parrotfish), (7) Sparisoma viride (stoplight parrotfish), (8) Stegastes dorsopunicans (damsel-fish). See Tables 2, 3, and 11 for data.

Table 4. P values from Mann-Whitney U test of stable isotopes between different groups of: Dictyota pinnatifida (D), Halimeda sp. (H), Thalassia testudinum (Th), turf algae (T), Rhizophora mangle (M). Shaded cells represent level of significance for δ15N and unshaded cells represent δ13C. Bold values are significant (P < 0.05).

D H Th S T MD – 0.000 0.000 0.000 0.627 0.000H 0.000 – 0.000 0.109 0.007 0.000Th 0.029 0.070 – 0.000 0.000 0.000S 0.000 0.000 0.000 – 0.018 0.000T 0.124 0.000 0.006 0.000 – 0.000M 0.000 0.103 0.009 0.000 0.000 –


Temporal Variations.—No significant differences were found in δ15N and δ13C val-ues and C:N ratios of the fishes collected in 2000 and those collected in 2005 (p > 0.05).

Water Column Concentrations of No2−, No3

−, Nh4+, and doN

No2− concentrations were the lowest for all measured species and were 0.07 µm (±

0.08, n = 87) for the entire study (table 15, fig. 4). mean No3− concentrations were

slightly higher at 0.60 µm (± 1.08, n = 96), while mean measured Nh4+ concentra-

tions were higher at 2.05 µm (± 2.15, n = 95). mean doN concentrations showed the highest measured nitrogen species in our study at 7.48 µm (± 4.53, n = 96). When as-sessed for seasonal variations, there were no significant differences between wet and dry season concentrations for any of the nitrogen species tested and no overarching seasonal patterns were observed through time (fig. 4). spatially, only No3

− and Nh4+

exhibited significant differences based on station position (p < 0.05), with higher concentrations in the canal and lower concentrations at non-canal sites. raw data can be accessed online (table s2).

Table 5. Mean temporal variations in δ15N of samples of mangroves, algae, sponges, and seagrass collected over a 30-mo period. All analyses represent the mean of two or more samples unless the standard deviation is zero (one sample). Empty cells indicate that no samples were collected. These data are presented graphically in Figure 3. Data in brackets represent one standard deviation.

Date MangroveDictyota

pinnatifida Halimeda sp.Thalassia testudinum Sponges Turf algae

Jul-03 0.9 (± 0.7) 1.5 (± 1.6) 2.3 (± 0.4) 0.9 (± 0.0) 3.5 (± 1.6) Aug-03 1.3 (± 0.8) 1.7 (± 0.6) 2.8 (± 0.0) 4.5 (± 1.5) Nov-03 0.3 (± 0.0) 1.5 (± 0.9) 1.7 (± 0.3) 3.0 (± 0.0) 2.9 (± 0.1)Dec-03 1.2 (± 0.0) 3.5 (± 0.5)Jan-04 −0.1 (± 0.0) 1.6 (± 0.2) 1.5 (± 1.0) 2.2 (± 0.0) 4.2 (± 1.5) 2.2 (± 0.3)Feb-04 2.0 (± 1.5) 2.2 (± 0.6) 2.6 (± 1.0) 2.2 (± 0.6) 4.3 (± 1.8) 1.0 (± 0.0)Mar-04 0.1 (± 0.7) 1.8 (± 0.9) 1.3 (± 0.5) 1.1 (± 0.3) 3.7 (± 0.6) 1.6 (± 0.0)Apr-04 1.6 (± 0.9) 2.6 (± 0.2) 2.2 (± 0.3) 0.6 (± 0.2) 3.4 (± 1.5) May-04 0.8 (± 0.4) 1.5 (± 1.3) 0.2 (± 1.9) 0.9 (± 1.3) 3.5 (± 2.2) 2.8 (± 0.2)Jun-04 3.4 (± 1.7) 2.2 (± 1.0) 1.2 (± 1.1) 1.7 (± 0.9) 3.9 (± 1.9) 2.4 (± 1.2)Jul-04 2.2 (± 1.3) 3.2 (± 0.9) 2.5 (± 1.6) 1.0 (± 0.0) 4.2 (± 0.6) 3.0 (± 0.0)Aug-04 0.9 (± 0.6) 2.4 (± 0.8) 1.6 (± 0.7) 2.1 (± 1.4) 3.7 (± 1.8) 2.8 (± 1.1)Oct-04 1.5 (± 2.5) 3.0 (± 0.0) 2.2 (± 0.7) 2.0 (± 0.7) 3.6 (± 1.7)Nov-04 −0.9 (± 0.0) 1.9 (± 0.6) 1.2 (± 1.2) 1.3 (± 0.6) 2.5 (± 2.5) Dec-04 1.4 (± 1.0) 4.0 (± 0.7) 1.8 (± 0.4) 1.8 (± 1.1) 4.9 (± 1.4) Jan-05 1.1 (± 0.2) 3.0 (± 1.4) 1.4 (± 0.4) 2.5 (± 1.3) 4.3 (± 2.2) Feb-05 1.8 (± 0.0) 2.3 (± 0.7) 0.9 (± 0.8) 1.3 (± 1.6) 3.7 (± 2.2) Apr-05 1.8 (± 0.4) 2.1 (± 0.9) 1.2 (± 0.6) 2.2 (± 0.5) 4.9 (± 1.6) May-05 1.3 (± 0.4) 2.7 (± 1.6) 1.6 (± 0.7) 1.6 (± 1.0) 5.8 (± 1.2) Jun-05 1.9 (± 0.6) 1.3 (± 1.3) 1.1 (± 0.4) 1.7 (± 1.2) 5.2 (± 1.8) Jul-05 2.4 (± 1.3) 3.4 (± 2.0) 1.8 (± 1.5) 3.4 (± 1.0) 5.6 (± 0.9) Aug-05 1.5 (± 0.5) 2.4 (± 1.0) 0.8 (± 1.2) 2.5 (± 0.5) 4.6 (± 2.1) Sep-05 1.9 (± 0.0) 3.5 (± 0.2) 4.0 (± 0.5) Oct-05 1.7 (± 0.6) 3.9 (± 1.6) 2.2 (± 2.0) 2.8 (± 1.8) 4.3 (± 1.4) 2.4 (± 0.0)Dec-05 1.5 (± 0.1) 4.5 (± 1.0) 2.5 (± 1.0) 2.2 (± 1.2) 4.8 (± 0.9) Mean 1.3 (± 0.9) 2.4 (± 0.9) 1.7 (± 0.7) 1.8 (± 0.7) 4.2 (± 0.8) 2.5 (± 0.7)


Water Column δ15N and δ18o of No3− and doN

mean δ15NNo3 and δ18oNo3 during the study period were +4.4‰ (± 3.5) and +20.4‰ (± 6.9), respectively. Water samples had a mean δ15NdoN value of −0.4‰ (± 3.2; table 15, figs. 5, 6). There were no significant differences between wet and dry seasons for δ15NNo3, δ

18oNo3, or δ15NdoN, and no temporal trends in δ15N were detected through-out the study period.

δ15NNo3 was significantly more positive (mean δ15N = +10.1‰ ± 4.6) in the canals relative to all other non-canal sites (mean δ15N = +4.0‰ ± 3.1, p < 0.05) while δ18oNo3 was heavier in the non-canal sites (mean δ18o = +21.3‰ ± 6.5) and lighter in the canals (mean δ18o = +10.1‰ ± 4.6, p < 0.05). δ15NdoN was significantly lighter in the inshore areas compared to the offshore areas and canals. raw data can be accessed online (table s3).

Figure 3. Temporal variations of δ15N and δ13C of algae, mangroves, and sponges from the upper Florida Keys. Black circles are the δ13C data and the diamonds the δ15N data. A = Dictyota pin-natifida, B = Halimeda sp., C = Rhizophora mangle, D = Thalassia testudinum, E = sponges, F = turf algae. No long term or seasonal trends were evident in either the δ15N or δ13C values in most of the data. The exceptions are δ15N for D. pinnatifida and sponges, and δ13C for R. mangle and sponges, which revealed significant increases over time (Spearman’s Rank: P < 0.05).


Table 6. P values from Mann-Whitney U test of stable isotopes in Dictyota pinnatifida from six upper Florida Keys sites: Pennekamp (PK), Rodriguez (R), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05).

PK R T WB SI PPK – 1.000 0.001 0.002 0.003 0.001R 1.000 – 0.002 0.000 0.000 0.000T 0.521 0.278 – 0.002 0.004 0.011WB 0.742 0.373 0.645 – 0.856 0.303SI 0.398 0.113 0.845 0.328 – 0.452P 0.351 0.129 0.912 0.436 0.973 –

Table 7. P values from Mann-Whitney U test of stable isotopes in Halimeda sp. from six upper Florida Keys sites: Pennekamp (PK), Rodriguez (R), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05).


Table 8. P values from Mann-Whitney U test of stable isotopes in Thalassia testudinum from six upper Florida Keys sites: Pennekamp (PK), Rodriguez (R), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05). Blacked out cells indicated that no samples were taken from these localities.

PK R T WB SI PPK – 0.001 0.000 0.000 0.012R 0.182 – 0.005 0.537 0.292T 0.021 0.631 – 0.000 0.732WB 0.024 0.442 0.937 – 0.139SI –P 0.749 0.573 0.261 0.260 –

Table 9. P values from Mann-Whitney U test of stable isotopes in turf algae from six upper Florida Keys sites: Pennekamp (PK), Rodriguez (R), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05). Blacked out cells indicated that no samples were taken from these localities.

PK R T WB SI PPK – 0.000 0.000 0.000 0.000R –T 0.857 – 0.111 0.052 0.393WB 0.533 0.556 – 0.352 1.000SI 0.643 0.247 0.914 – 0.714P 0.200 0.036 0.057 0.095 –


discussion

diN and doN distributionsour results show that water column nutrient concentrations sampled from man-

grove and reef sites within the fKNms were low (fig. 5), indicative of an ecosystem not frequently inundated with elevated nitrogen loads. measured concentrations of No3

− are similar in magnitude to those reported for other open marine reef sites, such as barbados (tomascik and sander 1985) and australia’s great barrier reef (gbr; hatcher and hatcher 1981, Costanzo et al. 2000, atkinson et al. 2001). The concentration of doN found in the present study (mean ~8 µm) is somewhat ele-vated over “pristine” reefs from australia’s gbr (doN = ~6 mm, furnas et al. 1997); however, these levels are not nearly as high as those from waters impacted by high wastewater loads (> 50 µm doN, homewood et al. 2004). The concentrations of ammonium reported in our study are also elevated (by ~1 µm) over reported Nh4

+ values from other reef studies, but are similar to values previously reported values for waters from the fK (szmant and forrester 1996, boyer and Jones 2002).

previously, it had been suggested that significantly more positive δ15N values were manifest during the wet season as a result of runoff contributing a greater amount of sewage derived nitrogen to the marine waters (lapointe et al. 2004). however, in the present study, we found no significant differences in the concentrations of diN or doN between the wet and dry seasons, suggesting that water column nutrient avail-ability is not subject to seasonal fluctuations. instead, it appears that the water col-umn diN and doN are supplied from processes or sources that operate consistently throughout the year. samples collected along inshore to offshore transects do not show significant spatial trends in No2

−, Nh4+, or doN concentrations, implying that

source(s) for these nitrogen species are relatively spatially homogeneous throughout the study area. The canals are an exception in this respect with samples clearly show-ing elevated No3

− concentrations. These findings are similar to a prior report for the fKNms, where concentrations of N species were elevated in canals, and dropped to oligotrophic levels within 1 km of the shoreline (szmant and forrester 1996). This reduction was attributed to rapid uptake of the nutrients by immediately-nearshore water column biota and benthos (szmant and forrester 1996). similarly, impacts from onshore activities in barbados, such as industry and tourism developments, were clearly seen in elevated No3

−, No2−, and Nh4

+ concentrations close to shore (tomascik and sander 1985).

Table 10. P values from Mann-Whitney U test of stable isotopes in sponges from six upper Florida Keys sites: Pennekamp (PK), Rodriguez (R), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05).



No3− and doN isotopic Composition

The canal water No3− values were consistently enriched in 15N and depleted in

18o when compared to all non-canal sites. previous studies, such as silva et al. (2002), have identified such isotopic signatures as indicative of sewage N (figure 6). although the mean δ15NNo3 of the non-canal sites and were significantly lighter and ranged between +3.5‰ and +4.5‰, there were instances of quite elevated δ15NNo3 values at all of the other sites, occasionally reaching as high as those values found in the canals. although these elevated values might be explained by being represen-tative of a sewage component, the δ15NNo3 values are not correlated with increases in nitrate concentration and are therefore probably best explained by fractionation during assimilation (Wada and hattori 1976, Kendall 1998, sammarco et al. 1999, pantoja et al. 2002, anderson and fourqurean 2003, mahaffey et al. 2004), operating on nitrate derived from a range of sources including upwelled waters (δ15N = +4‰ to +6‰), N2 fixation (δ15N = ~0‰), atmospheric deposition as either No3

− (−5‰ to +4‰) or Nh4

+ (−12‰ to ~0‰; dillon and Chanton 2005, Knapp et al. 2005, 2008), or fertilizers (δ15N = ~0‰). The δ18oNo3 of mangrove and reef waters are also very differ-ent from the canal waters and indicate a primary nutrient source different from that

Table 11. Mean δ13C, δ15N, and C:N ratios of fish muscle for eight fish species sampled from the upper Florida Keys.

Species δ15N (‰) δ13C (‰) C:N nAcanthurus bahianusOcean surgeonfish

+6.16 (± 1.24) −15.68 (± 1.62) 4.32 (± 0.97) 42

Kyphosus sectatrixChub

+6.42 (± 0.58) −15.88 (± 0.77) 5.03 (± 0.12) 10

Scarus croicensisStriped parrotfish

+5.18 (± 0.49) −13.69 (± 0.81) 3.87 (± 0.51) 31

Sparisoma atomariumGreenblotch parrotfish

+5.59 (± 0.34) −13.68 (± 0.44) 3.78 (± 0.03) 7

Sparisoma aurofrenatumRedband parrotfish

+5.91 (± 0.73) −14.66 (± 0.97) 4.16 (± 0.98) 14

Sparisoma chrysopterumRedtail parrotfish

+5.11 (± 1.22) −13.45 (± 0.47) 4.08 (± 1.24) 5

Sparisoma virideStoplight parrotfish

+4.92 (± 1.01) −13.40 (± 1.45) 3.80 (± 0.69) 191

Stegastes dorsopunicansDamselfish

+8.47 (± 0.19) −14.89 (± 0.60) 3.60 (± 0.68) 7

Table 12. P values from Mann-Whitney U test of isotopes in eight fish species collected from the upper Florida Keys. Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05). ABA= Acanthurus bahianus, SVI = Sparisoma viride, SCH = Sparisoma chrysopterum, SAU = Sparisoma aurofrenatum, SAT = Sparisoma atomarium, SDO = Stegates dorsopunicans, KSE = Kypohosus sectatrix, SCR = Scarus croicensis.

ABA KSA SCR SVI SAU SAT SDO SCHABA – 0.175 0.000 0.000 0.045 0.000 0.249 0.000KSA 0.788 – 0.000 0.000 0.004 0.000 0.025 0.002SCR 0.000 0.000 – 0.045 0.002 0.970 0.000 0.822SVI 0.000 0.000 0.225 – 0.000 0.228 0.000 0.479SAU 0.026 0.122 0.002 0.000 – 0.020 0.535 0.046SAT 0.000 0.005 0.027 0.010 0.197 – 0.000 0.927SDO 0.000 0.000 0.000 0.000 0.000 0.000 – 0.006SCH 0.037 0.054 0.233 0.136 0.382 0.527 0.006 –


in the canals. Non-canal δ18oNo3 values range between +19‰ and +23‰. although precipitation elevates δ18o values, it is likely that the measured δ18o do not reflect this input as the nitrate is rapidly consumed in the reef ecosystem.

The fK waters are unusual compared to open oceanic waters in that they have a relatively large pool of Nh4

+ compared to No3. This Nh4+ is derived from the decom-

position of organic material and is rapidly utilized by organisms and converted to No2

− and No3−. during this latter process, there is a reverse isotopic fractionation

of oxygen so that the resultant nitrate has an isotopically more positive δ18o value (Casciotti 2009). The latter process should produce an inverse correlation between δ18o and δ15N. however, this effect would be diminished if No2

− oxidation were ef-ficient as indicated by the low concentrations observed. indeed, the δ18o and δ15N of No3

− covary within each of the sample subsets with a slope close to unity. following the interpretation of Wankel et al. (2006), this association is suggested to indicate that both the δ18o and δ15N values are fractionated during the assimilation of nitrate by algae. if the slope is greater than unity, then the nitrate is thought to be influenced by surface nitrate regeneration. synthetic fertilizers as sources of No3

− to non-canal sites can probably be eliminated from the list of possible influences on the waters of the fK, simply because the only source of synthetic fertilizers used in noticeable quantities would be in the everglades agricultural areas (eaa). These fertilizers are considered to have minimal influence on the fK, as ~90% of the fertilizer-sourced N leaving the eaa is removed via biotic uptake before leaving the everglades (rudnick et al. 1999, sutula et al. 2003). The residual fertilizer-derived N that is still present in everglades discharge is utilized upon entry into florida bay and does not reach the fK nearshore communities (sutula et al. 2001).

Table 13. P values from Mann-Whitney U test of stable isotopes in Acanthurus bahianusin from six sites in the upper Florida Keys: Little Grecian (LG), Algal Reef (AR), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05). Blacked out cells indicate that no samples were taken from these localities.

LG AR SI P WB TLG – 0.148 0.023 0.036 0.004AR 0.303 – 0.556 0.530 0.151SI 0.477 0.190 – 0.412 0.286P 0.935 0.202 0.412 – 0.343WB 0.869 1.000 0.730 0.530 –T –

Table 14. P values from Mann-Whitney U test of stable isotopes in Sparisoma viride from six sites in the upper Florida Keys: Little Grecian (LG), Algal Reef (AR), Triangles (T), White Banks (WB), Sand Island (SI), and Pickles (P). Shaded cells represent level of significance for δ15N and unshaded cells for δ13C. Bold values are significant (P < 0.05).

LG AR SI P WB TLG – 0.086 0.000 0.025 0.004 0.205AR 0.156 – 0.000 0.008 0.000 0.033SI 0.543 0.053 – 0.033 0.080 0.223P 0.329 0.014 0.674 – 0.743 0.826WB 0.655 0.090 0.854 0.978 – 0.854T 0.317 1.000 0.223 0.209 0.374 –


Tabl

e 15

. Mea

n co

ncen

tratio

ns a

nd N

isot

opic

com

posi

tions

of D

IN a

t var

ious

site

s in

the

uppe

r Flo

rida

Key

s as

sho

wn

in F

igur

e 1

and

liste

d in

Tab

le 1

. Dat

a fr

om K

ey L

argo

can

als a

re a

lso

liste

d; d

ata

in b

rack

ets r

epre

sent

one

stan

dard

dev

iatio

n.

Stat

ion

NO

2 (µM

)N

H4 (

µM)

NO

3 (µM

)N

O3 δ

15N

NO

3 δ18

OD

ON

(µM

)D

ON

δ15

Nn

Can

al0.

1 (±

0.1

)2.

1 (±

0.1

)2.

0 (±

0.4

)10

.1 (±

3.8

)10

.1 (±

4.5

)8.

8 (±

1.8

)1.

4 (±

1.3

)6

Penn

ekam

p0.

1 (±

0.0

)1.

7 (±

0.7

)0.

3 (±

0.2

)3.

6 (±

3.7

)19

.8 (±

7.0

)8.

2 (±

1.4

)−0

.6 (±

2.6

)13

Whi

te B

anks

0.1

(± 0

.1)

4.0

(± 4

.8)

0.8

(± 1

.7)

3.3

(± 2

.9)

21.4

(± 5

.5)

10.0

(± 1

0.7)

−3.2

(± 5

.7)

13Sa

nd Is

land

0.1

(± 0

.2)

1.6

(± 0

.4)

0.7

(± 0

.1)

4.4

(± 2

.6)

20.2

(± 6

.9)

5.8

(± 1

.5)

−0.7

(± 1

.9)

15R

odrig

uez

0.1

(± 0

.1)

2.0

(± 0

.7)

0.4

(± 0

.4)

4.3

(± 3

.1)

26.3

(± 4

.7)

8.6

(± 2

.8)

0.9

(± 2

.9)

13Tr

iang

les

0.0

(± 0

.0)

1.6

(± 0

.4)

0.2

(± 0

.1)

3.8

(± 3

.5)

22.4

(± 6

.7)

6.1

(± 1

.0)

−0.3

(± 1

.4)

11Pi

ckle

s0.

1 (±

0.1

)1.

6 (±

0.7

)0.

5 (±

0.4

)4.

4 (±

3.1

)17

.9 (±

4.6

)6.

3 (±

2.8

)0.

7 (±

2.0

)19


mangroves, benthic algae, and seagrassesmangrove leaves sampled from the islands close to the fK have δ15N values indi-

cating a strong dependence on nutrients supplied from nitrogen fixation (muzuka and shunula 2006) and do not appear to be impacted by sources of isotopically posi-tive nitrogen. research conducted in australia indicates enriched δ15N signatures of mangrove leaves (> +9.0‰) in areas in close proximity to sewage treatment facilities, and depleted leaf δ15N signatures, as low as +1.6‰, in areas unaffected by sewage (Costanzo et al. 2005). hence, values reported in the present study (+1.5‰) indicate that anthropogenic nitrogen is not important and suggest that other sources, such as nitrogen fixation, play a more dominant role in nutrient supply to the nearshore ecosystem.

macroalgae gathered within the fKNms, including D. pinnatifida, turf algae, and Halimeda sp., also have low δ15N values, typical of macroalgae growing in shallow, oligotrophic systems that have experienced high irradiance and natural N sources such as nitrogen fixation, lapointe et al. (2005a). furthermore, T. testudinum col-lected during the present study displayed δ15N values similar in magnitude and range to previously reported values for T. testudinum (anderson and fourqurean 2003) from the fK, and did not show evidence of impacts from sewage related influences. in a study on T. testudinum from florida bay, very positive δ15N values were noted by Corbett et al. (1999) in the vicinity of taylor slough. as there were no obvious sewage sources in this area, these workers concluded that the δ15N was being enriched by the process of denitrification in the sediments. samples of seagrasses analyzed from the ocean side of Key largo were similar to our values.

Figure 4. Variations in the mean concentration of dissolved inorganic nitrogen (DIN) at all sites measured in this study as a function of time. Error bars represent 1 standard deviation of the data. The stippled area denotes the dry season, while the blank areas are wet season sampling periods.


Figure 5. δ15N and δ18O of nitrate measured in different areas off Key Largo. Within each area there is a correlation between δ18O and δ15N [inshore r = 0.65, P = 0.001; mangrove r = 0.38, P = 0.05; offshore r = 0.40, P = 0.05; Canals r = 0.48, P > 0.05 (non-significant due to low sample size)]. The solid line shows the relationship for the inshore samples. The slopes of the relation-ships are in each instance not significantly different from unity.

Figure 6. The δ15N of nitrate and the δ15N of the dissolved organic nitrogen (DON) from differ-ent sites sampled in the upper Florida Keys. No significant correlation was found between these values (P > 0.05).


δ13C values differed significantly among all genera collected during the study pe-riod (with the exception of T. testudinum and D. pinnatifida). These differences arise principally from variations in each organism’s specific Co2 fixation pathways. The δ13C values of T. testudinum samples collected in the present study were similar in magnitude to those previously reported (fourqurean et al. 2005). The δ13C values of D. pinnatifida, turf algae, and Halimeda sp. measured in our study are within report-ed ranges of δ13C for macroalgae reported in the literature (δ13C = −13‰ to −17‰; Currin et al. 1995, behringer and butler 2006, Corbisier et al. 2006). mangrove leaves had the lightest δ13C values and were similar to previously published values (−25.9‰ to −29.1‰, muzuka and shunula 2006).

all mean C:N values were found to be greater than the published values for each specific genera (patriquin 1972), suggesting that the reef tract is subsisting within a N-limiting environment. Halimeda sp. and T. testudinum had mean C:N values above published genera specific thresholds for nitrogen limitation (7.5 and 13.9, re-spectively; patriquin 1972, atkinson and smith 1983) and are evidence for a nutrient limiting environment in the fKNms. mangrove leaves showed the highest and most variable C:N ratios, which is attributed to the varying decomposition states of the leaves collected (davis et al. 2006).

The mean C:N value for D. pinnatifida measured during the present study is above the published D. pinnatifida thresholds for N-limitation (C:N = 17.9, atkinson and

Figure 7. Summary of δ15N and δ13C data from organic components measured in the present study combined with data from previous studies in the Florida Keys. Data from coral tissue and zooxan-thellae (triangles) were measured on Montastraea faveolata (Swart et al. 2005, Lamb and Swart 2008). POM = particulate organic material.


smith 1983), although many D. pinnatifida collected in our study had C:N values that fell below the 17.9 threshold, which may initially imply excess nitrogen available for uptake. however, it may be that C:N values generated from the samples of D. pin-natifida collected in hawaii and Queensland by atkinson and smith (1983) are not applicable in the fK ecosystem, or for every Dictyota species. furthermore, the mean C:N values for all other genera reported in the present study are well above their re-spective thresholds; if nitrogen was in excess in the water column, a majority of the genera sampled would be expected to have similarly lower C:N values.

in four of the groups analyzed, there were no significant temporal trends in biotic tissue δ15N throughout the duration of the study period. for D. pinnatifida, the δ15N showed a small but significant increase. south florida’s wet season brings substantial amounts of rainwater and, consequently, the highly permeable limestone bedrock experiences greater groundwater flushing, surface runoff, and thus possibly deliver-ing a greater load of sewage-influenced nutrients during the rainy season (lapointe et al. 1990, 2004, shinn et al. 1994). if land-based pollution were a major nutrient source to the benthic biota, then there should be enrichment in the δ15N of the sampled tis-sues during the wet season. however, combining the data into wet (June–November) and dry seasons (November–may) revealed no significant differences between the δ13C and δ15N in any of the biota analyzed between these time periods.

Spatial Variations.—The absence of distinct differences in the δ15N of tissues col-lected at the different sites is not consistent with the hypothesis that land-sourced anthropogenic wastes are the cause of the nitrogen variations. although there are some instances in which the δ15N values are different between the nearshore and offshore sites, in all instances, the inshore sites were more depleted in δ15N. it was expected that under a shore-based, sewage-loading hypothesis, the more enriched δ15N values would be closest to shore, and would become more depleted moving off-shore. instead, mangrove sites had mean δ15N values that were lighter than both the inshore and offshore reef sites. moreover, the δ15N values for all sites were lighter compared to sewage-impacted biota tissues and did not show evidence that the reef tract is dominantly influenced by anthropogenic inputs originating from nearshore environments.

examining the isotopic data for spatial patterns in δ13C revealed that the inshore to offshore landscape trends for δ13C were significant, but again, the patterns do not appear to be a result of anthropogenic influences. instead, the δ13C spatial patterns were controlled primarily by the relative abundance of the various types of sample found at each site. specifically, pennekamp and rodriguez were the only two sites that had mangrove leaves available for collection. Consequently, the R. mangle sam-ples skewed the average δ13C values for those two sites to be more depleted than the other four reef sites. likewise, T. testudinum was prevalent at White banks and triangles reef (inshore reefs), but not at sand island or pickles reef (offshore reefs); as such, the mean δ13C values at the inshore reef sites are skewed more positively than the offshore reef sites.

spatial patterns in reported C:N values are also a product of sample type, as man-grove leaf C:N skewed both pennekamp and rodriguez station C:N averages to be much higher than the other reef sites. in fact, when mangrove samples are not in-cluded in statistical comparisons of station mean C:N, all stations are statistically indistinguishable from each other.


spongessponges collected from fKNms had the most positive of all the measured tissue

δ15N (+4.13‰) values, but were actually within the “normal” range of nitrogen iso-topic composition for sponges (Weisz et al. 2007). Work on this and similar sponge species was described by Weisz et al. (2007), who gathered specimens from the fK, North Carolina, and papua New guinea. They reported the δ15N values for sponge tissues to be between +0‰ and approximately +5.5‰, and demonstrated that the δ15N of their samples showed no correlation to the proximity of the shore and were well within expected ranges for sponge δ15N. instead, the sponge δ15N values were linked to species-specific symbiotic associations with microbial communities, the subsequent exchanges of nitrogen between bacteria and the host sponge, and a diet further augmented by local pom sources (Weisz et al. 2007). These factors also like-ly account for the values we observed for the fK. our observations for the δ15N of sponges are also similar to those made by Ward-paige et al. (2005a) in the fK.

since sponges are not photosynthetic, there are no direct linkages between Co2 fixation pathways and tissue δ13C. however, because sponges are filter feeders, their tissue δ13C should approximate the δ13C of their diet, once a small enrichment of +0.5‰ to 1‰ is considered (deNiro and epstein 1981, fry and sherr 1984, Wada et al. 1991, michener and schell 1994). results from the present study indicate that sponges (mean δ13C = −17.19‰ ± 1.76) may be feeding on a mixture of seagrass- and macroalgae-derived carbon and pom, as was also reported by Weisz et al. (2007) for sponges in the fK. in fact, the δ13C and δ15N of the sponges directly overlap the values reported for pom from the florida Keys (lamb and swart 2008).

fishes The δ13C of muscle tissue collected from all herbivorous fishes during the pres-

ent study (−13.4‰ ± 1.5 to −15.9‰ ± 0.8) are well within the reported range of δ13C for fishes unaffected by anthropogenically-derived carbon (fry et al. 1982). The δ13C compositions reported in our study are representative of the dietary carbon source for each genus. specifically, ocean surgeonfish, chubs, and the damselfish follow the reported approximately +1‰ enrichment in C over the turf and macroalgae that they consume, and the fishes have δ13C values that are, respectively, +0.8‰, +0.6‰, and +1.6‰ enriched over their selected food sources. all parrotfishes, however, had δ13C values that were significantly more enriched (> +2‰) than their algal food sources. This apparent discrepancy can be explained when one considers the mode of feed-ing for parrotfishes, in comparison to surgeonfishes, damselfishes, and chubs. as mentioned previously, parrotfishes, with their fused-teeth beaks, actually bite into and remove a portion of the underlying calcium carbonate substrate when feeding on algae. Consequently, a percentage of each bite ingested consists of the limestone ma-trix, which is approximated to have an isotopic composition of approximately −3‰ in the fK (martin et al. 1986). since parrotfishes are able to dissolve calcium car-bonate in their gut (smith and paulson 1974, 1975), some assimilation of the carbon isotopic signature of the substrate into their tissues occurs.

The sampled muscle tissues from the parrotfishes, chubs, and ocean surgeonfish indicate that the primary control on the δ15N composition is diet (range from +4.9‰ ± 1.0 to +6.2‰ ± 1.2), and follow the reported approximately +3‰ enrichment in nitrogen from their food source. if sewage-derived nitrogen were a primary nutritive source in the marine environment, then appreciably enriched δ15N compositions (>


+10‰; schlacher et al. 2005, hadwen and arthington 2007) would be discernable in both the food source and the muscle tissues of the herbivorous fishes that consumed the algae (burnett and schaeffer 1980, gaston et al. 2004). instead, the muscle δ15N compositions we found are significantly depleted when compared to the reported δ15N values for marine herbivorous fishes exposed to anthropogenic wastes. instead, the δ15N signatures of the muscle tissues are indicative of the feeding preferences of the ocean surgeonfish, chubs, and parrotfishes. The components of turf and mac-roalgae can be clearly extrapolated from the δ15N values of the tissues, and show the appropriate trophic level enrichment in N compared to their food source.

based on the δ15N data, the damselfish from our study are almost a full trophic level above similar reef herbivores (damselfish = +8.5‰ ± 0.19 vs mean parrotfish = +5.3‰ ± 0.4). although many damselfishes are primarily herbivorous (gobler et al. 2006), some are planktivorous and some are known to be opportunistic feed-ers, consuming polycheates, copepods (emery 1973), and detritus. The specific diet composition of this species at our study sites is unknown. however, a study of a different species of damselfish from taiwan found that up to 75% of the gut con-tents consisted of coral polyps, the remainder consisting of filamentous algae (ho et al. 2009). although S. dorsopunicans may not consume coral in the fK, the δ15N of M. faveolata in the area is between +4.7‰ and +6.6‰ (swart et al. 2005). The δ15N values of the S. dorsopunicans are therefore consistent with a mixed food source in which both algae and corals, or other isotopically similar substances, form a portion of the diet. a similar δ15N enrichment in damselfish (δ15N = +8.6‰) over parrotfishes (δ15N = +6.3‰) has also been reported from the bahamas (Kieckbusch et al. 2004). damselfish δ13C is consistent with a mixed source of corals (−13.3‰) and turf algae (−16.6‰).

Spatial Variations.—Comparisons across various sites for S. viride and A. bahianus indicate that there is no trend in either δ13C or δ15N along the reef tract. however, values of δ13C were significantly more depleted in A. bahianus from little grecian and in S. viride from little grecian and algal reef. The precise explanation for this difference is unclear, but may relate to local variations in the δ13C of the diC.

reported C:N values of muscle tissues were lower at inshore reefs (3.55) when com-pared to offshore reef fish muscle tissues (4.09), implying a greater N availability at inshore reef sites relative to offshore reef sites. however, it is important to note that the C:N values are representative of nutrient presence and not nutrient origin. as such, when coupled with the δ15N values, the C:N values show that N is indeed present in greater amounts at inshore reef sites, but it is not being sourced from an-thropogenic wastes. instead, it is more likely that the source of N to these environs is natural, such as N fixation or breakdown of organic N.

implications for the sources of Nitrogen in the florida reef tractThere are a large number of internal and external sources which contribute N to

the fK. internal sources include contributions from the decay of organic material and fecal material from higher trophic organisms. These sources involve the recy-cling of existing N and do not provide new diN to the system. external sources of nitrogen in the fK can be considered to be derived from (1) land based sources of pollution including sewage and runoff, (2) upwelling, (3) atmospheric deposition, (4) input from florida bay and gulf of mexico, (5) nitrogen fixation, and (6) diffusion


from the sediments. The inputs are balanced by uptake of N by organisms and export to the open ocean and burial in the sediments.

land-based sources of pollution in the fK are mainly derived from sewage effluent leaking from septic systems, package plants, cesspits, and live-aboard boats. a small-er amount is contributed from stormwater runoff. a total of 487 mt yr−1 has been estimated to be delivered to the fK annually (Kruczynski and mcmanus 2002) from these sources (based on 1993 estimates from the environmental protection agency). Various workers have attempted to assess the influence of this contribution on the marine biota and nutrient concentrations (shinn et al. 1994, szmant and forrester 1996, lamb and swart 2008). While there was some evidence that geochemical and fecal tracers make their way rapidly from the septic tanks to the adjacent marine wa-ters (paul et al. 1995a,b, 1997), the nutrient flux from these sources appears to be rap-idly assimilated by nearshore organisms, leaving waters relatively low in the nutrient concentrations (szmant and forrester 1996, boyer and Jones 2002). our results are consistent with this conclusion in that there were no significant spatial patterns in the data, which would suggest enrichment in the δ15N of nearshore communities via anthropogenic input.

Upwelling has been reported to deliver nutrients to various sections of the fK reef tract, with water masses containing higher than ambient concentrations of nutrients (lee et al. 1994, leichter and miller 1999, leichter et al. 1996, 2003, 2007). in fact, it was estimated that more N was supplied to the entire reef tract during one upwelling event than from the entire output of the sewage treatment plant in Key West since it started operation (szmant and forrester 1996). based on frequency of upwelling an annual flux of between 1740 and 6260 mt yr−1 was estimated in 2000 (leichter et al. 2003). The δ15N values of upwelled nitrate have been measured in the fK to be approximately +4.8‰ (leichter et al. 2007), similar to the values we measured in the open marine waters. Nitrate supplied by upwelling is rapidly assimilated, leav-ing little evidence that the event occurred (szmant and forrester 1996). The δ18o of upwelled nitrogen has been reported to have values close to zero, hence a significant contribution of nitrate from an upwelling source would result in a reduction of the slope of δ18o and δ15N to below unity (Wankel et al. 2009).

another potential source of N is atmospheric deposition (wet and dry), processes that are active year round and are reported to produce No3

− with δ15N values of around zero (delwiche and steyn 1970, fogel and Cifuentes 1993). Using deposition values measured in miami (savoie 1987) and integrated over the area of the fKNms, it is estimated that ~376 mt yr−1 of N are contributed, a value comparable to the es-timated anthropogenic contribution. The δ15N of this source has not been measured in the fK, but based on other work in south florida, is likely to be highly variable (approximately −12‰ to +4‰, dillon and Chanton 2005).

input from the gulf of mexico and florida bay appears to be equivalent to that derived from upwelling. Values of 5500 mt yr−1 were estimated as being contribut-ed from the flow through the middle fK and about 500 mt yr−1 from the upper fK (Kruczynski and mcmanus 2002, gibson et al. 2008). The δ15N of this source is not constrained at the present time.

fixation of N2 is performed year round by microbial communities, present not only in the upper portion of the carbonate sediments, but also found growing as epiphytes on seagrasses and other aquatic vegetation (diaz and Ward 1997). moreover, it has been shown that microbial communities associated sponges are capable of fixing N2


and converting it into No3− at rates two to four orders of magnitude greater than pre-

viously reported for microbial communities found in unconsolidated tropical car-bonate sediments (diaz and Ward 1997). although there are few data on N fixation from the fKNms, the magnitude of the potential effect can be estimated by extrapo-lation of rates measured on the gbr (larkum et al. 1988) to the fKNms. This pro-vides an estimate of ~2500 mt yr−1. The δ15N of this source is likely to be close to 0‰.

These crude estimates of the mass balance of N being contributed to the fKNms confirm what is suggested by the N isotopic data presented here, namely that an-thropogenic derived N (3.5%) is a relatively small percentage of the total N budget. Consequently, its δ15N signal can be detected and its influence seen in only canal and very near-shore environments.

explanation for the patterns in the δ15N dataWhen our data are combined with the flux estimates and data from two previous

studies, in which the δ15N of pom (lamb and swart 2008) and coral tissues (swart et al. 2005) were measured, a clearer picture of the factors controlling the δ15N of the florida reef tract emerges (fig. 7). The limits of the δ13C values can be defined by mangroves with a δ13C of −30‰ at one extreme, and seagrasses with values of approximately −8‰ to −10‰ at the other. The δ13C of the pom falls more or less be-tween these two end members with values becoming more positive closer to the fK. This trend in δ13C was interpreted as being a result of a greater contribution to the pom of detrital material derived from seagrasses closer to the fK (lamb and swart 2008). The range of the δ15N is ~0‰ to +10‰ (with some data points more nega-tive than 0‰). The more negative values probably primarily reflect N derived from organisms, which fix atmospheric N2, or use N derived from atmospheric sources. in contrast, the most positive δ15N values correspond to the values measured in fish tis-sue. While more positive values could arise from sewage sources, there are abundant other processes which can also produce more positive δ15N values. These processes include fractionation during assimilation and fecal contributions from naturally oc-curring higher trophic feeders. all other samples fall within the boundaries of these end members and therefore there is no need to invoke more exotic sources of N to ex-plain the distributions observed in the different organisms investigated in the pres-ent study. subtle variation within the system occurs as diN species are fractionated by normal processes during the transformation from one species to another or dur-ing assimilation. in fact, the strong positive correlation between the δ18o and δ15N of the nitrate suggests that the patterns of these two isotopes are driven by fraction-ation during the assimilation of nitrate and therefore emphasizes the importance of this mechanism for producing isotopic variation in the system.

acknowledgments

We acknowledge support from the National Center for Coral reef research (NCore) and funded through the epa. additional support was provided by the stable isotope laboratories at the University of miami and University of massachusetts dartmouth. The following per-sons presently or previously at the University of miami are thanked for assistance: r Cowen, s sponaugle, m paddack, K grorud-Colvert, d pinkard, e d’alessandro, K denit, K huebert, J fell, a tallman, p ortner, t lee, l Williams, g mackenzie, a saied, C schroeder, g ellis, and C moses. Q devlin and a ohelert are thanked for help with the post facto experiments to prove that there were no artifacts in the preparation procedures. We also thank tX Wu and l Zhang from the University of massachusetts dartmouth. This paper was improved by discussions with a Knapp and the comments of three reviewers.


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date submitted: 28 december, 2010.date accepted: 19 august, 2011.available online: 6 september, 2011.

addresses: (lK, pKs) Marine Geology and Geophysics, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, Florida 33149. present address:(lK) ExxonMobil Exploration, 222 Benmar Drive, Houston, Texas 77060. (maa) Department of Estuarine and Ocean Sciences, School for Marine Science and Technology, University of Massachusetts, Dartmouth, Massachusetts 02744. Corresponding author: (pKs) E-mail: <[email protected]>.

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