Iron additions revert seagrass (Posidonia oceanica) decline in carbonate sediments

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1

2

Iron Additions Reduce Sulfide

3

Intrusion and Reverse Seagrass

4

(Posidonia oceanica) Decline in

5

Carbonate Sediments

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7

Nu´ria Marba`,

* Maria Ll. Calleja,

Carlos M. Duarte,

Elvira a´lvarez,

Elena

8

Dı´az-Almela,

and Marianne Holmer

9 ¹Grup d‘Oceanografia Interdisciplinar (GOI), Institut Mediterrani d‘Estudis Avanc¸ats (CSIC-UIB), Miquel Marques 21, 07190, Esporles

10 (Illes Balears), Spain;²Direccio´ General de Pesca, Conselleria d‘Agricultura i Pesca, Govern de les Illes Balears, Foners 10, 07006,

11 Palma de Mallorca (Illes Balears), Spain;³Institute of Biology, SDU-Odense University, Campusvej 55, 5230, Odense M, Denmark

1213

14

A

15 We conducted a 2-year in situ experiment to test 16 the capacity of iron additions to reverse the decline 17 experienced by a Posidonia oceanica meadow colo- 18 nizing carbonate, iron poor sediment. Iron addi- 19 tions improved the sediment conditions that 20 support seagrass growth by decreasing the sedi- 21 ment sulfide concentration and sulfate reduction 22 rates, and decreased sulfide intrusion into the 23 plants. Iron additions for 2 years did not signifi- 24 cantly change survivorship of shoots present at the 25 onset of the experiment, but significantly increased 26 shoot recruitment and survivorship of shoots 27 recruited during the experiment. After 2 years, iron

28 additions reversed seagrass decline and yielded

29 positive growth rates of shoots relative to control

30 populations where seagrass continued to decline.

31 This research demonstrates that seagrass decline in

32 carbonate sediments may be reversed by targeting

33 critical processes such are sediment sulfide pools

34 and seagrass nutritional status, controlling the

35 functioning of the ecosystem.

36 Key words: carbonate; sulfur; iron additions;

37 sediment;Posidonia oceanica; decline; demography ;

38 clonal growth.

39

4041

I

42 Seagrass meadows rank amongst the most valuable 43 ecosystems on Earth for both functions and services 44 (Duarte 2002), but are also amongst the most 45 threatened, with global decline estimated at 46 approximately 1.8% y⁾¹ (Green and Short 2003;

47 Duarte and others 2007). Increased organic and 48 nutrient input is recognized generally as the major

49 cause of worldwide seagrass decline (Duarte 2002;

50 Green and Short 2003; Duarte and others 2005).

51 Excess organic inputs deteriorate sediment condi-

52 tions that support seagrass growth by stimulating

53 sulfate reduction and production of sulfide that is toxic to seagrasses (Terrados and others1999; Hol- 54 mer and others 2003). The effects of sulfides are 55 buffered in iron-rich sediments by the precipitation 56 of pyrite as sulfides combine with iron (Berner 57

58 1984). Seagrasses growing in carbonate sediments

59 are particularly vulnerable to increased organic in-

60 puts because the sediments are iron-poor (Duarte

61 and others1995) and lack sulfide buffering capacity.

Received 18 April 2006; accepted 15 April 2007

*Corresponding author; e-mail:[email protected]

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62 Further, Mediterranean seagrass (Posidonia ocea- 63 nica) meadows growing on carbonate sediments 64 have been reported to continue to decline even 65 after suppression of organic inputs (compare Del- 66 gado and others 1999). No intervention has yet 67 been able to stop or reverse P. oceanica decline 68 once detected. P. oceanica meadows, which repre- 69 sent the dominant and most productive coastal 70 ecosystem in the Mediterranean, are experiencing 71 widespread decline throughout the region, with 72 current decline rates resulting, on average, in a 73 reduction of seagrass density to half in 6.8 years 74 (Marba` and others2005). Losses of Mediterranean 75 P. oceanicameadows are particularly concerning, as 76 the slow clonal growth (1–7 cm y<sup>)1</sup>, Marba` and 77 Duarte 1998) and sparse reproduction (Pergent 78 and others 1989) of this species results in 79 extraordinarily long recolonization rates (centuries 80 to millenium, Duarte 1995; Marba` and others 81 2002).

82 Short-term (1–8 months) iron addition experi- 83 ments to seagrass sediments have shown a stimu- 84 lation of seagrass leaf growth (Duarte and others 85 1995; Chambers and others 2001; Holmer and 86 others 2005), as well as a suppression of sulfate 87 reduction activity inP. oceanicasediments receiving 88 excess organic inputs (Holmer and others 2005).

89 These short-term experiments in impacted car- 90 bonate sediments, however, have not tested the 91 ability of iron inputs to discontinue or reverse 92 seagrass decline. Here we present the results of a 2- 93 year iron addition experiment testing whether iron 94 additions can increase the resistance of P. oceanica 95 meadows to organic inputs by buffering sulfide 96 production and stimulating clonal growth, thereby 97 reversing seagrass decline. The examination of the 98 demographic response to experimental manipula- 99 tions in P. oceanica is particularly challenging, be- 100 cause of the slow recruitment rates and shoot 101 turnover time (less than 10% y⁾¹ and up to few 102 decades, respectively, Marba` and others 1996, 103 2005) characteristic of this species, the slowest- 104 growing seagrass in the world (Marba` and Duarte 105 1998). In addition, shoot density in P. oceanica 106 meadows is heterogeneous as reflected by, on 107 average, a coefficient of variation of 15% (for 108 example, Marba` and others 2005). The slow 109 growth, and to some extent the spatial heteroge- 110 neity in P. oceanica structure, rules out spectacular 111 demographic responses to any experimental treat- 112 ment even if imposed over relatively long (2 years) 113 experimental periods, as substantial responses can 114 only be expressed over time scales of decades to 115 centuries.

M

The experiment was conducted on an impactedP. 117 oceanicameadow growing at 17 m depth in Es Port 118

119 de Cabrera, Cabrera Island, the largest of 19 islands

120 and islets forming the Cabrera Archipelago Na- tional Park (398.81¢N 255.86¢E, Balearic Islands, 121

122 Spanish Mediterranean). Es Port de Cabrera is a

123 sheltered bay traditionally used as a natural harbor.

124 Since the Archipelago was declared a national park

125 in 1991 it hosts the park‘s visitor center, facilities,

126 and moorings for 50 pleasure boats, and, thus,

127 supports substantial human pressure. The meadow

128 at Es Port de Cabrera has been in decline for the last

129 decade at an average rate exceeding 4% y⁾¹(Marba`

130 and others2002). The decline of the meadow at Es Port de Cabrera is attributed to enhanced sulfate 131 reduction rates (12.5 mmol sulfate m⁾²d⁾¹, Hol- 132 mer and others2003) and sulfide accumulation in 133

134 the sediments. Stable carbon-isotope ratios of bac-

135 terial biomarkers identified sedimentary inputs

136 (279 mg C m⁾²d⁾¹) as an important source of or-

137 ganic carbon support to bacterial activity at this site

138 (Holmer and others2004).

139 In July 2002 eight experimental 1.5 m· 1.5 m

140 permanent plots were installed in the meadow. The

141 plots were distributed along two rows separated by

142 a 4 m corridor, with neighboring plots within the

143 row separated by 2 m. One permanent

144 0.5 m·0.5 m quadrat, for seagrass shoot census, was delimited at the center of each plot, where 145 sampling of plants and sediments was prevented for 146 the entire duration of the experiment. The top 147

148 30 cm sediment layer of the 4 plots along 1 row

149 were enriched with iron pulses of 0.8 mol ir-

150 on m⁾², as Fe-chelate (Fe-EDDHA) dissolved in

151 seawater, comparable to the inputs in previous iron

152 addition experiments to seagrass sediments (Hol-

153 mer and others 2005), in July 2002, November

154 2002, July 2003, and March 2004. Iron pulses were

155 applied through 49 injections of 60 ml Fe-chelate

156 dissolved in seawater per plot, where 5 ml of

157 solution per injection were added at the top 5, 10,

158 15, 20, 25, and 30 cm of sediment. The other four

159 plots were kept as controls. The plots were visited every fourth month over 2 years. 160

At each visit, SCUBA divers collected two sedi- 161 ment cores per experimental plot, one of internal 162

163 diameter (i.d.) 2.6 cm and one of i.d. 4.3 cm. The

164 depth of all sediment cores was 10 cm, and cutting

165 of roots and rhizomes was avoided during the col-

166 lections. The sediment collected in the 2.6 cm

167 diameter cores was used to measure the sediment

168 sulfate reduction rate (SRR), acid volatile sulfides

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169 (AVS) and chromium reducible sulfur (CRS). The 170 sediment collected in the 4.3 cm diameter cores 171 was used for measuring pore-water concentrations 172 of sulfate, sulfides and total dissolved iron 173 (Fe²⁺+ Fe³⁺) and the solid phase characteristics 174 (sediment density, water content, porosity and or- 175 ganic matter content). During visits when iron 176 pulses were supplied to the Fe-enriched plots, all 177 sediment cores were collected prior to iron addi- 178 tions. In addition, at the beginning of the experi- 179 ment one sediment core per plot was collected 180 immediately after iron injections to assess the in- 181 crease in iron concentration resulting from the 182 injections.

183 Sulfate reduction rate were quantified by the 184 core-injection technique (Jørgensen 1978). Two 185 microliter of ³⁵S-sulfate (70 kBq) were injected 186 with 1-cm intervals through predrilled silicone fil- 187 led holes and the cores were incubated at in situ 188 temperature in darkness for 1–3 h. After the incu- 189 bation, the sediment was fixed in 1 M zinc acetate 190 (vol:vol). The samples were stored frozen until 191 distillation according to the two-step extraction 192 scheme; in the first step AVS was liberated by the 193 addition of 6 M HCl (in 50% ethanol) and in the 194 second step CRS was extracted by adding 1 M CrCl2

195 (in 0.5 HCl), both were trapped in zinc acetate, 196 following Fossing and Jørgensen (1989). Radioac- 197 tivity was counted on a Beckman LS-3801 scintil- 198 lation counter. Sulfate reduction rates (SRR, in 199 nmol SO42) m⁾³d⁾¹) were calculated for each 200 sediment core following Fossing and Jørgensen 201 (1989) as:

SRR¼ a

aþA

ð ÞtSO²₄ 1:06 203

203 wherea is the total radioactivity in the traps, Ais 204 the total radioactivity of the sulfate pool after 205 incubation, t is the incubation time (in days), 206 [SO42)] is the sulfate concentration in the sediment 207 (nmol cm⁾³) and 1.06 is the correction factor for 208 microbial isotope fractionation between ³²S and 209 ³⁵S. The concentrations of reduced sulfide pools 210 from the traps were determined spectrophotomet- 211 rically according to Cline (1969).

212 Porewater samples were obtained from sediment 213 cores sliced under N2 atmosphere to keep them 214 anoxic. The sediment was centrifuged and super- 215 natant was sampled for analysis of sulfate (SO4

2)), 216 sulfides (H2S), and porewater total dissolved iron 217 (Fe²⁺+ Fe³⁺). Sulfate was determined using the 218 turbidimetric assay described by Tabatabai (1974).

219 Sulfides were kept in zinc acetate and determined 220 spectrophotometrically according to Cline (1969) 221 and total dissolved iron was kept on HCl (pH 1) and

222 analyzed as Fe²⁺ after addition of hydroxylamine

223 for reduction of Fe³⁺ as described by Stookey

224 (1970). Sediment density was obtained by weight

225 of a known volume, and the water content was obtained after drying it overnight at 105C. Poros- 226

227 ity was calculated from sediment density and water

228 content. Organic matter content was obtained by ignition of the dried sediment overnight at 450C. 229

At the end of the experimental period one sedi- 230 ment core (i.d. 2.6 cm) from each plot was col- 231 lected to determine thed³⁴Ssulfidevalues in the AVS 232 and CRS pools. The sediment (0–10 cm) was dis- 233

234 tilled as described above according to Fossing and

235 Jørgensen (1989), but the trap content was ex-

236 changed with AgNO3 solution. The sulfides pre-

237 cipitated in the traps and Ag2S was collected on a

238 GF/F filter. Thed³⁴Ssulfidevalue was determined as

239 described below for the plants.

240 Seagrass shoot demographic parameters were

241 quantified by direct shoot census in the

242 0.5 m· 0.5 m quadrats installed inside the exper-

243 imental plots following the procedures described in

244 Short and Duarte (2001). At the beginning of the experiment, all shoots within the quadrats were 245 tagged, with a plastic cable tie, and counted. Every 246 eighth and every fourth month during the first and 247 second year, respectively, the number of surviving 248 shoots (that is, shoots tagged with a cable tie) and 249

250 the number of recruited shoots between consecu-

251 tive visits (that is, young untagged shoots) in each

252 permanent quadrat were counted. The number of

253 rhizome apexes in the quadrats was also recorded,

254 and the recruited shoots found were tagged with a

255 cable tie of a different color, allowing monitoring of

256 survival of the different shoot cohorts. Identifica-

257 tion of rhizome apexes in the permanent plots re-

258 quired minor sediment disturbance during visits.

259 Rhizome apexes ofP. oceanicawere easy to identify visually, as they had shorter and more curved leaf 260 shoots than those on vertical rhizomes. Occasion- 261 ally, rhizome apexes were identified by carefully 262 touching them by hand within the top 0–2 cm 263

264 sediment layer. These measurements provided

265 estimates of shoot and apex density, survival tra-

266 jectories for shoots older than 2 years and shoot

267 cohorts recruited during the experiment, curves of

268 cumulative recruitment during the experiment,

269 and the absolute and specific rates of shoot mor-

270 tality, recruitment and population growth in be-

271 tween consecutive visits. Absolute and relative

272 shoot mortality, recruitment and net population

273 growth rates were estimated as described in Marba`

and others (2005). 274

Leaf and horizontal rhizome elongation rates (in 275 cm shoot⁾¹y⁾¹ and cm rhizome apex⁾¹y⁾¹, 276

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277 respectively) were measured using marking tech- 278 niques, as described in Short and Duarte (2001), 279 whereas estimates of vertical rhizome growth were 280 quantified retrospectively (Duarte and others1994) 281 on three shoots of each plot harvested at the end of 282 the experiment. Leaf growth was estimated in be- 283 tween consecutive visits on eight shoots per 284 experimental plot. The horizontal rhizome elon- 285 gation rate was only estimated during the second 286 year. In July 2003, 14 rhizome apexes distributed 287 amongst the 0.5 m ·0.5 m quadrats were tagged 288 with a cable tie, and were harvested at the end of 289 the experiment. Leaf annual production (in 290 g DW m⁾²y⁾¹) was estimated as annual leaf elon- 291 gation rate multiplied by the specific leaf weight 292 (g DW cm leaf⁾¹) and shoot density. Similarly, 293 vertical (and horizontal) rhizome annual produc- 294 tion (in g DW m⁾²y⁾¹) was calculated as the 295 product of annual vertical (and horizontal) rhi- 296 zome elongation rate, specific vertical (and hori- 297 zontal) rhizome weight (g DW cm rhizome⁾¹) and 298 shoot (and apex) density.

299 Iron concentration, d³⁴S abundance and the 300 fraction of total sulfur in plant tissues (that is, 301 leaves, rhizomes, roots) derived from sedimentary 302 sulfides were measured onP. oceanica samples col- 303 lected from each experimental plot at the end of 304 the experiment. Sulfur isotope analyses were made 305 by the National Isotope Geosciences Facility (Not- 306 tingham, UK) using an automated, on-line facility 307 coupled to a Thermo Finnigan Delta XL. The sulfur 308 isotope composition of a sample is expressed in the 309 standardd notation given by:

34S¼ R_sample=R_standard 1

1000 311

311 where R=³⁴S/³²S. Values are expressed on a per 312 mil (&) basis and were calibrated to CDT (troilite 313 standard from the Canyon Diablo meteorite) using 314 IAEA standards S1 and S2. Replicate analyses of 315 internal standards (barium sulfate, silver sulfide 316 and an internal laboratory organic standard, broc- 317 coli) showed that reproducibility was ±0.4& or 318 better. To determine the relative contribution of 319 sediment sulfide to the sulfur composition in the 320 leaves, rhizomes and roots, the fraction of the total 321 sulfur pool derived from sedimentary sulfides 322 (Fsulfide) was estimated:

Fsulfide¼ d³⁴Stissued³⁴Ssulfate

d³⁴S_sulfated³⁴S_sulfate 324

324 where d³⁴Stissue is the value measured in the leaf, 325 rhizome or root,d³⁴Ssulfatewas the values measured 326 in the seawater (average +20.99&) and d³⁴Ssulfide

327 was the values measured in the sedimentary AVS

328 pools (average)17.15&).

Iron concentration in plant tissues was obtained 329 after acid hydrolysis (1 M HCl) for 1 h at 105C and 330 analyzed as described above for Fe²⁺. 331

332 Sediment and plant responses to iron additions,

333 per sampling visit and per grand mean (that is,

334 average across the entire experiment duration),

335 were examined using Student‘s t test. Similarly,

336 Student‘s t test was used to assess the changes in

337 sediment parameters between the beginning and

338 end of the experiment. The persistence of consis-

339 tent responses of sediment parameters to iron

340 additions during the experiment was identified

341 using Wilcoxon‘s signed-ranks test. The temporal

342 trend of plant responses to iron additions was

343 evaluated using regression analysis on log trans- formed variables, and differences between treat- 344 ments by comparing the slopes using Student‘s 345 t-test. Standard errors of mean values are always 346

347 provided.

R

No significant differences (t-test, P> 0.05) among 349 350 bulk sediment parameters (that is, sediment den-

351 sity, porosity and organic carbon content) between

352 control versus iron-enriched plots either initially or

353 after 1 year of measurement were observed (Ta-

354 ble1).

355 The sediments investigated were iron poor, with

356 porewater total dissolved iron in control plots

357 averaging 0.72 ± 0.16 mmol Fe m⁾² (range 0.29–

358 1.41 mmol Fe m⁾², Table1) during the experi-

359 ment. Iron additions raised the amount of pore-

360 water total dissolved iron three-orders of

361 magnitude following injections (808 ± 526 mmol Fe m⁾², Table1), but these declined rapidly, likely 362 through diffusive loss and benthic irrigation, to 363 average 1.75 ± 0.59 mmol Fe m⁾² 8 months fol- 364

365 lowing injections (Table1). Despite the losses, iron

366 injections maintained elevated porewater total

367 dissolved iron levels twofold above that in control

368 plots, at least 8 months following injections. The

369 amount of porewater total dissolved iron in iron-

370 enriched plots was maintained significantly (Wil-

371 coxon‘s test,P< 0.05) higher than that in control

372 plots for the entire duration of the experiment. The

373 porewater sulfide concentration in control plots

374 increased greatly, but not significantly (t-test,

375 P> 0.05), during the study, from low initial con-

376 centrations of 0.42 ± 0.12 mmol H2S m⁾² at the onset of the experiment to reach concentrations of 377 5.82 ± 3.66 mmol H2S m⁾² by the end of the 378 experiment (Table1). The iron-enriched sediments 379

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Table1.Average(andstandarderror,n=4)SedimentDensity,Porosity,OrganicCarbonContent,PorewaterTotalDissolvedFe,H2SPool, SulfateReductionRate(SRR),andAcidVolatileSulfides(AVS),ChromiumReducibleSulfur(CRS)andTotalReducibleSulfides(TRS)Pools,within theTop10cmSedimentLayer,inFe-EnrichedandControlPlotsduringExperimentVisits ParameterTreatmentJuly2002November2002March2003July2003November2003April2004July2004Average±SE Sedimentdensity (gcm)3 )Fe-enriched1.38±0.01 Control1.37±0.01 Porosity(gH2Ocm)3 )Fe-enriched1.38±0.011.39±0.041.37±0.01 Control0.72±0.030.73±0.020.72±0.031.39±0.021.35±0.021.38±0.03 Organiccarbon content(gm)2 )Fe-enriched4,657±1754,469±2024,486±1230.72±0.020.70±0.020.67±0.03 Control4,801±824,683±1534,535±1494,672±94 TotaldissolvedFe (mmolm)2)Fe-enriched808±526* 3.13±0.962.19±1.191.18±0.282.30±1.131.49±0.590.80±0.461.85±0.38 Control0.49±0.190.29±0.051.41±0.130.74±0.031.02±0.310.71±0.390.36±0.050.72±0.16 H2S(mmolm)2 )Fe-enriched0.33±0.181.28±1.090.22±0.012.68±1.011.43±0.380.28±0.031.21±0.451.06±0.36 Control0.42±0.120.35±0.130.27±0.071.16±0.681.82±1.420.56±0.275.82±3.661.49±0.81 SRR(mmolSm)2 d)1 )Fe-enriched13.47±2.608.91±2.853.94±1.1418.38±13.794.99±2.058.63±0.456.27±1.139.22±2.09 Control19.20±5.506.45±0.6812.00±6.934.12±0.284.30±1.898.12±6.6011.96±5.399.45±2.20 AVS(molSm)2 )Fe-enriched0.37±0.080.43±0.020.45±0.150.37±0.020.37±0.020.340.32±0.030.38±0.02 Control0.51±0.110.39±0.230.41±0.110.38±0.010.38±0.020.30±0.020.32±0.010.39±0.03 CRS(molSm)2 )Fe-enriched0.25±0.010.22±0.030.20±0.010.26±0.030.27±0.020.290.32±0.030.26±0.02 Control0.25±0.020.18±0.020.17±0.030.31±0.010.23±0.080.26±0.010.26±0.010.24±0.02 TRS(molSm)2 )Fe-enriched0.62±0.090.65±0.010.65±0.150.64±0.040.64±0.040.620.64±0.050.64±0.01 Control0.76±0.110.58±0.250.55±0.140.69±0.020.61±0.070.56±0.010.58±0.010.62±0.03 TotaldissolvedFeinFe-enrichedplotsinJuly2002(astericks)wasmeasuredafterironinjections.Average(andstandarderror;n=7,exceptforsedimentdensity,porosity,organiccarboncontentn=3,andporewatertotaldissolvedFe n=6)estimatesacrosstheentriredurationoftheexperimentinFe-enrichedandcontrolplotsarealsoprovided.Standarderrorofaveragevaluesarenotprovidedwhenn=1.

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380 did not show such an increase of porewater sulfide 381 concentration during the experiment (Table 1).

382 Despite the wide temporal fluctuations in pore- 383 water sulfide concentration in control and iron- 384 enriched sediments (Table 1), the average pore- 385 water sulfide concentration during the entire study 386 was 40% higher in control plots (1.49 ± 0.81 mmol 387 H2S m⁾², Table 1) than in iron-enriched ones 388 (1.06 ± 0.46 mmol H2S m⁾², Table1).

389 Sediment sulfate reduction rates fluctuated 390 widely over time in iron-enriched and control 391 plots, the highest rates being observed during 392 spring–summer (Table 1). However, similar (t-test,

393 P> 0.05) sediment sulfate reduction rates were

394 observed at the onset and end of the experiment in

395 control plots (Table1). Conversely, 2 years of iron

396 additions significantly (t-test, P < 0.05) decreased

397 sediment sulfate reduction rates by twofold (Ta-

398 ble1). Total pools of reduced sulfides (TRS) were

399 similar in sediments of fertilized and control plots,

400 averaging 0.63 ± 0.004 mol S m⁾²in iron-enriched plots and 0.62 ± 0.03 mol S m⁾² in control plots 401 (Table1). As a result, the turnover rate of total 402 reduced sulfides in the iron-enriched plots was half 403 of that in the control plots at the end of the 404

405 experiment, indicating lower oxygen consumption

406 for re-oxidation of sulfides. In addition, the total

407 sulfur pools shifted over the last year of the

408 experiment, in response to iron additions, towards

409 a slightly greater contribution of CRS (pyrite, 47%

410 in the iron-enriched plots, compared to an average

411 of 41% in the control plots by the end of the

412 experiment, Table1).

413 The iron concentration in tissues of control

414 plants was very low, with leaves having the lowest

415 iron concentrations (Figure1). Iron concentration in seagrass leaves tended to increase, although not 416 significantly (t-test, P > 0.05), in response to Fe 417 additions, with the average Fe concentration 418 increasing from 75.1 ± 11.5lg Fe (g DW)⁾¹ in 419 control plants to an average of 113.4 ± 28.3lg Fe 420

421 (g DW)⁾¹ in iron-enriched plots during the exper-

422 iment (Figure1). Iron concentrations were similar

423 (t-test, P> 0.05) in roots and rhizomes at Fe-en-

424 riched and control plots (Figure1). The ³⁴S abun-

425 dance varied across P. oceanica tissues of plants in

426 control plots (Table2). At the end of the experi-

427 ment, the ³⁴S abundances in leaves and roots of

428 iron-enriched plots were significantly (t-test,

429 P< 0.05) higher than those in similar tissues of

430 plants growing in control plots (Table2). Exami- nation, through the³⁴S abundance, of the fraction 431 of sedimentary sulfide in the S pool of the seag- 432 rasses showed a major (fivefold) and significant (t- 433 test, P< 0.05) reduction in the contribution of 434

435 sulfide to the S pool of leaves in iron-enriched plots

436 (Figure1). The contribution of sulfide to S pool of

437 roots was also significantly (t-test, P< 0.05) lower

438 in plants in iron-enriched than in control plots

439 (Figure1).

440 The shoot density declined during the experi-

441 ment, with an average net decline of 11.2% (Fig-

442 ure2), resulting in an average (±SE) specific

443 population growth rate of )5.6 ± 3.8% y⁾¹ (Ta-

444 ble3). Most of the decline occurred over the first 8 months of the experiment (Figure2). Shoot 445 censuses revealed a significant recruitment of new 446 shoots during the study period, but insufficient to 447 Figure 1. The average (±SE) iron concentration and the

fraction of total sulfur (Fsulfide) in leaves, rhizomes and roots derived from sedimentary sulfides (AVS pool) in control (empty bars) and Fe-enriched (grey bars) experi- mental plots. The average iron concentrations in leaves was computed as the average of seven sampling events during the experiment (n= 28), whereas the rest of the parameters were estimated at the end of the experiment.

Statistically significant differences (t-test, P< 0.05) be- tween treatments are indicated (astericks).

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448 compensate for shoot mortality (Figure2). Iron

449 additions did not result in a significant (t-test, P > 0.05) reduction in shoot mortality, but they 450 increased significantly (t-test, P< 0.05) by 2.5 fold 451 shoot recruitment during the experimental period 452

453 (Figure2; Table3). The average specific recruit-

454 ment rate in iron-enriched plots increased signifi-

455 cantly (regression analysis, P< 0.01, n= 4) over

456 time, whereas shoots recruited at similar (regres-

457 sion analysis, P> 0.5,n = 4) average rates in con-

458 trol plots during the experiment. Iron additions did

459 not change survival of shoots present in the mea-

460 dow at the onset of the experiment; depletion

461 curves were similar (t-test on the slopes, P> 0.05)

462 in iron-enriched and control plots (Figure 2).

463 Conversely, iron additions significantly increased

464 survival of shoots recruited during the experiment (Figure3). Although annual survival of recruits 465 was not significantly different from 100% (regres- 466 sion analysis, P> 0.05, n= 9) in iron-enriched 467

468 plots, annual survival of recruits significantly de-

469 clined to 68% (regression analysis,P< 0.01,n= 9)

470 in control plots (Figure3). As a result of these

471 combined responses, iron additions tended to re-

472 verse the decline of the meadow toward the end of

473 the experiment, with an increase in shoot density

474 by 7.6% (Figure 4). Responses of shoot population

475 growth rates to iron additions during the experi-

476 ment were not statistically (t-test, P > 0.05, Ta-

477 ble3) significant, due to the large error imposed by

478 the patchiness of the meadow. Examination of temporal trends revealed a significant (regression 479 analysis, P< 0.05, n= 4) increase in the average 480 shoot population growth rate in iron-enriched 481 plots, whereas no temporal changes were observed 482

483 (regression analysis,P> 0.5,n= 4) in control plots.

484 The increased shoot recruitment in iron-enriched

485 plots was sustained by stimulation, although not

486 significant (t-test, P > 0.05), of clonal growth. In

487 iron-enriched plots, the number of rhizome apices Table 2. Average Values ofd³⁴S inPosidonia oceanicaLeaves, Rhizomes and Roots fom Iron-Enriched and Control Plots at the End of the Experiment

Seagrass tissue Treatment d³⁴S (&) P

Shoot Iron enriched 20.45 ± 0.14 *

Control 18.15 ± 0.37

Rhizome Iron enriched 8.73 ± 1.42 n.s.

Control 7.94 ± 0.72

root Iron enriched 9.54 ± 0.98 *

Control 6.29 ± 0.23

Standard error of averaged³⁴S in shoots, rhizomes, and roots are provided (n = 4). The level of significance [t-test, P < 0.05(*); P > 0.05 (NS)] of tissued³⁴S signature response to iron additions is indicated.

Figure 2. Average (±SE; n= 4) shoot density, and tra- jectories (as % of the initial shoot density) of relative shoot survival and cumulative recruitment in control and Fe-enriched experimental plots during the experiment.

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Table3.Average(andStandardSrror,n=4)AbsoluteandApecificShootRecruitment,MortalityandPopulationGrowthRatesinFe-Enriched andControlPlotsDuringExperimentSamplings.Average(andstandarderror,n=20)EstimatesacrosstheEntrireDurationoftheExperimentin Fe-EnrichedandControlPlotsarealsoProvided,andtheProbabilityofSignificantDifferences(Student‘st-test)betweenTreatmentsisProvided Absolutedemographicrates(shootsm)2 d)1 )RecruitmentrateMortalityratepopulationgrowthrate DateFertilisedControlFertilisedControlFertilisedControl 11March20030.0341±0.02120.0169±0.00790.1066±0.06720.1609±0.0812-0.0725±0.0491-0.1439±0.0772 26November20030.0810±0.03190.0504±0.01990.0771±0.02180.0503±0.02660.0040±0.03660.0001±0.0319 13April20040.1572±0.10810.0708±0.03410.0858±0.04250.0638±0.04310.0715±0.07190.0070±0.0699 19July20040.3091±0.10720.0821±0.04330.0725±0.03020.0930±0.02310.2367±0.1136-0.0108±0.0627 Average±SE0.1454±0.06950.0551±0.01650.0855±0.00870.0920±0.02840.0599±0.0761-0.0369±0.0414 Relativedemographicrates(d)1 ) 11March20030.0001±0.00010.00005±0.000020.0003±0.00020.0006±0.0003-0.0002±0.0002-0.0005±0.0003 26November20030.0003±0.00010.0002±0.00010.0003±0.00010.0002±0.0001-0.00002±0.00010.00001±0.0001 13April20040.0005±0.00030.0002±0.00010.0003±0.00010.0002±0.00020.0002±0.0002-0.00003±0.0003 19July20040.0010±0.00040.0003±0.00010.0003±0.00010.0003±0.00010.0008±0.0004-0.0001±0.0002 Average±SE0.0005±0.00020.0002±0.00010.0003±0.000010.0003±0.00010.0002±0.0002-0.0002±0.001 ParameterTreatmentMarch2003November2003April2004July2004Average±SEP Absoluterecruitmentrate (shootsm-2 d-1 )Fe-enrichedcontrol0.03±0.020.08±0.030.16±0.110.31±0.110.15±0.07<0.05 0.02±0.010.05±0.020.07±0.030.08±0.040.06±0.02 Absolutemortalityrate (shootsm-2 d-1 )Fe-enrichedcontrol0.11±0.070.08±0.020.09±0.040.07±0.030.09±0.010.81 0.16±0.080.05±0.030.06±0.040.09±0.020.09±0.03 Absolutepopulation growthrate(shootsm-2 d-1 )Fe-enrichedcontrol-0.07±0.050.01±0.040.07±0.070.22±0.110.06±0.080.06 -0.14±0.080.00±0.030.01±0.07-0.01±0.06-0.04±0.04 Specificrecruitmentrate(%d-1 )Fe-enrichedcontrol0.01±0.010.03±0.010.05±0.030.10±0.040.05±0.02<0.05 0.01±0.000.02±0.010.02±0.010.03±0.010.02±0.01 Specificmortalityrate(%d-1 )Fe-enrichedcontrol0.03±0.020.03±0.010.03±0.010.03±0.010.03±0.000.79 0.06±0.030.02±0.010.02±0.020.03±0.010.03±0.01 Specificpopulation growthrate(%d-1 )Fe-enrichedcontrol-0.02±0.020.00±0.010.02±0.020.08±0.040.02±0.020.07 -0.05±0.030.00±0.010.00±0.03-0.01±0.02-0.02±0.01

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488 increased (Figure5), indicative of an increased 489 branching rate, and the rhizome elongation rate 490 tended to be twice that in control plots. The cal- 491 culated average net production rate increased in 492 iron-enriched plots relative to control plots for the

493 net production of horizontal rhizomes (Figure 6),

494 with the total (rhizome + leaf) net production in

495 iron-enriched plots increasing marginally (7.5% on

496 average) relative to that of control plots during the

497 experiment (Figure6).

D

499 The ecosystem studied was iron-poor, with iron

500 concentrations in seagrass leaves below the critical

501 values (100 lg Fe (g DW)⁾¹, Duarte and others

502 1995), the lowest yet reported forPosidonia oceanica,

503 and comparable to the lowest values, characteristic

504 of Fe-deficient plants, reported for seagrasses else-

505 where (Duarte and others 1995). This iron defi-

506 ciency renders this ecosystem highly vulnerable to

507 increased organic inputs from emissions of visitors

508 to the Bay, and have been identified as the cause for the severe decline of the meadow (Marba` and 509 others 2002; Holmer and others 2003). The accu- 510 mulation of toxic sulfides in the sediments, which 511 diffuse into plant tissues as reflected in the d³⁴S 512 isotope signals in plant tissues, compound with 513

514 iron-limitation of plant growth to yield the ob-

515 served seagrass decline (Holmer and others 2005).

516 Seagrass decline, in turn, might contribute to in-

517 crease sediment sulfide accumulation, because, as

518 the meadow thins, the amount of photosynthetic

519 oxygen released by roots (Borum and others 2006)

520 and, thus, the capacity of the system to reoxidize

521 sediment sulfide would decrease. Experimental

522 iron additions maintained elevated iron pools over

523 2 years, significantly decreased sediment sulfate reduction rates and tended to reduce sulfide pools, 524 Figure 3. Survival of different cohorts of shoots re-

cruited during the experiment in control (white symbols) and Fe-enriched (black symbols) experimental plots. Sur- vival was calculated as percentage of the total number of shoots per cohort recruited per treatment. The number of shoots recruited in control and enriched plots in cohort 1 (circles) was 4 and 8, respectivley; in cohort 2 (squares) 13 and 22 respectively; in cohort 3 (triangles) 10 and 22, respectively. The slopes ± SE of fitted depletion equa- tions in control (dashed line) and iron-enriched (solid line) plots were)0.08 ± 0.01 shoots d⁾¹(regression analysis, P< 0.0005, n= 9) and 0.003 ± 0.009 shoots d⁾¹ (regression analysis,P> 0.05,n= 9), respectively.

Figure 4. Average (±SE;n= 4) relative shoot population growth (as % of the initial shoot density) during the experiment in control and iron-enriched plots.

Figure 5. Average (±SE; n= 20) density of horizontal rhizome apexes in control and iron-enriched plots during the experiment.

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525 and thus released sulfide pressure on the plants. As 526 a result of these effects, the leaves and roots 527 showed a significant decrease in sulfide intrusion, 528 as reflected in changes ind³⁴S isotopic composition 529 in iron-enriched plants, which contributed to 530 accelerate clonal growth. Iron is involved in key

531 sediment and organism processes. Iron is an

532 essential nutrient for plant metabolism. At the

533 same time, iron modulates key ecosystem pro-

534 cesses, such as pyrite formation, which is a mech-

535 anism for renoval of sulfides from sediments,

536 thereby decreasing the likelihood of sulfide toxic-

537 ity. Pyrite formation, moreover removes feed back

538 processes between anoxic conditions and increas-

539 ing sulfate reduction, which in turn releases sul- fides acting as O2sinks, that act to preserve anoxic 540 conditions in iron-poor sediments (Chambers and 541 others2001; Holmer and others 2003,2005). 542

543 Increased clonal growth ofP. oceanicain response

544 to iron additions confirms the key role of iron in

545 plant nutrition, and as a factor alleviating stress

546 from increased organic inputs and associated high

547 sulfide production (Holmer and others2003,2005).

548 Iron additions had previously been shown to

549 stimulate seagrass growth on carbonate sediments

550 in the Caribbean (compare. Duarte and others

551 1995), Florida Bay (Chambers and others 2001)

552 and the Mediterranean meadow studied here

553 (Holmer and others 2005). However, all of these studies were conducted over time scales too short 554 to assess demographic responses, such as those 555 observed here. An increase in shoot recruitment 556 and net population growth of Posidonia oceanicain 557 response to 2 years of iron additions represents the 558

559 first demonstration that iron addition can improve

560 the status of seagrass populations. This observation

561 is particularly remarkable provided the exceedingly

562 slow demographic dynamics of this species (for

563 example, shoot turnover time in the control plots

564 23.3 ± 8.2 years), where direct observation is

565 challenging (Marba` and others2005).

566 Most importantly, the results presented here

567 demonstrate that sustained iron additions can re-

568 verse seagrass decline, as the meadow shifted from declining by about 7% y⁾¹to expanding at a rate of 569 7% y⁾¹ as a result of iron additions. This shift was 570 possible because of the stimulation of rhizome 571 growth, which is the basis for clonal growth, 572

573 resulting in a sizeable increase in the recruitment

574 rate. Despite no significant reduction in bulk shoot

575 mortality in response to iron additions, the increase

576 in shoot recruitment rate sufficed to drive the

577 population from net decline to net growth. The

578 observation that the mortality rate of new recruits

579 was reduced, in response to iron additions, with

580 survival of recruits in iron-enriched plots doubled

581 over that of recruits in control plots, suggests that

582 the improved demographic status evident already after 2 years of experimental iron additions, is likely 583 to improve even further as these vigorous recruits 584 replace shoots produced prior to iron additions. 585 Figure 6. Average (±SE) net leaf and rhizome produc-

tion in control and iron-enriched plots during the experiment.

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586 P. oceanica shoot population responses to iron 587 additions, however, exhibited large variability. The 588 high variance in the responses ofP. oceanicapopu- 589 lation dynamics to iron additions was due to the 590 slow population dynamics of P. oceanica, and, to 591 some extent, to the spatial heterogeneity of sea- 592 grass meadows. P. oceanica rhizomes produce 0.82 593 new shoots per year (Marba` and others 1996), 594 preventing detection of clear responses of shoot 595 recruitment to environmental change at time scales 596 shorter than a few years. In addition, because the 597 slow shoot turnover time for this population 598 (23 ± 8 years), the structure of the meadow 599 2 years after iron additions was similar to that in 600 control plots because most (78 ± 9%) shoots in the 601 population receiving iron were born prior to iron 602 additions, and hence not sensitive to iron additions.

603 Moreover, shoot density is highly heterogeneous in 604 P. oceanica meadows. Given the net population 605 growth rates during the experiment and shoot 606 densities at the beginning of the experiment, dif- 607 ferences in population structure (that is, shoot 608 density) between control and fertilized plots are 609 expected to be significant no earlier than after 610 5 years of iron additions. Hence, demographic re- 611 sponses are projected to display their full expres- 612 sion in decades, which defies the logistic demands 613 of underwater experimental ecology.

614 The observation that iron additions can improve 615 the status of impacted seagrass meadows growing 616 in carbonate sediments is, however, an impor- 617 tant one. Mediterranean P. oceanica meadows are 618 declining at rates in excess of 5% y⁾¹ across the 619 Mediterranean basin (Marba` and others2005), and 620 represent, therefore, the most threatened habitats 621 in the Mediterranean Sea. All attempts to reverse 622 this decline have failed to date, both at the regional 623 and even local scales. For instance, removal of a 624 fish farm following the observation of negative 625 impacts on the adjacent seagrass meadows (Del- 626 gado and others1999) failed to stop the decline of 627 the affected P. oceanica meadow, which continued 628 to decline years after the farming operation was 629 discontinued (Delgado and others 1999). The 630 demonstration that iron additions to organic-im- 631 pacted seagrass sediments can reverse seagrass de- 632 cline provided here represents, therefore, an 633 important finding pointing to avenues to reverse 634 this process, which is depleting seagrass ecosystems 635 in the Mediterranean and globally (Duarte and 636 others2002, 2007). Whether iron additions can be 637 safely applied at the ecosystem scale remains to be 638 assessed, but the fact that iron addition experi- 639 ments have already been conducted, for scientific 640 purposes, rather than to restore threatened eco-

641 systems, at a large scale over the ocean suggests

642 that it must be feasible.

643 In summary, this research shows, for the first

644 time, that seagrass decline can be reversed by iron

645 additions. We achieved this by targeting critical

646 nodes controlling the functioning of the system,

647 based on previous research aimed at elucidating the

648 demographic decline of the seagrass meadow (Marba` and others2002,2005), and the role of iron 649 in promoting seagrass growth (Duarte and others 650 1995; Chambers and others 2001) and controlling 651 sulfide dynamics (Holmer and others 2003, 2005) 652

653 in carbonate sediments. Because iron deficiency is

654 widespread in carbonate sediments across the

655 ocean (Duarte and others 1995), the role of iron

656 additions in reversing seagrass decline in this study

657 may well apply to seagrass decline caused by or-

658 ganic inputs to carbonate sediments elsewhere. As

659 seagrass meadows are suffering a global decline

660 (Duarte2002; Duarte and others 2007), the results

661 presented here offer an encouraging model to de-

662 velop effective strategies, together with regulatory

663 measures to reduce nutrient and organic matter inputs, to reverse decline and preserve seagrass 664 meadows. 665

666 6 6 7 A C K N O W L E D G E M E N T S

668 This study was funded by the EU project MedVeg

669 (Q5RS-2001-02456) and the project 055/2002 of

670 the Spanish Ministry of Environment. We thank

671 the company JAER for supplying the Fe-chelate

672 used in the experiment. We are grateful to the

673 officers and guards of Cabrera Archipelago National

674 Park for providing access to the study site and park

675 facilities, and to Miguel Angel for his delicious

676 cooking. We are indebted to Rocı´o Santiago and Regino Martı´nez for field and laboratory assistance. 677 Elena Dı´az-Almela and Maria Calleja were sup- 678 ported by PhD grants from the Balearic Govern- 679 ment and the Spanish Research Council, 680 respectively. We thank Antonio Tovar-Sa´nchez and 681

682 two anonymous reviewers for useful comments on

683 the manuscript.

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