Morphological responses of Posidonia oceanica to experimental nutrient enrichment of the canopy water

Journal of Experimental Marine Biology and Ecology 339 (2006) 1 – 14 www.elsevier.com/locate/jembe

Morphological responses of Posidonia oceanica to experimental nutrient enrichment of the canopy water Vanina Leoni, Vanina Pasqualini ⁎, Christine Pergent-Martini, Alexandre Vela, Gérard Pergent Equipe Ecosystèmes Littoraux, Faculty of Science, University of Corsica, BP 52, 20250 Corte, France Received 19 January 2006; received in revised form 26 April 2006; accepted 20 May 2006

Abstract To simulate an anthropogenic loading, experimental N and P enrichment was performed on a Posidonia oceanica meadow in the western Mediterranean Sea (Calvi, Corsica, France) during an annual cycle. The aim was to assess the morphological responses of the plant and the impact on the epiphytic index in order to test those parameters as indicators of coastal nutrient enrichment. Monthly monitoring of nutrient levels of the water (canopy and sediment) and of the epiphytic index was performed in parallel to investigations on plant morphology and leaf dynamic (leaf length, leaf surface, number of leaves per shoot, A coefficient: % of leaves that lost their apex, number of produced and fallen leaves, leaf longevity). To consider grazing interactions of Sarpa salpa, which can influence plant response, the reaction of the meadow was observed in an enriched zone, both unprotected and cageprotected, and compared with a reference zone. The epiphytic index and the leaf length are particularly impacted by nutrient enrichment, respectively, in summer and in spring, and in summer. However, data suggest that the epiphytic index increase is a specific but not systematic response, and that the reduced leaf length is more systematic in summer but not specific to nutrient enrichment. Their use is recommended to reveal nutrient enrichment including in association with other descriptors. © 2006 Elsevier B.V. All rights reserved. Keywords: Descriptors; Epiphytic index; Nutrient enrichment; Phenology

1. Introduction Worldwide, coastal zones are exposed to excessive N and P discharge due to human activities (e.g. artificial fertilizers, extensive breeding, deforestation, clearing, fish farming, waste water discharge; Larsson et al., 1985; Valiela et al., 1992; Soler Torres and del Rio, 1995; Short and Burdick, 1996; Short and Wyllie-

⁎ Corresponding author. Tel.: +33 4 95 45 06 52; fax: +33 4 95 45 01 62. E-mail address: [email protected] (V. Pasqualini). 0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2006.05.017

Echeverria, 1996; Howarth et al., 2000; Cancemi et al., 2003). Nutrient enrichment strongly affects coastal ecosystem mechanics, structure and productivity (Lapointe et al., 1994; Havens et al., 2001). An increase in phytoplanctonic production (Soler Torres and del Rio, 1995; Moncheva et al., 2001), even perhaps toxic species (toxic algae blooms; Glibert et al., 2001; Anderson et al., 2002; Heil et al., 2005) and in algae production (Lapointe et al., 1994; Howarth et al., 2000; Lapointe et al., 2004) can be observed. Consequences may include (i) an increase in turbidity leading to a reduction in the light available to benthic producers, (ii) anoxic events (Diaz and Rosenberg, 1995), (iii) a

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decreased biodiversity (Howarth et al., 2000) and (iv) fauna mortality likely to reduce fish stocks (Howarth et al., 2000; Verdelhos et al., 2005). The significant decline in seagrass meadows, which are essential ecosystems (Costanza et al., 1997), in response to nutrient enrichment has been reported worldwide (Orth and Moore, 1983; Cambridge and McComb, 1984; Valiela et al., 1992; Short and WyllieEcheverria, 1996; Pergent-Martini et al., 1996; Short and Burdick, 1996; Pergent et al., 1999; Ruiz et al., 2001; Cancemi et al., 2003). Because of their sensibility to nutrient enrichment, among others characteristics (e.g. extended distribution, benthic species), seagrasses are good indicators of that type of disturbance. Although considered as oligotrophic, the Mediterranean Sea is also affected by coastal nutrient enrichment (Turley, 1999) and Posidonia oceanica (L.) Delile meadows, as other seagrasses, appear to be very sensitive (Pérès and Picard, 1975; Pérès, 1984; Pergent-Martini, 1994; Pergent-Martini et al., 1996; Delgado et al., 1999; Pergent et al., 1999; Ruiz et al., 2001). Moreover, this species, as a Mediterranean seagrass species, is recognized as a determining element in assessing the biologic quality of Mediterranean coastal zones (EU Directive 2000/60/CE, of October 23rd, 2000). Nevertheless, the choice of specific descriptors to be used to reveal nutrient enrichment remains to be made.

The natural environment often makes it difficult to observe changes in seagrasses from a reference state to a nutrient-enriched state although such monitoring is necessary to identify early signs of disturbance. It can be performed through in situ addition of fertilizers (nitrogen and phosphorus at the canopy water level) in so far as it helps experimentally reproduce an enrichment process similar to those observed near an anthropogenic discharge. While many experimental enrichment processes with various purposes have been performed on seagrasses, very few of them have dealt with P. oceanica (Alcoverro et al., 1997; López and Duarte, 2004; Pérez and Romero, 1993; Invers et al., 2004; Pergent-Martini et al., 1996; Ruiz, 2001), including N and P enrichment of the canopy water (Pergent-Martini et al., 1996; Ruiz, 2001; Invers et al., 2004). Therefore, the aim of this study is to test if phenological responses and/or the epiphytic index can be used as descriptors of nutrient enrichment in the canopy water. 2. Materials and methods 2.1. Study site Experiments have been performed in the Revellata Bay (Calvi, Corsica, France), near the Submarine and Oceanographic Research Station (STARESO, Fig. 1).

Fig. 1. Studied area location ( ).

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They have been performed across a 2800 m2 area, whose topography is relatively homogeneous, with a bathymetry value from 9.7 m to 12.5 m. As an increase in nutrient levels can contribute to the intensification of grazing pressure (Ruiz, 2001), cages are used to limit that impact, especially that of the sparid herbivorous Sarpa salpa (L.). Six 4 m3 (2 × 2 × 1 m) fishing net cages, with 4 cm mesh–which makes it possible to intercept less than 5% of the related irradiance and to minimize the effects of light reduction (Ruiz, 2001)– were set up (Fig. 2). This type of mesh can provide protection against S. salpa; its diet changes depending on its size and only adult individuals (> 30 cm) graze high quantities of P. oceanica (Houziaux, 1993; Verlaque, 1985). Cages are cleaned on a monthly basis and replaced in spring. The site was randomly divided into four zones approximately 15 m apart from one another, where experiments were performed with triplicat: a cage-free non-enriched reference zone (T1 to T3, Fig. 3), a cage-free enriched zone (F1 to F3), a non-enriched cage zone (C1 to C3) and an enrichedcage zone (CF1 to CF3). 2.2. Enrichment The fertilizer used for the enrichment is a conifer fertilizer (N-P-K: 9-8-5) containing 2/3 ammonium (NH3) and 1/3 organic nitrogen, which is close to urban runoff ratios (Lesouef et al., 1991; Menesguen, 1991), and phosphates (PO5). The enrichment is calculated so as to increase the N and P concentrations of the canopy water about 10 times all the while contributing to an N/P

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ratio close to 10 (urban runoff; Aminot and Guillaud, 1991; Travers and Kim, 1997; Stapleton et al., 2000). The fertilizer granules are crushed and mixed in guar gum and water in fixed proportions (1/¼/2). Those mixtures were poured in mosquito net type bags (1 mm2 mesh) at the canopy level (80 cm) every month from August 2002 to September 2003 for continued diffusion. This nutrient enrichment process generated an average of 40 g of nitrogen compounds and 15 g of phosphorus a month. To monitor nutrient enrichment, canopy water and interstitial water samples were collected every month from August 2002 to September 2003. Orthophosphate, nitrate, nitrite and ammonium measurements were done by spectrophotometry (Spectrophotometer Hach DR/2000 DR/2000). 2.3. Study of P. oceanica shoots Fifteen orthotropic P. oceanica shoots were taken every month from September 2002 to September 2003 from each study zone. The shoots were fresh-dissected and measured according to the Giraud protocol (1977). The measured parameters included the following: length, leaf surface and number of adult and intermediate leaves per shoot. The apex state of adult and intermediate leaves (whole or broken) was also noted and helped calculate the A coefficient (or the percentage of leaves per shoot that lost their apex). After scratching the leaves with a razor blade, the epiphytic coverage was dried (lyophilisation, 72 h), weighed and the epiphytic index, or epiphyte dry weight, related to the leaf surface was determined. The lepidochronologic

Fig. 2. In situ photographs of cages.

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Fig. 3. Studied site location (T: no treatment, F: enrichment, C: cage, CF: enriched cages).

analysis (Pergent, 1990) helped determine the number of produced leaves and fallen leaves on a monthly basis, and to deduce their longevity (Pergent and PergentMartini, 1990). 2.4. Statistical analysis Analyses of variances (ANOVA) including one or more factors were used when the conditions of application were met (variance homogeneity and population normality). Otherwise, non-parametric median comparison tests were conducted (Kruskall Wallis and Student Newman Keuls). To be more specific, one-way ANOVAs were used to confirm homogeneity of the studied parameters at the different sites before the experiment. ANOVA with repeated measures (time factor), two-fixed-factor (enrichment and cage factor) and a nested random factor (quadrat factor) were used to compare the mean value of the various parameters under study. As regards the study of water nutrient concentrations, two-way ANOVAs were used (enrichment and cage factor). A regression line comparison was performed

for the number of leaves produced and fallen over the study. Non-parametric tests were performed to compare leaf longevities (Kruskall Wallis and Student Newman Keuls). 95% confidence intervals were applied on the graphs. The software that was used is STATISTICA®. 3. Results 3.1. Water nutrient concentrations The various mean annual nutrient concentrations were significantly superior in the interstitial water as compared with canopy water for orthophosphates (PO5−) and ammonium (NH4+) independent of treatment type (p < 0.05; Fig. 4). On the other hand, nitrate and nitrite contents were roughly similar in both compartments (Fig. 4). An enrichment effect with stronger nutrient concentrations in the enriched zones was demonstrated for orthophosphate and ammonium concentrations in interstitial water (p < 0.05 and p < 0.001, respectively), and for orthophosphate, nitrate and ammonium concentrations in the canopy water (p < 0.05).

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Fig. 4. Mean annual water nutrient concentrations depending on treatment (T: no treatment, F: enrichment, C: cage, CF: enriched cages).

3.2. Morphological parameter response Monthly variations have been typically recorded (Table 1) for each parameters. The mean annual leaf length varied from 431 ± 29.6 mm (F) to 505.5 ± 31.5 mm (C) for the adult leaves and from 275.4 ± 31.5 mm (F) to 330 ± 38.9 mm (T) for the intermediate leaves (Fig. 5). For adult leaves, an enrichment effect, with shorter leaf lengths, and a cage effect, with longer leaf lengths, were both noted (Table 1). During the year, the enrichment effect was obvious in July and August 2003 (Fig. 6, Table 2). The cage effect, which was noticeable all year long, was significant in September and October 2002 and in January 2003 (Table 2). For the intermediate leaves, changes relatively

similar to those noted for the adult leaves were recorded during the year. The enrichment effect was highly significant in July 2003 (Table 2) and seemed more noticeable in cage-free zones (distance between T and F; Fig. 7). The mean annual base lengths varied from 34.6 ± 0.8 mm (F) to 36.3 ± 0.9 mm (C) (Fig. 5). During the year, as with leaf lengths, the enrichment effect revealed smaller bases and the cage effect revealed longer bases (Table 1). The mean annual number of leaves varied from 3.8 ± 0.1 (CF) to 3.9 ± 0.1 (T) for the adult leaves and from 2.2 ± 0.2 (C and T) to 2.3 ± 0.2 (CF) for the intermediate leaves (Fig. 5). No cage or enrichment effect was noted on a yearly basis (Table 1). On the other hand, a highly

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Table 1 Anova values for cage and enrichment effects and their interactions regarding Posidonia oceanica morphological parameters and epiphytic index during the year (⁎ = significant values) Shoot parameters

Factors and interactions

F value

p

Adult leaves Length

Nb

F. S.

A coef.

Base lengths

I. E.⁎

Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time Enrich. Cage Time Enrich. × Cage Enrich. × Time Cage × Time Enrich. × Cage × Time

significant enrichment effect, which increased the number of intermediate leaves, was observed during the year, in June and July 2003 (Table 2). The mean annual leaf surface varied from 171.4 ± 10.9 (F) to 194.9 ± 14.6 cm2 fx− 1 (C) for the adult leaves and from 71.6 ± 8.7 (C) to 76.9 ± 9 cm2 fx− 1 (T) for the intermediate leaves (Fig. 5). An enrichment effect (Table 1) showing lower values in the fertilized zones was noted for the adults leaves. During the year, that

24.82 54.03 98.20 1.88 6.49 1.20 0.26 0.35 1.20 12.36 0.17 0.82 0.50 0.70 17.83 4.96 54.19 0.25 3.34 0.73 0.43 7.44 12.84 85.95 3.94 3.77 1.54 1.07 10.58 6.57 2.26 0.92 3.05 1.13 0.34 35.13 37.19 58.33 7.06 4.24 1.64 1.43

F value

p

Intermediate leaves 0.0011⁎ 0.0001⁎ 0.0000⁎ 0.2070 0.0000⁎ 0.2995 0.9920 0.5683 0.3050 0.0000⁎ 0.6946 0.6231 0.8960 0.7385 0.0029⁎ 0.0565 0.0000⁎ 0.6284 0.0007⁎ 0.7096 0.9369 0.0259⁎ 0.0072⁎ 0.0000⁎ 0.0824 0.0002⁎ 0.1328 0.3969 0.0117⁎ 0.0335⁎ 0.0176⁎ 0.3651 0.0017⁎ 0.3450 0.9748 0.0004⁎ 0.0003⁎ 0.0000⁎ 0.0290⁎ 0.0000⁎ 0.1007 0.1743

5.89 2.89 37.94 0.39 1.36 0.41 0.29 4.57 0.09 183.93 0.09 1.18 2.75 1.99 0.43 1.22 96.03 0.82 0.94 2.06 1.46 1.19 0.09 2.72 2.76 1.11 1.16 0.54

0.0414⁎ 0.1276 0.0000⁎ 0.5481 0.2038 0.9495 0.9855 0.0650 0.7678 0.0000⁎ 0.7678 0.3158 0.0042⁎ 0.0386⁎ 0.5284 0.3010 0.0000⁎ 0.3920 0.5074 0.0321⁎ 0.1622 0.3079 0.7718 0.0046⁎ 0.1352 0.3657 0.3294 0.8712

enrichment effect was significant in July and August 2003 (Table 2). The A coefficient mean annual value varied from 30.9 ± 4.4% (C) to 41.9 ± 5.1% (F) for the adult leaves and from 2.1 ± 1.9% (C) to 5.3 ± 2.9% (CF) for the intermediate leaves (Fig. 5). The adult leaf A coefficient alone seemed affected by cages and fertilization (Table 1), with a cage effect showing a lower A coefficient, and a fertilization effect showing a higher A coefficient.

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Fig. 5. Morphological parameter and epiphytic index annual average of Posidonia oceanica depending on treatment (T: no treatment, F: enrichment, C: cage, CF: enriched cages).

During the year, that cage effect was particularly visible from April until August 2003, mostly between the C and T zones (Fig. 6). Likewise, the enrichment effect was significant in April, July and August 2003 (Table 2). The epiphytic index mean annual value varied from 0.55 ± 0.06 mg cm− 2 (C) to 0.88 ± 0.09 mg cm− 2 (F; Fig. 5). A cage effect and an enrichment effect, both quite very highly significant, were demonstrated (Table 1);

the enrichment effect led to an increase in the epiphytic index while the cage effect led, on the contrary, to a decrease in the same. During the year, this enrichment effect was quite visible from May 2003 to September 2003 (Table 2, Fig. 6). The cage effect was significant in January and June 2003 (Table 2). The number of produced and fallen leaves (slope comparison, p = 0.95) as well as leaf longevity (Kruskall

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Fig. 6. Monthly evolution of mean adult leaf length, A coefficient and epiphytic index of Posidonia oceanica depending on treatment (T: no treatment, F: enrichment, C: cage, CF: enriched cages).

Wallis, p > 0.05) were not significantly different depending on treatment types (Table 3). 4. Discussion and conclusion The cage setup helped study P. oceanica behaviour in the absence of grazing, at least by S. salpa; indeed urchins are much more difficult to control, especially small size individuals. The presence of cages effectively

reduces grazing, which is demonstrated by longer adult leaves (more exposed to grazing due to their external position) in cages, and lower adult leaf A coefficient in non-fertilized cages than in reference zones, especially from April to August 2003. This last observation indicates a more intense grazing activity over that period of the year (Fig. 6). These results are in keeping with Houziaux's (1993) results that showed seasonal changes in S. salpa eating behaviour. Indeed, they

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Table 2 Anova values for cage and enrichment effects and their interactions regarding Posidonia oceanica morphological parameters and epiphytic index during the year (⁎ = significant values)

Enrich. Cage Enrich. × Cage Base Enrich. lengths Cage Enrich. × Cage Length Enrich. Int. Cage Enrich. × Cage Nb Ad. Enrich. Cage Enrich. × Cage Nb Int. Enrich. Cage Enrich. × Cage F.S. Ad. Enrich. Cage Enrich. × Cage F.S. Int. Enrich. Cage Enrich. × Cage A coef. Enrich. Ad. Cage Enrich. × Cage A coef. Enrich. A Int. Cage Enrich. × Cage E.I. Enrich. Cage Enrich. × Cage

Length. Ad.

sep-02

oct-02

nov-02

dec-02

jan-03

mar-03

apr-03

may-03 jun-03

jul-03

aug-03

0.1891 0.0320⁎ 0.5549 0.5596 0.6580 0.6027 0.8021 0.7164 0.4553 0.4419 0.3122 1.0000 0.2861 0.5834 0.0805 0.8711 0.1084 0.3628 0.5465 0.5865 0.3816 0.4752 0.1942 0.4161

0.5915 0.0086⁎ 0.9872 0.9616 0.9509 0.7095 0.9299 0.1058 0.9251 0.3169 0.3169 0.3169 0.2466 0.4747 0.4747 0.3416 0.2884 0.4803 0.4066 0.5760 0.4516 0.5694 0.9722 0.9861

0.6397 0.1175 0.4531 0.0841 0.1134 0.2931 0.1579 0.1094 0.1374 0.8232 0.6565 0.5080 0.2815 0.5796 0.2815 0.8657 0.6087 0.4668 0.2926 0.3581 0.3472 0.8103 0.4780 0.0177⁎

0.8544 0.0589 0.5628

0.7560 0.0025⁎ 0.9483 0.9764 0.0502 0.8930 0.3469 0.0097⁎ 0.6891 0.4635 0.0905 0.2815 0.8243 0.0356⁎ 0.2845 0.6098 0.2336 0.5678 0.3974 0.9699 0.8725 0.2096 0.7195 0.7195 0.4379 1.0000 0.4379 0.3922 0.0832 0.0415⁎

0.1667 0.3013 0.5752 0.4786 0.8300 0.5600 0.2209 0.5547 0.4004 0.0790 0.5212 0.0393⁎ 0.4876 0.4876 0.8145 0.8992 0.9314 0.1351 0.7106 0.7286 0.9333 0.3425 0.1604 0.2371 0.2075 0.2075 0.2075 0.2652 0.2825 0.6607

0.5383 0.3932 0.9229 0.4747 0.9795 0.5920 0.2455 0.3971 0.7503 0.6258 0.4226 0.6258 0.8852 0.8852 0.6666 0.4451 0.1753 0.4864 0.5345 0.4649 0.5735 0.0294⁎ 0.5456 0.3313 0.3169 0.0200⁎ 0.3169 0.1844 0.0033⁎ 0.3958

0.1098 0.2093 0.5952 0.0016⁎ 0.4533 0.6278 0.0941 0.9182 0.7153 0.4419 0.4419 0.6043 0.8243 0.1470 0.2845 0.5817 0.8880 0.8547 0.1615 0.2521 0.8252 0.9416 0.0555 0.5224 1.0000 0.0008⁎ 1.0000 0.0002⁎ 0.2212 0.4753

0.0033⁎ 0.3861 0.8847 0.0222⁎ 0.0820 0.8634 0.0040⁎ 0.4428 0.1251 0.2133 1.0000 0.3402 0.0068⁎ 0.2623 0.0167⁎ 0.0188⁎ 0.6069 0.5639 0.0040⁎ 0.4428 0.1251 0.0052⁎ 0.9262 0.1766 0.0147⁎ 0.6514 0.1969 0.0157⁎ 0.0104⁎ 0.4668

0.0025⁎ 0.2247 0.8762 0.0004⁎ 0.5435 0.4621 0.8641 0.3394 0.8178 0.7924 0.7924 0.6011 0.8145 0.0066⁎ 0.0606 0.0049⁎ 0.7721 0.7371 0.8641 0.3394 0.8178 0.0182⁎ 0.0012⁎ 0.0099⁎

0.6150 0.4577 0.1242

0.5701 0.9162 0.1359 0.7319 0.0185⁎ 0.8292 0.3221 0.1478 0.0501 0.7599 0.1525 0.1525 1.0000 0.0667 0.1950 0.9766 0.1799 0.1258 0.9580 0.0078⁎ 0.1495 0.4758 0.4252 0.6033 0.3466 0.3466 0.3466 0.8426 0.0669 0.7705

0.5344 0.0652 0.1597

would feed on sciaphiles algae beyond meadow limits during the winter (Verlaque, 1990), and that diet change in spring to summer, with predominance in grazed P. oceanica leaves (Houziaux, 1993). That phenomenon seems consistent in so far as S. salpa shows a preference for P. oceanica adult and epiphyted leaves (Peirano et al., 2001) and the epiphytic index is at its highest over that period. The presence of cages also seemed to have contributed to a decrease in the epiphytic index, conflicting with the results noted in Fong et al. (2000) and Keuskamp (2004), who, during their exclusion experiment, recorded a negative correlation between grazers and the epiphytic index. This index reduction could be the result of a light cage-induced decrease (Neverauska, 1988; Moore and Wetzel, 2000). However, in so far as the used net mesh intercepted less than 5% of the incident irradiance (Ruiz, 2001), a reduction in epiphyte fixation, resulting from a change in light, did not seem very likely. The cage structure may have served as an epiphyte substrate all the more as most of epiphyte

0.1585 0.2235 0.1167 0.9571 0.0941 0.1669 0.3451 0.6464 0.0379⁎ 0.2368 0.7363 0.9103 0.0020⁎ 0.6938 0.0755 0.9183 0.4345 0.7729 0.3451 0.6464 0.0379⁎ 0.6056 0.3616 0.5622 0.2466 0.8089 0.2466 0.0696 0.0741 0.0279⁎

sep-03

0.1416 0.0701 0.7448 0.5765 0.4315 0.8575 0.2134 0.0017⁎ 0.8386 0.6996 0.6996 0.6996 0.5024 0.0980 0.5024 0.3210 0.1992 0.4730 0.2204 0.0020⁎ 0.8544 0.8039 0.6349 0.8039 0.3270 0.3270 0.3270 0.0332⁎ 0.0014⁎ 0.7835 0.0127⁎ 0.6795 0.6045

species are found in other host plants but are also observed in non-living substrates (synthesis in Harlin, 1980). The regular fertilizer supply led to an increase in ambient nutrient levels as compared with those in nonfertilized zones; those differences were noted both in the interstitial water and the canopy water. That enrichment made at the canopy level resulted in a significant increase in interstitial water ammonium and orthophosphate levels. The increase in ammonium sediment levels seems consistent (i) in relation with the supply, mainly performed in this form, and (ii) due to the bacterial activity responsible for a higher ammonification during the sediment enrichment process (López et al., 1998). However, ammonium concentrations, more generally nitrogen concentrations, remained low compared with those noted in sites subjected to anthropogenic activities (e.g. fish farming, urbanization, Lapointe et al., 1994; Cancemi et al., 2003; Vela et al., 2004). Nutrient enrichment impact on P. oceanica meadows is especially visible in spring and summer, a period

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Fig. 7. Monthly evolution of mean length and number of Posidonia oceanica intermediate leaves depending on treatment (T: no treatment, F: enrichment, C: cage, CF: enriched cages).

marked by plant nutrient deficiency as indicated by Pedersen and Borum (1993). It is suggested that, as a result of that high deficiency, the light and temperaturebased growth control applied in winter would change to a nutrient-based growth control in summer (Alcoverro et al., 1997). When enrichment seems to strongly impact the plant (from May to September), another correlation between ammonium, nitrate and orthophosphate levels is found at the canopy level, which illustrates the achieved enrichment (R2 between 61.7 and 75.5). On the other hand, a negative correlation (R2 = 59.0) between sediment nitrate and ammonium levels is observed, expressing a bacterial activity in all likelihood (López et al., 1998). Table 3 Average number of produced and fallen leaves during the studied year and Posidonia oceanica mean leaf longevity (monthly basis)

Number of produced leaves Number of fallen leaves Longevity

T

F

C

CF

8.9 9.5 9.3

10.1 9.7 8.4

9.5 9.1 8.5

9.5 9.6 8.6

While nutrient enrichment seems to influence most of the studied morphological parameters, either occasionally (one given month) or in a more general way (over the year), the parameters that seem to be the most sensitive are the epiphytic index and the leaf length. From May to September 2003, this epiphytic index is correlated to ammonium, nitrate and phosphate levels as measured in the canopy water (R2 = 35.1, 35.2 and 54.8, respectively). Nutrient enrichment led to a massive epiphytic development mostly observed in the F zone, in spring and summer, and confirmed the results obtained through other experimental enrichments (Lapointe et al., 1994; Pergent-Martini et al., 1996; Wear et al., 1999; Moore and Wetzel, 2000) or observed in meadows subjected to high nutrient content human runoff (Cambridge et al., 1986; Silberstein et al., 1986; Delgado et al., 1999; Cancemi et al., 2003). Leaf length also seems strongly influenced by nutrient enrichment in spring and summer. Ruiz (2001) and Short et al. (1995) noted a comparable reduction in leaf length with experimental nutrient enrichment, for P. oceanica and Zostera marina, respectively. In a natural

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environment, shorter leaves were also observed near urban emissaries (Maggi et al., 1977) or fishing structures (Delgado et al., 1999; Ruiz et al., 2001). This decrease in leaf size may be mechanically induced. The epiphytic development may (i) make the leaf apex more fragile, leading to increased breakage and thus to a reduction in leaf length (Harlin, 1980), and (ii) generate an increase in grazing (e.g. superior leaf tissue and/or epiphyte nutritive quality; Ruiz, 2001; Ruiz et al., 2001; McGlathery, 1995). The reduced size can also be due to an indirect physiological cause. So, nutrient enrichment of the water column often leads to a decrease in plant growth (or mortality) associated with epiphytic development and to a decrease of available light (Sand-Jensen, 1977; Twilley et al., 1985; Tomasko and Lapointe, 1991; Pergent-Martini et al., 1996; Neundorfer and Kemp, 1993; Lapointe et al., 1994; Short et al., 1995; Moore and Wetzel, 2000). Nutrient enrichment may also indirectly and seriously impact seagrass functioning and physiology through the induction of sedimentary changes (e.g. anoxy, hypoxy, sulfides; Delgado et al., 1999). Finally, direct physiological mechanisms (i.e. imbalanced internal nutrient supply ratio, ammonium toxicity; Burkholder et al., 1992, 1994; Santamaría et al., 1994; Katwijk van et al., 1997; Touchette et al., 2003; Invers et al., 2004) unrelated to epiphytic development (Touchette et al., 2003) may be responsible for a reduced elongation (Bird et al., 1998). This study shows higher A coefficient values in fertilized zones in July and August 2003 (Table 2), especially significant differences between C and CF. These differences are explained by an increase in leaf breakage in response to an epiphytic development in the fertilized cages (Fig. 6). The mechanical effect of nutrient enrichment on leaf length as previously mentioned is therefore confirmed although it is difficult to identify the grazed and broken parts outside of the cages. While authors suggest an increase in grazing as a result to an improved leaf and/or epiphyte nutrient quality further to nutrient enrichment (McGlathery, 1995; Ruiz, 2001), we found that the A coefficient is not significantly different between the T and F sites, and the F and CF sites through the year. These results are in agreement with those of Delgado et al. (1999) who did not observe any A coefficient increase near fishing structures. A physiological effect also seems to exist apart from that mechanical effect. Indeed, a decrease in the mean length of adult leaf bases, generally speaking, and of the oldest adult leaf (rank 1) in particular, is observed. That first adult leaf base length is correlated with the total

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adult leaf length (Pergent and Pergent-Martini, 1991). The significant difference observed between reference shoots and fertilized shoots from May to September 2003 seems to indicate a growth disturbance associated with fertilization. This observation seems to be confirmed by the reduced length of the fertilized intermediate leaves naturally hardly grazed and with few epiphytes. Growth disturbance could be related to an epiphyte-induced light reduction. Indeed, we punctually observe an increase in the number of intermediate leaves per shoot, which might constitute an adaptive response of the plant aiming at increasing its photosynthetic surface to compensate for the epiphytic development-induced loss (fact observed with depth increase; Dalla Via et al., 1998). Moreover, direct toxicity mechanisms cannot be ruled out in so far as reduced leaf lengths are especially observed in summer, a period characterized by a decreased irradiance in leaves (related to a significant epiphytic development), but also by an increase in water temperature, yet temperature and shade are considered as aggravating parameters for toxicity (Burkholder et al., 1992, 1994; Katwijk van et al., 1997; Touchette et al., 2003). On the contrary, a sediment change impacting the plant seems somewhat unlikely given the relatively low concentrations as measured; for example, sediment ammonium values correspond to a third of the values observed in Corsica, 100 m from fishing cages (Cancemi et al., 2003). The use of morphological descriptors to reveal nutrient enrichment may thus be considered. Although several parameters show significant variations (Table 1), it may be wise to favor the epiphytic index and the leaf length, which seem to be the most relevant. Nevertheless, their use as nutrient enrichment descriptors requires considering their limits of use especially in terms of time of use–limited to spring and summer–and in terms of response specificity and reproducibility. Indeed, although it seems to be a specific response to nutrient enrichment, an increase in the epiphytic index is not systematically observed. Nutrient availability can lead to the fast development of others primary producers (i.e. epiphytes, phytoplankton, floating or fixed macroalgae). Lin et al. (1996) and Taylor et al. (1995) did not observe any increase in the epiphytic biomass but noted phytoplankton development in response to nutrient enrichment. The epiphytic biomass increase is thought to occur in response to nutrient enrichment only with sufficient irradiance (Moore and Wetzel, 2000), and phytoplankton development seemed to be observed only in response to a strong enrichment (Twilley et al., 1985). Community

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types which develop as a result may thus depend on the type of enrichment (ratio, quantity; Twilley et al., 1985), on environmental conditions, such as the quantity of available light (Sand Jensen and Borum, 1991; Moore and Wetzel, 2000), on water movement (Taylor et al., 1995; Harlin and Thorne Miller, 1981), on grazing interactions (Moore and Wetzel, 2000; Neckles et al., 1993) and on in situ experimental enrichment conditions (e.g. column vs. sediment enrichment). Reduced leaf lengths, unlike the epiphytic index, seem to be more widely observed (at least in summer, Short et al., 1995), as a result of physiological and/or mechanical changes independent of the developing community (Maggi et al., 1977; Ruiz et al., 2001; Delgado et al., 1999; Pergent-Martini et al., 1996). Indeed, nutrient enrichment-related intense grazing leads to reduced leaf lengths (Ruiz, 2001; Ruiz et al., 2001), while an epiphytic index increase is not always noticeable (Ruiz, 2001). It is however necessary to note that other environmental factors can lead to smaller leaf sizes, in particular a decreased irradiance (Neverauska, 1988; Dalla Via et al., 1998; Longstaff and Dennison, 1999). This observation underlines the non-specificity of that parameter, which can be relevant of both nutrient enrichment and any type of disturbances resulting in a decreased water transparency (e.g. shoreline development). To conclude, both parameters can be considered as good disturbance indicators even in the case of moderate nutrient enrichment as shown in our study. Furthermore, those descriptors are easy to implement, incur low application costs and use study protocols that are applied fairly consistently across the scientific community (Pergent-Martini et al., 2005). However, the resulting data needs to be matched with those of other descriptors to reduce potential diagnostic errors and obtain earlier information. Indeed, the use of physiological indicators would (i) help confirm the hypotheses regarding response-governing mechanisms and (ii) facilitate the coupling up of descriptors across the population and on an individual level (morphological and physiological) to try and obtain more accurate and quicker information on the trophic status of the environment. Acknowledgements This work benefited from the financial support of the European INTERREG IIIA program. The authors thank H. Demortier for help with the English translation. [SS]

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