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Aquatic Botany 96 (2012) 58–62

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Leaf vs. epiphyte nitrogen uptake in a nutrient enriched Mediterranean seagrass

(Posidonia oceanica) meadow
Eugenia T. Apostolaki a,b,∗ , Salvatrice Vizzini c , Ioannis Karakassis b
Institute of Oceanography, Hellenic Centre for Marine Research, PO Box 2214, 71003, Heraklion, Crete, Greece
Marine Ecology Laboratory, Biology Department, University of Crete, PO Box 2208, 71409, Heraklion, Crete, Greece
Department of Earth and Marine Science, University of Palermo, Via Archirafi 18, 90123 Palermo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In situ nitrogen uptake by leaves and epiphytes was studied in a Mediterranean seagrass (Posidonia ocean-
Received 11 February 2011 ica) meadow impacted from a fish farm and a pristine meadow, using 15 NH4 and 15 NO3 as tracers. In the
Received in revised form impacted meadow both leaves and epiphytes yielded higher N concentrations and showed higher spe-
20 September 2011
cific N uptake, suggesting a linkage between N uptake and its accumulation. Epiphytes took up N faster
Accepted 21 September 2011
than leaves in relation to their corresponding biomass, but when assessed per unit area, N uptake was
Available online 28 September 2011
higher in leaves. Leaf N uptake was negatively correlated with epiphyte N uptake. With increasing epi-
phyte load on leaves, N leaf uptake decreased while N epiphyte uptake increased, indicating that epiphyte
Nitrogen cycling
overgrowth hinders N uptake by P. oceanica leaves. Epiphyte contribution to total N uptake increased,
Nutrient uptake while that of leaves decreased at the impacted meadow. However, 2–3 times less N was transferred daily
Eutrophication from the water column to the benthic compartment, through seagrass and epiphyte uptake on total, at
Fish farm impact the impacted meadow. Therefore, it is probably still the loss of the key species – the seagrass – which
N tracer plays the most important role in N cycling in seagrass ecosystems.
Stable isotope © 2011 Elsevier B.V. All rights reserved.

1. Introduction significantly to total N uptake of seagrass beds by absorbing NH4 +

and NO3 − (Lepoint et al., 2007). Nevertheless, most studies have
Coastal human activity has increased nutrient loading world- measured N uptake only by leaves, neglecting uptake by epiphytes
wide, causing wide seagrass loss (Burkholder et al., 2007). Fish (e.g. Hemminga et al., 1994; Stapel et al., 1996; Pedersen et al.,
farming facilities have contributed to this loading, by discharging 1997; Terrados and Williams, 1997; Lee and Dunton, 1999; Vonk
solute wastes to the surroundings (Sarà, 2007). Posidonia oceanica et al., 2008; Alexandre et al., 2011). However, different morphol-
(L.) Delile, the endemic seagrass of the Mediterranean, is largely ogy and physiology (i.e. additional mechanisms such as root uptake
impacted by fish production (Boudouresque et al., 2009); the latter and internal storage and translocation in seagrasses; Romero et al.,
has increased exponentially around the basin in the past decades 2006) and complex interactions among leaves and epiphytes (i.e.
(UNEP, 2002). P. oceanica meadows are listed as priority natural competition for water column nutrients; Borowitzka et al., 2006)
habitats according to the Directive on the Conservation of Natural predispose for different N uptake rates.
Habitats and of Wild Fauna and Flora because they rank among The experimental addition of 15 N labelled compounds as exper-
the most valuable ecosystems along the Mediterranean coast- imental tracers has allowed to assess simultaneous uptake rate
line. However, P. oceanica meadows are detrimentally affected by of N by leaves and epiphytes (Lepoint et al., 2004) in a few sea-
human activities including fish farming due to water and sediment grass meadows. As far as we know, N uptake rates are given
quality deterioration, epiphyte overgrowth and herbivore pressure only for the few following seagrass species and their associated
(Pergent-Martini et al., 2006; Holmer et al., 2008). epiphytes; Thalassia testudinum (Cornelisen and Thomas, 2006),
Seagrass leaves take up both ammonium (NH4 + ) and nitrate P. oceanica (Lepoint et al., 2007), P. angustifolia and Amphibolis
(NO3 − ) from the water column, contributing to the N requirement antarctica (Nayar et al., 2010). However, to our knowledge, no effort
of the plant (Touchette and Burkholder, 2000). Recent findings has been made so far to quantify the N uptake in seagrass leaves
suggest that epiphytes covering seagrass leaves also contribute and epiphytes when the meadow is under nutrient enrichment.
Increased N availability usually favours the fast-growing epi-
phytes (Lepoint et al., 2007), shifting the competitive balance
∗ Corresponding author at: Hellenic Centre for Marine Research, PO Box 2214, from seagrass to epiphytes (Fourqurean et al., 2010). In nutri-
71003, Heraklion, Crete, Greece. Tel.: +30 2810337714; fax: +30 2810337822.
ent enriched seagrass meadows, epiphyte load usually increases
E-mail address: (E.T. Apostolaki). (Balata et al., 2010; Giovannetti et al., 2010; Apostolaki et al.,

0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62 59

2011) and it has been repeatedly related to a reduction in leaf pro- flexible and allow turbulence in the interior of the benthic cham-
duction (Borowitzka et al., 2006). Nevertheless, herbivores often bers, ensuring mixing.
increase in nutrient enriched meadows controlling down the epi- Solutions of ammonium sulphate (99.0% 15 N) or sodium nitrate
phyte abundance (Ruiz et al., 2009) and hence decreasing epiphyte (99.0% 15 N) (Eurisotop, France) of 0.06 ␮M concentration were
contribution to total N uptake of seagrass shoot. On the other hand, added in the chambers using syringes, in order to achieve around
Apostolaki et al. (2011) showed recently that, although herbivore 20% enrichment of the initial ambient concentration in 15 NH4 or
pressure increased, it did not counterbalance the effect of moderate 15 NO , respectively. The incubation lasted only for an hour, in
yet continuous nutrient input from fish farm discharges to the sur- accordance with similar studies (Lepoint et al., 2002, 2007), to avoid
roundings, which shifted the control of epiphytes from top-down nutrient depletion. At the end of the incubation, all seagrass shoots
to bottom-up and led to increased epiphyte load. from the chamber (∼10–15 shoots) were retrieved by hand and
Therefore, we hypothesized that the main primary producers of kept frozen.
the P. oceanica ecosystem (seagrass and epiphytes) react differently
to nutrient enrichment in terms of N uptake, based on their differ- 2.2. Laboratory analysis
ent physiology, their complex interaction and their interaction with
other components of the ecosystem. We aimed to study if nutrient In the laboratory, the water samples were analyzed manually
enrichment from fish farm waste affects N uptake by leaves and for NH4 + (Ivančič and Deggobis, 1984; detection limit 0.1–10 ␮M)
epiphytes in a Mediterranean seagrass (P. oceanica) meadow. We and NO3 − concentrations (Strickland and Parsons, 1972; detection
report leaf and epiphyte uptake of NH4 + and NO3 − in impacted and limit 0.05–4 ␮M). Epiphytes were gently removed from the leaves
pristine patchy seagrass meadows, using 15 NH4 and 15 NO3 as trac- using a razor blade and P. oceanica leaves and epiphytes were dried
ers in in situ incubations. We also examine the effect of epiphyte at 70 ◦ C for 48 h, weighed and grounded to a fine powder. Total
load on leaf N uptake. nitrogen and isotopic analysis in seagrass leaves and epiphytes
were performed using a Thermo Scientific Delta Plus XP Isotope-
Ratio Mass Spectrometry (IRMS) in continuous flow configuration.
Overall analytical error was always less than 0.2‰. Isotopic data is
2. Materials and methods reported in common delta (ı) units referred to atmospheric nitro-
gen standards.
2.1. Sampling strategy
2.3. Calculations
The experiment was conducted once in July 2007 in two patchy
P. oceanica meadows in Crete (Aegean Sea, Greece). The first site is
The excess 15 N incorporated in seagrass leaves and epiphytes
situated in a shallow bay in Sitia (35◦ 15.23 N, 26◦ 13.29 E). A fish
was calculated from the increase in the fraction (F) of 15 N after
farm in the bay has been operating for 12 years, producing annu-
labelling (Ft15 ) relative to the background F (Fb15 ), according to
ally 1000 tons of gilthead sea bream (Sparus aurata) and sea bass
Kiswara et al. (2005):
(Dicentrarchus labrax). The meadow is located as close to the fish
cages as possible, around 80 m away from both the fish farm and 15
N = Ft15 − Fb15 ,
the coast, at 4.5 m depth. It has been suggested that fish farming
impact is evident up to 400 m away from the fish cages (Holmer where F15 = R/(R + 1) and R = (ı/1000 + 1) × Rstandard , with
et al., 2008). Therefore, the meadow in Sitia falls within the area Rstandard = 0.0036765.
affected by fish farm waste. Further information on the study site Specific N uptake rate (sNU, ␮g N g−1 DW h−1 ) was estimated as
and fish farm characteristics can be found in Cook et al. (2006). The
second site is situated in a shallow bay in Psaromoura (35◦ 24.46 N,
(15 N × N)
25◦ 01.05 E) and represents a pristine site, where the meadow is sNU = ,
(W × t)
located 80 m away from the coast, at 4.9 m depth.
Background concentrations of NH4 + and NO3 − in water column where 15 N is the excess 15 N, N the concentration of nitrogen (in
and ␦15 N and N in leaves and epiphytes were measured before each % DW), W the dry weight of incubated tissue (in g DW) and t the
experiment. At each site and just before the incubations, divers col- duration of the incubation (h).
lected triplicate water samples (400 ml each) above the seagrass N uptake rate (NU, ␮g N m−2 sediment h−1 ) was computed as:
canopy, using acid-washed syringes. The samples were filtered
through pre-combusted glass fibre filters (Whatman GF/F), kept (15 N × N)
NU =
frozen and later analyzed for NH4 + and NO3 − . From each site around (W × t × s)
15 shoots of P. oceanica were retrieved by hand, kept frozen and
later used for measuring background ␦15 N and N concentration in where s is the sediment surface (in m2 ) that was incubated.
seagrass leaves and epiphytes, shoot and leaf biomass and epiphyte Epiphyte load (g DW g−1 DW leaf) was calculated by dividing the
load. dry weight of epiphytes with that of seagrass leaves at each site.
NH4 + and NO3 − uptake were assessed using 15 N as a tracer
which allowed quantifying in situ and simultaneously the N uptake 2.4. Data analysis
of leaves and epiphytes under natural conditions. At each site,
divers deployed benthic chambers (three replicates) in the morn- Differences between sites in background nitrogen concentra-
ing. Each chamber consisted of a PVC cylinder (10 cm internal tions in water column, leaves and epiphytes, in shoot and leaf
diameter), which penetrated approximately 15 cm into the sed- biomass and epiphyte load were assessed by means of t-Test.
iment, a gas-tight but flexible polyethylene plastic bag (Hansen Two-way Analysis of Variance (ANOVA) was used to test possible
et al., 2000) and a port to add the solution. Such bags have been differences in leaf or epiphyte N uptake between sites, N sources
used widely for studying nutrient cycling in seagrass ecosystems (i.e. NH4 + and NO3 − incubation) and types of primary producer (i.e.
(e.g. Calleja et al., 2006; Barrón and Duarte, 2009; Apostolaki et al., leaf and epiphyte). Regression analysis was used in order to detect
2010), because they allow experimental incubation without dilu- possible relationship between epiphyte load, leaf and epiphyte N
tion, continuous stirring, or gaseous headspace while they are uptake.
60 E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62

Table 1
Background NH4 + and NO3 − in water column, ␦15 N and N in leaves and epiphytes, shoot and leaf biomass and epiphyte load at both sites (mean ± standard error, n = 3).
p-Values of t-Tests related to differences between sites are given. Shoot biomass was log10 transformed.

Sitia (fish farm) Psaromoura (control) t-Test


NH4 + (␮M) 2.20 ±0.10 0.60 ±0.13 0.001

NO3 − (␮M) 0.47 ±0.08 0.24 ±0.08 0.225

Shoot biomass (g DW m−2 sediment) 100.37 ±5.04 255.67 ±34.17 0.038

Leaf ␦15 N 4.50 ±0.17 2.45 ±0.11 0.001

Leaf N (% DW) 1.85 ±0.04 1.55 ±0.04 0.010
Leaf biomass (g DW m−2 sediment) 58.25 ±2.71 162.82 ±22.19 0.042

Epiphyte ␦15 N 8.26 ±0.03 3.78 ±0.29 0.004

Epiphyte N (% DW) 1.27 ±0.03 1.46 ±0.13 0.354
Epiphyte load (g DW g−1 DW leaf) 0.23 ±0.02 0.05 ±0.01 0.019

Table 2
␦15 N, specific N uptake and N uptake (mean ± standard error, n = 3) in leaves and epiphytes after an hour of incubation with NH4 + and NO3 − at both sites.

Sitia (fish farm) Psaromoura (control)

NH4 incubation Leaf ␦ N
5.64 ±0.26 4.39 ±0.24
Specific N uptake (␮g 15 N g−1 DW h−1 ) 0.04 ±0.01 0.02 ±0.002
N uptake (␮g 15 N m−2 sediment h−1 ) 4.49 ±1.46 17.85 ±1.30
Epiphyte ␦15 N 11.41 ±0.66 5.86 ±0.50
Specific N uptake (␮g 15 N g−1 DW h−1 ) 0.36 ±0.08 0.41 ±0.15
N uptake (␮g 15 N m−2 sediment h−1 ) 1.98 ±0.42 0.99 ±0.36

NO3 − incubation Leaf ␦15 N 5.99 ±0.20 4.40 ±0.36

Specific N uptake (␮g 15 N g−1 DW h−1 ) 0.06 ±0.01 0.02 ±0.003
N uptake (␮g 15 N m−2 sediment h−1 ) 5.86 ±1.65 17.88 ±6.25
Epiphyte ␦15 N 13.42 ±0.34 3.88 ±0.07
Specific N uptake (␮g 15 N g−1 DW h−1 ) 0.59 ±0.04 0.04 ±0.19
N uptake (␮g 15 N m−2 sediment h−1 ) 3.23 ±4.65 0.11 ±0.11

3. Results significantly higher specific uptake rate in Sitia when incubated

with NO3 + (F = 114.37, p = 0.0004) but not when incubated with
Background concentration of NH4 + in the water column, back- NH4 + (F = 0.09, p = 0.78).
ground ␦15 N in leaves and epiphytes and N in leaves were Site had a significant effect on N uptake (per unit area) in both
significantly higher in Sitia than in Psaromoura (Table 1). Epiphyte NH4 + and NO3 − incubations, but the effect depended on type of
load was also significantly higher in Sitia, while shoot and leaf primary producer (i.e. leaves vs. epiphytes) (Table 3b). Leaf uptake
biomass showed the reverse pattern (Table 1). was significantly higher in Psaromoura for both incubations (1-way
At the end of the experiments, incubated leaves were enriched ANOVA, p = 0.002 in NH4 + incubation and p = 0.02 in NO3 − incu-
up to 77% and epiphytes up to 62% relative to natural (i.e. back- bation). Epiphyte uptake, on the contrary, increased in Sitia but
ground) isotopic composition (Tables 1 and 2). this increase was only significant in case of NO3 − incubation (1-
Leaf epiphyte specific N uptake rate differed significantly way ANOVA, p = 0.15 in NH4 + incubation and p = 0.0002 in NO3 −
between sites (Table 3a), being higher in Sitia (Table 2), regard- incubation).
less of N source. The effect of site on epiphyte specific N uptake On average, leaf N uptake was 5.18 (±1.24 SE) and 17.87 (±2.08
depended on N source (Table 3a). A 1-way ANOVA between sites SE) ␮g 15 N m−2 sediment h−1 , while epiphyte N uptake was 2.61
but separately on each N source revealed that epiphytes showed (±0.50 SE) and 0.55 (±0.36 SE) ␮g 15 N m−2 sediment h−1 in Sitia

Table 3
ANOVA results examining the effects of a) Site and N source on leaf and epiphyte specific N uptake and b) Site and Type of Primary Producer on NH4+ and NO3− uptake.

Variable Source of variability df %SS p-Value

(a) Effect of Site and N source on specific N uptake

Leaf specific uptake Site 1 51 0.016
N source 1 3 0.488
Site × N source 1 3 0.490
Error 8
Epiphyte specific uptake Site 1 29 0.021
N source 1 2 0.446
Site × N source 1 41 0.009
Error 8

(b) Effect of Site and Type of Primary Producer on N uptake

NH4 + uptake Site 1 20 0.0003
Type of Primary Producer 1 49 0.00001
Site × Type of Primary Producer 1 27 0.0001
Error 8
NO3 − uptake Site 1 10 0.025
Type of Primary Producer 1 51 0.0002
Site × Type of Primary Producer 1 28 0.002
Error 8
E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62 61

25 4

m-2 sediment h-1)

m-2 sediment h-1) Leaves
3 Leaves


Epiphyte uptake (µg

Leaf uptake (µg



0 0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

Epiphyte load (g DW g -1 DW leaf)

Fig. 1. Linear regression of leaf and epiphyte N uptake with epiphyte loading on seagrass leaves. Significance levels (p values) are given. Leaves: y = −40.34x + 19.0, R2 = 0.71,
p = 0.0006. Epiphytes: y = 7.43x + 0.21, R2 = 0.75, p = 0.0003.

and Psaromoura, respectively (Table 2). With increasing epiphyte where also epiphyte load on leaves increased, indicates that epi-
load on seagrass leaves, leaf N uptake decreased, while epiphyte phyte overgrowth hindered leaf N uptake. Epiphytes, apart from
N uptake increased (Fig. 1). Leaf N uptake rate was negatively cor- shading and direct competition for nutrients with leaves (Tomasko
related with epiphyte N uptake (Linear Regression, Slope = −3.75, and Lapointe, 1991), have been shown to block active uptake sites
R2 = 0.45, p = 0.017). of T. testudinum leaves (Cornelisen and Thomas, 2004).
Effects of eutrophication on P. oceanica epiphytes are usually not
4. Discussion only quantitative (change in biomass) but also qualitative (change
in species composition) (e.g. Balata et al., 2010; Ben Brahim et al.,
Seagrass leaves and epiphytes of P. oceanica ecosystem 2010; Giovannetti et al., 2010). Lepoint et al. (2007) showed that
responded differently to nutrient enrichment from fish farm facil- crustose corallines covering pristine P. oceanica leaves take up N
ities, in terms of N uptake. Nutrient enrichment from fish farm 4 times faster than brown algae, suggesting that epiflora N uptake
waste affected the meadow in Sitia, as shown by the increase depends on algal species. However, Balata et al. (2010) showed in
in epiphyte load and decrease in shoot and leaf biomass which nutrient enriched P. oceanica meadows that there was a general
are widely documented in P. oceanica meadows impacted by fish increase in macroalgae cover, both in encrusting corallines and
farming (Pergent-Martini et al., 2006; Holmer et al., 2008). Leaves filamentous forms. Prado et al. (2008) measured increase in epi-
and epiphytes of the impacted meadow showed higher specific flora to epifauna ratio in an artificially enriched P. oceanica meadow
N uptake and yielded higher N and ␦15 N concentrations, suggest- but only in summer, where water temperature, light and nutrient
ing direct linkage between N uptake and accumulation. Like in our offer counterbalanced natural nutrient deficit in the water column
study, increase in N (Pérez et al., 2008; Apostolaki et al., 2009) and and stimulated algal overgrowth. Recently, Apostolaki et al. (2011)
␦15 N (Dolenec et al., 2006; Vizzini and Mazzola, 2006; Pérez et al., showed that epiphyte load on fish farm impacted P. oceanica leaves
2008; Lassauque et al., 2010; Ruiz et al., 2010) in leaves and epi- is enhanced all year around, because nutrients are discharged from
phytes close to fish farms suggests assimilation and accumulation fish cages throughout the year and compensate for the natural
of aquaculture-derived N. Although seagrasses show higher affin- fluctuation in nutrient availability. Therefore, although we did not
ity for NH4 + than NO3 − (Touchette and Burkholder, 2000), many study epiphytic composition, we can assume that epiflora species
terrestrial and marine plants, including seagrasses, show increased increased in Sitia due to nutrient enrichment from fish farm activ-
activity of NO3 reductase (the key enzyme involved in NO3 assimi- ity, especially since the study was conducted in summer, being
lation) when they receive higher pulse of water column NO3 − in day the main responsible for the observed higher epiphyte N uptake
time (Touchette and Burkholder, 2007). This was possibly the case compared to that in Psaromoura.
in our study which led to NO3 − uptake rates similar to NH4 + rates In Sitia, the contribution of epiphytes to total above-
in case of leaves and or even higher in case of epiphytes. It can be ground N uptake (32–36%) increased, while that of leaves
expected that the impacted meadow is used to increased nutrient (64–88%) decreased in relation to Psaromoura, where leaves
availability derived from fish farm waste and therefore takes up the contributed the 95.0–99.5% and epiphytes only the 0.5–5.0%
excess nutrients. Seagrass often show luxury consumption, taking to the total. However, despite increase in epiphyte contri-
up more nutrients than they need when the latter are available in bution, 2–3 times less N (155–218 ␮g 15 N m−2 sediment d−1
excess (Romero et al., 2006). and 432–452 ␮g 15 N m−2 sediment d−1 , in Sitia and Psaromoura,
Being fast-growing primary producers, epiphytes incorporated respectively) was transferred daily from the water column to the
N faster than leaves (8–10 and 2–18 times in Sitia and Psaromoura, benthic compartment, through seagrass and epiphyte uptake on
respectively) in relation to corresponding biomass, as previously total, at the impacted meadow. Recent data support that increased
observed for other seagrass meadows (Cornelisen and Thomas, epiphyte load could not counterbalance the decrease in commu-
2006; Lepoint et al., 2007; Nayar et al., 2010). Nevertheless, when nity carbon production in a fish farm impacted P. oceanica meadow
the effect of biomass was eliminated and uptake was assessed per (Apostolaki et al., 2011). Hence, the decrease in total N uptake
unit area, leaf and epiphytes showed reverse patterns. The fact that should be mainly attributed to the extensive loss of P. oceanica. In
N leaf uptake decreased while N epiphyte uptake increased in Sitia, fact, the decrease in leaf biomass, despite increased N concentration
62 E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62

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seagrass Posidonia oceanica (Monocotyledons, Posidoniaceae). J. Phycol. 43,
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gen budget of the seagrass Posidonia oceanica as determined by in situ uptake
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