Aquatic Botany 96 (2012) 58–62

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Aquatic Botany
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Short communication

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
a
Institute of Oceanography, Hellenic Centre for Marine Research, PO Box 2214, 71003, Heraklion, Crete, Greece
b
Marine Ecology Laboratory, Biology Department, University of Crete, PO Box 2208, 71409, Heraklion, Crete, Greece
c
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
Keywords:
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
15
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 +

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

0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquabot.2011.09.008
 E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62
2011) and it has been repeatedly related to a reduction in leaf pro-
duction (Borowitzka et al., 2006). Nevertheless, herbivores often
increase in nutrient enriched meadows controlling down the epi-
phyte abundance (Ruiz et al., 2009) and hence decreasing epiphyte
contribution to total N uptake of seagrass shoot. On the other hand,
Apostolaki et al. (2011) showed recently that, although herbivore
pressure increased, it did not counterbalance the effect of moderate
yet continuous nutrient input from fish farm discharges to the sur-
roundings, which shifted the control of epiphytes from top-down
to bottom-up and led to increased epiphyte load.
Therefore, we hypothesized that the main primary producers of
the P. oceanica ecosystem (seagrass and epiphytes) react differently
to nutrient enrichment in terms of N uptake, based on their differ-
ent physiology, their complex interaction and their interaction with
other components of the ecosystem. We aimed to study if nutrient
enrichment from fish farm waste affects N uptake by leaves and
epiphytes in a Mediterranean seagrass (P. oceanica) meadow. We
report leaf and epiphyte uptake of NH4 + and NO3 − in impacted and
pristine patchy seagrass meadows, using 15 NH4 and 15 NO3 as trac-
ers in in situ incubations. We also examine the effect of epiphyte
load on leaf N uptake.
2. Materials and methods
2.1. Sampling strategy
The experiment was conducted once in July 2007 in two patchy
P. oceanica meadows in Crete (Aegean Sea, Greece). The first site is
situated in a shallow bay in Sitia (35◦ 15.23 N, 26◦ 13.29 E). A fish
farm in the bay has been operating for 12 years, producing annu-
ally 1000 tons of gilthead sea bream (Sparus aurata) and sea bass
(Dicentrarchus labrax). The meadow is located as close to the fish
cages as possible, around 80 m away from both the fish farm and
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
et al., 2008). Therefore, the meadow in Sitia falls within the area
affected by fish farm waste. Further information on the study site
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,
25◦ 01.05 E) and represents a pristine site, where the meadow is
located 80 m away from the coast, at 4.9 m depth.
Background concentrations of NH4 + and NO3 − in water column
and ␦15 N and N in leaves and epiphytes were measured before each
experiment. At each site and just before the incubations, divers col-
lected triplicate water samples (400 ml each) above the seagrass
canopy, using acid-washed syringes. The samples were filtered
through pre-combusted glass fibre filters (Whatman GF/F), kept
frozen and later analyzed for NH4 + and NO3 − . From each site around
15 shoots of P. oceanica were retrieved by hand, kept frozen and
later used for measuring background ␦15 N and N concentration in
seagrass leaves and epiphytes, shoot and leaf biomass and epiphyte
load.
NH4 + and NO3 − uptake were assessed using 15 N as a tracer
which allowed quantifying in situ and simultaneously the N uptake
of leaves and epiphytes under natural conditions. At each site,
divers deployed benthic chambers (three replicates) in the morn-
ing. Each chamber consisted of a PVC cylinder (10 cm internal
diameter), which penetrated approximately 15 cm into the sed-
iment, a gas-tight but flexible polyethylene plastic bag (Hansen
et al., 2000) and a port to add the solution. Such bags have been
used widely for studying nutrient cycling in seagrass ecosystems
(e.g. Calleja et al., 2006; Barrón and Duarte, 2009; Apostolaki et al.,
2010), because they allow experimental incubation without dilu-
tion, continuous stirring, or gaseous headspace while they are
59
flexible and allow turbulence in the interior of the benthic cham-
bers, ensuring mixing.
Solutions of ammonium sulphate (99.0% 15 N) or sodium nitrate
(99.0% 15 N) (Eurisotop, France) of 0.06 ␮M concentration were
added in the chambers using syringes, in order to achieve around
20% enrichment of the initial ambient concentration in 15 NH4 or
15 NO , respectively. The incubation lasted only for an hour, in
3
accordance with similar studies (Lepoint et al., 2002, 2007), to avoid
nutrient depletion. At the end of the incubation, all seagrass shoots
from the chamber (∼10–15 shoots) were retrieved by hand and
kept frozen.
2.2. Laboratory analysis
In the laboratory, the water samples were analyzed manually
for NH4 + (Ivančič and Deggobis, 1984; detection limit 0.1–10 ␮M)
and NO3 − concentrations (Strickland and Parsons, 1972; detection
limit 0.05–4 ␮M). Epiphytes were gently removed from the leaves
using a razor blade and P. oceanica leaves and epiphytes were dried
at 70 ◦ C for 48 h, weighed and grounded to a fine powder. Total
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
reported in common delta (ı) units referred to atmospheric nitro-
gen standards.
2.3. Calculations
The excess 15 N incorporated in seagrass leaves and epiphytes
was calculated from the increase in the fraction (F) of 15 N after
labelling (Ft15 ) relative to the background F (Fb15 ), according to
Kiswara et al. (2005):
15
N = Ft15 − Fb15 ,
where F15 = R/(R + 1) and R = (ı/1000 + 1) × Rstandard , with
Rstandard = 0.0036765.
Specific N uptake rate (sNU, ␮g N g−1 DW h−1 ) was estimated as
(15 N × N)
sNU = ,
(W × t)
where 15 N is the excess 15 N, N the concentration of nitrogen (in
% DW), W the dry weight of incubated tissue (in g DW) and t the
duration of the incubation (h).
N uptake rate (NU, ␮g N m−2 sediment h−1 ) was computed as:
(15 N × N)
NU =
(W × t × s)
where s is the sediment surface (in m2 ) that was incubated.
Epiphyte load (g DW g−1 DW leaf) was calculated by dividing the
dry weight of epiphytes with that of seagrass leaves at each site.
2.4. Data analysis
Differences between sites in background nitrogen concentra-
tions in water column, leaves and epiphytes, in shoot and leaf
biomass and epiphyte load were assessed by means of t-Test.
Two-way Analysis of Variance (ANOVA) was used to test possible
differences in leaf or epiphyte N uptake between sites, N sources
(i.e. NH4 + and NO3 − incubation) and types of primary producer (i.e.
leaf and epiphyte). Regression analysis was used in order to detect
possible relationship between epiphyte load, leaf and epiphyte N
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

p-Value

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
15
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

Background concentration of NH4 + in the water column, back-
ground ␦15 N in leaves and epiphytes and N in leaves were
significantly higher in Sitia than in Psaromoura (Table 1). Epiphyte
load was also significantly higher in Sitia, while shoot and leaf
biomass showed the reverse pattern (Table 1).
At the end of the experiments, incubated leaves were enriched
up to 77% and epiphytes up to 62% relative to natural (i.e. back-
ground) isotopic composition (Tables 1 and 2).
Leaf epiphyte specific N uptake rate differed significantly
between sites (Table 3a), being higher in Sitia (Table 2), regard-
less of N source. The effect of site on epiphyte specific N uptake
depended on N source (Table 3a). A 1-way ANOVA between sites
but separately on each N source revealed that epiphytes showed
with NO3 + (F = 114.37, p = 0.0004) but not when incubated with
NH4 + (F = 0.09, p = 0.78).
Site had a significant effect on N uptake (per unit area) in both
NH4 + and NO3 − incubations, but the effect depended on type of
primary producer (i.e. leaves vs. epiphytes) (Table 3b). Leaf uptake
was significantly higher in Psaromoura for both incubations (1-way
ANOVA, p = 0.002 in NH4 + incubation and p = 0.02 in NO3 − incu-
bation). Epiphyte uptake, on the contrary, increased in Sitia but
this increase was only significant in case of NO3 − incubation (1-
way ANOVA, p = 0.15 in NH4 + incubation and p = 0.0002 in NO3 −
incubation).
On average, leaf N uptake was 5.18 (±1.24 SE) and 17.87 (±2.08
SE) ␮g 15 N m−2 sediment h−1 , while epiphyte N uptake was 2.61
(±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
25
m-2 sediment h-1)
20
15
15N
Leaf uptake (µg
10
5
0 0
0.00 0.05 0.10 0.15 0.20
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
load on seagrass leaves, leaf N uptake decreased, while epiphyte
N uptake increased (Fig. 1). Leaf N uptake rate was negatively cor-
related with epiphyte N uptake (Linear Regression, Slope = −3.75,
R2 = 0.45, p = 0.017).
4. Discussion
Seagrass leaves and epiphytes of P. oceanica ecosystem
responded differently to nutrient enrichment from fish farm facil-
ities, in terms of N uptake. Nutrient enrichment from fish farm
waste affected the meadow in Sitia, as shown by the increase
in epiphyte load and decrease in shoot and leaf biomass which
are widely documented in P. oceanica meadows impacted by fish
farming (Pergent-Martini et al., 2006; Holmer et al., 2008). Leaves
and epiphytes of the impacted meadow showed higher specific
N uptake and yielded higher N and ␦15 N concentrations, suggest-
ing direct linkage between N uptake and accumulation. Like in our
study, increase in N (Pérez et al., 2008; Apostolaki et al., 2009) and
␦15 N (Dolenec et al., 2006; Vizzini and Mazzola, 2006; Pérez et al.,
2008; Lassauque et al., 2010; Ruiz et al., 2010) in leaves and epi-
phytes close to fish farms suggests assimilation and accumulation
of aquaculture-derived N. Although seagrasses show higher affin-
ity for NH4 + than NO3 − (Touchette and Burkholder, 2000), many
terrestrial and marine plants, including seagrasses, show increased
activity of NO3 reductase (the key enzyme involved in NO3 assimi-
lation) when they receive higher pulse of water column NO3 − in day
time (Touchette and Burkholder, 2007). This was possibly the case
in our study which led to NO3 − uptake rates similar to NH4 + rates
in case of leaves and or even higher in case of epiphytes. It can be
expected that the impacted meadow is used to increased nutrient
availability derived from fish farm waste and therefore takes up the
excess nutrients. Seagrass often show luxury consumption, taking
up more nutrients than they need when the latter are available in
excess (Romero et al., 2006).
Being fast-growing primary producers, epiphytes incorporated
N faster than leaves (8–10 and 2–18 times in Sitia and Psaromoura,
respectively) in relation to corresponding biomass, as previously
observed for other seagrass meadows (Cornelisen and Thomas,
2006; Lepoint et al., 2007; Nayar et al., 2010). Nevertheless, when
the effect of biomass was eliminated and uptake was assessed per
unit area, leaf and epiphytes showed reverse patterns. The fact that
N leaf uptake decreased while N epiphyte uptake increased in Sitia,
61
4
m-2 sediment h-1)
Leaves
Epiphytes
3 Leaves
Epiphytes
15N
Epiphyte uptake (µg
2
1
0.25 0.30 0.35 0.40 0.45 0.50
where also epiphyte load on leaves increased, indicates that epi-
phyte overgrowth hindered leaf N uptake. Epiphytes, apart from
shading and direct competition for nutrients with leaves (Tomasko
and Lapointe, 1991), have been shown to block active uptake sites
of T. testudinum leaves (Cornelisen and Thomas, 2004).
Effects of eutrophication on P. oceanica epiphytes are usually not
only quantitative (change in biomass) but also qualitative (change
in species composition) (e.g. Balata et al., 2010; Ben Brahim et al.,
2010; Giovannetti et al., 2010). Lepoint et al. (2007) showed that
crustose corallines covering pristine P. oceanica leaves take up N
4 times faster than brown algae, suggesting that epiflora N uptake
depends on algal species. However, Balata et al. (2010) showed in
nutrient enriched P. oceanica meadows that there was a general
increase in macroalgae cover, both in encrusting corallines and
filamentous forms. Prado et al. (2008) measured increase in epi-
flora to epifauna ratio in an artificially enriched P. oceanica meadow
but only in summer, where water temperature, light and nutrient
offer counterbalanced natural nutrient deficit in the water column
and stimulated algal overgrowth. Recently, Apostolaki et al. (2011)
showed that epiphyte load on fish farm impacted P. oceanica leaves
is enhanced all year around, because nutrients are discharged from
fish cages throughout the year and compensate for the natural
fluctuation in nutrient availability. Therefore, although we did not
study epiphytic composition, we can assume that epiflora species
increased in Sitia due to nutrient enrichment from fish farm activ-
ity, especially since the study was conducted in summer, being
the main responsible for the observed higher epiphyte N uptake
compared to that in Psaromoura.
In Sitia, the contribution of epiphytes to total above-
ground N uptake (32–36%) increased, while that of leaves
(64–88%) decreased in relation to Psaromoura, where leaves
contributed the 95.0–99.5% and epiphytes only the 0.5–5.0%
to the total. However, despite increase in epiphyte contri-
bution, 2–3 times less N (155–218 ␮g 15 N m−2 sediment d−1
and 432–452 ␮g 15 N m−2 sediment d−1 , in Sitia and Psaromoura,
respectively) was transferred daily from the water column to the
benthic compartment, through seagrass and epiphyte uptake on
total, at the impacted meadow. Recent data support that increased
epiphyte load could not counterbalance the decrease in commu-
nity carbon production in a fish farm impacted P. oceanica meadow
(Apostolaki et al., 2011). Hence, the decrease in total N uptake
should be mainly attributed to the extensive loss of P. oceanica. In
fact, the decrease in leaf biomass, despite increased N concentration
62 E.T. Apostolaki et al. / Aquatic Botany 96 (2012) 58–62
in leaves, implies that the seagrass was incapable of capitalizing N
into growth.
Our study showed the different response of key primary pro-
ducers, seagrass leaves and epiphytes, in a nutrient enriched P.
oceanica meadow in terms of N uptake. Nonetheless, further stud-
ies are needed to verify the above findings in other P. oceanica
meadows, as well as other seagrass species, under various anthro-
pogenic pressures. Additional experimentation is needed to detect
if N uptake by roots and relative contribution of leaves and roots to
total shoot uptake changes with anthropogenic pressure. Finally,
complex interactions with other components of the ecosystem,
such as herbivores that affect epiphytes (Prado et al., 2007), are
worth being explored in terms of N uptake in threatened seagrass
meadows.
Acknowledgments
This work was funded by Aqua-Planner Project (INTERREG III
Greece – Cyprus 2000–2006). Thanks are due to I. Glabedakis, S.
Dilliberto, M. Kanakakis, C. Fragiadaki and Z. Grigorakis for assis-
tance with sampling. We are grateful to Dr Jan Vermaat (Editor) and
two anonymous reviewers for helpful comments and criticism on
the manuscript.
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