ORIGINAL PAPER

Distinctive types of leaf tissue damage influence nutrient supplyto growing tissues within seagrass shoots

Patricia Prado • Catherine J. Collier •

Javier Romero • Teresa Alcoverro

Received: 20 September 2010 / Accepted: 28 February 2011 / Published online: 11 March 2011

� Springer-Verlag 2011

Abstract Herbivory is now recognized as an important

structuring agent in seagrass meadows but the attack pat-

tern and tissue damage of consumers are highly variable.

Nutritional preferences of herbivores and/or easy access to

resources may cause differences in biomass loss among

tissues that damage the plant in functionally distinctive

ways. The two main Mediterranean herbivores, the fish

Sarpa salpa (L.) and the sea urchin Paracentrotus lividus

(Lmk.), remove higher amounts of intermediate and

external shoot leaves, respectively. To test whether this

selective feeding can have different consequences on the

allocation patterns of nutrient within plants, we simulated

the effect of both herbivores by clipping external and

intermediate leaves (plus unclipped controls) of Posidonia

oceanica (L.) and we measured plant tolerance in terms

of shoot growth and leaf nutrient supply to new tissue

using isotopic markers. As expected, control treatments

displayed high carbon and nutrient supply from external

leaves (83% of the total 15N and 84% of the total 13C

incorporated by the shoot). When subjected to clipping, the

remaining leaves enhanced carbon and nitrogen supply

compared with the control by 16% of N and 36% of C—in

the intermediate clipping—and by over 100% of N and

200% of C—in the external clipping—to compensate for

the nutrient lost. However, only in the case of fish her-

bivory (intermediate clipping), enhanced supply alone was

able to fully compensate for the nutrient losses. In contrast,

this mechanism is not completely effective when external

leaves are clipped (urchin herbivory). Yet, the conse-

quences of this nutrient loss under sea urchin herbivory are

not apparent from the nutrient content of the new tissue,

suggesting that there are other sources of nitrogen income

(uptake or reallocation from rhizomes). Our study does

not only confirm the tolerance of P. oceanica to herbivory,

but also constitutes the first evidence of leaf-specific,

compensatory nutrient supply in seagrasses.

Introduction

Removal of tissue by grazers causes structural damage and

nutrient and carbohydrate losses, and may ultimately

reduce resource acquisition, growth, and reproduction

(Marquis 1992; Crawley 1997). These potential deleterious

effects can be alleviated through diverse mechanisms

including adaptive plant structure and growth habit (Liu

et al. 2007), reallocation of assimilates from remaining

leaves (Sosebee and Wiebe 1971; Ryle and Powell 1975)

and rhizomes (Harnett 1989; Verges et al. 2008), increased

photosynthetic rates in the remaining tissues (Detling et al.

1979; Caldwell et al. 1981), modifications of the hormonal

balance (Avery and Briggs 1968; Avery and Lacey 1968),

Communicated by P. Ralph.

P. Prado (&)

Institut de Recerca i Tecnologia Agroalimentaries,

Sant Carles de la Rapita, Carretera Poble Nou, km 5.5,

43540 Sant Carles de la Rapita, Tarragona, Spain

e-mail: patricia.prado@irta.cat

C. J. Collier

School of Marine and Tropical Biology,

James Cook University, Townsville, QLD 4811, Australia

J. Romero

Departamento de Ecologıa, Facultad de Biologıa,

Universidad de Barcelona, Avda. Diagonal 645,

08028 Barcelona, Spain

T. Alcoverro

Centre d’Estudis Avancats de Blanes, CSIC, Ctra. Acceso Cala

St. Francesc, 14, 17300 Blanes, Girona, Spain

123

Mar Biol (2011) 158:1473–1482

DOI 10.1007/s00227-011-1664-0

or increased nutrient uptake rates from roots (Caldwell

et al. 1981). The mechanisms in place are, in part, depen-

dent on the intensity of the grazing pressure. However,

nutritional preferences and behavioral aspects of herbivore

feeding (e.g. resource accessibility and/or aggregative

feeding behavior) may also damage the plant in function-

ally distinctive ways that can influence plant fitness as

much as the overall quantities of tissue lost (review by

Kotanen and Rosenthal 2000). Damage by vertebrates is

often spatially and temporally stochastic (Varnamkhasti

et al. 1995) and can be sudden and severe, a reflection of

their large size and basal metabolic requirements

(Demment and Soest 1985). In contrast, invertebrate

attacks often involve prolonged removal of small amounts

of tissue, which causes a continuous drain of resources (e.g.

Fay et al. 1996; Zimmerman et al. 2001). Tissue specificity

is generally greater for species of invertebrates than for

vertebrates (Strong et al. 1984; Crawley 1989) and,

depending on damage type, often causes indirect impacts

on plant physiology.

Seagrass beds have been shown to withstand intense

rates of herbivore activity (see review by Heck and

Valentine 2006). Several types of herbivores feed on

seagrasses (e.g. Sirenians, green turtles, fishes, and sea

urchins), and they often coexist but exhibit very distinctive

ways of removing plant material. Feeding by Sirenians (i.e.

dugongs and manatees) often involves the removal of the

entire plant, including roots and rhizomes, and favors the

dominance of early- or mid-successional species in fre-

quently grazed meadows (Aragones and Marsh 2000;

Aragones et al. 2006). Green turtles have a preference for

young, actively growing tissues but, by cropping the sea-

grass just a few centimeters above the surface of the sub-

strate, large blade areas above the cropped region are also

lost (Bjorndal 1980; Thayer et al. 1984). Since large sea-

grass vertebrates remove plant tissue in bulk, tolerance to

these herbivores may often be linked to more general

genotypic traits such as clonal morphology, high rates of

leaf turnover, and flexible allocation of resources (Nakaoka

and Aioi 1999; Aragones et al. 2006). In contrast, patterns

of grazing by fishes are more diverse and either show

relatively homogeneous leaf mowing (Tomas et al. 2005;

Ferrari et al. 2007) or are intensely patchy, causing the

formation of ‘‘halos’’ (Randall 1965; McAfee and Morgan

1996), while selective consumption of epiphytised blade

tips has also been reported (Lobel and Ogden 1981). All

these can depend on factors such as seagrass and herbivore

abundance, habitat heterogeneity and complexity and pat-

terns of movement of the species (Macia and Robbinson

2005; Unsworth et al. 2007; Prado et al. 2008a). Marine

invertebrates, such as sea urchins that are in permanent

contact with the sea bottom, tend to feed on the more down

leaning accessible sections of the plant such as external

leaves (Shepherd 1987) or basal meristems if they are

exposed (Alcoverro and Mariani 2002). The feeding

behaviors of fish and urchins damage plants in functionally

distinct ways, and as a result, the response of the plant,

including nutritional compensatory mechanisms, may also

differ. Growth-compensatory responses like enhanced

levels of N-metabolizing enzymes and/or post-damage

reallocation of internal resources may be common mech-

anisms to tolerate more idiosyncratic types of seagrass

damage (Zimmerman et al. 2001; Valentine et al. 2004;

Verges et al. 2008) but effects under tissue-specific herbi-

vore attack are still unknown.

Posidonia oceanica (L.) is the dominant seagrass spe-

cies in oligotrophic Mediterranean waters. It features an

exceptionally long leaf life span among seagrasses (up to

300 days, Romero 1989), which may account for some of

the highest contributions of nutrient resorption to meet

growth requirements ever reported (up to 40%, Alcoverro

et al. 2000). In addition, plant losses to herbivores may be

alleviated by enhanced reallocation from stored reserves,

but not by increasing the photosynthetic rate (Verges et al.

2008). In these ecosystems, two important types of leaf

macrograzers coexist, the Sparid fish Sarpa salpa (L.) and

the sea urchin Paracentrotus lividus (Lmk.). Regionally,

these herbivores may remove, on average, 57% of the

annual leaf primary production of the plant in shallow

(5 m) meadows (Prado et al. 2007). However, grazing

pressure and herbivore abundance are very heterogeneous

in both time and space. Some locations have very high sea

urchin abundances (up to ca. 20 ind. m-2), and during the

period of higher herbivory (i.e. August–September), they

can consume up to 139% of monthly leaf primary pro-

duction. In other locations, fishes are the dominant herbi-

vores with consumption rates that in summer may exceed

local leaf production by 250% (Prado et al. 2007). The fish

S. salpa is a mid-water forager and thus, usually removes

the older sections of intermediate, upright leaves whereas

sea urchins more commonly consume the older, down

leaning sections of external leaves (Shepherd 1987;

Cebrian et al. 1996a; Pinna et al. 2009), each together with

differential epiphytic communities developed with leaf age

(Prado et al. 2010). Hence, these distinctive grazing

behaviors may interact with the dynamic balance between

the loss of old leaves and the production of new ones

(Sand-Jensen et al. 1994). The objectives of this study were

to evaluate, by herbivory simulation experiments, the

effects of two types of herbivore damage (fish loss of the

upper part of the intermediate leaves and sea urchin loss of

the upper part of the external leaves) on seagrass perfor-

mance. More specifically, we used isotopic markers to

(1) identify possible changes in leaf nutrient supply to

young emergent leaves and (2) to assess tissue growth and

the nutritional status of young leaves.

1474 Mar Biol (2011) 158:1473–1482

123

Materials and methods

Study site and experimental design

We first aimed to confirm whether sea urchin and fish

displayed distinctive feeding, potentially causing differen-

tial leaf damage. To do this, we reanalyzed data of leaf

consumption by Prado et al. (2007) obtained in 10 different

shallow sites selected according to regional availability and

accessibility and at four different times during the year (i.e.

one per season), using tethered shoots (n = 20 per site and

season). Mean averages of external and intermediate leaf

loss to each grazer per site and season (n = 40) were tested

with paired t tests and confirmed higher feeding rates by

the sea urchin P. lividus in external leaves (t = 6.01,

df = 40, P \ 0.001) and higher rates in intermediate leaves

by the fish S. salpa (t = 5.41, df = 40, P \ 0.001; Fig. 1).

Plant responses (shoot elongation and nutrient supply)

of P. oceanica shoots subjected to these two types of

herbivore damage (sea urchin and fish) were then evaluated

by a clipping experiment in a seagrass meadow in Cala

Giverola (41�440 N, 002�570 E, NW Mediterranean) during

late summer (early to mid-September)—the period of

highest herbivory pressure (Prado et al. 2007). We worked

at 5 m depth, in a patchy, semi-exposed seagrass meadow.

Nutrient conditions were oligotrophic with concentrations

of about 0.07 lM NH4, 0.96 lM NO3, and 0.29 lM PO4

(annual averages), and seawater temperature ranged

between 22 and 24�C (Cebrian et al. 1996b).

A total of 60 seagrass shoots were carefully selected

underwater making sure that each had five leaves (i.e. two

external, two intermediate, and one internal), over an area of

20 m2. Treatments consisted of the factorial combination of

two labeling positions (external leaves labeled and inter-

mediate leaves labeled) and three clipping levels (no clip-

ping, clipping in intermediate leaves, clipping in external

leaves), thus resulting in six experimental conditions (see

Fig. 2), each with ten replicated shoots. The treatments were

assigned at random over the 20 m2 area. Clipping consisted

of the removal of up to ca. 45 cm of leaf (either intermediate

or external, generally less for intermediate leaves), which is

roughly equivalent to the average value removed per month

during the August–September herbivory peak (Prado et al.

2007). The remaining portion of the clipped leaf was around

15 cm, to ensure similar surface area for uptake during

incubation. All experimental shoots were hole-punched with

a needle to measure leaf production according to Romero

(1989) and then marked with a peg inserted in the sediment

for later retrieval and treatment identification.

Isotopic incubation

N and C stable isotopes were used as traces to elucidate the

effect of clipping of leaves on the uptake and reallocation

of these elements toward growing tissues. Natural levels of

these isotopes are low and quite consistent under uniform

environmental conditions, therefore, when supplied, they

can be easily detected and traced above background levels.

To this end, clipped or unclipped external and intermediate

leaves, depending on the treatment (see Fig. 2 for details),

were incubated within an isotopic media. The incubation

chamber was made of a plastic bag fitted with a 50-mm

syringe filter holder and a one-way plastic stopcock (both

from Cole–Parmer) that allowed closure after isotopic

injection. Bags were pulled over the incubated leaves and

then sealed at the base with a padded clip. The efficiency of

the seal was assessed by previous in situ trials using col-

orants and showed no visual leakage (Prado et al. 2008b).

For incubation of unclipped leaves, the chamber was

0.5 l, whereas for clipped leaves (ca. 15 cm), the chamber

was 0.25 l (Fig. 2). This ensured a more consistent leaf

area to volume ratio between the treatments and allowed a

more adequate fastening of the plastic bag to the base of

the leaf. The chambers were filled by injecting seawater

with a syringe to the desired volume, and then, the isotopic

solution was injected. For all treatments, the injected

solution contained NaH13CO3 and 15NH4Cl to obtain a

final concentration of 300 lM NaH13CO3 and 40 lM15NH4Cl within the chamber (Marba et al. 2002; Prado

et al. 2008b). Nitrogen was supplied as NH4?, for which

seagrasses have higher uptake affinity than for NO3, in

order to maximize enrichment (Touchette and Burkholder

2000). Leaves were left to incubate for 4 h, and then, the

chambers were removed by taking away the bag while

pinching at the base to retain the fluid within the bag.

Throughout removal, seawater was rapidly moved across

*

*

Def

olia

tion

rate

s (c

m2 s

hoot

-1 d

-1)

0.0

0.1

0.2

0.3

0.4

S. salpa P. lividus

External Intermediate External Intermediate

***

***

Fig. 1 Defoliation rate (cm2 shoot-1 day-1) of external and inter-

mediate leaves by the fish S. salpa and the sea urchin P. lividus(mean ± SE). Values were obtained from seasonal values in Prado

et al. (2007) across the study region (n = 40). Significant differences

in defoliation rates among leaf positions for each herbivore are

indicated with asterisks (***P \ 0.001)

Mar Biol (2011) 158:1473–1482 1475

123

the shoot to prevent possible external contamination of

unlabeled leaves. Isotope-marked shoots were left at the

study site for 15 days to allow for transport of nutrients

from the labeled leaves to the rest, including very young

leaves (primords) produced during the experimental period.

Then, all shoots, including leaves and a section of the

associated rhizome, were retrieved and carefully placed

into plastic bags for further processing in the laboratory.

Non-labeled shoots of P. oceanica were also collected

within the area in order to determine ambient values of

d15N and d13C in internal leaves. Additionally, ten shoots,

which were immediately adjacent to the labeled shoots, but

not connected by any rhizome, were collected in order to

further verify whether there were possible leakages from

the incubation chambers (see Table 1).

Sample processing

In the laboratory, epiphytes were gently removed from

the leaves with a razor blade. Leaves and rhizomes from

each shoot were separated and their length and width

measured. Leaf production was assessed according to

Romero (1989) for all leaves within a shoot. Newly

grown sections were cut out, measured, dried, and

weighed for each leaf (mg day-1). Internal leaves and

new growing primords (usually a single translucent leaf

of 0.5–1 cm length) were dried and ground together with

the original internal leaf to a fine powder in a Retsch

mixer mill (MM 200) and the combined material used

for isotopic determination.

d13C and d15N isotope values of samples were deter-

mined using an ANCA-NT (Europa Scientific, Crewe, UK)

interfaced with a 20–20-isotope ratio mass spectrometer

(Europa Scientific, Crewe, UK). Isotope ratios in the

samples are calculated from linear calibration curves con-

structed with standard reference materials of known com-

position and a blank correction. The average difference in

isotopic composition between samples and reference

material is determined by the equation:

½ðRsample � RstandardÞ=ðRstandardÞ� � 1000 ¼ dsample�standard

where Rsample is 13C/12C (or 15N/14N) in the sample;

Rstandard is 13C/12C (or 15N/14N) in the working reference

gas (PBD carbonate standard for d13C and N2 for d15N)

which is calibrated against an internal standard (Atropina,

IAEA, and/or UGS), and dsample-standard is the difference in

isotopic composition of the sample relative to that of the

reference, expressed in %.

Delta values for leaves and rhizome were first converted

to atom %15N and atom %13C using the following equa-

tions (Gonfiantini et al. 1995):

Atom %15Ni ¼ 100=½272=½1þ ðd15Ni=air=1000Þ� þ 1�Atom %13Ci ¼ 100=½89=½1þ ðd13Ci=standard=1000Þ� þ 1�

Atom % excess of d13C and d15N in internal leaves and

primords was calculated by subtracting Atom % values in

reference leaves and rhizomes and then, transforming them

in mg of 15N and 13C for each clipping and labeling

treatment. In addition, for each clipping treatment (i.e.

unclipped, external and intermediate leaves clipped), we

estimated total nutrient contribution that each shoot makes

to the growth of internal leaves and primords by adding the

mg of isotopic material (15N and 13C) supplied by

intermediate and external leaves.

Unclipped

LILE

External clipped

LILE

Intermediate clipped

EL IL

a b

c d

e f

Fig. 2 Illustration of the six different experimental treatments

accordingly to the position of the clipped and the labeled leaves.

a Unclipped, external labeled, b unclipped, intermediate labeled,

c external clipped, external labeled, d external clipped, intermediate

labeled, e intermediate clipped, external labeled, f intermediate

clipped, intermediate labeled. An arrow indicates the position of the

internal leaf

1476 Mar Biol (2011) 158:1473–1482

123

Data analyses

The effect of each clipping treatment (fixed factor, 3 levels:

unclipped, external leaves clipped, and intermediate leaves

clipped) on the mass of 15N and 13C supplied to the internal

leaf and new emerging primords from each leaf

position (fixed factor, 2 levels: external leaves marked and

intermediate leaves marked) was investigated with a two-

way factorial ANOVA. The effect of the clipping treat-

ment (fixed factor, 3 levels) on biomass production

(mg DW day-1) and N content of internal leaves and

primords and on the total production per shoot was

investigated with one-way ANOVA.

For all ANOVAs, data were first tested for homogeneity

of variances (Cochran’s test) and normality (Kolmogorov–

Smirnov distribution-fitting test of the residuals) and

transformed when necessary to satisfy these assumptions.

The existence of significantly different groupings was

investigated with Student–Newman–Keuls post hoc

comparisons.

Results

Patterns of 15N and 13C supply to growing tissues

Basal isotopic signatures (obtained from shoots far apart

from those labeled) in the internal leaf and primords were

6.67 ± 1.5 ppt for d15N and -11.72 ± 0.28 ppt for d13C.

Procedural controls (shoots collected near the labeled ones)

had signatures similar to these basal values (5.72 ± 0.36 ppt

for d15N and -11.93 ± 0.20 ppt for d13C) and were sub-

stantially lower than those of labeled plants (see Table 1),

indicating that leakage from the plastic bag during incuba-

tion was negligible.

The amounts of 15N and 13C that were supplied to the

internal leaf and primords varied significantly among the

six experimental combinations of clipping and labeling (i.e.

clipping 9 labeling interaction, Table 2a). The highest

contributions were observed in treatments with the inter-

mediate leaves clipped and the external leaves labeled and

in controls with the external leaves labeled; and the lowest

in treatments in which intermediate and external leaves had

been simultaneously clipped and labeled (see Fig. 3). Both

nutrients followed the same trend although the supply of

nitrogen from external leaves was not significantly differ-

ent from that of unclipped leaves whereas the supply of

carbon was increased when intermediate leaves were

clipped (Fig. 3a, b).

The effects of the clipping treatment alone (i.e. the

supply of external and intermediate leaves together)

showed that the capacity of intermediate and external

leaves to contribute to internal leaves and primords was

different (i.e. a significant clipping effect, Table 2b). The

amount of nitrogen and carbon supplied to young leaves

was higher in unclipped shoots (0.028 ± 0.007 mg of 15N

and 0.030 ± 0.005 mg of 13C), than in shoots with external

leaves clipped (0.010 ± 0.002 mg of 15N and 0.014 ±

0.004 mg of 13C) as increased supply from intermediate

leaves (109% and 208% increase in N and C supply,

respectively; see Fig. 3) could not compensate for the

reduced supply from old leaves. In contrast, clipping of

intermediate leaves did not change the overall amounts of

isotope material supplied to newly growing leaves

(0.028 ± 0.006 mg of 15N and 0.034 ± 0.015 mg of 13C),

as they have modest contributions and the supply from

external leaves increased by 16% of N and 36% of C

(Fig. 3). Hence, modest increases in the nutrient supply

from external leaves but not large increases in the supply

from intermediate leaves are able to compensate nutrient

contributions to the internal leaf and primords (Fig. 3). In

fact, unclipped external leaves supplied ca. fivefold more15N and 13C than unclipped intermediate ones, suggesting

that old leaves might have a major role as a source of C and

N of new growing leaves.

The reduced supply of 15N in shoots with external leaves

clipped did not result, however, in any significant decline in

the total nitrogen content in the internal leaf and primords

(Table 2c) since similar values of nitrogen content were

observed in all treatments (Unclipped: 2.26 ± 0.09%N;

External Clipped: 2.26 ± 0.08%N; Intermediate clipped:

2.39 ± 0.09%N).

Table 1 C:N ratio, d15N and d13C in the internal leaf and primords of P. oceanica (mean ± SE) by experimental treatment (Unclipped,

External clipped and Intermediate clipped) (n = 10)

Treatment Unclipped External clipped Intermediate clipped

EL IL EL IL EL IL

C:N 16.23 ± 0.45 19.85 ± 1.03 17.50 ± 0.71 19.15 ± 1.29 16.27 ± 0.83 18.02 ± 0.50

d15N 1528.8 ± 326 787.1 ± 130 110.2 ± 52 1797.7 ± 707 2267.8 ± 493 88.02 ± 41

d13C 29.73 ± 8.5 0.59 ± 2.9 -11.2 ± 0.5 29.91 ± 13.5 29.59 ± 12.6 -10.88 ± 0.8

EL External labeling, IL intermediate labeling

Mar Biol (2011) 158:1473–1482 1477

123

Leaf production

No significant effects of clipping were observed either in

the biomass production of internal leaf and primords

(Fig. 4; Table 2d) or in the total leaf biomass production

(mg DW day-1 shoot-1) as the values of these parameters

remained unaltered relative to control shoots (Fig. 4).

Table 2 ANOVA results for (a) 15N and 13C in the internal leaf and

primords per clipping treatment (unclipped, external clipped and

intermediate clipped) and leaf labeling (external and intermediate);

(b) total (i.e. isotopic mass from external and intermediate leaves

summed) 15N and 13C in the internal leaf and primords per clipping

treatment; (c) nitrogen content in the internal leaf and primords per

clipping treatment and; (d) leaf production of the internal leaves and

primords and of the whole shoot per clipping treatment

ANOVA

(a) Isotope mass per treatment

df 15N 13C

MS F ratio P MS F ratio P

Clipping = C 2 0.00 2.94 0.062 0.001 0.999 0.37

Labeling = L 1 0.002 12.08 0.01 0.003 5.060 0.029

C 9 L 2 0.002 10.05 0.000 0.003 5.784 0.005

Error 53 0.0002 0.00052

Transformation:

Cochran C = 0.45

Transformation:

Cochran C = 0.53

(b) Total isotope mass per clipping treatment

df 15N 13C

MS F ratio P MS F ratio P

Clipping = C 2 7.695 4.045 0.029 18.02 3.52 0.044

Error 27 0.0038 0.0056

Transformation: Hx

Cochran C = 0.43

Transformation: Hx

Cochran C = 0.55

(c) N content the internal leaf and primords

df MS F ratio P

Clipping = C 2 0.11 0.73 0.48

Error 56 0.1573

Transformation:

Cochran C = 0.36

Leaf production

(d) Internal leaf and primords Shoot

df MS F ratio P MS F ratio P

Clipping = C 2 0.03 1.56 0.22 0.01 0.89 0.41

Error 56 0.0017 0.0056

Transformation: Hx

Cochran C = 0.42

Transformation: HHx

Cochran C = 0.43

Significant P values are indicated in bold

0.00

0.01

0.02

0.03

0.04

0.05

0.06

a a

d c

d

b

External Clipped

Intermediate Clipped

Unclipped leaves

mg

15N

in th

e in

tern

al le

af a

nd p

rimor

ds

EL IL EL IL EL IL

Labeling treatment

0.00

0.01

0.02

0.03

0.04

0.05

0.06

b

b

e

d

c

a

e

mg

13C

in th

e in

tern

al le

af a

nd p

rimor

ds

EL IL EL IL EL IL

a

Fig. 3 a mg 15N and b mg 13C (mean ± SE), in the internal leaf and

primords of P. oceanica for each clipping and labeling treatment.

In SNK, significantly different groups are indicated with letters

(n = 10). Detail as in Fig. 2

Clipping Treatment

U IC EC

Leaf

pro

duct

ion

( mg

DW

d-1

)

0

1

2

3

4

5

6

7

Internal leaf and primords Total shoot

U IC EC

N.S.

N.S.

Fig. 4 Primary production (mg DW shoot-1) of the internal leaf and

primords (mean ± SE) and of the entire shoot (n = 10). U Unclipped,

EC external clipped, IC intermediate clipped, N�S. not significant

effect

1478 Mar Biol (2011) 158:1473–1482

123

Discussion

The seagrass P. oceanica is highly tolerant of herbivore

damage, sustaining leaf growth and maintaining constant

nutrient concentration in growing leaves when subjected to

considerable tissue losses that mimic the action of either

sea urchin or fish. Although tolerance to herbivory has been

previously reported for this species in terms of leaf growth

(Tomas et al. 2005; Verges et al. 2008), here we show that

plants are able to partially compensate for tissue losses by

increasing nutrient supply from both intermediate and

external leaves but that the importance of this process is

largely specific to leaf age features. When the overall shoot

supply could not be fully compensated, the total nutrient

content of the new tissue grown and growth rates of young

leaves were still unaltered during the experimental period

(Fig. 5), thus indicating that simultaneous contribution

from other sources also occurs (e.g. adjacent shoots or

rhizomes).

Isotope tracing studies have long been conducted to

assess the importance of resource sharing among adjacent

seagrass shoots (e.g. Harrison 1978; Libes and Boudour-

esque 1987; Marba et al. 2002; Prado et al. 2008b); how-

ever, to the best of our knowledge, never to address

compensation following distinctive leaf damage by marine

herbivores. Fish herbivory (simulated by clipping inter-

mediate leaves) caused either equal (for N) or even slightly

higher (for C) supply from external leaves to newly

growing leaves than that of control plants. In contrast,

when grazing by sea urchins occurs (simulated by clipping

external leaves), supply from intermediate leaves was also

stimulated, but was not effective enough to maintain the

overall supply of C and N, which decreased by 62% and

50% (N and C, respectively). These patterns of nutrient

supply can be explained by uneven basal nutrient

contributions among seagrass leaves as the oldest; most

external leaves are major sources of C and N whereas those

of intermediate age are only minor contributors (83% vs.

17% of the C supply and 84% vs. 16% of the N supply

from external and intermediate leaves, respectively).

External, fully grown leaves may have similar rates of N

uptake per unit weight compared with intermediate age

leaves and appear as important organs providing a con-

tinuous supply of nutrients and photosynthates to growing

tissues (Borum et al. 1989; Pedersen et al. 1997). External

leaves also have slightly larger surface areas than inter-

mediate leaves (ca. 10 cm2) and may achieve a higher

incorporation of nutrients from the media. Yet, unclipped

intermediate leaves provided 3.7 times less 15N and 4 times

less 13C than expected by area differences alone. Therefore,

leaf age features connected to the transition period from

sink to source status (Chabot and Hicks 1982) and, to a

lesser extent, higher rates of nutrient uptake induced by

greater leaf area appear to influence the enhanced contri-

bution of external leaves to the overall nutrient supply to

growing tissues. In fact, P. oceanica features the longest

leaf life span and the highest N resorption among sea-

grasses (ca. 40% of requirements; Alcoverro et al. 2000),

which provides further indirect evidence that nutrient

supply from external leaves is an important mechanism of

compensation in this species.

The influence of leaf age features in physiological

integration among plant parts and/or organs might also be

affected by other structural and developmental factors such

as plant anatomy and growth morphology as well as with

specific adaptations to the physical environment. For

instance, Honkanen et al. (1999) studied the effect of age-

needle defoliation on Pinus sylvestris trees and found that

the loss of 2-year-old needles of the branch leader shoot did

not affect productivity whereas loss of 1-year-old needless

reduced the mass and length of needles in the new shoots.

In contrast, plants without true specialized organs and

vascular connections, such as the alga Fucus vesiculosus,

appear to lack any ability to respond to any type of tissue

losses (Honkanen and Jormalainen 2002) and may persist

through herbivore grazing by adopting faster growth

strategies compared with higher plants. In seagrasses,

previous tracing studies suggest that sharing of nutrients

among shoots along the main rhizome is comparatively less

important (up to 5 times lower) than within-shoot reallo-

cation from external leaves (see Prado et al. 2008b) pos-

sibly, as a result of the large contribution of leaves to

nutrient acquisition (up to 60–70% of their total N uptake;

Hemminga et al. 1991). In our experiment, external leaves

were only able to increase nutrient supply to a small

amount under plant damage (16% for 15N and 36% for 13C

when intermediate leaves were clipped) but this was

enough to fully compensate the loss of intermediate leaves.

0

1

2

3

4

5

6

7

N.S.

N.S.

Clipping Treatment U IC EC U IC EC

Leaf

pro

duct

ion

(mg

DW

d-1

)

Total shootInternal leaf and primords

Fig. 5 Primary production (mg DW shoot-1) of the internal leaf and

primords (mean ± SE) and of the entire shoot (n = 10). Detail as in

Fig. 4. N.S. Not significant effect

Mar Biol (2011) 158:1473–1482 1479

123

In contrast, supply from intermediate seagrass leaves was

unable to compensate for the loss of external leaves,

despite an increase in the supply of over 100% 15N and

over 200% 13C, possibly because they are not yet fully

developed and have more active growth (ca. 11–17% of

total shoot production). Valentine et al. (2004) also found

that clipping of external leaves of Thalassia testudinum

increased the level of activity of nitrogen-metabolizing

enzymes, thus suggesting that they are fully active and may

be important organs sustaining growing tissues.

At first, given the absence of negative effects in the

nutrient budget when intermediate leaves were clipped, and

the decrease in the amounts of nutrients supplied when

external leaves were removed, it might appear that con-

tinuous sea urchin grazing could affect the plant nutrient

budget much more than fish grazing. Yet, this assertion has

several counterarguments. First, losing intermediate leaves

in summer could have negative consequences on mid-term

processes. In effect, after a few months, intermediate leaves

become external and, if part of their biomass has been lost,

their role as nutrient source could be curtailed. Second, and

probably more importantly, the seagrass shoot-specific

growth does not seem to be depressed by this tissue loss, or

at least not in the short to mid-term (Verges et al. 2008; this

study), which confirms the capacity of P. oceanica for

compensation and suggests that other sources of nitrogen

income (leaf uptake, reallocation from rhizomes, or adja-

cent ramets) might also be enhanced. For instance, Verges

et al. (2008) conducted a clipping experiment in which

treatments were applied independently of leaf age and

found that reallocation of nutrients stored in belowground

rhizomes is an important mechanism of compensation, at

least in P. oceanica. Shoots may also boost their nutrient

content by increasing leaf nutrient uptake, an important

mechanism of nutrient acquisition in seagrasses, even at

low nutrient concentrations (Pedersen et al. 1997; Lepoint

et al. 2002; Romero et al. 2007). During the experiment, we

measured the nutrient supply to internal, newly growing

leaves, and therefore, the possible effects of clipping on

nutrient uptake cannot be fully separated from that of

transport to new growing leaves. Yet, the large differences

in nutrient supply capacity between unclipped intermediate

leaves and expected amounts for external leaves with the

same area suggest that compensatory uptake was not the

main responsible mechanism.

Little is still known about mechanisms driving distinctive

leaf damage by macrograzers. A first hypothesis would be a

simple matter of accessibility, with more intermediate,

upright leaves being more reachable to fish while older,

external leaning ones more reachable to sea urchins. How-

ever, very recent work has shown that there is also a pref-

erential consumption, in part mediated by distinctive

epiphyte communities (Prado et al. 2010; Verges et al.,

submitted). Yet, the lack of other studies reporting leaf age-

dependent herbivory and mechanisms of tolerance to dif-

ferent types of leaf damage by seagrass consumers makes it

difficult to generalize our results. Traits causing herbivore

preference and conferring tolerance to herbivory may vary

among different groups of plants (Obeso 1993) and among

types of herbivores (Kotanen and Rosehental 2000). Seag-

rasses, however, gather a number of features that have been

associated with tolerance to both vertebrate (e.g. well-

developed clonality, stored reserves, presence of dormant

and basal meristems) and invertebrate herbivory (e.g. flex-

ible branching and post-damage resource allocation, Kota-

nen and Rosehental 2000). Such multiplicity of traits may

stem from seagrass evolution in systems with a rich diversity

of grazers (e.g. Domning 1981; Ivany et al. 1990; De Muizon

et al. 2004; Uhen 2007). In this case, compensation to her-

bivory, mostly by increased nutrient supply from external

leaves, also supports this view and suggests that it may be a

broad-scale mechanism to tolerate recurrent leaf damage by

common macroherbivores such as fish and sea urchins.

Acknowledgments This work was supported by a FI scholarship

from the Departament d’Universitats, Recerca i Societat de la

Informacio (DURSI, Generalitat de Catalunya) to P. Prado, and the

CGL2007-66771-C02 and CGL2009-12562 grants from the Spanish

Ministry of Science and Innovation. C. Collier was supported by a

short-term outgoing scholarship from the Australian Strategic Research

Fund for the Marine Environment and a travel grant from the Centre for

Ecosystem Management, Edith Cowan University, Perth, Western

Australia.

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