Eutrophication and trophic structure in response to the presence of the eelgrass Zostera noltii

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Mar Biol (2009) 156:2107–2120

DOI 10.1007/s00227-009-1241-y

ORIGINAL PAPER

Eutrophication and trophic structure in response to the presence of the eelgrass Zostera noltii

Alexandra Baeta · Ivan Valiela · Francesca Rossi · Rute Pinto · Pierre Richard · Nathalie Niquil · João C. Marques

Received: 8 August 2008 / Accepted: 17 June 2009 / Published online: 2 July 2009© Springer-Verlag 2009

Abstract In estuaries, eelgrass meadows contribute tofundamental ecosystem functions of estuaries, providingfood to several predators and buVering the negative eVectsof eutrophication. We asked whether the presence of theeelgrass Zostera noltii decreased the nitrogen concentrationin the overlying water, aVected the sources of nitrogensequestrated by primary producers and changed the benthicand pelagic food web structures. We also studied theimportance of these food webs in providing food to Wsh.We compared bare sediment to sediment covered by aZ. noltii meadow, and examined nutrient concentrations inthe water column and �15N in primary producers as indicatorsof anthropogenic inputs of nutrients. We then measuredboth �13C and �15N in the tissues of plants and consumersto establish food web structures. There were no diVerencesin the concentrations and sources of nitrogen between sites.Rather, �15N values indicated anthropogenic inputs of N(e.g. sewage discharges, agriculture) in both sites. Therewere no major diVerences in the structure of the planktonicfood web, which was in part sustained by particulateorganic matter and supported most predator Wsh, and in the

structure of the benthic food web. Nonetheless, there werediVerences in the sources of food for omnivore consumersand for the detritivore Scrobicularia plana. Overall, thebenthic food web did not use food derived from the eelgrassor macroalgae deposited on the substratum. Suspensionfeeders used particulate and sediment organic matter,whereas the �13C and �15N values of the other consumersindicated a likely contribution of benthic microalgae. Fur-thermore, in both habitats we found large variability in theisotope signatures of benthic macrofauna consumers, whichdid not allow distinguishing clearly diVerent trophic groupsand indicated a high level of omnivory and a mixed dietopportunistically making use of the availability of food inthe surroundings.

Introduction

Nutrient enrichment due to human activities may increaseeutrophication of aquatic habitats and severely impact theidentity and diversity of primary producers and consumerswith consequent alterations in the food-web structure, eco-system productivity and nutrient recycling. Estuaries,which are critical habitats for nutrient recycling and ecosys-tem productivity, often receive large inputs of nutrientsderived from human-activities and agricultural discharges,followed by severe eutrophication events (Valiela et al.1997; Cloern 2001; Bode et al. 2006; Lotze et al. 2006). Inthese habitats, one of the most relevant aspects of eutrophi-cation is the proliferation of fast-growing macroalgae,which may replace slow-growing macrophytes and signiW-cantly decrease areal extent of seagrass meadows and theecological value of the entire estuary (Short and Wyllie-Echeverria 1996; Howarth 1988; Bricker et al. 1999;Valiela 2006).

Communicated by U. Sommer.

A. Baeta (&) · F. Rossi · R. Pinto · J. C. MarquesDepartment of Zoology, IMAR, Institute of Marine Research, University of Coimbra, 3004-517 Coimbra, Portugale-mail: [email protected]

I. ValielaThe Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA

A. Baeta · P. Richard · N. NiquilCentre de Recherche sur les Ecosystèmes Littoraux Anthropisés, UMR6217, CNRS, IFREMER, Université de La Rochelle, Avenue Michel Crépeau, 17042 La Rochelle, France

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2108 Mar Biol (2009) 156:2107–2120

In estuaries, seagrass meadows as compared to bare sed-iment may largely contribute to the estuarine functioning,by sustaining longer and more complex food webs, whichmay aVect ecosystem productivity and stability under envi-ronmental change. Seagrasses increase sedimentation ofWne sediment particles and thus enhance quantity and qual-ity of food for many macrofauna invertebrates, which arethe fundamental trophic link between basal resources andpredators such as Wsh and seabirds. Seagrass meadows alsorepresent a nursery for Wsh of economic value and a refugeefrom predators (Duarte 2002). Furthermore, seagrasses,particularly Z. noltii, may contribute to carbon and nutrientsequestration and storage for longer periods than macro-algae, thereby keeping the water overlying the meadow ofhigher quality that the water overlying bare sediment andmitigating eutrophication eVects (Cebrian 1999).

Natural abundance of stable isotopes (�13C and �15N) ofdetritus, primary producers and consumers are extensivelyused to assess trophic structure of communities (Tieszenet al. 1983; Peterson and Fry 1987; Kwak and Zedler 1997;McClelland et al. 1997; Riera et al. 2000; Abreu et al.2006; Bode et al. 2006). Carbon stable isotope data are typ-ically used in ecological research to determine which pri-mary producer components are the ultimate carbon sourceassimilated by higher trophic level consumers (Fry andSherr 1984; Peterson and Fry 1987). Consumers typicallyhave �13C values similar (generally within 1‰) to theirfood source (DeNiro and Epstein 1978; Wada et al. 1991),yielding slightly more enriched values. Nitrogen stable iso-topes are more often used to study trophic levels in commu-nities (Wada et al. 1991). Organisms tend to fractionatenitrogen positively by 3–4‰ (Wada et al. 1991; Post 2002)

at each trophic level. Thus, higher trophic levels will havehigher �15N values due to the discriminatory retention ofthe heavy isotope over the light one (Post 2002). Further-more, high levels of �15N in primary producers can be usedas an alternative method to establish the level of humaneutrophication since anthropogenic sources of nitrogen aregenerally enriched in the heavy isotope compared to naturalsources (Cole et al. 2006).

In this study, we investigated whether nitrogen concen-trations of the water column, sources of nitrogen for pri-mary producers, and trophic relationships between primaryproducers, consumers and predators of the benthic and thewater column food web diVered in a site where Z. noltii wasabundant and a site characterised by bare sediment.

Materials and methods

Study site

The Mondego estuary, on the western coast of Portugal is arelatively small (1,600 ha), warm-temperate, polyhaline,intertidal system located on the Atlantic coast of Portugal,consisting of two arms, north and south (Fig. 1). The south-ern arm is characterised by intertidal mudXats (almost 75%of the area) exposed during low tide. The tidal range variesbetween 0.35 and 3.3 m depending on the site and tidecoeYcient, while water residence time varies between2 (northern arm) and 3 days (southern arm).

In the early 1990s, the communication between the twoarms of the estuary became totally interrupted in theupstream area. The combined eVect of an increased water

Fig. 1 Mondego estuary map showing sampling sites: Zostera and bare sediment sites (grey circles). Change in the area covered by Zostera noltii in the south arm of the Mondego estuary. Mapping of benthic vegetation is based on Weld observations, aerial photographs and GIS methodology (Arc View GIS version 8.2)

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Mar Biol (2009) 156:2107–2120 2109

residence time and of nutrient concentrations became majordriving forces behind the occurrence of seasonal Ulva spp.blooms and a concomitant severe reduction of the areaoccupied by Z. noltii beds, and also of Z. noltii biomass inthe areas where it still remained (Marques et al. 1997), as afunction of competition with macroalgae (Marques et al.2003). The shift in benthic primary producers has changedtrophic structure of benthos (Marques et al. 1997, 2003;Pardal et al. 2000, 2004; Cardoso et al. 2004, 2005; Patrícioand Marques 2006; Dolbeth et al. 2007).

Since 1998 the Z. noltii has been recovering and Ulva spp.blooms reducing (Lillebø et al. 2007), due to the decreasednutrient discharges from the river and the partial re-openingof the communication between the north and south arms(Neto 2004; Neto et al., submitted for publication).

Sample collection and preparation

To assess the relative nutrient enrichment at the two sites,we measured concentrations of nitrate (NO3

¡), ammonium(NH4

+), phosphate (PO43¡), and chlorophyll a in the water

column. We also compared the �15N in primary producerscollected in benthos and the bulk organic matter in the sedi-ment (sedimentary organic matter, SOM) and in the watercolumn (particulate organic matter, POM) between the twosites as a measure of the eutrophication level.

To examine and compare the food web structurebetween the two study sites, we measured �13C and �15N inprimary producers, bulk sediment and consumers. We sam-pled particulate organic matter (POM), sedimentary organicmatter (SOM), meiofauna, seagrass, macroalgae, macro-benthos and zooplankton at each of the two sites. Fish werecollected at the bare sediment site. Given their high mobil-ity, we retained collection in both sites irrelevant for thepurpose of this study. Samples for nutrient analyses andPOM were collected monthly from November 2005 to July2006. Plants and animals were collected in November2005, February, May and July 2006. At each site and sam-pling date, Wve samples were taken from an area of about4 £ 4 m and pooled before analyses. Samples taken at eachtime in each site are considered here as replicates sincethere were no temporal diVerences (Baeta et al. 2009).Water for nutrient concentration (700 ml) was Wlteredthrough a pre-combusted 0.45 �m pore size, 4.7-cm diame-ter glass Wbre Wlters (Whatman GFF Wlter). In addition,700 ml of water was passed through a Whatman GFF Wlterto measure Chl a concentration in the collected material(Parsons et al. 1985). All Wlters and water samples werestored on ice until arrival at the laboratory, when they weretransferred to the freezer (¡18°C). POM was obtained byWltering 0.5–1 l of seawater, from a depth of 0.5 m belowthe surface, onto pre-combusted (450°C, 4 h) WhatmanGFF Wlters with a low-pressure vacuum pump.

Sediment samples from the upper 1 cm were collectedwith an acrylic corer (31 mm of diameter). For the meio-fauna, sediment samples were collected, and the top 3 cmof each sediment core was then passed through 500 and 38 �msieves. Meiofauna were examined from the 38 �m fraction,and 50–300 individuals were collected for isotope analyses.Seagrass leaves and roots, and macroalgae were collectedby hand and gently cleaned of epiphytic material. Macro-invertebates were also taken manually from each site, andheld in Wltered seawater for 24 h to allow their guts to clear.

Zooplankton was collected at each site by towing aBongo net (0.5 m diameter, 200 �m mesh size) against thecurrent for 20 min. The zooplankton samples for isotopeanalysis were composites of 20–200 individuals. Resident(Atherina boyeri, Pomatoschistus microps, Pomatoschi-stus minutus, Syngnathus abaster, Syngnathus acus) andtransient (Dicentrarchus labrax, Diplodus vulgaris, Soleasolea) Wsh species were collected using a 2 m beam trawl,with a 5 mm stretched mesh size on the cod end. Thetrawls were carried out during the night at low-waterspring tides.

All samples were rinsed with Milli-Q water, beforefreeze-drying. Filters with POM were also rinsed with afew millilitres of ammonium formate to remove the saltsthat may damage the combustion columns of the IRMS ele-mental analyser. Despite ammonium formate may alter the�15N, no trace of ammonium formate is left in the Wlterbecause it sublimates when the Wlters are freeze-dried over-night (P. Richard, personal communication). When dry,samples were ground (Wlters with POM were kept whole)into homogenous powder using mortar and pestle,weighted, and loaded into tin capsules. For the macrofauna,we removed the shell from molluscs and skeleton fromcrustaceans. For the zooplankton, we analysed the entireanimals, given their small size. For Wsh, the muscle of thedorsal region was analysed.

Samples were analysed using an EA-IRMS (Isoprime,Micromass, UK). Isotopic values were expressed in the �unit notation as deviations from standards (Vienna Pee DeeBelemnite for �13C and N2 in air for �15N) following theformula: �13C or �15N = [(Rsample/Rstandard) ¡ 1] £ 103,where R is 13C/12C or 15N/14N. The precision of the mea-surement was 0.2‰ for both carbon and nitrogen.

Results

Eutrophication level

There were no signiWcant diVerences in NO3¡, NH4

+,PO4

3¡ and chlorophyll a concentrations, and in the totalnitrogen to phosphorus ration (DIN/PO4

3¡) between thetwo sites (Table 1).

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2110 Mar Biol (2009) 156:2107–2120

Nitrate concentrations ranged from 0.06 to 0.96 mg L¡1

(Fig. 2). Concentrations of NH4+ and PO4

3¡ were usuallymuch lower than those of NO3

¡ (Fig. 2a). N/P was gener-ally above the 16:1 RedWeld mass ratio during coldermonths, and below that in warmer months. This suggeststhat in winter P supply might be the limiting nutrient, whilein summer, N might limit producer growth nutrient(Fig. 2b). Chlorophyll a concentrations ranged between1.45 and 8.92 mg m¡3, and peaked in spring, perhaps fromdrawing down nitrate concentration during the warmermonths (Fig. 2c).

The �15N of primary producers ranged between 9.7 and13.2‰ in the bare sediment site and between 9.7 and 13.2in the Zostera site (Table 2). There were clearly no diVer-ences in �15N between the Zostera and the bare sedimentsite for green (F1,10 = 1.95, P = 0.20), red (F1,5 = 0.60,P = 0.48) or brown algae (F1,4 = 0.02, P = 0.98).

Food web structure

A total of 45 groups/species were identiWed, including5 species of primary producers, bulk POM, SOM and 38consumer groups. Among these consumers, there were 21species of macrofauna, 2 of meiofauna, 8 Wsh species, and7 zooplankton taxa (Table 2). The dual-stable isotope plots(Fig. 3) showed that both the benthic and plankton foodwebs were similar between sites. There were, however,some diVerences for the position of the detritivore bivalveScrobicularia plana, for the omnivore polychaetes Nepthyscirrosa and Hediste diversicolor, the omnivore crustaceansCyathura carinata, Crangon crangon and Carcinusmaenas. S. plana was closer to the suspension feederbivalves Cerastoderma edule, and Mytilus galloprovin-cialis, whereas the remaining species, especially H. diversi-color, were more enriched in 15N at the bare sediment thanthe Zostera site. Accordingly, there were signiWcant diVer-ences between the two sites in the �15N values of omni-vores (F1,10 = 13.85, P = 0.003).

At both sites, POM and SOM had the most depleted val-ues of �13C and �15N. Macroalgae and Z. noltii had a rela-tively variable range of values, similar to the �13C and �15Nvalues of macrofauna consumers (Table 2; Fig. 3). Amongthe macrofauna consumers, the isotopic signatures of thesuspension feeders M. galloprovincialis and C. edule werevery close to the planktonic food web and were 1–3‰enriched compared to POM (1–2‰ in 13C and 2–3‰ in15N, Fig. 3). The grazer macrofauna gastropod Hydrobiaulvae had the highest �13C values among all other macro-fauna consumers and very low �15N values. Only the macro-fauna suspension feeder C. edule and M. galloprovincialisshowed smaller values than H. ulvae. The highest �15Nvalues were found for the predator Glycera tridactyla andthe omnivore N. cirrosa, especially in the bare sediment(Table 2; Fig. 3).

The zooplankton consumers occupied a relatively nar-row area, as compared to the macrofauna consumers. Thosezooplankton species that are considered phyto- and micro-zooplanktivores were at an intermediate position betweenPOM and the zooplankton predator Sagitta friderici. Even-tually, the predator Wsh D. labrax, D. vulgaris and P. minu-tus, were 3–4‰ enriched in 15N and 1–2‰ in 13C ascompared to intermediate plankton consumers. The juve-niles of the predator Wsh S. solea and P. microps wereinstead very depleted in 13C, but not in 15N, whereas the

Table 1 Results of paired t tests of diVerences between the two sites for nutrient and chlorophyll concentrations in the Mondego estuary

t df

NO3¡ 0.068ns 8

NH4+ 1.385ns 8

PO43¡ 1.867ns 8

Chlorophyll a 0.701ns 8

DIN/PO43¡ 0.464ns 8

ns no signiWcant diVerences (P > 0.05)

Fig. 2 Nitrate, ammonium, and phosphate (a), DIN/PO43¡ (b), and

phytoplankton chlorophyll a (c) concentrations, at the Zostera site andbare sediment site, in the Mondego estuary, from November 2005 toJuly 2006

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Mar Biol (2009) 156:2107–2120 2111

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2112 Mar Biol (2009) 156:2107–2120

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123

Page 7

Mar Biol (2009) 156:2107–2120 2113

predator seahorse S. abaster and S. acus had �13C and �15Nvalues close to those of macrofauna omnivore.

To test statistically for diVerences between the trophicfood web of Zostera versus bare sediment, we Wrst identi-Wed all the taxa in common between the two sites withindiVerent trophic groups (e.g. macroalgae, benthic primaryconsumers such as detritivores and grazers, benthic second-ary consumers such as omnivores and predators, and zoo-plankton). Then, we correlated the natural abundance ofstable isotopes measured in the Zostera site versus the baresediment (Fig. 4; Table 3). We expected signiWcant correla-tion and values of the slope close to 1 if there were nodiVerences in the isotope signature of primary producersand consumers among the two sites. In all cases, except for�15N for the benthic secondary consumers, there was

signiWcant correlation (R values) of �13C and �15N valuesbetween the two sites and the slope was close to 1(Table 3). Heavier carbon isotopic signatures for benthicconsumers in the Zostera site were found, compared tothose from the site with no Z. noltii. �13C values of the baresediment site macrobenthos were 1.2‰ depleted, relative tothose of the Zostera site.

To further explore the relationship between the values of�15N and the trophic guild in which consumers are classi-Wed in the literature, we simpliWed published data into “pro-ducer”, “primary consumer”, and “secondary consumer”and plotted the �15N values of each category versus theothers in the Zostera (Fig. 5) and the bare sediment site(Fig. 6). Among macrofauna consumers, the diVerenttrophic groups were overlapped at both sites, whereas the

Fig. 3 �13C versus �15N of the benthic and water column food webs,in the Zostera site (left) and bare sediment site (right), in the Mondegoestuary. Benthos: (Wlled circle) primary producers; (Wlled triangle)detritivore; (Wlled diamond) grazer/detritivore; (open square) omni-

vore. Water column: (Wlled square) Wsh; (open diamond) zooplankton.Average is presented for biota collected from November 2005 to July2006. The abbreviations of the species/groups are shown on Table 2

ZOSTERA

SITE

BARE SEDIMENT SITE

δ15N

(‰

)

4

6

8

10

12

14

16

-26 -24 -22 -20 -18 -16 -14 -12 -10

POM

SOM BENTHOS

Mg

Ce

UE

Llev

Mp Ic

Em

F

G Sp

Hu

Hd

Nc

Av

Cc

Hf

Cm Ccr

Cca

SshCo

Ne

Ar

Fish

Gu

Ll

Zl Zr

Zooplankton

4

6

8

10

12

14

16

-26 -24 -22 -20 -18 -16 -14 -12 -10

POM

SOM BENTHOS

Mg

Ce

UE

Llev

Mp Ic

Em

F

G

Sp

Hu

HdNc

Av

CcHf

Cm

CcrCca

Ssh

CoNe

Ar

Fish

Zooplankton

4

6

8

10

12

14

16

-26 -24 -22 -20 -18 -16 -14 -12 -10

POM

SOM WATER COLUMN

Mg

Ce

Benthos

At ClPl

Sf

Zo

My

Asp

Sac

Sa

Ab

Pmi

Dl

Dv

Pm

Ss

4

6

8

10

12

14

16

-26 -24 -22 -20 -18 -16 -14 -12 -10

POM

SOM WATER COLUMN

Mg

Ce

At

Sf

Zo

Asp

Sac

SaAb

Pmi

Dl

Dv

Pm

Ss

Benthos

δ13C (‰)

123

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2114 Mar Biol (2009) 156:2107–2120

separation among planktonic groups was clear (Figs. 5, 6).The range of variability of each trophic group was alsoquite large, varying between 1.5 and 4.5‰, indicating thattrophic guilds are probably inadequate for invertebratemacrofauna (Figs. 5, 6).

Discussion

Eutrophication and anthropogenic input of N

The concentration of nitrogen in the overlying water andthe sources of nitrogen used by primary producers werevery similar between the two sites, despite expected diVer-ences in the level of eutrophication between sites, due topresence of Z. noltii that may buVer eVects of nutrient

enrichment, by storing nutrients in its tissue and by sup-porting a more diverse food web which can better recyclenutrients (Cebrian 1999; Duarte 2002).

The high nitrogen isotopic signatures of primary produc-ers could indicate that the sources of nitrogen were fromhuman activities. Indeed, �15N values ranging from +10 to+20‰ in primary producers strongly indicate anthropo-genic sources, whereas nitrate derived from atmosphericdeposition produce values smaller than 6‰ (Kendall 1998).In our study, the �15N for Z. noltii leaves (9.5 § 3.5‰), andgreen algae Ulva sp. (12.5 § 3.0‰) were about 3–4‰higher than values measured in Ria Formosa, Portugal(6.1‰; 8.8‰, respectively) (Machás et al. 2003), or inestuaries of the NW Iberian peninsula (5.6 § 0.4‰;8.4 § 0.3‰, respectively) (Bode et al. 2006). Seawaternitrogen signatures in the Atlantic coast have values

Fig. 4 Comparison of C (left) and N (right) stable isotope ratios, for the water column (top) [(open circle) POM; (Wlled cir-cle) zooplankton], and benthos (bottom) [(Wlled circle) primary consumers; (Wlled circle) sec-ondary consumers; (open circle) macroalgae; (open square) SOM], collected from the Zostera and bare sediment sites, in the Mondego estuary. Dashed line shows 1:1 correlation

δ13C (‰) δ15N (‰)

bare

sed

imen

t site

-26

-22

-18

-14

-10

-26 -22 -18 -14 -10

POM

Zooplankton

WATER COLUMN

2

6

10

14

18

2 6 10 14 18

WATER COLUMN

Zooplankton

POM

-26

-22

-18

-14

-10

-26 -22 -18 -14 -10

BENTHOS

Macroalgae

Benthic invertebratesSOM

2

6

10

14

18

2 6 10 14 18

Benthic invertebrates

Macroalgae

BENTHOS

SOM

Zostera site

Table 3 Correlation coeYcient test results testing diVerences, in the companion of Fig. 3, between sites for the groups benthic and water columnfood webs collected in the Mondego estuary on C and N isotope ratios

** P < 0.05; *** P < 0.001; nlr no linear relationship between the variables (P > 0.05)

Groups �13C �15C

R P N Slope R P N Slope

Macrophytes 0.920 *** 12 1.001 0.680 *** 12 0.790

Benthic primary consumers 0.880 *** 50 0.820 0.824 *** 50 0.820

Benthic secondary consumers 0.510 ** 21 0.420 0.068 nlr 21 0.091

POM 0.732 ** 9 0.778 0.778 *** 9 0.830

Zooplankton 0.923 *** 14 1.110 0.927 *** 14 0.776

123

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Mar Biol (2009) 156:2107–2120 2115

ranging from 5.2‰ (Liu and Kaplan 1989) and 6.5‰ (Bodeet al. 2003).

Furthermore, by comparing the nutrient concentration inthe water column to the values of 106 North American estu-aries (Tomasky et al., submitted for publication), we foundthat the Mondego estuary was more enriched than 90% ofAmerican estuaries. Despite mitigation procedures imple-mented in the Mondego estuary in 1998 and 2002 (Lillebøet al. 2007), high nitrogen loads are thus still entering thesystem and the eelgrass habitat is not able to buVer theseinputs of nutrients. The measures currently employed seemthus insuYcient to ensure high environment quality. Proba-bly larger eelgrass meadows could sequestrate part of thesenutrients and more environmental protection might beneeded to ensure a full recovery of the eelgrass in the system.

Food web structure

The presence of Zostera did not change considerably thetrophic structure of both the sediment and the water col-umn. There were, however, diVerences in the isotopic sig-natures of some benthic macrofauna consumers, such as thedetritivore S. plana and the omnivores N. cirrosa, H. diver-sicolor, C. carinata, C. crangon and C. maenas. ThesediVerences may suggest the occurrence of a diet shiftfollowing the characteristics of the habitat where theseanimals are. Zostera meadows has a more complex habitatstructure created by the presence of rooted macrophytes,and can oVer higher protection from predators and high lev-els of organic matter in the sediment (Duarte 2002). DiVer-ences in isotopic signatures of marine invertebrates often

Fig. 5 Nitrogen stable isotope signatures of producers and consumers for the benthic (top) and water column (bottom) food webs from the Zostera site, in the Mondego estuary. Black symbols represent species/groups collected in the benthos [(Wlled circle) producers; (Wlled triangle) primary consumers; (Wlled square) secondary con-sumers]. White symbols repre-sent species/groups collected in the water column [(open circle) producers; (open triangle) primary consumers; (open square) secondary consumers]. Average § standard error is pre-sented for biota collected from November 2005 to July 2006 SOM

Z. noltii (leaves)

Fucus sp. Gracilaria sp.

Z. noltii (roots)

Ulva sp.Enteromorpha sp.

L. leviiS. plana

H. ulvaeC. capitata

I. chelipesM. palmata

A. romijniG. umbilicalis

CopepodaE. marinus

S. shrubsoliiA. valida

H. filiformis

H. diversicolorNematoda

C. carinata

C. maenas

L. littorea

N. cirrosa

G. tridactyla

S. acus

S. abasterP. minutus

C. crangon

4 6 8 10 12 14 16

Pro

duce

rs

Prim

ary

cons

umer

sS

econ

dary

co

nsum

ers

BENTHOS

POM

M. galloprovincialis

C. edule

Mysidacea

Acartia sp.

Zoeae decapoda

Cladocera

A. tonsa

Pomatoschistus sp.

S. friderici

S. acus

S. solea (juv)

4 6 8 10 12 14 16

A. boyeri

S. abaster

D. labrax (juv)

D. vulgaris (juv)

P. microps (juv)

P. minutus

Sec

onda

ry

cons

umer

sP

rimar

y co

nsum

ers

WATER COLUMN

δ15N (‰)

123

Page 10

2116 Mar Biol (2009) 156:2107–2120

indicate a shift in feeding behaviour following speciesinteractions, when for instance feeding choice depends onthe availability of resources and the time spent handling thefood under high predation risk (Stephens and Krebs 1986;Burrows and Hughes 1991; Rossi et al. 2004; Sommer et al.2005). In our study, omnivore macrofauna showed to feedat lower levels of the food web at the Zostera than at thebare sediment site. Furthermore, the detritivore S. planashowed a diet close to that of the suspension feeders in thebare sediment, whereas in the Zostera site, the diet wasclose to that of other detritivores. By providing refuge andincreasing organic matter availability, Z. noltii might allowthese macrofauna species to rely on organic matter, ratherthan on other consumers or on the particles suspended inthe water column.

Nonetheless, the bulk organic matter seemed to not sup-port the macrofauna consumers, including the detritivoresand the omnivores that varied diet among habitats. In ourstudy, these consumers had �13C and 15N values (�13C:between ¡11 and ¡18‰ and �15N: higher than 8‰) toohigh to be feeding on SOM, if we consider the general frac-tionation values proposed in the literature of 1 and 3–4‰for �13C and �15N, respectively (Minagawa and Wada1984). Similarly, macroalgae or eelgrass did not seem to bethe main sources of food for the macrofauna consumers.The organic matter available in the sediment (SOM) ismade up of various terrestrial and marine sources, on whichmany macrofauna consumers may forage selectively. Itmay be possible that in both sites animals fed on a mix ofdiVerent sources. The values of SOM in both sites indicated

Fig. 6 Nitrogen stable isotope signatures of producers and con-sumers for the benthic (top) and water column (bottom) food webs from the bare sediment site, in the Mondego estuary. Black symbols represent species/groups collected in the benthos [(Wlled circle) producers; (Wlled triangle) primary consumers; (Wlled square) secondary con-sumers]. White symbols repre-sent species/groups collected in the water column [(open circle) producers; (open triangle) primary consumers; (open square) secondary consumers]. Average § standard error is pre-sented for biota collected from November 2005 to July 2006

SedimentGracilaria sp.

Fucus sp. Ulva sp.

Enteromorpha sp.S. plana

L. leviiH. ulvae

I. chelipesM. palmata

E. marinusS. shrubsolii

A. validaA. romijni

Copepoda

Nematoda

C. capitata

S. acusC. crangon

H. filiformis

C. carinataC. maenas

S. abasterH. diversicolor

N. cirrosaG. tridactyla

P. minutus

4 6 8 10 12 14 16

Prim

ary

cons

umer

sS

econ

dary

co

nsum

ers

Pro

duce

rs

BENTHOS

POM

M. galloprovincialis

C. edule

Acartia sp.

A. tonsa

Zoeae decapoda

S. friderici

S. acus

S. solea (juv)

4 6 8 10 12 14 16

A. boyeri

S. abaster

D. labrax (juv)

D. vulgaris (juv)

P. microps (juv)

P. minutus

Prim

ary

cons

umer

sS

econ

dary

co

nsum

ers

WATER COLUMN

δ15N (‰)

123

Page 11

Mar Biol (2009) 156:2107–2120 2117

a mix of terrestrial organic matter, very depleted in 13C(¡30 to ¡23‰, Fry and Sherr 1984), microalgae (between¡21 and ¡12‰ for �13C and between 5 and 8‰ for �15N,France 1995; Currin et al. 1995; Riera et al. 1996, 1999;Herman et al. 2000; Moens et al. 2002; Page and Lastra2003) and macroalgae (¡18 to ¡11‰, in this study). Micro-algae indeed represent a fundamental source of food forbenthic macrofauna in many estuaries, especially on inter-tidal bare sediment (Herman et al. 2000; Middelburg et al.2000; Rossi et al. 2009). In the bare site, sediment micro-algae were more abundant than in the Zostera site and theisotopic values of microalgae measured in November 2008ranged between ¡13 and ¡14‰ for �13C and 6‰ for �15N(Alexandra Baeta, personal observation). It may thus bepossible that animals foraged on a mix of surface detritus,macroalgae and benthic microalgae, selecting for benthicmicroalgae when available.

Recently, empirical studies have found variable �15Nvalues within trophic levels and demonstrated that the gen-erally recognised average 3–4‰ �15N increase at each tro-phic level (e.g. Minagawa and Wada 1984) does not alwaysapply to the marine system because food sources are tem-porally and spatially variable and because consumers varytheir metabolism depending on the source of food ingested(Sommer et al. 2005; Vanderklift and Ponsard 2003). Fur-thermore, omnivory is a very common feature in estuarinehabitats and it may further complicate the interpretation ofisotopic signatures of consumers and their food sources(Svensson et al. 2007; Jaschinski et al. 2008). In our study,it is possible that 15N enriched values of macrofauna spe-cies compared to the �15N of SOM or macroalgae were dueto a diVerent metabolism of these consumers with respect totheir nitrogen sources. In addition, the high frequency ofomnivory may explain why �15N values overlapped amongthe trophic groups in which macrofauna species are catego-rised. We indeed found that the �15N values for the herbi-vores and detritivores (primary consumers) of the benthosranged from 7.8 to 12.2‰ and overlapped to those of thepredators/omnivores (secondary consumers), which rangedfrom 10.8 to 14.2‰. This variability in trophic signaturesmay have important consequences for interpreting aquaticfood webs because it may undermine the estimates ofenergy or mass Xow through food webs, when such esti-mates are based on the assumption that trophic levels arediscrete. Indeed, considering discrete trophic levels cannotcapture the complex trophic interactions and the omnivorytypical of natural environment, underestimating the Xow ofenergy in the ecosystem (Vander Zanden and Rasmussen2001; Post 2002; Vanderklift and Ponsard 2003; Bode et al.2006).

Compared to the benthic food web, the food web of thewater column was less complex. The values of �13C for theorganic matter suspended in the water column (POM) were

in agreement with the literature values of surface waters inthe latitudinal range around 40°N (from ¡25 to ¡18‰,Goerick et al. 1994) and were within the range for estuarineand coastal marine phytoplankton from other temperateregions (see Gearing et al. 1984). Taking into account afractionation of 3–4‰ for �15N and of 1‰ for �13C, in bothsites the link between the food web of benthos and thewater column was clearly represented by the suspensionfeeders M. galloprovincialis and C. edule. The �13C ofC. edule (¡18‰) was similar to other studies that sug-gested C. edule has a diet based on plankton microalgaeand organic matter (POM) mixed with benthic microalgaeresuspended on the overlying water (Rossi et al. 2004). Inaddition, a part of the water column trophic web was sup-ported by POM. The primary consumer A. tonsa, a micro-zooplanktivore and other phyto-microzooplanktivorescould forage on POM and transfer part of this carbon andnitrogen up to the food web. Fish fed on a mixed diet thatmay include both allochthonous and autochthonous foodsources, from both the benthos and the plankton. Forinstance, the juveniles of S. solea and P. microps were toodepleted in 13C to indicate they only foraged on planktonconsumers. It may be possible they used other sourcesderived from the terrestrial food web, as found in the Rhoneriver (Darnaude 2005). Furthermore, the 13C enrichment ofother Wsh (A. boyeri, P. minutus, D. labrax, D. vulgaris)was up to 5‰ compared to the copepod A. tonsa and to theother phyto-microzooplanktivores. They were thus tooenriched in 13C to suggest a diet exclusive on these species.Rather, their diet could include benthic consumers andother plankton consumers (Acartia sp. and zoeae).

In conclusion, in the Mondego estuary the presence ofthe eelgrass Z. noltii did not alter nutrient enrichment andthe transfer of these nutrients and carbon in the food web,since we found very similar trophic structures between baresediment and sediment occupied by Z. noltii. The benthicfood web revealed more complex than the water columnfood web and evidenced high variability in the isotopic sig-natures, probably due to the variability of food sources andthe physiological characteristics of the macrofauna. Wesuggest that macrofauna could rely on a complex mix ofmicroalgae, macroalgae and detritus, actively selecting forthe most nutritive food sources when available. Other stud-ies are, however, needed to establish the trophic signiW-cance of the diVerent food sources and, in particular, ofbenthic microalgae.

Acknowledgments This study has beneWted from the constructivecomments of two anonymous reviewers. The authors are grateful toFilipe Martinho, João Neto, and Ana Bessa for Weld assistance duringsample collection, and Gaël Guillou for the isotope analysis. We alsothank Sophia Fox, and Mirta Teichberg, for helpful discussions andvaluable comments on the manuscript. This research was supported byFCT (Fundação para a Ciência e Tecnologia) through a Ph.D. Grant

123

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2118 Mar Biol (2009) 156:2107–2120

attributed to Alexandra Baeta (SFRH/BD/23574/2005), and a FLAD/IMAR Grant (04/06). Additional funding for stable isotope analysiswas provided by IMAR—Institute of Marine Research, through theR&D project RECONNECT (PTDC/MAR/64627/2006) and WISER(FP7-ENV-2008-1).

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