Sources of organic matter for flatfish juveniles in coastal and estuarine nursery grounds: A meta-analysis for the common sole (Solea solea) in contrasted systems of Western Europe

Journal of Sea Research 75 (2013) 85–95

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Sources of organic matter for flatfish juveniles in coastal and estuarine nurserygrounds: A meta-analysis for the common sole (Solea solea) in contrasted systems ofWestern Europe

O. Le Pape a,⁎, J. Modéran b, G. Beaunée a, P. Riera c, D. Nicolas d, N. Savoye e, M. Harmelin-Vivien f,A.M. Darnaude g, A. Brind'Amour h, H. Le Bris a, H. Cabral i, C. Vinagre i, S. Pasquaud i, S. França i, C. Kostecki a

a UMR 985 ESE, Agrocampus Ouest, Inra 65 rue de St Brieuc CS 84215 35042 Rennes Cedex, Franceb Romberg Tiburon Center, San Francisco State University, 3152 Paradise Drive, Tiburon, CA 94920, USAc UPMC Université Paris 06, UMR 7144, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, Franced Irstea, UR EPBX, 50 av. de Verdun, F-33612 Cestas, Francee Observatoire Aquitain des Sciences de l'Univers, UMR 5805 EPOC, Université Bordeaux 1/CNRS, Station Marine d'Arcachon, 2, rue du Pr. Jolyet, 33120 Arcachon, Francef Institut Méditerranéen d'Océanologie (MIO), UMR CNRS 7294, Aix-Marseille Université, rue Batterie des Lions, 13007 Marseille, Franceg Laboratoire ECOSYM, UMR 5119 UM2-CNRS-IRD-IFREMER-UM1, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier, Franceh Ifremer, Centre de Nantes, Rue de l'Île d'Yeu, B.P. 21105, 44311 Nantes, Francei Universidade de Lisboa, Faculdade de Ciências, Centro de Oceanografia, Campo Grande, 1749-016 Lisboa, Portugal

⁎ Corresponding author. Tel.: +33 2 23 48 55 31; faxE-mail address: [email protected]

1385-1101/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.seares.2012.05.003

a b s t r a c t

Article history:Received 21 October 2011Received in revised form 27 April 2012Accepted 6 May 2012Available online 14 May 2012

Keywords:Nursery groundFlatfishOrganic matter originStable isotopesMixing model SIAR

Coastal and estuarine nursery grounds are essential habitats for sustaining flatfish stocks since only theseshallow and productive areas provide the high food supply that allows maximizing juvenile growth and sur-vival in most flatfish species. However, the main organic matter sources at the basis of benthic food websmight differ drastically between estuarine nursery grounds under strong freshwater influences, where foodwebs are mainly supported by continental organic matter, and coastal ecosystems under limited freshwaterinfluence, where the local marine primary production is the main source of carbon for the benthos. To betterunderstand the links between continental inputs to the coastal zone and stock maintenance in the highlyprized common sole, Solea solea (L.), we investigated the variability in the organic matter sources supportingthe growth of its young-of-the-year (YoY) in five contrasted estuarine and coastal nursery grounds undervarying freshwater influence. Stable isotopes of carbon and nitrogen allowed tracing the origin of the organicmatter exploited by YoY soles in the very first months following their benthic settlement, i.e. when most ofthe juvenile mortality occurs in the species. A mixing model was run to unravel and quantify the contributionof all major potential sources of organic matter to sole food webs, with a sensitivity analysis allowing assess-ment of the impact of various trophic enrichment factors on model outputs. This meta-analysis demonstrateda relative robustness of the estimation of the respective contributions of the various organic matter sources.At the nursery scale, the upstream increase in freshwater organic matter exploitation by YoY soles and itspositive correlation with inter-annual variations in the river flow confirmed previous conclusions aboutthe importance of organic matter from continental origin for juvenile production. However, inter-site differ-ences in the organic matter sources exploited for growth showed that, although freshwater organic matteruse is significant in all nursery sites, it is never dominant, with especially high contributions of local primaryproduction by microphytobenthos or saltmarsh macrophytes to juvenile sole growth in tidal nursery ecosys-tems. These patterns stress the need for maintaining both the intensity of freshwater inputs to the coastalzone and of local autochthonous primary production (especially that of the intertidal microphytobenthos)to preserve the nursery function of coastal and estuarine ecosystems.

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1. Introduction

Estuarine and coastal ecosystems play a critical role for stockmaintenance in marine fish (Beck et al., 2001; Peterson, 2003). Dueto their high productivity and the consequent high availability ofprey for juvenile fish (Beck et al., 2001; Gibson, 1994; Le Pape andBonhommeau, submitted for publication), they are essential nursery

86 O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

habitats for many species (Beck et al., 2001; Woodland et al., 2012)and particularly flatfishes (Le Pape et al., 2003b; Van der Veer et al.,2000). Because flatfish juveniles preferentially settle in habitats thatenhance their fitness (Gibson, 1994), the capacity of coastal and estu-arine areas to sustain juvenile growth can greatly condition the re-newal of flatfish populations (Iles and Beverton, 2000; Rijnsdorp etal., 1992). Yet, although rich and productive estuarine and coastalecosystems favours the development of benthic invertebrates,which are suitable food for young flatfish (Gibson, 1994), the main or-igin of the organic matter that sustains flatfish food webs on theirnursery grounds is still unclear. In estuaries, many studies (Banaruand Harmelin-Vivien, 2009; Darnaude et al., 2004a,b; Kostecki et al.,2010; Leakey et al., 2008; Pasquaud et al., 2008; Vinagre et al.,2008b) pointed out a predominant incorporation of allochthonous or-ganic matter of continental origin into juvenile flatfish food webs.However, in systems under low freshwater influence, in situ primaryproduction can override other food sources and significantly contrib-ute to growth in young flatfish (Kostecki et al., 2012).

Although spatio-temporal variations in the origin of the organicmatter used were analysed locally in various flatfish nursery sites(Darnaude, 2005; Kostecki et al., 2010; Pasquaud et al., 2008;Vinagre et al., 2011), only very few studies so far tried to quantifythe relative contribution of different possible sources of organic mat-ter to the food webs sustaining flatfish juveniles (França et al., 2012;Kostecki et al., 2012). This work aimed at filling this gap by quantita-tively estimating the variability in the origin of the organic matterexploited for juvenile growth in the common sole, Solea solea (L.,1758), among five major nursery sites in Western Europe.

S. solea is a highly prized species. Like most flatfish, it has a com-plex life cycle. Across its whole distribution area, fromWestern Africaand the Mediterranean to the Baltic (Wheeler, 1978), it spawns off-shore in winter and spring (Koutsikopoulos and Lacroix, 1992;Rochette et al., 2012; Vinagre et al., 2008a). Larvae are pelagic anddrift for several weeks in the marine plankton (Koutsikopoulos etal., 1991; Rochette et al., 2012; Savina et al., 2009) before settlingon coastal and estuarine nursery grounds from spring to early sum-mer (Le Pape et al., 2003b; Millner and Whiting, 1990; Riou et al.,2001). The juveniles spend about two years in these habitats, beforemigrating to deeper marine areas to join the reproductive stock(Dorel et al., 1991). The profusion of data gathered across Europe onboth the diet and the food webs of S. solea during this juvenile stage(Darnaude et al., 2001; Kostecki et al., 2012; Vinagre and Cabral,2008) provided a rare opportunity for a comprehensive multi-siteevaluation of the origin of the organic matter sustaining the growthof flatfish juveniles on their nursery grounds and its variability.

For this, stable isotopes analysis (SIA), based on nitrogen (δ15N)and carbon (δ13C) signatures of various food webs compartments,can be a powerful tool. This technique, which allows identificationof trophic pathways and energy sources in varied ecosystems(DeNiro and Epstein, 1978, 1981; Fry, 2006; Peterson and Fry,1987), has proved particularly successful in coastal and estuarine sys-tems, where the fate of different sources of both freshwater and ma-rine Particulate Organic Matter (POM) can be distinguished (Fry,1999; Vander Zanden and Rasmussen, 2001; Yokoyama and Ishihi,2008). By applying a mixing model analysis (Parnell et al., 2010) toprevious measurements of the δ15N and δ13C of both sole juvenilesand the main sources of organic matter sustaining benthic foodwebs in contrasted flatfish nursery habitats spread over Western Eu-rope, our aim was to quantify the main origin of the organic matterused for juvenile growth in S. solea across its distribution range, andthe reasons for its variability. More specifically, we wanted to testthe now long standing hypothesis of a positive link between thefreshwater POM availability related to upstream-downstream gradi-ent and variations of river flow and its contribution to juvenilesoles' food webs (Darnaude et al., 2004b; Kostecki et al., 2010), bothat the intra- and inter-site scales, and to investigate the influence of

various environmental characteristics (i.e. freshwater discharge,tidal amplitude, total surface of inter-tidal productive mudflats) onthe relative contribution of various organic matter sources to juvenilesole growth.

2. Materials and methods

2.1. Collection of standardized data in different nursery systems

2.1.1. Site selectionSuch ameta-analysis required comparable and detailed stable isoto-

pic data on young-of-the-year (YoY) soles and their potential organicmatter sources from various major nursery grounds of the species, notonly spread over a vast part of its geographic range, but also with con-trasted environmental characteristics. Juvenile diet in S. solea can differhighly from site to site, following the inevitable geographic variations inthe composition of the assemblages of benthic invertebrates, which arethe major preys for YoY sole (Darnaude et al., 2001; Kostecki et al.,2012; Vinagre et al., 2008b). This spatial variability prevented the inclu-sion of intermediate trophic levels (i.e. sole's prey) in the present work.Therefore, sites with isotopic signatures (δ15N and δ13C) available bothfor fully settled YoY soles and for all the organic matter sources poten-tially sustaining benthic food webs were considered for inclusion inthe present comparisons. Five sites were finally retained (Fig. 1): theMont St. Michel Bay (MSMB), in theWestern English Channel, the estu-aries of the Vilaine, the Charente and the Tagus rivers, spread along theAtlantic European coasts, and the prodelta of the Rhone river in theMediterranean. These five sites have contrasted environmental charac-teristics (Table 1; Nicolas et al., 2010) and are representative of the va-riety of nursery habitats (estuaries, sheltered bays, prodeltas) colonisedby the species across its geographic distribution. Environmental differ-ences aremaximal between theMSMBand theRhone river prodelta, es-pecially in terms of intertidal zone area and freshwater inputs (Table 1):tidal amplitude is very low in the Mediterranean, resulting in a tidalrange of only a few centimetres in front of the Rhone river mouth,which strongly contrasts with the conditions in the MSMB, wheretidal range is the highest in Europe during spring tides (15.5 m) andthe area of the intertidal zone is estimated at 250 km2 (Arbach Leloupet al., 2008). Moreover, while in the MSMB the few existing riverine in-puts (27 m3.s−1) are rapidly diluted by the high hydrodynamics forceslinked to the mega-tidal regime (Cugier et al., 2010), the Rhone river dis-charge into the Mediterranean (1700 m3.s−1) is the main driver of theecosystem structure in front of the river delta (Auger et al., 2010). Envi-ronmental conditions in the three other habitats investigated are inter-mediate both in terms of freshwater inputs and intertidal zone area.

2.1.2. Standardisation/selection of dataIsotopic data from the five selected nursery sites were derived

from previous published studies (Darnaude et al., 2004b; Kostecki etal., 2010, 2012; Modéran, 2010; Vinagre et al., 2011). In each case,the same standardized subset of δ15N and δ13C signatures was select-ed to allow reliable comparisons between systems:

Ontogenetic changes in diet are common in S. solea during the firstyear of life, the YOY juveniles generally shifting from epibenthicmeiofauna to small benthic invertebrates during the first few monthsfollowing their benthic settlement (Marchand, 1993). Therefore, toavoid any confounding patterns, isotopic signatures of sole YOY inthe present meta-analysis were restricted to juveniles with compara-ble diets. In the common sole, the duration of the spawning seasonvaries with the latitude (Koutsikopoulos and Lacroix, 1992;Rochette et al., 2012; Vinagre et al., 2008a), and that of the larvaldrift depends on the population (Koutsikopoulos et al., 1991;Rochette et al., 2012; Savina et al., 2009). Accordingly, dates for larvalsettlement in the species fluctuate from spring to early summeramong the five selected sites. Thus, a common survey date wouldnot systematically sample YoY at the same life stage. To standardize

Mont St Michel Bay

Tagus

Vilaine

Charente

Rhone

U

U0 4km

0 5km45 °N

40 °N

10 ° W 5 °W 0 ° 5 °E

Fig. 1. Location of the five study sites along the North East Atlantic coast considered in this study. In the two upper panels, the upstream (U) and downstream (D) locations ofjuvenile sole data collection in the Vilaine and Charente estuaries are presented.

87O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

data among sites, only the isotopic signatures measured in YoY ofsimilar sizes (6 to 11 cm; Table 2) captured a few months after theirsettlement in each site (i.e. in late spring or summer depending onthe latitude) were selected for the present analysis. Thereby, all theYoY juveniles compared had approximately the same age and hadall entered the phase of fast growth sole juveniles experience aftertheir settlement in their nursery habitats. This allowed us to avoid bi-asing our conclusions by falsely including isotopic signatures fromfreshly settled post-larvae, with isotopic signatures still reflectingtheir pelagic life offshore (Darnaude, 2005), or from larger andolder fish, with a different diet (Kostecki et al., 2012; Marchand,1993). For the MSMB, the Tagus estuary and the Rhone river prodelta,S. solea juveniles' isotopic signatures were derived from YoY individ-uals all collected in the same year at their site of maximum abun-dance within the nursery habitat. In the other two nursery sites(Charente and Vilaine estuaries), two different locations were inves-tigated along the estuary (Fig. 1), during one year only for the Cha-rente estuary but for four different years in the Vilaine estuary. As aresult, a total of 13 different sets of isotopic signatures were exploited

Table 1Average annual river flow and percentage of intertidal area for the five estuarine sys-tems considered in this study (Nicolas et al., 2010).

Estuaries Average annual river flow(m3.s−1)

Proportion of intertidal area(%)

Mont St. Michel Bay 27 [60,80]Vilaine 73 [20,40]Charente 40 [40,60]Rhône 1700 [0,20]Tagus 300 [40,60]

for juvenile soles: 1 from the MSMB, 1 from the Tagus estuary and 1from the Rhone river prodelta, 2 from the Charente estuary and8 from the Vilaine estuary (Table 2).

Potential sources of organic matter at the base of benthic foodwebs are diverse in coastal and estuarine habitats (Kostecki et al.,2012). They include not only the in situ primary productions bylocal phytoplankton, microphytobenthos (MPB) and benthic macro-phytes (MPh), but also allochthonous seawater POM (SW) broughtby the currents and the freshwater POM discharged by local rivers(FW). In most cases, both the water POM and the sedimentary organ-ic matter (SOM) available locally in these areas result from a mix ofthese different sources in various proportions. It is thus difficult toidentify the ultimate origin of the organic matter ingested by the ben-thos from the isotopic signature of these two organic matter pools.Moreover, only a fraction of the sedimentary pool is labile and thetotal SOM is not an acceptable sample of fraction actually consumedby the benthos (Zetsche et al., 2011). The isotopic signatures of thelocal SOM and water POM were therefore not taken into account inthe meta-analysis. Similarly, the isotopic signature of in situ phytoplank-tonic production was not included, because this biotic compartment isvirtually impossible to separate from the POM of marine (SW) or fresh-water (FW) origin also permanently present in estuarine waters.

For the four sources of organic matter investigated (FW, SW, MPBand MPh), the isotopic signatures used at each site were collectedusing two alternative procedures (Table 2); if the sources wereanalysed within the same year and during the same season thanYoY, they were selected. If simultaneous data were not available, theywere replaced by values of time series data to take into account the po-tentially high temporal variability in the isotopic signatures of thesesources (Table 2). Especially, because seawater POM (SW) was not

Table 2Standardized stable isotopes data collected for the meta-analysis.

Nursery areas Intrasitesample

Young of the Year sole (YoY) Sources References

Date Number Mean size (mm) FW⁎ SW⁎ MPB⁎ MPh⁎

Mont St. Michel Aug. 08 51 78 Surv⁎ TS⁎ Surv Surv Kostecki et al. (2012)TS: Somlit, station “Astan”, Jun–Sep 2009

Vilaine Up⁎ July 04 5 80 Surv Surv Surv Surv Kostecki et al. (2010),excepted for SW:Nérot (2011), station “Est Hoedic”

Dw⁎ 5 65Up July 05 5 82Dw 5 95Up July 07 5 76Dw 17 68Up July 08 24 74Dw 5 69

Charente Up Sept. 07 17 97 Surv TS Surv No⁎ Surv: Modéran (2010); Modéran et al. (2012)TS: Riera (1995), station “Ré”, May–Sep 1998Dw 7 94

Rhône June 01 13 79 Surv TS No Surv Surv: Darnaude et al. (2004b)TS: Somlit, station “Frioul”, Mar–Jun 2010

Tagus July 09 6 83 TS Surv Surv Surv Vinagre et al. (2011)TS: Vinagre et al. (2008b), May–Sept 2001

⁎FW, freshwater POM, SW, seawater POM, MPB, microphytobenthos, MPh, macrophytes, Up, upstream, Dw, downstream of the estuary. Surv: sources are sampled in the same sur-vey period as YoY and data come from the same reference, TS: sources are taken from time series indicated in reference, period (month year) and sample stations are indicated, No:the source was not considered as significant.

Table 3Trophic enrichment factors (TEF) from sources to YoY.

Element Set of TEF Source-prey Prey-YoY Σ source-YoY

δ 13C TEF1 1 1 2TEF2 2 3

δ15N TEF1 2.2 3.4 5.6TEF2 −0.3 1.9

88 O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

often sampled simultaneously to fish collection in the ecosystems stud-ied, its isotopic signaturewas derived from time-series data available ei-ther from other local surveys (Vinagre et al., 2008b) or from the FrenchCoastal Monitoring Network SOMLIT (Service d'Observation en MilieuLITtoral; http://somlit.epoc.u-bordeaux1.fr/fr/). Benthic macrophyteisotopic signatures were not included in the analysis for the Charenteestuary, as they do not represent a significant organic matter source inthis nursery habitat, compared to the huge production from the inter-tidal microphytobenthos (Leguerrier et al., 2003; Modéran et al.,2012). Similarly, microphytobenthos was not considered a significantsource of organic matter for the Rhone river prodelta, because its pro-duction in this non-tidal turbid system is considered to be very limited.Detailed sampling and analytical protocols for each site can be found inthe references listed in Table 2, as well as more comprehensive detailson (i) variation in the isotopic signature of the freshwater POM from dif-ferent tributaries of the same area (MSMB, Kostecki et al., 2012), (ii) het-erogeneous isotopic signatures from different local macrophytes species(Kostecki et al., 2012; Vinagre et al., 2011) and, more generally, (iii) vari-ability in isotopic signatures among samples (Cloern et al., 2002).

2.2. Quantified estimates of organic matter sources in YoY trophic chain

The contributions of each organic matter source to YoY sole pro-duction in the five nursery habitats studied were quantified usingthe R (R Development Core Team, 2009) package SIAR (SIA in R) formixing model analysis (Parnell et al., 2010). The use of mixing modelsis necessary when dealing with more sources (in the present case,four) than the number of elements analysed (in the present case,two: δ15N and δ13C). In this case, only this kind of approach can pro-vide the user with the ranges of possible contribution for all sourcesthat could explain the isotopic signatures of the consumer consid-ered. Inputs to SIAR mixing model for each element (δ15N and δ13C)are the isotopic signatures of all the organic matter sources investi-gated with their associated standard errors, replicates samples of con-sumer isotopic signatures and the trophic enrichment factors (TEF),i.e. the differences in C and N isotopic signatures between consumersand their diets (Minagawa and Wada, 1984) for the food webs stud-ied with their standard errors. In SIAR, the application of Bayesianmethods to the mixing model allows accounting for both uncertainty(i.e. the variation associated to both the variability of the isotopic sig-nature of the sources and that of the TEFs) and variability (i.e. the var-iation linked to inter-individual differences in isotopic signatures inthe consumer) when generating the probability estimates of thesource contributions.

Uncertainty in δ15N and δ13C isotopic signatures for each organicmatter source was calculated from the datasets. Both mean and stan-dard error of isotopic signatures for sources and individual YoY isoto-pic signatures (from 5 to 51 fish per sample, Table 2) were included inSIAR. A sensitivity analysis was also performed to analyse the impactof different TEFs onmodel outputs. The trophic level of YoY sole beingclose to 3 (Darnaude et al., 2001; Kostecki et al., 2012; Vinagre et al.,2008b), the global TEF used for this analysis corresponded, for eachelement (N and C), to the sum of that from primary producers orPOM to benthic invertebrates (YoY preys) and that from inverte-brates to YoY soles. A wide range of TEF values is available for juvenileflatfish in the literature (Bucheister and Latour, 2010; Minagawa andWada, 1984). However, in the present work, only 2 different globalTEFs (Table 3) were compared: one (TEF1) corresponding to thesum of the mean TEF values commonly used in the literature for theprimary consumers and the predators (DeNiro and Epstein, 1978,1981; Fry, 2006), and one (TEF2) corresponding to the sum of themean TEF value commonly used for primary consumers (DeNiroand Epstein, 1978, 1981) and a mean TEF value derived from labora-tory investigations of TEF variability in the winter flounderPleuronectes americanus (Bosley et al., 2002), a flatfish with juvenilephysiology close to that of sole. These 2 global TEFs were among themost extreme values proposed between primary producers and sec-ondary consumers in the literature, others estimates being generallyintermediate (Fry, 2006; Minagawa and Wada, 1984). Therefore, thesensitivity analysis performed probably allowed reliable estimationof the variation in source contributions to be expected given the nat-ural variability of TEFs. For this, the SIAR model was run twice foreach site, with the standard error associated to both the global TEFvalues tested set by default at 0.5‰.

2.3. Calculation of sources' contribution from SIAR mixing model outputs

For each of the two global TEFs retained, the SIARmixingmodel wasrun on the 13 datasets collected for the 5 nursery sites studied (Table 2)

Fig. 2. First factorial plan of a Principal Component Analysis on the contributions (in %) of the 4 organic matter sources in YoY, calculated on the 13 samples collected on the fivenursery sectors (Table 2) with 2 different TEFs (Table 3). a) 26 individuals (13 samples∗2 TEFs) with their character code (First, cap. letter for the nursery sector, “M” MSMB, “V”Vilaine, “C” Charente, “R” Rhone and “T” Tagus; second, when needed, min. letter for spatial subsamples “u” for upstream and “d” for downstream; third, Vilaine only, number forthe sampling year in 2000s). Symbols depend on the TEF, filled circle for TEF1 and filled squares for TEF2. Sector barycentre is indicated as crossed squares. b) Variables (4 sources),dashed lines indicating the projection of supplementary variables, not included in the analysis.

Table 4Differences in source contributions for YoY (in %) between estimates realized with thetwo tested enrichment factors, calculated on the 13 samples.

SW FW Intertidal:MPB+Mph

Mean σ Mean σ Mean σ

TEF1 37.3 19.1 14.4 12.4 48.2 15.1TEF2 22.7 16.7 22.1 11.8 55.2 18.6TEF2–TEF1 −14.87 14.6 +7.7 8.6 +5.9 13.9

89O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

to estimate the ranges in contribution of the four different organic mat-ter sources (FW, SW, MPB, MPh) to YoY sole growth (in %; see Kosteckiet al. (2012) for an example of SIAR model outputs for MSMB). When,for one of these four sources, several isotopic signatures were includedin the analysis (i.e. from different rivers or different types of freshwaterloadings or for several macrophytes species), their contributions weresummed to obtain a global contribution range for the correspondingsource (FW, SW, MPB or MPh).

To visualize the influence of TEF variation on the model outputs andprovide a first overview of the general patterns in sources contributionamong the different sites, a Principal Component Analysis (PCA) wasrun on the model outputs (26 individuals: 13 samples×2 runs of themixingmodels for the two global TEFs; four variables: themean estimat-ed contribution of each source being expressed in %). The differences insource contribution estimates between paired points (i.e. between thetwo different estimates obtained with the two global TEFs for the samesample) were also calculated to provide additional information on thesensitivity of the SIAR mixing model to uncertainty on the TEFs.

2.4. Intra-site and inter-site variability

The results from themultiple stations analysed in the Vilaine and theCharente estuaries allowed investigation of the spatio-temporal varia-tions in source (especially freshwater POM) contribution to YoY solegrowth within nursery habitats, in link with the position of their site ofcapture siteswith respect to theupstream-downstream salinity gradientand to the inter-annual variability in river flow (in the Vilaine estuary).

Model outputs were finally compared among the five nursery sitesand the differences in sources contribution were analysed in relationto average river flows and the area covered by the intertidal zone ineach habitat (Table 1).

3. Results

3.1. Sensitivity of SIAR model outputs to TEF variations

The first factorial plan of the PCA performed on the SIAR modeloutputs (Fig. 2) reflected more than 78% of the total variability inthe dataset and therefore gave a reliable image of the variability insource contribution estimates, both among samples and accordingto the global TEF retained for the calculations. Source contribution

estimates for each sample were generally close for both TEF values,although the seawater POM contributions were systematically lower(of 15% in average) with TEF2 (Table 4). Differences between sampleswere higher than that linked to TEF choice, apart for the samples col-lected in the Vilaine estuary in consecutive years (in 2004 and 2005and in 2007 and 2008, Fig. 2). Model outputs therefore enabled toconfidently identify spatial differences in source contributions, notonly between the five nursery sites, but also between different partsof the Vilaine and Charente estuaries.

As contrasts between sites were not obscured by discrepancies linkedto TEFs, source contributions for each site were averaged between thetwo TEFs (TEF1 and TEF2) and the resulting averaged proportions wereused to interpret the variations observed among the 13 samples analysed.

3.2. Intra-habitat variations in food sources for YoY sole

SIAR model outputs always differed between the upstream anddownstream samples from the same nursery habitat (Vilaine or Cha-rente estuaries) and the same year (for the Vilaine estuary), with aconsistently higher contribution of seawater POM (SW) in the down-stream sampling sites (Fig. 2). This systematic change in source con-tributions along the estuarine salinity gradient was confirmed byFig. 3. For YoY sole located downstream (D; Fig. 1), the contributionof freshwater POM (FW) was lower and compensated by higher con-tributions of seawater POM and microphytobenthos (MPB). Averag-ing the contributions of both sampling sites for each year in theVilaine estuary also showed the existence of inter-annual variationsin the contributions of each source to YoY sole growth, with an in-crease in the contribution of FW and a reduction in that of SW andMPB (Fig. 4a) with increasing river flow (Fig. 4b).

SW FW MPB MPh-15

-10

-50

510

15SW FW MPB MPh

2004200520072008

diffe

renc

e in

con

trib

utio

n (d

owns

trea

m-u

pstr

eam

in %

)

-20

-10

010

2030

SW FW MPB-40

-30

-20

-10

010

20

a b c

Fig. 3. Differences in organic matter sources contribution (in %) for YoY between the outer and the inner parts of the estuaries a) for the 4 studied years in the Vilaine, b) in averageamong years in the Vilaine, c) in the Charente.

90 O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

3.3. Inter-habitat variations in food sources for YoY sole

The contributions of the four organic matter sources to YoY solegrowth differed between the five nursery habitats (Figs. 2 and 5). Es-timated contributions for freshwater POM (FW) varied between 14%and 35% according to the nursery site. FW contribution to YOY solegrowth was thus substantial everywhere but never dominant.

SW FW MPB

a

Pro

port

ion

(%)

010

2030

4050

Fig. 4. a) Sources' contribution in the Vilaine estuary with relation to the river flow on the 4 sand during YoY settlement, from February to June.

Primary production was the main source of organic matter foryoung soles irrespective of the nursery habitat: in the Rhone riverprodelta, the only non-tidal system, YoY soles' food webs appearedto be mainly supplied by phytoplankton, as indicated by the majorcontribution of SW (Fig. 5). In the tidal sites however, SW contribu-tion was low and intertidal primary production (by the micro-phytobenthos and saltmarsh macrophytes) provided the main part

MPh

2004

2005

2007

2008

bR

iver

flow

(m

3/s)

020

4060

8010

012

0

2004

2005

2007

2008

tudied years a) contributions (in %) of the 4 sources, b) mean river flow (m3.s−1) before

Pro

port

ion

(in %

)

020

4060

8010

0

MSM

B

Chare

nte

Rhone

Vilaine

Tagus

SWFWMPBMph

Fig. 5. Average contribution (in %) of organic matter sources to YoY trophic chain onthe 5 studied nursery sectors.

91O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

(>50%) of the organic matter exploited by young soles (Fig. 5). In theMSMB, and the Charente and Vilaine estuaries, macrophytes contri-bution was low and the microphytobenthos (MPB) was the mainsource of organic matter sustaining YoY sole growth.

Inter-habitat comparisons of the contributions of the differentsources to YoY sole growth revealed an unexpected overall reverse

4 5 6 7

1020

3040

5060

7080

SW

Pro

port

ion

(in %

)

4 5

1520

2530

35

Log(R

Fig. 6. Contribution (in %) of the different organic matter sources to YoY trop

relationship between FW exploitation by young soles and local riverflow (Fig. 6). However, this general tendency seemed to be mainlydriven by the particularly high river flow observed for the Rhoneriver prodelta when compared to the four other sectors studied(Table 1). A similar bias was observed when local source contribu-tions were related to intertidal zone area for the five habitats, thegeneral pattern observed for the four tidal sites contrasting with thesituation in the Rhone river prodelta (Fig. 7); apart from the Rhone,intertidal production appeared to be the dominant organic mattersource for YOY soles.

4. Discussion

4.1. Providing reliable estimates of organic matter source contributions:a difficult challenge

4.1.1. Estimates of source contribution at a critical juvenile life stageIn YoY juveniles of the common sole, muscle isotopic turnover

rates (i.e. the speed at which this tissue reaches a new isotopicsignature equilibrium after a shift to an isotopically distinct diet)can exceed one month (Vinagre et al., 2008b; Darnaude A.M., per-sonal communication). Therefore, the isotopic signature of some ofthe sole individuals used for the present meta-analysis (6–11 cmin size) might still partly reflect that acquired during their larvallife in the marine plankton. Hence, the isotopic signature forolder juveniles could suggest a much higher exploitation of fresh-water POM (Darnaude, 2005; Darnaude et al., 2004a). However,the most important trophic changes in sole occur during thevery first months of benthic life (Marchand, 1993), well beforethe YoY reach the sizes used in the present meta-analysis. Becausemortality in the species is maximum during this period and foodavailability is a major determinant in modulating final populationsize in sole (Juanes, 2007; Le Pape and Bonhommeau, submittedfor publication), the isotopic signature analysed here reflect that

6 7

FW

4 5 6 7

1020

3040

5060

Intertidal: MPB + MPh

iver flow)

hic chain with relation to log-transformed average river flow (m3.s−1).

1020

3040

5060

7080

SW

Pro

port

ion

(%)

1 2 3 4

1520

2530

35

FW

1 2 3 4

1020

3040

5060

Intertidal: MPB + MPh

1 2 3 4

Intertidal area (in classes)

Fig. 7. Contribution (in %) of the different organic matter sources to YoY trophic chain with relation to the proportion of intertidal surface (in classes of percentage: 1 [0%, 20%], 2[20%, 40%], 3 [40%, 60%], 4 [60%, 80%]).

92 O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

of the sources of organic matter exploited during the critical stagethat determines most of the recruitment success in the species.

4.1.2. Accounting for uncertainty on both isotopic signatures and TEFsusing mixing models coupled with a sensitivity analysis

In this study, a non-negligible level of uncertainty remains aroundthe local isotopic signatures used for the four organic matter sourcesinvestigated, especially in the habitats where isotopic signatures werederived from time-series data because measurements concomitant toYoY sole sampling were missing (Table 2). YoY sole isotopic signa-tures also presented an important variability for a given site anddate (Darnaude et al., 2004b; Kostecki et al., 2010, 2012; Modéran,2010; Vinagre et al., 2011). The SIAR software package, based onBayesian inference and thereby allowing integration in the analysisof both within sources and among samples variabilities (Parnell etal., 2010), was therefore particularly appropriate for this meta-analysis. However, stable isotopes mixing models are highly sensitiveto TEFs (Bond and Diamond, 2011). As very different values of TEFshave been previously estimated from prey to consumers (DeNiroand Epstein, 1981; Fry, 1999; McCutchan et al., 2003; Post, 2002)and more specifically from preys to juvenile flatfish (Bosley et al.,2002; Bucheister and Latour, 2010), the robustness of SIAR outputsto TEF variability had to be tested. Our results demonstrated thatSIAR estimates of source contributions were sensitive to variationsin the TEF values used for the predictions but the correspondingbias in source contribution estimates was too low to obscure inter-sites variations in source contributions. The only overlap in sourcecontribution estimates with the two extreme TEFs tested concernedsamples from the Vilaine estuary collected in the same part of the es-tuary (upstream or downstream) in couples of years (2004–2005 and2007–2008) with similar annual river flow conditions (Fig. 4b). Whenthe same area (upstream or downstream part of the estuary) is

sampled for fairly similar annual river flow conditions (2004–2005and 2007–2008; Fig. 4b); source contribution is also quite similar(Kostecki et al., 2010) and local interannual patterns in source contri-bution could not be dissociated from differences related to TEFs. Thus,despite the inevitable uncertainty in our estimates linked to the var-iability in the isotopic signatures (Cloern et al., 2002) dataset usedand that linked to inter-site possible variability in TEFs (Post, 2002),the spatio-temporal patterns in source contributions obtained canbe considered robust.

4.1.3. Towards more robust conclusions from a quantitative approachSIAR model outputs from the present work supplement the infor-

mation gathered from the site-specific studies previously conductedin the Rhone river prodelta and in the three estuarine habitats(Tagus, Charente and Vilaine) included in this meta-analysis(Darnaude et al., 2004a,b; Kostecki et al., 2010; Modéran, 2010;Vinagre et al., 2011). Hence, the sources of organic matter for youngsoles and their variations, formerly only identified graphically basedon δ15N vs. δ13C biplots, were quantified precisely for the first timein these nursery habitats. Our results confirmed the general down-stream decrease in freshwater POM exploitation by YoY soles intheir estuarine nurseries (Kopp et al., submitted for publication;Kostecki et al., 2010; Pasquaud et al., 2008; Vinagre et al., 2008b)and the positive link between river flow and freshwater POM incor-poration at different time scales (Kostecki et al., 2010; Vinagre et al.,2011). However, several previous studies, based on graphic interpre-tations of stable isotope signatures only, had concluded that YoY solesmainly rely on freshwater organic matter in their estuarine nurserygrounds of Western Europe: in front of the Rhone river (Darnaudeet al., 2004b) and in the Vilaine estuary (Kostecki et al., 2010) inFrance; in the Tagus estuary (Vinagre et al., 2008b) in Portugal andin the Thames estuary (Leakey et al., 2008) in the UK. Be re-

93O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

analysing quantitatively the data from 3 of these 4 sites, the presentstudy indicated that freshwater POM contributes to less than a thirdof the organic matter exploited for growth in YoY sole, at least duringthe first few months of benthic life investigated here. Despite thesources of uncertainty in our model predictions listed above, graphicmisinterpretations of trophic relationships from δ15N vs. δ13C biplots(Parnell et al., 2010) might have resulted in previous overestimationsof freshwater POM contribution to YoY sole growth. In any case, thesediscrepancies plead for a more thorough analysis of δ15N vs. δ13Cbiplots and the combined use of quantitative approaches like SIARmixing models for accurate estimation of source contributions fromstable isotopes data.

4.2. Variations of organic matter sources for YoY soles: from local spatio-temporal patterns in organic matter sources to differences among nurs-ery sectors

4.2.1. A systematic moderate influence of freshwater inputsAs confirmed by the outputs from our models, the organic matter

of freshwater origin incorporated into benthic food webs in coastaland estuarine ecosystems is transferred up to juvenile soles(Darnaude, 2003, 2005; Kostecki et al., 2010). It controls both theavailability of suitable prey for sole juveniles (Darnaude et al.,2004b) and the size of their nursery habitats (Le Pape et al., 2003a).Therefore freshwater inputs in organic matter modulate nursery ca-pacity and recruitment success in sole (Darnaude et al., 2004b; LePape et al., 2003b). However, although the influence of FW as a tro-phic source for YoY sole was notable in all the nursery sites testedin the present study and the results for the Charente and the Vilaineestuaries confirmed that its exploitation in sole increases with itsavailability over an upstream-downstream gradient and with theriver flow (Darnaude et al., 2004a; Kostecki et al., 2010), the contribu-tion of this organic matter source to YoY sole growth appeared to re-main quite low irrespective of the nursery site. The highestcontribution (35%) was surprisingly found in the MSMB, where fresh-water inputs are very limited (Cugier et al., 2010). The widespreadhypotheses of a larger exploitation of freshwater inputs by sole juve-niles in large estuaries and of a marked difference between the organ-ic matter sources sustaining their growth in their estuarine and intheir marine nursery sites (Kostecki et al., 2012) were therefore notconfirmed by the present meta-analysis. This can partly result fromthe size classes of YOY sole selected for inclusion in the analysis, espe-cially since isotopic signatures in older YoY sole (collected in late au-tumn) reflected a more pronounced freshwater influence in front ofthe Rhone river (Darnaude, 2003). However, YoY sole behaviouralso contributes to the lack of inter-site contrast evidenced here.Hence, the common sole is not considered a strictly estuarine depen-dent species (Araujo et al., 2000; Le Pape et al., 2003b). Its juvenilesdo not use the most oligohaline parts of the estuaries (Rochette etal., 2010) and, although YoY spatial distribution changes during de-velopment (Dorel et al., 1991), their salinity niche during their firstmonths of benthic life is quite similar among nursery sectors (LePape et al., 2003b; Rochette et al., 2010) and their distribution driftsdownstream with increasing river flow (Le Pape et al., 2003a). Thissuggests that YoY faces a similar range of freshwater influenceirrespective of the river discharge in estuarine habitats. It is thereforenot particularly surprising that the contribution of FW to their growthis not more important in larger estuaries. YoY sole behaviour also ex-plains the surprising high FW contribution to sole growth found inthe marine MSMB. In this bay, sole juveniles concentrate in front ofsmall tributaries (Kostecki et al., 2012), in restricted areas under no-ticeable estuarine influence (Cugier et al., 2010). Low salinity hasbeen shown to facilitate metamorphosis in the species and attractits larvae (Koutsikopoulos et al., 1989), which use selective tidalstream transport to reach appropriate nursery habitats (Amara etal., 1998; Boehlert and Mundy, 1988; Grioche et al., 2000; Ramzi et

al., 2001). Attraction by sediment or food chemical signatures wasalso suggested as an explanation for this estuarine tropism, althoughthe nature of the cues for their detection remains unclear (Amara etal., 1998; Vinagre et al., 2007). Anyhow, the resulting distribution ofYoY sole in the MSMB explains the relatively high freshwater POMcontribution to their growth in this marine zone despite low freshwa-ter inputs and reinforce the hypothesis of YoY sole being attracted to-ward areas under freshwater influence, where their growth isenhanced (Le Pape et al., 2003c).

4.2.2. A significant influence of intertidal productionThis meta-analysis revealed the major role of benthic intertidal

primary production in sustaining YoY food webs and maintainingthe nursery function of estuarine and coastal tidal ecosystems. Forthe four tidal habitats studied, the contribution of intertidal benthicproduction to YoY sole growth was shown to exceed 50%, in accor-dance with results on the main organic food sources sustaining fishproduction in tropical estuaries (Melville and Connolly, 2003). Intemperate estuaries, the production of various species of commercialinterest had been suggested to be predominantly sustained bysaltmarsh macrophytes detritus, either through direct ingestion orvia their incorporation in local benthic food webs (Boesch andTurner, 1984). This is nuanced by the results from the present analy-sis, which confirmed the contribution of organic matter fromsaltmarsh plants to fish production, notably in the MSMB (Laffailleet al., 1998), but stressed the prime role of the microphytobenthosin sustaining juvenile marine fish growth in tidal areas, as alreadysuggested in the Tagus estuary (França et al., 2012) and the MSMB(Kostecki et al., 2012). Our results suggest however that the contribu-tion of benthic primary production to YoY sole growth could be verylow in non-tidal nursery habitats. Indeed, in the Rhone river prodelta,the contribution of autochthonous phytoplankton prevails becausewater turbidity, linked to the presence of a particularly dense riverplume, limits benthic primary production (Bodoy and Plante-Cuny,1980). Supplementary analyses that include other types of Mediterra-nean nursery habitats (coastal lagoons, estuaries, sheltered bays) areneeded to confirm this observation and further investigate organicmatter sources for YoY sole in non-tidal systems.

Our conclusions echo previous calls for the preservation and eventhe restoration of the quality of intertidal zones in tidal areas (Elliottet al., 2007). Indeed, our results stress that juvenile growth in thecommon sole depend highly on intertidal food sources, as in othercoastal and estuarine dependent species (França et al., 2012;Kostecki et al., 2012). Maintaining the productivity of habitats thatsustain nursery function should be the primary focus for such fishspecies, whose population size depends on trophic resources at thejuvenile stage (Lamberth et al., 2009; Le Pape and Bonhommeau,submitted for publication; van de Wolfshaar et al., 2011). Neverthe-less, primary production in estuarine and coastal habitats is highlydependent on freshwater loadings in inorganic nutrients (Mallin etal., 1993). Because the latter enhance both micro- (Underwood andProvot, 2000) and macro-phytobenthic productions (Valiela andTeal, 1974) in the intertidal zone, the maintenance of river inputs tothe coastal zone is also of primary importance for flatfish stock main-tenance. Our results confirm that future modifications of river inputsto the coastal zone in Western Europe, under the combined influenceof climate change and anthropogenic freshwater use, could alter pro-foundly the nursery function of estuarine and coastal ecosystems(Dolbeth et al., 2008). At least in the common sole, the consequenceson fisheries production of such changes could prove dramatic if boththe direct (through benthos use of freshwater POM) and indirect(through nutrients use by local primary producers) enrichments ofYoY food webs by riverine inputs were altered. Appropriate measureswill have to be urgently taken to preserve the nursery function andcapacity of coastal and estuarine systems and, as a consequence, theexploited stocks and related fisheries.

94 O. Le Pape et al. / Journal of Sea Research 75 (2013) 85–95

Acknowledgements

This project used data from the French Coastal Monitoring Net-work SOMLIT (Service d'Observation en Milieu LITtoral; http://somlit.epoc.u-bordeaux1.fr/fr/; P.I. Benoit Sautour, UMR EPOC,OASU, Arcachon, France). We thank the SOMLIT teams of Roscoff(P.I. Pascal Morin) and Marseille (P.I. Patrick Raimbaut) for providingus with their data. Other data were obtained in the scope of projectsconducted in Portuguese estuaries, funded by Fundação para a Ciênciae para a Tecnologia and on French coastal and estuarine sectors,funded by the French national program for coastal environment(PNEC). We thank Caroline Nérot for her contribution to data provid-ing. Julien Moderan and Caroline Kostecki were funded by a PhDgrant from the French ministry of research. Catarina Vinagre ac-knowledges the grant SFRH/BPD/34934/2007 and Susana França thegrant SFRH/BD/27627/2006 from the Portuguese National ScienceFoundation (FCT). We are grateful to Sophie Pasquier for compilingreferences and to Rowena Stern (SAHFOS, Plymouth, UK) and ZaraReveley (Universidade de Lisboa, Portugal) for suggesting English im-provements. We would like to apologize to colleagues from variousresearch institutes (Valérie David, Valérie Ménage, Benoit Sautour)for removing some other study sectors from the analysis after theirdata collection because of the lack of standardized data. We alsowould like to thank the two anonymous reviewers for their helpfuladvice and significant improvements of the manuscript.

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