Estuarine habitats structure zooplankton communities: Implications for the pelagic trophic pathways

Accepted Manuscript Estuarine habitats structure zooplankton communities: implications for the pelagic trophic pathways Valérie David, Jonathan Selleslagh, Antoine Nowaczyk, Sophie Dubois, Guy Bachelet, Hugues Blanchet, Benoît Gouillieux, Nicolas Lavesque, Michel Leconte, Nicolas Savoye, Benoît Sautour, Jérémy Lobry PII:

S0272-7714(16)30022-1

DOI:

10.1016/j.ecss.2016.01.022

Reference:

YECSS 5020

To appear in:

Estuarine, Coastal and Shelf Science

Received Date: 11 October 2014 Revised Date:

9 January 2016

Accepted Date: 15 January 2016

Please cite this article as: David, V., Selleslagh, J., Nowaczyk, A., Dubois, S., Bachelet, G., Blanchet, H., Gouillieux, B., Lavesque, N., Leconte, M., Savoye, N., Sautour, B., Lobry, J., Estuarine habitats structure zooplankton communities: implications for the pelagic trophic pathways, Estuarine, Coastal and Shelf Science (2016), doi: 10.1016/j.ecss.2016.01.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Structuration of the zooplankton community during summer in the Gironde estuary: implications for zooplanktivorous fishes

ACCEPTED MANUSCRIPT Estuarine habitats structure zooplankton communities: implications for the pelagic trophic pathways Valérie David(1), Jonathan Selleslagh(1,2), Antoine Nowaczyk(1), Sophie Dubois(1), Guy Bachelet(1), Hugues Blanchet(1*), Benoît Gouillieux(1), Nicolas Lavesque(1), Michel Leconte(1), Nicolas Savoye(1), Benoît Sautour(1), Jérémy Lobry(2) Université de Bordeaux-CNRS, UMR 5805 EPOC, 2 Rue du Professeur Jolyet, 33120 Arcachon,

France. (2)

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(1)

Irstea, UR EABX, centre de Bordeaux, 50 avenue de Verdun-Gazinet, 33612 Cestas, France.

* Corresponding author: [email protected]

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Tel: +33 (0)5 56 22 39 07

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Fax: +33 (0)5 56 83 86 51

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ACCEPTED MANUSCRIPT Abstract Estuarine ecosystems have been described as a mosaic of habitats exhibiting different physical, biological and chemical properties and processes. These habitats are of primary importance for fishes, providing refuges and/or food for juveniles. While it is well known that habitats contribute also to the structuration of meio- and macrobenthic assemblages, this concept of habitat has never been associated to zooplankton communities, a major food resource for many pelagic fishes during summer

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in North-European estuaries. The objective of this work was thus to assess if estuarine habitats, in addition to the salinity gradient, structured zooplankton communities as well. Sampling was conducted at high tide during summer in a highly turbid system, the Gironde estuary, for which primary production and thus food resource at the basis of the food web is strongly limited. The results showed

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that even if the upstream-downstream estuarine gradient was the main factor structuring zooplankton at the scale of the estuary, there was a significant difference of zooplankton assemblages between samples collected over subtidal areas and those collected over intertidal areas. More particularly, the

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estuarine gradient was associated to the distribution pattern of species while difference between subtidal and intertidal samples were mainly due to difference in the level of abundance of species. Stable isotope analysis revealed that these zooplanktonic omnivorous species may be attracted to intertidal mudflats by microphytobenthos availability and that some planktivorous fishes, in particular Alosa fallax, preferentially fed on this zone. The role of intertidal habitats in structuring zooplankton assemblages suggests that this habitat strongly participates to the production of planktivorous species

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and that it represents a biotic vector of carbon resources toward subtidal areas. The loss of tidal flats habitats could thus have consequences on the functioning of pelagic system as well.

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Keywords: zooplankton ; estuary ; tidal flats; salinity gradients ; food web; fish

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ACCEPTED MANUSCRIPT 1. Introduction Estuaries provide a variety of ecosystem services and societal benefits due to their connections with the adjoining terrestrial, freshwater and marine systems (MEA, 2005; O'Higgins et al., 2010; Barbier et al., 2011). They face rapid and strong physico-chemical fluctuations, at both spatial and temporal scales, preventing the establishment of a complex organization of the estuarine food web. This, on the other hand, is allowing the system to be both very productive and dynamically stable in terms of

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energy fluxes and consumer population dynamics (Moore et al., 2004; Lobry et al., 2008; Selleslagh et al., 2012a). Many marine juveniles of fishes presenting an economical interest depend on estuaries to complete their life cycle (Ray, 2005): whatever the fish feeding preference these systems provide highly nutritive environments, good environmental conditions favouring growth and, shallow turbid

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refuges (Beck et al., 2001, Pasquaud et al., 2010; Selleslagh et al. 2011).

Human population growth in the vicinity of estuaries has altered their structure and functioning (Elliott

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and Whitfield, 2011) because of excess nutrient loads (Bianchi et al., 2000), toxic pollutants (Tomlinson et al., 1980), alteration of water flows (Nilsson et al., 2005) and the loss of habitat, in particular wetlands and intertidal areas (MEA, 2005, Lotze et al. 2006). Estuarine ecosystems have been for a long time described as a mosaic of habitats exhibiting various physical, biological and chemical properties and processes (O'Higgins et al., 2010). These different habitats include (e.g.) subtidal or intertidal soft substratum, saltmarshes, biogenic reefs and seagrass meadows. They play a primary role as nursery for fishes and macrocrustaceans, providing refuge and/or food for juveniles

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(Elliott, 2002, Dahlgren et al., 2006, Holsman et al., 2006, Wouters and Cabral, 2009). Intertidal habitats, for instance display much higher primary and secondary productivity than other estuarine habitats (McLusky and Elliott, 2004). In such habitats, high organic and nutrient loads are associated with high biomasses of meio- and macrobenthic invertebrates, which in turn provide food to higher

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trophic levels (McLusky and Elliott, 2004). Thus, habitat structure and tidal influence contribute to the structuration of meio- and macrobenthic communities (Hosack et al., 2006; Wouters and Cabral, 2009;

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Blanchet et al., 2014). As a result, habitats have to be considered as subsystems within a larger ecosystem, when estimating not only the estuarine functioning but also its ecological and service values (O'Higgins et al., 2010). This concept of habitat has never been associated to plankton communities. Ranking among the bases of aquatic food webs, plankton plays a key role in estuarine productivity, with phytoplankton turning inorganic carbon and nutrients into bioavailable organic matter and zooplankton contributing to the transfer of this primary production to higher trophic levels including commercial fishes (Lobry et al., 2008, Selleslagh et al., 2012a). In highly heterotrophic estuaries (such as the Gironde estuary), where primary production is low, zooplankton is also known to be the most important vector for carbon transfer from detritus to top predators (Tackx et al., 2003, David et al., 2006a, Lobry et al., 2008). At the estuary scale, zooplankton distribution is classically explained by the salinity gradient (Baretta and

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ACCEPTED MANUSCRIPT Malschaert, 1988, Mouny and Dauvin, 2002, Tackx et al., 2004, Marques et al., 2007, Modéran et al., 2010, Chaalali et al., 2013a). However, habitats (e.g. subtidal versus intertidal areas) exhibit different physical and chemical conditions associated with different types and quantity of food sources (e.g. phytoplankton vs microphytobenthos), which could in turn favor or inhibit some zooplankton species (De Jonge and Van Beuselom, 1992). In addition, the differences in benthic macrofauna assemblages between habitats (McLusky and Elliott, 2004) may imply differences in the density and assemblages

structuration of zooplankton assemblages according to habitats.

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of meroplankton in the above water column. As a consequence, these factors might induce further

In this context, the main objective of this work was to assess if estuarine habitats, in addition to salinity gradient, participate to the structuration of zooplankton communities. This study was focused

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on subtidal and intertidal soft substrata, ranking among the main habitats recognized of interest for fishes (Elliott, 2002), in a highly turbid system: the Gironde Estuary (SW France). A particular attention was paid to food resources (through carbon and nitrogen stable isotopes) as a potential

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explaining factor of the habitat-related structuration. Implications for the trophic pathways are

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discussed.

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ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Study area The Gironde estuary is the largest SW European estuary (Fig. 1). Its surface area is about 625 km2 at high tide and its watershed covers 81,000 km2. Intertidal mudflats are reduced, representing approximately 8% of the estuary total area. The estuary is 76 km long between the Ocean and the Bec

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d’Ambès, where the Dordogne and Garonne rivers meet. This macrotidal well-mixed estuary is characterized by a large Maximum Turbidity Zone (MTZ), generated by tide asymmetry, that moves along the upstream-downstream axis according to river flow and tidal cycles (Sottolichio and Castaing, 1999). Water residence time ranges between 20 and 86 days. Particles residence time has been estimated to range in between 1 and 2 years (Jouanneau and Latouche, 1981). In the MTZ,

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particulate organic carbon (POC) content is very low and constant (1.5%, Etcheber et al., 2007). It is one of the most turbid estuaries in Europe with a level of suspended particulate matter (SPM) >500mg L-1 (Sautour and Castel, 1995) in which primary production is strongly reduced due to light limitation.

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As a consequence the phytoplankton primary production has been estimated as 10gC m-2 year-1 by Irigoien and Castel (1997). POC is mainly of terrestrial origin in the inner estuary (Savoye et al., 2012).

2.2. Sampling design and analyses

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2.2.1. Zooplankton community structure

Sampling was conducted at high tide in 18 stations during July 2012 (Fig. 1). Stations were located along the salinity gradient and within two habitats: subtidal (11 stations) and intertidal (7 stations) areas. Among intertidal sampling stations, two consisted in a former artificial pond and saltmarsh

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system which connection with the estuary was restored 13-years ago after storm-induced collapse of the seawall. Subtidal stations were located in the 2 main channels (Médoc and Saintonge channels)

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and in the middle of the estuary. Intertidal stations were located along the western bank of the estuary and in the downstream Chant-Dorat flat. The former pond-saltmarsh system was located in the downstream area, in Mortagne (Fig. 1). Prior to each zooplanktonic sampling, salinity and temperature were measured at each station with a WTW LF 197 thermo-salinometer. Sampling stations were then classified into three categories of salinity according to the Venice system (McLusky, 1993): stations where measured salinity was <5 (oligohaline conditions) were used to define the “upstream” zone of the estuary in summer (Fig. 1). Stations where salinity was comprised between 5 and 15 (mesohaline conditions) were used to define a “median” zone and stations where the salinity level was higher than 15 defined a “downstream” sector (with polyhaline conditions) (Fig. 1). The terms “polyhaline”, “mesohaline” and “oligohaline”

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ACCEPTED MANUSCRIPT areas were not used for these three sectors since our salinity measures were performed at high tide and corresponded to summer conditions only. Zooplankton was collected in the top first two meters below the surface using horizontal tow using a standard 200µm WP2 net. The volume of water filtered through the net was recorded with a Hydrobios digital flowmeter (5 to 10 m3). Samples were fixed in 5% seawater/buffered formalin, sorted and identified to the lowest possible taxonomic level under a stereomicroscope. Species

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identification was performed on 200 individuals for “strict” zooplankton (Frontier, 1972) and on all sampled individuals for suprabenthos (i.e. mysids, amphipods, isopods, shrimps) and fish larvae. Abundances were expressed as number of individuals per m-3.

Mysids, amphipods, isopods, shrimps and fishes might have avoided the 200-µm net due to their

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swimming abilities. A 500-µm bongo net, would indeed be more appropriate for sampling of hyperbenthic organisms in the Gironde estuary. A preliminary work however showed that a significant linear regression existed between mysid abundances estimated with a 200-µm WP2 net and a 500-µm

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bongo net (n=23; R2=0.78; data not shown). WP2 nets thus give a good signal of the variability of mysid densities despite a systematic underestimation of real densities. For all these taxonomic groups, densities were thus probably underestimated, but nevertheless still brought information of both their spatial distribution and their relative abundances among stations. The characterization of zooplankton assemblages (i.e. “strict zooplankton” and hyperbenthos) was performed with a previous fourth-root transformation, not only to decrease the great differences of density range between taxa and thus to

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buffer the importance of the highly dominant taxa (i.e. copepods) while avoiding a great consideration of rather “rare” and underestimated hyperbenthic taxa compared to dominant “strict” zooplankton taxa. 2.2.2. Trophic pathways

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Copepods, mysids and three planktivorous fishes (Engraulis encrasicolus, Sprattus sprattus and Alosa fallax) were collected in the subtidal and intertidal habitats of the downstream area for stable isotopes

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analysis. Zooplankton

Zooplankton sampling was conducted during the same period but one month before the sampling of fishes in order to take into account the turnover rates of isotopic elements in fishes (weeks for young fishes with fast growth). It was collected by short horizontal traits (1 to 3 minutes) with a WP2 net. Samples were filtered through 500 µm mesh to remove large debris. Organisms were then placed in filtered estuarine water for 24 h at 15°C before being sorted in the laboratory. Copepods and mysids were separated under a stereomicroscope and immediately frozen at -20°C. Only organisms displaying empty guts were collected. Samples were then freeze-dried and ground in fine powder with mortar and pestle for stable isotope analysis. Planktivorous fishes

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ACCEPTED MANUSCRIPT Fishes were collected during daylight using beam trawls in both the intertidal and subtidal areas (Selleslagh and Amara, 2008). In subtidal areas, additional samples were collected near the water surface using rectangular frame nets fitted on both side of the boat. These sampling devices allowed to catch small fishes including both bentho-demersal and pelagic species. For the current study only planktivorous fish data were used, namely Engraulis encrasicolus, Sprattus sprattus and Alosa fallax. Captured fishes were immediately washed with milli-Q water, identified, counted, measured (total

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length with 0.5 mm precision), and frozen at -20°C. Before stable isotopes analyses, fish were thawed and their white dorsal muscle was dissected. Samples were then freeze-dried and ground into fine powder before being encapsulated for stabe isotopes analysis. In order to verify that samples were not contaminated by carbonates, powder subsamples of all sample types were observed under binocular

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microscope and acidified with 1% HCl. Stable isotope analysis

Approximately 0.4 mg of sample was accurately weighed and encapsulated into small tin cups for

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stable isotopes analysis. Dissection tools, mortar and pestle and other materials used for preparation of samples were washed with 10% HCl, rinsed with milli-Q water and dried at 60°C. Stable isotope analysis was carried out by continuous-flow isotope ratio mass spectrometry (CFIRMS) with a Delta V advantage Isotope Ratio Mass Spectrometer (Thermo Scientific, Bremen, Germany) coupled with a Flash EA 1112 Elemental Analyser (Thermo Scientific, Milan, Italy). According to new recommendations of the International Union of Pure and Applied Chemistry

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(IUPAC), all isotopic data are expressed in the conventional delta notation (Coplen 2011): δ13Csample or δ15Nsample = (Rsample/ Rreference – 1)

Reference is Pee Dee Belemnite for δ13C and atmospheric N2 for δ15N. Replicate analyses of international standards (IAEA-N-2, IAEANO-3, IAEA-600, USGS 24, NBS-21) gave analytical errors

2.3. Data analysis

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(standard deviation) of less than 0.1‰ and 0.2‰ for carbon and nitrogen respectively.

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The objective was to assess the effects of “haline zone” and “habitats” on zooplankton through (1) univariate indices (total abundance an abundance of the main phyla, number of species) and (2) community composition. Stations were affected to three different “haline zones”, namely “downstream zone”, “median zone” and “upstream zone”. Permutational Analyses of Variance (PERMANOVA) were used on unbalanced design (Anderson, 2001). As subtidal and intertidal habitats were sampled in the three haline zones whereas the three habitats were available only for the polyhaline zone, two different analyses were performed. A first series of PERMANOVA (one per univariate variables plus one for community composition) was conducted on a two-way crossed design for which the two fixed crossed factors were: (1) haline zones (3 levels: upstream, median and downstream) and (2) habitats (2 levels: subtidal and intertidal) and in which the interaction between haline zones and habitats was considered. The relative size-effect

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ACCEPTED MANUSCRIPT of both factors were compared using the estimated mean square for each term of the model according to Anderson et al. (2008). The second series of PERMANOVA was realized on a one-way design to test the habitat effect (3 levels: subtidal, intertidal flats and former pond) in the downstream zone. The response variables chosen for univariate descriptors of zooplankton structure were fourth-root transformed: total densities, number of species and densities of the main phyla. Euclidean distance matrix was used for

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each univariate index and PERMANOVA were based on 9999 permutations residuals under a reduced model. For zooplankton community composition, all abundances were fourth-root transformed and the association matrix was based on Bray-Curtis similarities. The statistical significance of permutational variance components was tested using 9999 permutations. Pairwise comparisons were performed

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whenever significant differences were detected for each factor or interaction. If the number of permutations was lower than 300, the Monte-Carlo permutation p was used.

The relative distances of all sampled stations based on their zooplankton assemblages (rank order of

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the Bray-Curtis dissimilarities) were represented in a two-dimensions space using a non-metric multidimensional scaling (nMDS) ordination (Kruskal and Wish, 1978). Zooplankton community compositions that presented significant differences based on PERMANOVA tests were characterized by the similarity percentage routine (SIMPER; Clarke, 1993) and the IndVal method (Dufrêne and Legendre, 1997). SIMPER indicated which taxa were mainly responsible for differences observed among identified communities using Bray-Curtis dissimilarities. It also provided

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the contribution of taxa to the average similarity of each community and the average dissimilarity of each pair of assemblage. In addition, IndVal index was computed for each taxa in each identified community taking into account measures of both fidelity and specificity. In contrast to the SIMPER routine, the IndVal index for a given taxon is independent of the relative abundance of any other taxa.

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It is maximal when all individuals of a taxon are found in a single community or when the taxon occurs in all stations for that community. The significance of the index was tested by random combination of stations within communities (Z test, n=9 999).

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Bilateral Wilcoxon rank sum test was used to compare isotopes ratios (carbon and nitrogen) between subtidal and intertidal habitats in the downstream zone for copepods, mysids and the three planktivorous species Engraulis encrasicolus, Alosa fallax and Sprattus sprattus. Unilateral Wilcoxon rank sum test was then used to test if each potential predators presented higher isotope ratio than its potential preys (mysids on copepods, planktivorous fish on copepods and mysids). For each prey, the tests were realized independently on subtidal and intertidal isotope ratios whenever significant differences were recorded with previous tests. Bayesian mixing models were used in the discussion to assess the relative importance of several food sources in the diet of the three planktivorous species based on carbon and nitrogen stable isotope analysis (package SIAR: Stable Isotope Analysis in R; Parnell et al., 2010). They allowed to incorporate uncertainties in (i) carbon and nitrogen signatures of sources and consumers and (ii)

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ACCEPTED MANUSCRIPT trophic enrichment factors within the model. They provide potential dietary solutions as true probability distributions. trophic fractionations of 1±0.5‰ for carbon and 3±1‰ for nitrogen (Vanderklift and Ponsard, 2003) were used. The choice of the sources considered in SIAR models was based on the results of this study and were thus detailed in the discussion. All tests were performed with the R software (R-Development-Core-Team 2008) or with the

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PERMANOVA+ v1 for PRIMER 6.1 package.

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ACCEPTED MANUSCRIPT 3. Results Thirty-seven taxa of zooplankton were recorded in the 18 stations, from genus to species excepted for larval stages of benthic species, with “strict” mesozooplankton taxa (9 copepods, 4 jellyfishes, 1 ctenophore, 5 larvae of benthic species and 7 fish larvae) and suprabenthic species (3 mysids, 4

3.1. Zooplankton structuration : haline zones versus habitats

3.1.1. Synthetic descriptors

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shrimps, 2 isopods, 1 amphipod; Table 1).

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Number of taxa was higher in the downstream zone compared to both intermediate and upstream zones (> 13 taxa for the downtsream area vs < 10 taxa, on average, in the intermediate and upstream areas; permutational pairwise tests p<0.05; Fig. 2 and Table 2). In addition the number of taxa per

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sample was significantly higher in intertidal (On average, >13 taxa) than subtidal habitats (<10 taxa, on average; permutational pair-wise tests p<0.05; Fig. 2 and Table 2).

The a posteriori Monte-Carlo test showed significant decreasing total densities from upstream- to downstream zones only for subtidal habitats (33 790±5 182 ind.m-3, 2 322±269 ind.m-3 and 408±198 ind.m-3, for upstream, median and downstream zones, respectively Fig. 2 and Table 2). Differences in total densities between subtidal and intertidal habitats were only observed in the downstream zone

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with higher densities in intertidal habitats (11 854±3 603 vs 408±198 ind m-3, respectively; MonteCarlo test with p<0.05; Fig. 2 and Table 2).

Copepods and mysids presented significantly different densities among haline zones and a significant interaction between haline zones and habitats (permutational pair-wise tests p<0.05, Table 2).

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Copepods showed a significant decrease of abundances from upstream to downstream zones in subtidal habitats (Fig. 2 and Table 2). Mysids presented significantly increasing densities from

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upstream to downstream zones for intertidal habitat (Fig. 2) and significantly higher densities for the intertidal habitat than for the subtidal one only in the downstream zone (886±53 ind.m-3 and 41±27 ind.m-3, respectively; Monte-Carlo test, Fig. 2). Gelatinous plankton presented significant spatial pattern according to haline zones with null densities recorded in the upstream zone vs median and downstream zones (permutational pair-wise tests p<0.05, Fig. 2 and Table 2). Benthic larvae showed significantly higher abundances for intertidal (4 852±3 108 ind.m-3) than for subtidal habitats (92±51 ind.m-3) whereas fish larvae presented the same trend but only in the downstream zone (Monte-Carlo test, Fig. 2 and Table 2).

3.1.2. Zooplankton community composition

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ACCEPTED MANUSCRIPT According to PERMNOVA results, there was a significant effect of both “haline zones” and “intertidal/subtidal habitat” on the zooplankton community structure (Table 2). In addition the effect of “intertidal/subtidal” situation of samples was similar across “haline zones” since the interaction term was not significant (Table 2). According to the estimates of the components of variation provided with the PERMANOVA results, the effect of “haline zone” on zooplankton community structure was stronger than the effect of “intertidal vs subtidal habitat” (Anderson and Gorley, 2008). The MDS-plot

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illustrates the clear difference of zooplankton assemblages sampled in the three haline zones (Fig 3). It also suggests an effect of the intertidal/subtidal situation of samples on the community structure (Fig. 3). In the downstream zone where the intertidal situation also included saltmarshes, our results showed that the zooplankton composition was significantly different only between subtidal and

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intertidal zones (PERMANOVA and a posteriori Monte Carlo test) (Table 2, Fig. 3).

3.2.1. Considering the salinity gradient

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3.2. Characterization of the different zooplankton assemblage structure

The upstream zone, where salinity level was lower than 5, presented 5 species reaching significant IndVal values: the copepod Eurytemora affinis (IV=96%, p=0.008), the mysid Neomysis integer (IV= 87%, p=0.009), the amphipod Gammarus zaddachi (IV=99.9%, p=0.001) and the shrimps, Palaemon longirostris (IV=100%, p=0.002), and P. macrodactylus (IV=65%, p=0.02). These species may be considered as representative due to their high specificity and fidelity to this zone. Due to its very high

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abundances in this zone (23 176±10 436 ind.m-3; Table 1), E. affinis was not only the most important species contributing to the average similarity of this group (60%). This low-salinity zone was also characterized by the cyclopoid Acanthocyclops robustus, which was only recorded here (Table 1).

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The median zone, gathering stations where salinity was comprised between 5 and 15, presented one significant indicator species: the jellyfish Blackfordia virginica (IV=97%, p=0.001) even if it contributed to only 10% to the average similarity of this zone (67.16% of total dissimilarity). The

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other highly contributing species were the copepods Acartia spp. (27%) and E. affinis (24%) and the mysid Mesopodopsis slabberi (20%). Acartia spp. gelatinous zooplankton (B. virginica, Nemopsis bachei, Rhizostoma pulmo) and marine copepods (Euterpina acutifrons, Paracalanus parvus) were not found upstream of this zone (Table 1). The downstream zone, where salinity was higher than 15 at the time of sampling, was characterized by the significant specificity and fidelity of the chaetognath Sagitta elegans (IV=88%, p=0.005) and the jellyfish Nemopsis bachei (IV=75%, p=0.002). Both species contributed to the average similarity of this zone (8% and 15%, respectively). The other taxa contributing highly to this community structuration were gastropod larvae (20%), Acartia spp. (19%) and Mesopodopsis slabberi (18%). (Table 1). This zone was also characterized by the highest species richness of copepods (8 species),

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ACCEPTED MANUSCRIPT gelatinous zooplankton species (5 species), benthic macrofauna larvae (5 taxa), mysids (3 species), isopods (2 species) and fish larvae (5 species; Table 1). 3.2.2. Considering the intertidal or subtidal habitat According to the SIMPER analysis, differences of zooplankton assemblages sampled in subtidal and intertidal areas were mainly due to the higher densities of the copepods Acartia spp. and Paracalanus

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parvus, the mysid Mesopodopsis slaberri and by several meroplanktonic stages of benthic organisms (bivalve, gastropod and decapods) in the intertidal samples (Table 1). Subtidal samples were mainly characterized by higher densities of the copepod Eurytemora affinis (Table 1). With the exception of bivalve larvae, which were only caught in intertidal samples, all these taxa were present in both intertidal and subtidal samples (Table 1). Hence the difference of zooplankton assemblage was mainly

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related to difference in the abundance level they reached according to habitat.

The two stations located in former ponds, which were only sampled in the downstream area, displayed

(PERMANOVA and pairwise test p>0.05).

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no significantly different assemblage of zooplankton compared to other intertidal habitat

3.3. Planktivorous consumers in the habitats of the downstream zone: Isotope signatures of copepods, mysids and planktivorous fishes

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Significant differences in carbon and nitrogen isotope ratio were recorded between subtidal and intertidal habitats for copepods (δ13C = -25.7±0.3‰ vs -24.3±0.2‰ and δ15N = 10.9±0.0‰ vs 10.2±0.1‰; Wilcoxon rank sum test, p-value<0.05, Fig. 4): intertidal habitats presented higher δ13C and lower δ15N. Carbon isotope ratio was also significantly different between habitats for mysids (-

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18.8±0.0‰ vs -21.9±0.3‰ for subtidal and intertidal habitats, respectively; Fig. 4) whereas no significant difference was observed for nitrogen isotope ratio (δ15N = 8.6±0.0‰ vs 9.0±0.4‰). Carbon and nitrogen isotope ratios of planktivorous fishes were not significantly different between

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habitats for Engraulis encrasicolus, Alosa fallax nor Sprattus sprattus (Fig. 4). Carbon isotope ratios were significantly higher for mysids than for copepods (unilateral Wilcoxon rank sum test, p-value<0.01). Likewise, nitrogen isotope ratios were significantly higher for copepods than for mysids (unilateral Wilcoxon rank sum test, p-value<0.01, Fig. 4). Carbon isotope ratios of fishes were significantly higher than δ13C of copepods (unilateral Wilcoxon rank sum test, p-value<0.05; Fig. 4) whereas their nitrogen isotope ratios were only significantly higher than intertidal copepods. Only the δ13C of E. encrasicolus and S. sprattus were significantly higher than intertidal mysid ones. δ15N was higher for all the fish species than for mysids (Fig. 4).

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ACCEPTED MANUSCRIPT 4. Discussion

4.1. Summer zooplankton community in the Gironde estuary The zooplankton community recorded in July 2012 in the Gironde estuary matched the classical summer distribution observed in other North European estuaries (Baretta and Malschaert, 1988, Mouny and Dauvin, 2002, Tackx et al., 2004, Modéran et al., 2010): total densities were highly

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dominated by calanoid copepods (mainly Eurytemora affinis and Acartia spp.) while meroplanktonic forms (benthic macrofauna and fish larvae) constituted a rather sizeable group at this season. Gelatinous zooplankton and mysids (mainly Mesopodopsis slabberi at juvenile stages) presented notable high densities compared to previous works in estuarine systems (Modéran et al., 2010; Primo

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et al., 2012; Selleslagh et al., 2012b).

Compared to other north-european estuaries such as the Seine, Charente or Scheldt estuaries (Mouny and Dauvin 2002; Tackx et al. 2004; Modéran et al. 2010), the taxa richness of the “strict”

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mesozooplankton community was exceptionally poor in the Gironde Estuary: cyclopoids and cladocerans were under-, or even not represented at all, in this system in July 2012. It is also the case during the others seasons (data not shown). The Gironde Estuary is one of the most heterotrophic systems where the balance between autotrophic processes (mainly primary production) and heterotrophic processes (mainly respiration) is in favor of heterotrophic processes compared to other European estuaries (Abril et al., 2002). Its very high turbidity indeed limits the phytoplankton

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production. In combination with a long residence time of particles, this results in POC-poor particles (Etcheber et al., 2007) and thus a reduced food availability for zooplankton species (Irigoien and Castel, 1997). In such a context, the absence of cladocerans, which are herbivores, may be simply explained by this lack of phytoplankton production (Barnett et al., 2007). In contrast, the low densities

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of cyclopoids, which have similar diet in upper estuaries than the dominant Eurytemora affinis (Azemar et al., 2007), may be more probably explained by a depletion exerted by either competition

1991).

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for food with E. affinis or predation on nauplii stages of cyclopoids by E. affinis (Soto and Hurlbert,

The high variability of physico-chemical conditions in estuaries requires that species have a greater tolerance to stress, resulting in ecological communities less diverse than in adjacent aquatic ecosystems (rivers and sea; Elliott and Whitfield, 2011). As for other biological compartments, zooplankton in estuaries might include more r-strategists (short-lived, high turnover, small bodied, fast colonizers) than K-strategists (Jerling and Wooldridge, 1991) and be more generalists than specialists in terms of physiological tolerances and feeding strategies (Elliott and Whitfield, 2011). However, all species do not present exactly the same life strategies which might explain the observed pattern. Previous studies showed that high environmental variability allows many species to coexist, explaining the so-called “paradox of the plankton” (Scheffer et al., 2003). As an example, the main

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ACCEPTED MANUSCRIPT estuarine copepods, Eurytemora affinis and Acartia bifilosa are known to be euryhaline species even if the former has a broader tolerance range (Irigoien and Castel, 1995, Devreker et al., 2009). However, the egg-carrying copepod E. affinis is qualified as K-strategist while the free-spawning copepod Acartia spp. are considered as an r-strategist (Hirche, 1992). Inversely, E. affinis has a broader food spectrum with a higher ability to select its prey among inorganic matter (Gasparini and Castel, 1997) and to feed on detrital vegetal matter in contrast to Acartia spp. (David et al., 2006a). This feeding

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strategy gives to E. affinis an advantage in the upstream zone where the maximum turbidity zone is localized, and more particularly in the Gironde Estuary where POC is mainly of terrestrial origin (Savoye et al., 2012). In contrast, Acartia spp. presents a competitive advantage in the downstream zone where phytoplankton contribution to POC is higher, especially during summer conditions

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(Savoye et al., 2012). The co-existence of both species in the same system could thus be explained by both spatial and seasonal segregations due to both differences in reproductive strategies and diet

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(Chaalali et al., 2013b).

4.2. Zooplankton structuration: haline gradient vs benthic habitat

In estuaries, zooplankton community structure is mainly driven by the salinity gradient (Telesh and Khlebovich, 2010) with different community composition along the upstream-downstream axis. Our study highlights this pattern but also a clear structuration of the community between subtidal and intertidal areas during high tide. During low tide, all pelagic organisms are concentrated in subtidal

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area while intertidal habitat is emerged. Our result suggest that during rising tide, some pelagic species are preferentially transported by the tide current over intertidal areas. Our observations highlights that even if zooplankton species cannot swim against the current, they have the ability to be retained in suitable ecological niche/habitat. Among several proposed mechanisms the most likely is tidal vertical

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migration in the water column. This swimming behaviour consists in part to remain near the bottom or move in the water column depending if currents are favourable or not (Morgan et al., 1997). Such

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swimming ability has already been highlighted for a wide variety of planktonic organisms (reviewed in Kimmerer et al. 2014, table1).

4.2.1. Zooplankton structuration according to the haline gradient The strong effect of salinity on zooplankton community structure has been highlighted in many descriptions of estuarine zooplankton assemblages (Baretta and Malschaert, 1988, Mouny and Dauvin, 2002, Tackx et al., 2004, Marques et al., 2007, Modéran et al., 2010, Chaalali et al., 2013a). However, the zooplankton distribution along the salinity gradient is more than a simple consequence of osmoregulations abilities among species since most of them are euryhaline species and might display a broader distribution than observed by the single effect of salinity (Sautour and Castel, 1995). This is

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ACCEPTED MANUSCRIPT valid for copepods (Irigoien and Castel 1995, Devreker et al. 2009), mysids and other hyperbenthic taxa (Mees et al., 1995), as well as fishes (Selleslagh et al., 2012b). Actually, the different haline zones also exhibit difference in environmental characteristics and hydrodynamic features (e.g. the maximum turbidity zone), which also have strong ecological consequences on organisms (e.g. food availability) (David et al., 2006a). In the upstream zone, zooplankton almost exclusively consisted in one species (97%): the copepod

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Eurytemora affinis, which displayed very high abundances due to the absence of inter-specific competition. This pattern characterizes the maximum turbidity zone in the Gironde estuary (Sautour and Castel, 1995) since E. affinis is the copepod which is best adapted to highly turbid conditions compared to Acartia species (Gasparini and Castel, 1997, David et al., 2006a). The other species

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characterizing this zooplankton community (Neomysis integer, Palaemon longirostris, P. macrodactylus, and Gammarus zaddachi) are either known to feed on E. affinis and thus benefit of its

Mees, 1999, David et al., 2006b).

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very high densities in this zone, and/or to directly feed on detritus (Sorbes 1979, 1983, Fockedey and

The downstream zone presented the highest diversity due to the presence of neritic species but the lowest total densities. This has been classically reported in the downstream area of other European estuaries (Baretta and Malschaert, 1988, Mees et al., 1995, Mouny and Dauvin, 2002, Marques et al., 2007, Modéran et al., 2010) for copepods (Centropages hamatus, Temora longicornis, Euterpina acutifrons, Labidocera wallastoni, Oithona nana), mysids (Schistomysis spiritus) and chaetognaths

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(Sagitta elegans). Meroplanktonic forms – benthic macrofauna and fish larvae- are also classically present in the polyhaline zone of estuaries (Soetaert and Van Rijswiijk, 1993; Mouny and Dauvin, 2002; Marques et al., 2007,;Modéran et al., 2010). This neritic community penetrates upstream in the estuary during summer conditions (low river flow) even if its maximum abundance is expected in the

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estuarine plume in the Bay of Biscay (Sautour and Castel, 1999). Gelatinous species diversity was also high in this zone. Their abundance was similarly high during summer in the polyhaline zone of the

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Mondego estuary, where the salinity intrusion favoured the marine intrusion of gelatinous species (Primo et al., 2012) such as R. pulmo and P. pileus in the Gironde estuary. This zone was also characterized by high densities of the estuarine species, Acartia spp and Mesopodopsis slabberi, which find in this zone a higher quality of food: phytoplankton production is allowed there thanks to the reduced turbidity compared to the MTZ area (Kerambrun et al., 1993, Irigoien and Castel, 1995, Irigoien and Castel 1997, David et al., 2006a). The median zone appeared as an intermediate area between oligo- and polyhaline zones, where the main species of both communities are found (E. affinis, Acartia spp, Mesopodopsis slabberi). The community was indeed co-dominated by the two copepods, Eurytemora affinis and Acartia spp. The jellyfish Blackfordia virginica appeared as an indicator species of this zone whereas this species has a large range of salinity tolerance (0 to 36). It is also the case in the San Francisco estuary where the

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ACCEPTED MANUSCRIPT highest abundances of this species occurred in between 6 to 20 of salinity (Wintzer et al., 2013). This zone thus represents a salinity gradient zone with a spatial succession of species assemblages, matching the concept of ecocline as proposed by Modéran et al. (2010). 4.2.2. Zooplankton structuration according to intertidal/subtidal aeas This study was performed during high tide when both subtidal and intertidal areas were flooded. Our

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results demonstrate that zooplankton community composition is significantly different over tidal flats. Different reasons could contribute to this spatial segregation. Firstly, the larger abundance of meroplanktonic larvae in intertidal habitats could be explained by the low water depth and the much higher densities of macrozoobenthos in this zone compared to subtidal habitats (Heip and Herman,

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1995). In addition, conspecific chemical cues are known to be an important factor for habitat selection of benthic larvae (Lecchini and Nakamura, 2013) that might explain the proximity of benthic larvae to their genitors. Moreover, food availability might explain the habitat structuration of some biological

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compartments (Sorbe, 1981). Only two species, the copepod E. affinis and the jellyfish Rhizostoma pulmo, were preferentially distributed over subtidal habitats during high tide. Though E. affinis is probably favored compared to Acartia spp. in subtidal area as discussed earlier, a reverse process might occur in intertidal area.

As well, during high tide, the abundances of fish larvae and juveniles are significantly higher over intertidal than over subtidal areas (Selleslagh, pers. obs.). Intertidal zones of estuaries are actually

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known to play the role of nursery for young fish for several reasons (Selleslagh et al., 2011): (1) fish growth is favored by the higher temperature recorded in intertidal habitats in summer during high tides compared to subtidal habitats, (2) lower hydrodynamism in intertidal compared to subtidal zone, and (3) the abundance of preys, particularly benthic macrofauna, which is more abundant in the intertidal

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areas. This work also reports higher densities of zooplankton in intertidal than in subtidal habitats at high tide suggesting that the nursery role of intertidal area would also be true for planktivorous fishes

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in the Gironde estuary.

Copepods and mysids isotope signatures can be confronted to local sources signatures: (i) terrestrial detritus (δ13C=-28.4±0.4‰ and δ15N=4.4±2.0‰; Savoye et al., 2012), (ii) phytoplankton (δ13C=21.5±1.9‰ and δ15N=5.8±0.9‰ according to Savoye et al. (2012) considering the local range of salinity in polyhaline zone), (iii) microphytobenthos (δ13C=-23.9±0.2‰ and δ15N=9.1±0.2‰; unpublished data), (iv) protozoa (δ13C=-24.2±0.3‰ and δ15N=8.5±0.4‰). Protozoa isotopic signature was estimated from the isotopic signature of the bacteria-colonized particulate organic matter from the maximum turbidity zone (Savoye et al. 2012) considering a) that protozoa feed on bacteria, b) that bacteria exhibited the same isotopic signature than the MTZ and c) a trophic fractionation of 1±0.5‰ for carbon and 3±1‰ for nitrogen (Vanderklift and Ponsard, 2003) between bacteria and protozoa.

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ACCEPTED MANUSCRIPT δ13C and δ15N discriminated the different food sources with the exception of protozoa and microphytobenthos that exhibited similar isotopic signatures (Fig. 4). Considering the carbon and nitrogen fractionation proposed by Vanderklift and Ponsard (2003), copepods preferentially located over subtidal area should preferentially feed on a mixed pool composed of terrestrial detritus and protozoa/microphytobenthos whereas copepods favouring intertidal area during high tide seemed to include a larger part of micorphytobenthos in their diet (Fig

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4). The availability of microphytobenthos on intertidal mudflats could explain the difference in contrbution of this food source in the diet of intertidal copepods, especially when diatoms are the preferred food item of the dominant species (Acartia bifilosa and A. clausi) (Kerambrun et al. 1993, Gasparini and Castel 1997). This does not exclude the probable importance of protozoa in the diet of

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copepods in intertidal mudflats since the nanograzer Paracalanus parvus represented 35% of total copepod densities (Pagano et al., 2012). In the Gironde estuary, particulate organic matter mainly consist in terrestrial detritus, which represents a very poor-quality food for Acartia spp. This might 3

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explain the very low densities of this species observed over subtidal areas during high tide (218 ind.mvs 1 500 ind.m-3 over subtidal and intertidal habitats, respectively).

ItMysids collected in subtidal areas seemed to preferentially feed on phytoplankton whereas mysids retrieved over intertidal areas were influenced by microphytobenthos/protozoa. In both habitats, mysids were largely dominated by Mesopodopsis slabberi and their abundance at high tide was 20 times higher over intertidal than over subtidal areas. Over intertidal habitat, its population was mainly

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constituted by juvenile stages (pers. obs.) in contrast to subtidal habitat where both adults and juveniles were found. This species is predominantly herbivorous at juvenile stages whereas carnivory increased in larger individuals (Froneman, 2001). Thus, microphytobenthos may be a more likely source of food than protozoa for Mesopodopsis slabberi in intertidal areas. It appears that

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phytoplankton was available for zooplankton but not exploited by Acartia spp., despite its preference for this food source. This can be explained by (1) a dominance of large cells in phytoplankton

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communities: M. slabberi preferentially consumes large cells >20 µm (Jerling and Wooldridge 1995) whereas copepods preferentially feed on nanophytoplankton (Gasparini and Castel, 1997) and (2) a low phytoplankton stock that limits its availability to passive species such as copepods (Gasparini and Castel, 1997).

For both copepods and mysids, carbon isotope signatures were significantly different between organisms retrieved over subtidal and over intertidal habitats. Considering that this tool gives information on diet at the time scale of tissue turnover (about one month for zooplankton (Modéran et al., 2012)), the difference observed further demonstrate the difference in both swimming and feeding behavior resulting in an ecological segregation among both mysids and copepods. Sorbe (1981) reported such a spatial segregation for mysid populations, with juveniles being distributed on the banks and adults in the main channels of the Gironde Estuary. For copepods or other strictly

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ACCEPTED MANUSCRIPT zooplanktonic taxa (i.e. meroplankton), diel vertical migrations could regulate horizontal transport of organisms and promote their retention in one particular habitat (Dauvin et al., 1998). In conclusion our results suggest that some planktonic organisms such as Acartia spp. and mysids have the ability to migrate over intertidal areas during high tide. These organisms are thus able to feed on microphytobenthos that grow over tidal flats.

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Finally, the former ponds localized in the downstream zone presented intermediate zooplanktonic assemblage between subtidal and intertidal habitat ones but closer than intertidal ones. Since their deembankment following a storm in 1999, such marshes are flooded at high tides and the sampling had been realized on an area that is now always submerged. The intermediate assemblage could thus be

4.3. Implications for the trophic pathways

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explained by the progressive return to a natural subtidal habitat of the sampling zone.

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Such spatial segregation between intertidal and subtidal habitats at high tide allows to optimise the use of the available carbon resources and thus helps the estuarine zooplankton communities to successfully cope with stressful conditions (Lobry et al., 2008): in such a highly heterotrophic system, herbivorous species (Mesopodopsis slabberi, Acartia spp.) find in intertidal habitats a suitable nutritive environment, allowing a release of competition with E. affinis. Spatial segregation may attenuate the constraint of being a generalist species considering feeding strategies in an estuarine

habitat.

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system (Elliott and Whitfield 2011), each taxa could actually find its own trophic niche in either

Such a spatial segregation pattern during high tide is not fixed in time since, for example, mysids may return in subtidal habitat at adult stage (Sorbe, 1981). Within this context, mysids can be considered as

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a “biotic vector” of intertidal primary production (microphytobenthos) to subtidal habitat, providing food to higher trophic levels. Zooplankton and hyperbenthic taxa have been previously reported as

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important biotic vectors of nutrients between systems (Lesutienė et al., 2008, Zhou et al., 2009). Zooplankton spatial segregation has also consequences for the higher trophic levels such as planktivorous fishes. Copepods and mysids represented the main food sources for the three species (Oesmann and Thiel, 2001, Pasquaud et al., 2010). As fishes are able to displace between the different habitats, one might consider that they feed in both subtidal and intertidal habitats. The projection, in the isotopic plan, of fish stable isotopes corrected for the trophic fractionation allowed to consider in the model only the most probable preys (Fig. 4). Alosa fallax mainly fed on intertidal preys (67.0±15.5% of mysids and 33.0±15.5% of copepods, Fig. 5). In contrast, Sprattus sprattus and Engraulis encrasicolus only fed on mysids but in both subtidal and intertidal habitats (60.3±11.1% and 70.8±12.8%, respectively for subtidal mysids). Considering that the surface of intertidal mudflat represents only ca. 23% of the total polyhaline zone and that S. sprattus diet is composed of ca. 40%

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ACCEPTED MANUSCRIPT of intertidal preys, this species may be considered as indirectlyretrieving most of its food from intertidal. In contrast, E. encrasicolus appeared independent of the two habitats for feeding. This apparent preference for intertidal habitat could be explained by a preference of fishes for zooplanktonic preys that feed over intertidal zone (Acartia spp., M. slabberi). Whatever the reasons (active behavior of fish feeding on intertidal habitat or feeding preference on species favoured in intertidal habitat), intertidal mudflats appear as a major habitats for planktivorous fishes. In the

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Gironde estuary, Lobry et al. (2008) suggested a summer trophic functioning associated with pelagic species that mostly feed on zooplankton. The present work shows that this functioning clearly depends on the availability of intertidal habitats in the polyhaline zone. The loss of such habitat under

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anthropogenic pressures could thus have dramatic consequences on the ecosystem functioning.

5. Conclusions

Estuarine habitats contribute significantly to zooplankton structuration. Intertidal habitats, despite their

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low surface area, represent a particularly important source of primary producers that fuels the pelagic food web via zooplankton communities consumed by planktivorous fishes. However, zooplankton communities are scarcely studied in these habitat. Future research should not disregard this compartment when studying the properties of intertidal habitat in the global ecosystem functioning and estimating its ecological and service values. Management actions consisting in restoring intertidal areas and saltmarshes are thus also useful to planktivorous fishes through the preservation of

6. Acknowledgements

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zooplankton communities (Zedler and Kercher, 2005, Mossman et al., 2012).

This study was supported by Irstea and the CPER “Estuaire” project with the support of European

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7. References

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fund FEDER and the Aquitaine region.

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Fig. 1: Map of the Gironde estuary showing the 18 sampling stations as well as the haline zones, namely the upstream zone, the median zone and the downstream zone as defined by salinity during high tide. CNPE indicates the location of the CNPE nuclear powerplant.

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Fig 2: Mean (±SD) of the community structure measures (total abundance, number of taxa) as well as densities of the main groups (copepods, jellyfishes, mysids and benthic larvae) according to haline zones and benthic habitats.

Fig 3: Non-metric multidimensional scaling based on the zooplankton composition (Bray-Curtis

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similarity) at the 18 stations.

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Fig. 4: Isotopes dual plot (mean ±SE) for copepods and mysids (A); and for planktivorous fishes collected in subtidal and intertidal stations (B). Potential sources signatures are also plotted (see text for details).

Fig. 5: Boxplot representing the relative importance of different preys in the diet of planktivorous

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fishes calculated by SIAR models.

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TABLES

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n.1 m-3

Chaetognaths

Sagitta elegans

Copepods

Acanthocyclops robustus Acartia spp. 1061 Centropages hamatus 29 Eurytemora affinis 5 Euterpina acutifrons 8 Labidocera wallastoni 2 Oithona nana 3 Paracalanus parvus 217 Temora longicornis <1

Meroplankton

n.100 m-3 Amphipod

<1

Bivalve larvae Cirriped larvae Decapod zoeae Gastropod larvae Polychaete larvae

Gammarus zaddachi

± 2143 ± 55 ± 9 ± 22 ± 5 ± 7 ± 357

Upstream (3 stations)

934 ± 676 ± 916

926 2 ± 6

23176

± 18075

5 ± 13

115 ± 152

1401 33 471 9 2 3 239 40 <1 57

± <1

EP

68 1 ± 2 6 ± 10 29

Intertidal (7 stations) 73 ± 121 ± 2266

± 69

23

± 39

± 1156 ± 24 ± 5 ± 8 ± 379

2

3 ± 6 118 ± 246 8 ± 8

179 74 ± 102

36 <1 25 4601 13

1 ± 4

1279 ± 1956

11

± <1

21 ± 36 3950 ± 5611 8 ± 17

± 98 ± <1 ± 56

0 ± 1 ± 6

± 192

Subtidal (11 stations) ± 4 1 26 474

± 86 ± 547

± 58

± <1

± 41

<1

Median (7 stations)

95 ± 165

4 ± 7

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Gelatinous zooplankton Blackfordia virginica Liriope tetraphylla Nemopsis bachei Pleurobrachia pileus Rhizostoma pulmo

Downstream (8 stations) 65 ± 114

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group

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Table 1: Mean densities (±standard deviation) per taxa by haline zones and by habitat (intertidal vs subtidal). Depending on taxa, densities are given as number of individuals per 1 m3 (n.1 m-3) or per 100 m3 (n.100 m-3). Values indicated in bold indicates species displaying significant (p<0.05) IndVal. Underlined values correspond to species/taxa contributing the most to the within-group similarity (up to 70% of cumulated contribution to similarity) according to the SIMPER analysis. To compare intertidal and subtidal habitats, italic value indicate taxa/species contributing the most (up to 70% of cumulated contribution) to the dissimilarity between subtidal and intertidal habitats according to SIMPER analysis.

6613 2 <1 <1 9 <1

± <1 ± 22 ± <1

± <1

± 1061

± 19

± 29

343

± 5736

± <1

± 116

<1 1 68 22

± 38

± 5

51 <1 28 <1 4

± 73 ± <1

± 13361

± <1 ± 34 ± 2 ± 9

± 4 ± 119 ± 56

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9

± 34

21 1 ± 3

2

9 ± 27 7 ± 19 57 ± 105 5 ± 8

37 ± 34 <1 ± <1

2 ± 4 70 ± 46

48783 ± 43136 33470 ± 30323 163 ± 388 31 ± 64 ± 52 26

Mesopodopsis slabberi Neomysis integer Schistomysis spiritus

Shrimps

Crangon crangon Palaemon longirostris Palaemon serratus Paleamon macrodactylus

13 ± 36 119

± 224

1 ± 2

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6782 ± 11693 1295 ± 1943 548 ± 950 142 ± 102

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Mysids

± 4

354 11

± 849

13

± 17

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Alosa fallax Argyrosomus regius Dicentrarchus labrax Engraulis encrasicolus Gasterosteus aculeatus Pomatoschistus minutus Sprattus sprattus

± 17

60 ± 159

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Fish larvae

310 ± 796 9 ± 27

Eurydice pulchra Sphaeroma rugicauda

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Isopod

31 ± 27

11 8 1 71 5

38

± 127

12 1

± 29

39 1

± 41

± 29

± 3

± 29 ± 20 ± 2 ± 108 ± 9

± 2

58845 ± 38484 21180 ± 27880 198 ± 408 365 ± 1055 ± 57 ± 12 22 5 249 37 136 7

± 617 ± 98

15

± 34

4

± 13

± 236 ± 19

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MS

F

MS

F

0.037 0.034 0.001 0.007

5.178* 4.778* 0.082

26.496 1.404 21.324 1.378

19.232*** 1.019 15.478**

0.011 0.007

1.521

10.189 3.123

3.263

9.229 0.363 0.991 1.189

7.759** 0.306 0.833

0.322 0.285

0.836

Mysids

Benthic larvae

Fish larvae

Community

F

MS

F

MS

F

MS

F

4.832 1.117 6.180 1.104

4.379* 1.013 5.600*

5.126 29.939 6.016 4.351

1.178 6.880* 1.383

0.092 0.084 0.328 0.038

2.440 2.220 8.683*

5961 1972 1189 710

8.392*** 2.778* 1.638

8.490 0.602

14.106**

32.154 6.308

5.097

0.472 0.248

1.902

2356 848

2.779*

MS

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Gelatinous zooplankton MS F

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Copepods

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df MS F Estuary, subtidal vs intertidal HZ 2 11.3 14.986* BH 1 0.19 0.257 HZ×BH 2 33.96 45.028*** Res 10 7.54 Total 15 Downstream sector BH 2 28.36 17.954** Res 5 1.58 Total 7

Species richness

EP

Total densities

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Table 2: Results of PERMANOVA tests performed on the community structure measures (total densities, taxa richness), densities of the main groups (copepods, gelatinous zooplankton, mysids, benthic macrofauna and fish larva) and community assemblages between haline zones (HZ), benthic habitats (BH) and interactions HZ×BH. Bold values indicate significant differences: * p<0.05, ** p<0.01, ***p<0.001. (df= degree of freedom; MS=mean square; F= F-statistic, Res= residuals)

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ACCEPTED MANUSCRIPT David et al. Estuarine habitats structure zooplankton communities: implications for the pelagic trophic pathways

Highlights: ● The effect of the estuarine gradient and of the position of samples over intertidal or over

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subtidal areas on zooplankton was investigated in the Gironde estuary.

● In addition to the estuarine gradient, subtidal or intertidal position is significant in discriminating summer zooplankton assemblages.

● Intertidal habitats exhibited higher taxa richness and abundances of the main plankton taxa.

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● Stables isotope investigation suggest that most planktivorous fishes fed preferentially on intertidal zooplankton assemblages.

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pelagic food web in estuaries.

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● This study highlights the importance of intertidal habitats for estuarine zooplankton and