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Feeding strategies and body condition of juvenile European flounder Platichthys flesus in a nursery habitat

Published online by Cambridge University Press:  28 July 2020

C. Mendes*
Affiliation:
Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira, 228, 4050-313Porto, Portugal Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208Matosinhos, Portugal Department of Biological and Marine Sciences, University of Hull, HullHU6 7RX, UK International Estuarine & Coastal Specialists Ltd, LevenHU17 5LQ, UK
S. Ramos
Affiliation:
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208Matosinhos, Portugal International Estuarine & Coastal Specialists Ltd, LevenHU17 5LQ, UK
M. Elliott
Affiliation:
Department of Biological and Marine Sciences, University of Hull, HullHU6 7RX, UK International Estuarine & Coastal Specialists Ltd, LevenHU17 5LQ, UK
A. A. Bordalo
Affiliation:
Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira, 228, 4050-313Porto, Portugal Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208Matosinhos, Portugal
*
Author for correspondence: C. Mendes, E-mail: claudiavrmendes@gmail.com
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Abstract

Estuarine habitats are major nurseries for the European flounder Platichthys flesus, with different year classes sharing food and space resources. Hence, an understanding of feeding strategies that optimize resource use and maintain carrying capacity is fundamental for sustainable and successful ecosystem management. The main feeding areas of juvenile European flounder (including 0-group and 1-group age classes) in the Lima estuary (northern Portugal) nursery ground were investigated by integrating stomach content analyses with stable isotopic values (δ13C and δ15N) and fish condition indices (Fulton K and RNA:DNA ratio). The 0-group flounder that were associated with the upstream section of the estuary presented the lowest δ13C value (−25.58 ± 1.86‰), while 1-group flounder exhibited a higher δ13C value (−22.59 ± 2.51‰), indicating use of the more saline areas of the estuary (lower and middle sections). The two age groups did not differ in terms of δ15N (0-group: 13.93 ± 0.29‰; 1-group: 13.50 ± 0.96‰), indicating similar trophic levels. The low salinity upper estuary was the main feeding area of 0-group flounder (74%), while 1-group flounder fed along the estuary both upstream (52%) and downstream (48%). Juvenile flounder showed high individual condition based on the Fulton K index (0-group: 1.05 ± 0.08; 1-group: 1.07 ± 0.05) and RNA:DNA (0-group: 1.70 ± 0.70; 1-group: 1.41 ± 0.47). These indices deal with fish health, and hence indicate nursery habitat quality. It is concluded that in this temperate nursery habitat, different feeding strategies sustained the condition of the European flounder juveniles, compared with other flounder populations.

Type
Research Article
Copyright
Copyright © Marine Biological Association of the United Kingdom 2020

Introduction

Estuaries are highly productive systems (McLusky & Elliott, Reference McLusky and Elliott2004) that support nursery areas for many marine fish species (Kerstan, Reference Kerstan1991; Beck et al., Reference Beck, Heck, Able, Childers, Eggleston, Gillanders, Halpern, Hays, Hoshino, Minello, Orth, Sheridan and Weinstein2001; Potter et al., Reference Potter, Tweedley, Elliott and Whitfield2015). Early life stages of fishes find suitable conditions in nursery habitats that enhance body condition, growth and survival through high food availability and shelter from predation (Boesch & Turner, Reference Boesch and Turner1984; Gibson, Reference Gibson1994; Pihl et al., Reference Pihl, Cattrijsse, Codling, Mathieson, McLusky and Roberts2007). Ultimately, nursery conditions promote recruitment, and therefore maintain the adult populations (Gibson, Reference Gibson1994; Rijnsdorp et al., Reference Rijnsdorp, Berghahn, Miller and Van Der Veer1995; Beck et al., Reference Beck, Heck, Able, Childers, Eggleston, Gillanders, Halpern, Hays, Hoshino, Minello, Orth, Sheridan and Weinstein2001). The quantity and quality of habitat together with food availability are major determinants of nursery carrying capacity (Gibson, Reference Gibson1994; Nunn et al., Reference Nunn, Tewson and Cowx2012; Le Pape & Bonhommeau, Reference Le Pape and Bonhommeau2015), in terms of the maximum number of individuals or biomass that can ecologically be supported by an area (Elliott et al., Reference Elliott, Burdon, Hemingway and Apitz2007). However, historically there has been a high loss of estuarine areas and habitats (Wolanski & Elliott, Reference Wolanski and Elliott2015; Amorim et al., Reference Amorim, Ramos, Elliott, Franco and Bordalo2017), and there is a need for management measures promoting remediation of these habitats (Elliott et al., Reference Elliott, Mander, Mazik, Simenstad, Valesini, Whitfield and Wolanski2016). Hence, it is important to know whether food and space resources have diminished for fishes, including economically valuable species, and whether this has in turn decreased the estuarine carrying capacity.

The dispersal of juveniles supports ecological interactions between the different units that comprise nursery habitats, ensuring connectivity (Sheaves, Reference Sheaves2009; Palmer et al., Reference Palmer, Coulon and Travis2014). Losses in connectivity may decrease trophic interactions and lead to food web fragmentation, compromising the system resilience and nursery function (Vinagre et al., Reference Vinagre, Salgado, Cabral and Costa2011; Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015). Similarly, a loss of connectivity would increase competition for scarce resources within an area. Moreover, distribution patterns will reflect abiotic factors, such as temperature, salinity, depth, sediment type (Power et al., Reference Power, Attrill and Thomas2000; Andersen et al., Reference Andersen, Schou, Sparrevohn, Nicolajsen and Støttrup2005a; Ramos et al., Reference Ramos, Ré and Bordalo2009; Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Maia, Fonseca, França, Wouters, Costa and Cabral2010) and biotic factors such as prey availability, predation and competition (van der Veer et al., Reference van der Veer, Berghahn, Miller and Rijnsdorp2000; Darnaude et al., Reference Darnaude, Harmelin-Vivien and Salen-Picard2001; Amara et al., Reference Amara, Selleslagh, Billon and Minier2009; Sheaves et al., Reference Sheaves, Baker, Nagelkerken and Connolly2015). In particular, prey availability is likely to affect the condition and growth of juveniles (Cabral et al., Reference Cabral, Lopes and Loeper2002; Amara et al., Reference Amara, Selleslagh, Billon and Minier2009; De Raedemaecker et al., Reference De Raedemaecker, Brophy, O'Connor and O'Neill2012b), and hence nursery value. Therefore, migrations and recruitment to areas with high prey availability may either optimize resource use or prevent food limitation, thus minimizing competition (Tableau et al., Reference Tableau, Brind'Amour, Woillez and Le Bris2016). These migrations may shape habitat and resource partitioning between different life stages and species in nursery habitats (Darnaude et al., Reference Darnaude, Harmelin-Vivien and Salen-Picard2001; Russo et al., Reference Russo, Pulcini, O'Leary, Cataudella and Mariani2008). Apart from migration, other resource use and resource partitioning strategies include feeding on highly abundant prey (Molinero & Flos, Reference Molinero and Flos1992; Vinagre et al., Reference Vinagre, França, Costa and Cabral2005), ontogenetic shifts in the diet (Aarnio et al., Reference Aarnio, Bonsdorff and Rosenback1996; Kopp et al., Reference Kopp, Le Bris, Grimaud, Nérot and Brind'Amour2013) and temporal differences in habitat use (Cabral & Costa, Reference Cabral and Costa1999).

Stomach contents analysis offers a snapshot of the fish diet (Hyslop, Reference Hyslop1980; Marshall & Elliott, Reference Marshall and Elliott1997), which can be combined with stable isotope information to give broader temporal information (DeNiro & Epstein, Reference DeNiro and Epstein1978; Minagawa & Wada, Reference Minagawa and Wada1984; Peterson & Fry, Reference Peterson and Fry1987) to gauge trophic relationships. Carbon (δ13C) stable isotope ratios help to explain the diet composition of consumers (DeNiro & Epstein, Reference DeNiro and Epstein1978; Fry & Sherr, Reference Fry, Sherr, Rundel, Ehleringer and Nagy1989), while δ15N can also indicate trophic position (DeNiro & Epstein, Reference DeNiro and Epstein1981; Post, Reference Post2002). Recently, stable isotope mixing models (Parnell et al., Reference Parnell, Phillips, Bearhop, Semmens, Ward, Moore, Jackson, Grey, Kelly and Inger2013; Phillips et al., Reference Phillips, Inger, Bearhop, Jackson, Moore, Parnell, Semmens and Ward2014) have been used to identify the main prey and organic matter sources (Le Pape et al., Reference Le Pape, Modéran, Beaunée, Riera, Nicolas, Savoye, Harmelin-Vivien, Darnaude, Brind'Amour, Le Bris, Cabral, Vinagre, Pasquaud, França and Kostecki2013; Hoffman et al., Reference Hoffman, Kelly, Peterson and Cotter2015; Dias et al., Reference Dias, Morais, Faria, Antunes and Hoffman2017), and to characterize trophic niche widths and niche overlap between species (Kostecki et al., Reference Kostecki, Roussel, Desroy, Roussel, Lanshere, Le Bris and Le Pape2012; Vaslet et al., Reference Vaslet, Phillips, France, Feller and Baldwin2015). This information may then be integrated to track movements over variable temporal and spatial scales, and identify key habitats regarding food use (Green et al., Reference Green, Smith, Grey and Underwood2012; Kopp et al., Reference Kopp, Le Bris, Grimaud, Nérot and Brind'Amour2013; Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015).

Indices of fish condition are valuable in assessing nursery habitat quality (Gilliers et al., Reference Gilliers, Amara and Bergeron2004; Fonseca et al., Reference Fonseca, Vinagre and Cabral2006; De Raedemaecker et al., Reference De Raedemaecker, Brophy, O'Connor and Comerford2012a), by measuring individual somatic nutritional status, and hence the potential for growth and reproduction. These indices, including morphometric and biochemical ones, respond to environmental conditions over various temporal scales (Suthers et al., Reference Suthers, Fraser and Frank1992; Fonseca et al., Reference Fonseca, Vinagre and Cabral2006). The Fulton condition factor, K (Ricker, Reference Ricker1975) is a morphometric index widely used to measure fish somatic condition (Amara et al., Reference Amara, Selleslagh, Billon and Minier2009; Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Fonseca, Ruano, Tanner, Costa and Cabral2009; De Raedemaecker et al., Reference De Raedemaecker, Brophy, O'Connor and Comerford2012a), considering that for a given length, the heavier fish are in better condition. The Fulton K is sensitive to the nutritional status of the juveniles (Ferron & Leggett, Reference Ferron and Leggett1994; Selleslagh & Amara, Reference Selleslagh and Amara2013) and integrates condition over the preceding weeks to months (e.g. Caldarone et al., Reference Caldarone, MacLean and Sharack2012; De Raedemaecker et al., Reference De Raedemaecker, Brophy, O'Connor and Comerford2012a). Complementary to this, the biochemical RNA:DNA ratio indicates recent nutritional condition and growth (Clemmesen, Reference Clemmesen1994; Malloy & Targett, Reference Malloy and Targett1994; Chícharo & Chícharo, Reference Chícharo and Chícharo2008). It assumes that the DNA content in a somatic cell remains constant, while the RNA concentration reflects changes in protein synthesis rates (Bulow, Reference Bulow1970; Buckley et al., Reference Buckley, Caldarone and Ong1999), i.e. in a non-stressful situation, higher RNA:DNA ratio reflects higher somatic growth of the fish. Furthermore, the RNA:DNA responds to changes in food availability within days (e.g. Clemmesen, Reference Clemmesen1994; Selleslagh & Amara, Reference Selleslagh and Amara2013; Foley et al., Reference Foley, Bradley and Höök2016).

The flatfish European flounder Platichthys flesus (L. 1758) uses estuarine habitats as nurseries (Henderson & Holmes, Reference Henderson and Holmes1991; Kerstan, Reference Kerstan1991; Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008), including the Lima estuary (northern Portugal) (Ramos et al., Reference Ramos, Ré and Bordalo2010) where they are an important component of the estuarine fish community. The NW Portuguese coast represents the southern limit of the distribution of flounder (Nielsen, Reference Nielsen, Whitehead, Bauchot, Hureau, Nielsen and Tortonese1986), with a recent decline in abundance associated with increasing seawater temperatures (Cabral et al., Reference Cabral, Vasconcelos, Vinagre, França, Fonseca, Maia, Reis-Santos, Lopes, Ruano, Campos, Freitas, Santos and Costa2007). Moreover, food limitation has been linked to interspecific competition and higher metabolic demands in southern European nurseries, resulting in reduced growth rates of flounder compared with higher latitudes (Freitas et al., Reference Freitas, Kooijman and van der Veer2012).

Flounder spawning typically occurs from winter to early spring (Summers, Reference Summers1979; Muus & Nielsen, Reference Muus and Nielsen1999; Primo et al., Reference Primo, Azeiteiro, Marques, Martinho, Baptista and Pardal2013) although the exact location of the spawning grounds off the Portuguese Coast remains unknown (Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2016). Larvae usually enter the Lima estuary between February and July (Ramos et al., Reference Ramos, Ré and Bordalo2010; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2016). Direct settlement occurs inside the estuary (Ramos et al., Reference Ramos, Ré and Bordalo2010; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2018), and most likely in the upper section (Ramos et al., Reference Ramos, Ré and Bordalo2010), as indicated by the high larval (Ramos et al., Reference Ramos, Paris and Angélico2017) and newly settled flounder abundances found in this area (Ramos et al., Reference Ramos, Ré and Bordalo2010; Mendes et al., Reference Mendes, Ramos and Bordalo2014; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2016). As in other estuaries (Elliott & Hemingway, Reference Elliott and Hemingway2002), the spatial distribution of flounder in the Lima estuary varies with age (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014, Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2018). Young-of-the-year flounder aggregate in the upper estuary, while older flounder juveniles are more abundant in the middle section (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2018). The juvenile flounder typically feed on common prey such as polychaetes (Summers, Reference Summers1980; Piet et al., Reference Piet, Pfisterer and Rijnsdorp1998; Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008) and amphipods (Aarnio et al., Reference Aarnio, Bonsdorff and Rosenback1996; Vinagre et al., Reference Vinagre, França, Costa and Cabral2005; Mendes et al., Reference Mendes, Ramos and Bordalo2014). These studies have led to the hypothesis that juvenile distribution and feeding in any nursery area will be structured to minimize competition through resource partitioning and ensure good growth and condition. In order to test this hypothesis, the present study aimed to: (1) determine the main feeding areas of 0-group (<1 year old) and 1-group (juveniles 1–2 year old) flounder, using dietary indices and stable isotope mixing models, and (2) assess flounder juveniles' condition through morphometric and RNA:DNA indices in the Lima estuary. This, in turn, is needed to indicate the feeding use of flounder in a nursery system near its southern geographic distribution limit, which is crucial information needed for better management decisions.

Materials and methods

Study area

The Lima estuary, NW Atlantic coast of Portugal, is a small open temperate estuary with a semidiurnal and mesotidal regime (3.7 m tidal range). Salt intrusion can extend up to 20 km upstream, with an average flushing rate of 0.4 m s−1, and a residence time of 9 days (Ramos et al., Reference Ramos, Cowen, Ré and Bordalo2006). This study sampled at five stations covering the lower, middle and upper estuary (Figure 1). The lower estuary (station 1, average depth of 7.0 m), located within the first 2.5 km, is a narrow, 9-m deep navigational channel, industrialized, with walled banks, including a shipyard, commercial seaport and fishing harbour; the middle estuary (stations 2–3, average depth 5.1 m) comprises a broad shallow intertidal saltmarsh zone, mainly colonized by the common rush (Juncus spp.); the upper estuary (Stations 4–5, average depth 2.1 m) is a narrow shallow channel, less disturbed, with natural banks and small exposed sandbanks (Ramos et al., Reference Ramos, Ré and Bordalo2010).

Fig. 1. The Lima estuary with the location of the sampling stations (1 – lower estuary; 2 and 3 – middle estuary; 4 and 5 – upper estuary).

Data collection

Juvenile European flounder Platichthys flesus, macroinvertebrates, sediment and water samples were collected in August 2013. This sampling date ensured that 0-group flounder should have reached isotopic equilibrium to the estuarine habitats, considering that this process could take weeks to months (Herzka, Reference Herzka2005), given the typical late spring estuarine colonization (Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008; Ramos et al., Reference Ramos, Ré and Bordalo2009). Vertical profiles of temperature and salinity were obtained at each site by means of a multi-parameter water quality probe YSI 6820. Bottom water samples for particulate organic matter (POM) analysis were collected at each sampling site with a Van Dorn bottle. Samples for sediment organic matter (SOM), and macroinvertebrate analysis were retrieved at each station with a Petite Ponar grab of area 0.023 m2. Juvenile flounder, as well as the shore crab Carcinus maenas were collected during two-nightly ebb tides with a 2 m beam trawl, with a cod-end of 5 mm mesh, and a tickler chain, and sorted immediately. All samples were kept on ice until further processing in the laboratory. The geographic location of each sampling station was recorded with a Magellan 315 GPS, and vertical profiles of water temperature and salinity were obtained using a multi-parameter probe YSI 6820.

Stomach content analysis

Juvenile flounder were measured for total length (TL; ±1 mm), and wet weight (W; ±0.01 g) and sorted according to total length at first sexual maturity (assumed to be 200 mm; Dinis, Reference Dinis1986). The stomachs were excised, and the contents removed and preserved in 70% alcohol. The prey items were identified to the highest taxonomic separation possible, using a binocular microscope (Leica MZ12-5), counted and weighed (wet weight; 0.001 g) after blotting on a tissue paper (Mendes et al., Reference Mendes, Ramos and Bordalo2014).

Stable isotope analysis

POM samples were obtained by pre-filtering 1 litre of water sample through a 200 μm nylon mesh to remove zooplankton and debris, and then through pre-combusted GF/F glass fibre filters (Harmelin-Vivien et al., Reference Harmelin-Vivien, Loizeau, Mellon, Beker, Arlhac, Bodiguel, Ferraton, Hermand, Philippon and Salen-Picard2008; Suzuki et al., Reference Suzuki, Kasai, Isoda, Nakayama and Tanaka2008). Filters were acidified with 10% HCl for carbonate removal (Vizzini et al., Reference Vizzini, Sarà, Michener and Mazzola2002; Kennedy et al., Reference Kennedy, Kennedy and Papadimitriou2005), which is δ13C enriched compared with organic carbon (DeNiro & Epstein, Reference DeNiro and Epstein1978), dried at 60°C for 48 h, and stored at −80°C. Sediment samples for SOM analysis were dried at 60°C, and ground to a fine powder. In addition, SOM samples for δ13C analysis were acidified with 10% HCl to remove carbonates, and dried at 60°C. Samples were stored in a desiccator until further analysis. Bivalvia, Carcinus maenas, Chironomidae, Corophium spp., Gastropoda, Isopoda and Polychaeta were considered as main prey groups (Vinagre et al., Reference Vinagre, França, Costa and Cabral2005; Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008; Mendes et al., Reference Mendes, Ramos and Bordalo2014) and sorted from the sediment samples. Samples from the upper estuary comprised Chironomidae, Corophium spp. and Polychaeta. The prey C. maenas and Polychaeta from the lower and middle sections were analysed, as well as Gastropoda from the lower estuary. Rare prey groups such as Bivalvia and Isopoda were not included due to insufficient material available for the stable isotope analysis. Whole individuals were used for prey stable isotope analysis, except for larger prey (C. maenas), for which muscle tissue was collected from the claws. Dorsal white muscle was removed from the flounder juveniles. Prey and fish samples were kept frozen (−80°C) until analysis. Prior to stable isotope analysis, animal tissue samples were dried and ground to a fine powder, using a mortar and pestle. Corophium spp. and gastropods were acidified (10% HCl) to remove carbonates (Ng et al., Reference Ng, Wai and Williams2007; Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015) from the samples for δ13C analysis. Carbon and nitrogen stable isotope analysis were performed on individual samples of fish and crabs. For other prey groups, several individuals were pooled in order to have sufficient material for analysis. Ratios of 13C/12C and 15N/14N in each sample were determined by continuous flow isotope mass spectrometry (CF-IRMS) (Preston & Owens, Reference Preston and Owens1983), using a Thermo Scientific Delta V Advantage IRMS via a Conflo IV interface. The delta (δ) notation was used to express the stable isotope ratios as ppt differences from a standard reference material:

$$\partial X\;\lpar \perthousand \rpar = \lpar {R_{{\rm sample}}\;\times R_{{\rm standard}}^{{-}1} -1} \rpar \times \;10^3$$

where X is 13C or 15N and R is the ratio of 13C/12C or 15N/14N. Isotope ratios were measured relative to the international standards of PeeDee Belemnite for carbon and atmospheric N2 for nitrogen. Analytical precision (standard deviation) was ±0.2‰ of reference material for carbon and nitrogen.

Condition analysis

The individual somatic condition of the European flounder juveniles was assessed through the Fulton index and RNA:DNA ratio indices. The Fulton condition factor, K (Ricker, Reference Ricker1975) was determined following the formula:

$$K = W \times \lpar {\rm T}{\rm L}^3\rpar ^{{-}1} \times 100\comma \;$$

where W is the wet weight (mg) and TL is the total length (mm). For the RNA:DNA, dorsal white muscle samples from European flounder juveniles were preserved in liquid nitrogen upon collection, and kept at −80°C until analysis. Prior to analysis, muscle samples were homogenized in 500 μl TEN-SDS buffer (0.05 M Tris, 0.01 M EDTA, 0.1 M NaCl, 0.01% SDS, pH 8). RNA:DNA was determined for three replicate samples of dorsal white muscle (10 mg) of each juvenile flounder, by the fluorometric method described in Caldarone et al. (Reference Caldarone, Wagner, Onge-Burns and Buckley2001), and modified by De Raedemaecker et al. (Reference De Raedemaecker, Brophy, O'Connor and O'Neill2012b). Fluorescence was measured on a FluoroSkan Ascent FL microplate reader with 535 nm excitation wavelength and 586 nm emission wavelength. RNA and DNA concentrations were determined based on standard curves prepared with baker's yeast RNA (Sigma) and pure calf-thymus DNA (Sigma). The RNA:DNA was determined as the ratio between average RNA and DNA concentrations of each sample. The ratio between the slopes of the RNA and DNA standard curve was 2.5, which can be used as a standardization factor for inter-calibration with other studies (Caldarone et al., Reference Caldarone, Clemmesen, Berdalet, Miller, Folkvord, Holt, Olivar and Suthers2006).

Data analysis

Temperature and salinity parameters of the bottom water column (1 m above bottom depth) were averaged for each location. European flounder juveniles were sorted according to length that would broadly equate with <150 mm for 0-group flounder and <200 mm for 1-group flounder in Portuguese populations (Dinis, Reference Dinis1986; Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Marques and Pardal2007; Primo et al., Reference Primo, Azeiteiro, Marques, Martinho, Baptista and Pardal2013). These are literature classifications based on size-frequency distributions which may present an associated error due to inter-individual and inter-annual variability in growth rates. For each group, feeding activity was evaluated by the vacuity index (Iv), defined as the per cent of empty stomachs (Hyslop, Reference Hyslop1980). Key prey were identified based on the numerical (NI) and weight (WI) percentages of each prey item in the diet (Hyslop, Reference Hyslop1980). The average δ13C of POM and SOM sources was determined for each estuarine sector. The high δ15N variability between replicates for POM and SOM sources, possibly linked to the low nitrogen content of the samples, did not allow the use of this isotope to assess POM and SOM sources. The stable isotope signatures of POM and SOM, and samples from each prey category were sorted into estuarine classification, namely upstream (comprising the upper estuarine sector), and downstream (including pooled data from the lower and middle sections, see Supplementary material for details). The main feeding locations of juvenile flounder were also investigated. Therefore, the relative contribution of each prey to the diet of 0-group and 1-group flounder were determined with stable isotope mixing models, using the SIAR package in R software (Parnell et al., Reference Parnell, Inger, Bearhop and Jackson2010). The stable isotope mixing models estimate probability distributions of food source contributions based on Bayesian inference and accounting for different levels of uncertainty (sources, consumer and contributions of individual sources, Parnell et al., Reference Parnell, Inger, Bearhop and Jackson2010). One general set of trophic enrichment factors (TEF), 1% for δ13C and 3.4% for δ15N per trophic level, was used based on a meta-analysis from the scientific literature, and with an associated standard error of ±0.5 (Kostecki et al., Reference Kostecki, Roussel, Desroy, Roussel, Lanshere, Le Bris and Le Pape2012). Differences of δ13C and δ15N between flounder groups were tested with a permutational multivariate analysis of variance (PERMANOVA). Multivariate dispersion was tested with the PERMDISP routine. The PERMANOVA and PERMDISP analyses were based on the Euclidean distance dissimilarity matrix, and performed with PRIMER v6.1 (Primer-E Ltd, UK) and PERMANOVA + 1.0.1 add-on software (Clarke & Gorley, Reference Clarke and Gorley2006; Anderson et al., Reference Anderson, Gorley and Clarke2008). Possible length effects were investigated through Pearson correlations between total length and (a) isotopic values of carbon and nitrogen, and (b) condition indices of the 0-group and 1-group flounder. The relationship between condition indices Fulton K and RNA:DNA was also investigated through Pearson correlations. Differences in condition indices between respective flounder age groups were investigated with a t-test. A significance level of P < 0.05 was used for all the statistical analyses that were performed using R software (R Development Core Team, 2007).

Results

Physical-chemical parameters

The bottom temperature ranged between 13.7 and 22.8°C, with an average of 17.1 ± 3.4°C. The temperature increased from the lower (14.6 ± 0.7°C) to the middle (14.8 ± 0.8°C) and upper (21.4 ± 1.1°C) sections. Salinity, as expected, decreased from the lower (29.9. ± 0.1) and middle (29.5 ± 0.3) sections to the upper estuary (7.2 ± 5.4).

Stomach contents analysis

A total of 42 European flounder juveniles were collected in the Lima estuary, which were allocated to two age groups based on total length (0-group flounder, N = 22; 1-group flounder, N = 20; Figure 2; Table 1). Most of the 0-group flounder were caught in the upper estuary (N = 20), but two 0-group flounder were retrieved from the middle estuary. The 1-group flounder were collected from the middle estuary. The 0-group flounder presented a lower vacuity index (8%), compared with the 70% vacuity of the 1-group flounder.

Fig. 2. Size frequency distribution of the sampled 0-group (N = 22) and 1-group (N = 20) European flounder in the Lima estuary.

Table 1. Number (N) of 0-group and 1-group European flounder sampled in the Lima estuary, mean total length (mm), total weight (g), Fulton K, RNA:DNA and muscle carbon (δ13C) and nitrogen (δ15N) stable isotope signatures (‰)

The diet of the 0-group flounder was dominated by the amphipod Corophium spp. (NI = 94%, WI = 88%), followed by the polychaete Hediste diversicolor that reached 12% of the WI (Figure 3A). Other prey such as Crangon crangon, Chironomidae, Mysidae and Oligochaeta were also identified as minor items (< 1%). The diet of the 1-group flounder comprised mainly polychaetes (NI = 48%; WI = 69%). Isopoda (NI = 20%, WI = 3%), and C. crangon (NI = 16%, WI = 17%) were also important prey items (Figure 3B). Other prey of 1-group flounder included Bivalvia, Carcinus maenas and Corophium spp. Both 0- and 1-groups also had plant debris and sand in their stomach contents.

Fig. 3. Numerical (NI) and weight (WI) indices for stomach contents of (A) 0-group, and (B) 1-group European flounder in the Lima estuary.

Stable isotope analysis

Organic matter sources

The carbon signature of the upstream areas ranged from −32.66 to −25.55‰, and was significantly lower (see Supplementary material) than downstream areas with δ13C varying from −24.57 to −19.45‰ (Table 2, Figure 4A).

Fig. 4. Carbon (δ13C), and nitrogen (δ15N) stable isotopes (‰) of (A) sediment (SOM, upstream and downstream), and water particulate (POM, upstream and downstream) organic matter sources; (B) 0-group and 1-group European flounder, and respective upstream and downstream prey. Trophic enrichment factors were applied to sources (δ13C: ± 2‰, i.e. ±1‰ per trophic level), and to prey (δ13C: ± 1‰, δ15N: ± 3.4‰).

Table 2. Mean carbon stable isotope δ13C (‰) of particulate organic matter (POM) and sediment organic matter (SOM) sources in the upstream and downstream areas of the Lima estuary

Prey

The upstream prey (Corophium spp., Polychaeta and Chironomidae) presented lower δ13C compared with other prey (Table 3). In upstream areas, Corophium spp. presented the lowest δ13C, while polychaetes presented enriched δ15N compared with other prey. In the downstream areas, C. maenas and gastropods presented the most enriched δ13C, while polychaetes presented the lowest δ15N values (Table 3, Figure 4B).

Table 3. Mean carbon (δ13C) and nitrogen (δ15N) stable isotope signatures of the main prey groups of European flounder juveniles in the upstream and downstream areas of Lima estuary

European flounder juveniles

European flounder juvenile carbon (0-group flounder: R = 0.16, P = 0.46; 1-group flounder: R = −0.16, P = 0.50) and nitrogen (0-group flounder: R = 0.04, P = 0.86; 1-group flounder: R = 0.14, P = 0.56) stable isotope ratios did not vary significantly with total length. Therefore, it was not necessary to correct the data for length effects. The stable isotopes varied significantly between flounder groups (PERMANOVA, Pseudo-F = 13.62, P = 0.02). The 1-group flounder had higher δ13C and lower δ15N values than the 0-group flounder (Table 1).

SIAR outputs

The diet of 0-group flounder depended primarily on Corophium spp. and other prey from the upper estuary (Figure 4B), with a total of 74% contribution according to SIAR (Figure 5A). However, some 0-group flounder had higher δ13C values (Figure 4B), between −24 and −18‰, than expected if the diet depended only on the upper estuary resources. Indeed, polychaetes from the downstream areas constituted 26% of the diet. The dual isotope plots displayed one cluster of 1-group flounder (Figure 4B), with isotopic signatures similar to Corophium spp., and sources from upstream, while the remaining individuals presented more scattered values consistent with the sources and prey from downstream areas. Indeed, the SIAR model applied to 1-group flounder data showed that their diet relied equally on prey both from upstream (48%), and downstream areas (52%) (Figure 5B). Specifically, downstream polychaetes (46%), and upstream Corophium spp. (42%), represented the main prey of 1-group flounder.

Fig. 5. Boxplots of the mixing models estimates of prey contribution to the diet of (A) 0-group and (B) 1-group European flounder. Prey groups divided according to upstream and downstream areas.

Condition analysis

There was no correlation between flounder juvenile total length and Fulton K (0-group flounder: R = 0.03, P = 0.87; 1-group flounder: R = −0.25, P = 0.28), or RNA:DNA (0-group flounder: R = 0.25, P = 0.24; 1-group flounder: R = 0.21, P = 0.40). Therefore, no corrections for length effect were applied to the indices. Also, there were no significant differences in condition between 0-group flounder and 1-group flounder (Table 1), in terms of Fulton K (t-test, t = −0.89, df = 40, P = 0.38), or in terms of RNA:DNA (t-test, t = 1.58, df = 40, P = 0.12). The two condition indices Fulton K and RNA:DNA were also not correlated (0-group flounder: R = 0.06, P = 0.78, 1-group flounder: R = −0.44, P = 0.06).

Discussion

Integrating stomach contents and stable isotope analysis

Stomach content analysis provided a first indication of the recent diet of juvenile flounder, enabling identification of the main prey (polychaetes, Corophium spp. and Carcinus maenas) that could be included in the stable isotope analysis. A discrimination between sources and prey signatures was necessary to reconstruct the consumer diet based on stable isotope analysis (Vander Zanden et al., Reference Vander Zanden, Cabana and Rasmussen1997; Post, Reference Post2002). The δ13C depletion usually increases along the horizontal salinity gradient from marine to estuarine and terrestrial sources (Fry, Reference Fry2002). In the Lima estuary, the δ13C similarity between lower and middle estuaries reflected the euhaline regime of both areas. The depleted δ13C signatures in the upper estuary were related to the increasing terrestrial input in the upstream areas (Darnaude et al., Reference Darnaude, Salen-Picard and Harmelin-Vivien2004; França et al., Reference França, Vasconcelos, Tanner, Máguas, Costa and Cabral2011). The POM and SOM presented similar δ13C values, providing good indicators of local δ13C signatures.

In general, prey from upstream stretches consistently presented higher δ15N values than the downstream prey, which may result from differences in the organic matter sources between estuarine sections. However, the high variability between replicates did not allow the determination of δ15N of the organic matter sources to corroborate this hypothesis. The δ15N variability between prey groups within each estuarine area may reflect differences in trophic position (Minagawa & Wada, Reference Minagawa and Wada1984; Peterson & Fry, Reference Peterson and Fry1987; Post, Reference Post2002). For example, the epibenthic predator C. maenas (Raffaelli et al., Reference Raffaelli, Conacher, McLachlan and Emes1989) showed the highest δ15N from the downstream prey, while the omnivorous polychaetes from upstream areas had higher δ15N than Chironomidae and Corophium, which are opportunistic omnivores (Armitage et al., Reference Armitage, Pinder and Cranston1995), and suspension and deposit feeders (Gerdol & Hughes, Reference Gerdol and Hughes1995), respectively. Prey from the upstream areas showed markedly depleted δ13C compared with the more brackish downstream areas, thus reflecting the use of local OM sources. This discrimination between upstream and downstream prey enabled the use of stable isotope mixed models to identify the main feeding locations, and trace movements of European flounder juveniles in the Lima estuary.

The stomach content results complemented those provided by the stable isotope analysis. Discrepancies between these two methods may be related to the fact that stomach contents reflect recent feeding (Hyslop, Reference Hyslop1980), while stable isotope analysis indicates and integrates long-term patterns (Vander Zanden et al., Reference Vander Zanden, Cabana and Rasmussen1997). Hence, easily digested prey, such as polychaetes, were probably underestimated in 0-group flounder dietary indices even though these were important prey, together with Corophium, according to SIAR results. Also, the near absence of the upper estuary prey Corophium spp. in 1-group flounder dietary indices contrary to SIAR, reflected the recent feeding on polychaetes in the middle estuary, where juveniles were caught.

The diet of 0-group flounder was mostly composed by the amphipod Corophium as observed in other nursery areas, including the Douro (Vinagre et al., Reference Vinagre, França, Costa and Cabral2005), Mondego (Martinho et al., Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008), Schelde (Hampel et al., Reference Hampel, Cattrijsse and Elliott2005) and Ythan estuaries (Summers Reference Summers1980). Corophium was highly abundant and mostly restricted to the Lima upper estuary where the 0-group flounder were also concentrated (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014). Corophium is highly active and may become energetically more advantageous to the European flounder (Andersen et al., Reference Andersen, Carl, Grønkjær and Støttrup2005b; Grønkjær et al., Reference Grønkjær, Carl, Rasmussen and Hansen2007) as a visual predator (De Groot, Reference De Groot1971), especially in the scarce vegetation conditions of the upper estuary (Mendes et al., Reference Mendes, Ramos and Bordalo2014). The polychaetes were the main prey of the 1-group flounder as observed in other estuaries (e.g. Summers et al., 1980; Doornbos & Twisk, Reference Doornbos and Twisk1984; Pasquaud et al., Reference Pasquaud, Elie, Jeantet, Billy, Martinez and Girardin2008). The 1-group flounder consumed less Corophium, reflecting their ability to handle larger prey such as polychaetes (Vinagre et al., Reference Vinagre, Cabral and Costa2008), and the increased use of downstream areas (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014) where amphipods were less abundant (Sousa et al., Reference Sousa, Dias and Antunes2006; Mendes et al., Reference Mendes, Ramos and Bordalo2014). Molluscs were more abundant in the lower estuary (Sousa et al., Reference Sousa, Dias and Antunes2006) and were only occasionally consumed by juvenile flounder that were distributed over other estuarine areas. Moreover, sedentary prey such as molluscs may become major prey in high turbidity systems such as the Severn estuary (Moore & Moore, Reference Moore and Moore1976), where reduced visibility does not favour the predation on mobile prey. This is not the case in the Lima estuary, where low turbidity can be found in the summer months (Ramos et al., Reference Ramos, Cowen, Ré and Bordalo2006), associated with the presence of higher salinity coastal waters. Shrimps, crabs and isopods were only occasionally consumed as observed in other estuaries (Hampel et al., Reference Hampel, Cattrijsse and Elliott2005; Vinagre et al., Reference Vinagre, França, Costa and Cabral2005).

European flounder juvenile movements and main feeding areas within the Lima estuary

The upper estuary was the main feeding area of 0-group flounder in line with the typical preference of early stage flounder for upstream areas (e.g. Kerstan, Reference Kerstan1991; van der Veer et al., Reference van der Veer, Bergman, Dapper and Witte1991; Freitas et al., Reference Freitas, Costa-Dias, Campos, Bio, Santos and Antunes2009). Moreover, the lower δ13C confirms the dependence on freshwater-derived sources observed from late larval (Dias et al., Reference Dias, Morais, Faria, Antunes and Hoffman2017) to juvenile stages (Pasquaud et al., Reference Pasquaud, Elie, Jeantet, Billy, Martinez and Girardin2008; Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015) of European flounder. Prey availability (Bos, Reference Bos1999; Florin & Lavados, Reference Florin and Lavados2010; Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Maia, Fonseca, França, Wouters, Costa and Cabral2010), and reduced competition for space and food (Beaumont & Mann, Reference Beaumont and Mann1984; Złoch & Sapota, Reference Złoch and Sapota2010; Souza et al., Reference Souza, Dias, Nogueira, Campos, Marques and Martins2013) may explain this preference for upstream areas, especially as prey availability and salinity have been previously correlated with the concentration of 0-group flounder in the Lima upper estuary (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014). Despite this, 0-group flounder feeding was not restricted to the upper estuary, since polychaetes from downstream areas were also identified as prey items.

Limited mobility, and consequent patchy segregation along upstream-downstream estuarine gradients, have been reported for 0-group flatfish (Raffaelli et al., Reference Raffaelli, Richner, Summers and Northcott1990; Le Pape & Cognez, Reference Le Pape and Cognez2016). However, these movements between feeding habitats (Summers, Reference Summers1980; Wirjoatmodjo & Pitcher, Reference Wirjoatmodjo and Pitcher1984; Dando, Reference Dando2011; Souza et al., Reference Souza, Dias, Nogueira, Campos, Marques and Martins2013) may be the result of the 0-group flounder's increased mobility given that the individuals sampled were approaching 1-group age class (≥150 mm). The risk from predation inherent in feeding in downstream areas was reduced as individuals attained size-based refuge from predation by the crustaceans C. maenas (>50 mm TL), and C. crangon (>30 mm TL) (van der Veer & Bergman, Reference van der Veer and Bergman1987; Burrows et al., Reference Burrows, Gontarek, Nash and Gibson2001). Laboratory experiments have showed that low salinity typically found in the upstream section may limit somatic condition and growth (Gutt, Reference Gutt1985; O'Neill et al., Reference O'Neill, De Raedemaecker, McGrath and Brophy2011) of the juvenile flounder. The predator–prey trade-offs mentioned above may explain why the juveniles remained upstream despite these limitations. In parallel, the downstream areas may have promoted higher condition as the juveniles approached 1-group age size, and became less vulnerable to predation, while consuming larger and more diverse prey (Mendes et al., Reference Mendes, Ramos and Bordalo2014).

The 1-group fed throughout the estuary, in contrast to the 0-group flounder that fed mostly on upstream prey. These differences between feeding patterns of 0-group and 1-group flounder were clear and consistent both in stomach content and stable isotope data, despite the low number of sampled individuals (N = 42). The 1-group flounder relied equally on prey from downstream where they were more abundant (Ramos et al., Reference Ramos, Ré and Bordalo2009), and upstream areas. It is of note that a limited home range and high site fidelity have been reported for juvenile flounder (Raffaelli et al., Reference Raffaelli, Richner, Summers and Northcott1990; Dando, Reference Dando2011). However, diel and tidal migrations (Edwards & Steele, Reference Edwards and Steele1968; Edwards et al., Reference Edwards, Steele and Trevallion1970; Gibson, Reference Gibson1973), to areas with high prey availability (Modin & Pihl, Reference Modin and Pihl1996), reflected movements between estuarine habitats. Moreover, a gradual use of the downstream areas throughout development (Kerstan, Reference Kerstan1991; Primo et al., Reference Primo, Azeiteiro, Marques, Martinho, Baptista and Pardal2013; Souza et al., Reference Souza, Dias, Nogueira, Campos, Marques and Martins2013), is typical of the flounder life cycle (Elliott & Hemingway, Reference Elliott and Hemingway2002). Thus, the wide range of 1-group flounder δ13C may indicate recent migration from upstream to downstream areas, where most 1-group flounder were caught, given that muscle tissue takes many weeks to reach isotopic equilibrium (Vander Zanden et al., Reference Vander Zanden, Cabana and Rasmussen1997; Herzka, Reference Herzka2005). Overall, connectivity between upstream and downstream estuarine habitats has also been observed for 1-group flounder in other estuarine habitats (Vinagre et al., Reference Vinagre, Salgado, Cabral and Costa2011; Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015). Such a connectivity may also allow the use of alternative resources if one of the habitats is compromised (i.e. resource partitioning), hence increasing the ability of flounder to tolerate environmental change (Selleslagh et al., Reference Selleslagh, Blanchet, Bachelet and Lobry2015). Overall, these feeding strategies corroborated the life cycle described for the European flounder in the Lima estuary (Ramos et al., Reference Ramos, Ré and Bordalo2010; Mendes et al., Reference Mendes, Ramos and Bordalo2014; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2018). The larvae settled in early summer in the upper estuary (Ramos et al., Reference Ramos, Ré and Bordalo2010; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2016) that was also the main feeding habitat of the 0-group flounder. The juveniles increasingly used the middle estuary as they grew (Ramos et al., Reference Ramos, Ré and Bordalo2009; Mendes et al., Reference Mendes, Ramos and Bordalo2014; Amorim et al., Reference Amorim, Ramos, Elliott and Bordalo2018), with the 1-group flounder feeding between upstream and downstream areas. Adults representing 23% of the total population were mostly found in the lower and middle estuaries (Ramos et al., Reference Ramos, Ré and Bordalo2010). These differences in habitat use and feeding strategies between life-stages may prevent intraspecific competition.

Feeding strategies promoting European flounder condition in the Lima estuary and management implications

The issue of food limitation in nursery habitats is still debated (Le Pape & Bonhommeau, Reference Le Pape and Bonhommeau2015), as several authors suggest that feeding on abundant prey (van der Veer et al., Reference van der Veer, Berghahn, Miller and Rijnsdorp2000; Amara et al., Reference Amara, Selleslagh, Billon and Minier2009; Selleslagh & Amara, Reference Selleslagh and Amara2013), and resource partitioning strategies (Evans, Reference Evans1983; Besyst et al., Reference Besyst, Cattrijsse and Mees1999; Hampel et al., Reference Hampel, Cattrijsse and Elliott2005; Haynes et al., Reference Haynes, Brophy, De Raedemaecker and McGrath2011) may reduce the effects of niche overlap and prevent competition. Accordingly, the distinct isotopic signatures of 0-group and 1-group flounder and their feeding on highly abundant prey (e.g. Corophium and polychaetes) in the Lima estuary (Sousa et al., Reference Sousa, Dias and Antunes2006), did not support evidence for competition. Moreover, the morphometric condition (mean = 1.06 ± 0.07) of European flounder juveniles in the Lima estuary was within the same range observed for this species in estuaries such as Canche (mean = 0.89), Authie (mean = 1.0) (Amara et al., Reference Amara, Selleslagh, Billon and Minier2009), Minho, Mondego and Douro (mean = 0.70, Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Fonseca, Ruano, Tanner, Costa and Cabral2009) suggesting good somatic condition. Thus, the differential distribution and feeding strategies of the 0-group and 1-group flounder may have minimized competition and sustained somatic condition over the summer months preceding collection of juveniles. Overall, these results did not suggest food limitation, i.e. the carrying capacity of the Lima estuary may not have been fully exploited, at least at the time scale integrated by the Fulton K and stable isotope analysis. However, it has been suggested that limitation of food resources may be underestimated by individual condition indices (Le Pape & Bonhommeau, Reference Le Pape and Bonhommeau2015), as size-selective mortality favours survival of fast-growing individuals (Sogard, Reference Sogard1997). The growth of the surviving individuals would be close to maximal values even when there was food limitation (Le Pape & Bonhommeau, Reference Le Pape and Bonhommeau2015). Thus, further studies are required to estimate the relative amounts of prey available to predators and carrying capacity.

The RNA:DNA of the European flounder in the Lima estuary (mean = 1.57 ± 0.62) was well above the critical starvation value (0.32), and close to optimal feeding values (1.71) defined for newly settled plaice Pleuronectes platessa L. 1758 (Selleslagh & Amara, Reference Selleslagh and Amara2013). However, it was lower than RNA:DNA of well-fed metamorphosing flounder (>2, O'Neill et al., Reference O'Neill, De Raedemaecker, McGrath and Brophy2011), common sole Solea solea L., 1758 (>1.5, Richard et al., Reference Richard, Bergeron, Boulhic, Galois and Person-Le Ruyet1991) and Japanese flounder Paralichthys olivaceus Temminck and Schlegel, 1846 (>2, Gwak & Tanaka, Reference Gwak and Tanaka2001). These comparisons must be taken with caution as RNA:DNA is species and size dependent (Ferron & Leggett, Reference Ferron and Leggett1994; Buckley et al., Reference Buckley, Caldarone and Clemmesen2008; Tanaka et al., Reference Tanaka, Satoh, Yamada, Takebe, Nikaido and Shiozawa2008). Decreased growth rates of juvenile flounder have been linked to food limitation, which may be caused by changes in prey quality and availability (Teal et al., Reference Teal, de Leeuw, van der Veer and Rijnsdorp2008), and intraspecific (Edwards et al., Reference Edwards, Steele and Trevallion1970; Laffargue et al., Reference Laffargue, Lagardère, Rijnsdorp, Fillon and Amara2007) and interspecific competition (Jager et al., Reference Jager, Kleef and Tydeman1995; van der Veer et al., Reference van der Veer, Freitas, Koot, Witte and Zuur2010; Freitas et al., Reference Freitas, Kooijman and van der Veer2012; Ciotti et al., Reference Ciotti, Targett, Nash and Burrows2013b). It is of note that European flounder juveniles were sampled in the late summer when sub-optimal growth has been suggested for juveniles of this species (Jager et al., Reference Jager, Kleef and Tydeman1995; Freitas et al., Reference Freitas, Kooijman and van der Veer2012), and similar to other flatfish such as plaice (Freitas et al., Reference Freitas, Kooijman and van der Veer2012; Ciotti et al., Reference Ciotti, Targett and Burrows2013a; van der Veer et al., Reference van der Veer, Jung, Freitas, Philippart and Witte2016) and common sole (Fonseca et al., Reference Fonseca, Vinagre and Cabral2006; Laffargue et al., Reference Laffargue, Lagardère, Rijnsdorp, Fillon and Amara2007; Teal et al., Reference Teal, de Leeuw, van der Veer and Rijnsdorp2008). However, Poiesz et al. (Reference Poiesz, de Vries, Cardoso, Witte, van der Veer and Freitas2019) found no trend in flounder growth over time in the Wadden Sea, and suggested that this species, as an epibenthic predator, was not affected by reduced infauna activity in late summer, and thus reduced prey availability, in contrast to plaice which is a benthic feeder (van der Veer et al., Reference van der Veer, Jung, Freitas, Philippart and Witte2016). The RNA:DNA ratio of European flounder in the Lima estuary was lower than in the nearby Minho and Douro (Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Fonseca, Ruano, Tanner, Costa and Cabral2009), while in the same range as Canche, Authie (Amara et al., Reference Amara, Selleslagh, Billon and Minier2009) and Mondego (Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Fonseca, Ruano, Tanner, Costa and Cabral2009). Although this may suggest suboptimal food conditions, differences in methodology may limit comparisons of the RNA:DNA ratio between studies (Caldarone et al., Reference Caldarone, Clemmesen, Berdalet, Miller, Folkvord, Holt, Olivar and Suthers2006), and temperature effects must also be considered. Indeed, temperature is a controller of enzymatic activity and metabolic reactions (Fry, Reference Fry, Hoar and Randall1971), and influences RNA synthesis and activity (Buckley et al., Reference Buckley, Caldarone and Clemmesen2008). Therefore, temperature affects RNA:DNA ratios and the relationship between RNA:DNA and growth (Ferron & Leggett, Reference Ferron and Leggett1994; Buckley et al., Reference Buckley, Caldarone and Ong1999). Lower temperatures were observed in Lima downstream areas (T = 14.7 ± 0.7) compared with upstream areas (T = 21.4 ± 1.1°C). Therefore, temperature differences between the Lima estuary (T = 17.1 ± 3.4°C) and other estuaries within the same geographic area (>20°C, Vasconcelos et al., Reference Vasconcelos, Reis-Santos, Fonseca, Ruano, Tanner, Costa and Cabral2009) may have contributed to the lower RNA:DNA observed in this study, even though temperature in the upper estuary was within the optimal range (18–22°C) for flounder growth (Fonds et al., Reference Fonds, Cronie, Vethaak and Van Der Puyl1992). The different lag response to environmental conditions of the Fulton K and the RNA:DNA ratio may explain the lack of a correlation between these indices (Suthers et al., Reference Suthers, Fraser and Frank1992; Ferron & Leggett, Reference Ferron and Leggett1994; De Raedemaecker et al., Reference De Raedemaecker, Brophy, O'Connor and O'Neill2012b). Hence, the Lima estuary appeared to promote good condition of the juveniles during summer, while short-term changes in environmental conditions or prey availability and competition may justify the low RNA:DNA as this index is sensitive to fish recent feeding (Clemmesen, Reference Clemmesen1994; Malloy & Targett, Reference Malloy and Targett1994), and condition (Buckley, Reference Buckley1984; Gwak & Tanaka, Reference Gwak and Tanaka2001). These condition patterns observed over a brief summer period may not be reflected at the seasonal and interannual scales.

Historical habitat loss is a major environmental problem in estuaries (Wolanski & Elliott, Reference Wolanski and Elliott2015; Amorim et al., Reference Amorim, Ramos, Elliott, Franco and Bordalo2017), and there are increasing measures to remedy that loss (Elliott et al., Reference Elliott, Mander, Mazik, Simenstad, Valesini, Whitfield and Wolanski2016). Coordinated management initiatives (e.g. Lonsdale et al., Reference Lonsdale, Weston, Barnard, Boyes and Elliott2015) aim to balance the effects of the various estuarine users, and their demands on the systems. The Lima estuary is located at the southern limit of the distribution of flounder where increased temperatures, especially in the shallow upstream areas, may limit the food and habitat available with effects on juvenile flounder condition and growth (Freitas et al., Reference Freitas, Kooijman and van der Veer2012). Hence, the present study is important in determining which species and stages use habitats within and adjacent to the estuary, thereby guiding the habitat management within the estuary. Further studies are required to assess how the juvenile flounder condition will respond to changes in food availability and temperature related to climate change.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/S0025315420000600.

Acknowledgements

The authors wish to thank two anonymous referees and the Editor for their valuable comments made on an earlier version of the paper.

Financial support

This research was partially supported by FCT – The Portuguese Foundation for Science and Technology through the PhD fellowship awarded to C. Mendes (SFRH/BD/86325/2012). This study was also partially funded through the project “Mytag – Integrating natural and artificial tags to reconstruct fish migrations and ontogenetic niche shifts” (PTDC/MAR-EST/2098/2014), under the Project 9471 – Reforçar a Investigação, o Desenvolvimento Tecnológico e a Inovação (Projeto 9471-RIDTI) and subsidized by the European Regional Development Fund (FEDER, POCI-01-0145-FEDER- 016787), and by the Strategic Funding UID/Multi/04423/2013 and UID/Multi/04423/2019 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of PT2020.

References

Aarnio, K, Bonsdorff, E and Rosenback, N (1996) Food and feeding habits of juvenile flounder Platichthys flesus (L.), and turbot Scophthalmus maximus L. in the Åland archipelago, northern Baltic Sea. Journal of Sea Research 36, 311320.CrossRefGoogle Scholar
Amara, R, Selleslagh, J, Billon, G and Minier, C (2009) Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality. Hydrobiologia 627, 87.CrossRefGoogle Scholar
Amorim, E, Ramos, S, Elliott, M and Bordalo, AA (2016) Immigration and early life stages recruitment of the European flounder (Platichthys flesus) to an estuarine nursery: the influence of environmental factors. Journal of Sea Research 107, 5666.CrossRefGoogle Scholar
Amorim, E, Ramos, S, Elliott, M, Franco, A and Bordalo, AA (2017) Habitat loss and gain: influence on habitat attractiveness for estuarine fish communities. Estuarine, Coastal and Shelf Science 197(Suppl. C), 244257.CrossRefGoogle Scholar
Amorim, E, Ramos, S, Elliott, M and Bordalo, AA (2018) Dynamic habitat use of an estuarine nursery seascape: ontogenetic shifts in habitat suitability of the European flounder (Platichthys flesus). Journal of Experimental Marine Biology and Ecology 506, 4960.CrossRefGoogle Scholar
Andersen, AK, Schou, J, Sparrevohn, CR, Nicolajsen, H and Støttrup, JG (2005 a) The quality of release habitat for reared juvenile flounder, Platichthys flesus, with respect to salinity and depth. Fisheries Management and Ecology 12, 211219.CrossRefGoogle Scholar
Andersen, BS, Carl, JD, Grønkjær, P and Støttrup, JG (2005 b) Feeding ecology and growth of age 0 year Platichthys flesus (L.) in a vegetated and a bare sand habitat in a nutrient rich fjord. Journal of Fish Biology 66, 531552.CrossRefGoogle Scholar
Anderson, MJ, Gorley, RN and Clarke, KR (2008) PERMANOVA + for PRIMER: Guide to Software and Statistical Methods. Plymouth: Primer-E.Google Scholar
Armitage, PD, Pinder, LC and Cranston, P (1995) The Chironomidae: Biology and Ecology of Non-Biting Midges. London: Chapman and Hall.Google Scholar
Beaumont, WRC and Mann, RHK (1984) The age, growth and diet of a freshwater population of the flounder, Platichthys flesus (L.), in southern England. Journal of Fish Biology 25, 607616.CrossRefGoogle Scholar
Beck, MW, Heck, KL, Able, KW, Childers, DL, Eggleston, DB, Gillanders, BM, Halpern, B, Hays, CG, Hoshino, K, Minello, TJ, Orth, RJ, Sheridan, PF and Weinstein, MP (2001) The identification, conservation, and management of estuarine and marine nurseries for fish and invertebrates. BioScience 51, 633641.CrossRefGoogle Scholar
Besyst, B, Cattrijsse, A and Mees, J (1999) Feeding ecology of juvenile flatfishes of the surf zone of a sandy beach. Journal of Fish Biology 55, 11711186.CrossRefGoogle Scholar
Boesch, DF and Turner, RE (1984) Dependence of fishery species on salt marshes: the role of food and refuge. Estuaries 7, 460468.CrossRefGoogle Scholar
Bos, AR (1999) Tidal transport of flounder larvae (Platichthys flesus) in the Elbe River, Germany. Archive of Fishery and Marine Research 47, 4760.Google Scholar
Buckley, LJ (1984) RNA-DNA ratio: an index of larval fish growth in the sea. Marine Biology 80, 291298.CrossRefGoogle Scholar
Buckley, L, Caldarone, E and Ong, T-L (1999) RNA–DNA ratio and other nucleic acid-based indicators for growth and condition of marine fishes. Hydrobiologia 401, 265277.CrossRefGoogle Scholar
Buckley, LJ, Caldarone, EM and Clemmesen, C (2008) Multi-species larval fish growth model based on temperature and fluorometrically derived RNA/DNA ratios: results from a meta-analysis. Marine Ecology Progress Series 371, 221232.CrossRefGoogle Scholar
Bulow, FJ (1970) RNA–DNA ratios as indicators of recent growth rates of a fish. Journal of the Fisheries Research Board of Canada 27, 23432349.CrossRefGoogle Scholar
Burrows, MT, Gontarek, SJ, Nash, RDM and Gibson, RN (2001) Shrimp predation on 0-group plaice: contrasts between field data and predictions of an individual-based model. Journal of Sea Research 45, 243254.CrossRefGoogle Scholar
Cabral, H and Costa, MJ (1999) Differential use of nursery areas within the Tagus Estuary by sympatric soles, Solea solea and Solea senegalensis. Environmental Biology of Fishes 56, 389397.CrossRefGoogle Scholar
Cabral, HN, Lopes, M and Loeper, R (2002) Trophic niche overlap between flatfishes in a nursery area on the Portuguese coast. Scientia Marina 66, 293300.CrossRefGoogle Scholar
Cabral, HN, Vasconcelos, R, Vinagre, C, França, S, Fonseca, V, Maia, A, Reis-Santos, P, Lopes, M, Ruano, M, Campos, J, Freitas, V, Santos, PT and Costa, MJ (2007) Relative importance of estuarine flatfish nurseries along the Portuguese coast. Journal of Sea Research 57, 209217.CrossRefGoogle Scholar
Caldarone, EM, Wagner, M, Onge-Burns, JS and Buckley, LJ (2001) Protocol and Guide for Estimating Nucleic Acids in Larval Fish Using a Fluorescence Microplate Reader. Northeast Fisheries Science Center (Reference Document) 01-11, 1–22.Google Scholar
Caldarone, EM, Clemmesen, CM, Berdalet, E, Miller, TJ, Folkvord, A, Holt, GJ, Olivar, MP and Suthers, IM (2006) Intercalibration of four spectrofluorometric protocols for measuring RNA/DNA ratios in larval and juvenile fish. Limnology and Oceanography: Methods 4, 153163.Google Scholar
Caldarone, EM, MacLean, SA and Sharack, B (2012) Evaluation of bioelectrical impedance analysis and Fulton's condition factor as nonlethal techniques for estimating short-term responses in postsmolt Atlantic salmon (Salmo salar) to food availability. Fishery Bulletin 110, 257270.Google Scholar
Chícharo, M and Chícharo, L (2008) RNA:DNA ratio and other nucleic acid derived indices in marine ecology. International Journal of Molecular Sciences 9, 14531471.CrossRefGoogle ScholarPubMed
Ciotti, BJ, Targett, TE and Burrows, MT (2013 a) Decline in growth rate of juvenile European plaice (Pleuronectes platessa) during summer at nursery beaches along the west coast of Scotland. Canadian Journal of Fisheries and Aquatic Sciences 70, 720734.CrossRefGoogle Scholar
Ciotti, BJ, Targett, TE, Nash, RDM and Burrows, MT (2013 b) Small-scale spatial and temporal heterogeneity in growth and condition of juvenile fish on sandy beaches. Journal of Experimental Marine Biology and Ecology 448, 346359.CrossRefGoogle Scholar
Clarke, KR and Gorley, RN (2006) Primer V6: User Manual – Tutorial. Plymouth: Plymouth Marine Laboratory.Google Scholar
Clemmesen, C (1994) The effect of food availability, age or size on the RNA/DNA ratio of individually measured herring larvae: laboratory calibration. Marine Biology 118, 377382.CrossRefGoogle Scholar
Dando, PR (2011) Site fidelity, homing and spawning migrations of flounder Platichthys flesus in the Tamar estuary, South West England. Marine Ecology Progress Series 430, 183196.CrossRefGoogle Scholar
Darnaude, AM, Harmelin-Vivien, ML and Salen-Picard, C (2001) Food partitioning among flatfish (Pisces: Pleuronectiforms) juveniles in a Mediterranean coastal shallow sandy area. Journal of the Marine Biological Association of the United Kingdom 81, 119127.CrossRefGoogle Scholar
Darnaude, AM, Salen-Picard, C and Harmelin-Vivien, ML (2004) Depth variation in terrestrial particulate organic matter exploitation by marine coastal benthic communities off the Rhone River delta (NW Mediterranean). Marine Ecology Progress Series 275, 4757.CrossRefGoogle Scholar
De Groot, SJ (1971) On the interrelationships between morphology of the alimentary tract, food and feeding behaviour in flatfishes (Pisces: Pleuronectiformes). Netherlands Journal of Sea Research 5, 121196.CrossRefGoogle Scholar
DeNiro, MJ and Epstein, S (1978) Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42, 495506.CrossRefGoogle Scholar
DeNiro, MJ and Epstein, S (1981) Influence of diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45, 341351.CrossRefGoogle Scholar
De Raedemaecker, F, Brophy, D, O'Connor, I and Comerford, S (2012 a) Habitat characteristics promoting high density and condition of juvenile flatfish at nursery grounds on the west coast of Ireland. Journal of Sea Research 73, 717.CrossRefGoogle Scholar
De Raedemaecker, F, Brophy, D, O'Connor, I and O'Neill, B (2012 b) Dependence of RNA:DNA ratios and Fulton's K condition indices on environmental characteristics of plaice and dab nursery grounds. Estuarine, Coastal and Shelf Science 98(Suppl. C), 6070.CrossRefGoogle Scholar
Dias, E, Morais, P, Faria, AM, Antunes, C and Hoffman, JC (2017) Benthic food webs support the production of sympatric flatfish larvae in estuarine nursery habitat. Fisheries Oceanography 26, 507512.CrossRefGoogle ScholarPubMed
Dinis, MT (1986) Quatre Soleidae de l'estuaire du Tage. Reproduction et croissance. Essai d'elevage de Solea senegalensis Kaup (Ph.D. dissertation). Université de Bretagne Occidentale, Brest, France.Google Scholar
Doornbos, G and Twisk, F (1984) Density, growth and annual food consumption of plaice (Pleuronectes platessa L.) and flounder (Platichthys flesus (L.)) in Lake Grevelingen, the Netherlands. Netherlands Journal of Sea Research 18, 434456.CrossRefGoogle Scholar
Edwards, R and Steele, JH (1968) The ecology of 0-group plaice and common dabs at Loch Ewe I. Population and food. Journal of Experimental Marine Biology and Ecology 2, 215238.CrossRefGoogle Scholar
Edwards, RRC, Steele, JH and Trevallion, A (1970) The ecology of 0-group plaice and common dabs in Loch Ewe. III. Prey-predator experiments with plaice. Journal of Experimental Marine Biology and Ecology 4, 156173.CrossRefGoogle Scholar
Elliott, M and Hemingway, KL (2002) Fishes in Estuaries. Oxford: Blackwell Science.CrossRefGoogle Scholar
Elliott, M, Burdon, D, Hemingway, KL and Apitz, SE (2007) Estuarine, coastal and marine ecosystem restoration: confusing management and science – a revision of concepts. Estuarine, Coastal and Shelf Science 74, 349366.Google Scholar
Elliott, M, Mander, L, Mazik, K, Simenstad, C, Valesini, F, Whitfield, A and Wolanski, E (2016) Ecoengineering with ecohydrology: successes and failures in estuarine restoration. Estuarine, Coastal and Shelf Science 176, 1235.CrossRefGoogle Scholar
Evans, S (1983) Production, predation and food niche segregation in a marine shallow soft-bottom community. Marine Ecology Progress Series 10, 147157.CrossRefGoogle Scholar
Ferron, A and Leggett, WC (1994) An appraisal of condition measures for marine fish larvae. Advances in Marine Biology 30, 217303.CrossRefGoogle Scholar
Florin, A-B and Lavados, G (2010) Feeding habits of juvenile flatfish in relation to habitat characteristics in the Baltic Sea. Estuarine, Coastal and Shelf Science 86, 607612.CrossRefGoogle Scholar
Foley, CJ, Bradley, DL and Höök, TO (2016) A review and assessment of the potential use of RNA:DNA ratios to assess the condition of entrained fish larvae. Ecological Indicators 60, 346357.CrossRefGoogle Scholar
Fonds, M, Cronie, R, Vethaak, AD and Van Der Puyl, P (1992) Metabolism, food consumption and growth of plaice (Pleuronectes platessa) and flounder (Platichthys flesus) in relation to fish size and temperature. Netherlands Journal of Sea Research 29, 127143.CrossRefGoogle Scholar
Fonseca, VF, Vinagre, C and Cabral, HN (2006) Growth variability of juvenile soles Solea solea and Solea senegalensis, and comparison with RNA:DNA ratios in the Tagus estuary, Portugal. Journal of Fish Biology 68, 15511562.CrossRefGoogle Scholar
França, S, Vasconcelos, RP, Tanner, S, Máguas, C, Costa, MJ and Cabral, HN (2011) Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: a stable isotope approach. Marine Environmental Research 72, 204215.CrossRefGoogle ScholarPubMed
Freitas, V, Costa-Dias, S, Campos, J, Bio, A, Santos, P and Antunes, C (2009) Patterns in abundance and distribution of juvenile flounder, Platichthys flesus, in Minho estuary (NW Iberian Peninsula). Aquatic Ecology 43, 1143.CrossRefGoogle Scholar
Freitas, V, Kooijman, S and van der Veer, HW (2012) Latitudinal trends in habitat quality of shallow-water flatfish nurseries. Marine Ecology Progress Series 471, 203214.CrossRefGoogle Scholar
Fry, FEJ (1971) 1 – The effect of environmental factors on the physiology of fish. In Hoar, WS and Randall, DJ (eds), Fish Physiology, vol. 6. London: Academic Press, pp. 198.Google Scholar
Fry, B (2002) Conservative mixing of stable isotopes across estuarine salinity gradients: a conceptual framework for monitoring watershed influences on downstream fisheries production. Estuaries 25, 264271.CrossRefGoogle Scholar
Fry, B and Sherr, EB (1989) Δ13C measurements as indicators of carbon flow in marine and freshwater ecosystems. In Rundel, PW, Ehleringer, JR and Nagy, KA (eds), Stable Isotopes in Ecological Research. New York, NY: Springer, pp. 196229.CrossRefGoogle Scholar
Gerdol, V and Hughes, RG (1995) Feeding behaviour and diet of Corophium volutator in an estuary in southeastern England. Oceanographic Literature Review 42, 391.Google Scholar
Gibson, RN (1973) Tidal and circadian activity rhythms in juvenile plaice, Pleuronectes platessa. Marine Biology 22, 379386.CrossRefGoogle Scholar
Gibson, RN (1994) Impact of habitat quality and quantity on the recruitment of juvenile flatfishes. Netherlands Journal of Sea Research 32, 191206.CrossRefGoogle Scholar
Gilliers, C, Amara, R and Bergeron, J-P (2004) Comparison of growth and condition indices of juvenile flatfish in different coastal nursery grounds. Environmental Biology of Fishes 71, 189198.CrossRefGoogle Scholar
Green, BC, Smith, DJ, Grey, J and Underwood, GJC (2012) High site fidelity and low site connectivity in temperate salt marsh fish populations: a stable isotope approach. Oecologia 168, 245255.CrossRefGoogle ScholarPubMed
Grønkjær, P, Carl, JD, Rasmussen, TH and Hansen, KW (2007) Effect of habitat shifts on feeding behaviour and growth of 0 year-group flounder Platichthys flesus (L.) transferred between macroalgae and bare sand habitats. Journal of Fish Biology 70, 15871605.CrossRefGoogle Scholar
Gutt, J (1985) The growth of juvenile flounders (Platichthys flesus L.) at salinities of 0, 5, 15 and 35%%. Journal of Applied Ichthyology 1, 1726.CrossRefGoogle Scholar
Gwak, WS and Tanaka, M (2001) Developmental change in RNA: DNA ratios of fed and starved laboratory-reared Japanese flounder larvae and juveniles, and its application to assessment of nutritional condition for wild fish. Journal of Fish Biology 59, 902915.Google Scholar
Hampel, H, Cattrijsse, A and Elliott, M (2005) Feeding habits of young predatory fishes in marsh creeks situated along the salinity gradient of the Schelde estuary, Belgium and the Netherlands. Helgoland Marine Research 59, 151162.CrossRefGoogle Scholar
Harmelin-Vivien, M, Loizeau, V, Mellon, C, Beker, B, Arlhac, D, Bodiguel, X, Ferraton, F, Hermand, R, Philippon, X and Salen-Picard, C (2008) Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean). Continental Shelf Research 28, 19111919.CrossRefGoogle Scholar
Haynes, PS, Brophy, D, De Raedemaecker, F and McGrath, D (2011) The feeding ecology of 0 year-group turbot Scophthalmus maximus and brill Scophthalmus rhombus on Irish west coast nursery grounds. Journal of Fish Biology 79, 18661882.CrossRefGoogle ScholarPubMed
Henderson, PA and Holmes, RHA (1991) On the population dynamics of dab, sole and flounder within Bridgwater Bay in the lower Severn Estuary, England. Netherlands Journal of Sea Research 27, 337344.CrossRefGoogle Scholar
Herzka, SZ (2005) Assessing connectivity of estuarine fishes based on stable isotope ratio analysis. Estuarine, Coastal and Shelf Science 64, 5869.CrossRefGoogle Scholar
Hoffman, JC, Kelly, JR, Peterson, GS and Cotter, AM (2015) Landscape-scale food webs of fish nursery habitat along a river-coast mixing zone. Estuaries and Coasts 38, 13351349.CrossRefGoogle Scholar
Hyslop, EJ (1980) Stomach contents analysis – a review of methods and their application. Journal of Fish Biology 17, 411429.CrossRefGoogle Scholar
Jager, Z, Kleef, HL and Tydeman, P (1995) Mortality and growth of 0-group flatfish in the brackish dollard (Ems Estuary, Wadden Sea). Netherlands Journal of Sea Research 34, 119129.CrossRefGoogle Scholar
Kennedy, P, Kennedy, H and Papadimitriou, S (2005) The effect of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment. Rapid Communications in Mass Spectrometry 19, 10631068.CrossRefGoogle ScholarPubMed
Kerstan, M (1991) The importance of rivers as nursery grounds for 0- and 1-group flounder (Platichthys flesus L.) in comparison to the Wadden Sea. Netherlands Journal of Sea Research 27, 353366.CrossRefGoogle Scholar
Kopp, D, Le Bris, H, Grimaud, L, Nérot, C and Brind'Amour, A (2013) Spatial analysis of the trophic interactions between two juvenile fish species and their preys along a coastal–estuarine gradient. Journal of Sea Research 81, 4048.CrossRefGoogle Scholar
Kostecki, C, Roussel, JM, Desroy, N, Roussel, G, Lanshere, J, Le Bris, H and Le Pape, O (2012) Trophic ecology of juvenile flatfish in a coastal nursery ground: contributions of intertidal primary production and freshwater particulate organic matter. Marine Ecology Progress Series 449, 221232.CrossRefGoogle Scholar
Laffargue, P, Lagardère, F, Rijnsdorp, AD, Fillon, A and Amara, R (2007) Growth performances of juvenile sole Solea solea under environmental constraints of embayed nursery areas. Aquatic Living Resources 20, 213221.CrossRefGoogle Scholar
Le Pape, O and Bonhommeau, S (2015) The food limitation hypothesis for juvenile marine fish. Fish and Fisheries 16, 373398.CrossRefGoogle Scholar
Le Pape, O and Cognez, N (2016) The range of juvenile movements of estuarine and coastal nursery dependent flatfishes: estimation from a meta-analytical approach. Journal of Sea Research 107(Part 1), 4355.CrossRefGoogle Scholar
Le Pape, O, Modéran, J, Beaunée, G, Riera, P, Nicolas, D, Savoye, N, Harmelin-Vivien, M, Darnaude, AM, Brind'Amour, A, Le Bris, H, Cabral, H, Vinagre, C, Pasquaud, S, França, S and Kostecki, C (2013) 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, 8595.CrossRefGoogle Scholar
Lonsdale, J-A, Weston, K, Barnard, S, Boyes, SJ and Elliott, M (2015) Integrating management tools and concepts to develop an estuarine planning support system: a case study of the Humber Estuary, eastern England. Marine Pollution Bulletin 100, 393405.CrossRefGoogle ScholarPubMed
Malloy, KD and Targett, TE (1994) The use of RNA:DNA ratios to predict growth limitation of juvenile summer flounder (Paralichthys dentatus) from Delaware and North Carolina estuaries. Marine Biology 118, 367375.CrossRefGoogle Scholar
Marshall, S and Elliott, M (1997) A comparison of univariate and multivariate numerical and graphical techniques for determining inter- and intraspecific feeding relationships in estuarine fish. Journal of Fish Biology 51, 526545.CrossRefGoogle Scholar
Martinho, F, Leitão, R, Neto, JM, Cabral, HN, Marques, JC and Pardal, MA (2007) The use of nursery areas by juvenile fish in a temperate estuary, Portugal. Hydrobiologia 587, 281290.CrossRefGoogle Scholar
Martinho, F, Leitão, R, Neto, JM, Cabral, H, Lagardère, F and Pardal, MA (2008) Estuarine colonization, population structure and nursery functioning for 0-group sea bass (Dicentrarchus labrax), flounder (Platichthys flesus) and sole (Solea solea) in a mesotidal temperate estuary. Journal of Applied Ichthyology 24, 229237.CrossRefGoogle Scholar
McLusky, DS and Elliott, M (2004) The Estuarine Ecosystem: Ecology, Threats and Management. Oxford: Oxford University Press.CrossRefGoogle Scholar
Mendes, C, Ramos, S and Bordalo, AA (2014) Feeding ecology of juvenile flounder Platichthys flesus in an estuarine nursery habitat: influence of prey–predator interactions. Journal of Experimental Marine Biology and Ecology 461, 458468.CrossRefGoogle Scholar
Minagawa, M and Wada, E (1984) Stepwise enrichment of 15N along food chains: further evidence and the relation between δ15N and animal age. Geochimica et Cosmochimica Acta 48, 11351140.CrossRefGoogle Scholar
Modin, J and Pihl, L (1996) Small-scale distribution of juvenile plaice and flounder in relation to predatory shrimp in a shallow Swedish bay. Journal of Fish Biology 49, 10701085.CrossRefGoogle Scholar
Molinero, A and Flos, R (1992) Influence of season on the feeding habits of the common sole Solea solea. Marine Biology 113, 499507.CrossRefGoogle Scholar
Moore, JW and Moore, IA (1976) The basis of food selection in flounders, Platichthys flesus (L.), in the Severn Estuary. Journal of Fish Biology 9, 139156.CrossRefGoogle Scholar
Muus, BJ and Nielsen, JG (1999) Sea Fish. Hedehusene: Scandinavian Fishing Year Book.Google Scholar
Ng, JSS, Wai, T-C and Williams, GA (2007) The effects of acidification on the stable isotope signatures of marine algae and molluscs. Marine Chemistry 103, 97102.CrossRefGoogle Scholar
Nielsen, JG (1986) Pleuronectidae. In Whitehead, PJP, Bauchot, ML, Hureau, JC, Nielsen, J and Tortonese, E (eds), Fishes of the North-Eastern Atlantic and Mediterranean. Paris: UNESCO, pp. 12991307.Google Scholar
Nunn, AD, Tewson, LH and Cowx, IG (2012) The foraging ecology of larval and juvenile fishes. Reviews in Fish Biology and Fisheries 22, 377408.CrossRefGoogle Scholar
O'Neill, B, De Raedemaecker, F, McGrath, D and Brophy, D (2011) An experimental investigation of salinity effects on growth, development and condition in the European flounder (Platichthys flesus L.). Journal of Experimental Marine Biology and Ecology 410, 3944.CrossRefGoogle Scholar
Palmer, SCF, Coulon, A and Travis, JMJ (2014) Inter-individual variability in dispersal behaviours impacts connectivity estimates. Oikos 123, 923932.CrossRefGoogle Scholar
Parnell, AC, Inger, R, Bearhop, S and Jackson, AL (2010) Source partitioning using stable isotopes: coping with too much variation. PLoS ONE 5, e9672.CrossRefGoogle ScholarPubMed
Parnell, AC, Phillips, DL, Bearhop, S, Semmens, BX, Ward, EJ, Moore, JW, Jackson, AL, Grey, J, Kelly, DJ and Inger, R (2013) Bayesian stable isotope mixing models. Environmetrics 24, 387399.Google Scholar
Pasquaud, S, Elie, P, Jeantet, C, Billy, I, Martinez, P and Girardin, M (2008) A preliminary investigation of the fish food web in the Gironde estuary, France, using dietary and stable isotope analyses. Estuarine, Coastal and Shelf Science 78, 267279.CrossRefGoogle Scholar
Peterson, BJ and Fry, B (1987) Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18, 293320.CrossRefGoogle Scholar
Phillips, DL, Inger, R, Bearhop, S, Jackson, AL, Moore, JW, Parnell, AC, Semmens, BX and Ward, EJ (2014) Best practices for use of stable isotope mixing models in food-web studies. Canadian Journal of Zoology 92, 823835.CrossRefGoogle Scholar
Piet, GJ, Pfisterer, AB and Rijnsdorp, AD (1998) On factors structuring the flatfish assemblage in the southern North Sea. Journal of Sea Research 40, 143152.CrossRefGoogle Scholar
Pihl, L, Cattrijsse, A, Codling, I, Mathieson, S, McLusky, DS and Roberts, C (2007) Habitat use by fishes in estuaries and other brackish areas. Fishes in Estuaries. Oxford: Blackwell Science, pp. 1053.Google Scholar
Poiesz, SSH, de Vries, A, Cardoso, JFMF, Witte, JIJ, van der Veer, HW and Freitas, V (2019) A comparison of growth in two juvenile flatfish species in the Dutch Wadden Sea: searching for a mechanism for summer growth reduction in flatfish nurseries. Journal of Sea Research 144, 3948.CrossRefGoogle Scholar
Post, DM (2002) Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83, 703718.CrossRefGoogle Scholar
Potter, IC, Tweedley, JR, Elliott, M and Whitfield, AK (2015) The ways in which fish use estuaries: a refinement and expansion of the guild approach. Fish and Fisheries 16, 230239.CrossRefGoogle Scholar
Power, M, Attrill, MJ and Thomas, RM (2000) Environmental factors and interactions affecting the temporal abundance of juvenile flatfish in the Thames Estuary. Journal of Sea Research 43, 135149.CrossRefGoogle Scholar
Preston, T and Owens, NJP (1983) Interfacing an automatic elemental analyser with an isotope ratio mass spectrometer: the potential for fully automated total nitrogen and nitrogen-15 analysis. The Analyst 108, 971977.CrossRefGoogle Scholar
Primo, AL, Azeiteiro, UM, Marques, SC, Martinho, F, Baptista, J and Pardal, MA (2013) Colonization and nursery habitat use patterns of larval and juvenile flatfish species in a small temperate estuary. Journal of Sea Research 76(Suppl. C), 126134.CrossRefGoogle Scholar
Raffaelli, D, Conacher, A, McLachlan, H and Emes, C (1989) The role of epibenthic crustacean predators in an estuarine food web. Estuarine, Coastal and Shelf Science 28, 149160.CrossRefGoogle Scholar
Raffaelli, D, Richner, H, Summers, R and Northcott, S (1990) Tidal migrations in the flounder (Platichthys flesus). Marine Behaviour and Physiology 16, 249260.CrossRefGoogle Scholar
Ramos, S, Cowen, RK, , P and Bordalo, AA (2006) Temporal and spatial distributions of larval fish assemblages in the Lima estuary (Portugal). Estuarine, Coastal and Shelf Science 66, 303314.CrossRefGoogle Scholar
Ramos, S, , P and Bordalo, AA (2009) Environmental control on early life stages of flatfishes in the Lima Estuary (NW Portugal). Estuarine. Coastal and Shelf Science 83, 252264.CrossRefGoogle Scholar
Ramos, S, , P and Bordalo, AA (2010) Recruitment of flatfish species to an estuarine nursery habitat (Lima Estuary, NW Iberian Peninsula). Journal of Sea Research 64, 473486.CrossRefGoogle Scholar
Ramos, S, Paris, CB and Angélico, MM (2017) Larval fish dispersal along an estuarine–ocean gradient. Canadian Journal of Fisheries and Aquatic Sciences 74, 14621473.CrossRefGoogle Scholar
R Development Core Team (2007) R: A Language and Environment for Statistical Computing. Vienna: R foundation for Statistical Computing.Google Scholar
Richard, P, Bergeron, J-P, Boulhic, M, Galois, R and Person-Le Ruyet, J (1991) Effect of starvation on RNA, DNA and protein content of laboratory-reared larvae and juveniles of Solea solea. Marine Ecology Progress Series 72, 6977.CrossRefGoogle Scholar
Ricker, WE (1975) Computation and Interpretation of Biological Statistics of Fish Populations. Ottawa: Bulletin of the Fisheries Research Board of Canada.Google Scholar
Rijnsdorp, AD, Berghahn, R, Miller, JM and Van Der Veer, HW (1995) Recruitment mechanisms in flatfish: what did we learn and where do we go? Netherlands Journal of Sea Research 34, 237242.CrossRefGoogle Scholar
Russo, T, Pulcini, D, O'Leary, Á, Cataudella, S and Mariani, S (2008) Relationship between body shape and trophic niche segregation in two closely related sympatric fishes. Journal of Fish Biology 73, 809828.CrossRefGoogle Scholar
Selleslagh, J and Amara, R (2013) Effect of starvation on condition and growth of juvenile plaice Pleuronectes platessa: nursery habitat quality assessment during the settlement period. Journal of the Marine Biological Association of the United Kingdom 93, 479488.CrossRefGoogle Scholar
Selleslagh, J, Blanchet, H, Bachelet, G and Lobry, J (2015) Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis. Estuaries and Coasts 38, 14311447.CrossRefGoogle Scholar
Sheaves, M (2009) Consequences of ecological connectivity: the coastal ecosystem mosaic. Marine Ecology Progress Series 391, 107115.CrossRefGoogle Scholar
Sheaves, M, Baker, R, Nagelkerken, I and Connolly, RM (2015) True value of estuarine and coastal nurseries for fish: incorporating complexity and dynamics. Estuaries and Coasts 38, 401414.CrossRefGoogle Scholar
Sogard, SM (1997) Size-selective mortality in the juvenile stage of teleost fishes: a review. Bulletin of Marine Science 60, 11291157.Google Scholar
Sousa, R, Dias, S and Antunes, JC (2006) Spatial subtidal macrobenthic distribution in relation to abiotic conditions in the Lima Estuary, NW of Portugal. Hydrobiologia 559, 135148.CrossRefGoogle Scholar
Souza, AT, Dias, E, Nogueira, A, Campos, J, Marques, JC and Martins, I (2013) Population ecology and habitat preferences of juvenile flounder Platichthys flesus (Actinopterygii: Pleuronectidae) in a temperate estuary. Journal of Sea Research 79(Suppl. C), 6069.CrossRefGoogle Scholar
Summers, RW (1979) Life cycle and population ecology of the flounder Platichthys flesus (L.) in the Ythan estuary, Scotland. Journal of Natural History 13, 703723.CrossRefGoogle Scholar
Summers, RW (1980) The diet and feeding behaviour of the flounder Platichthys flesus (L.) in the Ythan estuary, Aberdeenshire, Scotland. Estuarine and Coastal Marine Science 11, 217232.CrossRefGoogle Scholar
Suthers, IM, Fraser, A and Frank, KT (1992) Comparison of lipid, otolith and morphometric condition indices of pelagic juvenile cod Gadus morhua from the Canadian Atlantic. Marine Ecology Progress Series 84, 3140.CrossRefGoogle Scholar
Suzuki, KW, Kasai, A, Isoda, T, Nakayama, K and Tanaka, M (2008) Distinctive stable isotope ratios in important zooplankton species in relation to estuarine salinity gradients: potential tracer of fish migration. Estuarine, Coastal and Shelf Science 78, 541550.CrossRefGoogle Scholar
Tableau, A, Brind'Amour, A, Woillez, M and Le Bris, H (2016) Influence of food availability on the spatial distribution of juvenile fish within soft sediment nursery habitats. Journal of Sea Research 111, 7687.CrossRefGoogle Scholar
Tanaka, Y, Satoh, K, Yamada, H, Takebe, T, Nikaido, H and Shiozawa, S (2008) Assessment of the nutritional status of field-caught larval Pacific bluefin tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared fish. Journal of Experimental Marine Biology and Ecology 354, 5664.CrossRefGoogle Scholar
Teal, LR, de Leeuw, JJ, van der Veer, HW and Rijnsdorp, AD (2008) Effects of climate change on growth of 0-group sole and plaice. Marine Ecology Progress Series 358, 219230.CrossRefGoogle Scholar
van der Veer, HW and Bergman, MJN (1987) Predation by crustaceans on a newly settled 0-group plaice Pleuronectes platessa population in the western Wadden Sea. Marine Ecology Progress Series 35, 203215.CrossRefGoogle Scholar
van der Veer, HW, Bergman, MJN, Dapper, R and Witte, JIJ (1991) Population dynamics of an intertidal 0-group flounder Platichthys flesus population in the western Dutch Wadden Sea. Marine Ecology Progress Series 73, 141148.CrossRefGoogle Scholar
van der Veer, HW, Berghahn, R, Miller, JM and Rijnsdorp, AD (2000) Recruitment in flatfish, with special emphasis on North Atlantic species: progress made by the Flatfish Symposia. ICES Journal of Marine Science 57, 202215.CrossRefGoogle Scholar
van der Veer, HW, Freitas, V, Koot, J, Witte, JIJ and Zuur, AF (2010) Food limitation in epibenthic species in temperate intertidal systems in summer: analysis of 0-group plaice Pleuronectes platessa. Marine Ecology Progress Series 416, 215227.CrossRefGoogle Scholar
van der Veer, HW, Jung, AS, Freitas, V, Philippart, CJM and Witte, JIJ (2016) Possible causes for growth variability and summer growth reduction in juvenile plaice Pleuronectes platessa L. in the western Dutch Wadden Sea. Journal of Sea Research 111, 97106.CrossRefGoogle Scholar
Vander Zanden, MJ, Cabana, G and Rasmussen, JB (1997) Comparing trophic position of freshwater fish calculated using stable nitrogen isotope ratios (δ15N) and literature dietary data. Canadian Journal of Fisheries and Aquatic Sciences 54, 11421158.CrossRefGoogle Scholar
Vasconcelos, RP, Reis-Santos, P, Fonseca, V, Ruano, M, Tanner, S, Costa, MJ and Cabral, HN (2009) Juvenile fish condition in estuarine nurseries along the Portuguese coast. Estuarine, Coastal and Shelf Science 82, 128138.CrossRefGoogle Scholar
Vasconcelos, RP, Reis-Santos, P, Maia, A, Fonseca, V, França, S, Wouters, N, Costa, MJ and Cabral, HN (2010) Nursery use patterns of commercially important marine fish species in estuarine systems along the Portuguese coast. Estuarine, Coastal and Shelf Science 86, 613624.CrossRefGoogle Scholar
Vaslet, A, Phillips, DL, France, CAM, Feller, IC and Baldwin, CC (2015) Trophic behaviour of juvenile reef fishes inhabiting interlinked mangrove–seagrass habitats in offshore mangrove islets. Journal of Fish Biology 87, 256273.CrossRefGoogle ScholarPubMed
Vinagre, C, França, S, Costa, MJ and Cabral, HN (2005) Niche overlap between juvenile flatfishes, Platichthys flesus and Solea solea, in a southern European estuary and adjacent coastal waters. Journal of Applied Ichthyology 21, 114120.CrossRefGoogle Scholar
Vinagre, C, Cabral, H and Costa, MJ (2008) Prey selection by flounder, Platichthys flesus, in the Douro estuary, Portugal. Journal of Applied Ichthyology 24, 238243.CrossRefGoogle Scholar
Vinagre, C, Salgado, J, Cabral, HN and Costa, MJ (2011) Food web structure and habitat connectivity in fish estuarine nurseries – impact of river flow. Estuaries and Coasts 34, 663674.CrossRefGoogle Scholar
Vizzini, S, Sarà, G, Michener, RH and Mazzola, A (2002) The role and contribution of the seagrass Posidonia oceanica (L.) Delile organic matter for secondary consumers as revealed by carbon and nitrogen stable isotope analysis. Acta Oecologica 23, 277285.CrossRefGoogle Scholar
Wirjoatmodjo, S and Pitcher, TJ (1984) Flounders follow the tides to feed: evidence from ultrasonic tracking in an estuary. Estuarine, Coastal and Shelf Science 19, 231241.Google Scholar
Wolanski, E and Elliott, M (2015) Estuarine Ecohydrology: An Introduction. Boston, MA: Elsevier.Google Scholar
Złoch, I and Sapota, M (2010) Trophic interactions between preadult and adult Pomatoschistus minutus and Pomatoschistus microps and young Platichthys flesus occurring in inshore waters of the Gulf of Gdańsk (Southern Baltic). Oceanological and Hydrobiological Studies 39, 3753.CrossRefGoogle Scholar
Figure 0

Fig. 1. The Lima estuary with the location of the sampling stations (1 – lower estuary; 2 and 3 – middle estuary; 4 and 5 – upper estuary).

Figure 1

Fig. 2. Size frequency distribution of the sampled 0-group (N = 22) and 1-group (N = 20) European flounder in the Lima estuary.

Figure 2

Table 1. Number (N) of 0-group and 1-group European flounder sampled in the Lima estuary, mean total length (mm), total weight (g), Fulton K, RNA:DNA and muscle carbon (δ13C) and nitrogen (δ15N) stable isotope signatures (‰)

Figure 3

Fig. 3. Numerical (NI) and weight (WI) indices for stomach contents of (A) 0-group, and (B) 1-group European flounder in the Lima estuary.

Figure 4

Fig. 4. Carbon (δ13C), and nitrogen (δ15N) stable isotopes (‰) of (A) sediment (SOM, upstream and downstream), and water particulate (POM, upstream and downstream) organic matter sources; (B) 0-group and 1-group European flounder, and respective upstream and downstream prey. Trophic enrichment factors were applied to sources (δ13C: ± 2‰, i.e. ±1‰ per trophic level), and to prey (δ13C: ± 1‰, δ15N: ± 3.4‰).

Figure 5

Table 2. Mean carbon stable isotope δ13C (‰) of particulate organic matter (POM) and sediment organic matter (SOM) sources in the upstream and downstream areas of the Lima estuary

Figure 6

Table 3. Mean carbon (δ13C) and nitrogen (δ15N) stable isotope signatures of the main prey groups of European flounder juveniles in the upstream and downstream areas of Lima estuary

Figure 7

Fig. 5. Boxplots of the mixing models estimates of prey contribution to the diet of (A) 0-group and (B) 1-group European flounder. Prey groups divided according to upstream and downstream areas.

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