Habitat use and food sources of European flounder larvae (Platichthys flesus, L. 1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula)

Habitat use and food sources of European flounder larvae (Platichthys flesus, L. 1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula)

Journal Pre-proof Habitat use and food sources of European flounder larvae (Platichthys flesus, L. 1758) across the Minho River estuary salinity gradi...

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Journal Pre-proof Habitat use and food sources of European flounder larvae (Platichthys flesus, L. 1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula) Ester Dias, Ana Gabriela Barros, Joel C. Hoffman, Carlos Antunes, Pedro Morais

PII: DOI: Reference:

S2352-4855(19)30596-1 https://doi.org/10.1016/j.rsma.2020.101196 RSMA 101196

To appear in:

Regional Studies in Marine Science

Received date : 5 August 2019 Revised date : 2 January 2020 Accepted date : 20 February 2020 Please cite this article as: E. Dias, A.G. Barros, J.C. Hoffman et al., Habitat use and food sources of European flounder larvae (Platichthys flesus, L. 1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula). Regional Studies in Marine Science (2020), doi: https://doi.org/10.1016/j.rsma.2020.101196. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier B.V.

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Habitat use and food sources of European flounder larvae (Platichthys flesus, L.

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1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula)

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Ester Dias1*, Ana Gabriela Barros1,2, Joel C. Hoffman3, Carlos Antunes1,4, Pedro Morais5

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Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, 4450-208

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Matosinhos, Portugal.

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Minho, Campos de Gualtar, 4710-057 Braga, Portugal.

CIMAR/CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of

CBMA - Centre of Molecular and Environmental Biology, Department of Biology, University of

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Environmental Protection Agency, 6201 Congdon Blvd, Duluth, Minnesota, 55804, USA.

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Aquamuseu do Rio Minho, Parque do Castelinho s/n, 4920-290 Vila Nova de Cerveira, Portugal.

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CCMAR – Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro,

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Portugal.

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*corresponding author: [email protected]

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Mid-Continent Ecology Division, National Health and Environmental Effects Research Lab, US

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ABSTRACT The European flounder (Platichthys flesus Linnaeus, 1758) exhibits plasticity for several life

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traits throughout its distribution range, including ontogenetic habitat shifts during early life, as well as

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the timing and duration of spawning. Estuaries are preferred as nursery habitat; however, the

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importance of specific salinity zones for larval development is not well-understood. Therefore, we

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aimed to identify the significance of distinct estuarine salinity habitats (i.e., tidal freshwater, brackish)

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along the Minho River estuary (NW-Iberian Peninsula, Europe) for larval development by combining

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field observations with carbon (C) and nitrogen (N) stable isotope analysis. Sampling occurred between

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January 2015 and January 2016 in six sampling stations across the estuarine salinity gradient. A total of

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29 larvae were collected in the Minho River estuary from March till September 2015. Spawning likely

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occurred near the river mouth because the highest abundance of larvae occurred in the brackish estuary.

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Timing for migration towards freshwater was variable with metamorphosis likely occurring in both

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brackish and freshwater habitats. European flounder larvae obtained their diet from the benthic food

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web, indicating that benthic habitat is fundamental for larval development, including prior to

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settlement. This study provides further evidence on the behavioral plasticity of European flounder

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during early life regarding both habitat use and timing of migration towards freshwater habitats.

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Additionally, this study demonstrates the importance of preserving estuarine connectivity for this

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migratory species.

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KEYWORDS: stable isotopes, recruitment, nursery, estuaries.

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1. Introduction Phenotypic plasticity is a trait which enables a species to adapt to distinct ecological conditions

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along its distribution range, as well as to regional interannual variability (Price et al., 2003). The details

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of species-specific plasticity are important for both conservation and population management

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(Schindler et al., 2010; Wilson et al., 2018). The European flounder Platichthys flesus (Linnaeus, 1758)

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is among those species known to exhibit plasticity for several traits related to early life. This species is

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distributed along the European coast, from the White Sea to southern Portugal, along the western

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Mediterranean and eastwards until the Azov Sea (Nielsen, 1986). In estuaries, the European flounder

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uses brackish waters for extended periods (Radforth, 1940; Marchand et al., 2003) and migrates at an

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early stage to freshwater tidal areas (Bos, 1999; Morais et al., 2011; Daverat et al., 2012), possibly

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because reduced salinity triggers metamorphosis (Hutchinson and Hawkins, 2004). However, this

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species shows behavioral plasticity concerning the use of different salinity zones during ontogeny

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(Daverat et al., 2012), as well as the timing and duration of the spawning period (Martinho et al., 2013).

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It was proposed that European flounder may spawn in estuaries, either because of anecdotal reports

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from fishers (Elbe river, Germany- Bos 1999; Minho estuary, NW-Iberian Peninsula), sporadic

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observations made by scientists (pre-metamorphosed larvae were collected in the upper freshwater

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Dordogne River (France) at more than 100 km from the ocean – M. Lepage, IRSTEA, pers. comm.), or

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due to evidence derived from otolith chemistry studies showing that spawning can occur in low salinity

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environments (Morais et al., 2011). Indeed, some studies demonstrate that European flounder life-

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history plasticity is broader than previously acknowledged (Daverat et al., 2011, 2012). In the Minho

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estuary (Portugal), the European flounder were predominantly in brackish habitats during pre-

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metamorphosis and metamorphosis, whereas in the Gironde and Seine estuaries (France) they were

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mostly in coastal and freshwater environments, respectively (Daverat et al., 2012). Also, the age at

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which European flounder migrate into freshwater varies, mostly at age-0 in the Minho and Gironde

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estuaries, and at age-1 in the Seine estuary (Daverat et al., 2012). European flounder demonstrates facultative use of estuarine habitat, either using it as larvae or

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juveniles, or later in life as adults (Daverat et al., 2011; Morais et al., 2011; Daverat et al., 2012). Stable

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isotope ratios can be used to determine the nursery habitat function of estuarine habitats (e.g., salinity

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zones, or benthic versus pelagic zones) for fish larvae because they provide information about their

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basal sources of nutrition (Dias et al., 2017), and can be used to track ingress, settlement, and within-

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estuary movements when larvae move between habitats with isotopically distinct prey (Herzka, 2005;

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Hoffman et al., 2007). Nitrogen stable isotope ratios (δ15N: 15N/14N) indicate the trophic level (Vander

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Zanden et al., 1997), and may reflect regional variation in δ15N baselines (McMahon et al., 2013),

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which can vary with anthropogenic wastewater inputs (McClelland et al., 1997). Carbon stable isotope

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ratios (δ13C: 13C/12C) can distinguish among different autotrophs at the base of the food web, because

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they will differ among primary products with respect to C source and method of C fixation (Smith and

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Epstein, 1971; Fry and Sherr, 1984), and can be useful indicators of estuarine salinity gradients because

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freshwater autotrophs tend to differ significantly from marine autotrophs (Dias et al., 2014, 2016).

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Thus, stable isotope analysis can be used both to identify important trophic pathways supported by

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distinct habitats across the river-coast mixing zone, as well as track along-estuary movements during

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early life. Building upon prior knowledge (Morais et al., 2011), we hypothesize that pre-metamorphic

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larvae must be present in the low estuary but also further upstream, in freshwater habitats. Dias et al.

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(2017) found that European flounder larvae in the lower Minho estuary relied mostly on the benthic

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food web; however, this study analyzed larvae collected during a restricted period (fortnightly during

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one month) and along a limited spatial range (first 4 km of the estuary). Because detritus and the

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benthic food web play an important role in subsidizing the Minho estuary food web (Dias et al., 2016,

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2017), we expect they will contribute to production of European flounder larvae.

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Research on flounder larvae and juveniles present in estuaries has focused on the timing and

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location of estuarine habitats used and the relation to prevailing environmental conditions and transport

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mechanisms (Bos, 1999; Souza et al., 2013; Amorim et al., 2016). Fundamental research regarding the

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duration of reproduction, the timing of estuarine ingress, and larval trophic ecology remain scarce.

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Thus, the goals of this study were to obtain more detailed information about the timing of ingress,

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habitat use, and trophic niche variability during early life of the European flounder. To accomplish

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these goals larvae were sampled over the course of an entire year and throughout the Minho River

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estuary (NW-Iberian Peninsula, Europe), combining field observations with carbon and nitrogen stable

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isotope analysis of both larvae and the most likely basal organic matter (OM) sources to the estuarine

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food web. This estuary was chosen because it is considered a putative nursery area for this species

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(Freitas et al., 2009; Souza et al., 2013), and habitats used during early life were previously determined

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based solely on otolith chemistry analyses (Morais et al., 2011). Moreover, flounder fisheries have high

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social and economic relevance for local communities.

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2. Material and Methods

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2.1. Study area

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The Minho estuary is located in the NW-Iberian Peninsula (Europe; Fig. 1) and covers a total

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area of 23 km2, 9% of which is composed of intertidal areas. It is a mesotidal estuary with tides ranging

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between 0.7 m and 3.7 m (Alves, 1996). The limit of tidal influence is about 40 km inland (Vilas and

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Somoza, 1984), and the uppermost 30 km are tidal freshwater wetlands (Souza et al., 2013; Dias et al.,

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2016). This estuary is partially mixed, but during periods of high floods tends to evolve towards a salt

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wedge estuary (Sousa et al., 2005). The continental platform is narrow in the coastal area adjacent to

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the estuary (Palenzuela et al., 2004), varying between 30 km and 75 km in width, and is influenced by

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the NW-Iberian upwelling system (Alvarez et al., 2008, and references therein).

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2.2. Field sampling Samples were collected bimonthly from January to July 2015 and monthly from August 2015 to

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January 2016, during the high tide of spring tides. European flounder larvae and basal organic matter

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(OM) sources (or proxies) that could support larvae production were collected at six fixed stations that

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represent salinity zones along the estuarine salinity gradient (Fig. 1). Stations 1 and 2 (Coura saltmarsh)

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are located near the river mouth (mesohaline to euhaline, salinity varies with tides); stations 3 and 4 are

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located in the salinity transition zone (polyhaline to oligohaline; salinity varies with tides and river

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discharge) at 7 km and 9 km from the river mouth, respectively; stations 5 and 6 are located in the tidal

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freshwater (TFW; fresh to oligohaline) area at 15 km and 21 km from the river mouth, respectively

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(Souza et al., 2013; Dias et al., 2016).

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The potential basal OM sources for European flounder larvae included particulate organic matter

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(POM), sediment organic matter (SOM), aquatic plants (emergent and submerged), terrestrial plants,

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debris, epilithon, and macroalgae. At each station, surface and bottom water samples (ca. 0.5 m off the

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bottom) were collected using a 2-L Ruttner water sampler to determine the isotopic composition of

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POM (including particulate organic carbon [POC] δ13C, particulate nitrogen [PN] δ15N, and molar

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C/N). The POM samples were pre-filtered with a 150 μm sieve and filtered onto pre-combusted

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(500°C for 2 h) Whatman GF/F filters and kept frozen (-20°C) until analysis. Plants, debris, sediment,

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and drifting macroalgae were hand-collected using gloves. Epilithon samples were scraped with a soft

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brush from submerged stones. The abundance of European flounder larvae is low in Atlantic Iberian

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estuaries (Ramos et al., 2010; Vieira et al., 2015), and therefore to maximize the collection of larvae,

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triplicate sampling tows at each sampling station in each sampling event were performed. Larvae were

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collected with a plankton net (200 µm mesh size, 0.57 m mouth diameter) equipped with a Hydro-Bios

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flowmeter towed near the surface at an average tow speed of 2 knots for 3 minutes. Samples were

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preserved immediately in 70% ethanol. Water temperature and salinity were recorded using a

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multiparametric probe YSI EXO 2 at each sampling station.

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2.3. Laboratory analyses

Filters for POM and epilithon δ13C analyses were fumed with concentrated HCl to remove

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inorganic carbonates and dried at 60°C for 24 h (Lorrain et al., 2003). Sediment δ13C samples were

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rinsed with 10% HCl (also to remove carbonates) and dried at 60°C for 48 h. Filters for POM and

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epilithon δ15N analyses and sediment δ15N samples were dried at 60°C for 24 h and 48h, respectively.

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Macroalgae and vascular plants were cleaned with deionized water, dried (60°C), and ground to a fine

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powder with a mixer mill for stable isotope analysis. Larvae were sorted under a stereomicroscope

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(Leica S8 APO) and measured with Leica Application Suite V4.6 software. Additionally, the

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ontogenetic stage of each larva was assigned based on Ramos et al. (2010; Table 1). Briefly, stage 1

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larvae are newly hatched, with yolk sac and unpigmented eyes; stage 2 larvae have pigmented eyes and

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hypural plates are formed; stage 3 larvae still have bilateral symmetry, but the yolk sac is totally

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absorbed and rays from dorsal and caudal fins are formed; the loss of bilateral symmetry starts at stage

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4; by stage 5 the eye migration is completed. The number of larvae collected was standardized to

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number of larvae 100 m-3. Afterwards, whole larvae were dried at 60°C for stable isotope analysis

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(except September, for which OM sources were not collected).

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Stable isotope ratios were measured using a Thermo Scientific Delta V Advantage IRMS via

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Conflo IV interface (MARINNOVA, University of Porto). The raw data were normalized by three-

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point calibration using the international reference materials IAEA-N-1 (δ15N= +0.4‰), IAEA-NO-3

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(δ15N = +4.7‰), and IAEA-N-2 (δ15N= +20.3‰) for nitrogen isotopic composition, and two-point

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calibration using USGS-40 (δ13C= -26.39‰) and USGS-24 (δ13C= -16.05‰) for carbon isotopic

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composition. Stable isotope ratios are reported in δ notation, δX= (Rsample/Rstandard - 1) × 103, where X is

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the C or N stable isotope, R is the ratio of heavy/light stable isotopes. The δ13C and δ15N are expressed

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in units per mill (‰) relative to Vienna Pee Dee Belemnite and air, respectively. The analytical error,

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the mean standard deviation (SD) of the replicate reference material, was ± 0.1 ‰ for both δ13C and

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δ15N.

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2.4. Data analyses

The relationship between either larva 13C or 15N values and size (total length) was tested to

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check for potential maternal influence on stable isotope ratios. Further, the most likely OM sources

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supporting European flounder larvae production were identified using δ13C and δ15N bi-plots, where

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flounder larvae δ13C and δ15N mean values (after adjusting for trophic fractionation) were compared to

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OM sources δ13C and δ15N mean values. Several fractionation estimates have been reported for flatfish

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species derived from laboratory studies; however, those estimates varied according to the species

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analyzed, life stage, type of food, or water temperature (Witting et al., 2004; Gamboa-Delgado et al.,

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2008). Therefore, we decided to use the estimates from Vander Zanden and Rasmussen (2001) which

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were based on a review of aquatic animal studies: +0.47‰ or +0.94‰ δ13C (+0.47‰ per trophic level),

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and +2.5‰ or +5.9‰ δ15N (+2.5‰ for primary consumers and +3.4‰ for secondary consumers;

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Vander Zanden and Rasmussen, 2001). Small larvae (TL< 6.0 mm) can feed on phytoplankton and

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microzooplankton (Last, 1978). During spring-early summer, larvae collected in the lower estuary

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presented overall low δ15N values when compared to the most likely sources. For that reason, the stable

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isotope values of those larvae were adjusted for one trophic fractionation level, while for the remaining

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scenarios the adjustment was for two trophic levels (Vander Zanden and Rasmussen, 2001). Also,

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European flounder larvae δ13C values were corrected for lipid content based on tissue C/N ratio

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(Hoffman and Sutton, 2010), and δ13C and δ15N values for ethanol preservation (+0.4‰ δ13C, +0.6‰

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δ15N; Feuchtmayer and Grey, 2003).

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We used a two-way Permutational Multivariate Analysis of Variance (PERMANOVA) to test for

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differences in larvae stable isotope ratios between months and stations (fixed factors). PERMANOVA

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tests the simultaneous response of one or more variables to one or more factors in an ANOVA design,

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based on any distance measure, and using permutation methods (Anderson, 2001). In all

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PERMANOVA tests, the significance level was set at 0.05 and using 9999 permutations of residuals

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within a reduced model. When the number of permutations was lower than 150, the Monte Carlo p-

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value was considered (PRIMER v.6.1.6, PRIMER-E, with the PERMANOVA +1.0.1 add-on;

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Anderson et al., 2008).

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The contribution of OM sources to European flounder larvae tissues was quantified with a dual-

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stable isotope mixing model that uses Bayesian inference to solve indeterminate linear mixing

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equations (i.e., for two stable isotope ratios and more than three diet sources). Indeterminate linear

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mixing equations produce a probability distribution that represents the likelihood of a given source to

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contribute to the consumer (Parnell et al., 2010). We used the model Stable Isotope Analysis in R

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(SIAR), which allows each source and the trophic enrichment factor (TEF; or trophic fractionation) to

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be assigned a normal distribution (Parnell et al., 2010). SIAR produces a distribution of feasible

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solutions and estimates credibility intervals (95% CI in this study). In the SIAR mixing model, we

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adjusted the δ13C and δ15N values for one or two trophic levels using the TEF estimates from Vander

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Zanden and Rasmussen (2001) as described above. In middle estuary during winter-early spring and in

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the upper estuary during the two periods, larvae δ13C and δ15N were outside the range of the OM

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sources sampled and for that reason, the stable isotope mixing model analysis was not conducted to

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these larvae. Standard deviation (±SD) will be used as a measure of data dispersion when reporting

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mean values.

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3. Results 9

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3.1. Larvae density and distribution A total of 29 European flounder larvae were collected in the Minho river estuary from March to

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September 2015. Larvae started to ingress into the estuary in March, which coincided with an increase

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in the mean estuarine water temperature (Fig. 2) and salinity values (Fig. 3). The maximum monthly

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mean density occurred at station 1 in May 2015 (5.7 ± 8.0 larvae 100 m-3; Fig. 4). The highest mean

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density values in the estuary were observed during spring and early summer (April 2015: 1.7 ± 2.3

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larvae 100 m-3; June 2015: 1.4 ± 2.7 larvae 100 m-3) (Fig. 4). Station 1 always had the highest density

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of larvae, except in July and September 2015, when they were only present in the samples collected at

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stations 3 and 6, and at station 5, respectively (Fig. 4).

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The total length of European flounder larvae varied between 4.2 mm (station 1, June 2015) and

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9.3 mm (station 1, March 2015). The mean total length varied between 6.7 ± 1.4 mm (station 5) and 8.1

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± 0.5 mm (station 4) (Fig. 5A). The mean total length decreased from March to June 2015, and

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increased again in July 2015 (Fig. 5B). A total of 64% of larvae were at stage 3, 18% at stage 2, 11% at

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stage 4, and 7% at stage 5. No newly hatched larvae (stage 1) were collected, and stage 2 larvae were

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only present in station 1 in April and June 2015. Stage 3 and 4 larvae were found across the estuary,

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whereas stage 5 larvae were only collected in the TFW area at stations 5 (September 2015) and 6

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(March and July 2015).

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3.2 Stable isotope analysis

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The mean larvae δ13C and δ15N values were similar between stations (Pseudo-F= 0.84; P= 0.60);

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however, they differed between sampling periods (Pseudo-F= 5.10; P< 0.05). The δ13C values were ca.

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1.5‰ higher in March and April (mean δ13C: -17.31 ± 0.46 ‰) than in the following months (δ13C: -

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18.80 ± 0.77‰) (Pseudo-F= 8.73; P< 0.05). So, the different months were grouped into two major

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groups, one merging March and April (late winter-early spring), and the other merging data from May, 10

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June, and July (late spring-early summer). There was no relationship between size and δ15N (R2= 0.03;

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p> 0.05) and a significant relationship between size and δ13C values, though the R2 value was low (R2=

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0.2; p< 0.05), suggesting maternal influence was quite low in early stage larvae (data used in the

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regression in Fig. 6).

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Overall, the δ13C and δ15N values of larvae collected in the lower (stations 1 and 2) and middle

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estuary (stations 3 and 4) – after adjusting for trophic fractionation – were intermediate between several

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OM sources measured, indicating reliance on multiple sources (Fig. 7). The δ13C values of larvae

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suggest that OM sources were epilithon, macroalgae, or SAV (Fig. 7). Larvae collected in the middle

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estuary during winter-early spring and in the upper estuary during the two periods were 13C- enriched

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relative to the available OM sources and had values similar to those from larvae collected in the

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downstream stations (Figs. 6, 7).

The dual-stable isotope mixing model (95% CI) indicates that epilithon had the highest

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proportional contribution for European flounder larvae collected in the lower estuary during winter-

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early spring (21%-85%) and spring-early summer (12%-49%) (Table 1). In the middle estuary, during

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spring-early summer, larvae relied mostly on macroalgae detritus (2%-52%), followed by epilithon (up

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to 50%) (Table 1).

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4. Discussion

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A total of 29 European flounder larvae were collected during this study, which is more than those

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collected by other studies using similar sampling strategies (Ramos et al. 2010, Vieira et al. 2015),

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despite being a lower absolute number in comparison to other more abundant estuarine species (Faria et

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al., 2006). A previous study conducted in the lower Minho River estuary registered a low annual mean

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abundance (< 0.02 ind. m-3, Vieira et al., 2015), as well as another conducted in the nearby Lima

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estuary (25 km southward) where the same number of larvae were collected but over a 2-year period

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(Ramos et al., 2010). European flounder ingressed early in life into the Minho River estuary, before settlement (< 2

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months after fertilization; Hutchinson and Hawkins, 2004), and during late winter-early spring. Field

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observations and the results from stable isotope analyses reveal that European flounder migrated to the

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tidal freshwater area (TFW) during larval development, and that the stage at which occurred was

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variable. Stable isotope ratios indicate that European flounder larvae mostly rely on the benthic food

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web. The ingress of European flounder larvae into the Minho estuary and the importance of its

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estuarine habitats for larval development will be discussed below.

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4.1. Larval ingress

European flounder displayed a protracted recruitment period in the Minho River estuary in 2015–

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larvae were collected from March to September – which is longer than anywhere else in their

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distribution range (Grioche et al., 1997; Florin and Höglund, 2008; Martinho et al., 2013), including

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neighboring estuaries (Amorim et al., 2016). We hypothesize that reproduction was not restricted to

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February and March as described for the southern area of the species distribution range (Sobral, 2008).

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However, the spawning peak must have occurred during this period since the highest mean larvae

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density values occurred in April. Spawning likely started in January, and extended at least until July,

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since stage 5 larvae, i.e., post-metamorphic larvae older than two months (Hutchinson and Hawkins,

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2004), were collected in March and September. Also, the smallest larvae and in stage 2 were collected

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in June, supporting the hypothesis of a more extended reproduction period than previously described.

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This suggests that either late spawners of the 2014-2015 spawning season produced late recruits, or that

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European flounder exhibits bet-hedging strategies to cope with environmental uncertainty (Crean and

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Marshall, 2009). If bet-hedging strategies are used by European flounder, then reproductive

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phenological plasticity may increase the population stability and resilience towards environmental and

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climatic variability. Thus, this is a topic deserving further attention. The ingress of fish larvae hatched from pelagic eggs spawned in coastal habitats into estuarine

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nursery areas relies on two main factors: the location of the spawning site, and the ability of larvae to

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swim towards the nursery. Local oceanographic and hydrodynamic conditions may displace the eggs

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and the competent larvae away from nursery areas (Teodósio et al., 2016). In the case of the European

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flounder, the presence of pre-metamorphic and metamorphic larvae in the lower and upper Minho

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River estuary indicates that spawning may occur close to the entrance of the estuary. Four observations

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support this hypothesis. First, the highest density values of larvae were generally observed close to the

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river mouth. Second, the Minho River inflow is markedly high during the peak of European flounder

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spawning (mean January-March 2007-2016: 542 ± 407 m3 s-1; Confederación Hidrográfica del Miño-

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Sil, 2017), so the net transport of eggs and non-competent larvae would be offshore if spawning did not

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occur within the proximities of the estuary. Third, offshore spawning would likely result in the capture

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of only post-metamorphic larvae as observed for two flounder species in the Onslow Bay (North

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Carolina, USA) (Burke et al., 1998). Fourth, an otolith chemistry study on juveniles from the Minho

297

River estuary concluded that most of the specimens analyzed hatched in low salinity habitats (Morais et

298

al., 2011). Spawning close to the estuary entrance minimizes the inherent disadvantages of coastal

299

spawning, i.e., offshore or longshore advection, while it maximizes the chances of recruitment within

300

estuarine nursery areas. Thus, it may be possible that European flounder chooses spawning locations

301

according to local oceanographic and hydrological conditions (Burke et al., 1998). For example,

302

Japanese flounder Paralichthys olivaceus (Paralichthyidae) spawn in the vicinity of the nursery area to

303

maximize the chances of ingress into a non-tidal ecosystem, while in tidal ecosystems ingress will rely

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on active swimming strategies of post-metamorphic larvae (Burke et al., 1998).

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Several factors influence the timing of flounder movements into nursery areas. During this study,

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ingress began during late winter-early spring, specifically in March, when oceanic temperatures are

307

usually low in the southwestern Europe (SeaTemperature). This period coincided with the chlorophyll

308

a spring peak, as observed in the Lima estuary (Barros, 2015; Amorim et al., 2016), which is often

309

associated with high production of zooplankton, which are the main prey of flounder larvae (Last,

310

1978).

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4.2. Estuarine habitats used during larval development

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The presence of pre-metamorphosed European flounder larvae in estuaries is frequent along the

314

species distribution range – Minho estuary (this study), Lima estuary (Portugal; Amorim et al., 2016),

315

Seine estuary (France; Daverat et al., 2012), Ems estuary (Netherlands/Germany; Jagger, 1998), Elbe

316

River (Germany; Bos, 1999). However, there are exceptions. In the Mondego (Portugal) and Gironde

317

(France) estuaries, the adjacent coastal areas were the primary habitats used by European flounder until

318

metamorphosis (Daverat et al., 2012; Primo et al., 2013). Further, previous studies based on otolith

319

chemistry found that flounder larvae migration into freshwater might vary between estuaries as well as

320

the metamorphosing habitat (Daverat et al., 2012).

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The stable isotope data suggest rapid upriver movement from the estuary mouth into tidal

322

freshwater. This is indicated by the similar stable isotope ratios of larvae collected in the TFW area

323

(δ15N= 9.5 ± 0.8‰; δ13C= -17.9 ± 0.7‰) and near the river mouth (δ15N= 9.0 ± 0.6‰; δ13C= -17.5 ±

324

1.2‰). As such, upstream larvae were not in equilibrium with local OM sources because larvae were

325

more

326

from the lower Minho River estuary to the TFW area must have taken less than 20 days, which

327

corresponds to the isotopic turnover period of fish larvae (Hoffman et al., 2007). These metamorphic

328

and post-metamorphic larvae (stage 3 onwards) in the TFW area likely moved up-estuary using active

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C-enriched than the available basal OM sources. This movement of European flounder larvae

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swimming strategies rather than passive transport since flounder larvae can regulate their position in

330

the water column (Grioche et al., 1997, 2000). Selective tidal stream transport (STST), in which larvae

331

move up into the water column during the rising tide and down in the water column during ebb tides

332

(Forward Jr. et al., 1998), was described as the mechanism used by European flounder larvae to

333

accomplish up-estuary movement along the Elbe Estuary (Bos, 1999; Jager, 1998; Jager and Mulder,

334

1999). STST enabled Elbe European flounder larvae to reach an area located at 49 km from the river

335

mouth in less than ten days (Bos, 1999). If Minho European flounder larvae also use STST, then it

336

would be feasible to reach tidal freshwater areas (station 6, 21 km from the river mouth) within the

337

tissue turnover period (~10-20 days; Hoffman et al., 2007), and thereby account for

338

values of European flounder in comparison to local OM sources.

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329

13

C-enriched

Salinity influences larvae behavior and dictates the spatial distribution of juveniles along

340

estuaries (Hutchinson and Hawkins, 2004; Bos and Thiel, 2006; Souza et al., 2013). Indeed, there is an

341

increasing preference for low salinity habitats during the ontogenetic development of European

342

flounder (Bos and Thiel, 2006). Although the reasons for the migration towards low salinity areas are

343

not fully understood, the upstream migration of Elbe European flounder larvae coincided with the

344

increase in plankton concentrations in the limnetic area (Bos, 2000). Also, this migration might

345

decrease the competition for food with marine and anadromous species larvae due to spatial

346

displacement (Thiel, 2000), and with freshwater species larvae owing to temporal mismatch since they

347

usually develop later in the year (Barros, 2015).

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4.3. Estuarine food webs supporting the production of the European flounder larvae

350

We found that the origin of the European flounder larvae diet varied along the Minho River

351

estuary but relied mostly on sources with benthic origin (epilithon and SOM). There are no estimates

352

on the productivity of microphytobenthos (MPB) in the Minho River estuary, but this study along with 15

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others (Dias et al., 2016, 2017) demonstrate that benthic trophic pathways are more important for

354

flatfish larvae and other consumers’ biomass than phytoplankton. Plausibly, reliance on benthic trophic

355

pathways is opportunistic, because European flounder larvae are associated with the benthic

356

environment during their incursion to upstream areas, during ebb tides (Bos, 1999), as well as after

357

metamorphosis and settlement (Schreiber, 2006). In the middle portion of the estuary, larvae relied on

358

epilithon and SOM, but the source with the highest proportional contribution was macroalgae detritus

359

(2%-52%). Although unexpected, similar reliance on macroalgae detritus was previously found for

360

calanoid copepods (i.e., larvae potential prey; Last,1978) in the brackish portion of the estuary at the

361

end of the high river discharge periods (Dias et al., 2016). Unfortunately, it was not possible to estimate

362

OM source contributions for larvae collected in the TFW because their stable isotope values indicate

363

they were not yet equilibrated with OM sources available in that area.

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One critical assumption of the dual stable isotope mixing model was that larvae were feeding

365

directly on the basal OM sources during spring and early summer, instead of primary consumers such

366

as zooplankton (Last, 1978; Dias et al., 2017). As a result, we applied a trophic fractionation

367

corresponding to one trophic level. The larvae collected between May and July 2015 presented a mean

368

TL of 6.3 ± 1.4 mm, with the smallest larvae being collected in June 2015 with a mean TL of 5.5 ± 1.3

369

mm. Previous studies indicate that flatfish larvae can start to feed exogenously during the yolk-sac

370

stage and that small flounder larvae (TL< 6.0 mm) can feed on phytoplankton and microzooplankton

371

(Last, 1978). Nonetheless, given that the major isotopic difference among sources are their 13C values,

372

if the larvae stable isotopes were corrected for two trophic levels, the main conclusions would be

373

similar, although the importance of terrestrial detritus would increase. For the mixing model, it is also

374

assumed that larvae were in isotopic equilibrium with their diet. This assumption was supported by the

375

poor relationship between stable isotope values and size, indicating that maternal influence on the

376

stable isotope values must have been minimal.

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377 378

5. Conclusions The ingress of the European flounder occurred before settlement in the Minho River estuary. Pre-

380

and post-metamorphic larvae were collected between March and September 2015, and stage 2 larvae

381

were collected in April and June 2015 suggesting that the reproductive season may be longer than

382

previously described for the southern distribution area of the European flounder. The larval abundance

383

was highest in the brackish portion of the estuary, decreasing towards the TFW, which confirms that

384

brackish habitats are critical nursery areas; nonetheless, European flounder larvae can arrive to

385

freshwater habitats before metamorphosis is completed, which indicates intra-population plasticity in

386

habitat use. Thus, the present study shows that ecosystem connectivity is vital for this population, since

387

the European flounder relies on the benthic food web during larval development across a mosaic of

388

estuarine habitats. However, the causes and mechanisms responsible for the European flounder

389

plasticity is yet to be unraveled which will ultimately help developing effective management policies

390

along the species distribution range.

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Acknowledgments

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We would like to thank the staff at Aquamuseu do Rio Minho for their collaboration during the

394

fieldwork, to Ana M. Faria for confirming the identification of flounder larvae, to Jacinto Cunha for

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providing the map of the study area, and to an anonymous reviewer for valuable comments on an

396

earlier version of the manuscript. This work was partially supported by the Strategic Funding

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UID/Multi/04423/2019 through national funds provided by Fundação para a Ciência e a Tecnologia

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(FCT, Portugal) and European Regional Development Fund (ERDF), in the framework of the

399

programme PT 2020. ED [SFRH/BPD/104019/2014] was supported by post-doc scholarships financed

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by FCT. The contents of this material do not necessarily reflect the views and policies of the US EPA,

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nor does mention of trade names or commercial products constitute endorsement or recommendation

402

for use.

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Table 1. Proportion of each food source to European flounder Platichthys flesus (Linnaeus, 1758)

555

larvae collected in the lower and middle estuary between March and September 2015 in the Minho

556

River estuary. Food sources included in the model were phytoplankton, particulate organic matter

557

(POM), macroalgae, epilithon, and sediment organic matter (SOM). The upper value indicates the most

558

likely value (mode) and the ranges indicate the 95% Bayesian credibility intervals. Where no value is

559

shown, sources were not included as end-members in the model.

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(*) Values adjusted for one trophic level (Vander Zanden and Rasmussen, 2001).

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ORGANIC MATTER SOURCES POM Macroalgae Epilithon

SEASON

ESTUARY SECTION

Phytoplankton

Late Winter – Early Spring

Lower

17 (0-42)

1 (0-19)

-

67 (21-85)

4 (0-40)

Spring – Early Summer

Lower (*) Middle

29 (1-41) 20 (0-43)

17 (0-38) -

34 (2-52)

31 (12-49) 28 (0-50)

26 (0-43) 26 (0-44)

562

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25

SOM

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Fig. 1 Location of the sampling stations along the Minho River estuary (NW-Portugal, Europe).

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26

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567 568 569

Fig. 2 Surface (closed circles) and bottom (open circles) water temperature values (℃), recorded in the

570

Minho River estuary between January 2015 and January 2016.

571

574 575 576 577 578

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572

579 27

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Fig. 3 Surface and bottom water salinity values recorded in the Minho River estuary between January

582

2015 and January 2016, in the lower (stations 1 (closed circles) and 2 (open circles)), middle (stations 3

583

(closed squares) and 4 (open squares)), and upper estuary (stations 5 (closed triangles) and 6 (open

584

triangles)). Note that the y-axis scale differs between plots.

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Fig. 4 Mean density (± SD) of European flounder Platichthys flesus (Linnaeus, 1758) larvae collected

588

in the Minho River estuary, from station 1 (near the river mouth) up to station 6 (tidal freshwater area),

589

between January 2015 and January 2016.

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29

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Fig. 5 Mean (± SD) total length (TL) of European flounder Platichthys flesus (Linnaeus, 1758) larvae

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grouped by station (S; A) and month (B) collected in the Minho River estuary from station 1 (near the

594

mouth of the river) to station 6 (tidal freshwater) between March and September 2015.

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Fig. 6 Nitrogen and carbon stable isotope ratios (‰) of European flounder Platichthys flesus (Linnaeus,

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1758) larvae as a function of larvae size (total length, mm) collected during late winter-early spring

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(closed symbols) and spring-early summer (open symbols) in the lower (dots), middle (squares), and

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upper (inverted triangles) Minho River estuary.

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Fig. 7 Mean (± SD) δ13C and δ15N values of European flounder Platichthys flesus (Pf) (Linnaeus, 1758)

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larvae not adjusted for trophic fractionation. Potential organic matter (OM) sources include

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phytoplankton (Phyto; Dias et al. 2016), particulate OM (POM), epilithon (Epi), macroalgae (M),

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sediment OM (SOM), and submerged (SAV), emergent (EAV), and terrestrial (Terr) plants. Note that

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y-axis varies among plots.

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Journal Pre-proof 1Highlights 2The European flounder exhibits plasticity for several life traits. 3Flounder showed a protracted reproductive period in the Minho estuary.

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4Pre- and post-metamorphic larvae were collected in freshwater habitats.

5Flounder larvae relied on the estuarine benthic food web during ontogeny.

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6Estuarine connectivity is fundamental to preserve flounder populations.

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Declaration of interests

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xThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Journal Pre-proof Ester Dias: Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Writing- Original Draft, Writing- Review & Editing, Project administration, Funding acquisition Ana G. Barros: Investigation, Writing- Review & Editing Joel C. Hoffman: Writing- Review & Editing, Visualization Carlos Antunes: Investigation, Resources, Writing- Review & Editing, Funding acquisition

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Pedro Morais: Writing- Review & Editing, Visualization