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.
Journal Pre-proof
1
Habitat use and food sources of European flounder larvae (Platichthys flesus, L.
2
1758) across the Minho River estuary salinity gradient (NW Iberian Peninsula)
lP repro of
3
Ester Dias1*, Ana Gabriela Barros1,2, Joel C. Hoffman3, Carlos Antunes1,4, Pedro Morais5
4 5
1
6
Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, 4450-208
7
Matosinhos, Portugal.
8
2
9
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
10
3
11
Environmental Protection Agency, 6201 Congdon Blvd, Duluth, Minnesota, 55804, USA.
12
4
Aquamuseu do Rio Minho, Parque do Castelinho s/n, 4920-290 Vila Nova de Cerveira, Portugal.
13
5
CCMAR – Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro,
14
Portugal.
15
*corresponding author:
[email protected]
17 18 19 20
rna
Jou
16
Mid-Continent Ecology Division, National Health and Environmental Effects Research Lab, US
21 22 1
Journal Pre-proof
23
ABSTRACT The European flounder (Platichthys flesus Linnaeus, 1758) exhibits plasticity for several life
25
traits throughout its distribution range, including ontogenetic habitat shifts during early life, as well as
26
the timing and duration of spawning. Estuaries are preferred as nursery habitat; however, the
27
importance of specific salinity zones for larval development is not well-understood. Therefore, we
28
aimed to identify the significance of distinct estuarine salinity habitats (i.e., tidal freshwater, brackish)
29
along the Minho River estuary (NW-Iberian Peninsula, Europe) for larval development by combining
30
field observations with carbon (C) and nitrogen (N) stable isotope analysis. Sampling occurred between
31
January 2015 and January 2016 in six sampling stations across the estuarine salinity gradient. A total of
32
29 larvae were collected in the Minho River estuary from March till September 2015. Spawning likely
33
occurred near the river mouth because the highest abundance of larvae occurred in the brackish estuary.
34
Timing for migration towards freshwater was variable with metamorphosis likely occurring in both
35
brackish and freshwater habitats. European flounder larvae obtained their diet from the benthic food
36
web, indicating that benthic habitat is fundamental for larval development, including prior to
37
settlement. This study provides further evidence on the behavioral plasticity of European flounder
38
during early life regarding both habitat use and timing of migration towards freshwater habitats.
39
Additionally, this study demonstrates the importance of preserving estuarine connectivity for this
40
migratory species.
42 43
rna
Jou
41
lP repro of
24
KEYWORDS: stable isotopes, recruitment, nursery, estuaries.
2
Journal Pre-proof
44
1. Introduction Phenotypic plasticity is a trait which enables a species to adapt to distinct ecological conditions
46
along its distribution range, as well as to regional interannual variability (Price et al., 2003). The details
47
of species-specific plasticity are important for both conservation and population management
48
(Schindler et al., 2010; Wilson et al., 2018). The European flounder Platichthys flesus (Linnaeus, 1758)
49
is among those species known to exhibit plasticity for several traits related to early life. This species is
50
distributed along the European coast, from the White Sea to southern Portugal, along the western
51
Mediterranean and eastwards until the Azov Sea (Nielsen, 1986). In estuaries, the European flounder
52
uses brackish waters for extended periods (Radforth, 1940; Marchand et al., 2003) and migrates at an
53
early stage to freshwater tidal areas (Bos, 1999; Morais et al., 2011; Daverat et al., 2012), possibly
54
because reduced salinity triggers metamorphosis (Hutchinson and Hawkins, 2004). However, this
55
species shows behavioral plasticity concerning the use of different salinity zones during ontogeny
56
(Daverat et al., 2012), as well as the timing and duration of the spawning period (Martinho et al., 2013).
57
It was proposed that European flounder may spawn in estuaries, either because of anecdotal reports
58
from fishers (Elbe river, Germany- Bos 1999; Minho estuary, NW-Iberian Peninsula), sporadic
59
observations made by scientists (pre-metamorphosed larvae were collected in the upper freshwater
60
Dordogne River (France) at more than 100 km from the ocean – M. Lepage, IRSTEA, pers. comm.), or
61
due to evidence derived from otolith chemistry studies showing that spawning can occur in low salinity
62
environments (Morais et al., 2011). Indeed, some studies demonstrate that European flounder life-
63
history plasticity is broader than previously acknowledged (Daverat et al., 2011, 2012). In the Minho
64
estuary (Portugal), the European flounder were predominantly in brackish habitats during pre-
65
metamorphosis and metamorphosis, whereas in the Gironde and Seine estuaries (France) they were
66
mostly in coastal and freshwater environments, respectively (Daverat et al., 2012). Also, the age at
Jou
rna
lP repro of
45
3
Journal Pre-proof
67
which European flounder migrate into freshwater varies, mostly at age-0 in the Minho and Gironde
68
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
70
juveniles, or later in life as adults (Daverat et al., 2011; Morais et al., 2011; Daverat et al., 2012). Stable
71
isotope ratios can be used to determine the nursery habitat function of estuarine habitats (e.g., salinity
72
zones, or benthic versus pelagic zones) for fish larvae because they provide information about their
73
basal sources of nutrition (Dias et al., 2017), and can be used to track ingress, settlement, and within-
74
estuary movements when larvae move between habitats with isotopically distinct prey (Herzka, 2005;
75
Hoffman et al., 2007). Nitrogen stable isotope ratios (δ15N: 15N/14N) indicate the trophic level (Vander
76
Zanden et al., 1997), and may reflect regional variation in δ15N baselines (McMahon et al., 2013),
77
which can vary with anthropogenic wastewater inputs (McClelland et al., 1997). Carbon stable isotope
78
ratios (δ13C: 13C/12C) can distinguish among different autotrophs at the base of the food web, because
79
they will differ among primary products with respect to C source and method of C fixation (Smith and
80
Epstein, 1971; Fry and Sherr, 1984), and can be useful indicators of estuarine salinity gradients because
81
freshwater autotrophs tend to differ significantly from marine autotrophs (Dias et al., 2014, 2016).
82
Thus, stable isotope analysis can be used both to identify important trophic pathways supported by
83
distinct habitats across the river-coast mixing zone, as well as track along-estuary movements during
84
early life. Building upon prior knowledge (Morais et al., 2011), we hypothesize that pre-metamorphic
85
larvae must be present in the low estuary but also further upstream, in freshwater habitats. Dias et al.
86
(2017) found that European flounder larvae in the lower Minho estuary relied mostly on the benthic
87
food web; however, this study analyzed larvae collected during a restricted period (fortnightly during
88
one month) and along a limited spatial range (first 4 km of the estuary). Because detritus and the
89
benthic food web play an important role in subsidizing the Minho estuary food web (Dias et al., 2016,
90
2017), we expect they will contribute to production of European flounder larvae.
Jou
rna
lP repro of
69
4
Journal Pre-proof
Research on flounder larvae and juveniles present in estuaries has focused on the timing and
92
location of estuarine habitats used and the relation to prevailing environmental conditions and transport
93
mechanisms (Bos, 1999; Souza et al., 2013; Amorim et al., 2016). Fundamental research regarding the
94
duration of reproduction, the timing of estuarine ingress, and larval trophic ecology remain scarce.
95
Thus, the goals of this study were to obtain more detailed information about the timing of ingress,
96
habitat use, and trophic niche variability during early life of the European flounder. To accomplish
97
these goals larvae were sampled over the course of an entire year and throughout the Minho River
98
estuary (NW-Iberian Peninsula, Europe), combining field observations with carbon and nitrogen stable
99
isotope analysis of both larvae and the most likely basal organic matter (OM) sources to the estuarine
100
food web. This estuary was chosen because it is considered a putative nursery area for this species
101
(Freitas et al., 2009; Souza et al., 2013), and habitats used during early life were previously determined
102
based solely on otolith chemistry analyses (Morais et al., 2011). Moreover, flounder fisheries have high
103
social and economic relevance for local communities.
104
2. Material and Methods
106
2.1. Study area
rna
105
lP repro of
91
The Minho estuary is located in the NW-Iberian Peninsula (Europe; Fig. 1) and covers a total
108
area of 23 km2, 9% of which is composed of intertidal areas. It is a mesotidal estuary with tides ranging
109
between 0.7 m and 3.7 m (Alves, 1996). The limit of tidal influence is about 40 km inland (Vilas and
110
Somoza, 1984), and the uppermost 30 km are tidal freshwater wetlands (Souza et al., 2013; Dias et al.,
111
2016). This estuary is partially mixed, but during periods of high floods tends to evolve towards a salt
112
wedge estuary (Sousa et al., 2005). The continental platform is narrow in the coastal area adjacent to
113
the estuary (Palenzuela et al., 2004), varying between 30 km and 75 km in width, and is influenced by
114
the NW-Iberian upwelling system (Alvarez et al., 2008, and references therein).
Jou
107
5
Journal Pre-proof
115 116
2.2. Field sampling Samples were collected bimonthly from January to July 2015 and monthly from August 2015 to
118
January 2016, during the high tide of spring tides. European flounder larvae and basal organic matter
119
(OM) sources (or proxies) that could support larvae production were collected at six fixed stations that
120
represent salinity zones along the estuarine salinity gradient (Fig. 1). Stations 1 and 2 (Coura saltmarsh)
121
are located near the river mouth (mesohaline to euhaline, salinity varies with tides); stations 3 and 4 are
122
located in the salinity transition zone (polyhaline to oligohaline; salinity varies with tides and river
123
discharge) at 7 km and 9 km from the river mouth, respectively; stations 5 and 6 are located in the tidal
124
freshwater (TFW; fresh to oligohaline) area at 15 km and 21 km from the river mouth, respectively
125
(Souza et al., 2013; Dias et al., 2016).
lP repro of
117
The potential basal OM sources for European flounder larvae included particulate organic matter
127
(POM), sediment organic matter (SOM), aquatic plants (emergent and submerged), terrestrial plants,
128
debris, epilithon, and macroalgae. At each station, surface and bottom water samples (ca. 0.5 m off the
129
bottom) were collected using a 2-L Ruttner water sampler to determine the isotopic composition of
130
POM (including particulate organic carbon [POC] δ13C, particulate nitrogen [PN] δ15N, and molar
131
C/N). The POM samples were pre-filtered with a 150 μm sieve and filtered onto pre-combusted
132
(500°C for 2 h) Whatman GF/F filters and kept frozen (-20°C) until analysis. Plants, debris, sediment,
133
and drifting macroalgae were hand-collected using gloves. Epilithon samples were scraped with a soft
134
brush from submerged stones. The abundance of European flounder larvae is low in Atlantic Iberian
135
estuaries (Ramos et al., 2010; Vieira et al., 2015), and therefore to maximize the collection of larvae,
136
triplicate sampling tows at each sampling station in each sampling event were performed. Larvae were
137
collected with a plankton net (200 µm mesh size, 0.57 m mouth diameter) equipped with a Hydro-Bios
138
flowmeter towed near the surface at an average tow speed of 2 knots for 3 minutes. Samples were
Jou
rna
126
6
Journal Pre-proof
139
preserved immediately in 70% ethanol. Water temperature and salinity were recorded using a
140
multiparametric probe YSI EXO 2 at each sampling station.
142
lP repro of
141
2.3. Laboratory analyses
Filters for POM and epilithon δ13C analyses were fumed with concentrated HCl to remove
144
inorganic carbonates and dried at 60°C for 24 h (Lorrain et al., 2003). Sediment δ13C samples were
145
rinsed with 10% HCl (also to remove carbonates) and dried at 60°C for 48 h. Filters for POM and
146
epilithon δ15N analyses and sediment δ15N samples were dried at 60°C for 24 h and 48h, respectively.
147
Macroalgae and vascular plants were cleaned with deionized water, dried (60°C), and ground to a fine
148
powder with a mixer mill for stable isotope analysis. Larvae were sorted under a stereomicroscope
149
(Leica S8 APO) and measured with Leica Application Suite V4.6 software. Additionally, the
150
ontogenetic stage of each larva was assigned based on Ramos et al. (2010; Table 1). Briefly, stage 1
151
larvae are newly hatched, with yolk sac and unpigmented eyes; stage 2 larvae have pigmented eyes and
152
hypural plates are formed; stage 3 larvae still have bilateral symmetry, but the yolk sac is totally
153
absorbed and rays from dorsal and caudal fins are formed; the loss of bilateral symmetry starts at stage
154
4; by stage 5 the eye migration is completed. The number of larvae collected was standardized to
155
number of larvae 100 m-3. Afterwards, whole larvae were dried at 60°C for stable isotope analysis
156
(except September, for which OM sources were not collected).
rna
143
Stable isotope ratios were measured using a Thermo Scientific Delta V Advantage IRMS via
158
Conflo IV interface (MARINNOVA, University of Porto). The raw data were normalized by three-
159
point calibration using the international reference materials IAEA-N-1 (δ15N= +0.4‰), IAEA-NO-3
160
(δ15N = +4.7‰), and IAEA-N-2 (δ15N= +20.3‰) for nitrogen isotopic composition, and two-point
161
calibration using USGS-40 (δ13C= -26.39‰) and USGS-24 (δ13C= -16.05‰) for carbon isotopic
162
composition. Stable isotope ratios are reported in δ notation, δX= (Rsample/Rstandard - 1) × 103, where X is
Jou
157
7
Journal Pre-proof
the C or N stable isotope, R is the ratio of heavy/light stable isotopes. The δ13C and δ15N are expressed
164
in units per mill (‰) relative to Vienna Pee Dee Belemnite and air, respectively. The analytical error,
165
the mean standard deviation (SD) of the replicate reference material, was ± 0.1 ‰ for both δ13C and
166
δ15N.
lP repro of
163
167 168
2.4. Data analyses
The relationship between either larva 13C or 15N values and size (total length) was tested to
170
check for potential maternal influence on stable isotope ratios. Further, the most likely OM sources
171
supporting European flounder larvae production were identified using δ13C and δ15N bi-plots, where
172
flounder larvae δ13C and δ15N mean values (after adjusting for trophic fractionation) were compared to
173
OM sources δ13C and δ15N mean values. Several fractionation estimates have been reported for flatfish
174
species derived from laboratory studies; however, those estimates varied according to the species
175
analyzed, life stage, type of food, or water temperature (Witting et al., 2004; Gamboa-Delgado et al.,
176
2008). Therefore, we decided to use the estimates from Vander Zanden and Rasmussen (2001) which
177
were based on a review of aquatic animal studies: +0.47‰ or +0.94‰ δ13C (+0.47‰ per trophic level),
178
and +2.5‰ or +5.9‰ δ15N (+2.5‰ for primary consumers and +3.4‰ for secondary consumers;
179
Vander Zanden and Rasmussen, 2001). Small larvae (TL< 6.0 mm) can feed on phytoplankton and
180
microzooplankton (Last, 1978). During spring-early summer, larvae collected in the lower estuary
181
presented overall low δ15N values when compared to the most likely sources. For that reason, the stable
182
isotope values of those larvae were adjusted for one trophic fractionation level, while for the remaining
183
scenarios the adjustment was for two trophic levels (Vander Zanden and Rasmussen, 2001). Also,
184
European flounder larvae δ13C values were corrected for lipid content based on tissue C/N ratio
185
(Hoffman and Sutton, 2010), and δ13C and δ15N values for ethanol preservation (+0.4‰ δ13C, +0.6‰
186
δ15N; Feuchtmayer and Grey, 2003).
Jou
rna
169
8
Journal Pre-proof
We used a two-way Permutational Multivariate Analysis of Variance (PERMANOVA) to test for
188
differences in larvae stable isotope ratios between months and stations (fixed factors). PERMANOVA
189
tests the simultaneous response of one or more variables to one or more factors in an ANOVA design,
190
based on any distance measure, and using permutation methods (Anderson, 2001). In all
191
PERMANOVA tests, the significance level was set at 0.05 and using 9999 permutations of residuals
192
within a reduced model. When the number of permutations was lower than 150, the Monte Carlo p-
193
value was considered (PRIMER v.6.1.6, PRIMER-E, with the PERMANOVA +1.0.1 add-on;
194
Anderson et al., 2008).
lP repro of
187
The contribution of OM sources to European flounder larvae tissues was quantified with a dual-
196
stable isotope mixing model that uses Bayesian inference to solve indeterminate linear mixing
197
equations (i.e., for two stable isotope ratios and more than three diet sources). Indeterminate linear
198
mixing equations produce a probability distribution that represents the likelihood of a given source to
199
contribute to the consumer (Parnell et al., 2010). We used the model Stable Isotope Analysis in R
200
(SIAR), which allows each source and the trophic enrichment factor (TEF; or trophic fractionation) to
201
be assigned a normal distribution (Parnell et al., 2010). SIAR produces a distribution of feasible
202
solutions and estimates credibility intervals (95% CI in this study). In the SIAR mixing model, we
203
adjusted the δ13C and δ15N values for one or two trophic levels using the TEF estimates from Vander
204
Zanden and Rasmussen (2001) as described above. In middle estuary during winter-early spring and in
205
the upper estuary during the two periods, larvae δ13C and δ15N were outside the range of the OM
206
sources sampled and for that reason, the stable isotope mixing model analysis was not conducted to
207
these larvae. Standard deviation (±SD) will be used as a measure of data dispersion when reporting
208
mean values.
Jou
rna
195
209 210
3. Results 9
Journal Pre-proof
211
3.1. Larvae density and distribution A total of 29 European flounder larvae were collected in the Minho river estuary from March to
213
September 2015. Larvae started to ingress into the estuary in March, which coincided with an increase
214
in the mean estuarine water temperature (Fig. 2) and salinity values (Fig. 3). The maximum monthly
215
mean density occurred at station 1 in May 2015 (5.7 ± 8.0 larvae 100 m-3; Fig. 4). The highest mean
216
density values in the estuary were observed during spring and early summer (April 2015: 1.7 ± 2.3
217
larvae 100 m-3; June 2015: 1.4 ± 2.7 larvae 100 m-3) (Fig. 4). Station 1 always had the highest density
218
of larvae, except in July and September 2015, when they were only present in the samples collected at
219
stations 3 and 6, and at station 5, respectively (Fig. 4).
lP repro of
212
The total length of European flounder larvae varied between 4.2 mm (station 1, June 2015) and
221
9.3 mm (station 1, March 2015). The mean total length varied between 6.7 ± 1.4 mm (station 5) and 8.1
222
± 0.5 mm (station 4) (Fig. 5A). The mean total length decreased from March to June 2015, and
223
increased again in July 2015 (Fig. 5B). A total of 64% of larvae were at stage 3, 18% at stage 2, 11% at
224
stage 4, and 7% at stage 5. No newly hatched larvae (stage 1) were collected, and stage 2 larvae were
225
only present in station 1 in April and June 2015. Stage 3 and 4 larvae were found across the estuary,
226
whereas stage 5 larvae were only collected in the TFW area at stations 5 (September 2015) and 6
227
(March and July 2015).
229
3.2 Stable isotope analysis
Jou
228
rna
220
230
The mean larvae δ13C and δ15N values were similar between stations (Pseudo-F= 0.84; P= 0.60);
231
however, they differed between sampling periods (Pseudo-F= 5.10; P< 0.05). The δ13C values were ca.
232
1.5‰ higher in March and April (mean δ13C: -17.31 ± 0.46 ‰) than in the following months (δ13C: -
233
18.80 ± 0.77‰) (Pseudo-F= 8.73; P< 0.05). So, the different months were grouped into two major
234
groups, one merging March and April (late winter-early spring), and the other merging data from May, 10
Journal Pre-proof
June, and July (late spring-early summer). There was no relationship between size and δ15N (R2= 0.03;
236
p> 0.05) and a significant relationship between size and δ13C values, though the R2 value was low (R2=
237
0.2; p< 0.05), suggesting maternal influence was quite low in early stage larvae (data used in the
238
regression in Fig. 6).
lP repro of
235
239
Overall, the δ13C and δ15N values of larvae collected in the lower (stations 1 and 2) and middle
240
estuary (stations 3 and 4) – after adjusting for trophic fractionation – were intermediate between several
241
OM sources measured, indicating reliance on multiple sources (Fig. 7). The δ13C values of larvae
242
suggest that OM sources were epilithon, macroalgae, or SAV (Fig. 7). Larvae collected in the middle
243
estuary during winter-early spring and in the upper estuary during the two periods were 13C- enriched
244
relative to the available OM sources and had values similar to those from larvae collected in the
245
downstream stations (Figs. 6, 7).
The dual-stable isotope mixing model (95% CI) indicates that epilithon had the highest
247
proportional contribution for European flounder larvae collected in the lower estuary during winter-
248
early spring (21%-85%) and spring-early summer (12%-49%) (Table 1). In the middle estuary, during
249
spring-early summer, larvae relied mostly on macroalgae detritus (2%-52%), followed by epilithon (up
250
to 50%) (Table 1).
251 252
4. Discussion
rna
246
A total of 29 European flounder larvae were collected during this study, which is more than those
254
collected by other studies using similar sampling strategies (Ramos et al. 2010, Vieira et al. 2015),
255
despite being a lower absolute number in comparison to other more abundant estuarine species (Faria et
256
al., 2006). A previous study conducted in the lower Minho River estuary registered a low annual mean
257
abundance (< 0.02 ind. m-3, Vieira et al., 2015), as well as another conducted in the nearby Lima
Jou
253
11
Journal Pre-proof
258
estuary (25 km southward) where the same number of larvae were collected but over a 2-year period
259
(Ramos et al., 2010). European flounder ingressed early in life into the Minho River estuary, before settlement (< 2
261
months after fertilization; Hutchinson and Hawkins, 2004), and during late winter-early spring. Field
262
observations and the results from stable isotope analyses reveal that European flounder migrated to the
263
tidal freshwater area (TFW) during larval development, and that the stage at which occurred was
264
variable. Stable isotope ratios indicate that European flounder larvae mostly rely on the benthic food
265
web. The ingress of European flounder larvae into the Minho estuary and the importance of its
266
estuarine habitats for larval development will be discussed below.
lP repro of
260
267 268
4.1. Larval ingress
European flounder displayed a protracted recruitment period in the Minho River estuary in 2015–
270
larvae were collected from March to September – which is longer than anywhere else in their
271
distribution range (Grioche et al., 1997; Florin and Höglund, 2008; Martinho et al., 2013), including
272
neighboring estuaries (Amorim et al., 2016). We hypothesize that reproduction was not restricted to
273
February and March as described for the southern area of the species distribution range (Sobral, 2008).
274
However, the spawning peak must have occurred during this period since the highest mean larvae
275
density values occurred in April. Spawning likely started in January, and extended at least until July,
276
since stage 5 larvae, i.e., post-metamorphic larvae older than two months (Hutchinson and Hawkins,
277
2004), were collected in March and September. Also, the smallest larvae and in stage 2 were collected
278
in June, supporting the hypothesis of a more extended reproduction period than previously described.
279
This suggests that either late spawners of the 2014-2015 spawning season produced late recruits, or that
280
European flounder exhibits bet-hedging strategies to cope with environmental uncertainty (Crean and
281
Marshall, 2009). If bet-hedging strategies are used by European flounder, then reproductive
Jou
rna
269
12
Journal Pre-proof
282
phenological plasticity may increase the population stability and resilience towards environmental and
283
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
285
nursery areas relies on two main factors: the location of the spawning site, and the ability of larvae to
286
swim towards the nursery. Local oceanographic and hydrodynamic conditions may displace the eggs
287
and the competent larvae away from nursery areas (Teodósio et al., 2016). In the case of the European
288
flounder, the presence of pre-metamorphic and metamorphic larvae in the lower and upper Minho
289
River estuary indicates that spawning may occur close to the entrance of the estuary. Four observations
290
support this hypothesis. First, the highest density values of larvae were generally observed close to the
291
river mouth. Second, the Minho River inflow is markedly high during the peak of European flounder
292
spawning (mean January-March 2007-2016: 542 ± 407 m3 s-1; Confederación Hidrográfica del Miño-
293
Sil, 2017), so the net transport of eggs and non-competent larvae would be offshore if spawning did not
294
occur within the proximities of the estuary. Third, offshore spawning would likely result in the capture
295
of only post-metamorphic larvae as observed for two flounder species in the Onslow Bay (North
296
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
304
on active swimming strategies of post-metamorphic larvae (Burke et al., 1998).
Jou
rna
lP repro of
284
13
Journal Pre-proof
Several factors influence the timing of flounder movements into nursery areas. During this study,
306
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).
lP repro of
305
311
4.2. Estuarine habitats used during larval development
313
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).
rna
312
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
13
Jou
321
C-enriched than the available basal OM sources. This movement of European flounder larvae
14
Journal Pre-proof
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.
lP repro of
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).
Jou
348
rna
339
349
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
Journal Pre-proof
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.
lP repro of
353
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.
Jou
rna
364
16
Journal Pre-proof
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.
rna
391
lP repro of
379
Acknowledgments
393
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
395
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
397
UID/Multi/04423/2019 through national funds provided by Fundação para a Ciência e a Tecnologia
398
(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
400
by FCT. The contents of this material do not necessarily reflect the views and policies of the US EPA,
Jou
392
17
Journal Pre-proof
401
nor does mention of trade names or commercial products constitute endorsement or recommendation
402
for use.
404 405 406 407 408
lP repro of
403
References
Alvarez, I., Gomez-Gesteira, M., Castro, M., Dias, J.M., 2008. Spatio-temporal evolution of upwelling regime along the western coast of the Iberian Peninsula. J. Geophys. Res. 113, C07020. Alves, A.M., 1996. Causas e processos da dinâmica sedimentar na evolução actual do litoral do Alto Minho. PhD Dissertation, Universidade do Minho.
409
Amara, R., Laffargue, P., Dewarumez, J.M., Maryniak, C., Lagardére, F., Luczac, C., 2001.
410
Feeding ecology and growth of O-group flatfish (sole, dab and plaice) on a nursery ground (Southern
411
bight of the North Sea). J. Fish. Biol. 58, 788–803.
412
Amorim, E., Ramos, S., Elliott, M., Bordalo, A.A., 2016. Immigration and early life stages
413
recruitment of the European flounder (Platichthys flesus) to an estuarine nursery: The influence of
414
environmental factors. J. Sea. Res. 107, 56–66.
417 418 419 420 421 422 423 424
Austral Ecol. 26, 32–46.
rna
416
Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance.
Anderson, M.J., Gorley, R.N., Clarke, R.K., 2008. PERMANOVA + for PRIMER: guide to software and statistical methods. PRIMER-E, Plymouth. Barros, A.G., 2015. Caracterização das fontes de matéria orgânica que suportam a produção de
Jou
415
ictioplâncton no estuário do rio Minho. MSc dissertation, Universidade do Minho. Bos, A.R., 1999. Tidal transport of flounder larvae (Platichthys flesus) in the Elbe River, Germany. Arch. Fish. Mar. Res. 47, 47–60. Bos, A.R., 2000. Aspects of the life history of the European flounder (Pleuronectes flesus L. 1758) in the tidal River Elbe. PhD Dissertation, University of Hamburg. 18
Journal Pre-proof
425 426
Bos, A.R., Thiel, R., 2006. Influence of salinity on the migration of postlarval and juvenile flounder Pleuronectes flesus L. in a gradient experiment. J. Fish Biol. 68, 1411–1420. Burke, J.S., Ueno, M., Tanaka, Y., Walsh, H., Maeda, T., Kinoshita, I., Seikai, T., Hoss, D.E.,
428
Tanaka, M., 1998. The influence of environmental factors on early life history patterns of flounders. J.
429
Sea Res. 40, 19–32.
430 431
lP repro of
427
Crean, A.J., Marshall, D.J., 2009. Coping with environmental uncertainty: dynamic bet hedging as a maternal effect. Philos. T. Roy. Soc. B 364, 1087–1096.
432
Daverat, F., Martin, J., Fablet, R., Pécheyran, C., 2011. Colonisation tactics of three temperate
433
catadromous species, eel Anguilla anguilla, mullet Liza ramada and flounder Platichthys flesus,
434
revealed by Bayesian multielemental otolith microchemistry approach. Ecol. Freshw. Fish 20, 42−51.
435
Daverat, F., Morais, P., Dias, E., Babaluk, J., Martin, J., Eon, M., Fablet, R., Pécheyran, C.,
436
Antunes, C., 2012. Plasticity of European flounder life history patterns discloses alternatives to
437
catadromy. Mar. Ecol.-Prog. Ser. 147, 31–47.
Dias, E., Morais, P., Antunes, C., Hoffman, J.C., 2014. Linking terrestrial and benthic estuarine
439
ecosystems: organic matter sources supporting the high secondary production of a non-indigenous
440
bivalve. Biol. Invasions 16, 2163−2179.
rna
438
Dias, E., Morais, P., Cotter, A.M., Antunes, C., Hoffman, J.C., 2016. Estuarine consumers utilize
442
marine, estuarine and terrestrial organic matter and provide connectivity among these food webs. Mar.
443
Ecol.-Prog. Ser. 554, 21–34.
444 445 446 447
Jou
441
Dias, E., Morais, P., Faria, A.M., Antunes, C., Hoffman, J.C., 2017. Benthic food webs support the production of sympatric flatfish larvae in estuarine nursery habitat. Fish. Oceanogr. 26, 507–512. Faria, A.M., Morais, P., Chícharo, M.A., 2006. Dynamic of the ichthyoplankton in the Guadiana estuary and in the adjacent coastal area (SE-Portugal/SW-Spain). Estuar. Coast. Shelf S. 70, 85-97.
19
Journal Pre-proof
Feuchtmayer, H., Grey, J., 2003. Effect of preparation and preservation procedures on carbon
449
and nitrogen stable isotope determinations from zooplankton. Rapid Commun. Mass Sp. 1, 2605–2610.
450
Florin, A.-B., Höglund, J., 2008. Population structure of flounder (Platichthys flesus) in the
451
lP repro of
448
Baltic Sea: differences among demersal and pelagic spawners. Heredity 101, 27–38.
452
Forward Jr., R.B., Tankersley, R.A., Reinsel, K.A., 1998. Selective tidal stream transport of spot
453
(Leistomus xanthurus Lacepede) and pinfish [Lagodon rhomboides Linnaeus)] larvae: contribution of
454
circatidal rhythms in activity. J. Exp. Mar. Biol. Ecol. 226, 19–32.
455
Freitas, V., Costa-Dias, S., Campos, J., Bio, A., Santos, P., Antunes, C., 2009. Patterns in
456
abundance and distribution of juvenile flounder, Platichthys flesus, in Minho estuary (NW Iberian
457
Peninsula). Aquat. Ecol. 43, 1143–1153.
458 459
Fry, B., Sherr, E.B., 1984. δ13C measurements as indicators of carbon flow in marine and freshwater ecosystems. Contr. Mar. Sci. 27, 15−47.
Gamboa-Delgado, J., Cañavate, J.P., Zerolo, R., Le Vay, L., 2008. Natural carbon stable isotope
461
ratios as indicators of the relative contribution of live and inert diets to growth in larval Senegalese sole
462
(Solea senegalensis). Aquaculture 280, 190-197.
463 464
rna
460
Grioche, A., Koubbi, P., Sautour, B., 1997).Ontogenic migration of Pleuronectes flesus larvae in the eastern English Channel. J. Fish Biol, 51, 385–396. Grioche, A., Harlay, X., Koubbi, P., Lago, L.F., 2000. Vertical migrations of fish larvae:
466
Eulerian and Lagrangian observations in the Eastern English Channel. J. Plankton Res. 22, 1813–1828.
467
Herzka, S.Z., 2005. Assessing connectivity of estuarine fishes based on stable isotope ratio
468 469 470
Jou
465
analysis. Estuar. Coast. Shelf S. 64, 58–69. Hoffman, J.C., Bronk, D.A,, Olney, J.E., 2007. Tracking nursery habitat use by young American shad in the York River estuary, Virginia, using stable isotopes. T. Am. Fish Soc. 136, 1285–1297.
20
Journal Pre-proof
472 473 474 475 476 477 478 479 480
Hoffman, J.C., Sutton, T.T, 2010. Lipid correction for carbon stable isotope analysis of deep-sea fishes. Deep Sea Res Pt. I 57, 956–964. Hutchinson, S., Hawkins, L.E., 2004. The relationship between temperature and the size and age
lP repro of
471
of larvae and peri-metamorphic stages of Pleuronectus flesus. J. Fish Biol. 65, 445–459.
Jager, Z., 1998. Accumulation of flounder larvae (Platichthys flesus L.) in the Dollard (Ems estuary, Wadden Sea). J. Sea Res. 40, 43–57.
Jager, Z., Mulder, H.P.J., 1999. Transport velocity of flounder larvae (Platichthys flesus L.) in the Dollard (Ems Estuary). Estuar. Coast. Shelf S. 49, 327–346.
Last, J.M., 1978 The food of four species of Pleuronectiform larvae in the eastern English Channel and southern North Sea. Mar. Biol. 45, 359–368.
481
Lorrain, A., Savoye, N., Chauvaud, L., Paulet, Y.-M., Naulet, N., 2003. Decarbonation and
482
preservation method for the analysis of organic C and N contents and stable isotope ratios of low-
483
carbonated suspended particulate material. Anal. Chim. Acta 49, 125-133.
Marchand, J., Tanguy, A., Laroche, J., Quiniou, L., Moraga, D., 2003. Responses of European
485
flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast
486
of France. Mar. Ecol.-Prog. Ser. 260, 273–284.
487 488
rna
484
Martinho, F., van der Veer, H.W., Cabral, H., Pardal, M.A.,(2013. Juvenile nursery colonization patterns for the European flounder (Platichthys flesus): A latitudinal approach. J. Sea Res. 84, 61–69. McClelland, J.W., Valiela, I., Michener, R.H., 1997. Nitrogen-stable isotope signatures in
490
estuarine food webs: a record of increasing urbanization in coastal watersheds. Limnol. Oceanogr. 42,
491
930–937.
492 493
Jou
489
McMahon, K., Hamady, L.L., Thorrold, S.R., 2013. A review of ecogeochemistry approaches to estimating movements of marine animals. Limnol. Oceanogr. 58, 697−714.
21
Journal Pre-proof
Morais, P., Dias, E., Babaluk, J., Antunes, C., 2011. The migration patterns of the European
495
flounder Platichthys flesus (Linnaeus, 1758) (Pleuronectidae, Pisces) at the southern limit of its
496
distribution range: Ecological implications and fishery management. J. Sea Res. 65, 235–246.
lP repro of
494
497
Nicolas, D., Lobry, J., Lepage, M., Sautour, B., Le Pape, O., Cabral, H., Uriarte, A., Boët,
498
P.,2009. Fish under influence: A macroecological analysis of relations between fish species richness
499
and environmental gradients among European tidal estuaries. Estuar. Coast. Shelf S. 86, 137–147
500
Nielsen, J.G., 1986. Pleuronectidae. in: Whitehead, P.J.P,, Bauchot, M.-L., Hureau, J.-C.,
501
Nielsen, J., Tortonese, E. (Eds.), Fishes of the North-eastern Atlantic and the Mediterranean. UNESCO,
502
Paris, pp. 1299–1307.
504 505 506 507 508
Palenzuela, J.M.T., Iglesias, G.M., Vilas, L.G., 2004. Pelagic fisheries study using GIS and Remote Sensing imagery in Galicia (Spain). ICES CM 2004, 5.
Parnell, A.C., Inger, R., Bearhop, S., Jackson, A.L., 2010. Source partitioning using stable isotopes: coping with too much variation. PLoS One 5, e9672.
Price, T.D., Qvarnström, A., Irwin, D.E., 2003. The role of phenotypic plasticity in driving genetic evolution. P. Roy. Soc. B 270, 1433–1440.
rna
503
509
Primo, A.L., Azeiteiro, U.M., Marques, S.C., Martinho, F., Baptista, J., Pardal, M.A., 2013.
510
Colonization and nursery habitat use patterns of larval and juvenile flatfish species in a small temperate
511
estuary. J. Sea Res. 76, 126–134.
513 514 515 516 517
Radforth, I., 1940. The food of the grayling (Thymallus thymallus), flounder (Platichthys flesus),
Jou
512
roach (Rutilus rutilus) and Gudgeon (Gobio fluviatilis). J. Anim. Ecol. 9, 302–318. Ramos, S., Ré, P., Bordalo, A.A., 2010. Recruitment of flatfish species to an estuarine nursery habitat (Lima estuary, NW Iberian Peninsula). J. Sea Res. 64, 473–486. Schindler, D.E., Hilborn, R., Chasco, B., Boatright, C.P., Quinn, T.P., Rogers, L.A., Webster, M.S., 2010. Population diversity and the portfolio effect in an exploited species. Nature 465, 609-612 22
Journal Pre-proof
519 520 521 522 523
Schreiber, A.M., 2006. Asymmetric craniofacial remodeling and lateralized behavior in larval flatfish. J. Exp. Biol. 209, 610-621. SeaTemperature. World Sea Temperatures. https://www.seatemperature.org/ (accessed 12 December 2019)
lP repro of
518
Smith, B.N., Epstein, S., 1971. Two categories of 13C/12C ratios for higher plants. Plant Physiol. 47, 380−384.
524
Sobral, M.P., 2008. Aspectos relativos à biologia reprodutiva da solha, Platichthys flesus
525
(Linnaeus, 1758), da Ria de Aveiro e litoral adjacente. Relatórios Científicos e Técnicos IPIMAR.
526
Technical Report, 44, pp. 1–31.
527 528
Sousa, R., Guilhermino, L., Antunes, C., 2005. Molluscan fauna in the freshwater tidal area of the River Minho estuary, NW of Iberian Peninsula. Ann. Limnol. – Int. J. Lim. 41, 141–147.
529
Souza, A., Dias, E., Nogueira, A., Campos, J., Marques, J.C., Martins, I., 2013. Population
530
ecology and habitat preferences of juvenile flounder Platichthys flesus (Actinopterygii: Pleuronectidae)
531
in a temperate estuary. J. Sea Res. 79, 60–69.
Teodósio, M.A., Paris, C.B., Wolansk,i E., Morais, P., 2016. Biophysical processes leading to
533
the ingress of temperate fish larvae into estuarine nursery areas: A review. Estuar. Coast. Shelf S. 183,
534
187–202.
536
Thiel, R., 2000. Spatial gradients of food consumption and production of juvenile fish in the lower River Elbe. Arch. Hydrobiol. Supplement. 135, 441–462.
Jou
535
rna
532
537
Vander Zanden, M.J., Cabana, G., Rasmussen, J.B., 1997. Comparing trophic position of
538
freshwater fish calculated using stable nitrogen isotope ratios (δ15N) and literature dietary data. Can. J.
539
Fish. Aquat. Sci. 54, 1142-1158.
540 541
Vander Zanden, M.J., Rasmussen, J.B., 2001. Variation in δ15N and δ13C trophic fractionation: implications for aquatic food web studies. Limnol, Oceanogr. 46, 2061–2066. 23
Journal Pre-proof
Vieira, L.R., Guilhermino, L., Morgado, F., 2015. Zooplankton structure and dynamics in two
543
estuaries from the Atlantic coast in relation to multi-stressors exposure. Estuar. Coast. Shelf S. 167,
544
347-367.
545 546
lP repro of
542
Vilas, F., Somoza, L.,1984. El estuario del rio Miño: observaciones previas de su dinâmica. Thalassas, 2, 87-92.
547
Wilson, K.L., De Gisi, J., Cahill, C.L., Barker, O.E., Post, J.R., 2019. Life-history variation
548
along environmental and harvest clines of a northern freshwater fish: Plasticity and adaptation. J.
549
Anim. Ecol. 88, 717-733.
550
Witting, D.A., Chambers, R.C., Bosley, K.L., Wainright, S.C., 2004. Experimental evaluation of
551
ontogenetic diet transitions in summer flounder (Paralichthys dentatus), using stable isotopes as diet
552
tracers. Can. J. Fish. Aquat. Sci. 61, 2069–2084.
Jou
rna
553
24
Journal Pre-proof
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.
560
(*) Values adjusted for one trophic level (Vander Zanden and Rasmussen, 2001).
561
lP repro of
554
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
Jou
rna
563
25
SOM
lP repro of
Journal Pre-proof
564 565
Fig. 1 Location of the sampling stations along the Minho River estuary (NW-Portugal, Europe).
Jou
rna
566
26
lP repro of
Journal Pre-proof
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
Jou
573
rna
572
579 27
Jou
580
rna
lP repro of
Journal Pre-proof
581
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.
585 28
lP repro of
Journal Pre-proof
586 587
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.
Jou
rna
590
29
lP repro of
Journal Pre-proof
rna
591
Fig. 5 Mean (± SD) total length (TL) of European flounder Platichthys flesus (Linnaeus, 1758) larvae
593
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.
595
Jou
592
30
lP repro of
Journal Pre-proof
596
Fig. 6 Nitrogen and carbon stable isotope ratios (‰) of European flounder Platichthys flesus (Linnaeus,
598
1758) larvae as a function of larvae size (total length, mm) collected during late winter-early spring
599
(closed symbols) and spring-early summer (open symbols) in the lower (dots), middle (squares), and
600
upper (inverted triangles) Minho River estuary.
Jou
rna
597
31
601
rna
lP repro of
Journal Pre-proof
Fig. 7 Mean (± SD) δ13C and δ15N values of European flounder Platichthys flesus (Pf) (Linnaeus, 1758)
603
larvae not adjusted for trophic fractionation. Potential organic matter (OM) sources include
604
phytoplankton (Phyto; Dias et al. 2016), particulate OM (POM), epilithon (Epi), macroalgae (M),
605
sediment OM (SOM), and submerged (SAV), emergent (EAV), and terrestrial (Terr) plants. Note that
606
y-axis varies among plots.
Jou
602
607 32
Journal Pre-proof 1Highlights 2The European flounder exhibits plasticity for several life traits. 3Flounder showed a protracted reproductive period in the Minho estuary.
lP repro of
4Pre- and post-metamorphic larvae were collected in freshwater habitats.
5Flounder larvae relied on the estuarine benthic food web during ontogeny.
Jou
rna
6Estuarine connectivity is fundamental to preserve flounder populations.
Journal Pre-proof
Declaration of interests
lP repro of
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.
Jou
rna
☐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
Jou
rna
lP repro of
Pedro Morais: Writing- Review & Editing, Visualization