# Does reef structure affect oyster food resources? A stable isotope assessment

Brittany A Blomberg, Benoit Lebreton, Terence A. Palmer, Gaël Guillou, Jennifer Beseres Pollack, Paul A. Montagna

► To cite this version:

Brittany A Blomberg, Benoit Lebreton, Terence A. Palmer, Gaël Guillou, Jennifer Beseres Pollack, et al.. Does reef structure affect oyster food resources? A stable isotope assessment. Marine Environmental Research, 2017, 127, pp.32 - 40. 10.1016/j.marenvres.2017.03.003. hal-01661225

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1 **Title:** Does reef structure affect oyster food resources? A stable isotope assessment.

2

3 **Authors:** Blomberg, Brittany N.ª¹, Lebreton, Benoitᵇ; Palmer, Terence A.ª, Guillou, Gaëlᵇ;

4 Beseres Pollack, Jenniferᶜ; Montagna, Paul A.ª

5

6 **Affiliations:**

7 a Harte Research Institute for Gulf of Mexico Studies, Texas A&M University – Corpus Christi, 6300 Ocean Drive, Unit 5869, Corpus Christi, TX 78412-5869, USA.

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10 b Institut du Littoral et de l'Environnement, Université de La Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle, France.

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12 c Department of Life Sciences, Texas A&M University – Corpus Christi, 6300 Ocean Drive, Unit 5800, Corpus Christi, TX 78412-5800, USA.

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14 ¹Current Affiliation: Dauphin Island Sea Lab, 101 Bienville Boulevard, Dauphin Island, AL 36528, USA.

15

16 **E-mail addresses:**

20 BNB: bblomberg@disl.org

21 BL: benoit.lebreton@univ-lr.fr

22 TAP: terry.palmer@tamucc.edu

23 GG: gguill01@univ-lr.fr

24 JBP: Jennifer.pollack@tamucc.edu

25 PAM: paul.montagna@tamucc.edu

26

27 * Corresponding author: Brittany N. Blomberg

28 Mailing address: 101 Bienville Boulevard

29 Dauphin Island, AL 36528, USA

30 E-mail: bblomberg@disl.org

31 Telephone: +1 251 861 2141 x7535

32 Fax: +1 251 861 4646

33

34 **Running title:** Reef structure and oyster food resources.

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36
As ecosystem engineers, oysters create and maintain structured habitat and can influence trophodynamics and benthic-pelagic coupling in the surrounding landscape. The physical reef structure and associated biotic parameters can affect the availability of food resources for oysters. Oysters and potential composite food sources — suspended particulate organic matter (SPOM) and surface sediment organic matter (SSOM) — were assessed using a dual stable isotope (δ¹³C, δ¹⁵N) approach at three reef types (natural, restored, and unconsolidated) seasonally for two years to determine if changes in physical and/or biotic parameters affected the relative availability and/or use of food resources by oysters. SPOM was more depleted in ¹³C (-24.2 ± 0.6 ‰, mean ± SD) than SSOM (-21.2 ± 0.8 ‰). SPOM composition is likely dominated by autochthonous phytoplankton production, while SSOM includes trapped phytoplankton and benthic microalgae. SSOM was used by oysters in increasing proportions relative to SPOM over time at all reef types. This temporal trend is likely due to increased oyster biomass over time, promoting enhanced microphytobenthos growth through feedback effects related to oyster biodeposits. Structural differences between reef types observed in this study had no effect on food resource availability and use by oysters, indicating strong benthо-pelagic coupling likely due to shallow depths as well as strong and consistent winds. This study provides insights for restoration of oyster reefs as it highlights that food resources used by oysters remain similar among reef types despite changes in abiotic and biotic parameters among habitats and over time.

food source; oyster reef; benthо-pelagic coupling; stable isotopes; suspended particulate organic matter; sediment organic matter; Gulf of Mexico
## 1. Introduction

Eastern oysters, *Crassostrea virginica*, are ecosystem engineers that create and maintain structured biogenic habitat in estuarine areas and provide resources for other organisms. Oyster reefs serve as nursery grounds for a number of commercially and ecologically important species by enhancing survival and abundance (Beck et al. 2001). Oyster reefs substantially augment production (>200 g m⁻² y⁻¹) for associated fish and mobile crustaceans (Peterson et al. 2003, zu Ermgassen et al. 2015). Oysters help regulate nutrient dynamics and mediate benthic-pelagic coupling through their suspension-feeding activities (Beseres Pollack et al. 2013, Dame et al. 1984, Newell 2004). Oysters filter large amounts of particulate matter from the water column and excrete particles as biodeposits, thereby transferring organic matter from the water column to the benthos (Dame et al. 1984, Dame 2012, Newell 2004, Prins et al. 1998). Moreover, the three-dimensional structure of oyster reefs provides a large range of microhabitats, increasing the diversity of organisms and supporting complex trophic webs (Bateman and Bishop 2017, Grabowski and Peterson 2007, Summerhayes et al. 2009).

Oysters can rely on a large diversity of food resources, whose availability relates to both temporal and spatial variation. Food resources present in estuarine water columns can include phytoplankton, detritus, bacteria, and resuspended materials including benthic microalgae (Cranford et al. 2005, Dame 2012, Grant et al. 1997, Leal et al. 2008, Riera and Richard 1996). The composition of suspended particulate organic matter can vary spatially with relative location within an estuary (i.e., with relative influences of riverine and oceanic inputs; Leal et al. 2008, Quan et al. 2012, Riera and Richard 1996) and with relative proximity to other habitats (e.g., salt marshes, tidal flats; Kang et al. 2003, Quan et al. 2012, Riera and Richard 1996, Winemiller et al. 2007). Availability of food resources also varies temporally, with daily, seasonal and perhaps
longer-term climatic cycles (Cranford and Hill 1999, Grant et al. 1997, MacIntyre and Cullen 1996, Prins et al. 1998, Reyna et al. 2017). Tidal currents, wave action, high winds and human activities can cause resuspension of bottom sediments and organic matter, further influencing food resource composition available to suspension-feeders at varying spatial and temporal scales (Grant et al. 1997, Miller et al. 1996, Prins et al. 1998). Additionally, oysters can promote the growth of microphytobenthos through their biodeposits, effectively transferring nutrients to the sediments where they can fuel microalgae growth (Hargrave et al. 2008, Haven and Morales-Alamo 1966, Leal et al. 2008, Prins et al. 1998).

The physical structure of the habitat (e.g. sediment grain size, vertical relief) and the biotic parameters (e.g., oyster density, biomass) themselves can affect the availability of food resources for oysters (Dame et al. 1984), and therefore the production of oyster reefs (Soniat et al. 2004). Indeed, resuspension of sediment and organic matter is higher in open, bare habitats or where sediment grain size is small—increasing the availability of benthic food resources for oysters—than in enclosed or structured habitats or coarse sediment (Grant et al. 1997, Sundbäck 1984). However, extraordinarily high sediment resuspension can also have negative effects on suspension-feeders. High turbidity caused by resuspended sediments can decrease filtration ability and dilute food concentrations (Dame 2012, Grant et al. 1990, Newell and Langdon 1996). Higher biomass of oysters, leading to a higher three-dimensional complexity, may decrease water flow speed over the reef and increase deposition of particles from the water column (Colden et al. 2016).

The vertical relief of reefs may also influence the availability of sediment food sources to oysters. Because large amounts of suspended sediment can contribute to the burial of oyster reefs, approaches for reef restoration have moved to constructing higher vertical relief reefs to
prevent sedimentation and burial (Bartol et al. 1999, Grabowski 2004, Lenihan 1999, Powers et al. 2009, Schulte et al. 2009). However, high vertical relief could subsequently alter the degree of benthic-pelagic coupling and flow of organic matter through reef ecosystems (Fréchette et al. 1989, Quan et al. 2012). Thus, as movement continues toward designing and building higher vertical relief reefs, it is important to understand how the relative availability of different food sources may change with reef height.

The aim of this study is to compare the food resources used by oysters in several oyster reef types to determine if changes in physical and/or biotic parameters affect (1) the availability of potential food sources to oysters, and (2) the use of pelagic vs. benthic food sources by oysters. We examined the relative contributions of water column and benthic food resources to subtidal oysters (*Crassostrea virginica* Gmelin) in a shallow Texas estuary using a dual stable isotope (δ¹³C, δ¹⁵N) approach. Analysis of stable isotope composition, particularly carbon isotope composition, can be used to elucidate the different food resources utilized by consumers in natural settings (Fry and Sherr 1984, Fry 2006, Peterson 1999). Three reef types with different characteristics were compared: (1) a natural reef, (2) a restored reef, and (3) an unconsolidated reef. Field sampling was conducted over a two year period to examine how the use of food resources by oysters may differ among these reef types depending on their structure, as well as during development of the restored reef.

## 2. Materials and methods

### 2.1 STUDY AREA AND EXPERIMENTAL DESIGN

The study is a 2-way design, where the first independent variable compares three reef types (natural, restored, unconsolidated), and the second independent variable compares response
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over six time periods. The dependent variables are C and N stable isotope compositions in potential water column and sediment composite food resources and oysters.

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The Mission-Aransas Estuary is a bar-built system in the Texas Coastal Bend (Fig. 1a).

The estuary occupies approximately 463 km² and is composed of several shallow (average 2 m depth) bays (Armstrong 1987). The area has a semi-arid, subtropical climate with infrequent rain events. Evaporation typically exceeds precipitation (62.7 cm year⁻¹ net water loss) (Armstrong 1987). Two rivers feed the system: the Mission River flows through Mission Bay into Copano Bay and the Aransas River flows directly into Copano Bay. Average freshwater inflow to the estuary is low (< 500 million m³ year⁻¹) (Armstrong 1987, Montagna et al. 1996). The estuary is microtidal (0.15 m tidal range) and water movement is predominantly wind-influenced (Evans and Morehead Palmer 2012). Mixing with the Gulf of Mexico occurs via a tidal inlet at the southern end of the system. High winds characteristic of the region maintain well-mixed conditions throughout the system (Evans and Morehead Palmer 2012). Average water residence time is 360 days, but can be as long as three years (Beseres Pollack et al. 2011, Montagna et al. 1996). Oyster reefs are common in the estuary (Fig. 1a). Reefs are primarily subtidal and are more prominent in areas of low to moderate salinity.

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The study was conducted in Copano Bay (Fig. 1b; 28.135 °N, 97.053 °W), where average depth is 1.1 m. Oysters and potential composite food resources — suspended particulate organic matter (SPOM) and surface sediment organic matter (SSOM) — were sampled at three reef types, which had the following characteristics (see also supplementary material 1):

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- Natural reef: Low vertical relief (< 0.1 m), with densities (mean ± standard deviation) of adult and spat oysters of 196 ± 134 and 859 ± 922 individuals.m⁻², respectively.
150 - Unconsolidated reef: Dense shell hash and mud with scattered oysters (97 ± 35 adult
151 individuals.m⁻², 652 ± 912 spat individuals.m⁻²). No vertical relief (0 m; oysters directly on the
152 sediment).

153 - Restored reef: Eight higher vertical relief (0.3 m) reef mounds (600 m² each)
154 constructed in summer 2011 with a crushed concrete base and reclaimed oyster shell veneer.
155 Oyster densities were 280 ± 164 and 1462 ± 1595 individuals.m⁻² for adults and for spat,
156 respectively.

157 Sampling commenced in February 2012 (approximately six months post-reef
158 construction) and continued seasonally through September 2013 for a total of six sampling
159 periods (February 2012, June 2012, September 2012, March 2013, June 2013 and September
160 2013).

161 2.2 CONTINUOUS ENVIRONMENTAL DATA

162 Salinity, chlorophyll-a (chl-a, µg L⁻¹) and wind speed (m s⁻¹) were collected near the
163 study site (Copano Bay East station; 28.132 °N, 97.034 °W) as part of the National Estuarine
164 Research Reserve System monitoring program (NERRS 2014). All data were collected for the
165 entire study period, starting in July 2011 when reef construction began and continuing until
166 October 2013 when the study was complete.

167 2.3 SAMPLING AND PREPARATION OF WATER CHLOROPHYLL-A AND COMPOSITE FOOD SOURCE
168 SAMPLES

169 Bottom water samples were collected just above the sediment-water interface at each reef
170 type using a horizontal van Dorn water sampler for quantification of chl-a and stable isotope
171 analysis (δ¹³C, δ¹⁵N) of SPOM. Water was filtered on Whatman GF/F glass fiber filters (0.7 µm
172 porosity) to collect SPOM for chl-a analysis. Filters were stored at -20 °C until analysis. SPOM
samples for stable isotope analyses were sieved on a 300-µm screen to eliminate large detrital particles and zooplankton, and then filtered on two precombusted Whatman GF/F glass fiber filters (0.7 µm porosity). Filters were frozen at -20 °C and freeze-dried. Carbonates were removed from filters for δ¹³C and %C analyses by contact with HCl fumes in a vacuum-enclosed system. Untreated freeze-dried filters were used for δ¹⁵N and %N analyses.

Three cylindrical sediment cores (37.4 cm²) were collected by divers from each reef type for stable isotope analysis (δ¹³C, δ¹⁵N) of SSOM. Cores were collected and handled with minimal disturbance and the top 2 cm were sliced and stored at -20 °C until processing. Samples were thawed and sieved wet on a 500 µm mesh screen to eliminate macrofauna, shell pieces and large detrital particles. Sieved sediment was freeze-dried and ground using a mortar and pestle. Carbonates were removed from sediment for δ¹³C and %C analyses using 2 mol L⁻¹ HCl. HCl was added drop-by-drop until cessation of bubbling. Samples were then dried at 60 °C using a dry block heater under air flow. Dried samples were re-homogenized into ultrapure water using an ultrasonic bath, freeze-dried and ground again. Untreated ground sediment was used for δ¹⁵N and %N analyses.

## 2.4 SAMPLING AND PREPARATION OF OYSTERS

Oysters were collected from each reef type by divers and stored on ice for transport to the laboratory. They were cleaned of epibionts and kept alive for up to 36 h in filtered seawater to allow for gut content evacuation (Dubois et al. 2007). Oysters were frozen at -20 °C and then dissected to collect digestive gland material. We aimed to collect three spat oyster (≤ 25 mm shell height) samples and three adult oyster (> 25 mm) samples from each reef type during each sampling event. Spat oysters were pooled in groups (2-4 individuals) when possible to obtain enough material for stable isotope analysis. Low oyster density in the unconsolidated reef often
limited the number of oysters collected. In total, 52 adult oysters and 44 spat groups (111 individual spat) were analyzed, ranging in size from 25.4 to 76.8 mm (47.2 ± 13.4 mm, mean ± standard deviation (SD)) and from 11.5 to 25.0 mm (18.4 ± 3.4 mm), respectively.

Digestive gland samples were freeze-dried and ground to a homogenous powder.

Digestive glands contain high amounts of lipids, which are depleted in ¹³C relative to other tissues due to the different biochemical pathways involved in their synthesis (DeNiro and Epstein 1977). As a result, the δ¹³C value of a raw sample does not only reflect the food resources of a consumer. Thus, lipids were extracted from samples for δ¹³C and %C analyses using two successive extractions with cyclohexane. Samples were then dried at 45 °C and ground again. Untreated ground samples were used for δ¹⁵N and %N analyses.

## 2.5 CHLOROPHYLL-A AND STABLE ISOTOPE RATIO MEASUREMENTS

Chl-*a* was extracted from filters overnight using a non-acidification technique and read on a Turner Trilogy fluorometer (Turner Designs, Sunnyvale, USA) (Welschmeyer 1994, EPA method 445.0). Elemental and stable isotope compositions were determined using an elemental analyzer (Flash EA 1112, Thermo Scientific, Milan, Italy) coupled to an isotope ratio mass spectrometer (Delta V Advantage with a Conflo IV interface, Thermo Scientific, Bremen, Germany). Analyses were conducted at the Littoral, Environment and Societies Joint Research Unit stable isotope facility (University of La Rochelle, France). Results are expressed in δ notation as deviations from standards (Vienna Pee Dee Belemnite for δ¹³C and N₂ in air for δ¹⁵N) following the formula: δ¹³C or δ¹⁵N = [(Rsample/Rstandard) - 1] x 10³, where R is ¹³C/¹²C or ¹⁵N/¹⁴N. Calibration was done using reference materials (USGS-24, IAEA-CH6, -600 for carbon; IAEA-N2, -NO-3, -600 for nitrogen). Analytical precision was < 0.15 ‰ based on the analyses of acetanilide (Thermo Scientific) used as laboratory internal standard.
219 2.6 STATISTICAL ANALYSES

220 Statistical analyses were performed using R 2.12.2 (R Foundation for Statistical
221 Computing 2011). Comparisons between measured parameters among sampling dates and reef
222 types were conducted using nonparametric procedures (Zar 2010). Kruskal-Wallis tests
223 (kruskal.test) were used to compare isotope compositions and C:N ratios of SPOM, SSOM and
224 oysters, and bottom water chl-a concentrations and C:chl-a ratios of SPOM. Kruskal-Wallis
tests were followed by multiple comparisons of means (kruskalmc in R pgirmess package)
225 (Giraudoux 2011). Isotope compositions of oysters and food sources were compared considering
226 trophic fractionation factors of 0.3 ± 1.3 ‰ (mean ± SD) for δ¹³C values and of 2.3 ± 1.6 ‰ for
227 δ¹⁵N values (Vander Zanden and Rasmussen 2001). Theoretical oyster food resource use was
228 computed by subtracting the trophic fractionation values from observed δ¹³C and δ¹⁵N values of
229 oysters to correct for fractionation. Contributions of SPOM and SSOM as food resources to
230 oysters were estimated by solving mixing models within a Bayesian framework
231 (siarmcmcdirichlety4 in R siar package) (Parnell et al. 2010, Parnell and Jackson 2014). Models
232 were run for 500,000 iterations and the first 50,000 iterations were discarded. Means and
233 credibility intervals (CI) of 0.95 and 0.25 were computed and displayed on figures.
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235 3. Results

236 3.1 CONTINUOUS ENVIRONMENTAL PARAMETERS

237 Monthly average salinity ranged from 25.2 to 38.8, with lowest values observed during
238 May 2012 (Fig. 2). Monthly chl-a concentrations ranged from 2.0 to 16.1 µg L⁻¹, and varied
239 throughout the study (Fig. 2). Monthly average wind speed ranged from 1.1 to 7.3 m s⁻¹, with
240 lowest values observed from August through October 2012 (Fig. 2).
241 3.2 COMPOSITION OF POTENTIAL FOOD RESOURCES

242 Mean bottom water chl-a concentrations of collected SPOM samples ranged from 0.7 ±
243 0.6 to 4.3 ± 1.3 µg L⁻¹ (mean ± SD) (Fig. 2). Chl-a concentrations were similar among reef types
244 (P > 0.173), except during March 2013, when chl-a was higher at the unconsolidated reef
245 compared to the restored reef (P = 0.024). Across all reef types, chl-a concentration was lower
246 in February 2012 than in September 2012 and 2013 (P < 0.001). Mean C:chl-a ratios ranged
247 from 58.8 ± 9.2 to 328.8 ± 67.5; ratios were similar among reef types and were greater in
248 February 2012 than in September 2012 and 2013 (P = 0.023; Fig. 3).

249 Mean δ¹³C values of SPOM ranged from -24.8 ± 0.3 ‰ (mean ± SD) to -23.5 ± 0.3 ‰;
250 mean δ¹⁵N values ranged from 7.3 ± 0.3 to 8.7 ± 0.2 ‰ (Fig. 4). Mean δ¹³C and δ¹⁵N values of
251 SPOM were similar among reef types. δ¹³C values were different among some sampling periods
252 (P = 0.016), but no clear temporal patterns were evident for δ¹³C values. The δ¹⁵N values were
253 generally more enriched during September sampling periods (P = 0.010). Mean C:N ratios of
254 SPOM ranged from 6.4 ± 0.5 to 12.8 ± 2.5 and were similar among reef types. C:N ratios of
255 SPOM were lowest during September sampling periods in both 2012 and 2013 compared to all
256 other sampling periods (Fig. 3), though not significantly different (P = 0.086).

257 Mean δ¹³C values of SSOM ranged from -22.6 ± 1.0 to -20.1 ± 0.2 ‰ (mean ± SD) (Fig. 4), and followed a general pattern of enrichment over time at all reef types (P < 0.001). Mean δ¹⁵N values ranged from 7.5 ± 0.3 to 8.5 ± 0.5 ‰ (Fig. 4). Mean δ¹³C and δ¹⁵N values of SSOM were similar among reef types (P > 0.066 and P > 0.099, respectively). Mean C:N ratios of SSOM ranged from 7.7 ± 0.5 to 10.6 ± 1.3 and were similar among reef types (P > 0.077). δ¹⁵N values and C:N ratios were similar among sampling periods (P = 0.186 and P = 0.228, respectively).263
264 3.3 STABLE ISOTOPE COMPOSITION OF OYSTERS

265 Oyster mean δ¹³C values ranged from -23.3 ± 0.2 to -20.1 ± 0.5 ‰ (mean ± SD) and
266 mean δ¹⁵N values ranged from 8.2 ± < 0.1 to 11.1 ± 0.6 ‰ (Fig. 4). Mean δ¹³C and δ¹⁵N values
267 were similar among reef types except in the first sampling period (February 2012); oysters from
268 the natural reef were more enriched in both ¹³C (*P* = 0.010) and ¹⁵N (*P* = 0.044) than oysters at
269 unconsolidated reef. Overall, spat and adult oysters had similar δ¹³C values (*P* = 0.478), while
270 spat were more ¹⁵N-enriched (10.2 ± 1.0 ‰) compared to adult oysters (9.5 ± 1.0 ‰) (*P* =
271 0.003). Oysters were more enriched in both ¹³C and ¹⁵N during June and September 2013 than
272 during other months (*P* < 0.001 for both δ¹³C and δ¹⁵N values). Theoretical oyster food resource
273 use, corrected for trophic fractionation by subtracting 0.3 ‰ from δ¹³C values and 2.3 ‰ from
274 δ¹⁵N values of oysters, overlaps predominantly with SSOM food resources (Fig. 4).

275 3.4 MIXING MODEL ESTIMATIONS OF FOOD SOURCE CONTRIBUTIONS TO OYSTERS

276 Mixing model results revealed a general trend of decreasing SPOM and increasing SSOM
277 food resource contributions to oysters over time, among all reef types (Fig. 5). In February 2012,
278 SPOM contributed from 20 to 98 % (95% CI range) and SSOM contributed from 2 to 80 %. By
279 the end of the study (September 2013), the relative importance of food sources to oysters
280 switched with SPOM contributions ranging from 2 to 54 % and SSOM from 47 to 98 %.
281 Slightly higher proportions of SPOM contribution was observed in September 2012 than in
282 adjacent sampling dates. The same temporal patterns of food source contributions were observed
283 among the three reef types.
284 **4. Discussion**

285 **4.1 ORIGIN AND COMPOSITION OF FOOD RESOURCES**

286 Average δ¹³C values of SPOM were more depleted (-24.2 ± 0.6 ‰) than SSOM (-21.2 ±
287 0.8 ‰) throughout the study (mean ± SD). The range of δ¹³C values observed from SPOM (-
288 25.1 to -23.6 ‰) fall within ranges typical of river-estuarine phytoplankton (-30 to -24 ‰), and
289 C₃ marsh (-26 to -23 ‰) and terrestrial (-30 to -23 ‰) plants (Fry and Sherr 1984). Recent work
290 (2010-2011) in the Mission-Aransas Estuary determined δ¹³C values for C₃ marsh plants (-29 to -
291 27 ‰) and estuarine SPOM (-27 to -21 ‰) (Lebreton et al. 2016). C:N ratios less than 20 and
292 C:chl-a ratios less than 200 indicate a dominant influence of fresh organic matter (e.g., fresh
293 phytoplankton) rather than degraded matter (e.g., highly decayed phytoplankton, terrestrial or
294 saltmarsh plant detritus) (Cifuentes et al.1988, Leal et al. 2008). Considered together, the δ¹³C
295 values of SPOM, and low C:N and C:chl-a ratios observed throughout much of the study indicate
296 a dominant influence of autochthonous phytoplankton production on SPOM (Fry and Sherr
297 1984, Lebreton et al. 2016).

298 Mean δ¹³C values of SSOM (ranging from -22.6 to -20.1 ‰) indicate potential influences
299 from a variety of sources: trapped phytoplankton, benthic microalgae, as well as a mixture of
300 material of marsh (C₄ plants) and of riverine and terrestrial (C₃ plants) origin (Fry and Sherr
301 1984). C:N ratios remain low (generally < 10), indicative of fresh organic matter (Kang et al.
302 2003) and thus indicate that marsh, riverine and terrestrial sources of organic matter play only a
303 minor role in the composition of SSOM. We infer that SSOM is largely composed of fresh
304 organic matter sources, such as trapped phytoplankton (-24 ‰; Fry and Sherr 1984), but also
305 benthic microalgae, which are more ¹³C-enriched, with δ¹³C values of -17 ‰ in Fry and Sherr
306 (1984), and ranging from -21.7 to -12.0 ‰ in Lebreton et al. (2016). The distinct ranges of δ¹³C
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values observed for SSOM and SPOM further indicate that SSOM is not only composed of settled SPOM, but likely contains a large amount of benthic microalgae.

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## 4.2 SPATIO-TEMPORAL VARIATIONS OF SPOM AND SSOM COMPOSITIONS

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At the spatial scale, the origin and composition of SPOM and of SSOM were generally similar among reef types (natural, restored, unconsolidated), demonstrating that differences of structure (i.e., vertical relief, bottom composition, oyster biomass and densities) among reefs do not affect the quantity and the quality of the food sources available for oysters at the local scale (i.e., reef scale). Rezek et al. (2017) demonstrated that changes of food web structure in oyster reefs under development are related to changes of benthic macrofauna community but not to changes in food source uses by macrofauna.

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At the temporal scale, SPOM was of higher quality in late summer (i.e., September) as indicated by the lower C:N and C:chl-*a* ratios and the higher chl-*a* concentrations in the bottom water. This indicates that the proportion of autochthonous phytoplankton in the bottom water SPOM is higher during this period. Local primary productivity in the water column was probably enhanced during this period due to seasonally low wind speeds (NCDC 2017), leading to lower resuspension of sediment-trapped organic and to higher penetration of light into the water column. Lower primary productivity during winter months, and thus less fresh organic matter in the SPOM pool, explains the higher C:chl-*a* ratios observed among all reef types during February 2012. The lack of clear relationship between chl-*a* concentrations measured in the bottom water (i.e., our samples) and in the water column (i.e., NERRS measurements) indicates that SPOM composition in bottom water can differ from that in the water column, highlighting the importance of sampling SPOM as close as possible to the oyster reefs.
In the sediment, a temporal increase of δ¹³C values was observed for SSOM, with values most depleted in ¹³C during February 2012 and becoming more enriched through September 2013 among all reef types. This pattern likely reflects an increasing influence of benthic microalgae, which are enriched in ¹³C, and/or a decreasing influence of more ¹³C-depleted organic matter (i.e., trapped phytoplankton) on SSOM across the period. As this development is observed at the scale of the three reef types, it can be related to a large change, at the scale of the ecosystem, or to a change at a local scale (i.e., one reef type) affecting the functioning of the whole ecosystem. We therefore suggest two hypotheses to explain this change: (1) At the scale of the ecosystem, a gradual decrease of SPOM trapping into the sediment. Nevertheless, such a trend would probably be linked with a similar trend of some abiotic parameters, such as wind velocity or turbidity, which is not the case, making this hypothesis less likely. (2) At a local scale, increasing oyster biomass at the restored reef may have enhanced growth of microphytobenthos through feedback effects related to oyster biodeposits. As suspension-feeders, oysters can have a strong influence on the degree of benthic-pelagic coupling within a system (Dame 2012, Newell 2004, Prins et al. 1998). Oysters remove suspended particles from the water column, including those resuspended from the sediment, and deposit feces and pseudofeces, or biodeposits, onto the sediment, thereby linking organic matter and nutrient flows between pelagic and benthic habitats (Hsieh et al. 2000, Newell 2004, Prins et al. 1998, Quan et al. 2012). Feces and pseudofeces excreted by oysters may stimulate benthic production as this organic matter is remineralized by bacteria and releases nutrients that can be used by microphytobenthos (Miller et al. 1996, Prins et al. 1998). Indeed, after the introduction and intensification of oyster culture, benthic primary production has been shown to increase (Leguerrier et al. 2004), as has enrichment of sediments due to biodeposits, resulting in high
sediment chl-*a* concentrations (Castel et al. 1989). In our study, the restoration of the reef does affect the SSOM composition in this reef type, as well as in the adjacent habitats (i.e., natural and unconsolidated reefs) highlighting the importance of the connectivity between these different habitats at the ecosystem scale. This highlights the potential benefits of reef restoration, not only for the oyster reef itself, but also for the adjacent systems.

### 4.3 OYSTER UTILIZATION OF WATER COLUMN AND SEDIMENT FOOD RESOURCES

The range of δ¹³C values observed in oysters overlaps mostly with SSOM δ¹³C values, indicating that oysters use food resources from both the SPOM and SSOM pools, regardless of reef type. The strong role of SSOM as a food resource for oysters is confirmed by the similar shift of δ¹³C values between oysters and SSOM. As SSOM becomes more ¹³C-enriched at all reef types over the duration of the study—probably due to an increase of benthic microalgae into SSOM composition—so do oysters. Additionally, mixing model results indicate that the contribution of SSOM to oysters increases over the duration of the study across all reef types, and that proportions of SSOM as a food source for oysters are similar among the three reef types. SSOM becomes accessible to oysters via resuspension during water movement (Kang et al. 2003, Miller et al. 1996). Minimal tidal ranges and freshwater inflows, coupled with consistently strong winds in the region, indicate that wind-induced waves are the predominant forcing factor causing resuspension in this system (Armstrong 1987, Evans and Morehead Palmer 2012).

The restored reef in this study was constructed with relatively high vertical relief (0.3 m). Oyster reef restoration as a whole has become increasingly focused on providing vertical structure (i.e., >0.2 m above the bottom) to minimize sedimentation and improve temporal sustainability (Powers et al. 2009, Schulte et al. 2009). The vertical biogenic structure interacts with hydrodynamic boundary-layer flow over the bottom (Butman et al. 1994, Green et al. 1998),
influencing resuspension and food availability (Fréchette et al. 1989, Grant et al. 1990, Quan et al. 2012, Wildish and Kristmanson 1984), larval settlement (Hills et al. 1999, Lapointe and Bourget 1999), and secondary production (Lenihan 1999). In contrast, the unconsolidated and natural reefs in the current study had relatively low vertical relief (0m; < 0.1 m, respectively).
Although it could be predicted that SSOM food resources are more accessible to suspension feeders lower in the water column (Hsieh et al. 2000), results indicate food resource use was similar between reef types, even during periods of low wind. Temporal trends observed in SSOM of increased δ¹³C values over time are reflected in oyster δ¹³C values across reef types, indicating that small-scale differences in vertical relief are not sufficient to limit consumption of SSOM in Copano Bay. This highlights the very strong benthopelagic coupling in this ecosystem, probably due to shallow depths as well as strong and consistent winds. Further research is warranted to examine whether oysters on higher-vertical relief reefs (≥1-2 m; e.g. Dunn et al. 2014, Puckett and Eggleston 2012) or from reefs restored in deeper or less wind driven systems become decoupled from SSOM food resources compared to lower-vertical relief counterparts.

## 4.4 TEMPORAL INCREASE OF SSOM AS A FOOD RESOURCE FOR OYSTERS

There are two probable explanations for the consistent temporal change in relative contribution of SPOM and SSOM food resources to oysters among reef-types: (1) a decrease of the role of SPOM, potentially connected to changes in environmental drivers, and/or (2) an increase of the role of SSOM, linked to feedback effects of oysters on SSOM composition and availability. Composition of organic matter resources can be influenced by various environmental factors, and may also affect the assimilation of food resources by consumers (Fry 2006, Michener and Kaufman 2007).
In the Copano-Aransas estuary, SPOM composition is highly connected to environmental drivers (Lebreton et al. 2016, Mooney and McClelland 2012, Reyna et al. 2017). The higher concentrations in chl-*a* and better quality (i.e., higher C:N and C:chl-*a* ratios) of SPOM in late summer, likely related to lower wind speeds, may affect its use by oysters. Indeed, in September 2012, the proportions of SPOM as a food source to oysters is slightly higher than the general observed pattern. Nevertheless, this slight discrepancy is relatively minor compared to the general temporal pattern observed, which shows a general increase of the SSOM as a food source over the course of the study, highlighting the importance of benthic food resources into the diet of oysters (Dame 2012, Riera and Richard 1996).

The alternative explanation for the higher use of SSOM over time is that increasing oyster biomass at the restored reef enhanced growth of microphytobenthos through feedback effects related to oyster biodeposits (see section 4.2). Enhanced benthic production can provide important food sources to suspension-feeders such as oysters and other benthic organisms (Leal et al. 2008, Miller et al. 1996, Ubertini et al. 2012), particularly in shallow systems where SSOM is resuspended on a regular basis. In addition, South Texas embayments are characterized by very low freshwater inflows, limiting nutrient inputs (Bianchi et al. 1999), and therefore production of phytoplankton (Mooney and McClelland 2012). As a result, the feedback of oysters on benthic primary production may be critical in supporting oysters and reef-associated fauna (Leguerrier et al. 2004, Prins et al. 1998, Ubertini et al. 2012). Drought conditions persisted throughout the study period, probably limiting phytoplankton production (Reyna et al. 2017). Thus, the increasing influence of SSOM on oysters over the study period may be related to observed increases in oyster biomass following reef restoration, coincident with prolonged drought (Blomberg 2015).
421 **5. Conclusion**

422 Our study demonstrated that, in a shallow and wind-driven ecosystem like the Mission-
423 Aransas Estuary, differences in oyster reef structure do not affect the composition of food
424 resources available to oysters and their use by oysters. This is probably related to the regular
425 resuspension of the organic matter stored in the sediment and to the strong connectivity between
426 the different studied reef types. Similar studies should be carried out in ecosystems
427 characterized by different physical properties (e.g., deeper systems, higher stratification) to
428 determine how these physical properties can affect flows of organic matter in natural and
429 restored oyster reefs. In terms of restoration ecology, it may also be of strong interest to
430 determine how vertical relief is affecting availability of the food resources from the benthos.

431 Another major conclusion of our study is that oysters rely on large amounts of sediment
432 organic matter as food resources, highlighting the importance of benthо-pelagic coupling in
433 oyster reefs, and supporting the positive feedback role of oyster reefs on the production of
434 benthic microalgae and/or trapping of organic matter. Better determination of the composition of
435 sediment organic matter in and around oyster reefs would provide significant knowledge toward
436 a better understanding of the role of this material in oyster reefs, and flows of organic matter in
437 these habitats.

438

439 **Acknowledgements**

440 B.N.B. was supported by the National Oceanic and Atmospheric Administration, Office
441 of Education Educational Partnership Program (Award NA11SEC4810001). The contents of this
442 manuscript are solely the responsibility of the award recipient and do not necessarily represent
443 the official views of the U.S. Department of Commerce, National Oceanic and Atmospheric
Administration. Research trip of B.N.B. at the UMR LIENSs and stable isotope analyses were funded in the framework of the "CNRS research and higher education chair" granted to B.L.

Reef construction was funded by the Gulf of Mexico Foundation-NOAA Community Based
Restoration Partnership (Award GCRP#10-01). We thank Texas A&M University – Corpus

Christi and the Harte Research Institute for support, and the many volunteers who assisted in the
field and lab. We thank three anonymous reviewers whose comments have helped improve this
manuscript. We declare no competing interests.

451

452 Author Contributions

Conceived and designed the experiments: BNB, BL, JBP. Performed the experiments: BNB, BL, TAP, GG, JBP. Analyzed the data: BNB, BL, GG. Contributed reagents/materials/analysis tools:

BL, JBP, PAM. Wrote the paper: BNB. Extensive editorial comments, insight into results: BL, TAP, GG, JBP, PAM.

457

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653 **Figure Captions:**

654

655 Figure 1. Study area. (a) Location of study site within the Mission-Aransas Estuary, Texas,

656 USA. (b) Extent of sampling area among three reef types.

657

658 Figure 2. Continuous environmental observations over the course of the study (July 2011

659 through October 2013): monthly average wind speed (m s⁻¹), salinity and monthly grab samples

660 of chlorophyll-a (µg L⁻¹) from the Mission-Aransas National Estuarine Research Reserve

661 (MANERR) Copano Bay East station. Bottom water chlorophyll-a (µg L⁻¹) samples are

662 indicated with dots during each sampling period. Reef construction is indicated with the dark

663 gray bar; sampling periods are indicated with light shaded bars.

664

665 Figure 3. C:N and C:chl-a ratios (mean ± SD) observed for suspended particulate organic matter

666 sampled across the study area during each sampling period.

667

668 Figure 4. δ¹³C and δ¹⁵N values (mean ± SD) of suspended particulate organic matter (SPOM),

669 surface sediment organic matter (SSOM) and oysters (all sizes) during each sampling date. (a)

670 Unconsolidated reef. (b) Natural reef. (c) Restored reef. Grey areas represent the range of

671 theoretical oyster food source stable isotope composition accounting for trophic fractionation.

672

673 Figure 5. Seasonal comparison of contributions (%) of potential food resources (SPOM:

674 suspended particulate organic matter, SSOM: surface sediment organic matter) to oyster diets

675 resulting from the mixing model SIAR. (a) Unconsolidated reef. (b) Natural reef. (c) Restored
676 reef. Mean, 0.25 and 0.95 credibility intervals are shown with horizontal bar, grey box and

677 whiskers, respectively.
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