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Estuarine intertidal sandflat benthic microalgal responses to in situ and mesocosm nitrogen additions
Journal of Experimental Marine Biology and Ecology 390 (2010) 99–105
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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Estuarine intertidal sandflat benthic microalgal responses to in situ and mesocosm nitrogen additions Michael F. Piehler a,⁎, Carolyn A. Currin b, Nathan S. Hall a a b
UNC-CH Institute of Marine Sciences, 3431 Arendell Street, Morehead City, NC 28557, United States NOAA NOS, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, NC 28516, United States
a r t i c l e
i n f o
Article history: Received 30 April 2009 Received in revised form 20 May 2010 Accepted 21 May 2010 Keywords: Benthic microalgae Nitrogen Intertidal flat Eutrophication Primary productivity Microphytobenthos
a b s t r a c t Benthic microalgal communities are important components of estuarine food webs and make substantial contributions to coastal materials cycling. Nitrogen is generally the limiting factor for marine primary production; however other factors can limit benthic primary producers because of their access to the additional nutrients found in sediment porewater. Field and laboratory experiments were conducted to test the hypothesis that water column nitrogen supply affects estuarine sandflat benthic microalgal community structure and function. Our field and mesocosm experiments assessed changes at both the population and functional group levels. Simulated water column nitrogen additions increased maximum community photosynthesis in most cases (Pbmax from photosynthesis vs. irradiance curves). Additional changes that resulted from nitrogen additions were decreases in porewater phosphate, increases in porewater ammonium, shifts in community composition from N2 fixing cyanobacteria toward diatoms, and detectable, though not statistically significant increases in biomass (as chlorophyll a). Results from field and laboratory experiments were quite similar, suggesting that laboratory experiments support accurate predictions of the response of intertidal benthic microalgae to changes in water column nutrient conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Coastal primary productivity is most often limited by nitrogen availability (Howarth, 1988; Valiela, 1995), whereas bacterial activity is increasingly believed to be limited, at least in part, by the availability of phosphorus (Sundareshwar et al., 2003). Human activities have led to increases in the amount of many forms of nutrients transported to coastal waters (National Research Council, 2000). This has fueled well documented acceleration of carbon loading to coastal waters through stimulation of primary productivity (Nixon, 1995). Excessive primary productivity is often attributable to phytoplankton (Paerl, 1998), but examples of excessive macroalgal growth have also been documented (Hauxwell et al., 2001), as has the potential for stimulation of subtidal benthic microalgae (BMA) (Lever and Valiela, 2005). BMA have the potential to sequester nutrients in the sediments and reduce the potential for algal blooms in the water column (MacIntyre et al. 2004). Nitrogen-limitation of primary productivity in BMA has been documented in both subtidal (Sundbäck et al., 1991) and intertidal environments (Lever and Valiela, 2005); while other studies have found nitrogen (N) does not limit primary productivity by BMA (Underwood et al., 1998). Generally, nutrient-limitation of BMA is
⁎ Corresponding author. E-mail address: [email protected] (M.F. Piehler). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.05.012
considered more prevalent in sandy sediments, as these environments have lower organic matter concentrations and remineralization rates (Lever and Valiela, 2005). Because of their location at the sediment–water interface, estuarine intertidal sandflat BMA may be affected by changing external supplies of nitrogen delivered from a variety of sources, including atmospheric, riverine and groundwater inputs. The balance of productivity between BMA and phytoplankton communities has been shown to be affected by nutrient regimes in both experimental mesocosm (Fong et al., 1993) and modeling studies (MacIntyre et al., 2004). BMA in euphotic coastal sediments have been identified as important transformers of organic matter in estuarine ecosystems. Because of their position at the sediment–water and terrestrial– aquatic interfaces, intertidal BMA play an important role in estuarine benthic–pelagic coupling (MacIntyre et al., 2004). BMA fix inorganic C and N from both overlying- and porewater to labile organic matter at the sediment surface (Underwood and Kromkamp, 1999), and are an important component of estuarine foodwebs (Sullivan and Currin, 2000). Understanding the factors that control the structure and function of the intertidal flat BMA may be particularly important for predicting their success in an environment with significant human and climate driven stressors. Changes in nutrient regime can alter coastal BMA communities at various levels of organization including changes in total BMA biomass and shifts in community structure at levels ranging from functional groups to species (Pinckney et al., 1995; Underwood and Barnett,
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2006). Functional groups are defined here according to Thingstad et al. (2010) as a group of microalgae that share important properties such as size, substrate utilization, common predators, and motility that govern their role in food web and biogeochemical processes. By definition, changes in algal functional groups are expected to impart changes in the way BMA communities function through impacts on primary productivity (van Raalte et al., 1976), trophic transfer, and nutrient cycling (Tyler et al., 2003; Sundbäck et al., 2004). Both top down and bottom up control of BMA have been demonstrated to be important in intertidal and shallow subtidal coastal systems (Posey et al., 2002; Lever and Valiela, 2005; Heck and Valentine, 2007). In order to accurately gauge the impacts of modified nutrient regimes on BMA communities it is important to consider the potential for changes at a variety of levels of ecosystem organization. In this study we employed in situ field and laboratory experiments to test the hypothesis that nitrogen limits sandflat intertidal BMA primary productivity and that nitrogen additions will alter BMA community structure. We analyzed community structure at both the population and functional group levels. Community function was measured by assessing primary productivity of the BMA. By coupling the field and laboratory studies we were able to benefit both from the rigor of an in situ manipulation and the controlled environment of experimental mesocosms. We were also able to compare results from field and laboratory experiments to assess the utility and applicability of data from laboratory mesocosms.
2. Materials and methods 2.1. Site The Newport River forms a mesohaline estuary near Beaufort, NC. In the lower portion of the estuary where this study was conducted (34.5N, 76.5W) (Fig. 1), the intertidal zone is mostly sandy, and significant BMA communities have been found (Piehler et al., 2003). Both the field experiment and collection of surface sediments for the laboratory mesocosm experiment were conducted on Kirby-Smith Island (KSI), a tidal flat adjacent to the Duke Marine Laboratory and the NOAA Beaufort Lab and to the west of Piver's Island, NC. The salinity during this study was between 34 and 36 psu, sediment grain size was predominantly (87–93%) within the sand classification (2 mm–0.0625 mm) and sediment organic content was less than 1%. Water depth varied with tidal stage (maximum observed depth was 0.75 m), and the flats were exposed for approximately 2 h at each semi-diurnal low tide.
2.2. Field experiment In June 2001, 8 plots of 1 m2 were marked with PVC pipe for control and nitrogen treatments (four for each treatment, n = 4) on KSI. The plots were distributed in a randomized block design. Nitrogen was added twice weekly at slack low tide by spraying 0.5 L evenly over treatment plots filtered seawater containing 70 µM N (20 µM NH+ 4 -N, 50 µM NO3N). Control plots were sprayed with an equal amount of filtered seawater without added N. Samples were taken after 1, 2 and 5 weeks of nutrient treatments. Details of sampling and analysis are described below. 2.3. Laboratory experiment Surface sediments were obtained using a flat shovel that minimized disruption to the upper 10 cm of the tidal flat. Six sections measuring 0.5 m × 0.5 m × 0.1 m deep were placed in a PVC tank and transported intact from the tidal flat described above to the University of North Carolina at Chapel Hill, Institute of Marine Sciences (IMS), 8 km west of the field site. At IMS the sediment sections were subdivided into 24 (0.2 m × 0.2 m × 0.1 m deep) perforated incubation baskets. Three baskets were placed in each of eight incubation tanks at random. The tanks (0.9 m × 0.9 m × 0.25 deep) were fed with individual water supply lines from Bogue Sound, which is 100 m south of IMS. Treatments included control and nitrogen additions. There were four replicate tanks for each treatment; each tank had three perforated baskets of sediment. Values for each replicate tank were the result of taking the mean of the three perforated baskets in each tank (Fig. 7). Diurnal tides were simulated in the separate treatment tanks using flowing seawater and standpipes throughout the experiment. Water levels at simulated high tide were 40 cm above the sediment surface and sediments were exposed during simulated low tide. Additions of seawater containing 20 µM NH+ 4 - and 50 µM NO3 were made twice weekly at the beginning of the simulated slack tide in identical volume:area ratios as in the field experiment. 2.4. Sampling and analysis Cores (1 cm deep) for BMA biomass determination as estimated by chlorophyll a were taken from sediment in situ (field experiment) or from each perforated basket (mesocosm experiment) using a cut off 3 ml syringe. Two depth sections were analyzed from each core (0–0.5 cm and 0.5–1 cm depth). For algal chlorophyll a determination, sediment cores were placed in polypropylene centrifuge tubes with 10 ml methanol:acetone:water (45:45:10) solution. Samples
Fig. 1. Field site location with an inset of North Carolina on the United States Atlantic coast.
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were sonicated in ice for 30 seconds and extracted for ∼ 12 h at 0 °C prior to chlorophyll a analysis by fluorometry (Turner Model TD-700) (Welschmeyer, 1994). Determination of photosynthesis versus irradiance (P–I) relationships was made using a modified version of the Lewis and Smith (1983) photosynthetron method (Piehler et al., 2003). Controls were run with each treatment to assess the P–I relationships for each treatment because diurnal changes in sandflat BMA photophysiology make interpretation of treatment effects from treatments incubated at different times of day problematic. This approach was taken to avoid mis-characterization of diurnal changes as being the result of experimental manipulation. Sediment samples were collected from the photic zone (top 3 mm) of the field or mesocosm experimental units and homogenized. The pooled homogenized sediment was then subsampled using cut off 1 ml syringes (inner diameter 0.45 cm) to produce core volumes of 0.1 cm3 and placed in 20 ml borosilicate incubation vials. Each vial contained a 0.1 cm3 core of sediment and 10 ml of GF/F filtered seawater from the sampling site spiked with 14C-bicarbonate (Amersham, Inc.) to a final concentration of 0.8 µCi ml− 1. Three samples for time zero (T0) radioactivity control treatments contained 0.1 cm3 of sediment and 500 µl of buffered formalin that was added to each vial immediately after the addition of labeled seawater. T0 controls were used to correct for uptake of 14C label that occurred during experimental setup. Three vials for measurement of total radioactivity added (Tc) were prepared by adding 500 μl of phenethylamine (PEA) and 50 µl labeled sample water into a 20 ml scintillation vial. A Cool-Lux 75W projector lamp generated varied irradiances using a combination of neutral density filters, distance from light source, and angle of incidence [see Lewis and Smith (1983) for further detail]. Forty-five min incubations were terminated by adding 500 µl of formalin to the vials. One ml of 50% HCl was added to the samples which were then placed on a shaker table overnight to purge unincorporated 14C. Ten ml of Ecolume (ICN Inc.) scintillation cocktail was added to each vial, which were stored in the dark for 12 h and were then counted in a Beckman model LS 5000TD liquid scintillation counter. Counts per minute were converted to disintegrations per minute using quench curves from a calibrated 14C-toluene standard. Dissolved inorganic carbon concentration in seawater in all treatments was determined by a Shimadzu TOC 5000A total carbon analyzer. Ambient irradiances during the mid-day incubations generally ranged from 1200 to 1600 µmol photons m− 2 s− 1. BMA organisms were identified and enumerated following the field and laboratory experiments. Surface sediment samples (top 3 mm) were collected from each of the three perforated baskets with syringe corers within each replicate tank. The three samples from each tank were subsequently homogenized in a 15 mL centrifuge vial containing 1 mL of Lugol's solution to produce a single sample for each experimental tank unit. Cell abundance was determined by suspending a measured mass (∼50 mg) of sediment in 50 ml of filtered water, settling the slurry in an Utermöhl chamber (1.35 mL, 0.5 cm tall) for 24 h, and enumerating the BMA by inverted microscopy (Utermöhl 1958). One hundred random Whipple grids, containing between 357 and 770 BMA cells, were counted on a Leica DMIRB inverted microscope (Wetzlar, Germany) under phase contrast at a magnification of 400×. The BMA in this study were dominated by diatoms and filamentous cyanobacteria. We divided these groups into microalgal functional groups based on size, N2 fixation potential, and motility. Diatoms were divided into pennate diatoms that likely had the ability to vertically migrate and non-migrating centric diatoms. Previous research has shown that the pennate diatoms of the tidal flats in this area are dominated by highly motile species (Kingston 1999). Pennate and centric diatoms were further divided into two size classes, N or b20 µm diameter for centric diatoms and N or b20 µm apical axis length for pennate diatoms. The two dominant filamentous cyanobacteria, Lyngbya sp. and Anabaena sp. were separated based on N2
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fixation potential. Only diatoms with visible chloroplasts were counted. Cell concentrations were normalized to the wet weight of the sediment sample. Porewater nutrient samples were obtained in the field experiment by centrifugation of cored sediment subsections from various depths and collection of the supernatant. Samples were analyzed on an Alpkem nutrient autoanalyzer using standard methods. The limits of detection were 0.26 μM for nitrate, 0.31 μM for ammonium and 0.02 μM for phosphate. Porewater nutrient samples from the mesocosm experiment were lost due to a power outage. Experimental results were analyzed using a one-way analysis of variance (ANOVA) with treatment as the factors (α = 0.05). Chlorophyll a data from the field experiment were analyzed using a two-way ANOVA (treatment and depth as factors). Because there were no significant interactions between factors, a one-way ANOVA was used to compare the treatments and depths. A-posteriori comparisons of means were made using the Bonferroni procedure (Moore and McCabe, 1993). 3. Results 3.1. Chlorophyll a During the field experiment, there were no significant changes in benthic chlorophyll a in the nitrogen amendments relative to the controls in either the surface (0–0.5 cm) samples or the depth (0.5–1 cm) samples (p b 0.05, Bonferroni) (Fig. 2). Mean chlorophyll a was generally lower at depth than at the surface, but there were times when the chlorophyll a at depth was comparable to surface levels (Fig. 2). Chlorophyll a was highest in week 2 in all treatments and depths (Fig. 2). In contrast, mean values of benthic chlorophyll a during the mesocosm experiment were always higher in the nitrogen treatment than in the control; however, the difference was not statistically significant (Fig. 3). There was a large increase in mean benthic chlorophyll a in both the surface and depth samples after 1 week in the nitrogen treatment (Fig. 3). Chlorophyll a levels were comparable in the surface and depth samples throughout the experiment (Fig. 3). 3.2. Porewater nutrients Nitrate was below detection in all samples collected during the field experiment. Ammonium was highest in the deepest sediments (4 cm) and lowest in the surface sample (0.5 cm) (Fig. 4). Nitrogen amendment yielded somewhat higher mean ammonium concentrations at depth; however, this result was not statistically significant (p N 0.05 Bonferroni). Phosphate concentrations increased with depth
Fig. 2. Biomass of BMA as estimated by chlorophyll a in the field experiment. Pairs of bars are from 0–0.5 cm and 0.5–1 cm respectively. Bars are mean values of four replicates and error bars are one standard deviation (n = 4).
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Fig. 3. Biomass of BMA as estimated by chlorophyll a in the mesocosm experiment. Pairs of bars are from 0–0.5 cm and 0.5–1 cm, respectively. Bars are mean values of four replicates and error bars are one standard deviation (n = 4). C = control, N = nitrogen addition treatment.
in all treatments and sampling times (Fig. 5). However, the nitrogen treatment had a significant negative effect on porewater phosphate concentration from samples collected after 2 weeks, at 0.5 cm depth, and again after 5 weeks, at both 0.5 cm and 2.0 cm depths (Fig. 5).
Fig. 5. Depth profiles of phosphate at three time points in the field experiment. Samples were taken at 1, 2 and 5 weeks (n = 4). Asterisks indicate differences between the control and nitrogen treatment (p b 0.05). Error bars are one standard deviation.
Porewater nutrient values were not obtained from the mesocosm experiment due to sample loss following a power outage. 3.3. Cell counts Microscopic counts of BMA following the field experiment revealed several significant changes in community composition at the functional group level. The nitrogen treatment had significantly higher counts of both small pennate diatoms and small centric diatoms compared to the control (Fig. 6A). There were also significantly fewer Anabaena sp. in the nitrogen treatment than in the control (p b 0.05, Bonferroni) (Fig. 6A). There were increases in several mean values for cell counts of diatoms in the nitrogen treatment in the mesocosm experiment; however none of the differences between the nitrogen treatment and the control were statistically significant (Fig. 6B). As in the field experiment, there were significantly fewer Anabaena sp. in the nitrogen treatment compared to the control (p b 0.05, Bonferroni) (Fig. 6B). 3.4. Photosynthesis vs. irradiance Fig. 4. Depth profiles of ammonium at three time points in the field experiment. Samples were taken at 1, 2 and 5 weeks (n = 4). Error bars are one standard deviation.
Photophysiological parameters were measured after 1 and 5 weeks in both sets of experiments. During the field experiment,
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Fig. 7. Experimental setup for the mesocosm experiment. Each tank had individual seawater supplies and 3 smaller baskets were placed in each tank with intertidal sediment sections from the field site. Mean values for the 3 baskets in each tank were used to calculate a single mean for each tank. There were 4 tanks randomly distributed for each treatment (n = 4).
Fig. 6. Cell counts following the field (A) and mesocosm (B) experiments. Bars are means of three replicates and error bars are one standard deviation (n = 3). One asterisk indicates differences between the control and nitrogen treatment (p b 0.05), two indicates difference at p b 0.01. Note difference in scale of y-axis between panels.
there were no significant differences between alpha (α) and Pbmax for the control and nitrogen treatment after 1 week (Table 1). After 5 weeks, α was significantly lower in the nitrogen treatment as compared to the control (Fig. 7). Pbmax was significantly higher in the nitrogen treatment than in the control after 5 weeks (Table 1). In the mesocosm experiments, there were no significant differences between Pbmax the treatments after either 1 or 5 weeks (Table 1). The nitrogen treatment had a significantly lower α than the control after 5 weeks (Table 1).
4. Discussion We assessed the effects of nitrogen (N) additions on intertidal sandflat benthic microalgal communities in field and laboratory experiments. These data are among the few documenting the effects of N additions on intertidal sandflats. Additionally, our response variables covered multiple levels of community structure and important measures of community function. This multi-tiered design allowed us to assess impacts of N additions beyond conventionally measured metrics.
Biomass (as estimated by chlorophyll a) is the most common metric used in microalgal research, but showed the least change in response to N enrichment in this study. The effects of nutrient additions may be less likely to be manifest in biomass changes because of other factors that affect biomass of BMA; including grazing, resuspension, burial and light limitation (MacIntyre et al., 1996; Lever and Valiela, 2005). However, microalgal biomass is an important metric for estuarine modeling and is the target for many restoration and regulatory programs (Peterson et al., 2008). In these experiments, N additions did not lead to significant increases in BMA biomass as chlorophyll a. There were increases in mean biomass as a result of both the field and laboratory N amendments, but variability was high enough to preclude statistical significance. Mean biomass of BMA also increased (non-significantly) in the measurements from depth (0.5–1 cm). In contrast to the results from these 5 week long N enrichment experiments, biomass of BMA in shallow subtidal sandy sediments in Massachusetts did increase significantly in response to N + P additions (Lever and Valiela, 2005). In that study, the positive response of microalgal biomass to nutrient (N + P) additions increased with increasing ecosystem N loading. Our field experiment site is located in the lower Newport River Estuary, where water column N concentrations in summer months are typically less than 1 μΜ and phytoplankton growth is N limited (Litaker et al., 1993). The lack of significant response of BMA biomass to nutrient (N) addition we observed is consistent with results from brackish shallow waters in the Baltic Sea (Hillebrand and Kahlert, 2002) . However, our nitrogen amendments decreased porewater phosphate concentrations and increased porewater ammonium concentrations; evidence that the N added to the sediment surface was being utilized and that increased N consumption drew down P pools. It is possible that secondary limitation by P limited the biomass increases in our experiments. In contrast to total biomass of BMA, measures of primary productivity by BMA were significantly affected by the experimental
Table 1 Photophysiological parameters for the field and laboratory experiments. Treatment
Alpha
95 lower
95 upper
Pbmax C Chl a− 1 h− 1
95 lower
95 upper
F
p
Field Control-1 Nitrogen-1 Control-5 Nitrogen-5
0.011 0.013 0.017 0.011
0.009 0.011 0.016 0.010
0.012 0.014 0.017 0.012
3.451 4.282 1.810 2.817
3.160 3.965 1.709 2.662
3.742 4.599 1.911 2.973
640.85 848.58 215.13 326.32
b0.0001 b0.0001 b0.0001 b0.0001
Mesocosm Control-1 Nitrogen-1 Control-5 Nitrogen-5
0.013 0.022 0.012 0.009
0.009 0.010 0.011 0.008
0.017 0.033 0.014 0.010
2.295 1.787 2.722 2.926
1.990 1.521 2.590 2.751
2.601 2.053 2.853 3.101
229.18 121.9 433.14 293.79
b0.0001 b0.0001 b0.0001 b0.0001
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N additions. Maximum primary productivity rates (Pbmax) significantly increased in the field experiment, but not in the mesocosm experiment. There were also significant decreases in α following N addition in both field and mesocosm experiments. The reduction in α meant that higher levels of light were required to saturate community photosynthesis, and could have resulted from community composition changes. The observed reductions in α could have been attributable to the shift toward pennate diatoms (higher light adapted) and away from cyanobacteria (lower light adapted). However, because of their small size, the total contribution of cyanobacteria to cell volume was relatively small in all treatments. Mean values of BMA biomass were often much higher in the N additions, and though not statistically significant, suggested some increase in the BMA community at the surface. Because of the high irradiance at the sediment surface, an increase in the proportion of the community living there could have caused a shift to high light adaptation and thus lower α. As discussed above, community composition of BMA shifted away from cyanobacteria toward diatoms in the N additions treatments. Shifts in community composition in response to changes in nutrient conditions have been described in field studies of BMA before (Pinckney et al., 1995). Underwood and Barnett (2006) underscored the difficulty of separating nutrient effects from the multiple covariables that affect estuarine BMA. In this study, measuring changes at the functional group level had the advantage of providing information about changes in particular BMA with different ecological roles. The shift away from N2 fixing cyanobacteria and toward diatoms in the field experiment was statistically significant and in the laboratory experiment it was nearly so (p = 0.07). In the field experiment the decrease in N2 fixing cyanobacteria (Anabaena sp.) corresponded with increases in the counts of both b20 μm pennate and b20 centric diatoms. Our data suggest that diatoms compete better with cyanobacteria in conditions of nutrient enrichment. The higher growth rate potential of diatoms likely allowed them to increase their relative abundance with the availability of additional N. Alterations in the composition of the BMA community may affect the functional roles of BMA in the intertidal ecosystem, including food webs and nutrient cycling. Although some research has suggested that diatoms are generally more palatable than cyanobacteria, there is also evidence that benthic cyanobacteria respond positively to a reduction in grazing (Carman et al., 1997; Sullivan and Currin, 2000). Cyanobacteria capable of N2 fixation may fill an important and distinctive role in the sandflat N cycle by providing a new source of N to the sediment surface (Piehler et al., 1998). Nitrogen additions not only induced a significant shift in BMA functional groups, but also affected centric diatoms, a group of microalgae that are most often associated with the phytoplankton. This result provides another example of the difficulty of distinguishing phytoplankton and BMA in shallow ecosystems, as it is unclear whether the increase in centric diatoms represented a real contribution to the BMA community or only an increased retention of phytoplankton in surface sediments (Rusch and Huettel, 2000). This effect was only observed in the field experiment, perhaps lending support to the phytoplankton retention in surface sediments explanation. Changes in BMA communities that resulted from N additions led to changes in the sandflat environment. In the field experiment, N additions led to increases in pennate diatom abundance along with decreased porewater phosphate concentration and increased porewater ammonium concentration. Although N additions were applied to the sediment surface, the largest differences in porewater ammonium and phosphate concentrations were seen at depths 2 or 4 cm below the surface, 2 and 5 weeks after the experiment began. We speculate that observed changes in nutrient concentrations at depth were due to reduced uptake of ammonium and increased uptake of phosphate at depth by vertically migrating pennate diatoms whose nitrogen but not phosphorous requirements were fulfilled through the surface N additions. While we did not specifically determine
whether vertical migration occurred in this study, pennate diatoms on nearby (∼ 400 m away) sandflats are dominated by vertically migrating pennate diatoms that can descend to N5 cm over a tidal cycle to reach deep nutrient pools (Kingston 1999). Thus, it seems reasonable to assume that the observed increase in pennate diatoms, a functional group defined by the ability to vertically migrate, was responsible for the reduction in P at depths well within their vertical migration amplitudes. Reduction in porewater P that may result from BMA responses to enhanced external N loading could have cascading effects on coastal biogeochemistry. Sundareshwar et al. (2003) found P to limit many bacterially mediated conversions of N. Among their findings was that denitrification was favored in P depleted conditions as compared to P replete conditions. Impacts of modified P supply on N biogeochemistry are likely to be complex, but our results provide an example of the indirect impacts that change in N supply could have on N cycling. Comparing the in situ field manipulation and the laboratory mesocosm experiments utilized in this study provided some insight into the applicability of mesocosm experiments for research on BMA. Our results were quite similar for field and laboratory experiments. There were small but insignificant increases in mean biomass), significant changes in photosynthetic parameters (Pbmax and α) and significant shifts in the abundance of functional groups of BMA. The factors that varied between the field and mesocosm experiments included hydrodynamics, presence of transient predators and the presence of deep burrowing predators. Given the compelling evidence for both top down and bottom up control of BMA communities (Lever and Valiela, 2005; Heck and Valentine, 2007) it is notable that our two experiments yielded similar results. In weighing the costs and benefits of field versus laboratory manipulations the typical considerations are cost, feasibility and representativeness. In this case the logistics of the field experiment were slightly more complicated, the costs were fairly similar and the representativeness of the incubations appeared to be comparable. This study provides evidence of N driven changes in intertidal sandflat BMA communities and demonstrates the potential for BMA to transfer water column nutrients to the sediment. In intertidal sandflats, augmented N supply may lead to higher rates of BMA primary productivity and changes in the composition of the community to include a larger proportion of diatoms than cyanobacteria. The implications of these changes include the potential for higher trophic level impacts and influences from the alteration of other resource levels (e.g. porewater phosphate concentrations). These results suggest that increased N supply to shallow coastal systems could affect processes that control important sources and sinks of N such as denitrification and N2-fixation. Responses in the field and mesocosm experiments were similar, suggesting that container experiments are an appropriate experimental approach for research on estuarine BMA. Acknowledgements We thank V. Winklemann and J. Brewer for field and laboratory assistance. We acknowledge the NOAA Ecological Effects of Sea Level Rise Program and North Carolina Sea Grant for funding that contributed to the generation of this manuscript. We appreciate the editorial comments provided by W. Sunda and R. Waggett and the input of our anonymous reviewers. [SS] References Carman, K.R., Fleeger, J.W., Pomarico, S.M., 1997. Response of a benthic food web to hydrocarbon contamination. Limnol. Oceanogr. 42, 561–571. Fong, P., Donohoe, R.M., Zedler, J.B., 1993. Competition with macroalgae and benthic cyanobacterial mats limits phytoplankton abundance in experimental mesocosms. Mar. Ecol. Prog. Ser. 100, 97–102. Hauxwell, J., Cebrian, J., Furlong, C., Valiela, I., 2001. Macroalgal canopies contribute to eelgrass (Zostera marina) decline in temperate estuarine ecosystems. Ecology 82, 1007–1022.
M.F. Piehler et al. / Journal of Experimental Marine Biology and Ecology 390 (2010) 99–105 Heck, K.L., Valentine, J.F., 2007. The primacy of consumers in benthic ecosystems of the coastal zone. Estuar. Coasts 30, 371–381. Hillebrand, H., Kahlert, M., 2002. Effect of grazing and water column nutrient supply on biomass and nutrient content of sediment microalgae. Aquat. Bot. 72, 143–159. Howarth, R.W., 1988. Nutrient limitation of net primary production in marine ecosystems. Annu. Rev. Ecol. Syst. 19, 89–110. Kingston, M.B., 1999. Wave effects on the vertical migration of two benthic microalgae: Hantzchia virgata var. intermedia and Euglena proxima. Estuaries 22, 81–91. Lever, M.A., Valiela, I., 2005. Response of microphytobenthic biomass to experimental nutrient enrichment and grazer exclusion at different land-derived nitrogen loads. Mar. Ecol. Prog. Ser. 294, 117–129. Lewis, M.R., Smith, J.C., 1983. A small volume, short-incubation-time method for measurement of photosynthesis as a function of incident irradiance. Mar. Ecol. Prog. Ser. 13, 99–102. Litaker, W., Duke, C., Kenney, B., Ramus, J., 1993. Short-term environmental variability and phytoplankton abundances in a shallow tidal estuary. II. Spring and fall. Mar. Ecol. Prog. Ser. 94, 141–154. MacIntyre, H.L., Geider, R.J., Miller, D.C., 1996. Microphytobenthos: the ecological role of the “secret garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance, and primary production. Estuaries 19, 186–201. MacIntyre, H.L., Lomas, M.W., Cornwell, J.C., Suggett, D.J., Koch, E.W., Gobler, C.J., Kana, T.M., 2004. Mediation of benthic-pelagic coupling by microphytobenthos: an energy- and material-based model for initiation of blooms of Aureococcus anophagefferens. Harm. Alga. 3, 403–438. Moore, D.S., McCabe, G.P., 1993. Introduction to the Practice of Statistics. W.H. Freeman and Co., New York. National Research Council, 2000. Clean Coastal Waters: Understanding and Reducing the Effects of Nutrient Pollution. National Academy Press, Washington, D.C. Nixon, S.W., 1995. Coastal marine eutrophication: a definition, social causes, and future concerns. Ophelia 41, 199–219. Paerl, H.W., 1998. Structure and function of anthropogenically altered microbial communities in coastal waters. Curr. Opin. Microbiol. 1, 296–302. Peterson, C.H., Able, K.W., Frieswyk DeJong, C., Piehler, M.F., Simenstad, C.A., Zedler, J.B., 2008. Practical proxies for tidal marsh ecosystem services. Adv. Mar. Biol. 54, 221–266. Piehler, M.F., Currin, C.A., Casanova, R., Paerl, H.W., 1998. Development and N2-fixing activity of the benthic microalgal community in transplanted Spartina alterniflora marshes in North Carolina. Rest. Ecol. 6, 290–296. Piehler, M.F., Winkelmann, V., Twomey, L.J., Hall, N.S., Currin, C.A., Paerl, H.W., 2003. Impacts of diesel fuel exposure on the microphytobenthos of an intertidal sandflat. J. Exp. Mar. Biol. Ecol. 297, 217–235.
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Pinckney, J., Paerl, H., Fitzpatrick, M., 1995. The impacts of seasonality and nutrients on microbial mat community structure and function. Mar. Ecol. Prog. Ser. 123, 207–216. Posey, M.H., Alphin, T.D., Cahoon, L.B., Lindquist, D.G., Mallin, M.A., Nevers, M.B., 2002. Top-down versus bottom-up limitation in benthic infaunal communities: direct and indirect effects. Estuaries 25, 999–1014. Rusch, A., Huettel, M., 2000. Advective particle transport into permeable sediments — evidence from experiments in an intertidal sandflat. Limnol. Oceanogr. 45, 525–533. Sullivan, M.J., Currin, C.A., 2000. Community structure and functional dynamics of benthic microalgae in salt marshes. In: Weinstein, M.P., Kreeger, D.A. (Eds.), Concept and Controversies in Tidal Marsh Ecology. Kluwer Academic Publishers, Dordrecht, pp. 81–106. Sundareshwar, P.V., Morris, J.T., Koepfler, E.K., Fornwalt, B., 2003. Phosphorus limitation of coastal ecosystem processes. Science 299, 563–565. Sundbäck, K., Enoksson, V., Granéli, W., Pettersson, K., 1991. Influence of sublittoral microphytobenthos on the oxygen and nutrient flux between sediment and water: a laboratory continuous-flow study. Mar. Ecol. Prog. Ser. 74, 263–279. Sundbäck, K., Linares, F., Larson, F., Wulff, A., Engelsen, A., 2004. Benthic nitrogen fluxes along a depth gradient in a microtidal fjord: the role of denitrification and microphytobenthos. Limnol. Oceanogr. 49, 1095–1107. Thingstad, T.F., Strand, E., Larsen, A., 2010. Stepwise building of plankton functional type (PFT) models: a feasible route to complex models? Prog. Oceanogr. 84, 6–15. Tyler, A.C., McGlathery, K.J., Anderson, I.C., 2003. Benthic algae control sediment–water column fluxes of organic and inorganic nitrogen compounds in a temperate lagoon. Limnol. Oceanogr. 48, 2125–2137. Underwood, G.J.C., Barnett, M., 2006. What determines species composition in microphytobenthic biofilms? In: Kromkamp, J.C., de Brouwer, J.F.C., Blanchard, G.F., Forster, R.M., Créach, V. (Eds.), Functioning of Microphytobenthos in Estuaries. Royal Netherlands Academy of Arts and Sciences, Amsterdam. Underwood, G.J.C., Kromkamp, J., 1999. Primary production by phytoplankton and microphytobenthos in estuaries. Adv. Ecol. Res. 29, 93–153. Underwood, G.J.C., Phillips, J., Saunders, K., 1998. Distribution of estuarine benthic diatom species along salinity and nutrient gradients. Eur. J. Phycol. 33, 173–183. Utermöhl, H., 1958. Zur Vervollkommning der quantitativen Phytoplankton-Methodik. Mitt. Int. Verein. Theor. Agnew. Limnol. 9, 1–38. Valiela, I.A., 1995. Marine Ecological Processes Second Edition Springer. van Raalte, C.D., Valiela, I., Teal, J.M., 1976. Production of epibenthic salt marsh algae: light and nutrient limitation. Limnol. Oceanogr. 21, 862–872. Welschmeyer, N.A., 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnol. Oceanogr. 39, 1985–1992.
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Estuarine intertidal sandflat benthic microalgal responses to in situ and mesocosm nitrogen additions Michael F. Piehler a,⁎, Carolyn A. Currin b, Nathan S. Hall a a b
UNC-CH Institute of Marine Sciences, 3431 Arendell Street, Morehead City, NC 28557, United States NOAA NOS, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, NC 28516, United States
a r t i c l e
i n f o
Article history: Received 30 April 2009 Received in revised form 20 May 2010 Accepted 21 May 2010 Keywords: Benthic microalgae Nitrogen Intertidal flat Eutrophication Primary productivity Microphytobenthos
a b s t r a c t Benthic microalgal communities are important components of estuarine food webs and make substantial contributions to coastal materials cycling. Nitrogen is generally the limiting factor for marine primary production; however other factors can limit benthic primary producers because of their access to the additional nutrients found in sediment porewater. Field and laboratory experiments were conducted to test the hypothesis that water column nitrogen supply affects estuarine sandflat benthic microalgal community structure and function. Our field and mesocosm experiments assessed changes at both the population and functional group levels. Simulated water column nitrogen additions increased maximum community photosynthesis in most cases (Pbmax from photosynthesis vs. irradiance curves). Additional changes that resulted from nitrogen additions were decreases in porewater phosphate, increases in porewater ammonium, shifts in community composition from N2 fixing cyanobacteria toward diatoms, and detectable, though not statistically significant increases in biomass (as chlorophyll a). Results from field and laboratory experiments were quite similar, suggesting that laboratory experiments support accurate predictions of the response of intertidal benthic microalgae to changes in water column nutrient conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Coastal primary productivity is most often limited by nitrogen availability (Howarth, 1988; Valiela, 1995), whereas bacterial activity is increasingly believed to be limited, at least in part, by the availability of phosphorus (Sundareshwar et al., 2003). Human activities have led to increases in the amount of many forms of nutrients transported to coastal waters (National Research Council, 2000). This has fueled well documented acceleration of carbon loading to coastal waters through stimulation of primary productivity (Nixon, 1995). Excessive primary productivity is often attributable to phytoplankton (Paerl, 1998), but examples of excessive macroalgal growth have also been documented (Hauxwell et al., 2001), as has the potential for stimulation of subtidal benthic microalgae (BMA) (Lever and Valiela, 2005). BMA have the potential to sequester nutrients in the sediments and reduce the potential for algal blooms in the water column (MacIntyre et al. 2004). Nitrogen-limitation of primary productivity in BMA has been documented in both subtidal (Sundbäck et al., 1991) and intertidal environments (Lever and Valiela, 2005); while other studies have found nitrogen (N) does not limit primary productivity by BMA (Underwood et al., 1998). Generally, nutrient-limitation of BMA is
⁎ Corresponding author. E-mail address: [email protected] (M.F. Piehler). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.05.012
considered more prevalent in sandy sediments, as these environments have lower organic matter concentrations and remineralization rates (Lever and Valiela, 2005). Because of their location at the sediment–water interface, estuarine intertidal sandflat BMA may be affected by changing external supplies of nitrogen delivered from a variety of sources, including atmospheric, riverine and groundwater inputs. The balance of productivity between BMA and phytoplankton communities has been shown to be affected by nutrient regimes in both experimental mesocosm (Fong et al., 1993) and modeling studies (MacIntyre et al., 2004). BMA in euphotic coastal sediments have been identified as important transformers of organic matter in estuarine ecosystems. Because of their position at the sediment–water and terrestrial– aquatic interfaces, intertidal BMA play an important role in estuarine benthic–pelagic coupling (MacIntyre et al., 2004). BMA fix inorganic C and N from both overlying- and porewater to labile organic matter at the sediment surface (Underwood and Kromkamp, 1999), and are an important component of estuarine foodwebs (Sullivan and Currin, 2000). Understanding the factors that control the structure and function of the intertidal flat BMA may be particularly important for predicting their success in an environment with significant human and climate driven stressors. Changes in nutrient regime can alter coastal BMA communities at various levels of organization including changes in total BMA biomass and shifts in community structure at levels ranging from functional groups to species (Pinckney et al., 1995; Underwood and Barnett,
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2006). Functional groups are defined here according to Thingstad et al. (2010) as a group of microalgae that share important properties such as size, substrate utilization, common predators, and motility that govern their role in food web and biogeochemical processes. By definition, changes in algal functional groups are expected to impart changes in the way BMA communities function through impacts on primary productivity (van Raalte et al., 1976), trophic transfer, and nutrient cycling (Tyler et al., 2003; Sundbäck et al., 2004). Both top down and bottom up control of BMA have been demonstrated to be important in intertidal and shallow subtidal coastal systems (Posey et al., 2002; Lever and Valiela, 2005; Heck and Valentine, 2007). In order to accurately gauge the impacts of modified nutrient regimes on BMA communities it is important to consider the potential for changes at a variety of levels of ecosystem organization. In this study we employed in situ field and laboratory experiments to test the hypothesis that nitrogen limits sandflat intertidal BMA primary productivity and that nitrogen additions will alter BMA community structure. We analyzed community structure at both the population and functional group levels. Community function was measured by assessing primary productivity of the BMA. By coupling the field and laboratory studies we were able to benefit both from the rigor of an in situ manipulation and the controlled environment of experimental mesocosms. We were also able to compare results from field and laboratory experiments to assess the utility and applicability of data from laboratory mesocosms.
2. Materials and methods 2.1. Site The Newport River forms a mesohaline estuary near Beaufort, NC. In the lower portion of the estuary where this study was conducted (34.5N, 76.5W) (Fig. 1), the intertidal zone is mostly sandy, and significant BMA communities have been found (Piehler et al., 2003). Both the field experiment and collection of surface sediments for the laboratory mesocosm experiment were conducted on Kirby-Smith Island (KSI), a tidal flat adjacent to the Duke Marine Laboratory and the NOAA Beaufort Lab and to the west of Piver's Island, NC. The salinity during this study was between 34 and 36 psu, sediment grain size was predominantly (87–93%) within the sand classification (2 mm–0.0625 mm) and sediment organic content was less than 1%. Water depth varied with tidal stage (maximum observed depth was 0.75 m), and the flats were exposed for approximately 2 h at each semi-diurnal low tide.
2.2. Field experiment In June 2001, 8 plots of 1 m2 were marked with PVC pipe for control and nitrogen treatments (four for each treatment, n = 4) on KSI. The plots were distributed in a randomized block design. Nitrogen was added twice weekly at slack low tide by spraying 0.5 L evenly over treatment plots filtered seawater containing 70 µM N (20 µM NH+ 4 -N, 50 µM NO3N). Control plots were sprayed with an equal amount of filtered seawater without added N. Samples were taken after 1, 2 and 5 weeks of nutrient treatments. Details of sampling and analysis are described below. 2.3. Laboratory experiment Surface sediments were obtained using a flat shovel that minimized disruption to the upper 10 cm of the tidal flat. Six sections measuring 0.5 m × 0.5 m × 0.1 m deep were placed in a PVC tank and transported intact from the tidal flat described above to the University of North Carolina at Chapel Hill, Institute of Marine Sciences (IMS), 8 km west of the field site. At IMS the sediment sections were subdivided into 24 (0.2 m × 0.2 m × 0.1 m deep) perforated incubation baskets. Three baskets were placed in each of eight incubation tanks at random. The tanks (0.9 m × 0.9 m × 0.25 deep) were fed with individual water supply lines from Bogue Sound, which is 100 m south of IMS. Treatments included control and nitrogen additions. There were four replicate tanks for each treatment; each tank had three perforated baskets of sediment. Values for each replicate tank were the result of taking the mean of the three perforated baskets in each tank (Fig. 7). Diurnal tides were simulated in the separate treatment tanks using flowing seawater and standpipes throughout the experiment. Water levels at simulated high tide were 40 cm above the sediment surface and sediments were exposed during simulated low tide. Additions of seawater containing 20 µM NH+ 4 - and 50 µM NO3 were made twice weekly at the beginning of the simulated slack tide in identical volume:area ratios as in the field experiment. 2.4. Sampling and analysis Cores (1 cm deep) for BMA biomass determination as estimated by chlorophyll a were taken from sediment in situ (field experiment) or from each perforated basket (mesocosm experiment) using a cut off 3 ml syringe. Two depth sections were analyzed from each core (0–0.5 cm and 0.5–1 cm depth). For algal chlorophyll a determination, sediment cores were placed in polypropylene centrifuge tubes with 10 ml methanol:acetone:water (45:45:10) solution. Samples
Fig. 1. Field site location with an inset of North Carolina on the United States Atlantic coast.
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were sonicated in ice for 30 seconds and extracted for ∼ 12 h at 0 °C prior to chlorophyll a analysis by fluorometry (Turner Model TD-700) (Welschmeyer, 1994). Determination of photosynthesis versus irradiance (P–I) relationships was made using a modified version of the Lewis and Smith (1983) photosynthetron method (Piehler et al., 2003). Controls were run with each treatment to assess the P–I relationships for each treatment because diurnal changes in sandflat BMA photophysiology make interpretation of treatment effects from treatments incubated at different times of day problematic. This approach was taken to avoid mis-characterization of diurnal changes as being the result of experimental manipulation. Sediment samples were collected from the photic zone (top 3 mm) of the field or mesocosm experimental units and homogenized. The pooled homogenized sediment was then subsampled using cut off 1 ml syringes (inner diameter 0.45 cm) to produce core volumes of 0.1 cm3 and placed in 20 ml borosilicate incubation vials. Each vial contained a 0.1 cm3 core of sediment and 10 ml of GF/F filtered seawater from the sampling site spiked with 14C-bicarbonate (Amersham, Inc.) to a final concentration of 0.8 µCi ml− 1. Three samples for time zero (T0) radioactivity control treatments contained 0.1 cm3 of sediment and 500 µl of buffered formalin that was added to each vial immediately after the addition of labeled seawater. T0 controls were used to correct for uptake of 14C label that occurred during experimental setup. Three vials for measurement of total radioactivity added (Tc) were prepared by adding 500 μl of phenethylamine (PEA) and 50 µl labeled sample water into a 20 ml scintillation vial. A Cool-Lux 75W projector lamp generated varied irradiances using a combination of neutral density filters, distance from light source, and angle of incidence [see Lewis and Smith (1983) for further detail]. Forty-five min incubations were terminated by adding 500 µl of formalin to the vials. One ml of 50% HCl was added to the samples which were then placed on a shaker table overnight to purge unincorporated 14C. Ten ml of Ecolume (ICN Inc.) scintillation cocktail was added to each vial, which were stored in the dark for 12 h and were then counted in a Beckman model LS 5000TD liquid scintillation counter. Counts per minute were converted to disintegrations per minute using quench curves from a calibrated 14C-toluene standard. Dissolved inorganic carbon concentration in seawater in all treatments was determined by a Shimadzu TOC 5000A total carbon analyzer. Ambient irradiances during the mid-day incubations generally ranged from 1200 to 1600 µmol photons m− 2 s− 1. BMA organisms were identified and enumerated following the field and laboratory experiments. Surface sediment samples (top 3 mm) were collected from each of the three perforated baskets with syringe corers within each replicate tank. The three samples from each tank were subsequently homogenized in a 15 mL centrifuge vial containing 1 mL of Lugol's solution to produce a single sample for each experimental tank unit. Cell abundance was determined by suspending a measured mass (∼50 mg) of sediment in 50 ml of filtered water, settling the slurry in an Utermöhl chamber (1.35 mL, 0.5 cm tall) for 24 h, and enumerating the BMA by inverted microscopy (Utermöhl 1958). One hundred random Whipple grids, containing between 357 and 770 BMA cells, were counted on a Leica DMIRB inverted microscope (Wetzlar, Germany) under phase contrast at a magnification of 400×. The BMA in this study were dominated by diatoms and filamentous cyanobacteria. We divided these groups into microalgal functional groups based on size, N2 fixation potential, and motility. Diatoms were divided into pennate diatoms that likely had the ability to vertically migrate and non-migrating centric diatoms. Previous research has shown that the pennate diatoms of the tidal flats in this area are dominated by highly motile species (Kingston 1999). Pennate and centric diatoms were further divided into two size classes, N or b20 µm diameter for centric diatoms and N or b20 µm apical axis length for pennate diatoms. The two dominant filamentous cyanobacteria, Lyngbya sp. and Anabaena sp. were separated based on N2
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fixation potential. Only diatoms with visible chloroplasts were counted. Cell concentrations were normalized to the wet weight of the sediment sample. Porewater nutrient samples were obtained in the field experiment by centrifugation of cored sediment subsections from various depths and collection of the supernatant. Samples were analyzed on an Alpkem nutrient autoanalyzer using standard methods. The limits of detection were 0.26 μM for nitrate, 0.31 μM for ammonium and 0.02 μM for phosphate. Porewater nutrient samples from the mesocosm experiment were lost due to a power outage. Experimental results were analyzed using a one-way analysis of variance (ANOVA) with treatment as the factors (α = 0.05). Chlorophyll a data from the field experiment were analyzed using a two-way ANOVA (treatment and depth as factors). Because there were no significant interactions between factors, a one-way ANOVA was used to compare the treatments and depths. A-posteriori comparisons of means were made using the Bonferroni procedure (Moore and McCabe, 1993). 3. Results 3.1. Chlorophyll a During the field experiment, there were no significant changes in benthic chlorophyll a in the nitrogen amendments relative to the controls in either the surface (0–0.5 cm) samples or the depth (0.5–1 cm) samples (p b 0.05, Bonferroni) (Fig. 2). Mean chlorophyll a was generally lower at depth than at the surface, but there were times when the chlorophyll a at depth was comparable to surface levels (Fig. 2). Chlorophyll a was highest in week 2 in all treatments and depths (Fig. 2). In contrast, mean values of benthic chlorophyll a during the mesocosm experiment were always higher in the nitrogen treatment than in the control; however, the difference was not statistically significant (Fig. 3). There was a large increase in mean benthic chlorophyll a in both the surface and depth samples after 1 week in the nitrogen treatment (Fig. 3). Chlorophyll a levels were comparable in the surface and depth samples throughout the experiment (Fig. 3). 3.2. Porewater nutrients Nitrate was below detection in all samples collected during the field experiment. Ammonium was highest in the deepest sediments (4 cm) and lowest in the surface sample (0.5 cm) (Fig. 4). Nitrogen amendment yielded somewhat higher mean ammonium concentrations at depth; however, this result was not statistically significant (p N 0.05 Bonferroni). Phosphate concentrations increased with depth
Fig. 2. Biomass of BMA as estimated by chlorophyll a in the field experiment. Pairs of bars are from 0–0.5 cm and 0.5–1 cm respectively. Bars are mean values of four replicates and error bars are one standard deviation (n = 4).
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Fig. 3. Biomass of BMA as estimated by chlorophyll a in the mesocosm experiment. Pairs of bars are from 0–0.5 cm and 0.5–1 cm, respectively. Bars are mean values of four replicates and error bars are one standard deviation (n = 4). C = control, N = nitrogen addition treatment.
in all treatments and sampling times (Fig. 5). However, the nitrogen treatment had a significant negative effect on porewater phosphate concentration from samples collected after 2 weeks, at 0.5 cm depth, and again after 5 weeks, at both 0.5 cm and 2.0 cm depths (Fig. 5).
Fig. 5. Depth profiles of phosphate at three time points in the field experiment. Samples were taken at 1, 2 and 5 weeks (n = 4). Asterisks indicate differences between the control and nitrogen treatment (p b 0.05). Error bars are one standard deviation.
Porewater nutrient values were not obtained from the mesocosm experiment due to sample loss following a power outage. 3.3. Cell counts Microscopic counts of BMA following the field experiment revealed several significant changes in community composition at the functional group level. The nitrogen treatment had significantly higher counts of both small pennate diatoms and small centric diatoms compared to the control (Fig. 6A). There were also significantly fewer Anabaena sp. in the nitrogen treatment than in the control (p b 0.05, Bonferroni) (Fig. 6A). There were increases in several mean values for cell counts of diatoms in the nitrogen treatment in the mesocosm experiment; however none of the differences between the nitrogen treatment and the control were statistically significant (Fig. 6B). As in the field experiment, there were significantly fewer Anabaena sp. in the nitrogen treatment compared to the control (p b 0.05, Bonferroni) (Fig. 6B). 3.4. Photosynthesis vs. irradiance Fig. 4. Depth profiles of ammonium at three time points in the field experiment. Samples were taken at 1, 2 and 5 weeks (n = 4). Error bars are one standard deviation.
Photophysiological parameters were measured after 1 and 5 weeks in both sets of experiments. During the field experiment,
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Fig. 7. Experimental setup for the mesocosm experiment. Each tank had individual seawater supplies and 3 smaller baskets were placed in each tank with intertidal sediment sections from the field site. Mean values for the 3 baskets in each tank were used to calculate a single mean for each tank. There were 4 tanks randomly distributed for each treatment (n = 4).
Fig. 6. Cell counts following the field (A) and mesocosm (B) experiments. Bars are means of three replicates and error bars are one standard deviation (n = 3). One asterisk indicates differences between the control and nitrogen treatment (p b 0.05), two indicates difference at p b 0.01. Note difference in scale of y-axis between panels.
there were no significant differences between alpha (α) and Pbmax for the control and nitrogen treatment after 1 week (Table 1). After 5 weeks, α was significantly lower in the nitrogen treatment as compared to the control (Fig. 7). Pbmax was significantly higher in the nitrogen treatment than in the control after 5 weeks (Table 1). In the mesocosm experiments, there were no significant differences between Pbmax the treatments after either 1 or 5 weeks (Table 1). The nitrogen treatment had a significantly lower α than the control after 5 weeks (Table 1).
4. Discussion We assessed the effects of nitrogen (N) additions on intertidal sandflat benthic microalgal communities in field and laboratory experiments. These data are among the few documenting the effects of N additions on intertidal sandflats. Additionally, our response variables covered multiple levels of community structure and important measures of community function. This multi-tiered design allowed us to assess impacts of N additions beyond conventionally measured metrics.
Biomass (as estimated by chlorophyll a) is the most common metric used in microalgal research, but showed the least change in response to N enrichment in this study. The effects of nutrient additions may be less likely to be manifest in biomass changes because of other factors that affect biomass of BMA; including grazing, resuspension, burial and light limitation (MacIntyre et al., 1996; Lever and Valiela, 2005). However, microalgal biomass is an important metric for estuarine modeling and is the target for many restoration and regulatory programs (Peterson et al., 2008). In these experiments, N additions did not lead to significant increases in BMA biomass as chlorophyll a. There were increases in mean biomass as a result of both the field and laboratory N amendments, but variability was high enough to preclude statistical significance. Mean biomass of BMA also increased (non-significantly) in the measurements from depth (0.5–1 cm). In contrast to the results from these 5 week long N enrichment experiments, biomass of BMA in shallow subtidal sandy sediments in Massachusetts did increase significantly in response to N + P additions (Lever and Valiela, 2005). In that study, the positive response of microalgal biomass to nutrient (N + P) additions increased with increasing ecosystem N loading. Our field experiment site is located in the lower Newport River Estuary, where water column N concentrations in summer months are typically less than 1 μΜ and phytoplankton growth is N limited (Litaker et al., 1993). The lack of significant response of BMA biomass to nutrient (N) addition we observed is consistent with results from brackish shallow waters in the Baltic Sea (Hillebrand and Kahlert, 2002) . However, our nitrogen amendments decreased porewater phosphate concentrations and increased porewater ammonium concentrations; evidence that the N added to the sediment surface was being utilized and that increased N consumption drew down P pools. It is possible that secondary limitation by P limited the biomass increases in our experiments. In contrast to total biomass of BMA, measures of primary productivity by BMA were significantly affected by the experimental
Table 1 Photophysiological parameters for the field and laboratory experiments. Treatment
Alpha
95 lower
95 upper
Pbmax C Chl a− 1 h− 1
95 lower
95 upper
F
p
Field Control-1 Nitrogen-1 Control-5 Nitrogen-5
0.011 0.013 0.017 0.011
0.009 0.011 0.016 0.010
0.012 0.014 0.017 0.012
3.451 4.282 1.810 2.817
3.160 3.965 1.709 2.662
3.742 4.599 1.911 2.973
640.85 848.58 215.13 326.32
b0.0001 b0.0001 b0.0001 b0.0001
Mesocosm Control-1 Nitrogen-1 Control-5 Nitrogen-5
0.013 0.022 0.012 0.009
0.009 0.010 0.011 0.008
0.017 0.033 0.014 0.010
2.295 1.787 2.722 2.926
1.990 1.521 2.590 2.751
2.601 2.053 2.853 3.101
229.18 121.9 433.14 293.79
b0.0001 b0.0001 b0.0001 b0.0001
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M.F. Piehler et al. / Journal of Experimental Marine Biology and Ecology 390 (2010) 99–105
N additions. Maximum primary productivity rates (Pbmax) significantly increased in the field experiment, but not in the mesocosm experiment. There were also significant decreases in α following N addition in both field and mesocosm experiments. The reduction in α meant that higher levels of light were required to saturate community photosynthesis, and could have resulted from community composition changes. The observed reductions in α could have been attributable to the shift toward pennate diatoms (higher light adapted) and away from cyanobacteria (lower light adapted). However, because of their small size, the total contribution of cyanobacteria to cell volume was relatively small in all treatments. Mean values of BMA biomass were often much higher in the N additions, and though not statistically significant, suggested some increase in the BMA community at the surface. Because of the high irradiance at the sediment surface, an increase in the proportion of the community living there could have caused a shift to high light adaptation and thus lower α. As discussed above, community composition of BMA shifted away from cyanobacteria toward diatoms in the N additions treatments. Shifts in community composition in response to changes in nutrient conditions have been described in field studies of BMA before (Pinckney et al., 1995). Underwood and Barnett (2006) underscored the difficulty of separating nutrient effects from the multiple covariables that affect estuarine BMA. In this study, measuring changes at the functional group level had the advantage of providing information about changes in particular BMA with different ecological roles. The shift away from N2 fixing cyanobacteria and toward diatoms in the field experiment was statistically significant and in the laboratory experiment it was nearly so (p = 0.07). In the field experiment the decrease in N2 fixing cyanobacteria (Anabaena sp.) corresponded with increases in the counts of both b20 μm pennate and b20 centric diatoms. Our data suggest that diatoms compete better with cyanobacteria in conditions of nutrient enrichment. The higher growth rate potential of diatoms likely allowed them to increase their relative abundance with the availability of additional N. Alterations in the composition of the BMA community may affect the functional roles of BMA in the intertidal ecosystem, including food webs and nutrient cycling. Although some research has suggested that diatoms are generally more palatable than cyanobacteria, there is also evidence that benthic cyanobacteria respond positively to a reduction in grazing (Carman et al., 1997; Sullivan and Currin, 2000). Cyanobacteria capable of N2 fixation may fill an important and distinctive role in the sandflat N cycle by providing a new source of N to the sediment surface (Piehler et al., 1998). Nitrogen additions not only induced a significant shift in BMA functional groups, but also affected centric diatoms, a group of microalgae that are most often associated with the phytoplankton. This result provides another example of the difficulty of distinguishing phytoplankton and BMA in shallow ecosystems, as it is unclear whether the increase in centric diatoms represented a real contribution to the BMA community or only an increased retention of phytoplankton in surface sediments (Rusch and Huettel, 2000). This effect was only observed in the field experiment, perhaps lending support to the phytoplankton retention in surface sediments explanation. Changes in BMA communities that resulted from N additions led to changes in the sandflat environment. In the field experiment, N additions led to increases in pennate diatom abundance along with decreased porewater phosphate concentration and increased porewater ammonium concentration. Although N additions were applied to the sediment surface, the largest differences in porewater ammonium and phosphate concentrations were seen at depths 2 or 4 cm below the surface, 2 and 5 weeks after the experiment began. We speculate that observed changes in nutrient concentrations at depth were due to reduced uptake of ammonium and increased uptake of phosphate at depth by vertically migrating pennate diatoms whose nitrogen but not phosphorous requirements were fulfilled through the surface N additions. While we did not specifically determine
whether vertical migration occurred in this study, pennate diatoms on nearby (∼ 400 m away) sandflats are dominated by vertically migrating pennate diatoms that can descend to N5 cm over a tidal cycle to reach deep nutrient pools (Kingston 1999). Thus, it seems reasonable to assume that the observed increase in pennate diatoms, a functional group defined by the ability to vertically migrate, was responsible for the reduction in P at depths well within their vertical migration amplitudes. Reduction in porewater P that may result from BMA responses to enhanced external N loading could have cascading effects on coastal biogeochemistry. Sundareshwar et al. (2003) found P to limit many bacterially mediated conversions of N. Among their findings was that denitrification was favored in P depleted conditions as compared to P replete conditions. Impacts of modified P supply on N biogeochemistry are likely to be complex, but our results provide an example of the indirect impacts that change in N supply could have on N cycling. Comparing the in situ field manipulation and the laboratory mesocosm experiments utilized in this study provided some insight into the applicability of mesocosm experiments for research on BMA. Our results were quite similar for field and laboratory experiments. There were small but insignificant increases in mean biomass), significant changes in photosynthetic parameters (Pbmax and α) and significant shifts in the abundance of functional groups of BMA. The factors that varied between the field and mesocosm experiments included hydrodynamics, presence of transient predators and the presence of deep burrowing predators. Given the compelling evidence for both top down and bottom up control of BMA communities (Lever and Valiela, 2005; Heck and Valentine, 2007) it is notable that our two experiments yielded similar results. In weighing the costs and benefits of field versus laboratory manipulations the typical considerations are cost, feasibility and representativeness. In this case the logistics of the field experiment were slightly more complicated, the costs were fairly similar and the representativeness of the incubations appeared to be comparable. This study provides evidence of N driven changes in intertidal sandflat BMA communities and demonstrates the potential for BMA to transfer water column nutrients to the sediment. In intertidal sandflats, augmented N supply may lead to higher rates of BMA primary productivity and changes in the composition of the community to include a larger proportion of diatoms than cyanobacteria. The implications of these changes include the potential for higher trophic level impacts and influences from the alteration of other resource levels (e.g. porewater phosphate concentrations). These results suggest that increased N supply to shallow coastal systems could affect processes that control important sources and sinks of N such as denitrification and N2-fixation. Responses in the field and mesocosm experiments were similar, suggesting that container experiments are an appropriate experimental approach for research on estuarine BMA. Acknowledgements We thank V. Winklemann and J. Brewer for field and laboratory assistance. We acknowledge the NOAA Ecological Effects of Sea Level Rise Program and North Carolina Sea Grant for funding that contributed to the generation of this manuscript. We appreciate the editorial comments provided by W. Sunda and R. Waggett and the input of our anonymous reviewers. [SS] References Carman, K.R., Fleeger, J.W., Pomarico, S.M., 1997. Response of a benthic food web to hydrocarbon contamination. Limnol. Oceanogr. 42, 561–571. Fong, P., Donohoe, R.M., Zedler, J.B., 1993. Competition with macroalgae and benthic cyanobacterial mats limits phytoplankton abundance in experimental mesocosms. Mar. Ecol. Prog. Ser. 100, 97–102. Hauxwell, J., Cebrian, J., Furlong, C., Valiela, I., 2001. Macroalgal canopies contribute to eelgrass (Zostera marina) decline in temperate estuarine ecosystems. Ecology 82, 1007–1022.
M.F. Piehler et al. / Journal of Experimental Marine Biology and Ecology 390 (2010) 99–105 Heck, K.L., Valentine, J.F., 2007. The primacy of consumers in benthic ecosystems of the coastal zone. Estuar. Coasts 30, 371–381. Hillebrand, H., Kahlert, M., 2002. Effect of grazing and water column nutrient supply on biomass and nutrient content of sediment microalgae. Aquat. Bot. 72, 143–159. Howarth, R.W., 1988. Nutrient limitation of net primary production in marine ecosystems. Annu. Rev. Ecol. Syst. 19, 89–110. Kingston, M.B., 1999. Wave effects on the vertical migration of two benthic microalgae: Hantzchia virgata var. intermedia and Euglena proxima. Estuaries 22, 81–91. Lever, M.A., Valiela, I., 2005. Response of microphytobenthic biomass to experimental nutrient enrichment and grazer exclusion at different land-derived nitrogen loads. Mar. Ecol. Prog. Ser. 294, 117–129. Lewis, M.R., Smith, J.C., 1983. A small volume, short-incubation-time method for measurement of photosynthesis as a function of incident irradiance. Mar. Ecol. Prog. Ser. 13, 99–102. Litaker, W., Duke, C., Kenney, B., Ramus, J., 1993. Short-term environmental variability and phytoplankton abundances in a shallow tidal estuary. II. Spring and fall. Mar. Ecol. Prog. Ser. 94, 141–154. MacIntyre, H.L., Geider, R.J., Miller, D.C., 1996. Microphytobenthos: the ecological role of the “secret garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance, and primary production. Estuaries 19, 186–201. MacIntyre, H.L., Lomas, M.W., Cornwell, J.C., Suggett, D.J., Koch, E.W., Gobler, C.J., Kana, T.M., 2004. Mediation of benthic-pelagic coupling by microphytobenthos: an energy- and material-based model for initiation of blooms of Aureococcus anophagefferens. Harm. Alga. 3, 403–438. Moore, D.S., McCabe, G.P., 1993. Introduction to the Practice of Statistics. W.H. Freeman and Co., New York. National Research Council, 2000. Clean Coastal Waters: Understanding and Reducing the Effects of Nutrient Pollution. National Academy Press, Washington, D.C. Nixon, S.W., 1995. Coastal marine eutrophication: a definition, social causes, and future concerns. Ophelia 41, 199–219. Paerl, H.W., 1998. Structure and function of anthropogenically altered microbial communities in coastal waters. Curr. Opin. Microbiol. 1, 296–302. Peterson, C.H., Able, K.W., Frieswyk DeJong, C., Piehler, M.F., Simenstad, C.A., Zedler, J.B., 2008. Practical proxies for tidal marsh ecosystem services. Adv. Mar. Biol. 54, 221–266. Piehler, M.F., Currin, C.A., Casanova, R., Paerl, H.W., 1998. Development and N2-fixing activity of the benthic microalgal community in transplanted Spartina alterniflora marshes in North Carolina. Rest. Ecol. 6, 290–296. Piehler, M.F., Winkelmann, V., Twomey, L.J., Hall, N.S., Currin, C.A., Paerl, H.W., 2003. Impacts of diesel fuel exposure on the microphytobenthos of an intertidal sandflat. J. Exp. Mar. Biol. Ecol. 297, 217–235.
105
Pinckney, J., Paerl, H., Fitzpatrick, M., 1995. The impacts of seasonality and nutrients on microbial mat community structure and function. Mar. Ecol. Prog. Ser. 123, 207–216. Posey, M.H., Alphin, T.D., Cahoon, L.B., Lindquist, D.G., Mallin, M.A., Nevers, M.B., 2002. Top-down versus bottom-up limitation in benthic infaunal communities: direct and indirect effects. Estuaries 25, 999–1014. Rusch, A., Huettel, M., 2000. Advective particle transport into permeable sediments — evidence from experiments in an intertidal sandflat. Limnol. Oceanogr. 45, 525–533. Sullivan, M.J., Currin, C.A., 2000. Community structure and functional dynamics of benthic microalgae in salt marshes. In: Weinstein, M.P., Kreeger, D.A. (Eds.), Concept and Controversies in Tidal Marsh Ecology. Kluwer Academic Publishers, Dordrecht, pp. 81–106. Sundareshwar, P.V., Morris, J.T., Koepfler, E.K., Fornwalt, B., 2003. Phosphorus limitation of coastal ecosystem processes. Science 299, 563–565. Sundbäck, K., Enoksson, V., Granéli, W., Pettersson, K., 1991. Influence of sublittoral microphytobenthos on the oxygen and nutrient flux between sediment and water: a laboratory continuous-flow study. Mar. Ecol. Prog. Ser. 74, 263–279. Sundbäck, K., Linares, F., Larson, F., Wulff, A., Engelsen, A., 2004. Benthic nitrogen fluxes along a depth gradient in a microtidal fjord: the role of denitrification and microphytobenthos. Limnol. Oceanogr. 49, 1095–1107. Thingstad, T.F., Strand, E., Larsen, A., 2010. Stepwise building of plankton functional type (PFT) models: a feasible route to complex models? Prog. Oceanogr. 84, 6–15. Tyler, A.C., McGlathery, K.J., Anderson, I.C., 2003. Benthic algae control sediment–water column fluxes of organic and inorganic nitrogen compounds in a temperate lagoon. Limnol. Oceanogr. 48, 2125–2137. Underwood, G.J.C., Barnett, M., 2006. What determines species composition in microphytobenthic biofilms? In: Kromkamp, J.C., de Brouwer, J.F.C., Blanchard, G.F., Forster, R.M., Créach, V. (Eds.), Functioning of Microphytobenthos in Estuaries. Royal Netherlands Academy of Arts and Sciences, Amsterdam. Underwood, G.J.C., Kromkamp, J., 1999. Primary production by phytoplankton and microphytobenthos in estuaries. Adv. Ecol. Res. 29, 93–153. Underwood, G.J.C., Phillips, J., Saunders, K., 1998. Distribution of estuarine benthic diatom species along salinity and nutrient gradients. Eur. J. Phycol. 33, 173–183. Utermöhl, H., 1958. Zur Vervollkommning der quantitativen Phytoplankton-Methodik. Mitt. Int. Verein. Theor. Agnew. Limnol. 9, 1–38. Valiela, I.A., 1995. Marine Ecological Processes Second Edition Springer. van Raalte, C.D., Valiela, I., Teal, J.M., 1976. Production of epibenthic salt marsh algae: light and nutrient limitation. Limnol. Oceanogr. 21, 862–872. Welschmeyer, N.A., 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnol. Oceanogr. 39, 1985–1992.