Effects of Nutrient Enrichment on Distichlis Spicata and Salicornia Bigelovii in a Marsh Salt Pan

WETLANDS, Vol. 28, No. 3, September 2008, pp. 760–775 ’ 2008, The Society of Wetland Scientists

EFFECTS OF NUTRIENT ENRICHMENT ON DISTICHLIS SPICATA AND SALICORNIA BIGELOVII IN A MARSH SALT PAN

Amy Hunter1,2,4,5,NicoleM.B.Morris1,5,Ce´line Lafabrie1,3, and Just Cebrian1,3,6 1Dauphin Island Sea Lab 101 Bienville Blvd. Dauphin Island, Alabama, USA 36528

2Department of Biological Sciences, University of Alabama Tuscaloosa, Alabama, USA 35487

3Department of Marine Sciences, University of South Alabama Mobile, Alabama, USA 36688

4Present address: Toxicological and Environmental Associates, Inc. 775 North University Boulevard, Suite 260 Mobile, Alabama, USA 36608

Abstract: We investigated how nutrient addition affects the abundance, nutrient storage, and competition between Distichlis spicata and Salicornia bigelovii, two dominant species in salt pans of Northern Gulf of Mexico marshes. Namely, we compared fertilized and unfertilized plots in monospecific areas colonized respectively by D. spicata or S. bigelovii, and in a mixed area colonized by the two species. Nutrient addition generally increased the aboveground biomass and percent cover of the two species, and those increases were moderate to large in relation to the increases found for other marsh plant species. Nutrient addition also generally decreased the carbon:nitrogen and carbon:phosphorus ratios of aboveground and belowground tissues of the two species. Our results provide evidence that, under enhanced nutrient availability, D. spicata is a superior competitor over S. bigelovii in the mixed zone of the salt pan where the two species grow together. However, we did not detect large changes in biomass dominance by D. spicata following fertilization, possibly because the experiment only lasted 10 months. Our results suggest that nutrient addition, by increasing the structural complexity of the leaf canopy and the nutritional quality of plant tissues for first-order consumers, may enhance the value of salt pans as habitat for organisms

Key Words: competition, eutrophication, Gulf of Mexico, nutrient storage

INTRODUCTION clonal succulent plants. Clonal plants have rhizomes and therefore should better compete for nutrients. Plant community structure in salt marshes is However, succulent plants store water in their leaves affected by competition for light and nutrients and and thus may be more tolerant to water stress. stress induced by varying flooding frequency and Clonal and non-clonal plants in marsh salt pans salinity levels (Bertness 1991, Pennings and Call- utilize different strategies of growth, water conser- away 1992, Levine et al. 1998, Emery et al. 2001). vation, and nutrient storage. Ecological theory suggests that the interaction As human populations continue to grow and alter between competitive success and stress tolerance coastal watersheds, the delivery of nutrients into defines the niche for many plant species (Grime coastal marshes also increases (Valiela et al. 1992, 1979, Wilson and Keddy 1986). For example, in the Nixon 1995). This phenomenon, known as anthro- harsh environment of a marsh salt pan, character- pogenic eutrophication, is one of the most pervasive ized by low rates of tidal recharge and high human-induced stressors in coastal ecosystems evaporation rates, clonal plants coexist with non- world-wide (Jackson et al. 2000, Tilman et al. 2001). Previous research has examined how in- 5 These two authors contributed equally to this paper creased nutrient availability affects marsh plants, 6 Email: [email protected] but few studies have focused on the species that

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grow in salt pans (Valiela et al. 1975, Jefferies and with S. bigelovii to then give way to a stand of S. Perkins 1977, Covin and Zedler 1988). Salt pans bigelovii highest in the salt pan that fringes the provide a good setting to test the effects of increased upland maritime forest. nutrient availability on interactions between co- occurring species with different competitive abilities Experimental Design and stress tolerance. It is also of importance to examine whether salt pans, by partially absorbing Within each of the three zones (i.e., monospecific nutrient inputs from the surrounding watershed, can D. spicata,mixedD. spicata- S. bigelovii,and moderate the impacts of anthropogenic eutrophica- monospecific S. bigelovii) we fertilized six plots tion on coastal waters (Stout 1984, Tobias et al. and left six more unfertilized (controls). We paired 2001b, Mitsch and Gosselink 2007). the plots in each zone to minimize the influence of Here we focus on Distichlis spicata and Salicornia environmental patchiness within the zone, with each bigelovii, two plant species that co-occur abundantly pair having one fertilized and one control plot in salt pans of the Northern Gulf of Mexico. selected randomly. Plots were 1 3 1m2 and Distichlis spicata is a perennial clonal plant that separated 2 m from each other within a pair. occupies the seaward zone of the salt pan and Adjacent pairs were 10 m apart. spreads vegetatively through runners (Bertness To fertilize the plots, we applied 56 g nitrogen (N) 1991). Salt glands, leaf morphology, and water m22 every two weeks from early October to late reallocation via rhizomes are all thought to play a November 2003, and from early February to mid role in D. spicata’s salt tolerance (Hansen et al. 1976, April 2004. Based on measurements taken before Alpert 1990). Salicornia bigelovii is usually situated starting the experiment, we estimated this addition upland of the D. spicata zone and thrives in the salt would increase the ambient N concentration in the pan environment through succulence (Stout 1984). soil porewater approximately seven-fold, which Like other annual Salicornia species, S. bigelovii reflects a realistic level of human-induced nutrient colonizes bare areas by spreading seeds (Ungar enrichment in coastal marshes while not being large 1987, Alexander and Dunton 2002). In many salt enough to produce ammonium toxicity (Levine et al. pans, as the one studied in this paper, a mixed zone 1998, Valiela et al. 2000, 2001). In addition, and also where the two species grow together can be found. based on the same preliminary measurements, we 22 Our goals were to evaluate the effects of nutrient applied 7 g phosphorus (P) m simultaneously with addition on the abundance, nutrient storage and the N in order to reach an atomic N:P ratio in the competition between D. spicata and S. bigelovii in a porewater of the fertilized plots that approximated marsh salt pan in the Northern Gulf of Mexico. We the ratio found in the tissues of marsh plants (mean expected that nutrient addition, by relieving nutrient value of 16:1; Wetzel 1983, Thorman and Bayley limitation, would promote plant abundance and 1997). Thus, this procedure represents a realistic, nutrient accumulation as plant biomass. We also stoichiometrically balanced N and P enrichment. expected that D. spicata, owing to its higher storage Nutrients were added by hand-casting NH4NO3 and capacity and perennial life cycle, would be a superior P2O5. In April 2004, a few months before concluding competitor under enhanced nutrient availability. the experiment, we stopped fertilizing the plots because we found incipient signs of deleterious impacts due to overfertilization (e.g., necrotic or METHODS yellow leaf tips). Nevertheless, we continued to find Study Site increased nutrient concentrations in the porewater of fertilized plots in relation to control plots for the The salt pan studied is part of a larger marsh remainder of the experiment (see results). community located in the Northern Gulf of Mexico at Point Aux Pins, Bayou le Batre, Alabama, USA Variables Measured (30u229280 N, 88u189520 W). The lower marsh is dominated by Juncus roemerianus, with a band of Sediment porewater was collected in each plot five Spartina alterniflora at the water edge. The J. times over the course of the experiment (October roemerianus zone gives way to an extensive salt and November 2003; March, April, and June 2004) pan at the upper edge of the marsh that borders from wells made of 2.5 cm-diameter PVC pipe with maritime pine forest. There are three distinct zones mesh-covered holes in the lower 10 cm of the pipe. in the salt pan studied. Lowest in the salt pan, a At the beginning of the experiment, one well was stand of D. spicata borders the J. roemerianus zone. secured 15 cm into the sediment at the center of each Higher in the salt pan, D. spicata becomes mixed plot and left in place throughout the duration of the

Wetlands wetl-28-03-21.3d 23/6/08 14:32:23 761 Cust # 06-149 762 WETLANDS, Volume 28, No. 3, 2008 experiment. On each sampling date, the porewater aboveground S. bigelovii was separated into succu- within the well was pumped out with a syringe into lent young stems (fleshy cortex tissue that is green or acid-washed plastic vials and stored on ice. Upon red in color) and the woody cork layer (Fahn and return to the laboratory, all samples were filtered Arzee 1959, Boyer et al. 2001). Plant tissues were through 25 mm Whatman GF/F filters and frozen dried at 90uC for a minimum of 48 hours and 2 2 until analysis for nitrite (NO2 ), nitrate (NO3 ), ground using a Wiley Mill. A Carlo Erba Auto + + ammonium (NH4 ), and phosphate (PO4 ), which Analyzer (NA1500 N/C/S) and Costech Elemental was conducted following standard wet chemical Combustion System (ECS 4010 CHNS-O Analyzer) techniques (Strickland and Parsons 1972) modified were used to determine C:N ratios. Phosphorus for the Skalar SAN+ Autoanalyzer. concentrations were determined by measuring the In November 2003 and July 2004 we measured phosphorus-specific absorbance of the samples in a above- and belowground plant biomass in each plot Shimadzu UV-160 spectrophotometer (Strickland (grams dry weight 400 cm22) by haphazardly and Parsons 1972, Solorzano and Sharp 1980, tossing a 20 3 20 cm2 quadrat in the plot once Fourqurean et al. 1992). To derive C:P ratios, P and harvesting all enclosed above- and belowground concentrations were combined with the C concen- material. Samples were returned to the laboratory trations obtained with the Carlo Erba Auto and washed over a sieve. For each of the two species, Analyzer and Costech Elemental Combustion Sys- plant tissue was separated into above- and below- tem. ground components and dried at 90uCfora minimum of 48 hours. Data Analysis Percent plant cover was estimated from early October through late November 2003 and again We repeatedly sampled the same plants for our from early February through late June 2004. To measurements of percent cover. In addition, the estimate percent plant cover, a 1 3 1m2 quadrat plants sampled for biomass and nutrient content at was divided into 25 equal subquadrats, the quadrat different times, particularly for D. spicata,could was placed over each plot, and plant dominance have been connected and corresponded to the same within each subquadrat was visually estimated. The genet (i.e., genetic individual). Thus, we used three- subquadrat was considered to be dominated by a way repeated measures ANOVA to analyze the species if the species occupied . 50% of the data. To confirm that nutrient concentrations in the subquadrat area. Thus, each subquadrat was cate- sediment porewater were higher for fertilized than gorized as ‘‘dominated by D. spicata’’, ‘‘dominated for control plots, we ran three-way repeated by S. bigelovii’’, or ‘‘unvegetated’’. From the number measures ANOVA with treatment (fertilized or of subquadrats in each category, values of percent control plots) and zone (D. spicata monospecific, cover for the entire 1 3 1m2 quadrat were S. bigelovii monospecific, or mixed) as the between- calculated for the given species in monospecific subject factors and time as the within-subject factor. plots and for each species in mixed plots (i.e., To examine the effects of nutrient addition on the percent cover by a species in a plot 5 (number of abundance and nutrient storage in D. spicata and S. subquadrats dominated by the species/total number bigelovii, we ran three-way repeated measures of subquadrats) 3 100). This technique may ANOVA for each species separately with treatment underestimate percent plant cover (i.e., subquadrats and zone (monospecific or mixed) as between- where no species covered . 50% of the area were subject factors and time as the within-subject factor. considered unvegetated) and it can only detect To examine the effects of nutrient addition on the relatively large changes in percent plant cover (e.g., competition between the two species, we ran three- achangefrom10% to 40% cover by a species in a way repeated measures ANOVA with treatment and subquadrat is not recorded). However, these limita- species (D. spicata or S. bigelovii) as the between- tions did not affect the conclusions reached in this subject factors and time as the within-subject factor. study (see first paragraph in discussion section). Normality was evaluated using normal probabil- Carbon:nitrogen (C:N) and carbon:phosphorus ity plots and the Shapiro-Wilk statistic (Zar 1999). (C:P) atomic ratios in the above- and belowground Homogeneity of variance was determined using components of each species were measured four Levene’s test. Data were log transformed to meet times during the course of the experiment: October the above requirements except for percent cover and November 2003 and April and July 2004. We data which were arcsine transformed. In addition, haphazardly collected three individuals of each Mauchly’s criterion was used to confirm that species from each plot on each date and separated sphericity, a requirement of repeated measures the above- and belowground components. In turn, analysis, was met (Crowder and Hand 1990). All

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Salicornia bigelovii belowground biomass did not differ between fertilized and control plots at the beginning of the experiment. At the end of the experiment, fertilized plots showed higher S. bigelovii belowground biomass than control plots, but only in the monospecific zone (Figure 2, P , 0.05 for interaction term between treatment, zone, and time). Distichlis spicata aboveground biomass did not differ between control and fertilized plots at the beginning of the experiment, but was higher in fertilized plots at the end of the experiment (Figure 2, P , 0.05 for interaction term between treatment and time). The increase in D. spicata aboveground biomass with fertilization was similar in the monospecific and mixed zones (P $ 0.05 for interaction term between treatment and zone). In contrast, nutrient addition did not increase the belowground biomass of D. spicata (Figure 2, P $ 0.05 for all nutrient effects). Figure 1. Mean (6 SE) porewater dissolved inorganic nitrogen (DIN) and phosphate concentrations. Percent Cover. During the middle stages of the experiment, nutrient addition increased the percent statistical analyses were done using SYSTAT 10 and cover of S. bigelovii in the monospecific zone, but JMP 5.0.1 and results were considered significant at not in the mixed zone (Figure 3, P , 0.05 for p , 0.05. interaction term between treatment, zone, and time). Specifically, S. bigelovii covered 100% of the ground in control and fertilized plots in the monospecific RESULTS zone at the beginning of the experiment. One month Fertilization later senescence began and S. bigelovii covered ca. 90% of the ground in both types of plots. We For both control and fertilized plots, N and P resumed percent cover measurements in February concentrations in the sediment porewater were 2004 and, from that date through mid April 2004, higher during the last stage than at the beginning fertilized plots had higher S. bigelovii percent cover of the experiment (Figure 1; P , 0.05 for main time values than control plots in the monospecific zone effect, P $ 0.05 for interaction term between (one post-hoc comparison between control and treatment and time), probably due to little rainfall fertilized plots for each of those sampling dates, P from March 2004 through the end of the experi- , 0.05 for all comparisons). From May 2004 to the ment.Regardlessofthistemporaltrend,sediment end of the experiment, control and fertilized plots in porewater N and P concentrations were higher in the the monospecific zone showed similar S. bigelovii fertilized than in the control plots throughout the percent cover. We did not find any significant experiment (P , 0.05 for main treatment effect), differences in S. bigelovii percent cover between thereby demonstrating that we successfully enriched control and fertilized plots in the mixed zone. the fertilized plots. Fertilized plots showed higher D. spicata percent cover than control plots from mid March to early Effects of Fertilization on the Abundance and April 2004 in the mixed zone (Figure 3, P , 0.05 for Nutrient Storage by D. spicata and S. bigelovii interaction term between treatment, zone, and time; one post-hoc comparison between control and Biomass. Salicornia bigelovii aboveground biomass fertilized plots for each of those sampling dates, P did not differ between control and fertilized plots at , 0.05 for all comparisons). Control and fertilized the beginning of the experiment. Fertilized plots plots in the monospecific zone featured ca. 100% D. showed higher S. bigelovii aboveground biomass spicata cover throughout the duration of the than control plots at the end of the experiment, and experiment. that increase was much larger in the monospecific than in the mixed zone (Figure 2, P , 0.05 for Carbon:Nitrogen Ratio. The Carbon:Nitrogen interaction term between treatment, zone, and time). (C:N) ratio of the succulent tissue of S. bigelovii

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Figure 2. Mean (6 SE) above- and belowground biomass of Salicornia bigelovii and Distichlis spicata.

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Figure 3. Mean (6 SE) percent cover of Salicornia bigelovii and Distichlis spicata. In February, March and April 2004 percent cover was measured twice per month.

Wetlands wetl-28-03-21.3d 23/6/08 14:32:41 765 Cust # 06-149 766 WETLANDS, Volume 28, No. 3, 2008 did not differ between fertilized and control plots on throughout the experiment (Figure 5; P , 0.05 for the first two sampling dates (October and November main fertilization effect, P $ 0.05 for interaction 2003, Figure 4), but the ratio was lower in the term between treatment and time), and that decrease fertilized plots on the last two sampling dates (April was similar in the monospecific and mixed zones (P and July 2004; P , 0.05 for interaction term between $ 0.05 for interaction term between treatment and treatment and time, P , 0.05 for the post-hoc zone). Nutrient addition also decreased the C:P ratio comparisons between control and fertilized plots in of D. spicata belowground tissues throughout the April and July 2004, P $ 0.05 for the post-hoc experiment, but only in the mixed zone (Figure 5; P comparisons in October and November 2003). The , 0.05 for the interaction term between treatment decrease in the ratio due to fertilization was similar and zone). in the monospecific and mixed zones (P $ 0.05 for interaction term between treatment and zone). Effects of Fertilization on the Competition between We could only analyze the C:N ratios in S. bigelovii D. spicata and S. bigelovii woody tissue in October and November 2003 because sufficient woody tissue had not yet formed Biomass. Distichlis spicata reached a higher above- by April and May 2004. We did not find a significant ground and belowground biomass than S. bigelovii impact of nutrient addition for that restricted data in the mixed zone (Figure 2). By the end of the set (Figure 4, P $ 0.05 for all fertilization effects). experiment, fertilization seemed to have increased We did not sample enough S. bigelovii belowground aboveground biomass to a greater extent for D. tissue for nutrient analysis in mixed plots in July spicata than for S. bigelovii in that zone. That 2004. For the other three sampling dates, lower C:N observation, however, was not significant (P , 0.05 ratios were found in fertilized than in control plots for main fertilization effect, P $ 0.05 for interaction (Figure 4; P , 0.05 for main fertilization effect, P $ term between treatment, species, and time). Nutrient 0.05 for interaction term between treatment and addition did not have any significant effects on the time). That decrease was similar in the monospecific belowground biomass of either species in the mixed and mixed zones (P $ 0.05 for interaction term zone (Figure 2, P $ 0.05 for all treatment effects). between treatment and zone). Percent cover. Distichlis spicata had higher percent Nutrient addition decreased the C:N ratio of D. cover than did S. bigelovii in the mixed zone spicata aboveground tissue, and that decrease was (Figure 3). When comparing the impact of nutrient greater as the experiment progressed (Figure 4; P , addition on the percent cover by the two species in 0.05 for interaction term between treatment and the mixed zone, we found no effect on S. bigelovii time). In addition, the decrease was greater in the and an apparent, nearly significant increase in the mixed than in the monospecific zone (P , 0.05 for percent cover by D. spicata from mid March to early interaction term between treatment and zone). April 2004 (Figure 3; P 5 0.057 for interaction term Nutrient addition decreased the C:N ratio of D. between treatment, species, and time). spicata belowground tissue to a similar extent throughout the experiment (Figure 4; P , 0.05 for Carbon:Nitrogen Ratio. Nutrient addition de- main fertilization effect, P $ 0.05 for interaction creased the C:N ratios of the aboveground (succu- term between treatment and time) and in monospe- lent for S. bigelovii)tissuesofD. spicata and S. cific and mixed plots (P $ 0.05 for interaction term bigelovii in the mixed zone, with that decrease being between treatment and zone). almost significantly higher for the former than for the latter species (Figure 4; P , 0.05 for main Carbon:Phosphorus Ratio. Nutrient addition did fertilization effect, P 5 0.052 for interaction term not have any significant impacts on the C:P ratios of between treatment and species). Nutrient addition S. bigelovii succulent or woody tissues (Figure 5, P also decreased the C:N ratios of the belowground $ 0.05 for all fertilization effects). However, S. tissues of D. spicata and S. bigelovii in the mixed bigelovii belowground tissues showed lower C:P zone (comparison only includes October 2003, ratios in fertilized than in control plots in October November 2003, and April 2004), and the extent and November 2003, and that decrease was greater of the decrease was similar in the two species in the monospecific than in the mixed zone in (Figure 4; P , 0.05 for main fertilization effect, P October 2003, but vice-versa in November 2003 $ 0.05 for interaction term between treatment and (Figure 5, P , 0.05 for interaction term between species). treatment, zone, and time). Nutrient addition decreased the C:P ratio of D. Carbon:Phosphorus Ratio. When comparing the spicata aboveground tissues to a similar extent two species in the mixed zone, we found no effect

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Figure 4. Mean (6 SE) C:N ratios in Salicornia bigelovii and Distichlis spicata. Woody tissue of S. bigelovii had not yet formed in April and July 2004. We did not sample enough S. bigelovii belowground tissue for nutrient analysis in mixed plots in July 2004.

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Figure 5. Mean (6 SE) C:P ratios in Salicornia bigelovii and Distichlis spicata.

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of nutrient addition on the C:P ratio of S. bigelovii We compiled data from the literature to compare succulent tissues, but an apparent, nearly significant the increases in aboveground plant biomass due to decrease in the C:P ratio of D. spicata aboveground nutrient addition found here with the results of other tissues, (Figure 5, P 5 0.069 for interaction term fertilization experiments in marsh habitats (Table 1). between treatment and species). Despite the signif- The compilation includes many plant species, icant effects of fertilization on the belowground C:P elevations (i.e., low, middle, and high marsh), and ratios found for the two species when analyzed geographical areas. Increases found in our salt pan separately, fertilization did not have any significant varied from moderate to large in relation to changes effects on these ratios when the two species were reported in other studies, as indicated by the ratio of analyzed concomitantly in the mixed zone (Figure 5, fertilized to unfertilized mean aboveground biomass. comparison only includes October 2003, November In relation to the other ratios in Table 1, the ratios 2003, and April 2004; P $ 0.05 for all treatment for D. spicata in the monospecific zone and for S. effects). bigelovii in the mixed zone were intermediate (i.e., approximately 50% of the ratios compiled lie below those two ratios), and the ratios for D. spicata in the DISCUSSION mixed zone and for S. bigelovii in the monospecific zone were high (i.e., approximately 85% of the ratios As expected, nutrient addition often increased the compiled lie below those two ratios). In general, aboveground biomass and percent cover of S. these two species showed ratios similar to or higher bigelovii and D. spicata in the marsh salt pan than most other species, except for species known to studied. We also found that nutrient addition respond quickly to nutrient addition such as generally decreased the carbon:nitrogen and car- Spartina alterniflora (Levine et al. 1998, Pennings bon:phosphorus ratios of D. spicata and S. bigelovii. et al. 2002). The only exceptions were the C:N ratios and C:P As it could be expected based on its rhizomatous ratios of S. bigelovii woody tissue, and the C:P ratios structure and perennial life cycle, our experiment of S. bigelovii succulent tissue, which remained suggests D. spicata isasuperiorcompetitorfor unaffected by fertilization. Our measurements of nutrient uptake and storage under enhanced nutri- percent cover may be underestimates and do not ent availability. This is based on two results. First, detect small changes. These limitations, however, do the aboveground C:N ratio and the belowground not affect our conclusions. It is clear that fertiliza- C:P ratio of D. spicata decreased with nutrient tion only increased D. spicata cover in mixed plots, addition to a greater extent in mixed plots than they since the species already covered 100% of the ground did in monospecific plots. This indicates that, when in monospecific plots. Fertilization could have D. spicata grew in the company of S. bigelovii,it somewhat increased S. bigelovii cover in mixed absorbed and stored nutrient inputs to a greater plots, but our results demonstrate that such a extent than it did when growing in the monospecific potential increase would certainly have been lower stand. Second, it appears that nutrient addition to than the increase in S. bigelovii cover in monospe- the mixed zone may decrease at times the above- cific plots and in D. spicata cover in mixed plots. ground C:N and C:P ratios to a greater extent for D. Increases in aboveground biomass due to fertil- spicata than for S. bigelovii. ization were not observed in November 2003, Sustained higher rates of nutrient uptake and probably because plants senesce in the fall and only storage for D. spicata than for S. bigelovii in the one month had elapsed since the onset of fertiliza- mixed zone under fertilized conditions should have tion. In accordance with our results, Boyer et al. led to an increase in the biomass dominance of the (2001) began fertilizing Salicornia virginica in the fall former over the latter species. Accordingly, 10 and did not find an impact until the following months after starting our experiment, the increase spring. The only increase in belowground biomass in aboveground biomass in mixed plots due to due to fertilization was for S. bigelovii in the nutrient addition seemed to be larger for D. spicata monospecific zone. We may not have found an than for S. bigelovii, although that observation was increase in D. spicata belowground biomass under not statistically significant. Some of our other results enhanced nutrient availability because rhizomes may also suggest that D. spicata is a superior competitor absorb and concentrate extra nutrients for above- under enhanced nutrient availability and, given ground translocation rather than developing new enough time, should outgrow S. bigelovii.For belowground biomass and diluting the nutrients instance, fertilization did increase at times the cover (Pitelka and Ashmun 1985, Silvertown and Lovett- of D. spicata, but never increased the cover of S. Doust 1993). bigelovii, in the mixed zone. Salicornia bigelovii only

Wetlands wetl-28-03-21.3d 23/6/08 14:33:28 769 Cust # 06-149 770 WETLANDS, Volume 28, No. 3, 2008 ed and plants. All responds to : Russia; SE: MM 0.02 0.88 MM 0.49 0.51 M0.44* M0.98* M 2.98 M 5.88 M4.30* M2.87* MM 2.91 1.28 M 0.99 ) m 1.93 This work 5 PO 2 ;P 3 NO 4 ))M0.96 M 0.77 Jefferies and Perkins 1977 )M0.77 )M0.60 4 4 3 3 NPK m 0.84 Emery et al. 2001 NPK m 1.03 Emery et al. 2001 NPKNPK m m 1.00 1.06* Van der Graaf et al. 2007 Levine et al. 1998 NPK m 3.45 Emery et al. 2001 NPK m 1.97 Emery et al. 2001 NPK m 2.36* Levine et al. 1998 PM1.54 N(NH PM0.92 N(NO N(NO NPK M 1.40 Pennings et al. 2002 :tallform. uu England, USA England, USA Sea, NL England, USA England, USA England, USA England, USA Rhode Island (Nag Cove West), New Rhode Island (Nag Cove East), New Sapelo Island, Georgia, USAIsland of Gotland, BalticIsland Sea, of SE Schiermonnikoog, Wadden Rhode Island (Rumstick Cove), New NPK NPK M m 2.00 1.00 Pennings Van et der al. Graaf 2002 et al. 2007 Rhode Island (Nag Cove West), New Rhode Island (Nag Cove East), New Rhode Island (Rumstick Cove), New Sapelo Island, Georgia, USASapelo Island, Georgia, USA NPK NPK M M 3.39 0.42 Pennings et al. 2002 Pennings et al. 2002 Stiffkey, Norfolk, UK N (NH Kolokolkova Bay, Tobseda, RUGraveline Bayou, Mississipi, USAPoint aux Pins, Alabama, USA NPK NPK NP (NH M M 0.83 2.83 Van der Graaf et al. Pennings 2007 et al. 2002 : short form; u Species Location Nutrients MT Ratio Reference phryganodes Festuca rubra Juncus gerardi Batis maritima Borrichia frutescens Armeria maritima Carex subspathacea-Puccinellia Distichlis spicata Table 1. The ratio of increase in aboveground biomass with fertilization (biomass in fertilized conditions/biomass in control conditions) in marsh experiments were done in theunfertilized field conditions and at encompassed the atmonocultures end (m) least the or one growing mixed growing season. plots season.Sweden; Fertilized (M). The UK: Nutrients and ratio United refer unfertilized has to Kingdom; plots beenmean the USA: were calculated for specific open United with nutrients 1971, to the States added 1972 grazers mean to of and peak in the America; fertilized biomass 1973; all NL: plots. under experiments. The Other fertiliz Marsh abbreviations Netherlands; type and HD: (MT) symbols High cor are: RU Dosage; LD: Low Dosage; *: mean for 1993 and 1994; **:

Wetlands wetl-28-03-21.3d 23/6/08 14:33:28 770 Cust # 06-149 Hunter et al., EFFECTS OF FERTILIZATION ON SALT PAN PLANTS 771 Gallagher 1975 u u uu m2.58 m1.19 M4.42 M7.00 M0.75 M4.12 M6.00 M2.85 M1.38 M2.34* M3.60 M 1.66 Gough and Grace 1998 M0.89 ) m 3.96 This work 5 PO 2 ;P 3 ) m 1.11 Gallagher 1975 )m1.70 3 3 NO 4 NO ))M2.17 ) M) 1.67 Jefferies M and Perkins 1977 1.11 M Jefferies and Perkins 1977 2.00 Jefferies and Perkins 1977 NO )M1.26 )M2.33 )M1.30 )M2.00 4 4 4 4 4 4 3 3 3 3 NPK m 2.25 Emery et al. 2001 NPK m 1.85 Emery et al. 2001 N(NO N(NO N(NO N(NO NPK M 0.56 Pennings et al. 2002 NPK M 5.23 Pennings et al. 2002 NPK m 2.17* Levine et al. 1998 PM1.26 N(NH PM0.97 PM0.59 PM0.67 England, USA England, USA England, USA Sapelo Island, Georgia, USA N (NH Rhode Island (Nag Cove West), New Weeks Bayou, Mississipi, USAStiffkey, Norfolk, UK NPK N (NH M 2.24 Pennings et al. 2002 Weeks Bayou, Mississipi, USA NPK M 3.73 Pennings et al. 2002 Rhode Island (Nag Cove East), New Sapelo Island, Georgia, USATijuana Estuary, California, USAEast Pearl River, Louisiana, USAPoint aux Pins, Alabama, USARhode Island N (Rumstick Cove), New NPK NPK NPK M M M M 2.02 1.47 1.20 Pennings et Covin Gough al. and and 7.50 2002 Zedler Grace 1998 1988 Pennings et al. 2002 Stiffkey, Norfolk, UKEast Pearl River, Louisiana,Stiffkey, USA Norfolk, UKEast Pearl River, Louisiana, USA NPKMiddle Pearl N River, (NH Louisiana,Point USA aux Pins, Alabama, USA NPK N NPK (NH NP (NH M 3.95 M M Gough and Grace 1997 1.43 1.69 Gough and Grace 1997 Gough and Grace 1998 Graveline Bayou, Mississipi, USAPoint aux Pins, Alabama, USA NPK NPK M M 2.00 1.02 Pennings et al. 2002 Pennings et al. 2002 Sapelo Island, Georgia, USA N (NH Species Location Nutrients MT Ratio Reference spp. Graveline Bayou, Mississipi, USA NPK M 0.33 Pennings et al. 2002 Limonium vulgare Salicornia virginica Scirpus americanus Scirpus Spartina alterniflora Plantago maritima Polygonum punctatum Puccinellia maritima Sagittaria lancifolia Salicornia bigelovii Juncus roemerianus Table 1. Continued.

Wetlands wetl-28-03-21.3d 23/6/08 14:33:29 771 Cust # 06-149 772 WETLANDS, Volume 28, No. 3, 2008 MM0.81 10.02 M6.22 M0.38 M1.08 M0.69* M1.94* M0.92* M 3.00 Gough and Grace 1998 ) M 0.67 Jefferies and Perkins 1977 )M0.67 4 3 NPKLD NPK m M 2.02 Emery 2.20** et al. 2001 N(NO PM1.33 LD NPK M 2.31** NPK m 1.20 Emery et al. 2001 NPK m 2.00* Levine et al. 1998 England, USA England, USA England, USA Rhode Island (Nag Cove West), New Great Sippewissett, Massachusetts, USAStiffkey, Norfolk, UK HD NPK M N (NH 2.27** Valiela et al. 1975 Great Sippewissett, Massachusetts, USATijuana Estuary, California, USA HD NPK N M 3.69** Valiela et al. 1975 m 1.56 Covin and Zedler 1988 Rhode Island (Nag Cove East), New East Pearl River, Louisiana, USAGraveline Bayou, Mississipi, USAMiddle Pearl River, Louisiana,Rhode USA Island (Rumstick Cove), New NPK NPK NPK M M M 1.26 1.15 1.08 Gough and Grace 1997 Pennings et al. Gough 2002 and Grace 1998 Salicornia -( Species Location Nutrients MT Ratio Reference spp.) Spartina patens-Distichlis spicata Triglochin maritima Spartina foliosa Spartina alterniflora Spartina patens Table 1. Continued.

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Table 2. Changes in nitrogen (N) storage from September 2003 to July 2004. Samples of plant biomass and C:N ratios were also obtained in September 2003 before the start of fertilization. Nitrogen storage was calculated by multiplying mean biomass (gDW m22) times the mean ratio of N per DW. Geometric means were used in these calculations. The change in storage was then expressed as a percentage of the total quantity of N applied throughout the experiment (504 g N m22 6 month21). The belowground compartment of Salicornia bigelovii in the mixed area was not considered due to insufficient sampling for nutrient analysis.

gNm22 gNm22 Change in N Percentage of added N retained as Plant tissue and marsh zone September July storage plant biomass S bigelovii succulent tissue 0.36 7.61 7.25 1.4 (monospecific zone) S bigelovii belowground tissue 0.06 3.65 3.59 0.7 (monospecific zone) D. spicata aboveground tissue 4.36 14.60 10.24 2.0 (monospecific zone) D. spicata belowground tissue 20.72 25.24 4.52 0.9 (monospecific zone) S bigelovii succulent tissue 0.14 0.67 0.53 0.1 (mixed zone) D. spicata aboveground tissue 1.31 12.74 11.43 2.3 (mixed zone) D. spicata belowground tissue 3.54 15.33 11.79 2.3 (mixed zone)

increased its cover in response to fertilization in the fertilized conditions despite higher rates of nutrient monospecific zone. In addition, when the response uptake and storage by the former species. of S. bigelovii biomass to fertilization was compared Observed increases in aboveground plant biomass between the monospecific and mixed zones, fertil- and cover due to fertilization suggest that increased ization increased its aboveground biomass to a anthropogenic nutrient loading may increase the much greater extent in the monospecific than in the structural complexity of the plant canopy in salt mixed zone, and it increased its belowground pans, thereby providing better refuge to permanent biomass in the monospecific zone but not in the and transient residents (Stout 1984). In addition, mixed zone. higher plant biomass production could entail higher At any rate, we did not find a large change in the levels of food availability for the several species of biomass dominance of D. spicata over S. bigelovii in herbivores that feed on D. spicata (Pennings et al. the mixed zone as a result of nutrient addition, in 2001) and also for the invertebrate detritivores and contrast with the results of other studies. In a New microbial decomposers that inhabit the marsh soil England marsh, Levine et al (1998) showed that and whose primary food source is senesced marsh Spartina patents and Juncus gerardi dominated over plants (Zimmer et al. 2004). Higher nutrient S. alterniflora and D. spicata under unfertilized availability may also enhance seed production in conditions, but vice-versa under fertilized condi- salt pan plants (Boyer and Zedler 1999). Therefore, tions. In a comparative study including marshes in increased nutrient delivery could improve the value Georgia, Alabama, and Mississippi, Pennings et al. of marsh salt pans as habitat for organisms in the (2002) documented changes in biomass dominance Northern Gulf of Mexico. in four out of seven species mixtures following The general decrease in carbon:nutrient ratios in fertilization, with S. alterniflora and D. spicata often the tissues of the two species following fertilization becoming dominant over other marsh plant species suggests two important implications. First, the under fertilized conditions. Working in a salt marsh nutritional quality of D. spicata and S. bigelovii for in the Tijuany estuary, Covin and Zedler (1998) first-order consumers should increase with fertiliza- found that nutrient addition increased the biomass tion since the tissues of these two plants have a dominance of Salicornia virginica over Spartina larger quantity of nutrients per carbon unit when foliosa. Here, it may be that the duration of the nutrients are added. In turn, enhanced plant experiment (i.e., 10 months) was not long enough to nutritional quality following fertilization could induce large changes in the biomass dominance of promote the growth rates of the herbivores, D. spicata over S. bigelovii in the mixed zone under detritivores, and decomposers that feed on the

Wetlands wetl-28-03-21.3d 23/6/08 14:33:30 773 Cust # 06-149 774 WETLANDS, Volume 28, No. 3, 2008 plants (Sterner and Elser 2002, Cebrian and Lartigue Kiene for the use of his spectrophotometer, and 2004). Kate Sheehan for assistance with figure preparation. Second, higher nutrient concentrations in the Alina Corcoran, Laura Gough, Julie Olson, Mark tissues following fertilization, in conjunction with Hester, Darold Batzer, and two anonymous review- higher levels of biomass, suggests the plants studied ers provided valuable comments on this manuscript. can absorb a fraction of the nutrients added and, This research was funded by the Alabama Center for thus, potentially mitigate the negative impacts of Estuarine Studies (ACES) grant # 5-21854. A. anthropogenic eutrophication on coastal waters. To Hunter acknowledges funding from a University of further explore this, we estimated the percentage of Alabama Alumni Association Fellowship in Aquatic nitrogen added to the fertilized plots that was stored Biology and a Mississippi-Alabama Sea Grant as plant biomass over the course of the experiment. student fellowship. N. Morris acknowledges funding We did these calculations from the changes observed from the National Science Foundation Research in plant biomass and C:N ratios (Table 2). From Experience for Undergraduates (REU). October 2003 to April 2004, we added a total of 504 g nitrogen to each fertilized plot. On average, a LITERATURE CITED fertilized monospecific plot of S. bigelovii retained 2.1% of that total quantity as plant biomass-bound Alexander, H. D. and K. H. Dunton. 2002. Freshwater nitrogen, a monospecific plot of D. spicata retained inundation effects on emergent vegetation of a hypersaline marsh. Estuaries 25:1426–35. 2.9%, and a mixed plot retained a minimum of 4.7% Alpert, P. 1990. Water sharing among ramets in a desert (i.e., without including the S. bigelovii belowground population of Distichlis spicata (Poaceae). American Journal compartment, see Table 2). These numbers are of Botany 77:1648–51. Bertness, M. D. 1991. Interspecific interactions among high small. Nevertheless, the total percentage of added marsh perennials in a New England salt marsh. Ecology nitrogen intercepted by the salt pan before it moves 72:125–37. into lower areas of the marsh should be much Boyer, K. E., P. Fong, R. R. Vance, and R. F. Ambrose. 2001. Salicornia virginica in a Southern California salt marsh: higher. Our calculations do not account for plant seasonal patterns and a nutrient-enrichment experiment. biomass senescence and turnover throughout the Wetlands 21:315–26. experiment and, thus, they do not include the Boyer, K. E. and J. B. Zedler. 1999. Nitrogen addition could shift plant community composition in a restored California salt nitrogen retained as plant detritus in the salt pan. marsh. Restoration Ecology 7:74–85. In addition, a major fraction of nitrogen inputs into Cebrian, J. and J. Lartigue. 2004. Patterns of herbivory and marsh soils may be immobilized into particulate decomposition in aquatic and terrestrial ecosystems. Ecological organic matter or lost through bacterial denitrifica- Monographs 74:237–59. Covin, J. D. and J. B. Zedler. 1988. Nitrogen effects on Spartina tion (Tobias et al. 2001a,b). When these processes foliosa and Salicornia virginica in the salt marsh at Tijuana are invoked, the percentage of nitrogen inputs Estuary, California. Wetlands 8:51–65. intercepted by the salt pan studied could well be Crowder, M. J. and D. J. Hand. 1990. Analysis of Repeated Measures. Chapman and Hall, London, UK. within the range of estimates for other marshes (i.e., Emery, N. C., P. J. Ewanchuk, and M. D. Bertness. 2001. 40%–90%, Valiela et al. 1973, Howard et al. 1986, Competition and salt-marsh plant zonation: stress tolerators Tobias et al. 2001b). may be dominant competitors. Ecology 82:2471–85. Fahn, A. and T. Arzee. 1959. Vascularization of articulated This paper clearly shows that nutrient addition Chenopodiaceae and the nature of their fleshy cortex. increases the abundance and nutrient content of D. American Journal of Botany 46:313–26. spicata and S. bigelovii, and suggests a superior Fourqurean, J. W., J. C. Zieman, and G. V. N. Powell. 1992. Phosphorus limitation of primary production in Florida Bay: competitive ability for the former species under evidence from C:N:P ratios of the dominant seagrass. enhanced nutrient availability in marsh salt pans of Limnology and Oceanography 37:162–71. the Northern Gulf of Mexico. However, a longer Gallagher, J. L. 1975. Effect of an ammonium nitrate pulse on the growth and elemental composition of natural stands of study could have demonstrated a larger impact on Spartina alterniflora and Juncus roemerianus. American Journal plant abundance, higher nutrient retention as plant of Botany 62:644–48. biomass, and provided more support for the Gough, L. and J. B. Grace. 1997. The influence of vines on an superior competitive ability of D. spicata under oligohaline marsh community: results of a removal and fertilization study. Oecologia 112:403–11. fertilized conditions. Gough, L. and J. B. Grace. 1998. Herbivore effects on plant species density at varying productivity levels. Ecology 79:1586–94. ACKNOWLEDGMENTS Grime, J. P. 1979. Plant Strategies and Vegetation Processes. John Wiley & Sons, Inc., New York, NY, USA. We thank Alina Corcoran, Mairi Miller, Ryan Hansen, D. J., P. Dayanandan, P. B. Kaufmand, and J. D. Moody, Dustin Addis, David Patterson, and Sa- Brotherson. 1976. Ecological adaptations of salt marsh grass, Distichlis spicata (Gramineae), and environmental factors vannah Williams for assistance with field work, affecting its growth and distribution. American Journal of Laura Linn for help with nutrient analysis, Ron Botany 63:635–50.

Wetlands wetl-28-03-21.3d 23/6/08 14:33:31 774 Cust # 06-149 Hunter et al., EFFECTS OF FERTILIZATION ON SALT PAN PLANTS 775

Howard, R., R. Mikulak, R. Lord, J. Stout, M. Dardeau, C. Tilman, D., J. Fargione, B. Wolff, C. D’Antonio, A. Dobson, R. Lutz, E. Blancher, S. Massey, and J. L. Marsh. 1986. Howarth, D. Schindler, W. H. Schlesinger, D. Simberloff, and Utilization of a saltwater marsh ecosystem for the management D. Swackhamer. 2001. Forecasting agriculturally driven global of seafood processing wastewater. US Environmental Protec- environmental change. Science 292:281–84. tion Agency 904/9-86 142. Tobias, C. R., I. C. Anderson, E. A. Canuel, and S. A. Macko. Jackson, L. E., J. C. Kurtz, and W. S. Fisher (eds.). 2000. 2001a. Nitrogen cycling through a fringing marsh-aquifer Evaluation Guidelines for Ecological Indicators. US Environ- ecotone. Marine Ecology Progress Series 210:25–39. mental Protection Agency, Office of Research and Develop- Tobias, C. R., S. A. Macko, I. C. Anderson, E. A. Canuel, and J. ment, EPA/620/R-99/005. Research Triangle Park, North W. Harvey. 2001b. Tracking the fate of a high concentration Carolina. groundwater nitrate plume through a fringing marsh: a Jefferies, R. L. and N. Perkins. 1977. The effects on the vegetation combined groundwater tracer and in situ isotope enrichment of the additions of inorganic nutrients to salt marsh soils at study. Limnology and Oceanography 46:1977–89. Stiffkey, Norfolk. Journal of Ecology 65:867–82. Ungar, I. A. 1987. Population characteristics, growth, and Levine, J. M., J. S. Brewer, and M. D. Bertness. 1998. Nutrients, survival of the halophyte Salicornia europaea. Ecology competition, and plant zonation in a New England salt marsh. 68:569–75. Journal of Ecology 86:285–92. Valiela, I., M. L. Cole, J. McClelland, J. Hauxwell, J. Cebrian, Mitsch, W. J. and J. G. Gosselink. 2007. Wetlands. John Wiley & and S. Joye. 2001. Role of salt marshes as part of coastal Sons, Inc., New York, NY, USA. landscapes. p. 23–28. In M. P. Weinstein and D. A. Kreeger Nixon, S. W. 1995. Coastal marine eutrophication: a definition, (eds.) Concepts and Controversies in Tidal Marsh Ecology. social causes, and future concerns. Ophelia 41:199–219. Kluwer Academic Publishers, Dordrecht, The Netherlands. Pennings, S. C. and R. M. Callaway. 1992. Salt marsh plant zonation: the relative importance of competition and physical Valiela, I., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P. factors. Ecology 73:681–90. Peckol, B. DeMeo-Anderson, C. D’Avanzo, M. Babione, C.-H. Pennings, S. C., E. L. Siska, and M. D. Bertness. 2001. Sham, J. Brawley, and K. Lajtha. 1992. Couplings of coastal Latitudinal differences in plant palatability in Atlantic Coast watersheds and coastal waters: sources and consequences of salt marshes. Ecology 82:1344–59. nutrient enrichment in Waquoit Bay, Massachusetts. Estuaries Pennings, S. C., L. E. Stanton, and J. S. Brewer. 2002. Nutrient 15:443–57. effects on the composition of salt marsh plant communities Valiela, I., J. M. Teal, and W. J. Sass. 1973. Nutrient retention in along the Southern Atlantic and Gulf Coasts of the United salt marsh plots experimentally fertilized with sewage sludge. States. Estuaries 25:1164–73. Estuarine and Coastal Marine Science 1:261–69. Pitelka, L. F. and F. W. Ashmun. 1985. Physiology and Valiela, I., J. M. Teal, and W. J. Sass. 1975. Production and integration of ramets in clonal plants. p. 399–435. In J. B. C. dynamics of salt marsh vegetation and the effects of Jackson, L. W. Buss, and R. E. Cook (eds.) Population Biology experimental treatment with sewage sludge. Journal of Applied and Evolution of Clonal Organisms. Yale University Press, Ecology 12:973–82. New Haven, CT, USA and London, UK. Valiela, I., G. Tomasky, J. Hauxwell, M. L. Cole, J. Cebrian, and Silvertown, J. W. and J. Lovett-Doust. 1993. Introduction to K. D. Kroeger. 2000. Operationalizing sustainability: manage- Plant Population Biology. Blackwell Scientific, Oxford, UK. ment and risk assessment of land-derived nitrogen loads to Solorzano, L. and J. H. Sharp. 1980. Determination of total estuaries. Ecological Applications 10:1006–23. phosphorus and particulate phosphorus in natural water. Van der Graaf, A. J., J. Stahl, G. F. Veen, R. M. Havinga, and R. Limnology and Oceanography 25:756–60. H. Drent. 2007. Patchchoice of avian herbivores along a Sterner, R. W. and J. J. Elser. 2002. Ecological Stoichiometry: migration trajectory - from temperate to arctic. Basic and The Biology of Elements from Molecules to the Biosphere. Applied Ecology 8:354–63. Princeton University Press, Princeton, NJ, USA. Wetzel, R. G. 1983. Limnology. W. B. Sanders Company, Stout, J. P. 1984. The ecology of irregularly flooded salt marshes Philadelphia, PA, USA. of the northeastern Gulf of Mexico: a community profile. U. S. Wilson, S. D. and P. A. Keddy. 1986. Species competitive ability Fish and Wildlife Service, Biological Report 85 (7.1). Wa- and position along a natural stress/disturbance gradient. shington, DC, USA. Ecology 67:1236–42. Strickland, J. D. H. and T. R. Parsons. 1972. A practical Zar, J. H. 1999. Biostatistical Analysis, fourth edition. Prentice- handbook of seawater analysis. Bulletin of the Fisheries Hall, Englewood Cliffs, NJ, USA. Research Board of Canada 167:1–310. Zimmer, M., S. C. Pennings, T. L. Buck, and T. H. Carefoot. Thorman, M. N. and S. E. Bayley. 1997. Response of 2004. Salt marsh litter and detritivores: a closer look at aboveground net primary plant production to nitrogen and redundancy. Estuaries 27:753–69. phosphorus fertilization in peatlands in Southern Boreal Alberta, Canada. Wetlands 17:502–12. Manuscript received 6 October 2006; accepted 30 April 2008.

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