2009 NORTHEASTERN NATURALIST 16(3):395–408 Nutrient Limitation of Periphyton and Phytoplankton in Cape Cod Coastal Plain Ponds Maribeth Kniffin1, Christopher Neill2,*, Richard MCHorney2, and George Gregory3 Abstract - We investigated nutrient limitation of periphyton and phytoplankton in Cape Cod, MA coastal plain freshwater ponds. We assayed periphyton growth response to nitrogen (N) and phosphorus (P) in situ, assessed phytoplankton growth in growth chambers, and measured ratios of dissolved N and P in surface waters to determine if nutrient ratios were accurate predictors of nutrient limitation. In ponds receiving low groundwater nutrient inputs, periphyton responded weakly to addition of N or P alone, but responded strongly to addition of N+P. In these ponds, increases in both N and P were also required to increase phytoplankton growth. In ponds receiving high groundwater nutrient inputs, increased N+P were also required to increase phytoplankton growth. We found no indication that high nutrient inputs shifted pond phytoplankton to P limitation. There was no consistent correlation between dissolved N:P and whether periphyton was limited by either N or P or co-limited by N and P. Strong and widespread co-limitation by N and P suggested that greater attention should be given to both N and P when assessing the threat of nutrient enrichment to fresh waters. Effects of increased periphyton and phytoplankton growth on the unique flora of coastal plain ponds are not known, but bear increased attention given large increases of N in groundwater in many locations, the sensitivity of pond algae to nutrient supply, and the status of coastal plain ponds as high conservation priorities. Introduction Human-derived inputs of nutrients now alter rates of primary production and the trophic status of freshwater ecosystems in many regions on earth (Battarbee et al. 2005, Carpenter et al. 1998). Although the availability of phosphorus has been widely thought to limit algal productivity in most lakes (Dillon and Rigler 1974, Schindler 1977, Vollenweider 1976), evidence now indicates that the availability of nitrogen plays an important and underappreciated role as a control of primary production in fresh waters. Limitation by N or co-limitation by N and P may be widespread where natural inputs of both N and P historically have been low (Bergström et al. 2005, Elser et al. 1990, Maberly et al. 2002). There is also increasing evidence that even small increases in N supply caused by atmospheric N deposition can increase lake productivity and shift the trophic structure or the dominant limiting nutrient in oligotrophic lakes (Elser et al. 2007, Goldman et al. 1993, Maberly et al. 2002, Nydick et al. 2004, Sickman et al. 2003, Smith and Lee 2006, Wolfe et al. 2003). Because releases of reactive N to the biosphere from fossil fuel combustion, fertilizers, and leguminous crops have increased dramatically 1Smith College, Northampton, MA 01063. 2The Ecosystems Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543. 3Bates College, 2 Andrews Road, Lewiston, ME 04240. *Corresponding author - cneill@mbl.edu. 396 Northeastern Naturalist Vol. 16, No. 3 in recent decades (Galloway 2002, Vitousek et al. 1997), it is likely that the effects of increased N supply on the trophic status of lakes and ponds in many regions have been underestimated. Cape Cod and the coastal plain of southeastern Massachusetts contain naturally oligotrophic ponds and lakes which lie on nutrient-poor outwash deposits (LeBlanc et al. 1986). Many of these water bodies are kettle holes that have no inlet or outlet and undergo seasonal and inter-annual variations in water level of up to 3 m (McHorney and Neill 2007). Because periodic flooding of shoreline habitats maintains a diverse shoreline herbaceous flora (Keddy and Reznicek 1986, Sorrie 1994), coastal plain pond shorelines are one of the most restricted and imperiled plant communities in the northeastern US and Canada and are high priorities for conservation (Wisheu and Keddy 1989, Zaremba and Lamont 1993). Cape Cod and the coastal plain of southeastern Massachusetts are regions where residential development has expanded dramatically since the 1970s (Breunig 2003). Higher density of residences with on-site septic systems has increased the dissolved N concentrations of regional groundwater, increased the dissolved N and P concentrations of pond waters, and increased the number of ponds now classified as eutrophic (Cape Cod Commission 2003). Transport of N in groundwater occurs because the coastal plain lies over coarse-textured sediments that make up a single aquifer (LeBlanc et al. 1986, Oldale and Barlow 1986). Nitrate concentrations in groundwater in heavily residential watersheds now commonly exceed 300 μM and are 1 to 2 orders of magnitude greater than concentrations in undeveloped woodlands (Cape Cod Commission 2003, Kroeger et al. 2006, Portnoy et al. 1998). Caraco et al. (1987) found that phytoplankton in one Cape Cod freshwater pond was primarily P limited. However, Smith and Lee (2006) recently showed that periphyton in 12 ponds in the Cape Cod National Seashore were predominantly limited either by N or co-limited by N and P. The ratio of dissolved N to P and its deviation from an average value in algae of 16 (Redfield et al. 1963) has been used as a general indicator of the relative availability of N and P in surface waters, and the likelihood that N or P limits algal growth. The ability to predict N or P limitation from dissolved concentrations of N and P would have considerable utility for managing nutrient inputs to lakes and ponds. However, correlations between dissolved N:P and N or P limitation determined by bioassays are often variable, and the predictive ability of the ratio of the easily measured dissolved inorganic N (NH4 + + NO3 -) to soluble reactive P (SRP) is often poor (Dodds 2003). We examined the relative importance of N and P as the proximate controls on the growth of periphyton and phytoplankton in several Cape Cod coastal plain ponds. We addressed the following questions: 1) does N or P limit periphyton and phytoplankton growth? 2) does enrichment by chronic nutrient inputs from groundwater shift limitation of phytoplankton growth from N to P? and 3) do ratios of dissolved inorganic and organic N and P predict N or P limitation of periphyton? We tested periphyton response by measuring chlorophyll-a in growth in situ on diffusing disk bioassays that contained added N, P, or N+P. We tested phytoplankton responses to the 2009 M. Kniffin, C. Neill, R. MCHorney, and G. Gregory 397 same factors by incubating pond water in mesocosms placed in controlled environment chambers in the laboratory and measuring chlorophyll-a in particulate organic matter (POM) filtered from the mesocosms. We conducted two phytoplankton enrichment experiments. One compared responses to different treatments in 2 ponds with unimpaired water quality that receive low nutrient inputs. The other compared responses in 3 ponds that receive low nutrient inputs with 3 ponds that receive high nutrient inputs. We also measured NH4 +, NO3 -, SRP, total dissolved N (TDN), and total dissolved P (TDP) and used these concentrations to examine relationships among nutrient ratios and periphyton production measured in the bioassays. Methods Study ponds Periphyton nutrient enrichment bioassays and water chemistry sampling were conducted during June–July, 2006 in 6 ponds with low nutrient inputs (Israel Pond, Lamson Pond, Mary Dunn Pond, Shallow Pond, Crooked Pond, and Long Pondlet, Table 1). At the same time, phytoplankton bioassays were conducted in water collected from Mary Dunn and Crooked ponds. All ponds were in outwash deposits (Oldale and Barlow 1981). These ponds were chosen because they represent excellent examples of coastal plain ponds in locations where surrounding residential development was low. Long Pondlet dried during occasional years with low ground water levels. Israel and Lamson ponds dried during years of particularly low water. Mary Dunn Pond, Crooked Pond, and Shallow Pond retained water in all years. Phytoplankton nutrient enrichment bioassays were also conducted in November 2007 in 3 ponds with low nutrient inputs (Mary Dunn, Pine, and Slough ponds) and 3 ponds with high nutrient inputs (Aunt Betty’s, Cedar, and Emery ponds) (Table 1). Low-nutrient ponds had low numbers of houses in their watersheds and had low concentrations of chlorophyll-a (mean = 0.5 μg/L), total N (mean = 0.2 mg/L), and total P (mean = 7 μg/L) (Cape Cod Commission 2003). High-nutrient ponds had a high density of houses in their watersheds and high concentrations of chlorophyll-a (mean = 38.8 μg/L), total N (mean = 0.9 mg/L), and total P (mean = 35 μg/L) (Cape Cod Commission Table 1. Location and characteristics of study ponds on Cape Cod. Pond Location Area (ha) Nutrient class Latitude Longitude Israel Barnstable 3.4 Low 41o40'54" 70o16'45" Lamson Barnstable 5.2 Low 41o40'58" 70o16'05" Mary Dunn Barnstable 7.2 Low 41o40'30" 70o16'43" Shallow Falmouth 4.9 Low 41o36'31" 70o35'12" Crooked Falmouth 14.3 Low 41o35'54" 70o35'07" Long Pondlet Falmouth 1.0 Low 41o33'24" 70o36'27" Aunt Betty’s Barnstable 3.0 High 41o38'51" 70o17'49" Cedar Falmouth 8.5 High 41o38'55" 70o37'29" Emery Chatham 5.9 High 41o42'10" 69o58'51" Pine Brewster 10.0 Low 41o43'15" 70o07'57" Slough Brewster 13.1 Low 41o43'31" 70o08'14" 398 Northeastern Naturalist Vol. 16, No. 3 2003). These ponds retained water in all years. All ponds had no inlet or outlet, and pond water levels fluctuated with seasonal and inter-annual variations in groundwater level. Ponds ranged in size from 1 to 14 ha. Water levels in all ponds were above their long-term averages during this study. Periphyton nutrient-enrichment bioassays The in situ periphyton samplers were constructed by removing the ends of 50-mL centrifuge tubes and recapping them with disks (2.5 cm diameter, 0.31 cm thick, 70-μm pore size) made of porous polyethylene (Smith and Lee 2006). A mixture of 2% agar and either N (0.5 M NaNO3), P (0.05 M Na2HPO4), N+P (0.5 M NaNO3 and 0.05 M Na2HPO4), or no nutrient addition (control) were added to the centrifuge tubes and then topped with the porous lids. This allowed nutrients to diffuse from an area of high nutrient concentration in the tube to an area of low nutrient concentration in the water surrounding the surface of the polyethylene disk. The nutrient concentrations added to the agar were equivalent to those of other studies in freshwater ecosystems (Biggs and Lowe 1994, Fairchild et al. 1985, Higley et al. 2001, Pillsbury et al. 2002) and allowed for continuous diffusion of nutrients through the disk. Twelve 50-mL centrifuge tubes (3 replicates for each treatment) were randomly placed into holes drilled 5 cm apart on 1 m segments of 8.5-cm diameter PVC pipe. The pipes were attached to a float, anchored by a cement block, and submerged to a depth of 25 cm below the surface of the water, keeping all of the treatments at equal depth. Two pipes were placed in all 6 ponds, approximately 5 m from the shoreline in 1 to 4 m water depth and 2 to 4 m from each other. The periphyton nutrient enrichment bioassays were deployed in the field in 2006 for 22 days beginning on June 23 (Falmouth ponds) and June 26 (Barnstable ponds). After 22 days, the disks were removed from ponds, wrapped in aluminum foil, and transported on ice to the laboratory where they were stored at 4 °C in the dark. We analyzed chlorophyll-a on the disks after extraction with acetone (EPA method 445.0). Each disk was placed in a 50-mL centrifuge tube with 25 mL of 90% acetone in which chlorophyll-a was extracted overnight at 4 °C. The disk and acetone were sonicated, and chlorophyll was quantified on a Turner Designs 111 fluorometer calibrated with standards of known chlorophyll-a concentrations. Concentrations were calculated as μg of pigment/100 cm2 of substrate. Phytoplankton nutrient-enrichment bioassays The first phytoplankton nutrient enrichment bioassay was conducted in June–July 2006 in Mary Dunn and Crooked ponds in conjunction with the periphyton bioassays. Twelve 3.7-L plastic bottles were rinsed twice, filled with pond water, and transported to the laboratory. Pond water was then filtered through a 200-μm mesh to remove zooplankton. Two liters of the filtered water were placed in 3.7-L open-top plastic containers. Each container received one of four treatments, N (500 μM NaNO3), P (50 μM Na2HPO4), N+P (500 μM NaNO3 and 50 μM Na2HPO4), or a control with no nutrient addition (n = 3) (Caraco et al. 1987). The containers were placed randomly in a growth chamber that simulated daily temperature and 2009 M. Kniffin, C. Neill, R. MCHorney, and G. Gregory 399 light fluctuations of July days at the latitude of Cape Cod. Containers were bubbled with aquarium airstones to insure they were well mixed. A 200-ml water sample was collected from each container on days 0, 3, 6, 9, 12, 15, and 18. Water was filtered through GFF filters and stored in the dark at 4 °C. The filters were then placed in centrifuge tubes, and chlorophyll-a was extracted as described above. The second phytoplankton enrichment bioassay was conducted on water collected November 12, 2007 from Mary Dunn, Pine, Slough, Aunt Betty’s, Cedar, and Emery Ponds. Two 2-L samples of water from each pond were assigned control, N, P, and N+P treatments and incubated on a rotary shaker in a laboratory growth chamber (as above). Controls received no nutrient addition. The N treatments received 200 μM N as KNO3, the P treatments received 18 μM P as KH2PO4, and the N+P treatment was enriched in both N and P. After 14 d, water was filtered through GFF filters, and chlorophyll-a was extracted as described above. Water chemistry We examined the N-to-P ratios in the ponds sampled in 2006 to determine if the ratios of dissolved N and P could be used to predict periphyton nutrient limitation measured in the field. Our hypotheses were that ratios that included total dissolved P (TDP) would be better predictors of algal growth response than ratios that included just SRP (Dodds 2003, Smith and Lee 2006). We calculated the ratio of total dissolved N to total dissolved P (TDN:TDP), dissolved inorganic N (DIN) to total dissolved P (DIN:TDP), and dissolved inorganic N to dissolved inorganic P (DIN:SRP). Two replicate surface water samples were collected from Israel, Lamson, and Mary Dunn ponds on June 23 and July 19, 2006; from Shallow Pond, Crooked Pond, and Long Pondlet on June 26 and July 17, 2006; and from Cedar, Aunt Betty’s, Emery, Pine, and Slough ponds on November 14, 2007. Samples were filtered in the field through GFF filters into acid-washed 60-mL plastic bottles, placed on ice, and stored at 4 °C in the laboratory. Two additional 2-L samples of unfiltered water were collected for analysis of chlorophyll-a on the same dates. We measured NH4 + colorimetrically using the phenol-hypochlorite method (Strickland and Parsons 1972). Absorbance was read on a Cary 50 Scan UV-visible spectrophotometer. Nitrate was measured colorimetrically by Cd reduction on a Lachat flow injection analyzer (Method 31-107-04-1-C). SRP was determined colorimetrically by the method of Murphy and Riley (1962). Samples for TDN and TDP were digested with persulfate oxidizing reagent at 12 to15 psi for 1 h in an autoclave at 240 to250 °C (D’Elia et al. 1977, Koroleff 1983). In the digests, NO3 - was measured on a Lachat flow injection analyzer (Method 10-107-04-2B), and SRP was measured colorimetrically as described above. We calculated dissolved organic N (DON) as TDN - (NH4 + + NO3 -). Statistical analysis In the periphyton bioassay, we used a one-way analysis of variance (ANOVA) in SAS (Ver. 9.1) to test the effect of nutrient treatment in each 400 Northeastern Naturalist Vol. 16, No. 3 pond. When significant treatment effects were found, we used SAS REGWQ to determine significant differences among individual treatment means. We also conducted a three-way ANOVA with pond, treatment, and block (PVC rack) as main effects. Because the effect of block was never significant, blocks were subsequently pooled, and a two-way analysis of variance conducted with pond and treatment as main effects. For the 2006 phytoplankton bioassay, we tested the effect of nutrient treatment within each pond with a repeated-measures ANOVA using replicate containers as separate subjects. We also conducted a two-way ANOVA using the final chlorophyll-a concentration on day 18 as the dependent variable and pond and nutrient treatment as the main effects. For the 2007 phytoplankton assays, we tested the effect of nutrient treatment within each pond using one-way ANOVAs. We then performed a two-way ANOVA with pond nutrient input class and nutrient treatment as main effects. Results The pH of pond water ranged from 4.4 in Long Pondlet to 6.9 in Mary Dunn, Crooked, Shallow, Cedar, and Emery ponds. Conductivities ranged from 27 μS/cm in Long Pondlet to 100 μS/cm in Israel Pond. In the periphyton bioassay, nutrient treatment had a significant effect on chlorophyll-a concentration (df = 3,120; F = 40.7; P < 0.0001), but the effects of pond and pond-by-nutrient interactions were not significant. The N+P treatment increased periphyton chlorophyll-a in 5 of 6 ponds (Fig. 1), and chlorophyll-a in the N+P treatment was significantly greater than the control in all but Crooked Pond (Fig. 1). Periphyton chlorophyll in the Nonly treatments was higher than the control in all ponds and significantly greater in two ponds (Long Pondlet and Israel Pond). Periphyton chlorophyll in the P-only treatment was greater than the control in all ponds and signifi- cantly greater in one pond (Israel Pond). In the first phytoplankton bioassay, nutrient treatment increased chlorophyll- a in both Crooked Pond (df = 3, 8; F = 1332; P < 0.0001) and Mary Dunn Pond (df = 3, 8; F = 35.9; P < 0.0001). Chlorophyll-a in both ponds increased in the N+P treatment and responded only slightly to the P-only treatment in Mary Dunn Pond (Fig. 2). On day 18 of the experiment, there were significant effects of nutrient treatment (df = 3, 16; F = 59.1; P < 0.0001), pond (df = 1, 16; F = 25.8; P < 0.0001), and pond-by-treatment interaction (df = 3, 16; F = 25.8; P < 0.0001). The pond-by-treatment interaction reflected primarily a greater growth response to the N+P treatment in Mary Dunn Pond compared with Crooked Pond. In the second phytoplankton bioassay experiment, there was a significant effect of nutrient treatment on phytoplankton chlorophyll-a (df = 3, 40; F = 163.6; P < 0.0001). Phytoplankton growth also increased in the N+P treatment, but not in the P-only or N-only treatments (Fig. 3). There was a significant effect of pond nutrient class on phytoplankton response (df = 1, 40; F = 289.2; P < 0.0001) and a significant interaction between pond nutrient input class and nutrient treatment (df = 3, 40; F = 73.5; P < 0.0001). The 2009 M. Kniffin, C. Neill, R. MCHorney, and G. Gregory 401 interaction was caused by a greater growth response in the pond receiving high nutrient inputs (Fig. 3). There was no evidence that increased nutrient inputs shifted ponds toward P limitation. Concentrations of NH4 + were ≤1.4 μM for all ponds (Table 2), and NO3 - concentrations were <1.0 μM, except for Shallow Pond, which had a NO3 - concentration of 2.8 μM. DON represented 87 to 99% of TDN in all ponds (Table 2). Concentrations of SRP were generally similar among ponds and ranged from 0.04 μM in Slough Pond to 0.22 μM in Long Pondlet and Pine Pond, except in Emery Pond, where SRP was 3.22 μM. (Table 2). SRP constituted a minimum of 22% of TDP in Lamson Pond to a maximum of 46% in Mary Dunn Pond. The ratio of TDN:TDP in surface waters ranged from 73 in Crooked Pond to 108 in Lamson Pond (Table 2), and all incorrectly suggested P limitation. Figure 1. Concentrations of chlorophyll-a measured in periphyton bioassays in Cape Cod coastal plain ponds. F and P values are for one-way analysis of variance (df = 3, 20) of the effect of nutrient treatment within each pond. Error bars are ± 1 standard error. Treatments with the same letters within each pond were not significantly different (PROC REGWQ in SAS). 402 Northeastern Naturalist Vol. 16, No. 3 The ratio of DIN:TDP ranged from 1.2 in Lamson Pond to 9.3 in Shallow Pond (Table 2). Based on the assumption that DIN:TP of <0.5 predicts N limitation, DIN:TP of >4.0 predicts P limitation, and intermediate ratios are associated with co-limitation by N and P (Lafrancois et al. 2003), DIN:TDP predicted periphyton response in only 3 of 6 ponds (Israel, Lamson, and Mary Dunn). DIN:SRP ranged from 4.9 in Mary Dunn Pond to 35.4 in Shallow Pond, and based on the Redfield ratio of 16:1, came close to predicting limitation only in Crooked Pond, where N and P were co-limiting and DIN:DIP was 11.9. Discussion Modest responses to N or P alone, coupled with large responses to N and P in combination, indicated that N and P were generally strongly co-limiting to both periphyton and phytoplankton in most ponds. This result implies an important role for N in the control of periphyton and phytoplankton in Cape Cod coastal plain ponds, and supports recent work that also found co-limitation of periphyton by N and P to be the dominant pattern in other ponds in the Cape Cod region (Smith and Lee 2006). Similar responses of periphyton Figure 2. Concentrations of w a t e r- c o l u m n c h l o r o p h y l l - a in 2006 growthchamber bioassays in two low nutrient input ponds. Error bars are ± 1 standard error. Treatments with the same letters within each pond were not significantly different on day 18 (PROC REGWQ in SAS). 2009 M. Kniffin, C. Neill, R. MCHorney, and G. Gregory 403 and phytoplankton to N and P in Crooked and Mary Dunn ponds, where assays of both types of algae were measured, also suggested that periphyton and phytoplankton responded similarly to N and P. Our finding of strong N and P co-limitation differed from previous work on Cape Cod that examined N and P limitation along salinity gradients from fresh water to estuaries and concluded that P limitation predominated in fresh water (Caraco et al. 1987). Although DIN:TDP was the best predictor of periphyton response to nutrient enrichment, we found that no ratios consistently predicted periphyton nutrient limitation. This finding indicated that nutrient ratios should be Figure 3. Concentrations of water column chlorophyll-a in growth chamber bioassays. Aunt Betty’s, Cedar, and Emery ponds receive high nutrient inputs, Mary Dunn, Pine, and Slough ponds receive low nutrient inputs. F and P values are for one-way analysis of variance (df = 3, 7) of the effect of nutrient treatment within each pond. Error bars are ±1 standard error. Treatments with the same letters within each pond were not significantly different (PROC REGWQ in SAS). Table 2. Water chemistry variables (in μM) and molar N:P ratios for surface water samples taken in the six ponds during June and July 2006. Pond NO3 - NH4 + DON TDN SRP TDP TDN:TDP DIN:TDP DIN:SRP Israel 0.06 0.58 30.72 31.36 0.12 0.36 86.7 1.8 5.5 Lamson 0.16 0.30 40.01 40.47 0.08 0.37 108.4 1.2 6.0 Mary Dunn 0.01 0.79 19.67 20.47 0.16 0.35 58.2 2.3 4.9 Shallow 2.79 1.40 29.02 33.21 0.12 0.45 73.7 9.3 35.4 Crooked 0.97 0.39 17.15 18.51 0.11 0.25 73.3 5.4 11.9 Long Pondlet 0.74 0.88 60.86 62.48 0.22 0.58 107.9 2.8 7.4 Aunt Betty’s 11.33 0.06 0.13 87.6 Cedar 6.26 0.04 0.15 42.0 Emery 8.14 0.28 3.22 2.6 Pine 2.62 0.04 0.22 12.1 Slough 2.60 0.04 0.04 66.0 404 Northeastern Naturalist Vol. 16, No. 3 used with caution to predict the relative importance of N versus P limitation of periphyton growth. We lacked a complete set of ratios to assess their effectiveness in predicting phytoplankton growth. In contrast to our results, Smith and Lee (2006) found that the ratio of DIN:total P generally predicted N or P limitation of periphyton in ponds in the Cape Cod National Seashore. Maberly et al. (2002) found the same for English lakes. We did not measure total P on unfiltered water that would have allowed a direct comparison with these studies. We did, however, also find that TDN:TDP and DIN:SRP were poor predictors of the results of periphyton bioassays. The failure of DIN:SRP ratios to predict periphyton nutrient limitation was consistent with Dodds’ (2003) argument that DIN:SRP is not a reliable predictor of nutrient limitation or trophic status, especially when SRP concentrations are low. Our results support a growing body of evidence that co-limitation of lake algae by N and P occur frequently and that co-limitation of N and P predominates over P limitation. Elser et al. (1990) found that across a survey of 62 lakes, the frequency and degree of algal response did not differ for N or P enrichment and that N and P together were required to produce a substantial algal response. Other evidence for the importance of N limitation or strong N and P co-limitation comes from a range of lakes in Sweden (Bergström et al. 2005), the Rocky Mountains (Lafrancois et al. 2003, Nydick et al. 2004, Wolfe et al. 2003) and the Sierra Nevada (Goldman et al. 1993, Sickman et al. 2003) of North America, upland English lakes (Maberly et al. 2002), and Spanish lakes (Camacho et al. 2003). There is also growing evidence that 1) low levels of chronic inputs of atmospheric N deposition have shifted algal species composition in lakes in regions that historically received relatively little N deposition, and 2) most of the changes have taken place during the last 40 years (Baron et al. 2000, Saros et al. 2003, Sickman et al. 2003, Wolfe et al. 2001). Over time, even these low levels of annual anthropogenic N inputs can shift lakes from N limitation to P limitation (Bergström et al. 2005, Goldman et al. 1993). We found no evidence that chronic N inputs have shifted phytoplankton to P limitation in Cape Cod ponds. In fact, we found greater total phytoplankton growth in response to the N+P treatment in the high-nutrient ponds, although, relative to the control, growth in the N+P treatment was similar in both low-nutrient and high-nutrient ponds. High-nutrient ponds apparently receive inputs of P from their watersheds even though the area of farmland in all pond watersheds was negligible and all sewage treatment was by on-site septic systems that efficiently remove P (Valiela et al. 1997). The high-nutrient ponds had higher total P (Cape Cod Commission 2003), and we measured higher mean SRP in the highnutrient ponds (1.17 ± 0.13 μM) compared with the low-nutrient ponds (0.13 ± 0.02 μM). Together, these pieces of evidence indicate that greater attention should be given to N and to N and P in combination when assessing the controls and threats to nutrient enrichment of fresh waters in the Cape Cod region. The common co-limitation of periphyton and phytoplankton by N and P has important implications for the ecological function and conservation of 2009 M. Kniffin, C. Neill, R. MCHorney, and G. Gregory 405 the roughly 1000 freshwater ponds and more than 300 coastal plain ponds that occur on Cape Cod and in southeastern New England (Cape Cod Commission 2003, Corcoran 2002). First, because periphyton in all ponds responded modestly to additions of N alone, it is likely that increased delivery of dissolved N to ponds from the regional groundwater increases periphyton growth. The same general pattern of co-limitation by N and P was reported by Smith and Lee (2006). Second, N from atmospheric deposition and septic systems are the dominant sources of N to groundwater on Cape Cod because N deposition and residential development are relatively high, but land area in agriculture is very low. This deposition and land use, together with sandy soils that result in rapid soil water infiltration, minimal surficial runoff, and high retention of P relative to N in iron-rich soils, typically lead to relatively high N loading but low P loading via regional groundwater (Valiela et al. 1997). The combination of both high N loading and the sensitivity of periphyton to increased N supply indicate that regional increases in watershed N loads constitute an important and underappreciated influence on the trophic status of coastal plain ponds. Strong co-limitation of both periphyton and phytoplankton by N and P suggests that the overall magnitude of the effects of increased N loading to ponds will be limited by the supply of P. Because periodic water-level fluctuations in wetlands can lead to P release from recently flooded sediments (DeLaune et al. 1976, Kadlec 1986), the natural re-flooding of coastal plain pond shorelines may lead to greater stimulation of periphyton and phytoplankton if it is now accompanied by higher N supply caused by N enrichment of the regional ground-water. While algae blooms in Cape Cod ponds are now common (Cape Cod Commission 2003), changes in the magnitude or frequency of blooms and their relationship to water-level fluctuations have not been documented. Whether increased periphyton or phytoplankton growth in coastal plain ponds constitutes a threat to the diverse assemblage of shoreline and submersed species of conservation concern is not known. However, increased N supply is associated with lower species diversity in many ecosystems (Bobbink et al. 1998, Gross et al. 2005, Tilman 1987). Greater periphyton or phytoplankton production could interact in a number of ways with the life history of shoreline or submersed species to reduce species cover or diversity. These potential effects include increased seed mortality, decreased seedling survival caused by greater algal cover, or competitive exclusion during the seedling stage caused by higher growth of a small number of fast-growing species. These interactions deserve increased attention as specific mechanisms that link plant diversity and N loading in coastal plain ponds and in other fresh water bodies that harbor significant reservoirs of plant diversity and where increased N and P loading may result in substantial changes to algal growth and trophic status. Acknowledgments Financial support was provided by the Massachusetts Environmental Trust, the National Science Foundation Research Experience for Undergraduates, and the MBL’s Semester in Environmental Science. 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