Nitrogen versus phosphorus limitation for growth of an estuarine population of eelgrass (Zostera marina L.)

Aquatic Botany, 44 (1992) 83-100 Elsevier Science Publishers B.V., Amsterdam

83

Nitrogen versus phosphorus limitation for growth of a Ji estuarinc popalation of eelg 'a s (Zostera marina L. ) i' ?

L a u r a M u r r a y ~,b, William C. D e n n i s o n "~ a n d W. Michael K e m f ~University of Maryland System, Center for Environmental and Estuarine Studies, Horn Point Environmental Laboratories, Cambridge, MD 21613, USA bSalisbury State University, Biology Department, Salisbury, MD 21801, USA (Accepted 10 July 1992) ABSTRACT Murray, L., Dennison, W.C. and Kemp, W.M., 1992. Nitrogen versus phospborus limitation for growth of an estuarine population of eelgrass (Zostera marina L.). Aquat. Bot., 44: 83-100. The relative importance of nitrogen (N) and phosphorus (P) l"mitation for growth and biomass accumulation in an estuarine population of eelgrass (Zostera marina L.) was examined by in sitn additions of nitrogen (+N), phosphorus (+P) and nitrogen plus phosphorus ( + N + P ) to sediments at low and high loading rates. Nitrogen treatments resulted in no significant increases in leaf tissue N levels and only a small increase in the N content of root plus rhizome tissues. Phosphorus concentrations were, however, significantly (P< 0.05) higher in both leaf and root plus rhizome tissues with + P and + N + P enrichment. Eelgrassgrowth and biomass exhibited statistically significant (P<0.05) increases in response to high +P. Similar increases in mean plant growth and biomass were observed with + N and + N + P enrichment, but large variabilities rendered these responses nonsignificant. Our results are in contrast with those reported for a previous sediment fertilization ( + N + P ) study at the same site and we attribute this difference to a change in the nutrient status of the study area. Comparison with other sediment fertilization experiments for both freshwater and ma~ne pla,t s ~cies revealed a clear relationship bet ~'een relative plant growth rates and tissue nutrient concentrations for both N and P enrichment. Tt~jsrelationship suggests a uniformity of submersed plant nutrition, wherein responses to changes in nutrient availability are regulated by alterations in both growth rates and tissue nutrient content.

INTRODUCTION Seagrasses a n d r e l a t e d s u b m e r s e d f r e s h w a t e r m a c r o p h y t e s f o r m h i g h l y p r o d u c t i v e c o m m u n i t i e s in a q u a t i c h a b i t a t s ( S t e v e n s o n , 1988). P r i m a r y p r o d u c t i o n o f these e c o s y s t e m s is largely regulated by availabilities o f light ( B a r k o a n d S m a r t , 1981; S a n d - J e n s e n a n d B o r u m , 1983; D e n n i s o n , 1987) a n d / o r

Correspondence to: L. Murray, University of Maryland System, Center for Environmental and Estuarine Studies, Horn Point Environmental Laboratories, Cambridge, MD 21613, USA. ~Present address: Department of Botany, University of Queensland, St. Lucia, Qld. 4072, Australia.

84

L.MURRAYL~rAL

nutrients (Barko and Smart, 1986; Short, 1987). While light availability controls the depth penetration (Chambers and Kalff, 1985; Duarte, 1991 ) and biomass accumulation at depth (Dennison and Alberte, 1986), nutrient availability can also be a major limiting factor for the growth of submersed plants. Relative availabilities of the inorganic nutrients, nitrogen (N) and phosphorus (P), appear to control the growth of subme~'sed plants in freshwater (e.g. Christiansen et al., 1985 ) and both temperate (Iizumi et al., 1982) and tropical (Short et al., 1990) marine habitats. Nutrient limitation of growth even appea, s to be important in certain low-light envir,~,:tments, where ambient irradiance falls below that needed to saturate plant pbotosynthesi; (Orth, 1977; Wetzel and Penhale, 1983). A wide variety of approaches has been used for estimating tl~e extent to which the growth of submersed plants is regulated by nutrient deficiencies in a given environment. Several investigators have inferred nutrient limitation by comparing nutrient concentrations in the field with nutrient uptake parameters obtained from laboratory kinetic experiments f Iizumi and Hattori, 1982; Short and McRoy, 1984). Nutrient limitation has also been assessed indirectly by comparing nutrient uptake needed for observed plant growth rates with nutrient pools and regeneration rates in the sediments (Patriquin, 1972; Short et vl., 1985). Statistical correlations between sediment nutrient concentrations and plant abundance from various field sites have been used to infer nutrient control on plant growth (Anderson and Kalff, 1986b; Duarte and Kalff, 1988). Others have suggested nutrient limitation when plant tissue concentrations of N and P fall below the 'critical levels' at which laboratoryreared plants exhibit no further increase in growth with increases in tissue nutrients (Gerloff and Krombhclz, 1966; Van Wijk, 1989 ). Finally, nutrient status can be assessed by monitoring plant responses, in terms of biomass inc,rease (Orth, 1977) or growth (Short et al., 1990), to direct in situ appli= cation of fertilizers to sediments. Although the question of the relative importance of N vs. P limitation on plan~ grov~th in aquatic ecosystems has received much attention over recent decades, most of the focus has been directed to phytoplankton. This question is of general interest both in relation to understanding nutrient cycling processes in aquatic ecosystems, and in relation to practical problems of developing cost-effective nutrient control measu~:cs for reducing impacts of cultural eutrophication (D'Elia et al., 1986). The general pattern which seems to have emerged from such work is that P limits phytoplankton growth more often in fresh waters (Schindler, 1977), while N tends to be more important in marine waters (Ryther and Dunstan, 1971 ). In estuaries, P limitation appears to be predominant at the brackish end, while N limitation is more prevalent at the saline end (Caraco et al., 1987). Seasonal shifts in the relative importance of N and P limitation are also evident in estuarine ecosystems (D'Elia et al., 1986). Most of these generalizations are based on relatively

NITROGEN VERSUSPHOSPHORUSLIMITATION FOR GROWTH IN ZOSTERAMARINA

85

short-term, small-scale experiments and the patterns may differ "when assessed at the level of the whole ecosystem (Smith and Atkinson, 1984; Howarth, 1988). Far fewer studies have addressed the question of the relative importance of N vs. P limitation for the growth of seagrasses and submersed freshwater plants. Although the wide diversity of methods used to investigate submersed plant nutrition co~tributes to the variabilities in results and interpretations, several broad patterns of N and P regulation of plant growth are emerging. In oligotrophic and mesotrophic lakes, both field correlations (Anderson and Kalff, 1986a) and direct sediment fertilizations (Christiansen et al., 1985) indicated stronger responses of plant growth to P compared with N amendments. However, studies with the freshwater plant, Myriophyllum spicatum L., planted at field sites, suggested that N limitation can also prevail under some circumstances (Anderson and Kalff, 1986b). In temperate marine environments, sediment manipulation studies have indicated significant plant growth responses to N additions (as compared with P) for two related seagrasses (Bulthuis a~d Woelkerling, 1981; Short, 1987). In another study, however, no significant increase in the growth of Zostera marina L. was observed with the addition of ammonium to sediment pore waters (Dennison et al., 1987). Later experiments with plants at this site (Roth and Pregnall, 1989) suggest the possibility that high nitrate inputs via groundwater may tend to alleviate N deficiencies in this nearshore seagrass bed. The question of N and P nutrition for submersed plants in estuarine environments has yet to be addressed. The purpose of the present study was to examine the relative importance of N vs. P limitation for Z. marina growing in the large temperate estuary, Chesapeake Bay. Here we consider the response of plant growth, biomass and tissue nutriet~t concentrations to direct in situ fertilization of sediments. The study site selected was identical to one used 12 years earlier to demonstrate significant enhancement of Z. marina biomass with addition of N plus P to the sediments (Orth, 1977). METHODS

Description of study site This investigation was conducted in a seagrass bed, approxima:eiy 140 ha in size, located on the southeastern shore of Chesapeake Bay, Virginia ( 37 ° 25' N, 75 ° 59' W), known locally as Vaucluse Shores (Orth, 1977; Wetzel and Penhale, 1983; Murray and Wetzel, 1987). The s~agr~s bed is dominated by Z. marina in the deeper areas and by Ruppia maritima L. m the nearshore areas. The bed is protected by an offshore sand bar. The experimental site for this study was located in the middle of the area containing only

86

L. MURRAYEl" AL

Z. marina (mean depth of 1.4 m), which was characterized by sandy sediments with an organic carbon content (0.23%) intermediate for this region. The temperature during the study period (April-June) ranged from 15 to 23 °C and the salinity averaged 19%o. Experimental design and fertilization method Triplicate plots, within an experimental area of approximately 20 m × 20 m, were randomly selected and established for each of six treatments: Low +N; Low +P; LOw + N + P ; High +N; High +P; High + N + P . LOw fertilization rates of nitrogen (Low + N ) and phosphorus (LOw + P ) were approximately I00 g N m -2 and 20 g P m -2, while high rates were five times the respective Low + N and + P values. Three control plots received the same experimental manipulations without the m, trient additions. All plots were separated from each other by 2 m or more. The experiment was initiated in mid-spring (29 April 1988). Intact plantsediment cores ( 15 cm deep) were removed from each experimental plot using a sediment corer (0.012 m 2) and plants were marked with a rhizome tag for growth measurements (Dennison et al., 1987). In each hole created by removal of the plant-sediment core, the bottom half of a plastic Petri dish (8.5 cm in diameter) was placed facing upward. Each dish contained 2% (w/ w) agar into which NH4CI and/or NaHPO4 crystals had been dissolved to create the desired N and P treatment levels. The plant-sediment core was re"turned to the original plots within 30 min, the location of which had been marked with labeled surveyor flags. The experiment was conducted for a period of 54 days (until 20 June), when plants and sediments were harvested.

Growth and biomass measurements The plant-sediment cores were removed from each plot and immediately placed in a cooler containing water from the site. Ten plants were randomly selected from each core and marked for growth by placing a plastic-coated wire loop at the terminal rhizome segment just below the meristem (Dennison et al., 19[;7). After the experimental incubation period, the plant-sediment cores were collected, washed free of sediments and returned to the laboratory for processing. Three shoots were randomly selected from each plot for leaf area and weight determinations, and for rhizome length and weight analysis. The second youngest leaf (Number 2) from each turion was dried to a constant weight and saved for subsequent nutrient analysis. The total number of shoots was determincd tbr each sample, and plant material was separated into aboveground and belowground biomass. Each sample was dried at 60°C to a con-

NITROGEN VERSUSPHOSPHORUSLIMITATION FOR GROWTH IN ZOSTERA MARINA

87

stant weight (24-48 h) and weighed to the nearest milligram. Zostera marina biomass was calculated as g dry weight m -2. Growth of Z. marina was estimated from the number of new rhizome nodes formed during the experimental period, which is equal to the number of new leaves formed (Dennison and Alberte, 1986; Dennison, i990). Leaf growth rates were calculated by dividing the number of new leaves formed per incubation period by the number of leaves per shoot at the end of the experiment. These values were converted to dry weight by establishing a regression between the dry weight of leaves and chat of rhizomes plus roots. Growth rates are reported as mg growth m-2 day- ~and as mg growth per shoot day- ~.

Sediment nutr.fent sampling Sediment nutrient concentrations were determined by collecting intact sediments in clear acrylic cores (,4.9 cm 2) from the study site prior to treatment and following the 54 day incvbation period before the collection of plants. Extractions of pore water from all sediment cores were performed in an oxygen-free environment (glove b:tl~purged with N2 gas). The top 10 cm of sediment were homogenized with a mortar and pcstlc~ a~,d t.n aliquot (approximately 30 g) was weighed out into a centrifuge tube. ~diment nutrients (ammonium and phosphate) were extracted with 20 ml water which contained I N KCI and which had been deoxygenated by bubbling with N2 gas. Centrifuge tubes were capped and shaken vigorously for 2 rain, and then centrifuged ( 10 000×g) for 10 rain. The supernatant was decanted into a syringe filtration apparatus and filtered (GF/F, 1.2/an). Concentrations of NH~ and PO43- were determined with standard colorometric techniques using a Technicon Autoanalyzer, with values reported per liter of pore water. Concentrations reported Ibr NH~ include both the dissolved traction in pore waters and the KCl-extractable fractiou sorbed to sediments (Kemp et al., 1990).

Plant tissue nutrient analysis Plant tissues were prepared for nutrient analysis by selecting one individual leaf (Number 2) from each of the replicate experimental units and drying at 60°C to a constant weight (24-48 h). The plant tissue was then ground with a mortar and pestle, and separated into two parts. Approximately one half of the sample was used for carbon, hydrogen, N analysis (Perldn-Elmer CHN Model 240B Element Analyzer ); the remainder (about 3 mg) of each sample was used for tissue P analysis. These samples (approximately 3 mg) were analyzed for phosphate content (Technicon Autoanalyzer) after digestion and extraction with potassium persulfate for 4 h (Strickland and Parsons, 1972; Short et al., 1985).

8$

L.M U R k ' , ' Er AL

RESULTS

Sediment nutrient concentrations The pattern of sediment N and P concentrations measured at the end of the experimental fertilization period generally reflected the respective nutrient treatments (Fig. 1 ). The only treatment condition that did not demonstrate large increases in sediment nutrient pools was the Low + N treatment. In comparison with controls, NH4+ concentrations doubled at High + N enrichment and PO 3- concentrations increased by 13-fold at High + P enrichment. W'aen N and P were added in combination, NH~ increased by 30% at Low + N + P and 50% at High + N + P , and PO 3- increased by 3-fold at Low 4- N + P and 4-t6i6 at High + N + P. S:',diment N and especially P concentrations in unamended controls were relvtively higher at the end of the experiments (June) compared with at the be~nning (April, where NH + concentrations were 398/~mol and PO43- concentrations were 8/~mol per liter of pore water). Sediment NH~ concentrations were comparable with those reported previously at the same study site (Ca,~ey and Kemp, 1990), with the same seasonal trend of increasing nutrient concentrations with increasing temperature. The N:P ratios of sediment: KCI extracts were responsive to amendments. In + P and + N + P treatments, the N: P ratio was 8 or less, while ratios for controls and + N treatments were higher (12-50). Residual NH + and PO~- remained in sediments after fertilization (Fig. /

~ / / N : P = 6.1 ~I~ 8oo

~-~N~

z. ~

600

~"

(Y = ld5 + 2.03 X, r2 = 0.86)

_W

.Q

~

~

,400

:,,

.ITI,~.J.NPH /J-NPL

~2oo~

.,~

~

o

,

0

I

i

200

400

| 600

Sediment Pore Water [PO: ] Hg. 1. Scatter diagramof NH+ and PO~- concentrationsmeasuredin sedimentpore waters for eachtreatmentat the end of experimentalincubationsand for controlsat the beginningand end. Valuesgivenare meansof triplicate samples __standard error (SE). Abbreviationsare as follows:Cx,initialcontrol;C~,finalcontrol;NL,lownitrogentreatments;Nr~,highnitrogen;PL, lOWphosphorus;PH, highphosphorus;NIL, low nitrogenplus phosphorus;NPH,highnitrogen plus phosphorus.

NITROGEN VERSUS PHOSPHORUS LIMITATION FOR GROWTH IN ZOSTERA MARINA

89

1), indicating that these nutrients were not fully taken up by the seagrass. The balance between NH~ and PO]- in sediments in all treatments where N and P were added together or at low dosages, however, reflects a degree of interaction between sediments, nutrients and plant uptake processes (Fig. 1). Only the treatments with high N or P loadings when added separately showed a deviation from the balance of sediment N: P ratio of 6: i. A scatter diagram of sediment NH~ versus PO]- for each treatment reveals a significant correlation (r 2-- 0.80; slope = 6.1 N: P) for all but the High + N and High + P treatments (Fig. 1 ). For High + N treatments, PO 3- concentrations were deficient relative to NH~; alternatively, for High + P treatments, NH~" concentrations were deficient relative to PO]-. The consistency in the N: P ratio of sediment pore waters in the control, Low + N and +P, Low and High + N + P treatments indicates that microbial and/or plant processes act to maintain the sediment water N: P balances. Plant tissue nutrient concentrations

The storage and accumulation of nutrients in plant tissues exhibited distinct patterns in leaves compared with roots plus rhizomes. In Z. marina leaves, N ~ontent was not affected by treatments, with a consistent N content of about 1/~mol N g-m (Fig. 2). In contrast, leafP content was significantly (P<0.05) increased in + P and + N + P treatments. Leaf tissue P content increased by 78% in Low + P and by 122% in High + P treatments compared with controls. Even higher levels of P content were observed in + N + P Lreatmerits, with significant (P< 0.05) increases of 156% i~ Low + N + P and 219% in High + N + P treatments. Both N and P concentrations were lower in Z. marina root plus rhizome tissue than in leaves (Fig. 3). The N content ofbelowground tissues increased by 15-25% with + N and + N + P enrichments, but these increases were only marginally significant compared with controls (P<0.15). As in leaf tissue responses, the P content of root plus rhizome tissue we,s increased in + P and + N + P treatments compared with controls, but only + P increases were significant (P< 0.05). Atomic ratios of both leaf and root plus rhizome tissue responded to nutrient enrichments with increasing tissue ratios as pore water ratios increased (Fig. 4). Significant correlations were observed between both leaf N:P (r2=0.87) and root plus rhizome N:P (r2=0,63) and pore water N:P. The slopes were similar (0.61 and 0.63) for both regressions, indicating that both leaves and roots plus rhizomes responded in a similar manner. In all treatments, N: P ratios were higher in root~ plus rhizomes ( ~3.8-27.4) than leaves (5. l - 14.7). The significavt correlations between m e ~ values of N: P ratios in plant tissues vs. N:P ratios of sediment pore water (Fig. 4) suggest that

90

L. MURRAYET AL N Enrichment

5[

I~ Z

o

Control

LowN

E

"!t

Z

F-~ i~

o

t 04

HighN

E v

i o=

P Enrichment

a

I

P

Control

Control

,

LOwP

LowH+P TREATMENT

o ~"

HighP

U.

HighN*P

Fig. 2. Leaf tissue content o f N and P for Z. marina plants treated with low and high sediment

fertilizationrates for N, P and N pl,ls P comparedto untreatedcontrolsat the end of 7 weeks incubation(averageof triplicatesamples + SE; letters (a and b) indicatesignificantdifferences amonggroups). variations in plant nutrient status are balanced with changes in se4iment nutrient availabilities.

Biomass and growth responses The response of Z. marina to nutrient enrichment was measured in terms of changes in plant biomass and growth rates (Fig. 5). Total shoot biomass (leaves, roots and rhizomes) was unaffected by Low and High + N and Low + P treatments, but was higher in High + P treatments compared with controls. Biomass in High + P had a significant (P<0.10) 32% increase. Combined + N + P treatments had larger increases in biomass, with a 31% in-

NITROGEN VERSUS PHOSPHORUS LIMITATION FOR GROWTH IN ZOSTERA MARINA

91

Z I:=

00

Control

Low N

High N

E v

io. Control

LOW N

High N

~" +

+

,.o

,,

/ ~° 4-

O.5

o.o

~

s

oo Control

Low N

High N

TREATMENT

Fig. 3. Root tissue content of N and P for Z. marina plants treated with low and high sediment fertilization rates for N, P and N plus P compared to un~reated controls at the end of 7 weeks incubation (average of triplicate samples + SE; letters (a and b) indicate significant differences among groups).

crease ;n ~he Low + N + P treatment and a 41% increase in the High + N + P U:eatmen~L High variability among replicate plots, however, rendered these differences statistically insignificant. Shoot growth rates (g per shoot d~y- l) were increased in N, P and combi:l~d N plus P enrichments (Fig. 5). As in biomass responses, growth was uLaffected by Low + N and Low + P treatments, but increased in High + N and + P treatments compared with controls. The increase in growth in the High + N treatment was relatively high (41%), but marginally signifi~aat (P<0.15). A significant (P<0.05), but less substantial increase (26%) in gyowth was observed in the High + P treat;merit. Combined + N + P enrich-

L. MURRAYEl"AL

92

30

20

~

/

"tissue (Y= 13"93+ 0"63x' r2= 0"63)

~ / ' ' ~

lO

m

"~'~/

~

1Y=3.00+0.61X, r2=0.871

I o

Pore Water N:P Fig. 4. Correlation between mean molar N: P ratios in sedimentpore watersand in Z. marina leaf tissue (closedsymbols)and roots and rhizomestissue (open symbols)at the end of the fertilizationexperiment.

ments increased growth rates, up to 30% in High + N + P , but high variability resulted in non-significant differences. Total plant biomass values were 950 g dry weight (dw) m -2 in controls and exceeded 1300 g dw m -2 in nutrient enrichment plots. The majority of the biomass was below ground, with 680-1250 g dw m -2 of root and rhizome :issue. Aboveground to belowground biomass ratios ranged from 0.33 to 0.54 for all treatments. Shoot growth rates ranged from 2.7 to 3.0 mg dw per shoot day-~, and combining these growth rates with biomass measurements leads to areal integrated growth rates ranging from 2.6 to 4.8 g dw m -2 day- l, comparable with previously reported rates for Z. marina (cf. Zieman and Wetzel, 1980). Comparison of leaf tissue N and P (mmol N or P g-~ ) contents with areal plant growth rates (g dw m -2 day- l ) in fertilized treatments reveals a strong relationship to P, but not to N. A significant correlation (r 2-- 0.70; P < 0.05 ) of leaf P vs. areal growth rate in + P and + N + P treatments contrasts with a non-significant correlation (r 2= 0.38 ) of leaf N vs. areal growth rate in + N and + N + P . Overall, Z. marina growth and biomass responded to high levels of N and P enrichments; however, experimental variability muted the statistical significance of these responses. The only fertilization effects on growth that were statistically significant (P< 0.05) were those for High.+ P enrichment. Yet, growth increases for High + N and + N + P treatments were proportionately higher (41% and 31%, respectively) than the + P treatment (26%). Similarly, increases in biomass were only significant (P< 0. I 0) for High + P, with

NITROGEN VERSUS PHOSPHORUS LIMITATION FOR GROWTH IN ZOSTERA MARINA

Control

Low N

High N

93

"0

,e-.

"....

6

of

Control

.

LOw N,,P

.

High N~.P

Io

TREATMENT

Fig. 5. Total biomass and shoot growth for Z. marina plants treated with low and high sediment

fertilizationrates for N, P and N plus P compared with umreated contrGlsat the end of 7 weeks incubation (averageof triplicatesamples + SE; letters (a and b) indicatesignificantdifferences amonggroups). non-significant, but consistent increases in biomass in fertilization treatments ranging from 20 to 41% compared with controls. DISCUSSION The fertilization responses of Z. marina observed in the present study were markedly different from responses elicited in a fertilization study 12 years previously, despite conducting the experiments at the same site, at the same time ofthe year and using similar techniques (Orth, 1977). The relative magnitude of the biomass and growth responses to fertilization in the present study (20-40% increase) contrast with sediment fertilization responses in the Orth (1977) study (300-500% increases). Nutrient addition rates in the present study were chosen to approximate those used in the previous study (Orth,

94

L. MURRAY ET AL

1977), with the objective of distinguishing the relative importance of N vs. P. Yet, the overall importance of sediment nutrients appears to have changed at this study site. Subtle differences in methodology may also have contributed to different responses. Orth (1977) added nutrients to the sediments using a commercial fertilizer that was gently worked into the sediment surface. In contrast, we introduced fertilizer by burying nutrients in an agar dish below a sediment plug that was temporarily removed. Our nutrient additions were compared with controls (agar without nutrient~ added), while Orth compared fertilization responses with controls that were unaltered in any way. However, these differences in the method of nutrient addition are unlikely to account for the magnitude of the difference in responses between the two studies. Data on sediment nutrient concentrations and plant tissue nutrient contelits in the present study provide support for the interpretation that the ~mall fertilization responses were a result of different nutritional status of Z. marina in 1988 compared with 1976. There are several possible explanations for the difference in fertilization response between Orth's (1977) study and the present study. There is no evidence for major differences in insolation, water temperature or other environmental factors. Various lines of evidence suggest that this site has become progressively eutrophicated over the past decade. Pore water nutrient concentrations appear to have increased since 1980 (cf. Wetzel personal communication vs. Fig. 1 ). Abundant communities of epiphytic diatoms and macroalgae (especially Ulva sp. ) observed in 1989 were not present in 1981 (Murray and Wetzel, 1987). This eutrophication could be a result of a natural successional development of the seagrass bed in this area, but could also be associated with the general increase in nutrient loading which has occurred throughout Chesapeake Bay over the past several decades (Kemp et al., 1983). To compare our results with those of previous studies, we grouped the magnitude of biomass and growth responses to fertilization of submersed plants into two general categories: ( 1 ) modest effects (0-60% increases); (2) large effects (greater than 100% increases) compared with unamended controls. Out of a total of 29 fertilization studies that we found for submersed plants, one-third reported large responses to fertilization; in general, these occurred in oligotrophic environments. For example, Short et al. (1990) found marked increases in the growth (250%) of Syringodium filiforme Kiitz. with P additions in carbonate, P-deficient sediments of the Bahamas. Similar impressive increases in the growth of Halodule wrightii Aschers. (greater than 1000% increases) and Thalassia testudinum Banks ex K~nig (about 100% increases) were observed in response to fertilization with bird guano in carbonate sediments of Florida Bay. These same tropical seagrass species also exhibited large increases (80-100%) in growth with the addition of sediment nutrients in various Caribbean sites (Williams, 1987, 1990). Similar dramatic increases

NITROGEN VERSUS PHOSPHORUS LIMITATION FOR GROWTH IN ZOST£RA MARINA

95

in submersed pl~Lntbiomass and growth (100-200°/o) have been observed with fertilization studies in oligotrophic freshwater environments (Moeller, 1983; Duarte and Kalff, 1988). More frequently, however, smaller plant responses to sediment fertilization (as in the presen~ study) have been reported, mostly in areas characterized by relatively organic-rich sediments. Ascertaining statistical significance for these smaller effects can be difficult under field conditions due to natural variability. In studies in which Z. marina was fertilized, relatively small (Roberts et al., 1984) or no (Dennison et al., 1987) growth responses were observed. Bulthuis and Woelkerling (1981) were able to detect small growth responses to N fertilization (20% increase ) in a related species, Heterozostera tasmanica (Martens ex Aschers. ) den Hartog, by making repeated time-course measurements. They could not detect growth responses to P in the same study, however. Small (15%), but significant increases in the growth of a freshwater submersed plant, Littorella uniflora (L.) Aschers., were observed in response to N fertilization (Christiansen et al., 1985). Similarly small but significant responses of ~Lnother freshwater species, M. spicatum, were also reported (Anderson and Kalff, 1986a). In many cases, these muted responses to fertilization result from growth limitation by factors other than N and P. For example, inorganic carbon availability in soft water lakes (Christiansen et al., 1985) and light at deeper sites (e.g. Dennison and Alberte, 1986; Duarte and Kalff, 1988) can preclude a response to N or P. The relatively small growth and biomass responses to fertilization in the present study ,~an also be interpreted with respect to the relatively high sediment pore wa~er nutrient concentrations observed. Sediment pore water NH + concentrations were estimated from KCl-extractable [NH + ] to be about 200/zM, much higher than the 100/~M saturation level for growth responses postulated by Dennison et al. (1987). Seasonal measurements of sediment NH~ at this site indicate that these high pore water concentrations are maintained throughout the growing season (Caffrey and Kemp, 1990), leaving little opportunity for N limitation, in contrast, PO~- levels varied between April and June, with the possibility of some P limitation of growth early in the season. Comparison of nutrient half-saturation constants (Ks) for Z. marina with sediment nutrient pools provides another way to analyze possible nutrient limitation. If pore water concentrations are near or below observed K~values, nutrient limitation can be inferred. Hydroponic experiments with Z. marina roots separated from leaves indicate that root u p t ~ e of N has a Ks value of about 75-150/~M (Iizumi and Hattori, 1982; Thursby and Harlin, 1982; Short and McRoy, 1984). Similar values of Ks have been reported for several submersed freshwater plants (e.g. Toetz, 1973). Unfortunately, Ks values for P uptake of Z. marina are not available, but the values for other submersed plants appear to fall in the range of 5-! 5/dvl (Twilley et al., 1977; Smith and Adams, 1986; Hill, 1989). These Ks ranges for N (75-150/~M) and P (5-15

96

L.MURRAYETAL

/~b.) are less than the pore water nutrient concentrations (Fig. 1 ), with the exception of April PO 3- concentrations in control plots. This supports the conclusion that little nutrient limitation of Z. marina growth is evident, with the possible exception of slight P limitation early in the growing season. Another way of interpreting the nutritional status of plants is to analyze plant tissue nutrient content. Previous investigators have used the accumulation of a particular nutrient (N or P) in plant tissue after enrichment to infer plant compensation for a deficiency of that nutrient (Bulthuis and Woelkerling, 1981; Short, 1983). By this rationale, our data support a predominance of P limitation over N limitation, based on a significant pattern of P accumulation in both leaf and root plus rhizome tissue. The absence of N accumulation in leaves could reflect an inability of Z. marina to translocate N basipetally, although translocation studies with tracers do not support this hypothesis (Iizumi and Hattori, 1982; Short and McRoy, 1984). An alternative hypothesis for the accumulation of P, but not N, in plant tissues is the 'luxury' uptake of P, whereby plants are able to store excess P (when available) for subsequent use when P is in short supply. The general pattern of greater increases in tissue P compared with N (e.g. Moeller, 1983; Christiansen et al., 1985; Powell et al., 1989; Short et al., 1990) in fertilization studies tends to support this hypothesis. The relative importance of N vs. P limitation of plant productivity is often inferred from the deviation of tissue N : P ratios from a global median value (Redfield et al., 1963). This so-called 'Redfield approach' has been applied to macroalgae and seagrasses by Atkinson and Smith (1983), who calculated a global median N : P of ~pproximately 30:1 for all reported observations. Separating the seagras~ values from macroalgae values reported by Atkinson and Smith results in a median N : P value for seagrasses of about 18:1. N : P ratios of Z. marina in our controls were slightly less than this value ( 14-18:1 ), possibly indicative of modest N limitation. The significant correlation between tissue N: P and pore water N: P (Fig. 4) suggests that plant accumulation of N and P is strongly dependent on nutrient availability. It would seem that deviations of tissue N: P from the relative availabilities of N and P would be more suggestive of limitation of a particular nutrient (e.g. Short et al., 1990). Combining the results from the present investigation with other sediment fertilization studies in which both plant growth and tissue nutrients were measured provides a basis for comparing plant/nutrient interactions for a range of submersed plant species. Using dimensionless measures of growth and tissue nutrient responses to fertilizations allows for direct comparison of different species and sites (Fig. 6). The scatter plot reveals a positive relationship between growth responses and tissue nutrient responses. This positive relationship, developed from a variety of sub~nersed plant species in tropical to temperate and marine to lacustrine habitats, indicates a generic

NITROGEN VERSUS PHOSPHORUS LIMITATIONFOR GROWTH IN ZOSTERA MARINA

97

Growth vs. Tissue Nutrients (Y = 1.07 X - 1.49, r2 = 0.61)

•

Syr

-~jo= 200

"~

° L°DJ

1o0

"

o

<3

13Thai j Zos ~ Hzos,~o 0

• Thai Zos

o _Litt ~O0

200

A Leaf Tissue N or P (% Control)

Fig. 6. Relative growth and tissue nutrient responses (increases compared to comrois) with sediment fertilizationfor six submersed plant speciesfrom diverse environments:Syr, Syringodium filiforme (Short et al., 1990); Lob,Lobelia dortmanna L. (Moeller, 1983); Thai, Thalassia testudinum (Powellet al., 1988); Hzos,Heterozostera tasmanica (Bulthuisand Woclkerling, 1981); LitLLittorella uniflora ( Christiansenet al., 1985); Zos,Zostera mar/na (this study).

Open symbolsindicateleafN contentand dosed symbolsindicateneafPcontent. model of submersed plant/nutrient interaction. These plants all appear to respond to fertilizations by increasing both growth and tissue nutrient content. This overall relationship suggests that submersed plants sequester available nutrients in their tissues which directly influences their overall growth rates. There is no evidence here for a general submersed plant 'critical nutrient level' (Gerloff and Krombholz, 1966) beyond which plants continue to increase tissue nutrients without increasing growth. While such relationships have been demonstrated for several plant species (e.g. Thursby, 1984; Van Wijk, 1989), there is obviously a limit to the extent to which plants may increase tissue nutrients (especially N ), and there may be no reason to expect a universal relationship for all plants. In any case, the relative magnitude of the responses of either growth or tissue nutrient content could be used as an index of nutrient limitation. In conclusion, the relative importance of N and P as limiting nutrients for an estuarine population of Z. marina appears to have changed substantially over a 12 year period. Comparison of the modest fertilization effects in the present study with the much larger responses observed previously at the same site (Orth, 1977) indicates that progressive eutrophication, successional changes, or some other factor(s) have led to differences in the degree ofvutrient limitation in this Chesapeake Bay eelgrass bed. Plant growth and biomass responses in control and fertilized treatments have been used in the

98

v. MURRAYETAL

present study, along witi2 nutrient concentrations in sediment pore water and plant tissue nutrient content, to infer generally m o d e s t nutrient limitation for this eelgrass population. C o m p a r i s o n o f various fertilization responses in the literature indicates that m o s t studies c o n d u c t e d at mesotrophic to eutrophic sites reported similar m o d e s t responses. An overall positive relationship between growth increases a n d tissue nutrient concentration changes for fertilization studies with submersed plants in a wide diversity o f e n v i r o n m e n t s indicates a uniformity o f p l a n t / n u t r i e n t interaction. Submersed plants, thus, appear to generally respond to sediment nutrient availability by regulating both growth rates a n d tissue nutrient content. ACKNOWLEDGMENTS F u n d i n g for this project was provided by the U.S. Geological Survey, Water Resources Research Center o f Maryland a n d the State o f Maryland Chesapeake Bay Trust. We would like to t h a n k all those whose help m a d e this project possible. We would especially like to t h a n k Marilyn Mayer for her advice on methodology, Janet Neundorfer, David Poule, Tina Heister, T o m Randall a n d Mike Owens for their field a n d laboratory assistance, a n d Rick Bartleson for his c o m p u t e r expertise. Finally, we would like to t h a n k Jack Watts for the use o f his property a n d facilities as access to the study site.

REFERENCES Anderson, R.M. and Kalff, J., 1986a. Nutrient limitation ofMyriophyllum spicatum growth in situ. Freshwater Ecol., 16: 733-743. Anderson, R.M. and Kalff, J., 1986b. Regulation of submerged aquatic plant distribution in a uniform area ofa weedbed. J. Eco1.,74:953-961. Atkinson, M.J. and Smith, S.V., 1983. C:N:P ratios of benthic marine plants. Limnol. Oceanogr., 28: 568-574. Barko, J.W. and Smart, R.M., 1981. Comparative influence of light and temperature on growth and metabolism of selected submersed freshwater macrophytes. Ecol. Monogr., 51:219235. Barko, J.W. and Smart, K.M., 1986. Sediment-related mechanisms of growth limitation in submersed macrophytes. Ecology,6: 1328-1340. Bulthuis, D.A. and Woelkerling,W.J., 1981. Effects of in situ nitrogen and phosphorus enrichment of the sediments on the seagrass Heterozostera tasmanica (Martens ex Aschers.) den Hartog in Western Port, Victoria, Australia. J. Exp. Mar. Biol. Ecol., 53:193-207. Caffrey, J.M. and Kemp, W.M., 1990. Nitrogen cyclingin sediments with estuarine populations ofPotamogeton perfoliatus and Zostera marina. Mar. Ecol. Prog. Ser., 66: 147-160. Caraco, N., Tamse, A., Boutros, O. and Valiela, I., 1987. Nutrient limitation of phytoplankton growth in brackish coastal ponds. Can. J. Fish Aquat. Sci., 44: 473-476. Chambers, P.A. and Kalff, J., 1985. Depth distribution and biomass of submersed aquatic macrophyte communities in relation to secchi depth. Can. J. Fish. Aquat. Sci., 42: 701-709. Christiansen, R., Flus, N.J.S. and Szndergaard, M., 1985. Leaf production and nitrogen and

NITROGENVERSUSPHOSPHORUSLIMITATIONFORGROWTHINZOSTERAMARINA

99

phosphorus tissue content of Littorella un~.?.ora (L.) Aschers. in relation to nitrogen and phosphorus enrichment of the sediment in :)ligotrophic Lake Hampen, Denmark. Aquat. Bot., 23:1-11. D'Elia, C.F., Sanders, J.G. and Boynton, W.R., 1!86. Nutrient enrichment studies in a coastal plain estuary: Phytoplankton growth in large.scale, continuous cultures. Can. J. Fish. Aquat. Sci., 43: 397-406. Dennison, W.C., 1987. Effects of light on seagrass photosynthesis and depth distribution. Aquat. Bot., 27:15-26. Dennison, W.C., 1990. Rhizome/root production. In: R.C. McRoy and R.C. Phillips (Editors), Seagrass Research Methods. UNESCO, Paris, 210 pp. Dennison, W.C. and Alberte, R.S., 1986. Photosynthetic responses of Zostera marina L. (eelgrass) to in situ manipulations of light intensity. Oecologia (Berlin), 55:137-144. Dennison, W.C., AUer, R.C. and Alberte, R.S., 1987. Sediment ammonium availability and eelgrass (Zostera marina) growth. Mar. Biol., 94: 469-477. Duarte, C.M., 1991. Seagrass depth limits. Aquat. Bot., 40: 363-377. Duarte, C.M. and Kalff, J., 1988. Influence of lake morphometry on the response of submerged macrophytes to sediment fertilization. Can. J. Fish. Aquat. Sci., 4~: 216-221. Gerloff, G.C. and Krumbholz, P.H., 1966. Tissue analysis as a measure of nutrient availability for the growth of angiosperm aquatic plants. Limnol. Oceanogr., 11: 529-537. Hill, S.M., 1989. Phosphorus uptake and translocation in the submersed aquatic, Vallisneria americana. MS Thesis, University of Maryland System, College Park, 53 pp. Howarth, R.W., 1988. Nutrient limitation of net primary production in marine ecosystems. Annu. Rev. Ecol., 19:89-110. lizumi, H. and Hattori, A., 1982. Growth and organic production ofeelgrass (Zostera marina L.) in temperate waters of the Pacific Coast of Japan. III. The kinetics of nitrogen uptake. Aquat. Bot., 12: 245-256. lizumi, H., Hattori, A. and McRoy, C.P., 1982. Ammonium regeneration and assimilation in eelgrass (Zostera marina) beds. Mar. Biol., 66: 59-65. Kemp, W.M., Boynton, W.R., Twilley, R.R., Stevenson, J.C. and Means, J.C., 1983. The decline of submerged vascular plants in upper Chesapeake Bay: Summary of results concerning possible causes. Mar. Tech. Soc. J., 17 (2): 78-89. Kemp, W.M., Sampou, P., Caffrey, J., Mayer, M., Henriksen, K. and Boynton, W.R., 1990. Ammonium recycling versus denitrification in Chesapeake Bay sediments. Limnol. Oceanogr., 35: 1545-1563. Moeller, R.E., 1983. Nutrient-enrichment of rhizospbere sediments: An experimental approach to the ecology of submersed macrovegetation. Proc. Int. Symp. Aquatic Macrophytes, 18-23 ,September, Catholic University ofNijmegen, N~megen, Netherlands, pp. 145-149. Murray, L. and Wetzel, R.L., 1987. Oxygen production and consumption associated with the major autotrophic components in two temperate seagrass communities. Mar. Ecol. Prog. Ser., 38: 231-239. Orth, R.3., 1977. Effect of nutrient enrichment on growth of the eelgrass Zostera marina in the Chesapeake Bay, Virginia, USA. Mar. Biol., 44: 187-194. Patriquin, D.G., 1972. The origin of nitrogen and phosphorus for growth of the marine angiosperm, Thalassia testudinum. Mar. Biol., 15: 35-46. Powell, G.V.N., Kenworthy, W.J. and Fourqurean, J.W., 1989. Experimental evidence for nutrient limitation of seagrass growth in a tropical estuary with restricted circulation. Bull. Mar. Sci., 44: 324-340. Redfield, A.C., Ketchum, B.H. and Richards, F.A., 1963. The influence of organisms on the composition of sea-water. In: M.N. Hill (Editor), The Sea, vol 2. Wiley, New York, pp. 2677.

100

L.MURRAYETAL

Robel~.s, M.H., Orth, R.J. and Moore, K.A., 1984. Growth of Zostera marina L. seedlingsunder labor,~toryconditions of nutrient enrichment. Aquat. Bot., 20:321-328. Roth, N.C. and Pregnall, A.M., 1989. Nitrate reductase activity in Zostera marina. Mar. Biol., 99: 457-463. Ryther, J.H. ana Dunstan, ~V.M., 1971. Nitrogen, phosphorus and eutrophication in ~he coastal marne environment. Science, 117: 1008-1013. Sand4ensen, K. anO Borum, J., 1983. Regulation of growth in eelgrass (Zostera marina L.) in Daniish coastal waters. Mar. Tech. Soc. J., 17 (2): ! 5-21. Schindl~er,D.W., 1977. Evolution of phosphorus limitation in lakes. Science, 195: 260-762. Short, ET., 1983. The response of interstitial ammonium in eelgrass (Zostera mariszo L. ) beds to ,environmentalperturbations. J. Exp. Mar. Biol. Ecol., 68: 195-208. Short. F.T., 1987. Effects of sediment nutrients on seagrasses: literature review and mesocosm expeJfiment.Aquat. Bot., 27:41-57. Short, F.T. and McRoy, C.P., 1984. N;.trogenuptake by leaves and roots of the seagrass Zostera marina L. Bot. Mar., 27: 547-555. Sh,~rt, F.T., Davis, M.W., Gibson, R.A. and Zimmermann, C.E, 1985. Evidence for phosphorus limitation in carbonate sediments ofthe seagrassSyringodiumfiliforme. Estuarine Coastal Shelf Sci., 20:419-430. Short, ET., Dennison, W.C. and Capone, D.G., 1990. Phosphorus-limited growth of the tropical seagrass Syringodium filiforme in carbonate sediments. Mar. Ecol. Prog. Ser., 62:169174. Smith, C.S. and Adams, M.S., 1986. Phosphorus transfer from sediments by Myriophyllum spi. catum. Limnol. Oceanogr., 31: 1312-1321. Smith, S.V. and Atkinson, M.J., 1984. Phosphorus limitation of net production in a confined aquatic ecosystem. Nature, 307: 626-627. Stevenson, J.C., 1988. Comparative ecology of submersed grass beds in freshwate~r,estuarine, and marine environments. Limnol. Oceanogr., 33: 867-893. Strickland, J.D.H. and Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Ottawa, Canada, 310 pp. Thursby, G.B., 1984. Nutritional requirements of the submerged angiosperm Ruppia maritima in algae-freeculture. Mar. Ecol. Prog. Ser., 16: 45-50. Thursby, G.B. and Harlin, M.M., 1982. Leaf-root interaction in the uptake of ammonia by Zostera marina. Mar. Biol., 72:109-112. Toetz, D.W., 1973. The kinetics of NH4 uptake by Ceratophyllum. Hydrobiologia, 41: 275-290. Twilley, R.R., Brinson, M.M. and Davis, G.J., 1977. Phosphorus absorption, translocat.ionand secretion in Nuphar luwum. Limnol. Oceanogr., 22: 1022-1032. Van Wijk, R.J., 1989. Ecologicalstudies on PotamogeWn pectinatus L. V. Nuiritional ecology, in vitro uptake of nutrients and growth limitation. Aquat. Bot., 35:319-335. Williams, S., 1987. Competition between the seagrasses Thalassia testudinum and Syringodium filiforme in a Caribbean lagoon. Mar. Ecol. Prog. Ser., 35: 91-98. Williams, S., 1990. Experimental studies of Caribbean seagrass bed develgpment. Ecol. Monogr., 60: 449-469. Wetzel, R.L. and Penhale, P.A., 1983. Production ecologyof seagrass communiti,:s in the lower Chesapeake Bay. Mar. Tech. Soc. J., 17 (2): 22-31. Zieman, J.C. and Wetzel, R.G., 1980. Productivity in seagrasses: Methods and rates° In: R.C. Phillips and C.P. McRoy (Editors), Handbook of Seagrass Biology:An Ecosyste~aPerspective. Garland STPM Press, New York, pp. 87-116.