THE UNIVERSITY OF TEXAS AT AUSTIN THE GENERAL LIBRARIES PERRY-CASTANEDA LIBRARY LIMITED CIRCULATION

Nitrogen budget of the seagrass Thalassia testudinum in the western Gulf of Mexico

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Copyright by Kun-Seop Lee 1998

Nitrogen budget of the seagrass Thalassia testudinum in the western Gulf of Mexico

by Kun-Seop Lee, 8.5., M.S.

Dissertation

Presented to the Faculty of the Graduate School of the University of Texas at Austin in Partial Fulfillment for the Requirements

for the Degree of Doctor of Philosophy

The University of Texas at Austin August 1998

This work is dedicated to my wife, Mi Youn.

Acknowledgments

The fear of the LORD is the beginning of know ledge (Proverbs 1: 7).

Completion of this work would not have been possible without the guidance and support of my supervisor, Ken Dunton. He provided opportunities, encouragement, financial support as well as friendship. My committee members, Ron Benner, Chris Onuf, Ellery Ingall and Paul Montagna provided useful comments and advice on this research. Special thanks to Sharon Herzka, Lanny Miller and Christine Weilhoefer for their countless hours of reading, discussions and useful comments as well as their friendship. I am very grateful to Jim Kaldy and Kelly Machalek-Major for their discussions and friendship. Susan Schonberg and Kim Jackson were very helpfill in the field and lab, and for everything. Joe Kowalski, Chris Krull and Randy Pritchard provided excellent help in the field.

I especially thank Drs Kye Chil Oh and Jueson Maeng for their counsel, encouragement and guidance. Very special thanks to pastor Bong Kee Huh, all church members, and Korean friends, Jae-Kyun Lee, Joon Won Yoon, Minpyo Hong and Sung Ho Cho who improved my quality of life in Texas. Finally, I would like to thank my parents, my wife Mi Youn, daughter Jin and son Joshua for their love and patience.

This research was supported by the U.S. Army Corps of Engineers award # 96-92-03. Additional salary support was provided by E. J. Lund Fellowship to K-S Lee.

Nitrogen budget of the seagrass Thalassia testudinum in the western Gulf of Mexico

Publication No.

Kun-Seop Lee, Ph.D. The University of Texas at Austin, 1998

Supervisor: Kenneth H. Dunton

The nitrogen (N) budget of the seagrass Thalassia testudinum was examined with respect to inorganic-N acquisition and the effects of sediment NH 4 + enrichment on two distinct populations in south Texas. The two populations exhibit different biomass allocation patterns at Corpus Christi Bay (CCB) and lower Laguna Madre (LLM): plants at CCB have a higher above-ground biomass while plants at LLM have a higher below-ground biomass. Ambient sediment pore water NH 4 + concentrations at CCB (ca. 100 pM) were significantly higher than at LLM (ca. 30 pM). Therefore, it was hypothesized that 1) differences in biomass allocation are a result of the differential sediment N availability, 2) sediment NH 4 + enrichment will affect growth, leaf morphology and tissue nutritional content of T. testudinum to a greater degree at low sediment N conditions, and 3) the relative contributions by leaf and root tissues to total N acquisition will differ between the two study sites. To examine the effects of sediment NH 4 + enrichment, the seagrass bed sediments were fertilized with commercial N fertilizer, and changes in production, biomass, leaf morphology, tissue nutritional content and carbon (C) reserves were monitored. Additionally, N uptake by leaves and roots of T. testudinum from the two sites were measured seasonally. After fertilization, leaf production rates and shoot height at LLM increased to reach levels equivalent to CCB. However, sediment NH 4 + enrichment had little effect on production and leaf size of T. testudinum at CCB. These results suggest that sediment N availability at LLM limits seagrass production. Rhizome non-structural carbohydrates (NSC) decreased in response to sediment NH 4 + enrichment during the early periods of the experiment which suggests that C was reallocated from rhizome to leaf tissues to support the stimulated leaf growth. Thus, the NH 4 + enrichment affected concentration and allocation of Cas well as N. Root NH 4 + uptake accounted for about 52 %of total N acquisition, while leaf NH 4 + uptake contributed about 38 % and leaf NO 3 ’ uptake accounted for the remaining 10 %at both sites. The high biomass, chlorophyll, and C content in leaf tissues at CCB and the high biomass, C and NSC content in rhizome tissues at LLM demonstrated that plants responded to high sediment N conditions by enhancing leaf function, and to low N conditions by enhancing function of below-ground tissues.

Table of Contents

List of Table xi

List of Figure xii

Chapter 1: Effects of nitrogen enrichment on growth, biomass and leaf morphology of turtle grass Thalassia testudinum in the western Gulf of Mexico .... 1

Abstract 1

Introduction 3

Material and Methods 7

Results 14

Discussion 29

Chapter 2 : Influence of sediment nitrogen availability on carbon and nitrogen dynamics of the seagrass Thalassia testudinum 37

Abstract 37

Introduction 39

Material and Methods 42

Results 48

Discussion 61

Chapter 3 : Inorganic nitrogen acquisition in the seagrass Thalassia testudinum'. development of a whole-plant nitrogen budget 71

Abstract 71

Introduction 73

Material and Methods 76

Results 83

Discussion 99

Literature cited 114

Vita 125

List of Tables

Table 1.1. Biological, physical and chemical characteristics of study sites in Corpus Christi Bay and lower Laguna Madre, Texas 9

Table 1.2. Average sediment pore water NO 3 '+NO 2 ’ and PO 4 3 ’ concentrations in control and NH 4 + enriched Thalassia testudinum plots in Corpus Christi Bay and lower Laguna 16

Table 1.3. Species specific response to nitrogen fertilization and nitrogen limitaiion reported in in situ seagrass beds during growing season 31

Table 2.1. Sediment pore water ammonium concentrations in control and ammoniumfertilized Thalassia testudinum plots at Corpus Christi Bay and lower Laguna Madre between May and October 1997 44

Table 2.2. Thalassia testudinum. Differences in biomass, production, chlorophyll and tissue nutritional content between high and low sediment nitrogen availability 69

Table 3.1. Parameters (V max and KJ of the Michaelis-Menten model and uptake affinity (V max /KJ for leaf and root nitrogen uptake of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 89

Table 3.2. Parameters (V max and KJ of the Michaelis-Menten model for light and dark nitrogen uptake by leaf and root tissues 95

Table 3.3. Parameters (V max and KJ of the Michaelis-Menten model for tissue nitrogen uptake of various seagrass species 100

Table 3.4. Estimated DIN pool size in water column and sediment, annual mean nitrogen uptake rate by seagrass and DIN turnover time in water column and sediment in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) . lll

List of Figures

Figure 1.1. Location of study sites in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM), Texas, USA 8

Figure 1.2. Sediment pore water NH 4 + concentrations in control and NH 4 + enriched Thalassia testudinum plots in Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997 15

Figure 1.3. Thalassia testudinum. Shoot density (A, B), shoot height (C, D) and blade width (E, F) in control and sediment NH 4 + enriched plots in Corpus Christi Bay and lower Laguna Madre between May and October 1997 . 18

Figure 1.4. Thalassia testudinum. Leaf production rate (g dry wt m’ 2 d’ 1 ) in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997 20

Figure 1.5. Thalassia testudinum. Biomass of total and different plant components in control and sediment NH 4 + enriched plots at Corpus Christi Bay between May and October 1997 23

Figure 1.6. Thalassia testudinum. Biomass of total and different plant components in control and sediment NH 4 + enriched plots at lower Laguna Madre between May and October 1997 25

Figure 1.7. Thalassia testudinum. Above and below-ground biomass and below/above-ground biomass ratios in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997 28

Figure 2.1. Thalassia testudinum. Leaf growth rates (g dry wt shoot’ 1 d’ 1 ) and leaf turnover time (days) in control and sediment NH 4 + enriched plots in Corpus Christi Bay (A, B) and lower Laguna Madre (C, D) between May and October 5O

Figure 2.2. Thalassia testudinum. Total blade chlorophyll (mg chi g’ 1 dry wt) and chi A:B ratios in control and sediment NH 4 + enriched plots in Corpus Christi Bay

(A, B) and lower Laguna Madre (C, D) between May and October 1997 53

Figure 2.3. Thalassia testudinum. Carbon and nitrogen content and C/N molar ratios of leaf tissues in control and sediment NH 4 + enriched plots in Corpus Christi Bay (A, B, C) and lower Laguna Madre (D, E, F) between May and October 1997 55

Figure 2.4. Thalassia testudinum. Carbon and nitrogen content and C/N molar ratios of rhizome tissues in control and sediment NH 4 + enriched plots in Corpus Christi Bay (A, B, C) and lower Laguna Madre (D, E, F) between May and October 1997 56

Figure 2.5. Thalassia testudinum. Rhizome non-structural carbohydrate (NSC) content and persent of rhizome NSC carbon to total rhizome carbon in control and sediment NH 4 + enriched plots in Corpus Christi Bay (A, B) and lower Laguna Madre (C, D) between May and October 1997 59

Figure 2.6. Schematic diagram illustrating the effect of sediment NH 4 + enrichment on seagrass growth and mobilization of reserve NSC in rhizome tissues one month following fertilization 67

Figure 3.1. Water column NH 4 + (A), NO 3 +NO 2 ' (B) and sediment NH 4 + (C) concentrations in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) from October 1996 to November 1997 85

Figure 3.2. Thalassia testudinum. Seasonal changes in leaf and root biomass in Corpus Christi Bay (CCB; A) and lower Laguna Madre (LLM; B) 86

Figure 3.3. Thalassia testudinum. Leaf NH 4 + uptakes of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 as a function ofNH 4 + concentration 88

Figure 3.4. Thalassia testudinum. Leaf NO 3 ’uptakes of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 as a function of NO 3 ’ concentration 90

Figure 3.5. Thalassia testudinum. Root NH 4 + uptakes of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and

October 1997 as a function of NH 4 + concentration 92

Figure 3.6. Thalassia testudinum. Comparisons of light and dark nitrogen uptake rates for leaf NH 4 + (A), leaf NO 3 ' (B) and root NH 4 + (C) 94

Figure 3.7. Thalassia testudinum. Seasonal changes in daily nitrogen acquisition by leaves from water column NH 4 + (A) and NO 3 ' (B) and by roots from sediment NH 4 + (C) 97

Figure 3.8. Monthly and annual N acquisition by leaf and root tissues from water column and sediment in Corpus Christi Bay (A) and lower Laguna Madre (B) 9B

Figure 3.9. Nitrogen budget for Thalassia testudinum in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) 107

Chapter 1: Effects of nitrogen enrichment on growth, biomass and leaf morphology of turtle grass Thalassia testudinum in the western Gulf of Mexico

Abstract

The effects of sediment ammonium (NH 4 + ) enrichment on growth and leaf morphology of the seagrass Thalassia testudinum in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM), Texas were examined from May to October 1997. Prior studies had shown that shoot height and leaf biomass at CCB were significantly higher than those at LLM and ambient sediment NH 4 + concentrations in CCB (ca. 100 pM) were significantly higher than those in LLM (ca. 30 pM). It was hypothesized that the differences in plant morphology and biomass between the two areas could be related to differences in sediment nitrogen levels between two sites. To test this hypothesis, I conducted an in situ fertilization experiment at both sites over a six-month period. Results of this experiment revealed that seagrass growth, biomass and leaf size significantly increased as a result of sediment NH 4 + enrichment at LLM, but had little effect on plant density, biomass and leaf morphology at CCB. In control plots, average leaf production rate (7.4 g dry wt m’ 2 d' 1 ) and shoot height (43.3 cm) at CCB were significantly higher than those at LLM (2.5 g dry wt m’ 2 d’ 1 and 18.8 cm, respectively). After fertilization, leaf production rates and leaf size at LLM increased to reach equivalent levels of the CCB site. Leaf biomass at LLM increased significantly as a result of sediment NH 4 + enrichment, but there was little change in below-ground biomass. The below to above-ground biomass ratio at LLM (4.66) was about 3-fold higher than that at CCB (1.63) in control plots, but decreased significantly at LLM with sediment NH 4 + enrichment, while the ratio at CCB remained unchanged. I conclude, based on seagrass growth responses to increases in sediment NH 4 + , that sediment nitrogen availability at LLM limits seagrass productivity. An ambient sediment NH 4 + level of about 100 pM at CCB was considered to be the threshold concentration for nitrogen limitation of T. testudinum growth.

Introduction

Seagrasses are important primary producers in coastal and estuarine ecosystems. As autotrophs, seagrasses require light, inorganic carbon sources, and inorganic nutrients (e.g. nitrogen and phosphate). Because of their high production rates, e.g. 1000 g dry wt m' 2 yr' 1 (Mcßoy and McMillan, 1977; Lee and Dunton, 1996; Kaldy, 1997), seagrass beds may assimilate and sequester large amounts of inorganic nitrogen (N) and phosphorus (P), dominating estuarine nutrient cycling (Kenworthy et al., 1982; Romero et al., 1994). Thus, nutrient availability may play a significant role in regulating seagrass production in shallow and clear areas where light is plentiful. Fertilization studies have shown that the addition of Nor P can stimulate seagrass growth resulting in increased biomass (Orth, 1977; Harlin and Thome-Miller, 1981; lizumi et al., 1982; Dennison et al., 1987; Short et al., 1990; Perez et al., 1991; Murray et al., 1992, Williams and Ruckelshaus, 1993), suggesting nutrient limitation for plant growth.

The potential existence and extent of nutrient limitation for seagrass growth has frequently been evaluated through fertilization experiments (Orth, 1977; Bulthuis and Woelkerling, 1981; Bulthuis et al., 1992; Agawin et al., 1996; Udy and Dennison 1997). Other approaches have also been used, including comparison of uptake rates with plant nutrient requirements (Pedersen and Borum, 1993), elemental analysis of plant tissues (Duarte, 1990; Fourqurean et al., 1992 a, 1992 b; Gerloff and Krombholtz, 1996), and examination of the relationship between environmental nutrient levels and plant status (Short, 1983; Fourqurean et al., 1992 b). However, in situ fertilization experiments remain the most conclusive and direct measures of assessing nutrient limitation for plant growth as reflected by increases in biomass, growth, shoot height and density (Orth, 1977; Bulthuis and Woelkerling, 1981; Bulthuis et al., 1992; Agawin et al., 1996; Udy and Dennison 1997), tissue N and P content (Udy and Dennison, 1997), and photosynthetic activity (Agawin et al., 1996).

Seagrasses have access to inorganic-N sources in both the sediment and the water column (lizumi and Hattori, 1982; Thursby and Harlin, 1982; Short and Mcßoy, 1984; Stapel and Hemminga, 1997; Terrados and Williams, 1997). N enrichment of the water column can lead to decreased underwater light availability as a result of epiphyte growth and phytoplankton blooms that can lead to seagrass decline (Orth and Moore, 1983; Silberstein et al., 1986; Giesen et al., 1990; Tomasko and Lapointe, 1991). In addition, Burkholder et al. (1992, 1994) demonstrated that water column nitrate (NO 3 ’) enrichment caused death of Zostera marina though a direct physiological effect unrelated to algal light attenuation. In most cases, however, sediment N enrichment enhanced seagrass growth, and no harmful effects have been reported (Bulthuis and Woelkerling, 1981; Agawin et al., 1996; Alcoverro et al., 1997; Udy and Dennison, 1997). Therefore, although a high N requirement has been demonstrated for seagrass growth, the plant responses to increased N availability may vary depending on the N sources.

Due to the high concentration of NH 4 + in pore waters relative to the overlying water column, seagrasses obtain a large fraction of their N from the sediment via root tissues (lizumi and Hattori, 1982; Short and Mcßoy, 1984; Zimmerman et al., 1987). Sediment NH 4 + is the most abundant source of Nin seagrass beds (Agawin et al., 1996; Dunton, 1996; Terrados and Williams, 1997) and consequently plays an important role in regulation of seagrass growth. The concentration of sediment NH 4 + is highly variable, ranging from less than 20 pM (Bulthuis et al., 1992; Agawin et al., 1996; Udy and Dennison, 1997) to well over 100 pM (Bulthuis and Woelkerling, 1981; lizumi et al., 1982; Fourqurean et al., 1992 b; Czerny and Dunton, 1995; Dunton, 1996). Thus, existence of N limitation in each seagrass bed may depend on in situ sediment NH 4 + level, and the sediment NH 4 + concentration may be used as a indicator of the N status for seagrass growth.

Seagrasses living under varying conditions of N availability may respond differently to sediment N enrichment, and N limitation can be predicted from plant responses. The present study focused on plant responses to sediment N fertilization in two distinct T. testudinum populations in Texas: Corpus Christi Bay (CCB) and lower Laguna Madre (LLM). Previous studies demonstrated that plant height and leaf biomass in Corpus Christi Bay were significantly higher than in the lower Laguna Madre (Lee and Dunton, 1996; Herzka and Dunton, 1997, Kaldy, 1997). These differences were hypothesized to result from measured differences in pore water NH 4 + levels in the two areas. The present study tested the hypothesis that while N availability at LLM is limiting for plant growth, the N requirements of T. testudinum at CCB are adequately met. Changes in density, biomass and leaf morphology were evaluated in response to in situ sediment NH 4 + enrichment at the two sites. Pore water NH 4 + , nitrate+nitrite (NO 3 '+NO 2 ) and phosphate (PO 4 3 ’)levels were monitored to assess sediment nutrient availability for T. testudinum growth.

Materials and methods

Study site

The study was conducted in monotypic meadows of Thalassia testudinum in Corpus Christi Bay (CCB; 2T 49' N, 97° 07' W) and lower Laguna Madre (LLM; 26° 09' N, 97° 12' W), Texas, USA. The study sites are located about 200 km apart at similar water depth (1.2 m; Fig. 1.1), and have been the focus of several recent studies on south Texas seagrasses (Dunton 1990, 1994; Quammen and Onuf, 1993; Czerny and Dunton 1995, Lee and Dunton 1996, 1997, Herzka and Dunton 1997, 1998, Kaldy 1997). The CCB population is located on the eastern side of Corpus Christi Bay and is characterized by high pore water NH 4 + concentration (ca. 100 pM) and high sand content in the sediment (Table 1.1). In contrast, the sediments at the LLM site, located 200 km to the south, have lower pore water NH 4 + concentration (ca. 30 pM) and sand content. T. testudinum plants growing at CCB are much taller and have higher leaf biomass. Water column NH 4 + and NO 3 +NO 2 ’ at both sites were about 1 pM and were not significantly different. Underwater photosynthetically active radiation (PAR) was significantly higher at LLM than CCB (Table 1.1). The T. testudinum bed in LLM represents a relatively young population

which replaced Syringodium filiforme in the 1970 s and 1980 s (Quammen and Onuf, 1993).

Sediment NH/ enrichment

At each site, six experimental plots (1.0 x 1.0 m) were established in a homogeneous Thalassia testudinum meadow. Three served as controls and three were enriched with ammonium sulfate (21-0-0, Esco Corp. Dallas, TX). The fertilizer was wrapped in several layers of cheesecloth and in each plot, 25 packets consisting of 20 g of fertilizer were buried at 20 cm intervals and at about 10 cm sediment depth. The amount of N applied to each enriched plot was 105 g N m‘ 2 based on manufacturer’s nominal values. The rhizome connections at the perimeter of each plot were cut to a sediment depth of about 30 cm to physiologically separate plants located within and outside the plots. Ammonium enrichment was initiated in early May 1997; plant and sediment samples were collected monthly from May to October from both control and treatment plots.

Sediment nutrient analyses

On each sampling date, three replicate sediment samples were collected randomly from each plot to a depth of ca. 13 cm with a 60 ml syringe corer. Samples were placed on ice and frozen pending lab analyses. Sediment pore water was obtained by centrifugation (5000 g for 15 min) and used for determination of pore water NH 4 + , NO 3 +NO 2 ’ and PO 4 3 ' concentrations. Concentrations of NH 4 + and PO 4 3 " were determined using standard colorimetric techniques following the methods of Parsons et al. (1984) after dilution (1:5; v/v) with low nutrient sea water collected offshore in the Gulf of Mexico. Concentrations of NO 3 +NO 2 ’ were determined colorimetrically after running through a column containing copper coated cadmium which reduces NO 3 ' to NO 2 ‘ (Parsons et al. 1984).

Biological measurements

Measurements of shoot density, biomass, shoot height and blade width were completed monthly from May to October 1997. At each sampling date, one core sample from each plot was collected with a 9 cm diameter corer driven about 20 cm into the sediment. Samples were thoroughly cleaned of epiphytes and sediments, separated into leaf (blade and sheath), rhizome (vertical and horizontal) and root tissues, and dried at 60°C to a constant weight. Samples were weighed and biomass was converted to an areal estimate (g dry wt m' 2 ). Shoot density was estimated by counting the number of shoots inside the core. Shoot height was estimated by measuring the longest leaf length. Blade width was measured to the nearest 0.5 mm using the mature green leaves in cores collected for biomass.

Leaf production rates were measured using a modified blade marking technique (Zieman, 1974; Kentula and Mclntire, 1986). Five randomly chosen shoots from each plot were marked just above the bundle sheath with a hypodermic needle and then harvested after a period of 3 to 4 weeks. Leaf material was separated into leaf tissue produced before and after marking and dried at 60°C to a constant weight. The leaf production rate per shoot was determined by dividing the dry weight of new leaf tissue produced after marking by the number of days since marking. Areal production rates were obtained by multiplying the average leaf production per shoot in a given plot by its corresponding shoot density.

Statistics

All values are reported as means ± 1 standard error (SE). Statistical analyses were performed on a microcomputer using a general linear procedure (SAS Institute, 1989). Data were tested for normality and homogeneity of variance to meet the assumptions of parametric statistics, and assumptions were satisfied for all data tested. Differences in sediment nutrients, shoot density, shoot height, blade width and biomass among sampling time and between treatments were tested for significance using a two-way ANOVA, with time as a block. When a significant difference among variables was observed, the means were analyzed by a Tukey multiple comparison test to determine where the significant differences occurred among variables.



Figure 1.1. Location of study sites in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM), Texas, USA. Stippled areas represent wind-tidal flats, which are periodically flooded.

Parameter Site CCB LLM Sediment Pore water ammonium (pM) 115.08 ± 11.12 (51) 24.27 ±2.42 (19) *** Sand content (%) 91.19 ±0.47 (58) 71.44 ± 1.27 (35) *** Clay+silt (%) 8.81 ±0.47 (58) 28.60 ± 1.26 (35) *** Water column Ammonium 1.16 ±0.06 (58) 1.22 ±0.08 (38) Nitrate+nitrite 0.84 ± 0.05 (58) 0.84 ±0.06 (38) Thalassia testudinum Height (cm) 41.42 ±0.63 (149) 18.64 ±0.28 (196) *** Leaf biomass (g dry wt m’ 2 ) 227.76 ±24.85 (15) 159.42 ± 11.32 (7) * Root/shoot ratio 1.35 ±0.10 (15) 4.73 ± 0.27 (7) *** Underwater PAR (mol photons m' 2 d’ 1 ) 12.07 ± 0.42 (362) 15.44 ±0.50 (362) *** Mean ± standard error (sample size)

Table 1.1. Biological, physical and chemical characteristics of study sites in Corpus Christi Bay and lower Laguna Madre, Texas. Values are from a long-term data set collected since 1990 (Herzka and Dunton, 1997, 1998; Lee and Dunton, 1997; Kaldy, 1998; Dunton, unpubl. data). Asterisks represent significant differences between the two study sites based on t-tests: * < 0.05; ** < 0.01; *** < 0.001

Results

Sediment nutrients

Pore water NH 4 + concentrations in control plots at CCB (ca. 100 pM NH 4 + ) were significantly higher than at LLM (ca. 30 pM NH 4 + ) over the six-month experimental period (P < 0.001). During the first month following NH 4 + enrichment, the concentrations of sediment pore water NH 4 + increased to 2000 pM NH 4 + at both study sites. By the end of experiment, five months after NH 4 + addition, it declined to about 200 pM which was still significantly higher than control plots (Fig. 1.2).

Since pore water NO 3 +NO 2 ' and PO 4 3 ' concentrations did not exhibit significant seasonal trends, average values throughout the experiment are presented in Table 1.2. There were no significant differences in pore water NO 3 +NO 2 ‘ concentrations between study sites (P = 0.66) and between NH 4 + enriched and control plots (P = 0.48 and P = 0.91 at CCB and LLM, respectively). Pore water PO 4 3 ' concentration in control plots at CCB (ca. 24 pM PO 4 3 ) was significantly higher than at LLM (ca. 15 pM PO 4 3 '; P < 0.001). However, the pore water PO 4 3 ‘ concentrations were not significantly different between NH 4 + enriched and control plots at both CCB (P = 0.94) and LLM (P = 0.79; Table 1.2).

Shoot density and leaf morphology

The average shoot density in control plots at LLM (ca. 2000 m’ 2 ) was significantly higher than at CCB (ca. 1700 m’ 2 ; P < 0.001). Shoot densities at CCB ranged from 1200 m' 2 in July to 2100 m' 2 in August and did not change as a result of sediment NH 4 + enrichment (Fig. 1.3 A). In LLM, shoot densities in the enriched plots also did not increase relative to controls during the first four months following NH 4 + enrichment, but were significantly higher in the enriched plots by October, five months after fertilization (P = 0.022; Fig. 1,3 B).

In control plots, shoot height was significantly higher at CCB than at LLM (P < 0.001). Sediment NH 4 + enrichment had no significant effect on shoot height at CCB (P = 0.226). Shoot height at CCB showed a significant seasonal trend (P < 0.001); values ranged from 30.3 cm in May to 49.5 cm in August (Fig. 1.3 C). In contrast, shoot height at LLM significantly increased as a result of sediment NH 4 + enrichment (P < 0.001), increasing continuously in enriched plots from 17.7 cm in May to a maximum of 34.3 cm in September (Fig. 1.3 D). Shoot height in control plots at LLM remained at ca. 18 cm throughout the experiment.

Average blade widths in control plots at CCB (6.3 mm) were significantly higher than at LLM (5.2 mm; P < 0.001). At CCB, blade width in control plots ranged from 5.9 mm in June to 6.6 mm in October. There was no significant difference in blade width in enriched plots relative to controls (Fig. 1.3 E). In contrast, blade width at LLM increased significantly relative to controls as a result ofNH 4 + enrichment (P < 0.001; Fig. 1.3 F).

Leaf production and plant biomass

Leaf production rates in control plots at CCB (7.35 g dry wt m’ 2 d’ 1 ) were significantly higher than those at LLM (2.50 g dry wt m' 2 d' 1 ; P < 0.001). The rate of leaf production at CCB was about 2-fold higher in fertilized plots relative to controls during the first month following NH 4 + enrichment, although there were no significant differences between fertilized and control plots during subsequent months (P = 0.109; Fig. 1.4). Leaf production rates in control plots at CCB showed a strong seasonal trend ranging from 4.86 g dry wt m' 2 d' 1 in June to 9.37 g dry wt m' 2 d' 1 in August. Sediment NH 4 + enrichment significantly stimulated leaf production at LLM (P < 0.001; Fig. 1.4). The average leaf production rate in control plots at LLM was 2.50 g dry wt m' 2 d' 1 and 8.21 g dry wt m’ 2 d’ 1 in fertilized plots.

There was no significant effect of NH 4 + enrichment on total biomass as well as the biomass of different tissues at CCB (P > 0.05; Fig. 1.5). However, leaf biomass at LLM site significantly increased as a result of sediment NH 4 + enrichment (P < 0.001). Leaf biomass in control plots remained at a weight of about 150 g dry wt m‘ 2 throughout the experiment, while continuously increasing from 143 g dry wt m' 2 in May to 468 g dry wt m’ 2 in September in fertilized plots (Fig. 1.6). Root and total biomass at LLM did not change during the first three months following NH 4 + enrichment, although a significant increase was observed during subsequent months (P = 0.013, root and P < 0.001, total biomass; Fig. 1.6). Sediment NH 4 + enrichment had no significant effect on rhizome biomass at LLM (P = 0.54).

Average leaf biomass in control plots at CCB (281 g dry wt m’ 2 ) was significantly higher than that at LLM (163 g dry wt m' 2 ; P < 0.001; Fig. 1.5, 1.6). In contrast, average total and rhizome biomass at LLM (889 and 663 g dry wt m" 2 , respectively) were significantly higher than those at CCB (712 and 358 g dry wt m’ 2 , respectively) (P < 0.001). Root biomass did not differ significantly between study sites (P = 0.092).

Average below/above-ground biomass ratio in control plots at LLM (4.66) was significantly higher than that at CCB (1.63; P < 0.001). About 60% of total biomass was apportioned below ground in control plots at CCB. At LLM, about

82% of total biomass corresponded to below ground biomass in control plots and 72% of total biomass in fertilized plots. At CCB, there was no significant effect of NH 4 + enrichment on below/above-ground biomass ratio (P = 0.133; Fig. 1.7). However, below/above-ground biomass ratio in enriched plots (2.53) at LLM was significantly lower than that in control plots (4.66; P < 0.001; Fig. 1.7).



Figure 1.2. Sediment pore water NH 4 + concentrations in control and NH 4 + enriched Thalassia testudinum plots in Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997. Triangle on the x-axis denotes the date of fertilizer additions. Values are means ± SE (n=3).



Figure 1.3. Thalassia testudinum. Shoot density (A, B), shoot height (C, D) and blade width (E, F) in control and sediment NH 4 + enriched plots at Corpus Christi Bay and lower Laguna Madre between May and October 1997. Triangle on the x-axis denotes the date of fertilizer additions. Values are means ± SE (n=3).



Figure 1.4. Thalassia testudinum. Leaf production rate (g dry wt m’ 2 d' 1 ) in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997. Triangle on the x-axis denotes the date of fertilizer additions. Values are means ± SE (n=3).



Figure 1.5. Thalassia testudinum. Biomass of total and different plant components in control and sediment NH 4 + enriched plots at Corpus Christi Bay between May and October 1997. Triangle on the x-axis denotes the date of fertilizer additions. Values are means ± SE (n=3).



Figure 1.6. Thalassia testudinum. Biomass of total and different plant components in control and sediment NH 4 + enriched plots at lower Laguna Madre between May and October 1997. Triangle on the x-axis denotes the date of fertilizer additions. Values are means ± SE (n=3).



Figure 1.7. Thalassia testudinum. Above and below-ground biomass and below/above-ground biomass ratios in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A) and lower Laguna Madre (B) between May and October 1997.

CCB LLM Control Fertilized Control Fertilized NO 3 ±NO 2 ‘ (gM) 3.42 ±0.38 3.05 ±0.24 3.07 ±0.23 3.01 ±0.24 PO? (nM) 23.80 ±3.71 24.20 ±3.54 15.08 ±2.25 15.48 ± 1.86

Table 1.2. Average sediment pore water NO 3 +NO 2 ' and PO 4 3 ’concentrations in control and NH 4 enriched Thalassia testudinum plots in Corpus Christi Bay and lower Laguna Madre. Values are means ± SE (n=6)

Discussion

N availability for seagrass growth

Increased N availability as a result of NH 4 + fertilization had little effect on seagrass growth and leaf morphology at site CCB, which is characterized by having the higher ambient sediment pore water NH 4 + level (ca. 100 pM). Despite a 20-fold increase in N availability as a result of the fertilization, no differences in shoot density, shoot height, blade width and biomass were observed between control and fertilized plots. A similar lack of change in leaf biomass and shoot density in response to enrichment was reported by Bulthuis and Woelkerling (1981) in a Heterozostera tasmanica bed. They hypothesized that the high ambient pore water NH 4 + level in their study area (200 - 1700 pM NH 4 + ) provided an adequate reserve of N for seagrass growth. Zostera marina in Great Harbor, Massachusetts showed little response in leaf production and biomass to sediment NH 4 + manipulation which resulted in concentrations of 100 to 1000 pM NH 4 + (Dennison et al., 1987). Therefore, sediment NH 4 + availability was considered to be in excess of seagrass N demand.

However, as found in this study, Short (1983) and Dennison et al. (1987) also reported significantly smaller eelgrass leaf size and lower leaf production rates when sediment pore water NH 4 + concentrations dropped below ca. 100 pM, the suggested threshold concentration for N limitation for Z marina. The proposed threshold value of 100 pM is similar to saturation concentrations reported for Z. marina NH 4 + uptake by root tissues (lizumi and Hattori, 1982; Thursby and Harlin, 1982). In the present study, changes in sediment N availability from 100 to 2000 pM NH 4 + had no significant effect on shoot density, leaf morphology and biomass of T. testudinum. The lack of response to sediment NH 4 + enrichment by T. testudinum plants at CCB suggests that ambient pore water NH 4 + concentrations of 100 pM may provide an adequate pool of inorganic N for T. testudinum growth. This suggestion is consistent with reports for various seagrass species (Table 1.3). Seagrass growth has been limited at the range of ambient pore water NH 4 + concentration from 7.4 to 137 pM, while seagrasses growing at the range from 100 to 1000 pM have been demonstrated to be provided with sufficient N for growth (Table 1.3).

Although sediment NH 4 + concentrations in seagrass beds are indicative of N limitation of seagrass growth, turnover rates of the sediment NH 4 + pool and the regeneration of sediment NH 4 + are also important factors that determine the degree of N limitation. Sediment NH 4 + pools in seagrass beds have been reported to have rapid turnover rates ranging from 0.4 to 6 days (Capone, 1982; Moriarty et al., 1985; Boon et al., 1986). Bacterial N 2 fixation provides substantial inputs of Nin seagrass beds (Capone and Taylor, 1980; Capone, 1982; Moriarty and O’Donohue, 1993). Additionally, bacterial sulfate reduction in sediments is important in terms of the regeneration of sediment nutrients (Hines and Lyons, 1982; Holmer and Nielsen, 1997). Since NH 4 + regeneration rates vary spatially and temporally (Jorgensen, 1977; Moriarty and O’Donohue, 1993), the sediment NH 4 + pool size for N limitation of seagrass growth should also vary spatially and temporally.

Nitrogen availability in muddy sediments is usually higher than that in sandy sediments (Short, 1983, 1987). In the present study, however, the sandy sediments at CCB site were characterized by higher N availability. Processes of N input into sediments of seagrass meadows are N fixation in the rhizosphere and phyllosphere (Capone et al., 1979; Capone and Taylor, 1980; Capone, 1982; Moriarty and O’Donohue, 1993), settlement of particulate organic matter (Kenworthy and Thayer, 1984; Cooper, 1989) and N uptake from water column (Hemminga et al., 1991). Since there are no available values on N fixation and sedimentation rates at these study areas, limited riverine input into lower Laguna Madre (Texas Department of Water Resources, 1983) is presumed to be one of the factors for low sediment N availability in the LLM.

Plant responses to sediment NH/ enrichmeni

Although there was no significant change in shoot density, leaf morphology and biomass, there was a significant increase in the leaf production rate during the first month of sediment NH 4 + enrichment at CCB. However, this increased leaf production rate was not reflected as an increase in leaf biomass. The lack of correlation between leaf production and biomass has been attributed to high variation in biomass samples and differences in leaf turnover rate induced by nutrient enrichment (Bulthuis and Woelkerling, 1981). If leaf turnover rates increase as a result of N enrichment, leaf standing crop may remain constant, despite an increase in the rate of leaf production. However, there was no effect of N enrichment on leaf turnover time at CCB (Chapter 2). Leaf biomass in the first month of the experiment was highly variable and this variation probably obscured expected biomass increases, as observed in other studies (Erftemeijer et al., 1994; Agawin et al., 1996).

Ambient sediment pore water NH 4 + concentration at LLM (ca. 30 pM) was approximately one third that at CCB (ca. 100 pM), and NH 4 + fertilization significantly increased N availability. Leaf production rate, leaf morphology and biomass significantly responded to sediment NH 4 + enrichment at LLM. Short (1983) reported a strong correlation between sediment NH 4 + availability and leaf morphology for Zostera marina. Plants characterized by short and narrow leaves grew in low sediment N areas, while plants exhibiting long and wide leaves were found in high sediment N areas. In the present study, T. testudinum plants in LLM (low sediment NH 4 + ) had significantly shorter and narrower leaves than those at CCB (high sediment NH 4 + ). Shoot length and blade width at LLM increased significantly as a result of sediment NH 4 + enrichment. The increased leaf size in enriched plots at LLM was equivalent to that at CCB four months after the NH 4 + enrichment.

Increased leaf production rate as a result ofN enrichment has been shown to be the most pronounced response of seagrass in a N limited environment (Bulthuis and Woelkerling, 1981; Agawin et al., 1996; Alcoverro et al., 1997; Udy and Dennison, 1997). At ambient sediment NH 4 + concentrations, leaf production rates at CCB were about 3-fold higher than those at LLM. In fertilized plots at LLM, however, leaf production rates increased significantly to about 3 times that of the control plots. Hence, leaf production rates at LLM after sediment NH 4 + enrichment were similar to those at CCB. In LLM, increases in leaf production rate, shoot length and blade width in enriched plots to values similar to those observed at CCB, suggest that T. testudinum growth in the LLM site is limited by low sediment N availability, while plants at CCB are exposed to N sufficient conditions.

Significant increases in leaf biomass in response to nutrient additions have been reported from various seagrass species (Orth, 1977; Bulthuis et al., 1992; Agawin et al., 1996; Udy and Dennison, 1997). In this study, leaf biomass at LLM increased significantly as a result of sediment NH 4 + enrichment, although there was little change in below-ground biomass. Similar biomass responses have been reported for Zostera marina (Orth, 1977), Halodule uninervis and Zostera capricorni (Udy and Dennison, 1997). These responses are indicative of a stimulating effect of sediment NH 4 + enrichment on above-ground biomass. Increased NH 4 + assimilation due to sediment NH 4 + enrichment would require more carbon to incorporate assimilated NH 4 + into amino acids (Turpin, 1990; Huppe and Turpin, 1994). The increased carbon demand must be supplied by photosynthetic carbon fixation that is conducted in leaf tissues. Consequently, increases in leaf surface area are often associated with enhancement of leaf production rates and biomass to meet the increased carbon demand resulting from increased inorganic N assimilation during NH 4 + enrichment.

In contrast, plants under sediment nutrient deficient conditions increase biomass allocation to below-ground tissues to expand surface area for nutrient uptake (Gleeson, 1993; Vogt et al., 1993). In the present study, the below/above- ground biomass ratio at LLM was about 3-fold higher than that at CCB. This biomass allocation trend agrees with general plant response to sediment nutrient availability. Since below-ground biomass did not respond to short term NH 4 + enrichment, the significant decrease in below/above-ground biomass ratios at LLM due to sediment NH 4 + enrichment occurred as a result of enhanced leaf biomass. Increased carbon allocation to the shoots as a result of N fertilization and enhancement of below-ground carbon allocation as a result of elevated CO 2 concentrations have been reported in terrestrial grass species (Cotrufo and Gorissen, 1997). Therefore, C and N availability may have a reverse effect on biomass allocation to above or below-ground tissues: increasing root biomass at high C and/or low N availability and increasing leaf biomass at low C and/or high N availability. Changes in plant biomass allocation appear to reflect differences in N availability as the plant responds to imbalances in the ratio of nutrient versus carbon supplies.

Species Ambient pore water NH 4 + (pM) 1 Responses to N fertilization N limitation Area Source Leaf biomas Shoot ;s height Shoot density Growth Tissue N rate content Heterozostera 11 - 19 + + + + L Port Phillip Bay, Bulthuis et al (1992) tasmanica Australia Halodule 7.4 + + + + + L Moreton Bay, Udy & Dennison (1997) uninervis Australia Zostera 7.4 ns ns ns ns 4- L Moreton Bay, Udy & Dennison (1997) capricorni Australia Zostera 30 - 137 + L Puget Sound, WA Williams & marina USA Ruckelshaus (1993) Thalassia ca. 100 ns ns ns + N Indonesia Erftemeijer et al (1994) hempri chi i Zostera ca. 400 ns ns ns N Chesapeake Bay, Murray et al (1992) marina USA Zostera 100 - 1000 ns ns N Great Harbor, MA Dennison et al (1987) marina USA Thalassia 100 ns ns ns ns + N CCB, TX, USA Present study testudinum 30 + + ns + + L LLM, TX, USA

Table 1.3. Species specific response to nitrogen fertilization and nitrogen limitation reported in in situ seagrass beds during growing season. (+) - increases from controls due to sediment nitrogen fertilization; (ns) - no significant change; (L) - nitrogen limitation for seagrass growth; (N) - no nitrogen limitation

Chapter 2: Influence of sediment nitrogen availability on carbon and nitrogen dynamics of the seagrass Thalassia testudinum

Abstract

Responses of tissue carbon (C) and nitrogen (N) content and carbohydrate reserves to changes in sediment N availability were examined for two Thalassia testudinum populations in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM), Texas, USA from May to October 1997. A commercial fertilizer was utilized to increase sediment pore water NH 4 + concentrations at both study sites, which are characterized by NH 4 + concentrations that normally average 30 pM at LLM and 100 pM at CCB. Samples for leaf growth, leaf turnover time, leaf and rhizome N and C content and rhizome non-structural carbohydrate (NSC) content were collected monthly. At the high sediment-N site (CCB), leaf and rhizome N content was significantly higher than at LLM. Tissue N content significantly increased relative to controls as a result of sediment NH 4 + enrichment at both study sites. Leaf C content also increased significantly at both sites following fertilization, although rhizome C content decreased at CCB and did not change at LLM. C:N ratios of leaf and rhizome tissues were significantly higher at the low sediment-N site (LLM) than at CCB, and these ratios were significantly lower in fertilized plots than in control plots at both study sites. Leaf growth rates increased and leaf turnover time decreased as a result of sediment NH 4 + enrichment in LLM; at CCB an increase in the growth rate was observed only during the first month following fertilization and no changes in leaf turnover time were observed. Total chlorophyll concentration in control plots at CCB was significantly higher than that at LLM. Chlorophyll concentrations at CCB did not change significantly, while at LLM the concentrations were significantly lower in fertilized plots than in control plots during the first month following sediment NH 4 + fertilization. Rhizome NSC decreased significantly in response to sediment NH 4 + enrichment during the early periods of experiment. The decrease in rhizome NSC suggests C was reallocated from rhizomes to leaves to support the stimulated leaf growth resulting from sediment NH 4 + enrichment. Hence, the NH 4 + enrichment had an influence on concentration and allocation of C as well as N. Plants enhanced leaf function at high sediment N conditions, while plants enhanced function of below-ground tissues at low N conditions.

Introduction

High nutrient incorporation is required to support the high rates of production in seagrasses (Mcßoy and McMillan, 1977; Lee and Dunton, 1996). The high nutrient demand necessary to support high production suggests that in situ nutrient availability may limit seagrass growth in some seagrass beds. Fertilizer additions to sediments have been shown to enhance plant growth and biomass (Orth, 1977; Harlin and Thorne-Miller, 1981; Dennison et al., 1987; Short et al., 1990; Perez et al., 1991; Bulthuis et al., 1992; Agawin et al., 1996; Alcoverro et al., 1997; Udy and Dennison, 1997) by stimulating plant C production through physiological changes in the maximum rate of photosynthesis and photosynthetic efficiency, or increased chlorophyll concentrations (Agawin et al., 1996). Such responses in photosynthetic performance and chlorophyll concentrations following nutrient enrichment to a nutrient-limited population suggest an interaction between photosynthetic C fixation and nutrient availability.

Increased tissue N content as a result of N enrichment has been reported for Heterozostera tasmanica (Bulthuis and Woelkerling, 1981; Bulthuis et al., 1992), Thalassia hemprichii (Erftemeijer et al., 1994), Posidonia oceanica (Alcoverro et al., 1997), Halodule uninervis, Zoster a capricorni and Cymodocea serrulata (Udy and Dennison 1997). The assimilation of dissolved inorganic nitrogen (DIN) requires C skeletons to incorporate ammonium into amino acids (Turpin et al., 1990; Huppe and Turpin 1994). The energy for N metabolism is supplied from photosynthetic electron transport or the respiration of fixed C (Huppe and Turpin 1994). Therefore, increased N assimilation caused by N enrichment may require increases in photosynthetic C fixation and/or C metabolism.

Responses of seagrass above-ground tissues to nutrient enrichment are generally well documented, but the physiological characteristics and functional roles of below-ground tissues are less well known. Seagrass rhizomes serve as photosynthetic storage tissues (Dawes and Lawrence 1979; Durako and Moffler 1985; Pirc, 1985, 1989; Lee and Dunton 1996). Non-structural carbohydrates (NSC) in rhizome tissues can be reallocated to meet respiratory demands and to support leaf growth during low photosynthetic C fixation periods (Dawes and Lawrence, 1979; Durako and Moffler, 1985; Pirc, 1985, 1989; Dawes and Guiry, 1992; Lee and Dunton, 1996, 1997). Since NSC in rhizome tissues constitutes the major C reserve in seagrasses, it is hypothesized that the metabolically available C in rhizome tissues can be used to support enhanced leaf growth as a consequence of sediment N enrichment.

The nutritional content of various seagrass tissues have been used to infer nutrient limitation for seagrass growth (Atkinson and Smith, 1983; Duarte, 1990; Fourqurean et al., 1992 a), although there is no accepted C:N ratio for seagrasses that compares to the Redfield ratio commonly used for phytoplankton. However, nutrient limitation for seagrass growth can be inferred from comparison of tissue nutrient content among sites that have significantly different in situ nutrient concentrations, such as exists on the south Texas coast between Corpus Christi Bay (CCB) and lower Laguna Madre (LLM). Biomass allocation and sediment NH 4 + concentrations in monotypic meadows of Thalassia testudinum at both locations are significantly different (Lee and Dunton 1996, 1997; Herzka and Dunton 1997, 1998; Kaldy 1997; Chapter 1). Below-ground tissue biomass at the low sediment NH 4 + site (LLM) is significantly higher than at high sediment NH 4 + site (CCB), but leaf biomass is high at CCB and low at LLM (Dunton, 1990, 1994; Lee and Dunton 1996; Herzka and Dunton 1997; Kaldy 1997; Chapter 1). In this study, the effect of sediment NH 4 + enrichment on tissue C and N content and the reallocation of C among various tissues were examined at CCB and LLM. Nitrogen limitation for seagrass growth was evaluated though comparison of tissue C and N content between study sites. I also examined changes in leaf growth rate, chlorophyll concentrations and rhizome NSC content as a result of sediment NH 4 + enrichment over a six-month experimental period.

Materials and methods

Study sites

Two study sites, one in Corpus Christi Bay (CCB; 27° 49' N, 97° 07' W), and the second in the lower Laguna Madre (LLM; 26° 09' N, 97° 12' W), 200 km south, were chosen for this study. Both contained monotypic meadows of Thalassia testudinum at similar water depths (1.2 m; Dunton 1990, 1994; Quammen and Onuf 1993; Czerny and Dunton 1995; Lee and Dunton 1996, 1997; Herzka and Dunton 1997, 1998; Kaldy 1997). The CCB population is characterized by high pore water NH 4 + concentration (ca. 100 pM), while the average pore water NH 4 + concentration at LLM is ca. 30 pM (Chapter 1). Water column NH 4 + and NO 3 +NO 2 ’ at both sites are about 1 pM and are not significantly different (Chapter 3).

N enrichment

Six 1 m 2 experimental plots were established at each site. Three served as controls and others were fertilized using a commercial fertilizer, ammonium sulfate (21-0-0, Esco Corp. Dallas, Texas). In each fertilized plot, 25 packets of 20 g fertilizer wrapped in several layers of cheesecloth were buried at 20 cm intervals at a depth of 10 cm below the sediment surface. The amount of N applied to each N enrichment plot was 105 g N m' 2 based on the manufacturer’s nominal values. The rhizome connections at the perimeter of each plot were cut to a depth of about 30 cm. Sediment NH 4 + enrichment was initiated in early May 1997. Plant and sediment samples were collected monthly from May to October 1997 from both control and treatment plots. Sediment pore water NH 4 + concentrations in control and fertilized plots at both study sites are shown in Table 2.1. Data in the table were adapted from Chapter 1.

Leaf production

Leaf production rates were measured using a modified blade marking technique (Zieman, 1974; Kentula and Mclntire, 1986). Five randomly chosen shoots from each plot were marked just above the bundle sheath with a hypodermic needle and collected after 3 to 4 weeks. Leaf material was separated into tissue produced before and after marking and dried at 60°C to a constant weight. The rate of leaf production per shoot was determined by dividing the dry weight of new leaf tissue produced after marking by the number of days elapsed since marking. The leaf turnover time in days was determined using the following equation:

Turnover time (days) = (DW Total * # days) / DW New

Where DW New is the dry weight of new leaf tissue produced after marking, DW Total is the total leaf dry weight and # days is the number of days since marking.

Sediment chemistry

On each sampling date, three replicate sediment samples were collected to a depth of 13 cm from each plot with a6O ml syringe corer. Sediment pore water was obtained by centrifugation (5000 g for 15 min) and then diluted (1:5, v/v) with low NH 4 + seawater (< 1 pM) collected offshore in the Gulf of Mexico. Concentrations of NH 4 + were determined using standard colorimetric techniques following the alternative method of Parsons et al. (1984).

Tissue constituent analyses

For determination of leaf chlorophyll (chi) content, five to seven shoots from each plot were collected and then cleaned of epiphytes in the laboratory. Six replicate pre-weighed center portions of mature green leaves in each plot were ground in 90 % cold acetone buffered with 0.05 % MgCO 3 using a chilled mortar and pestle with washed sea sand. The extract was centrifuged and absorbances measured at 750, 664 and 647 nm on a spectrophotometer (Shimadzu UV 160 U). Chi a and b content was determined using the equations of Jeffrey and Humphrey (1975).

Plant tissue constituent analyses (C, N and NSC) were determined from core samples collected with a 9 cm diameter coring device driven about 20 cm into the sediments from each plot. Samples were thoroughly cleaned of epiphytes and sediments, separated into leaf (blade and sheath), rhizome (vertical and horizontal) and root tissues and dried at 60°C to a constant weight. Dried plant material was ground using a mortar and pestle and used for determination of total tissue C and N and NSC content. Total C and Nin leaf and rhizome tissues were measured using an elemental analyzer (Carlo Erba EA 1108) and C/N molar ratios were calculated. The content of NSC in rhizome tissues was determined using the MB TH (3-methyl-2-benzothiazolinone hydrazone hydrochroride) analysis method (Parsons et al., 1984; Pakulski and Benner, 1992; Lee and Dunton, 1996, 1997). Ground rhizome samples were hydrolyzed with 0.1 N HCI for 24 hat 100°C in a water bath. NSC content was expressed as the equivalent glucose weight (g glu g' 1 dry wt). To estimate the proportion of NSC carbon to total tissue C, NSC content was converted to the equivalent C values using a 0.4 conversion factor, and divided by total C content determined using elemental analysis.

Statistics

All values are reported as means ± ISE. Statistical analyses were performed on a microcomputer using a general linear procedure (SAS Institute, 1989). Data were tested for normality and homogeneity of variance to meet the assumptions of parametric statistics, and assumptions were satisfied for all data tested. To examine the effect of N enrichment and time, significant differences in leaf growth rate, leaf turnover time, blade chlorophyll content as well as C, N and NSC content between sediment N treatments and among sampling times were tested using a two-way ANOVA with time as a block. Significant differences in these parameters at control plots both among sites and sampling times were also tested using a two-way ANOVA. When a significant difference among variables was observed, the means were analyzed with a Tukey multiple comparison test to determine where the significant differences occurred among variables.

CCB LLM Control Fertilized Control Fertilized May (Initial 109.3 ±5.6 - 45.2 ± 10.4 - June 59.5 ±5.9 2211.4 ±369.2 35.4±3.8 1664.0 ±200.9 July 85.9 ±6.5 699.2 ± 165.0 42.0 ±5.2 762.7 ±227.6 August 106.7 ±23.4 446.3 ± 126.7 29.2 ±3.2 504.9 ± 119.1 September 79.8 ±20.6 299.4 ± 123.2 15.1 ±2.0 470.9 ± 187.2 October 37.4 ±3.4 124.9 ±37.1 21.0 ±2.2 292.1 ± 127.0

Table 2.1. Sediment pore water ammonium concentrations in control and ammonium-fertilized Thalassia testudinum plots at Corpus Christi Bay and lower Laguna Madre between May and October 1997. Values are means ± SE (n = 3). Table adapted from Chapter 1.

Results

Leaf growth rate

Throughout the experiment, leaf growth rates in control plots were significantly higher at CCB than at LLM (4.34 and 1.37 g dry wt shoot’ 1 d' 1 , respectively; P < 0.001). At CCB, leaf growth rates were significantly higher in fertilized plots than in control plots only during the first month of experiment (P = 0.011); no significant differences were observed between control and fertilized plots during subsequent months (P = 0.13; Fig. 2.1). On the other hand, at LLM leaf growth rates were significantly higher in fertilized plots than in control plots throughout the experiment (P < 0.001). Growth rates in control plots at LLM ranged from 1.90 g dry wt shoot' 1 d' 1 in June to 0.71 g dry wt shoot' 1 d' 1 in October, compared to growth in fertilized plots, which ranged between 4.62 g dry wt shoot' 1 d’ 1 in July to 2.64 g dry wt shoot' 1 d' 1 in October.

Average leaf turnover time in control plots at LLM (44.6 days) was significantly longer than that at CCB (38.1 days; P < 0.001; Fig. 2.1). Leaf turnover time at CCB did not significantly change as a result of sediment NH 4 + enrichment (P = 0.38; Fig. 2.1). However, leaf turnover time in fertilized plots at LLM (36.7 days)

was significantly shorter than that in control plots (44.6 days; P < 0.001; Fig. 2.1).

Blade chlorophyll

Total chlorophyll concentration in control plots at CCB was significantly higher than that at LLM (8.02 and 5.35 mg chi g’ 1 dry wt, respectively; P < 0.001) with plants at CCB also having a higher percentage of chi b as reflected in an average chi a.b ratio at CCB that was significantly lower than at LLM (2.72 and 3.05, respectively; P < 0.001; Fig. 2.2). There was no significant effect of sediment N enrichment on total chlorophyll content and chi a.b ratio at CCB (P =0.38 and P = 0.10, respectively; Fig. 2.2). At LLM, total chlorophyll content was significantly lower in fertilized plots than in control plots during the first month following sediment NH 4 + fertilization (P = 0.002), but after 3 months of NH 4 + enrichment, chlorophyll content was significantly higher in fertilized plots than in control plots (P < 0.001; Fig. 2.2). Chi a.b ratios in fertilized plots at LLM were significantly higher than those in control plots during the first month of experiment (P < 0.001), but then were lower significantly than in control plots during the subsequent months (P< 0.001; Fig. 2.2).

Tissue C and N content

C and N content of leaf tissues collected from control plots at CCB (35.51 % and 2.64 %, respectively) were significantly higher than those at LLM (34.82 % and 1.75 %; P = 0.013 and P < 0.001, respectively; Fig. 2.3). C:N molar ratios of leaf tissues in control plots were significantly higher at LLM than at CCB (23.32 and 15.97, respectively; P < 0.001). C content in leaf tissues was significantly higher in fertilized plots than in control plots at both study sites (P = 0.002 at CCB and P < 0.001 at LLM) as was N content (P < 0.001 for both sites), which was reflected in lower C:N ratios of leaf tissues in fertilized plots (Fig. 2.3).

Rhizome C content in control plots at LLM (36.89 %) was significantly higher than at CCB (35.21 %; P < 0.001), while rhizome N content at CCB (1.08 %) was significantly higher than at LLM (0.78 %; P < 0.001; Fig. 2.4). The C:N ratios of rhizomes were significantly higher at LLM than at CCB (55.72 and 38.95, respectively; P < 0.001). At CCB, rhizome C content was significantly lower in fertilized plots than in control plots during the first month of the experiment (P = 0.043) and subsequently there was no effect of enrichment on rhizome C content (Fig. 2.4). Rhizome C content at LLM was not significantly changed as a result of sediment NH 4 + enrichment (P = 0.26). Total N content in rhizome tissues were significantly higher in fertilized plots than in control plots at both study sites (P < 0.001 for both sites). C:N ratios in rhizome tissues were significantly lower in fertilized plots than in control plots at both study sites (P < 0.001 at CCB and P = 0.0012 at LLM; Fig. 2.4).

Non-structural carbohydrates

At CCB, rhizome non-structural carbohydrate (NSC) content and percent rhizome NSC carbon to total rhizome C (% NSC) were significantly lower in fertilized plots than in control plots in June (P = 0.006 and P = 0.005, respectively), but then there were no significant differences between control and fertilized plots during subsequent months (P = 0.47 and P = 0.23, respectively; Fig. 2.5). At LLM, the rhizome NSC contents and % NSC were significantly lower in fertilized plots than in control plots during the first 2 months of the experiment (P = 0.01 and P = 0.006, respectively), but there was no significant difference during subsequent months (P = 0.158 and P = 0.08, respectively; Fig. 2.5).

Rhizome NSC content and % NSC exhibited similar trends at both sites (Fig. 2.5). The rhizome NSC content and % NSC in control plots were significantly higher at LLM (0.46 g glu g 4 dry wt and 49.54 %, respectively) than at CCB (0.38 g glu g’ 1 dry wt and 43.14 %, respectively; P < 0.001; Fig. 2.5).



Figure 2.1. Thalassia testudinum. Leaf growth rates (g dry wt shoot' 1 d' 1 ) and leaf turnover time (days) in control and sediment ammonium-enriched plots in Corpus Christi Bay (A,B) and lower Laguna Madre (C,D) between May and October 1997. Triangle on the x-axis represents date of fertilization. Values are means ± SE (n=3).



Figure 2.2. Thalassia testudinum. Total blade chlorophyll (mg chi g' 1 dry wt) and chi a.b ratios in control and sediment ammonium-enriched plots in Corpus Christi Bay (A,B) and lower Laguna Madre (C,D) between May and October 1997. Triangle on the x-axis represents date of fertilization. Values are means ± SE (n=3).



Figure 2.3. Thalassia testudinum. Carbon and nitrogen content and C/N molar ratios of leaf tissues in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A, B, C) and lower Laguna Madre (D, E, F) between May and October 1997. Triangle on the x-axis represents date of fertilization. Values are means ± SE (n=3).



Figure 2.4. Thalassia testudinum. Carbon and nitrogen content and C/N molar ratios of rhizome tissues in control and sediment NH 4 + enriched plots at Corpus Christi Bay (A, B, C) and lower Laguna Madre (D, E, F) between May and October 1997. Triangle on the x-axis represents date of fertilization. Values are means ± SE (n=3).



Figure 2.5. Thalassia testudinum. Rhizome non-structural carbohydrates (NSC) content and percent of rhizome NSC carbon to total rhizome carbon in control and sediment ammonium-enriched plots in Corpus Christi Bay (A,B) and lower Laguna Madre (C, D) between May and October 1997. Triangle on the x-axis represents date of fertilization. Values are means ± SE (n=3). Asterisks indicate significant difference between enriched and control plots.

Discussion

Tissue C and N contents

The nutrient content of seagrass tissues can reflect in situ nutritional conditions of the sites the plants were grown. Seagrasses in low nutrient conditions have been shown to exhibit a greater increase in tissue nutrient content as a result of nutrient enrichment than those in environments where nutritional requirement are met (Duarte, 1990). Plants from low nutrient environments have significantly higher C:N and C:P ratios than plants from high nutrient conditions (Atkinson and Smith, 1983). In the present study, the leaf and rhizome N content of Thalassia testudinum at site CCB, which is characterized by high sediment NH 4 + concentrations, were significantly higher than at LLM. C:N ratios of leaf and rhizome tissues at LLM were significantly higher than those at CCB and as a result of fertilization, leaf and rhizome N content of T. testudinum at LLM showed a greater absolute increase than those at CCB. Likewise, C:N ratios decreased to a greater extent as a result of sediment NH 4 + enrichment at LLM than at CCB; following sediment NH 4 + enrichment, the leaf and rhizome N content and C:N ratios at LLM were similar to those at CCB. The observed plant responses to N enrichment suggest that in LLM seagrass growth is limited by N, while plants in CCB have a sufficient N supply for growth. This is in agreement with the demonstration of N limitation at LLM from the results of increases in biomass and leaf width and length as a result of N enrichment (Chapter 1).

There have been attempts to infer nutrient limitation from tissue nutritional ratios of seagrasses, similar to the Redfield ratio utilized for phytoplankton (Atkinson and Smith, 1984; Duarte, 1990; Fourqurean et al., 1992 a). Duarte (1990) suggested that seagrass plants with a leaf nutrient content below the median of literature reported values (1.82 % dry wt for N and 0.20 % dry wt for P) are likely to be nutrient-limited. In this study, the 1.75 % leaf N content of dry weight for control plants in LLM was similar to the median leaf N content calculated by Duarte (1990). However, in this study, leaf N content increased significantly as a result of sediment NH 4 + enrichment, suggesting N limitation. Therefore, the median value of literature-driven leaf N content may not übiquitously serve as a threshold value to predict N limitation for seagrass growth, and suggests that other factors may be involved.

In CCB, tissue C and N content and C:N ratios showed strong temporal trends. Tissue N content exhibited a reverse trend to leaf growth rate: N content was higher during periods of low growth, while lower N content was observed during periods of high growth. High growth rates can cause N depletion from the root zone in the sediments, which may result in nutrient limitation and consequently lower tissue nutrient content (Short et al., 1993). However, the temporal variation in the N content observed in the present study probably cannot be explained by nutrient limitation, because the observed seasonal patterns of N content did not change as a result of sediment NH 4 + enrichment (e.g. CCB leaf tissue; Fig. 2.3). If low tissue N content during periods of high growth were caused by depletion of available N in the sediments, then low tissue N content should have increased as a result of N enrichment, which was not the case. Hence , the seasonal patterns in tissue N content observed in this study suggest that they may not be controlled strictly by N availability. Thus, tissue nutrient content may not always be representative of the ambient nutrient conditions for plant growth.

Similar seasonal trends in tissue N content (low and high N content during the summer and winter, respectively) have been observed in several seagrass species (Harrison and Mann, 1975; Pellikaan and Nienhuis, 1988; Pirc and Wollenweber, 1988; Perez-Llorens and Niell, 1993; Short et al., 1993). Such seasonal variations have been explained by seasonal differences in N uptake rates and dilution processes (Stocker, 1980; Pellikaan and Nienhuis, 1988; Pirc and Wollenweber, 1988; Perez- Llorens and Niell). It has been proposed that during periods of low growth, N uptake may exceed plant N requirements (Thursby and Harlin, 1982), and that excess N will be stored as amino acids or protein. Higher protein and free amino acid levels during winter and spring relative to the summer have in fact been reported for several seagrass species (Pirc, 1985; Dawes, 1986; Dawes and Guiry, 1992). Such seasonal variations in amino acid and protein content would be in accordance with the seasonal trends in tissue N content observed in this study. Additionally, low tissue N content during periods of high growth can be a product of N dilution during rapid plant growth (Stocker, 1980; Pirc and Wollenweber, 1988; Perez-Llorens and Niell, 1993).

Carbon reallocation

Leaf growth rates were stimulated as a result of sediment N enrichment during the first month of the experiment at CCB and during the entire experimental period at LLM. In addition, leaf turnover time decreased with sediment N enrichment at LLM. The increase in leaf growth rates and decrease in leaf turnover time reflects an increase in leaf C acquisition, which can be met through increases in photosynthetic C fixation. Increases in blade chlorophyll concentrations and photosynthetic efficiency of seagrasses and terrestrial plants have been reported as a result of sediment N enrichment (O’Neill et al., 1984; Huber et al., 1989; Lopez- Cantarero et al., 1994; Agawin et al., 1996). These responses may cause an increase in photosynthetic C fixation to meet the increased C demand resulting from sediment NH 4 + enrichment. However, if photosynthetic C fixation in leaf tissues cannot meet the increased C demand, stored C in plant tissues must be used.

In seagrasses, non-structural carbohydrate (NSC) content is highest in rhizome tissues, serving as an energy and C reserve when the C demand exceeds photosynthetic C fixation (Dawes and Lawrence, 1979; Durako and Moffler, 1985; Pirc, 1985, 1989; Dawes and Guiry, 1992; Lee and Dunton, 1996, 1997). Stored C in rhizomes can be used for leaf growth and maintenance during winter and early spring, and can be accumulated during periods of high photosynthetic C fixation (Pirc, 1985, 1989; Lee and Dunton, 1996). This stored C can also be used to meet the C demands of plants during periods of low photosynthetic C fixation caused by severe environmental disturbance such as underwater light reduction (Burke et al., 1996; Rey and Stephens, 1996; Lee and Dunton, 1997) and during periods of leaf regrowth after defoliation (Dawes and Lawrence, 1979; Dawes and Guiry, 1992). Decreases in rhizome stored carbohydrates as a result of defoliation by blade clipping have suggested that rhizome C reserves are transported from the rhizome to leaf tissues and used for leaf regeneration (Dawes and Lawrence, 1979; Dawes and Guiry, 1992).

In the present study, rhizome NSC content decreased as a result of sediment NH 4 + enrichment during the early period of the experiment. These decreases in rhizome NSC are probably caused by C reallocation from rhizome to leaf tissues to support stimulated leaf growth. Additionally, blade chlorophyll concentrations at CCB did not change significantly, while chlorophyll was significantly lower in control plots than in fertilized plots at LLM during the first month of the experiment. Although photosynthetic performance was not quantified, these patterns in chlorophyll concentrations suggest that photosynthetic activity, which is associated with chlorophyll concentration, either decreased (LLM) or did not change (CCB) as a result of N enrichment during the early part of the experiment. Therefore, during the initial periods ofN enrichment, photosynthetic C fixation in leaf tissues was probably insufficient to meet the increased C demand associated with the enhancement of leaf growth as reflected by decreases in rhizome NSC levels (Fig. 2.6).

The decrease in chlorophyll content during the first month following N enrichment at LLM was probably a consequence of the rapid increase in leaf production. If the rate of chlorophyll production does not match rapid increases in leaf expansion, dilution of chlorophyll in leaf tissues may result. However, three

months following N enrichment, T. testudinum showed an increase in chlorophyll content, as reported for other seagrass species and terrestrial plants (O’Neill et al., 1984; Lopez-Cantarero et al., 1994 Agawin et al., 1996).

In control plots, leaf C content was higher at CCB, which reflects the 3-fold difference in sediment pore water N levels at CCB compared to LLM, while rhizome C content was significantly higher at LLM. These allocation patterns of tissue C content suggest that C allocation to leaf tissues is higher under sufficient sediment N conditions, but under low sediment N availability, the rhizomes become a sink for fixed C (Table 2.2). These C allocation patterns are in accordance with records of higher leaf biomass at CCB and higher rhizome biomass at LLM (Chapter 1). Such patterns are indicative of plant adaptations to prevent imbalances in the ratio of C and N supplies (Gleeson, 1993; Chapter 1). In low nutrient soils, vascular plants increase below-ground tissue biomass to maximize the surface area for nutrient uptake, while allocating more biomass into above-ground tissues (e.g. leaves) to increase C fixation under conditions of high soil nutrient conditions (Gleeson, 1993; Vogt et al., 1993; Cotrufo and Gorissen, 1997; Chapter 1). Additionally, higher chlorophyll content, which implies higher photosynthetic C fixation, was exhibited under high sediment N availability (Table 2.2).

In conclusion, leaf biomass, blade width and length, chlorophyll and leaf C content were higher under high sediment N availability, while biomass, C and nonstructural carbohydrate content of below-ground tissues were higher at low sediment N availability. Additionally, sediment N fertilization caused mobilization of reserved rhizome Cto leaf tissues to support enhanced leaf growth. These results suggest that seagrass plants respond to high sediment N conditions by enhancing leaf function and metabolism, while plants respond to low N conditions by enhancing function and metabolism of below-ground tissues.



Figure 2.6. Schematic diagram illustrating the effect of sediment NH4+enrichment on seagrass growth and mobilization of reserve NSC inrhizome tissues one month following fertilization.

High sediment N Low sediment N Biomass (g DW m-2) Leaf biomass High (202-394) Low (134-205) Below-ground biomass Low (318-550) High (625-809) Below/above-ground ratio Low (1.63) High (4.66) Leaf size and production Leaf width (mm) Wide (5.9-6.6) Narrow (5.1-5.3) Shoot height (cm) Tall (30.3-49.5) Short (17.7-21.8) Leaf productivity (g DW m-2 d-1) High (7.35) Low (2.50) Leaf turn-over time (days) Fast (38) Slow (45) Blade chlorophyll Total chi (mg chi g-1 DW) High (8.02) Low (5.35) Chi a:b ratio Low (2.7) High (3.1) Tissue nutritional content (%) Leaf C High (35.51) Low (34.82) Rhizome C Low (35.21) High (36.89) LeafN High (2.64) Low (1.75) Rhizome N High (1.08) Low (0.78) Leaf C/N ratio Low (16.0) High (23.3) Rhizome C/N ratio Low (43.0) High (55.7) Rhizome carbohydrate content (% NSC Low (43) High (50)

Table 2.2. Thalassia testudinum . Differences in biomass, production, blade chlorophyll and tissue nutritional content between high and low sediment nitrogen availability. Data for biomass and leaf size were adapted from Chapter 1

Chapter 3: Inorganic nitrogen acquisition in the seagrass Thalassia testudinum: development of a whole-plant nitrogen budget

Abstract

Whole plant nitrogen (N) uptake experiments were used to quantify the N budget of Thalassia testudinum growing under different sediment nutrient regimes in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM), Texas. Concurrent measurements of plant biomass and levels of dissolved inorganic nitrogen (DIN) in the water column and sediments were also made over a 12-month period (October 1996 - October 1997). Sediment NH 4 + concentrations in CCB (87 pM) were significantly higher than in LLM (26 pM), but water column NH 4 + and NO 3 '+NO 2 ’ concentrations were not significantly different between study sites (1.18 and 0.74 pM in CCB and 1.11 and 0.72 pM in LLM, respectively). Leaf biomass in CCB was significantly higher than in LLM (197 vs 130 g dry wt m‘ 2 , respectively), but there was no difference in root biomass between study sites (91 vs 92 g dry wt m‘ 2 in CCB and LLM, respectively). Leaf NH 4 + uptake showed clear seasonal variation: V max was highest in summer and fall, but K rn was highest in winter. V max of leaf NO 3 ’ uptake did not change with season, but K m decreased with increasing incubation temperature. There were no clear differences in leaf NH 4 + and NO 3 ' uptake rates between study sites although leaf NH 4 + uptake affinity was higher than that of NO 3 -. Root NH 4 + uptake was variable with season and did not saturate at the experimental NH 4 + concentrations (0 to 300 pM). Based on these measurements, N acquisition was highest during summer and fall and lowest during winter and spring. Annual N acquisition in CCB was double that in LLM (97.03 and 53.49 g N m’ 2 y* 1 , respectively). Root NH 4 + uptake accounted for about 52 %of total N acquisition, while leaf NH 4 + uptake accounted for about 38 % and leaf NO 3 ' uptake accounted for about 10 %. There were no differences in the contributions of leaf and root uptake to total N acquisition between the two study sites. The similarity in total tissue N acquisition by plants at both sites, despite significant difference in sediment NH 4 + pool sizes, results from the higher fraction of biomass allocated to below-ground tissues in plants living under low sediment N conditions (LLM). In N-sufficient sediments, overall plant productivity is greater as T. testudinum is able to allocate a greater proportion of its biomass into photosynthetic above-ground tissues.

Introduction

In contrast to terrestrial plants, aquatic vascular plants, including seagrasses, can take up inorganic N through both leaf and root tissues (lizumi and Hattori, 1982; Thursby and Harlin, 1982, 1984; Short and Mcßoy, 1984; Stapel et al., 1996; Pedersen et al., 1997; Terrados and Williams, 1997). The major N sources for seagrasses are NH 4 + and NO 3 ' in the water column for leaves, and NH 4 + in sediment pore waters for roots. With the exception of areas characterized by high river inflow, water column NH 4 + and NO 3 ’ concentrations in seagrass beds are usually less than 3 pM (Tomasko and Lapointe, 1991; Dunton, 1996; Stapel et al., 1996; Terrados and Williams, 1997). On the other hand, pore water NH 4 + concentrations range from less than 20 pM to well over 200 pM, significantly higher than that of the water column (Bulthuis and Woelkerling, 1981; lizumi et al., 1982; Fourqurean et al., 1992 b; Dunton, 1996). Thus, sediment pore waters are often considered the main source ofN for seagrass growth (lizumi and Hattori, 1982; Short and Mcßoy, 1984; Zimmerman et al., 1987).

Nevertheless, there is a evidence that leaf tissues have higher N uptake affinities than root tissues at low dissolved inorganic nitrogen (DIN) concentrations (Pedersen et al., 1997), and several studies have indicated that uptake by leaves can contribute considerably to the total N acquisition of seagrasses (Short and Mcßoy, 1984; Perdersen and Borum, 1992; Stapel et al., 1996; Terrados and Williams, 1997). In addition, Zimmerman et al. (1987) developed a numerical model of N uptake for Zostera marina, and predicted that N acquisition by root tissues would not exceed 80% of the total plant N demands, even when pore water NH 4 + concentrations were higher than 500 pM. Work by lizumi and Hattori (1982) indicated that 55 % of the N required for growth in Z. marina was supplied by the NH 4 + in the sediment pore water. However, no in situ quantitative work has directly addressed or confirmed the relative importance of water column vs sediment pore water DIN in the annual N budgets of seagrasses.

Tissue N uptake rates are dependent on photosynthesis since the energy for tissue N uptake is supplied by photosynthesis (Turpin et al., 1990; Huppe and Turpin, 1994). Assimilation of N also requires carbon (C) skeletons produced by photosynthesis for NH 4 + incorporation into amino acids. Since seagrass photosynthetic performance is closely correlated with water temperature and underwater irradiance, both which exhibit seasonal trends (Herzka and Dunton, 1997), N uptake rates should also vary seasonally. Leaf and root N uptake kinetics have been quantified for several seagrass species (lizumi and Hattori, 1982; Thursby and Harlin, 1982, 1984; Short and Mcßoy, 1984; Stapel et al., 1996; Pedersen et al., 1997; Terrados and Williams, 1997), but there have been no studies of seasonal N uptake kinetics, which are required for calculation of whole-plant N budgets.

Two monotypic meadows of T. testudinum characterized by significantly different pore water NH 4 + concentrations, but similar water column DIN levels, were chosen as study sites in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM),Texas, USA. The present study addresses seasonal differences in NH 4 + and NO 3 ’ uptake by leaves and NH 4 + uptake by roots to generate annual whole-plant N budgets based on field measurements of DIN levels and plant biomass collected within two seagrass beds in the southwestern Gulf of Mexico. Since previous work had demonstrated that seagrass growth in the lower Laguna Madre is strongly N regulated (Chapters 1,2), I hypothesized that the relative contributions by leaf and root tissues to total N acquisition varied between the two study areas.

Materials and Methods

Study sites

Experimental plants were collected from monotypic meadows of Thalassia testudinum in Corpus Christi Bay (CCB; 27° 49' N, 97° 07' W) and lower Laguna Madre (LLM; 26° 09' N, 97° 12' W). These study sites, about 200 km apart, are located at a similar water depth (1.2 m) and have been the focus of several recent investigations on south Texas seagrasses (Dunton, 1990, 1994; Quammen and Onuf, 1993; Czerny and Dunton 1995; Lee and Dunton 1996, 1997; Herzka and Dunton, 1997, 1998; Kaldy, 1997). A greater percentage of the plant biomass in LLM is allocated to below-ground tissues, as reflected in below/above-ground biomass ratios at LLM (4.66) that are significantly higher than at CCB (1.63; Chapter 1).

Plant collection

Plants were collected seasonally (February, May, July and October 1997) using a 15 cm diameter core driven about 20 cm into the sediments. Intact cores (n =8 to 12) were placed in 201 buckets for transport to the laboratory. Upon arrival, plants were gently cleaned of sediment using filtered seawater. The plants were separated into individual shoot units that included healthy leaves, roots and a 3 cm horizontal rhizome. Epiphytes were removed by gently scraping. The plants were maintained in aerated filtered seawater at an ambient field temperature in preparation for incubations the following day.

Biomass samples (n = 4 -5) were collected monthly with a 9-cm diameter core as described above. Plants were thoroughly cleaned of epiphytes and sediments, separated into leaf, rhizome and root tissues, and dried at 60°C to a constant weight. Biomass was expressed as areal estimates (g dry wt m‘ 2 ).

Water column and sediment DIN

To determine water column NH 4 + and NO ’ 2 +NO four replicate surface water samples were collected monthly at the two study sites from October 1996 to November 1997 and frozen pending chemical analysis. In addition, sediment pore water NH 4 + concentrations were determined from four replicate sediment samples collected with a6O ml syringe corer to a depth of 13 cm. Pore water from sediment samples was obtained by centrifugation (5000 x g for 15 min) and then diluted (1:5, v/v) with low NH 4 + seawater (< 1 pM) collected offshore in the Gulf of Mexico. Concentrations ofNH 4 + and NO 2 '+NO 3 ' were measured using standard colorimetric techniques following the methods of Parsons et al. (1984).

Incubation chamber

Four plants were placed in a specially constructed 11.5-cm diameter cylindrical plexiglass, two-compartment, incubation chamber. The chamber spatially separated above and below-ground tissues (hereafter leaf and root compartments, respectively). To prevent leakage, shoots were fitted through a rubber stopper covered in thin rubber tubing. The rubber tubing was sealed tightly to the short shoot by a rubber band. The root compartment was screened from light and was fitted with two 1-cm diameter ports to inject nutrients and collect water samples. In addition, a ventilation tube was placed in the root compartment to prevent a vacuum from forming when water samples were withdrawn. During the experiments, leakage was monitored by checking the water level in the ventilation tubing; an increase in water level indicated leakage between the two compartments.

Seawater used during incubations was collected from the study sites. Phytoplankton and bacteria were removed by filtering through a glass fiber filter followed by a 0.2-pm pore size polycarbonate filter. Circulation in the leaf compartment was provided by gentle aeration; water in the root compartment was not continuously mixed, but was magnetically stirred prior to sample collection.

Nitrogen uptake experiment

The experiments were conducted in a controlled environmental room with constant temperature and light intensity. Light was supplied on a 12h light : 12h dark cycle; fluorescent lights provided about 300 pmol photons m' 2 s' 1 to shoot tissues as measured by an LI-193 SA spherical quantum sensor in conjunction with an LI-1000 datalogger (LI-COR Inc., Lincoln, NB, USA). DIN uptake rates were determined for leaves and roots collected from both study sites in February, May, July and October 1997 at ambient temperatures of 16, 25, 30 and 25 °C, respectively. Incubations were also completed at night (20:00 to 05:00) in February and May to compare N uptake under dark and light conditions.

For leaf N uptake experiments, NH 4 + concentrations in the root compartment were maintained at ambient levels, 30 and 120 pM for LLM and CCB plants, respectively. Similarly, NH 4 + and NO 3 '+NO 2 ' concentrations in the leaf compartment were less than 2pM during measurement of root NH 4 + uptake rates.

In order to obtain uptake rates at a variety of DIN concentrations for leaf or root tissues, experimental runs lasted approximately 10 h. At the start of a run, the target compartment in each of nine chambers was spiked with concentrated NaNO 3 or NH 4 CI solutions to yield a representative range of NO 3 ’ or NH 4 + concentrations. For example, for leaf uptake determinations, leaf compartments were spiked to achieve NH 4 + concentrations of 3to 200 pM. One chamber without plants was incubated during each run as a control.

To eliminate a high initial uptake rate by adsorption, sampling for DIN measurements was started 1 h after addition of NH 4 + and NO 3 ' (Short and Mcßoy, 1984). Water samples were collected from the chambers at consistent time intervals (1 to 2h) and analyzed immediately to determine NH 4 + and NO 3 ’ concentrations following the colorimetric techniques of Parsons et al. (1984).

The NH 4 + and NO 3 ' uptake rates by leaf tissues and NH 4 + uptake rates by roots were assumed to be represented by decreases in the nutrient concentrations in target compartments over time. The NO 3 " or NH 4 + concentrations were corrected for volume to determine the total N species content within a chamber. Total N content per compartment was then normalized to per gram dry wt of root or leaf tissue within a chamber, and plotted against time. Dry weight of rhizome tissues was not included in the uptake rate calculation since N uptake by rhizome tissues is assumed to be negligible (Barnabas, 1991, 1994; Stapel et al., 1996). The slope of

the linear regression represents the uptake rate (pmol g' 1 dry wt hr' 1 ). Because uptake rates vary as a function of nutrient concentrations, two to five measurements from a given chamber were used to calculate an uptake rate.

Uptake rates were plotted as a function of nutrient concentration. The NH 4 + and NO 3 ‘ uptake kinetics were derived using the Michaelis-Menten equation:

V=V max *S/(K m + S)

where V(g mol g' 1 dry wt h' 1 ) represents actual uptake rate, V max is the maximum uptake rate, S (pM) is the nutrient concentration and K m (pM) is the half saturation constant, numerically equal to S at !4 Uptake affinity is equivalent to V tnax /K m .

Contribution of leaves and roots to total N acquisition

Daily N acquisition (N D ; mmole DIN m' 2 d' 1 ) by leaves and roots were calculated using the following equations:

V = V *S /fK + S 1 ’ ambient v max ‘“’ambient' \ m N D = V mfat *M tlt „ rw * 24/1000

where is the N uptake rate at ambient N concentration, S ambient is the ambient water column or pore water DIN concentration, and M leaforroot is leaf or root biomass (g dry wt m‘ 2 ). Based on observed in situ water temperature, V max and K m values calculated for February, May, July and October experiments were applied to daily N acquisition calculations for the periods of December to February, March to May, June to August and September to November, respectively.

Statistics

Statistical analyses were performed on a microcomputer using SAS (SAS Institute, 1989). Significant differences in water column and sediment pore water DIN, leaf and root biomass among sampling times and study sites were tested using a 2-way ANOVA. To test significant differences between light and dark N uptake rates, the Michaelis-Menten equation was rearranged to yield the linear equation for Hanes-Woolf plot (Segel, 1976): S/V = (l/V max )S + Nitrogen uptake rates were replotted using the linear equation, and significant differences between the two linear relationships for light and dark N uptake were tested using an analysis of covariance (ANCOVA). and K m were compared among sampling times and study sites using 95 % confidence intervals for these parameters.

Results

DIN and biomass

Water column NH 4 + concentrations ranged from 0.79 to 1.53 pM in CCB and 0.70 to 1.43 pM in LLM. Levels of NH 4 + varied significantly as a function of sampling date (P < 0.001), but no significant differences were observed between study sites (P = 0.28; Fig. 3.1 A). Water column NO 3 +NO 2 ' concentrations at both sites ranged from 0.26 to 1.19 pM; no significant difference was found between sites (P = 0.164), although significant seasonal variations were evident (P < 0.001; Fig. 3.IB). Sediment pore water NH 4 + concentrations ranged from 37 to 180 pM in CCB and from Bto4spM in LLM. Average pore water NH 4 + concentrations in CCB (87 pM) were significantly higher than in LLM (26 pM; P < 0.001; Fig. 3. IC).

Leaf biomass in CCB ranged from 128.5 g dry wt m’ 2 in January to 394.2 g dry wt m’ 2 in August, while root biomass ranged from 59.9 g dry wt m' 2 in August to 126.0 g dry wt m' 2 in October (Fig. 3.2 A). Leaf and root biomass in LLM were also seasonally variable; leaf biomass ranged from 65.1 g dry wt m' 2 in January to 204.9 g dry wt m' 2 in September, and root biomass ranged from 46.0 g dry wt m’ 2 in October to 178.0 g dry wt nf 2 in February (Fig. 3.28). Leaf biomass in CCB was

significantly higher than that in LLM (P < 0.001), while root biomass was not significantly different between study sites (P = 0.205).

Leaf NH/ and N0 3 ' uptake

Leaf NH 4 + uptake showed clear seasonal variation (Fig. 3.3). For CCB plants, the maximum uptake rate was highest in October (16.39 pmol g' 1 dry wt h' 1 ) and lowest in May (8.33 pmol g' 1 dry wt h’ 1 ), while the half saturation constant (KJ was highest in February (15.01 pM) and lowest in October (7.59 pM) (Table 3.1). V max and K m of leaf NH 4 + uptake in LLM showed a similar trend to CCB, with high V max and low K rn values in October and lowest V max and high K m values in February. Uptake affinity (V max /KJ was lowest in winter (February) and highest in early fall at both sites (Table 3.1).

Leaf N0 3 ' uptake also exhibited seasonal variation (Fig. 3.4, Table 3.1). V max did not change significantly with season, but K in decreased with increasing incubation temperature at both sites. Uptake affinity was lowest in February at both sites, and highest during July in LLM (1.095) and during October in CCB (1.678). There were no obvious differences in V niax and K m of leaf NO 3 ‘ uptake between study sites. Leaf NH 4 + uptake had higher V max values and similar or lower K m values than those of leafNO 3 - uptake; consequently leaf NH 4 + uptake resulted in higher uptake affinity than leaf NO 3 ' uptake.

Root NH/ uptake

In most cases root NH 4 + uptake did not saturate at the highest experimental NH 4 + concentrations utilized (ca. 300 pM). Therefore, V max and K m of root NH 4 + uptake estimated by the Michaelis-Menten model were much higher than those estimates for leaf uptake (Table 3.1). Root NH 4 + uptake was highly variable with season (Fig. 3.5, Table 3.1). For CCB plants, V max and K m values were highest in October, while lowest in May for V max and February for K tn . For LLM plants, V max and values were highest in May and lowest in February. There was no significant difference in root NH 4 + uptake rates between sites. Root NH 4 + uptake generally showed much lower uptake affinity than leaf NH 4 + and NO 3 ‘ uptake.

Light and dark N uptake

There were no differences between light and dark N uptake, as reflected by and K m , by leaf and root tissues (Fig. 3.6; Table 3.2). The uptake lines of the

Hanes-Woolf plot corresponding to light and dark were also not significantly different (P = 0.672, 0.154 and 0.375 for leaf NH 4 + , leaf NO 3 ' and root NH 4 + uptake, respectively; Fig. 3.6).

Nitrogen acquisition

Daily N acquisition from water column NH 4 + and NO 3 ‘ by leaf tissues was highest during summer and fall and lowest during winter and spring, compared to sediment pore water NH 4 + acquisition by root tissues, which was variable and exhibited no clear seasonal trend (Fig. 3.7). In summer, leaf N uptake accounted for about 80 % (CCB) and 65 % (LLM) of total N acquisition, but N acquisition in winter was dominated by root uptake, which accounted for about 80 % (CCB) and 70 % (LLM; Fig. 3.8). Nitrogen acquisition was much higher in CCB than that in LLM; the calculated annual N acquisition was 97.03 and 53.49 g N m' 2 y' 1 in CCB and LLM, respectively (Fig. 3.8). At both sites, NH 4 + uptake by root tissues accounted for about 52 % of total N acquisition, while leaf tissues assimilated about 38 %of total Nas NH 4 + , and about 10 %as NO 3 *.



Figure 3.1. Water column NH 4 + (A), NO 3 +NO 2 ‘ (B) and sediment NH 4 + (C) concentrations in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) from October 1996 to November 1997. Values are mean ± SE (n=4). When no error bars appear, SE is less than the size of the symbol.



Figure 3.2. Thalassia testudinum. Seasonal changes in leaf and root biomass in Corpus Christi Bay (CCB, A) and lower Laguna Madre (LLM, B). Values are mean ± SE (n=4). When no error bars appear, SE is less than the size of the symbol.



Figure 3.3. Thalassia testudinum. Leaf NH 4 + uptake rates of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 as a function of NH 4 + concentration. The curves represent the best fits of the Michaelis-Menten equation.



Figure 3.4. Thalassia testudinum. Leaf NO 3 ‘ uptake rates of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 as a function of NO 3 ' concentration. The curves represent the best fits of the Michaelis-Menten equation.



Figure 3.5. Thalassia testudinum. Root NH 4 + uptake rates of plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997 as a function ofNH 4 + concentration. The curves represent the best fits of the Michaelis-Menten equation.



Figure 3.6. Thalassia testudinum. Comparisons of light and dark nitrogen uptake rates for leaf NH 4 + (A), leaf NO 3 ’ (B) and root NH 4 + (C). The curves represent the best fits of the Michaelis-Menten equation. All results were rearranged to yield the linear equation for the Hanes-Woolf plot (right inset) to test significant differences between light and dark nitrogen uptake using an analysis of covariance (ANCOVA). The experiments were conducted in February for leaf NH 4 + uptake and for leaf NO 3 ’ uptake and root NH 4 + uptake in May.



Figure 3.7. Seasonal changes in daily nitrogen acquisition by leaves from water column NH 4 + (A) and NO 3 ‘ (B) and by roots from sediment NH 4 + (C).



Figure 3.8. Monthly and annual N acquisition by leaf and root tissue from water column and sediment in Corpus Christi Bay (A) and lower Laguna Madre (B).

v v max (gmol g 1 dry wt h 1 ) K m (gM) R 2 Affinity (V max /K m ) LeafNH 4 + uptake CCB Feb 8.50 (7.78-9.22) 15.01 (11.11-18.91) 0.876 0.567 May 8.33 (7.91-8.75) 11.67 (9.11-14.23) 0.889 0.714 July 12.78 (12.06-13.50) 7.65 (5.63-9.67) 0.941 1.671 Oct 16.39 (15.69-17.09) 7.59 (6.29-8.89) 0.960 2.160 LLM Feb 11.70 (10.92-12.48) 15.23 (12.23-18.23) 0.922 0.768 May 14.01 (13.01-15.01) 14.74 (11.74-17.74) 0.879 0.950 July 16.45 (15.41-17.49) 19.41 (15.51-23.31) 0.962 0.848 Oct 14.39 (13.53-15.25) 5.10 (3.88-6.32) 0.930 2.821 Leaf NO 3 ' uptake CCB Feb 5.85 (5.13-6.57) 38.45 (26.47-50.43) 0.911 0.152 May 3.81 (3.53-4.09) 5.32 (3.52-7.12) 0.721 0.717 July 3.74 (3.38-4.10) 6.33 (2.99-9.67) 0.723 0.591 Oct 3.67 (3.35-3.99) 2.19 (0.85-3.53) 0.657 1.678 LLM Feb 5.33 (4.69-5.97) 33.87 (24.31-43.43) 0.900 0.157 May 4.39 (4.03-4.75) 21.34 (14.86-27.82) 0.899 0.206 July 3.40 (3.14-3.66) 3.11 (1.71-4.51) 0.777 1.095 Oct 6.45 (5.95-6.95) 12.66 (8.94-16.38) 0.867 0.509 RootNH 4 + uptake CCB Feb 14.00 (10.82-17.18) 89.16 (42.38-135.94) 0.783 0.157 May 10.92 (9.82-10.20) 172.07 (136.83-207.31) 0.966 0.063 July 25.57 (8.94-42.20) 757.10 (128.90-1385.3 0.885 0.034 Oct 73.32 (35.69-110.9 765.52 (296.12-1234.9 0.878 0.096 LLM Feb 7.85 (6.37-9.33) 34.35 (14.97-53.73) 0.740 0.228 May 52.58 (32.52-72.64) 649.51 (351.51-947.51) 0.916 0.081 July 27.81 (11.53-44.09) 399.52 (233.82-565.22) 0.917 0.070 Oct 18.35 (15.53-21.17) 60.83 (40.93-80.73) 0.742 0.302

Table 3.1. Parameters (V max and K m ) of the Michaelis-Menten model and uptake affinity (V max /K m ) for leaf and root nitrogen uptake for plants from Corpus Christi Bay (CCB) and lower Laguna Madre (LLM) in February, May, July and October 1997. Values in parenthesesare 95 % confidence intervals for V max and K m .

v v max (gmol g’ 1 dry wt h’ 1 ) Km (gM) R 2 LeafNH 4 + uptake Day 8.11 (7.29-8.93) 12.57 (8.51-16.63) 0.854 Night 9.27 (8.29-10.25) 19.92 (13.80-26.04) 0.938 Leaf NO 3' uptake Day 4.03 (3.77-4.29) 2.31 (1.35-3.27) 0.751 Night 3.97 (3.61-4.33) 6.55 (3.33-9.77) 0.887 RootNH 4 + uptake Day 10.94 (9.32-12.56) 178.13 (124.51-231.75) 0.964 Night 10.94 (9.40-12.48) 167.55 (119.19-215.91) 0.970

Table 3.2. Parameters (V max and K m ) of the Michaelis-Menten model for light and darknitrogen uptake by leaf and root tissues. Values in a parenthesis are 95 % confidence intervals for V max and K m .

Discussion

Uptake kinetics

The results presented here demonstrated that both leaf and root tissues of Thalassia testudinum are capable of significant N uptake as shown in previous studies on other seagrass species (lizumi and Hattori, 1982; Thursby and Harlin, 1982, 1984; Short and Mcßoy, 1984; Stapel et al., 1996; Pedersen et al., 1997; Terrados and Williams, 1997). However, uptake kinetics can be highly variable among seagrass species (Table 3.3). Values of V max and K nl for NH 4 + and NO 3 ‘ uptake by T. testudinum leaf tissues were much lower than reported values for most other seagrass species (Thursby and Harlin, 1982, 1984; Stapel et al., 1996; Pedersen et al., 1997). These results suggest that T. testudinum has a lower capacity for leaf N uptake, but has a higher uptake affinity than other seagrass species at low water column DIN concentrations. The of root NH 4 + uptake was lower than that of Zostera marina (Thursby and Harlin, 1982) but is similar to that of Ruppia maritima (Thursby and Harlin, 1984) and Amphibolis antarctica (Pedersen et al., 1997). The K in of root NH 4 + uptake was higher than that of R. maritima but was within the range reported for Z. marina and A.

antarctica (lizumi and Hattori, 1982; Thursby and Harlin, 1982; Pedersen et al., 1997).

Higher leaf uptake rates have been reported for NH 4 + than NO 3 ‘ in Phyllospadix torreyi and Zostera marina (Short and Mcßoy, 1984; Terrados and Williams, 1997). In the present study, leaf NH 4 + uptake kinetics indicated higher V mix and uptake affinity than those for NO 3 ‘ uptake. Assimilation of NO 3 ’ by plants is influenced by the availability of photosynthate or stored carbohydrate, and is energetically expensive (Thacker and Syrett, 1972; Lara et al., 1987; Turpin, 1991). Burkholder et al. (1992, 1994) demonstrated that NO 3 * utilization is an energetically costly process in Z. marina as chronic water column NO 3 ‘ enrichment leads to plant decline. Therefore, seagrass leaf tissues probably prefer the reduced N source (NH 4 + ) to NO 3 ‘, and take up NH 4 + with higher affinity.

Pedersen et al. (1997) reported that leaf NH 4 + uptake was much faster than root NH 4 + uptake mAmphibolis antarctica. Similarly, in the present study, both leaf NH 4 + and NO 3 ' uptake affinities for Thalassia testudinum were much higher than for root NH 4 + . Seagrass leaf tissues are usually exposed to considerably lower NH 4 + or NO 3 ’ concentrations than root tissues, which are surrounded by high NH 4 + concentrations in pore water. Therefore, seagrass leaf tissues may have the ability to assimilate N under low DIN conditions, and the uptake may saturate at lower concentrations. Conversely, root NH 4 + uptake rates increased continuously as a function of NH 4 + concentration and exhibited saturation .u ' uvh higher NH 4 + concentrations than leaves. These N uptake patterns of leaf and root tissues likely reflect plant adaptations to life in oligotrophic like waters of low DIN and relatively higher levels of NH 4 + in sediment pore waters.

Nitrogen acquisition

In the present study, T. testudinum root NH 4 + uptake from the sediment accounted for about 50 %of total plant N acquisition, with leaf NH 4 + and NO 3 ' uptake from the water column comprising the remaining 50 % on an annual basis (Fig. 3.9). lizumi and Hattori (1982) calculated N acquisition in Zostera marina using average values of DIN concentrations, root/shoot ratio indices, and N uptake rates measured during spring, and demonstrated that about 55 % of the total N requirement for Z. marina growth was supplied by sediment NH 4 + . From Zimmerman’s model study (1987), contribution of root NH 4 + uptake varied from 0 % to 70 % as function of day length, and N uptake by roots accounted for 60 % of total N acquisition for a photoperiod of 12 h.

This work demonstrates however, that the contributions of leaves and roots to the total N budget of the plant changes seasonally. In summer, leaf N uptake accounted for about 80 % (CCB) and 65 % (LLM) of total N acquisition. In contrast, N acquisition in winter was dominated by root uptake, which accounted for about 80 % (CCB) and 70 % (LLM). The distinct seasonal difference was driven by changes in leaf tissue acquisition, which was highest during summer and fall and lowest during winter and spring. Nitrogen acquisition by root tissues was relatively constant with season. The distinct seasonal variations in leaf N acquisition were caused by higher leaf uptake rates and leaf biomass during summer and fall.

Total N acquisition by leaf and root tissues was high during fall and low during spring. This N acquisition pattern is consistent with previous reports of seasonal trends in tissue N content, protein and amino acid levels (Harrison and Mann, 1975; Pirc, 1985; Dawes, 1986; Pellikaan and Nienhuis, 1988; Pirc and Wollenweber, 1988; Dawes and Guiry, 1992; Perez-Lloens and Niell, 1993; Short et al., 1993; Chapter 2). Tissue N contents, protein and amino acid levels were high during fall and winter and low during spring and summer. This suggests that during periods of high N uptake, uptake may exceed the N requirements, and the excess N will be stored as amino acids or proteins to meet high N demands during periods of high production (Pirc, 1985; Dawes, 1986; Dawes and Guiry, 1992).

Surprisingly, significant differences in pore water NH 4 + concentrations between the two sites (CCB vs LLM) did not affect the contributions of leaves and roots to total N budget in T. testudinum. In contrast, Zimmerman et al. (1987) showed the effects of water column and sediment DIN concentrations on patterns of N assimilation with a numerical model, and noted that the contributions of leaf and root N uptake to total N acquisition varied as a function of DIN concentrations. However, in the present study, there was no difference in the relative contributions of leaf and root uptake to total N acquisition between the two study sites, even though the sites differed significantly with respect to sediment NH 4 + concentrations. This disagreement is probably a product of biomass allocation. Zimmerman et al. (1987) used a single root: shoot biomass ratio (0.20) for all calculations. However, biomass allocation into above or below-ground tissues has been shown to change with sediment nutrient availability (Chapter 1). Seagrasses allocate more biomass into below-ground tissues under low sediment N availability in order to increase the root surface area for N uptake; conversely, more biomass occurs in above-ground tissues under high sediment N availability to increase C fixation (Chapter 1). The average below/above-ground biomass ratio (Chapter 1) at the low sediment NH 4 + site (LLM, 4.66) was significantly higher than that at the high sediment NH 4 + site (CCB, 1.63). Since tissue N uptake rates on a dry weight basis were not different for T. testudinum from the two study sites, the leaf N acquisition calculated for CCB was higher than for LLM due to the larger leaf biomass which produced higher surface area for N uptake. (Fig. 3.9). Root N acquisition was also higher in CCB than LLM (51 and 28 g N m' 2 y 4, respectively) due to higher sediment NH 4 + concentrations at CCB (Fig. 3.9). Consequently, contributions of leaf and root N uptake to total N acquisition did not change in T. testudinum with sediment NH 4 + concentrations.

Although contributions of leaf and root N uptakes to total N acquisition were similar at both study sites, total annual N acquisition in CCB was significantly higher than that in LLM (97.03 and 53.49 g N m’ 2 y’ 1 , respectively). Carbon and nitrogen comprise about 35 and 2 %, respectively, of Thalassia testudinum on a dry weight basis (Chapter 2). Thus, the annual N acquisition can support an annual production 0f4851 g dry wt m‘ 2 y 4 (1698 g C m' 2 y' 1 ) in CCB and 2674 g dry wt m‘ 2 y 4 (936 g C m' 2 y 4) in LLM. Lee and Dunton (1996) estimated an annual blade production of 792 g dry wt m’ 2 y 4 (253 g C m' 2 y 4) in

CCB, while Kaldy (1997) estimated an annual total production of 953 g dry wt m' 2 y' 1 (340 g C m’ 2 y' 1 ) in LLM. These results show that the N acquisition by leaf and root tissues are much higher than N utilized for biomass production at both sites. The difference between N acquisition and utilization suggests that not all N acquired by plant tissues is incorporated into biomass.

Consequently, although T. testudinum growth in LLM has been demonstrated to be N limited (Chapter 1), over 50 % of the absorbed DIN was not incorporated into biomass. The difference between N acquisition and utilization may be explained by low efficiency of N assimilation into biomass. Excess N uptake over N utilization for biomass production has been reported for bacteria (Keil and Kirchman, 1991; Jorgensen et al., 1993; Kirchman, 1994). Kirchman (1994) calculated that bacterial production estimated from DIN uptake was about 10-fold higher than the microscopic-based estimate, suggesting that about 10 % of the acquired DIN was incorporated in bacterial biomass. The imbalance between DIN uptake and bacterial production has been explained by excretion of dissolved organic nitrogen (DON; Kirchman, 1994). Excretion of amino acids (Wood and Hayasaka, 1981) and dissolved organic carbon (DOC; Penhale and Smith, 1977; Wetzel and Penhale, 1979; Moriarty et al., 1986; Ziegler, 1998) by seagrass tissues has been reported, and it is possible that some inorganic N acquired by seagrass tissues is also released as DON. It has been demonstrated that pore water NH 4 + concentrations in seagrass beds were maintained by the continual deamination of amino acids by bacteria in the rhizosphere of seagrasses (Smith et al., 1984; Boon et al., 1986). The amino acids to maintain this NH 4 + regeneration are provided as exudates from seagrass roots (Wood and Hayasaka, 1981; Smith et al., 1984; Boon et al., 1986).

Since root tissues were incubated in pure seawater instead of sediments, there is a possibility that I overestimated root DIN uptake rates due to increases in surface area contact with pore water and in rates of DIN diffusion from pore water to the root surface (Stapel et al., 1996). Additionally, epiphytes or epifauna on leaf tissues were removed to exclude nitrogen uptake by these organisms. Epiphyte and epifaunal removal may cause increases in leaf surface area for nitrogen uptake, and consequently cause an overestimate of leaf DIN uptake during this experiment.

DIN pool and turnover rates

Root N uptake rates may be affected by the rates of N diffusion in the sediment. By comparing root uptake capacity with nutrient diffusion rates, Stapel et al. (1996) demonstrated that root uptake depends primarily on nutrient diffusion rates from pore water to the root surface. However, N for root uptake can be supplied by remineralization in the sediments in addition to diffusion (Hines and Lyons, 1982; Holmer and Nielsen, 1997). Thus, in situ N acquisition by roots probably depends on the combination of the uptake capacity of the roots, N diffusion and remineralization rates in sediment. Rapid sediment DIN turnover rates have been reported in various seagrass beds (Capone, 1982; Moriarty et al., 1985; Boon et al., 1986). In the present study, the DIN pool size in the water column and sediment was calculated from mean DIN concentrations and volume of water (Table 3.4). It was assumed that DIN in the entire water column (1.2 m depth) was available for leaf uptake. Since most T. testudinum roots in CCB and LLM penetrate to a depth of only 20 cm in the sediment (Lee and Dunton, in prep), pore water DIN in the top 20 cm of sediment was assumed to be available for root uptake. Sediment porosity at both sites is about 0.5 (Lee, unpubl. data). Assuming seagrass dominates DIN uptake in this system, DIN turnover time ranged from 0.21 to 0.91 day in water column and 0.95 to 1.75 days in sediment pore water (Table 3.4). The sediment DIN turnover time in this study is within the range found in other seagrass beds (0.4 to 6 days; Capone, 1982; Moriarty et al., 1985; Boon et al., 1986). The water column DIN turnover time calculated in this study is similar to other estimates for LLM (0.21 to 1.49 days) that were calculated using DIN remineralization rates (Ziegler, 1998). However, bacteria can account for a large portion of total DIN uptake in marine environments (Kirchman, 1994). Thus, actual DIN turnover rates at the study sites are probably faster than estimates calculated by only seagrass uptake. These rapid DIN turnover rates in seagrass beds indicate the importance of DIN regeneration processes for supporting seagrass production (Moriarty et al., 1985; Boon et al., 1986).

In conclusion, root and leaf tissues contributed equally to the N budget of Thalassia testudinum. There were no differences in the contributions of leaf and root uptake to total N acquisition between the two study sites, despite significant difference in sediment pore water NH 4 + levels. The similar contribution patterns in tissue N acquisition at both sites were caused by biomass allocation patterns: high leaf biomass at high sediment N conditions and high below-ground biomass at low sediment N conditions. Total N acquisition by leaf and root tissues was highest during fall and lowest during spring. Annual N acquisition in CCB was double that of LLM due to the significantly higher leaf biomass and higher sediment pore water NH 4 + concentrations in CCB. Nitrogen acquisition by leaf and root tissues did not match the amount incorporated into plant tissues; even at LLM, where the low DIN levels in the water column and sediments are believed to limit seagrass production, over 50 % of taken up N was not incorporated into biomass. The reason for the low assimilation efficiency must be addressed in future research to further understanding the N budget in seagrass beds.



Figure 3.9. Nitrogen budget for Thalassia testudinum in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM). Values represent annual means: nitrogen budget calculations were derived using seasonal uptake rates, biomass and ambient nitrogen availability. Size of boxes for plants corresponds to the average biomass of each plant part.

Species Tissue DIN v v max (p mol g 1 dw h' 1 ) K m (pM) Area Time Source Thalassia hemprichii Leaf NH/ 32-37 21-60 Indonesia April - June Stapel et al. (1996) Zostera marina Leaf NO 3 ' - 23 Japan March Iizumi and Root NH/ - 30 May Hattori (1982) Zostera marina Leaf nh 4 + 20.5 9.2 Rhode Island, USA Late summer Thursby and Root NH/ 211 104 Harlin (1982) Amphibolis antarctica Leaf nh/ 82 - 604 133 - 1041 Australia January Pedersen et al (1997) Root nh 4 + 16 66 Phyllospadix torreyi Leaf nh 4 + 9.30 - 33.97 95.69 - 204.32 California, USA July - August Terrados and Leaf NO 3 ‘ 4.39 - 16.98 24.97 - 75.47 Williams (1997) Ruppia maritima Leaf nh/ 243 - 270 9.0-17.7 - - Thursby and Root nh 4 + 48-56 2.8-12.6 Harlin (1984) Thalassia testudinum Leaf nh 4 + 8.33 - 16.45 5.10 - 19.41 Texas, USA Seasonal Present study Leaf NO 3 ' 3.40-6.45 2.19-38.45 Root nh/ 7.85 -73.32 34.35 -765.52

Table 3.3. Parameters (V max and K m ) of the Michaelis-Menten model for tissue nitrogen uptake of various seagrass species

Pool size (m mol N m' Uptake rate 2 ) (m mol rn 2 d' 1 ) Turnover time (d) CCB Water column NH 4 + 1.42 6.80 0.21 Water column NO 3 '+NO 2 0.88 2.27 0.39 Sediment NH 4 17.40 9.93 1.75 LLM Water column NH 4 + 1.33 4.12 0.32 Water column NO 3 +NO 2 ’ 0.86 0.95 0.91 Sediment NH 4 + 5.20 5.45 0.95

Table 3.4. Estimated DIN pool size in water column and sediment, annual mean nitrogen uptake rate by seagrass and DIN turnover time in water column and sediment in Corpus Christi Bay (CCB) and lower Laguna Madre (LLM)

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The vita has been removed from the digitized version of this document.