ABUNDANCE, PRODUCTION AND CARBON DYNAMICS OF THE SEAGRASS, THALASSIA TESTUDINUM IN CORPUS CHRISTI BAY, TEXAS

by KUN-SEOP LEE, M.S.

THESIS

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

MASTER OF ARTS

THE UNIVERSITY OF TEXAS AT AUSTIN

August 1995

ABUNDANCE, PRODUCTION AND CARBON DYNAMICS OF THE SEAGRASS, THALASSIA TESTUDINUM IN CORPUS CHRISTI BAY, TEXAS

APPROVED BY SUPERVISING COMMITTEE:

Kenneth H. Dunton, Supervisor

Ronald H. Benner

Paul A. Montagna

Acknowledgements

I would like to thank my advisor, Ken Dunton, for his encouragement, trust and tireless support. He gave me a chance and caring help. It was amy good fortune to meet him. I also thank my committee members, Ronald Benner and Paul Montagna for their time and valuable comments. I am very grateful to Jim Kaldy and Sharon Herzka for their countless hours of reading and discussions and extremely helpful comments. They were the best readers and reviewers. I thank Susan Schonberg and Kim Jackson for providing solutions for computational problems and excellent assistance in the lab. Finally, I would like to thank my wife Mi Youn and daughter Jin for their love and patience. This work was supported by the Texas Higher Education Coordinating Board Advanced Technology Program (Grant No. 3658-419 and 3658-426) and Grant No. X-996025-01-1 (EPA, Region 6) and a Coastal Submerged Aquatic Vegetation Initiative Grant from the U.S. Environmental Protection Agency.

Submitted to thesis committee on June 20, 1995.

ABSTRACT ABUNDANCE, PRODUCTION AND CARBON DYNAMICS OF THE SEAGRASS, THALASSIA TESTUDINUM IN CORPUS CHRISTI BAY, TEXAS

by Kun-Seop Lee, M.A. The University of Texas at Austin, 1995 SUPERVISOR: Kenneth H. Dunton

The seasonal production dynamics of the subtropical seagrass, Thalassia testudinum, were examined through measurements of biomass, leaf growth and carbohydrate carbon content from plants collected in Corpus Christi Bay from December 1993 to March 1995. Daily photon flux densities (PFD) showed strong seasonal variations, ranging from 9.6 mol m' 2 d 1 in April to 21.7 mol m“ d 1 in July. Shoot density and biomass changed significantly with season; values ranged from 321 shoots m’ 2 (454 g dry wt in 2 ) in March to 531 shoots m’ 2 (885 g dry wt m’ 2 ) in September. Rhizome tissues tended to have the highest biomass while root tissue had the lowest. Leaf productivities showed significant seasonal variation that were strongly correlated with temperature, ranging from 0.07 g dry wt m’ 2 d’ 1 in December to 5.6 g dry wt m’ 2 d' 1 in July. Chlorophyll (chi) concentrations were significantly higher and chi a:b ratios lowest during the spring/summer period of maximum photosynthetic production and growth than during winter. Soluble carbohydrate carbon content was highest in rhizome tissues (111-203 mg Cg 1 dry wt) and lowest in leaf tissues (46-70 mg C g’ 1 dry wt), which is consistent with the rhizome’s role as a carbon storage tissue. Soluble rhizome carbohydrate carbon content increased rapidly during June and July, which coincided with high water temperatures, underwater irradiance and blade chlorophyll concentrations. During winter and early spring, rhizome carbohydrate carbon content dropped nearly 50%, suggesting that these reserves were mobilized for maintenance and growth. Estimated annual biomass production of Thalassia testudinum in Corpus Christi Bay over the period of this study was 1320 g dry wt m' 2 yr' 1 , equivalent to 422 g C m' 2 yr’ 1 .

To assess the effects of light reduction on Thalassia testudinum, shade screens were used to reduce underwater light to 1628 mol m' 2 yr' 1 (14% of surface irradiance, SI) and 864 mol m' 2 yr' 1 (5% SI) starting in April 1993. All plants subjected to 5% SI died after 200 days and over 99% of plants receiving 14% SI died by the end of the experiment (490 days). Blade widths of plants in the controls ranged from 6.4 to 7.0 mm, and decreased to 4.7 mm as a result of light reduction. Leaf production rates were significantly higher in control plants compared to plants within the 14% and 5% SI treatments, with all plants showing a seasonal trend with high productivity in July and low productivity in April. Blade chlorophyll concentrations increased, while the chi a.b ratio decreased with reduced light level. In both light treatments rhizome soluble carbohydrate carbon content was 50% lower and leaf carbohydrate carbon content was about 15% lower than controls, while the root carbohydrate content did not differ significantly between treatments and controls (no decrease in structural carbohydrate carbon content was noted between treatments). Pore water ammonium and sulfide concentrations in the shaded cages were significantly higher than in control cages. Thalassia testudinum in Corpus Christi Bay exhibited a strong seasonal growth cycle in which changes in rhizome carbohydrate reserves and chlorophyll content may be under endogenous control as triggered by a combination of temperature and/or light period. In contrast to the seagrass Halodule, Thalassia maintained a larger carbohydrate reserve and exhibited a stronger physiological response to light reduction, which may contribute to its competitive superiority.

Table of Contents

List of Table

List of Figure ,x

Chapter 1: Seasonal changes in biomass, leaf productivity and carbohydrate carbon content of Thalassia testudinum in Corpus Christi Bay, Texas 1

Abstract 1

Introduction 2

Material and Methods 6

Results 9

Discussion

Chapter 2 : Effects of in situ light reduction on the maintenance, growth and partitioning of carbon resources in Thalassia testudinum 29

Abstract 29

Introduction 30

Material and Methods 34

Results 40

Discussion 58

Literature cited 65

Vita 77

List of Tables

Table 1.1: Thalassia testudinum. Seasonal variation in biomass partitioning to different plant parts 13

Table 2.1: Daily average photon flux density (PFD), % of in situ ambient (% ISA), % of surface irradiance (% SI) and the daily period of light saturated photosynthesis (H sat ) in control and light manipulation cages 42

Table 2.2: Chlorophyll a:b ratio of Thalassia testudinum leaves from control, 14% SI and 5% SI treatment cages at four different sampling times 49

Table 2.3: Biomass changes in total and individual plant parts as a result of light manipulation in May (initial sampling date) and August 1993 and April and July 1994 52

Table 2.4: Below- to above-ground ratios of Thalassia testudinum at 46% SI (control), 14% SI and 5% SI in August 1993 and April and July 1994 53

Table 2.5: Total, soluble and structural carbohydrate carbon content of different plant tissues of Thalassia testudinum from control and 14% SI cages at April 1994 57

List of Figures

Figure 1.1: Average daily irradiance, on a monthly basis, recorded at canopy level in a Thalassia testudinum bed in Corpus Christi Bay from October 1993 to January 1995 10

Figure 1.2: Thalassia testudinum. Seasonal changes in density and total biomass (A) and biomass of different plant components (B) 12

Figure 1.3: Thalassia testudinum. Seasonal changes in leaf production rates per shoot and areal leaf production rate from February 1994 to January 1995 15

Figure 1.4: Thalassia testudinum. Seasonal changes in total chlorophyll concentration (A) and chi a.b ratio in blades of plants collected from January 1994 to March 1995 16

Figure 1.5: Thalassia testudinum. Seasonal changes of soluble carbohydrate carbon content in leaf, short stem, rhizome and root tissues • 18

Figure 1.6: Thalassia testudinum. Correlations between leaf productivity and water temperature (A) and underwater irradiance (B) from February 1994 to January 1995 21

Figure 1.7: Thalassia testudinum. Regression between leaf productivity plotted on a log scale and water temperature 22

Figure 2.1: Average daily photon flux density (PFD) collected underwater (control, 14% SI ans 5% SI treatment cages) and at the surface (The University of Texas Marine Science Institute in Port Aransas) .... 41

Figure 2.2: Pore water ammonium concentration in sediments (A) and shoot densities (B) of control, 14% and 5% SI treatment cages 44

Figure 2.3: Blade widths of Thalassia testudinum from control, 14% SI and 5% SI treatment cages 46

Figure 2.4: Chlorophyll a, chlorophyll b and total (chi a+b) concentrations of Thalassia testudinum leaves from control, 14% SI and 5% SI treatment cages 47

Figure 2.5: Daily leaf production on a shoot (A) and areal (B) basis in control and treatment cages 50

Figure 2.6: Changes of biomass patitioning of Thalassia testudinum into different plant parts (leaf, short stem, rhizome and roots) as a result of light manipulation between August 1993 and July 1994 . . 55

Figure 2.7: Carbohydrate carbon concentration in different plant tissues of Thalssia testudinum from control and light treatment cages in August and December 1993 and April 1994 56

CHAPTER 1: Seasonal changes in biomass, leaf productivity and carbohydrate carbon content of Thalassia testudinum in Corpus Christi Bay, Texas.

Abstract

The seasonal production dynamics of Thalassia testudinum in Corpus Christi Bay were evaluated through measurements of biomass, leaf growth and carbohydrate carbon content from December 1993 to March 1995. Daily photon flux densities (PFD) showed strong seasonal variations, ranging from 9.6 mol m' 2 d 1 in April to 21.7 mol m‘ 2 d' 1 in July. Shoot density and biomass changed significantly with season; values ranged from 321 shoots m' 2 (454 g dry wt m’ 2 ) in March to 531 shoots m’ 2 (885 g dry wt m' 2 ) in September. Biomass of individual plant parts were significantly different over the sampling period; rhizome tissues tended to have the highest biomass while root tissue had the lowest. However, leaf biomass was higher than that of rhizome tissues during summer. Leaf productivities showed significant seasonal variation that were strongly correlated with temperature, ranging from 0.07 g dry wt m‘ 2 d 1 in December to 5.6 g dry wt m’ 2 d 1 in July. Chlorophyll (chi) concentrations were significantly higher and chi a.b ratios lowest during the spring/summer period of maximum photosynthetic production and growth than during winter. Soluble carbohydrate carbon content was highest in rhizome tissues (111-203 mg C g' 1 dry wt) and lowest in leaf tissues (46-70 mg C g’ 1 dry wt), which is consistent with the rhizome's role as a carbon storage tissue. Rhizome carbohydrate carbon content increased rapidly during June and July, which coincided with high water temperatures, underwater irradiance and blade chlorophyll concentrations. During winter and early spring, rhizome carbohydrate carbon content dropped nearly 50%, suggesting that these reserves were mobilized for maintenance and growth. Estimated annual biomass production of Thalassia testudinum in Corpus Christi Bay over the period of this study was 1320 g dry wt m' 2 yr 1 , equivalent to 422 g C m' 2 yr' 1 . Overall, annual productivity appears to be primarily regulated by temperature and secondarily by irradiance, as reflected in the timing and magnitude of the strong seasonal variations in leaf productivity, total chlorophyll content, chi a.b ratios and rhizome carbohydrate carbon content in Thalassia testudinum.

Introduction

Seagrass meadows are among the most productive of plant communities (Mcßoy and McMillan, 1977), providing habitat and food for a wide variety of flora and fauna (Heck and Westone, 1977; Orth et al., 1984; Summerson and Peterson, 1984; Huh and Kitting, 1985). Although few herbivores consume seagrass directly (Ogden, 1980; Mann, 1988), a substantial fraction of seagrass carbon enters coastal and estuarine food webs through microbial transformation of litter and particulate detritus (Kenworthy and Thayer, 1984; Mann, 1988; Chin-Leo and Benner, 1991; Koepfler et al., 1993; Opsahl and Benner, 1993; Peduzzi and Hemdl, 1991).

Accurate assessment of total seagrass production is difficult since a substantial fraction of plant biomass is below-ground. However, net above-ground production of seagrasses having strap-like leaves can be easily estimated using leaf marking techniques (Zieman, 1974; Vermaat et al., 1987). Leaf production shows clear seasonal trends with rates increasing in spring and summer and decreasing in fall and winter (Vermaat et al., 1987; Dunton, 1994). In spring, when water temperature and daylength increase, leaf production rates increase primarily due to the creation of new leaves which are thought to originate mainly from stored carbon in the rhizomes, while higher leaf production rate in summer is due to growth of existing leaves (Tussenbroek,l99s).

Seasonal growth of seagrasses is probably regulated by insolation and temperature or an interaction of both (Phillips, 1974; Dunton, 1994). Some researchers have considered temperature as the primary factor controlling seasonal growth (Setchell, 1929; Tutin, 1942; Phillips et al., 1983). However, Sand-Jensen and Borum (1983) stated that water temperature did not correlate with seasonal trends in leaf productivities, while others have suggested that light and temperature interact in controlling seagrass seasonal growth (Phillips, 1974; Wetzel and Penhale, 1983). In addition, endogenous circannual rhythms of leaf elongation have been suggested for Thalassia testudinum an&Halodule wrightii (Ott, 1979; Dunton, 1994; Czerny and Dunton, 1995). Therefore, factors controlling seasonal growth patterns of seagrasses remain an unresolved issue.

Production dynamics of above-ground tissues of seagrasses are generally well documented, but the metabolic features and functional role of below-ground tissues in whole plant production dynamics are less well known (Kraemer and Alberte, 1993). The below-ground portion of Thalassia testudinum can account for over 50% of the total biomass (Powell et al., 1989; Fourqurean and Zieman, 1991), and is supported by photosynthetically-derived carbon and oxygen (Smith et al., 1984; Caffrey and Kemp, 1991; Ralph et al., 1992). Seagrass rhizomes serve as carbohydrate storage tissues in form of soluble carbohydrate carbon that support growth and maintenance of other plant parts during periods of low photosynthetic production (Dawes and Lawrence, 1979, 1980; Pirc, 1985; Durako and Moffler, 1985; Dawes and Guiry, 1992). Therefore, production, metabolism and stored carbon content of below-ground tissues must be considered when deriving estimates of whole plant carbon balance (Fourqurean and Zieman, 1991; Kraemer and Alberte, 1993).

Pirc (1985, 1989) found that seasonal changes of rhizome carbohydrate concentrations in Posidonia oceanica and Cymodocea nodosa were characterized by seasonal maxima in summer and fall, with winter growth and maintenance supported by the mobilization of starch from the rhizome. In Thalassia testudinum, increases in rhizome carbohydrates in summer have been attributed to the production and storage of starch, while winter and spring decreases were linked to the utilization of these stored carbohydrates for growth and maintenance (Dawes and Lawrence, 1979, 1980; Durako and Moffler, 1985). Although carbohydrate carbon in below-ground tissues plays an important role in growth and survival of seagrass, little research has been conducted on carbohydrate carbon partitioning into different plant parts, particularly on a seasonal basis.

Most of the work on Thalassia testudinum in the Gulf of Mexico has conducted in Florida, and thus little is known about the basic characteristics of this species in Texas; in particular, a basic knowledge of the plant's seasonal changes in biomass, carbohydrate carbon content and leaf productivity is lacking. The major objectives of this study were to examine seasonal changes in parameters relating to the production ecology of Thalassia testudinum in Texas. I hypothesize that the seasonal production of Thalassia testudinum is regulated primarily by temperature with changes in irradiance having the greatest significance during the late spring/summer period when water temperatures are highest. These included measurements of density, chlorophyll content, chi a : b ratios, biomass, carbohydrate carbon content, leaf productivity and continuous in situ measurements of photosynthetically active radiation (PAR). I also examined seasonal changes in carbohydrate carbon partitioning into different plant parts encompassing leaves, rhizomes, short stems and roots.

Materials and Methods

Study Site

The study site is located in eastern side of Corpus Christi Bay (27° 49' N, 97° 7’ W). This site has been the focus of several recent investigations on south Texas seagrasses (Dunton, 1990, 1994; Czerny and Dunton, 1995). Thalassia testudinum, Halodule wrightii and Syringodium filiforme are the dominant seagrass species in this area. This study was conducted on a monotypic meadow of Thalassia testudinum with an average water depth of 1.2 m. Water temperature ranged from 34°C in July and August to 13°C in January, while salinity varied from 27 to 32 %o during the experimental period.

Production and Biomass Measurements

Monthly measurements of shoot density, biomass, leaf chlorophyll content, carbohydrate carbon content and leaf elongation rates were made from December 1993 to March 1995. Four replicate biomass samples were collected with a 9 cm diameter coring device driven 15-20 cm into the sediments. Samples were thoroughly cleaned of epiphytes and sediments, separated into leaf (blade and sheath), short stem(including vertical rhizome), rhizome and root tissues and dried at 60°C to a constant weight. Shoot density was estimated by counting the number of shoots inside a randomly thrown quadrat (0.05 m' 2 ; n=4 to 8).

Leaf production rates were obtained using the blade marking technique (Zieman, 1974). Fifteen to twenty randomly chosen shoots were marked just above the bundle sheath with a hypodermic needle and collected after 12-16 days. Leaf production rate per shoot was determined by dividing the dry weight of new leaf tissue produced after marking by shoot density and the number of days since marking. Areal leaf production rates were obtained by multiplying shoot leaf production rates by the shoot density.

Chemical Analyses

For determination of blade chlorophyll (chi) content, six replicate samples from each visit were collected and cleaned of epiphytes by gentle scraping in the laboratory. Pre-weighed leaf tissue was ground in 90% cold acetone buffered with 0.05% MgCO 3 using chilled pestles and mortars with washed sea sand. The extract was made up to a known volume, centrifuged, and absorbances measured at 750, 664 and 647 nm on a Shimadzu UV 160 U spectrophotometer. Chi a and b content was determined using the equations of Jeffrey and Humphrey (1975) for 90% acetone extractions.

Dried plant material from biomass samples was used to determine soluble carbohydrate carbon content in different plant parts. Total dissolved carbohydrates from leaf, horizontal rhizome, vertical rhizome and root were determined using the MB TH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) analysis (Parson et al., 1984, Pakulski and Benner, 1992). Ground plant samples were hydrolyzed with dilute HCI. The hydrolyzed samples were neutralized with NaOH solution, followed by reduction to alditols with KBH 4 . The alditols were oxidized with periodic acid solution to form two moles of formaldehyde per mole of monosaccharide. The aldehyde content was determined spectrophotometrically with MBTH. Absorbances were compared with a glucose standard and converted to equivalent carbon values (mg Cg 1 dry wt).

Photon Flux Measurement

Photosynthetically active radiation (PAR, 400-700 nm) was collected continuously using a LI-193 SA spherical quantum sensor that recorded PAR at canopy level in conjunction with a LI-1000 datalogger (LI-COR Inc.) enclosed in underwater housing. The underwater quantum sensor was cleaned regularly to minimize fouling. Photon flux density (pmol m' 2 s' 1 ) was measured at 1-min intervals and integrated hourly.

Statistics

All values are reported as means ± 1 SE. Statistical analyses were performed on a microcomputer using a general linear model procedure (SAS Institute, 1989). Significant differences in underwater irradiance, density, chlorophyll content, chi a : b ratios and leaf productivity among sampling times were tested using a one-way ANOVA. A two-way ANOVA was used to test significant differences in biomass and carbohydrate carbon content among sampling times and plant parts. 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.

Results

Underwater Irradiance

Underwater photon flux density (PFD) , which was collected on a continuous basis from October 1993 to January 1995, showed strong seasonal differences ranging from 9.6 mol m’ 2 d 1 in April to 21.7 mol m' 2 d 1 in July (Fig. 1.1). The annual quantum flux at the seagrass canopy was 5382 mol m’ 2 yr 1 , which corresponded to 47% of surface irradiance (SI).

Density, biomass and leaf production

Shoot (sht) density and total biomass showed significant seasonal variations (P<o.ool and P=0.019, respectively); values ranged from 321 shts m’ 2 (454 g dry wt m’ 2 ) in March to 531 shts m’ 2 (885 g dry wt m' 2 ) in September (Fig. 1.2 A). Biomass of individual plant parts was significantly different (P<0.001; Fig. 1.2 B). Averaged on an annual basis, rhizome tissues had the highest biomass (249 g dry wt m’ 2 ) while root tissues had the lowest (72 g dry wt m’ 2 ). During summer, however, leaf biomass was higher than rhizome biomass. Leaf and root biomass also showed significant seasonal variation (P<o.ool, and P=0.0026, respectively), but rhizomes and short stems did not (P=0.777 and P=0.418, respectively). Leaf biomass was highest in September (355 g dry wt m' 2 ) and lowest in early April (81 g dry wt m' 2 ), while root biomass was highest in September (103 g dry wt nf 2 ) and lowest in late February (43 g dry wt m’ 2 ). Seasonal changes in total biomass closely correlated with variations in leaf biomass which showed higher annual variability as compared to that of other plant parts (Fig. 1.2 A, B).

On an annual basis, rhizome tissues accounted for about 40% of total biomass, while root tissues accounted for about 12% of total biomass (Table 1.1). Biomass partitioning into leaf tissues significantly changed with season (P<0.001); leaf tissues accounted for 17% of total biomass in April and 41% in July. Biomass partitioning into rhizome tissues did not change with season (P=0.08).

Shoot production, as reflected by leaf growth (mg dry wt shf 1 d 1), and areal leaf production (g dry wt m’ 2 d 1) changed significantly with sampling time (P<0.001), increasing during spring and summer and decreasing during fall and winter to become nearly zero between December and February (Fig. 1.3). Leaf productivities were highest in July (12 mg dry wt sht 1 d 1 or 5.6 g dry wt m' 2 d ) and lowest in December (0.2 mg dry wt shf 1 d' 2 or 0.07 g dry wt m' 2 d 1). Leaf production per shoot and per area basis also showed a strong seasonal trend.

Chlorophyll

Total chlorophyll content from Thalassia testudinum leaf tissue changed significantly with sampling time (P=0.004), ranging from 8.3 mg chi g' 1 dry wt in June to 6.3 mg chi g' 1 dry wt in February (Fig. 1.4 A). Chlorophyll content increased during spring, remained constant until early winter and decreased later in the winter. Chi a.b ratios also changed significantly with sampling time (P<0.001), ranging from 2.9 in September to 3.2 in December (Fig. 1.4 B). In general, chi a.b ratios varied inversely with respect to total chlorophyll content, with highest values of total chlorophyll associated with higher proportions of chi b relative to chi a.

Soluble carbohydrate carbon content

Soluble carbohydrate carbon content after 0.1 N HCI hydrolysis varied significantly different with sampling date (P<0.001) and among different plant parts (P<0.001). Thalassia had highest soluble carbohydrate carbon content in mid-July and lowest in early-June (Fig. 1.5). On an annual basis, average soluble carbohydrate carbon content was highest in rhizome tissues (159 mg C g’ 1 dry wt) and lowest in leaf tissues (59 mg C g 1 dry wt). Rhizome and short stem carbohydrate carbon content increased rapidly during June and July, nearly doubling over a two month period. Levels decreased slightly during fall and decreased rapidly during winter and early spring. Soluble carbohydrate carbon content in leaves and roots showed little seasonal variation, with small increases during late spring and early summer, constant values in fall and early winter, and small decreases during late winter and early spring.

Soluble carbohydrate carbon estimated on an areal basis (g C m’ 2 ) also varied significantly with sampling time (P<0.001) and among different plant parts (P<0.001). Total seagrass soluble carbohydrate carbon was highest in September (115 g C m’ 2 ) and lowest in April (51 g C m’ 2 ). Soluble carbohydrate carbon in rhizome ranged from 25 g C m' 2 in April to 58 g C m' 2 in September, and accounted for about 50% of total seagrass soluble carbohydrate carbon throughout the year. Leaf carbohydrate carbon accounted for 8% of total seagrass carbohydrate carbon in April and 23% in June, while short stem and root carbohydrate carbon accounted for an average of 25% and 8%, respectively, of total seagrass carbohydrate carbon in the seagrass bed.



Fig. 1.1. Average daily irradiance, on a monthly basis, recorded at canopy level in a Thalassia testudinum bed in Corpus Christi Bay from October 1993 to January 1995. Values are x ± SE (n=2B to 31).



Fig. 1.2. Thalassia testudinum. Seasonal changes in density and total biomass (A) and biomass of different plant components (B). Values are x ± SE (n=4 to 8).



Fig. 1.3. Thalassiatestudinum. Seasonal changes in leaf production rates per shoot and areal leaf production rate from February 1994 to January 1995. Values are x ± SE (n=s to 16).



Fig. 1.4. Thalassiatestudinum. Seasonal changes in total chlorophyll concentration (A) and chi a.b ratio in blades of plants collected from January 1994 to March 1995. Values are x ± SE (N=6).



Fig. 1.5. Thalassia testudinum. Seasonal changes of soluble carbohydrate carbon content in leaf, short stem, rhizome and root tissues. Values are x ± SE (n=4 to 5).

Date % of total biomass Leaf Short stem Rhizome Root Feb. 1994 23.1 ± 1.9 25.3 ±2.1 43.1 ±1.5 8.5 ±0.8 April 1994 16.8 ± 1.6 30.3 ±3.5 39.5 ±3.9 13.4 ± 1.0 June 1994 33.8 ±3.0 14.7 ± 3.1 42.7 ± 1.6 8.8 ± 1.5 July 1994 41.3 ±3.3 18.1 ±0.9 32.5 ±3.9 8.1 ±0.3 Sept. 1994 40.4 ± 1.7 15.0 ± 1.7 32.8 ±3.9 11.7 ± 1.3 Nov. 1994 32.1 ±4.3 18.3 ± 1.7 37.6 ±2.3 12.1 ± 1.1 Dec. 1994 24.2 ± 2.6 19.0± 1.5 41.4 ± 1.8 15.4 ± 1.1 Mar. 1995 22.9 ±2.9 18.3 ± 1.2 44.2 ±4.4 14.6 ±2.6 Annual mean 29.3 ±3.2 19.9± 1.9 39.2 ± 1.6 11.6 ± 1.0

Table. 1. 1 Seasonal variation in biomass partitioning to different plant parts. Values are x ± SE (n=4)

Discussion

Biomass and production

Thalassia testudinum in Corpus Christi Bay exhibited strong seasonal variations in density, biomass and leaf productivity, increasing during spring and summer and decreasing during fall and winter. Similar trends in seagrass standing crop and production have been reported for Thalassia and other species earlier by several authors (Macauley et al., 1988; Dunton, 1990; Thom, 1990), and were attributed to changes in water temperature and insolation. Temperature has been considered as a major factor controlling seasonal growth (Tutin, 1942; Phillips et al., 1983) because of its significant effect on the biochemical process involved in photosynthesis and growth (Bulthuis, 1987). However, some researchers reported a poor correlation between water temperature and leaf productivity (Sand-Jensen and Borum, 1983). In this study, leaf productivities were closely correlated with water temperature changes throughout the year Productivity increased rapidly with increasing temperature during spring and summer and decreased rapidly with falling temperatures during fall and winter (Fig. 1.6 A). If plotted on a log scale, leaf productivity displayed an even stronger correlation with temperature Fig. 1.7), suggesting exponential changes in leaf productivity as a function of temperature. In contrast, with the exception of the late spring and summer period, no relationship was apparent between leaf productivity and underwater irradiance on an annual basis Fig. 1.6 B). For example, leaf productivities increased from 0.4 to 2.9 mg dry wt sht 1 d 1 during early spring (February to April 1994) when underwater irradiance decreased from 12 to 4 mol m’ 2 d l . Based on these data, water temperature appears to control leaf productivity during fall to earlyspring, while both water temperature and underwater irradiance play a role during late-spring and summer. Since early-spring and winter growth appears to be supported by stored reserves in the below-ground tissues, temperature is probably a more important factor controlling seasonal growth than insolation because of its significant role in plant metabolism (Bulthuis, 1987). Accordingly, underwater light reduction during the winter and early-spring may affect seagrass production less than during summer (Czerny and Dunton, 1995).

Barber and Behrens (1985) found that Thalassia testudinum exhibited greatest productivity between 23 and 31 °C, but above 31 °C, increases in respiration and decreases in photosynthesis resulted in lower growth rates. However, in this study leaf production increased continuously with increasing in situ temperature from 23° C to 29°C, and no high temperature inhibition effects were observed.

Leaf production reflected seasonal changes in leaf biomass, increasing during spring and summer and decreasing during fall and winter. On the other hand, rhizome biomass was constant throughout the year. Additionally, although root tissues exhibited significant seasonal variation, it accounts for small portion of total seagrass biomass and hence does not have a noticeable effect on total biomass. Therefore, seasonal changes in Thalassia total biomass occurred as a consequence of variations in leaf biomass. More metabolically-active plant tissues, namely the leaf (photosynthetic organ) and root (nutrient absorption site), showed significant seasonal variations, while the rhizome storage tissue did not.

To accurately estimate carbon balance, better understanding of belowground tissue dynamics is necessary, since below-ground tissues can constitute a significant portion of total plant biomass. However, few complete annual measurements of the Thalassia biomass divided into different plant parts have been reported; most research has been concerned with standing crop and conducted only for a short time period. Zieman (1975) reported that Thalassia testudinum leaf tissues accounted for 15 to 22% of total biomass and the remainder was root and rhizome. As a result of this study, however, the partitioning of biomass to aboveand below-ground tissues showed seasonal trends. During summer, leaf tissues

accounted for about 40% of total biomass, but only 15 to 20% during winter, while rhizome biomass composed approximately 40% of total biomass throughout the entire year. Therefore, these seasonal variations in biomass partitioning must be considered to accurately estimate an annual carbon balance.

Seagrass communities contribute significantly to the primary production of the shallow coastal ecosystems (Kentula and Mclntire, 1986; Roman and Able, 1988; Peduzzi and Hemdl, 1991). To estimate the role of seagrasses in the carbon budget of a coastal ecosystem, the annual primary production of seagrasses has been estimated (Roman and Able, 1988; Pergent and Pergent-Martini, 1991; Gallegos et al., 1993). In this study, an annual blade production of 792 g dry wt m’ 2 yr’ 1 was calculated by adding measurements of leaf productivity for the entire annual period. It must be noted that blade production did not include sheath production. Preliminary elemental analysis indicated that about 32% of total dry weight was attributed to carbon. Thus, the annual blade production of 792 g dry wt m’ 2 yr’ 1 is equivalent to 253 g C m’ 2 yr’ 1 . Assuming sheath and rhizome production account for 30 and 10% of total Thalassia production (Patriquin, 1973; Gallegos et al., 1993), the annual biomass production of Thalassia testudinum in this study area was estimated to be 1320 g dry wt m’ 2 yr’ 1 , equivalent to 422 g C m’ 2 yr’ 1 . This estimate is the low end of annual biomass production estimates of Thalassia in the Mexican Caribbean region, which range from 1500 to 4500 g dry wt m' 2 yr’ 1 (Gallegos,

1993).

Chlorophyll

Chlorophyll concentrations in seagrasses fluctuate with variations in temperature and light regime (Wiginton and McMillan, 1979; Dennison and Alberte, 1982, 1985; Macauley, 1988; Abai et al., 1994). Seagrasses typically respond to light stress by increasing chlorophyll content and decreasing chi a.b ratios (Wiginton and McMillan, 1979; Dennison and Alberte, 1982, 1985; Abai et al., 1994). However, we observed high chlorophyll concentrations and low chi a.b ratios during the period of high underwater irradiance. These measurements are not in agreement with seagrass photoadatation in response to underwater light stress, suggesting plants in this study area are not light limited.

Instead, the seasonal changes in total chlorophyll content and chi a.b ratios noted in this study are similar to those noted by Macauley et al. (1988), who noted that Thalassia blade chlorophyll concentrations were highest and chi a.b ratios lowest during summer and early-fall. These results provide evidence that variations in chlorophyll were probably related to water temperature and not to photoadaptative responses to changes in underwater irradiance.

Leaf productivity and rhizome soluble carbohydrate carbon content were closely correlated with chlorophyll content. The maximum chlorophyll concentration during June and July coincided with peaks of leaf productivity and rhizome carbohydrate carbon content. Photosynthesis was directly proportional to chlorophyll content in Posidonia (Drew, 1978) and the highest P max corresponded with a peak in total leaf chlorophyll in Halodule w rightii (Dunton and Tomasko, 1994). It is likely that increases in leaf productivity and rhizome carbohydrate carbon content are a consequence, at least in part, of increased photosynthesis in response to higher blade chlorophyll content.

Soluble carbohydrate carbon dynamics

Measurement of the distinct seasonal changes in soluble carbohydrate carbon content, as reported in this study, provides valuable information for modeling the whole plant carbon balance in Thalassia testudinum. Many researchers have calculated whole plant carbon balance based on estimates from laboratory measured photosynthesis vs irradiance (P vs. I) curves and the respiration rates of different plant parts to understand seagrass growth and survival under different environmental conditions (Zimmermann et al., 1989; Fourqurean and Zieman, 1991; Zimmermann et al., 1991). However, laboratory measurements of seagrass photosynthesis with leaf segments cannot always be extrapolated into the field (Dunton and Tomasko, 1994). In experiments with Zostera marina, Zimmermann et al. (1989) noted that respiration rates calculated from the incubation of root and rhizome tissues in the lab vary as a function of oxygen concentration. Consequently, total plant respiration can be overestimated since rhizome and root tissues are usually located in anoxic sediments, not in a well ventilated water column. Recent experiments comparing the respiration rates of whole Thalassia seedlings with that of separated above- and below-ground tissues have also shown that cut plants have much higher respiration rates, possibly because of wounding effects (Kaldy, unpubl.).

Seagrass rhizome tissues usually act as photosynthate storage tissue (Dawes and Lawrence, 1979; Durako and Moffler, 1985; Pirc, 1985, 1989). Stored carbon can be used to meet respiratory demands and can contribute to plant growth when carbon demands exceed photosynthesis. Photosynthetic carbon accumulates as soluble carbohydrate in the rhizome tissues when photosynthesis exceeds the carbon demands from respiration and growth. Therefore, the increase in rhizome carbohydrate carbon content indicates a positive whole plant carbon balance against respiration and growth, while a decreased rhizome soluble carbohydrate carbon indicates a negative carbon balance. In this study, rhizome carbohydrate carbon content increased dramatically during June and July, indicating periods of positive carbon balance. This rapid increase in rhizome carbohydrate carbon content coincided with high water temperatures and underwater irradiance, as well as high chlorophyll concentrations, low chi a.b ratios and high leaf biomass. Since the plants experienced optimal environmental and biological conditions for high photosynthetic rates, there was a net storage of soluble carbohydrate carbon in the rhizome tissues. During winter and spring, the plants showed a negative whole plant carbon balance as rhizome carbohydrate carbon content decreased rapidly. The rapid decrease of carbohydrate carbon contents during spring were probably due to spring leaf growth (Dawes and Lawrence, 1979; Durako and Moffler, 1985; Pirc, 1985, 1989). However, during fall the whole plant carbon balance was balanced suggesting that soluble carbohydrate carbon reserves were being maintained. Tissues not used for storage of carbon, particularly leaf and root material, did not show a strong seasonal trends in carbohydrate carbon content.

In summary, shoot densities, biomass and leaf production of Thalassia testudinum in Corpus Christi Bay showed significant seasonal trends and these correlated closely with water temperature and partially with underwater irradiance on an annual basis. Partitioning of seagrass biomass into different plant parts also changed seasonally; leaf tissues accounted for 40% of total biomass during the intensive growing season, but only 15 to 20% during the winter. The estimated annual biomass production of Thalassia testudinum in Corpus Christi Bay was 1320 g dry wt m' 2 yr' 1 . Positive whole-plant carbon balance occurred during June and July and coincided with optimum environmental (high temperature and underwater irradiance) and biological (high chlorophyll content and leaf biomass) conditions.



Fig. 1.6. Thalassiatestudinum. Correlations between leaf productivity and water temperature (A) and underwater irradiance (B) from February 1994 to January 1995.



Fig. 1.7. Regression between leaf productivity plotted on a log scale and temperature.

CHAPTER 2: Effects of in situ light reduction on the maintenance, growth and partitioning of carbon resources in Thalassia testudinum

Abstract

The effects of in situ light reduction on the subtropical seagrass, Thalassia testudinum in Corpus Christi Bay, Texas were examined from April 1993 to August 1994. The annual quantum flux at the seagrass canopy level was 5207 mol m’ 2 yr 1 or 46% of surface irradiance (SI) compared with two manipulated treatments that reduced underwater light to 1628 mol m' 2 yr 1 (14% SI) and 864 mol m' 2 yr’ 1 (5% SI). Shoot densities in the control cages (46% SI) were 785 shoots m' 2 in August 1993 and 457 shoots m’ 2 in July 1994. All plants subjected to 5% SI died after 200 days and over 99% of plants receiving 14% SI died by the end of the experiment (490 days). Blade widths of plants in the controls ranged from 6.4 to 7.0 mm, and decreased to 4.7 mm as a result of light reduction. Leaf production rates were significantly higher in control plants compared to plants within the 14% and 5% SI treatments, with all plants showing a seasonal trend with high productivity in July and low productivity in April. Blade chlorophyll concentrations increased, while the chi a : b ratio decreased with reduced light level. Soluble carbohydrate carbon content of controls were highest in rhizomes (102-152 mg Cg 1 dry wt), and were relatively low in leaves (50-66 mg Cg 1 dry wt) and in roots (57-74 mg C g' 1 dry wt). In both light treatments rhizome carbohydrate carbon content was 50% lower and leaf carbohydrate carbon content was about 15% lower than controls, while the root carbon content did not differ significantly between treatments and controls. Pore water ammonium and sulfide concentrations in the shaded cages were significantly higher than in control cages. We conclude that indices of shoot density, blade width, leaf growth, chi a : b ratio and blade chlorophyll content may be important indicators of chronic underwater light stress in Thalassia testudinum. A quantum flux of 1628 mol m' 2 yr 1 (14% SI) was insufficient to maintain a positive carbon balance of Thalassia testudinum in this bay system.

Introduction

There have been significant declines in seagrass coverage in many parts of the world related to reduced water quality and increased turbidity (Orth and Moore, 1983; Cambridge and McComb, 1984; Geisen et al., 1990; Larkum and West, 1990). The primary causes for increased turbidity are erosion of silt substrates, pollution, algal growth, ship and barge traffic and dredging activities (Cambridge et al., 1986; Peres and Picard, 1975; Onuf, 1994). Although seagrass growth and survival is largely related to light availability, there is a considerable amount of variability among species. Duarte (1991) reported an average minimum light requirement of 10.8% of surface irradiance (SI) for seagrasses from a worldwide survey of their maximum colonization depth. However, Dennison et al. (1993) reported that the estimated minimum light requirements for various seagrass species probably ranges from 4% to 25% SI . These variations are probably a result of the unique physiological and morphological adaptations among species and locations. Photoadaptive responses of seagrasses to reductions in irradiance have been reflected in some species by increases in chlorophyll (chi) content and decreases in biomass, growth rate, shoot density and chi a : b ratios (Backman and Barilotti, 1976; Wiginton and McMillan, 1979; Dennison and Alberte, 1982,1985, 1986; Bay, 1984; Neverauskas, 1988; Tomasko and Dawes, 1989; Abai et al., 1994).

However, despite the recognized importance of reduced irradiance in controlling the distribution and abundance of seagrasses, there are few studies that have detailed the physiological responses of these plants to measured decreases in underwater irradiance. The carbon balance of seagrasses is more complex than that of phytoplankton or macroalgae due to the structural complexity of seagrasses (Fourqurean and Zieman, 1991). Root/shoot ratios are very important to seagrass carbon budgets because below-ground non-photosynthetic tissues must be supported by photosynthetic carbon production in the leaves. The below-ground portion of Thalassia testudinum can account for over 50% of the total biomass (Powell et al., 1989; Fourqurean and Zieman, 1991). Below-ground tissue is generally a photosynthate reservoir that supports growth and maintenance of other tissues during periods of low photosynthetic production (Dawes and Lawrence, 1980; Pirc, 1985).

The photosynthate produced in leaf tissues and transported to subterranean tissues is critical in processes involving new shoot growth, carbohydrate storage and respiration (Ralph et al., 1992). Pirc (1985) found that winter leaf growth in Posidonia oceanica was supported by mobilization of starch in the rhizome, resulting in seasonal changes in carbohydrate levels. In Thalassia testudinum, an increase in rhizome carbohydrates during summer were due to the production and storage of starch, while winter and spring decreases were attributed to the utilization of stored carbohydrates for growth and respiration (Dawes and Lawrence, 1980).

Oxygen to support aerobic metabolism in seagrass roots is derived from shoot photosynthesis through the lacunal system, allowing roots to grow in an anoxic environment (Smith et al., 1984; Caffrey and Kemp, 1991). In addition, roots of submerged vascular macrophytes secrete photosynthetically produced oxygen into the sediments, creating an oxidized zone around the roots where sulfides are oxidized, reducing potential toxicity (Thursby, 1984; Flessa, 1994). Sulfides suppress the activity of alcohol dehydrogenase (ADH), the enzyme that catalyzes anaerobic fermentation, an important pathway in anoxic metabolism (Koch et al., 1990). Significant reductions in photosynthesis can induce anaerobic metabolism in below-ground tissues and result in the accumulation of toxic levels of sulfides. Reduced oxygen transport into below-ground tissues and accumulated sulfides inhibit aerobic and anaerobic respiration respectively, and consequently, nutrient uptake and growth diminishes (Smith et al., 1988; Pregnall et al., 1987; Koch et al., 1990).

Thalassia testudinum is one of the most important seagrass species along the coasts of the Caribbean and the Gulf of Mexico. Thalassia testudinum consists of horizontal rhizomes which branch at regular intervals, and erect short shoots (vertical rhizome) bearing foliage leaves and roots (Tomlinson and Vargo, 1966). This species constructs very dense rhizome systems and has differentiated vertical rhizome tissue (Duarte et al., 1994). In this study, I examined changes in leaf elongation, biomass, carbohydrate carbon content, blade width, chlorophyll content and chi a : b ratios in response to in situ light manipulations in Thalassia testudinum. I also investigated changes in biomass and carbohydrate carbon partitioning to different plant parts as a result of changes in underwater irradiance to determine the effect of light reduction on the patitioning of carbon in Thalassia testudinum. Continuous measurements of underwater quantum irradiance were made to document the amount of light received by plants in each shaded treatment. I also monitored changes in pore water ammonium and sulfide levels to assess sediment anoxia.

Materials and Methods

Study Site

The study site, East Flats, is located in eastern side of Corpus Christi Bay (27° 49' N, 97° 7' W). This site has been the focus of several recent investigations on south Texas seagrasses (Czerny and Dunton, 1995; Dunton, 1990, 1994). Thalassia testudinum, Halodule wrightii and Syringodium filiforme are the dominant seagrass species in this area. This study was conducted on a monotypic meadow of Thalassia testudinum with an average water depth of 1.2 m. Water temperature ranged from 34°C in July and August to 13°C in January, while salinity varied from 27 to 32 %o.

In situ Light Manipulation

Light shading cages (1.5 m x 1.5 m x 0.5 m) were placed in a monotypic meadow of Thalassia testudinum to achieve artificial in situ light reduction. Coarse mesh (1.91 x 1.91 cm) reduced irradiance to 14% of surface irradiance (SI) while fine mesh (0.64 x 0.64 cm) reduced irradiance to 5% SI. Cages without screen were placed on the seagrass bed as controls. Three replicated cages for each treatment were located randomly. The shading mesh was replaced every one or two weeks to minimize the effects of fouling on light transmissivity. The perimeter of each cage was cut to a sediment depth of about 30 cm to physiologically isolate plants located within and outside of the cages. Shading was initiated in late April 1993 and terminated in August 1994. The experiment lasted a total of 490 days. No data exist for fine mesh cages (5% SI) after November 1993, since all plants within these cages died by that date.

Photon Flux Measurement

Photosynthetically active radiation (PAR, 400-700 nm) was collected continuously using a LI-193 SA spherical quantum sensor at canopy level, which provided input to a LI-1000 datalogger (LI-COR Inc.) enclosed in underwater housing. An underwater sensor was placed within one replicate of each of the three treatments and cleaned regularly to minimize fouling. Photon flux was measured at 1-min intervals and integrated hourly. Coincident measurements of incident surface PAR were made at The University of Texas Marine Science Institute (UTMSI) in Port Aransas, approximately 8 km from the study site, using a LI-190S A cosine corrected quantum sensor and LI-1000 datalogger.

Biological Measurements

Quarterly measurements of plant density, biomass, leaf chlorophyll content, blade width and leaf elongation rates were made in the experimental cages. For biomass, three replicate samples from each cage were collected with a 9 cm diameter coring device. Samples were thoroughly cleaned of epiphytes and sediments, separated into leaf (blade and sheath), short stem(including vertical rhizome), rhizome and root and dried at 60°C to a constant weight. Shoot density was estimated by counting the number of shoots inside a randomly thrown quadrat (0.05 m 2).

For determination of blade chlorophyll content, six replicate samples from each cage were collected and then cleaned of epiphytes in the laboratory. Preweighed leaf tissues were extracted for 4-5 days in glass screw cap tubes with 5 ml N,N-dimethyl formamide (DMF) following Dunton and Tomasko (1994). Absorbance of the extracts was measured at 750, 664 and 647 nm on a Shimadzu UV 160 U spectrophotometer. Chi a and b contents were determined using the equations of Porra et al. (1989).

Leaf production rates were obtained using the blade marking technique (Zieman, 1974). Twelve randomly chosen shoots from each cage were marked just above the bundle sheath with a hypodermic needle and collected after about 2 weeks. Leaf production rate per shoot was determined by dividing the dry weight of new leaf tissue produced after marking by the number of shoots and days since marking. Areal leaf production rates were obtained by multiplying the leaf production rate per shoot by the shoot density.

Chemical Analyses

On each sampling date four replicate sediment samples were collected to 10 cm depth from each shading treatment cage with a6O ml syringe. Sediment pore water was obtained by centrifugation (5,000 xg for 15 min) and then diluted (1 : 5) with ammonium free seawater. Concentrations of NH 4 + were determined using standard colorimetric techniques following the alternative method of Parsons et al. (1984).

Sediment pore water samples to determine sulfide concentration were collected with a pore water sampler under anaerobic conditions (Zimmermann et al., 1978) at the end of the experiment (August 1994). Samplers filled with nitrogen gas were inserted in the sediment and pore water surrounding the porous polyethylene frit was collected by the vacuum created with a 50 ml syringe. Dissolved sulfide content of pore water was determined colorimetrically according to Cline (1969). A 5-ml pore water sample was transferred to a test tube, to which 0.4 ml of the mixed diamine reagent was added under a nitrogen atmosphere. Color development was allowed to proceed in the dark for 30 min, after which the absorbance was determined spectrophotometrically at 670 nm. Dilutions were made after color development with distilled water. The concentrations of sulfide in the samples were calculated by standardization with known sulfide concentrations.

Dried plant materials from biomass samples were used to determine carbohydrate carbon content in different plant parts. Soluble carbohydrates from leaf, horizontal rhizome, vertical rhizome and root were determined using the MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) analysis (Parson et al., 1984, Pakulski and Benner, 1992). Ground plant samples of 10 mg were hydrolyzed with 10 ml of 0.1 N HCI for 24 h at 100°C in a water bath to determine soluble carbohydrates. For determination of total carbohydrates, a hydrolysis using 12 M H 2 SO 4 was conducted. The hydrolyzed samples were neutralized with 2 ml of 0.5 N NaOH, and 0.1 ml of the sample was diluted with 10 ml of persulfate distilled water in a serum vial. This sample was reduced with 0.25 ml of 10% KBH 4 for at least 4 hours in the dark, and acidified with 1 ml of 2N HCI to allow hydrogen gas to evolve. Triplicate 1 ml aliquots of hydrolysate from each sample serum vial were transferred to acid-washed and combusted (500°C, 4 hours) screw cap test tubes. Two additional 1 ml aliquots of hydrolysate were transferred to serve as blanks. Periodic acid solution (0.1 ml) was added to each of the three sample tubes and incubated for 10 min in the dark at room temperature. Sodium arsenite solution (0.1 ml) was added to each sample tube in order to stop the oxidation reaction. For analytical blanks, 0.2 ml of the sodium arsenite and periodic acid mixture was pipetted into each of the two additional 1 ml of hydrolysate serving as blanks. The triplicate samples and duplicate blanks were acidified with 0.2 ml of 2 N HCI. Freshly prepared 0.2 ml of MBTH solution was added to both samples and blanks, after which the tightly-capped tubes were incubated for 3 min in a boiling water bath. The tubes were cooled to room temperature with tap water. Once cooled, 0.2 ml of ferric chloride solution was added to the tubes, followed by a 30 min incubation at room temperature in the dark for color development. After color development 1 ml of acetone was added to each tube and absorbances were measured immediately at 635 nm with a spectrophotometer. Mean corrected absorbances calculated by subtracting analytical blanks were compared with a glucose standard and converted to equivalent carbon values.

Statistics

All values are reported as means ± 1 SE. Statistical analyses were performed on a microcomputer using a general linear model procedure (SAS Institute, 1989). Significant differences in underwater irradiances, chlorophyll content, chi a : b ratios, leaf elongation rate, leaf width and pore water ammonium concentrations among different shading treatments and sampling times were tested using a two-way ANOVA using time as a block. A three-way ANOVA using a split-plot design with time as a block was used to test significant differences in biomass and carbohydrate carbon content among light treatments, sampling times and plant parts. 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 treatments. Because data from 5% SI cages was not available, a r-test was used to test for significant differences in pore water sulfide concentrations between controls and 14% SI treatment cages.

Results

Underwater Irradiance

The annual quantum flux at the surface was 12983 mol m’ 2 yr’ 1 , and ranged from an average of 55.7 in July to 18.4 mol m' 2 day’ 1 in December 1993 (Fig 2.1). The annual quantum flux at the seagrass canopy was 5207 mol m’ 2 yr’ 1 , which corresponded to 46% SI. Two light manipulation treatments using coarse and fine mesh significantly reduced (P<0.001) underwater irradiance to 1628 (14% SI) and 864 mol m’ 2 yr’ 1 (5% SI) respectively. In control cages, underwater photon flux density (PFD) ranged from 9 to 22 mol m’ 2 day’ 1 (average 14.3 mol m’ 2 day’ 1 ). Average PFD in the cages shaded with coarse and fine mesh was 4.5 and 2.4 mol m^d’ 1 respectively (Table 2.1). Unlike surface measurements of PAR, underwater PFD did not exhibit a seasonal sigmoidal curve.

Pore water Ammonium and Sulfide

Sediment pore water ammonium concentrations were measured three times

(September 1993 and April and July 1994) during the course of this study. Pore water ammonium concentrations in 14% and 5% SI cages were significantly (P<0.001) higher than controls (46% SI). Pore water ammonium concentrations for control cages ranged from 62 /zM NH 4 in April 1994 to 101 /zM NH 4 in July 1994. The concentrations in 14% SI treatment cages ranged from 141 /zM NH 4 in April 1994 to 179 /zM NH 4 in July 1994 (Fig. 2.2 A). There was no significant difference in pore water ammonium concentrations between sites receiving 14% SI and 5% SI in September 1993, the time at which both treatment cages were measured.

Pore water sulfide concentrations of the control and 14% SI cages in August 1994 were 107 //M and 179 //M sulfide, respectively. The concentration of sulfides in the shaded cages was significantly (P=0.01) higher than in the controls.

Shoot Density and Blade Width

Shoot densities in control cages (46% SI) ranged from 457 to 785 m‘ 2 . Shoot densities in 14% and 5% SI cages were significantly (P<0.001) lower than controls throughout the experiment (Fig. 2.28). In August 1993, after 116 days shading, shoot densities in the various treatments were 785 m‘ 2 (control), 296 m' 2 (14% SI), and 168 m' 2 (5% SI). All plants exposed to 5% SI died after 200 days of shading treatment and over 99% of plants receiving 14% SI died by the end of the experiment (490 days).

Blade widths in Thalassia testudinum decreased significantly (P<0.001) as a result of light reduction. Blade width decreased more rapidly in plants at 5% SI than those at 14% SI. Blade widths of plants receiving 5% SI were 6.0 mm after 36 days of shading (May 1993) and 4.7 mm after 128 days of shading (August 1993) compared to plants at 14% SI, which were 6.6 mm in May 1993, but decreased to 4.8 mm in April and July 1994 (Fig. 2.3). Blade widths of control plants ranged from 6.4 to 7.0 mm during the entire period.

Chlorophyll Content

Total chlorophyll (chi a + chi b) from control plants ranged from 5.0 mg chi g' 1 dry wt in July 1993 to 6.7 mg chi g 1 dry wt in July 1994. Total blade chlorophyll and chi b content increased significantly (P=0.019 and P<o.ool respectively) with decreased levels of PAR. Chi a levels also showed an increasing trend with light reduction, but it was not statistically significant (P=0.11). Blade chlorophyll concentrations in 5% SI plants ranged from 5.4 mg chi g 1 dry wt in September 1993 to 6.5 mg chi g' 1 dry wt in July 1993, while chlorophyll concentrations were lowest (6.0 mg chi g 1 dry wt) in July 1993 and highest (8.3 mg chi g’ 1 dry wt) in July 1994 for plants at 14% SI (Fig. 2.4).

The chi a : b ratios of blades from control cages ranged from 2.7 in September 1993 to 3.4 in July 1994. Chi a : b ratios of blade tissue decreased significantly (P<0.001) as a result of the two light reduction treatments. The ratios were highest in the plants receiving 46% SI and lowest in the plants receiving 5% SI. Chi a: b ratios of 14% SI plants was highest (2.7) in April 1994 and lowest (2.1) in July 1994, while plants at 5% SI showed chi a : b ratios of 2.4 in July 1993 and 2.5 in September 1993 (Table 2.2).

Leaf Production Rates

Leaf production rates were highest during summer and lowest during the cooler months (Fig. 2.5), ranging from 1.5 mg shoot 1 d 1 (0.7 g m' 2 d 1) in April 1994 to 5.0 mg shoot' 1 d' 1 (2.4 g m' 2 d' 1 ) in July 1994 in control plants. Leaf production rate per shoot (mg shoot' 1 d 1) and areal leaf production rate (g m' 2 d' 1 ) decreased significantly (P<0.001) with shading. In May and August 1993, leaf productivities of plants receiving 14% SI were 3.8 and 2.5 mg shoot' 1 d' 1 , compared to plants at 5% SI, which were 1.9 and 1.2 mg shoot' 1 d' 1 . Areal leaf productivity at 5 and 14% SI dropped to nearly zero after about one year of shading as a result of extremely low shoot densities.

Biomass

Biomass decreased significantly (P<0.001) with light reduction. In August 1993, 116 days after shading, the biomass in cages at 14% SI decreased to less than half that of controls and was less than a third of control biomass within the 5% SI treatments (Table 2.3). All plants receiving 5% SI died by November 1993, after 200 days of reduced PAR. The biomass of the plants receiving 14% SI decreased to 20% of control biomass by April 1994 (after 345 days of shading), and to 1.4% of control biomass by July 1994 (after 457 days of shading).

Relative to controls, leaf biomass decreased more rapidly than biomass of below-ground tissues under light reduction. In August 1993, after 116 days of shading, the leaf biomass of plants at 5% SI decreased 95% compared to a corresponding drop of 50% in below-ground biomass. After loss of leaf material, root biomass decreased rapidly. In April 1994, after 345 days of shading, root biomass decreased 90% at 14% SI, while short stem and rhizomes maintained 20- 30% of their biomass relative to controls. Although rhizome material was the most durable plant part, biomass of this component dropped 98% by the end of experiment.

Below/above-ground ratios changed significantly with season (P=0.0015). The ratio was lowest (1.3) in August and highest (5.8) in April (Table 2.4). Percentage of leaf (above-ground) biomass as a function of total biomass was highest in August 1993, while that of rhizome was highest in April 1994. The percentages of short stem and root biomass were fairly constant. Leaf biomass accounted for 45% of total biomass in August, while accounting for only 17% in April. Rhizome biomass was 20-40% of total biomass in July and August and about 50% of total biomass in April (Fig. 2.6).

Below/above-ground ratios significantly (p<0.001) increased with light reduction (Table 2.4). In August 1993 the ratio of plants receiving 46% SI was 1.3 while those of plants receiving 14% and 5% SI were 3.0 and 14.7, respectively. Percentage of leaf biomass decreased while that of rhizome increased with light reduction (Fig. 2.6).

Carbohydrate Carbon

Soluble carbohydrate carbon content of plants at 46% SI was highest in rhizomes (130-136 mg carbon g 1 dry wt) and in short stem (102-152 mg Cg 1 dry wt), and relatively low in leaf (50-66 mg Cg 1 dry wt) and in root tissue (57-74 mg C g' 1 dry wt) (Fig. 2.7). Shading treatments significantly (P<0.001) lowered the soluble carbohydrate carbon content of leaf, rhizomes and short stem. However, the content of root tissue did not change significantly (P=0.53) with light reduction. Soluble carbohydrate levels in rhizomes and short stem decreased more rapidly with reduced light than that of leaf material. In both shading treatments rhizome carbohydrate carbon content was 50% lower and leaf carbon content was about 15% lower than controls. In April 1994, total carbohydrate carbon content in control plants ranged from 107 mg Cg 1 dry wt in leaf to 158 mg C g' 1 dry wt in rhizome tissues (Table 2.5). Total carbohydrate carbon content decreased to 91 mg Cg 1 dry wt in leaf and 127 mg C g' 1 dry wt in rhizome tissues with light reduction. Structural carbohydrate carbon content was estimated by subtraction of soluble carbohydrate carbon content from total carbohydrate carbon content. In leaf tissues of control plants, about 50% of total carbohydrates was attributed to structural carbohydrate, while only 20% in rhizome tissues was structural. Structural carbohydrate carbon content in plant tissues did not decrease with light reduction.



Fig. 2.1. Average daily photon flux density (PFD) collected underwater (control, 14% SI and 5% SI treatment cages) and at the surface (The University of Texas Marine Science Institute in Port Aransas). Data collection at the 5% SI treatment was terminated in November 1993 following the death of all plants in these cages.



Fig. 2.2. Pore water ammonium concentration in sediments (A) and shoot densities (B) of control, 14% and 5% SI treatment cages.



Fig. 2.3. Blade widths of Thalassia testudinum from control, 14% SI and 5% SI treatment cages.



Fig. 2.4. Chlorophyll a, chlorophyll b and total (chi a+b) concentrations of Thalassia testudinum leaves from control, 14% SI and 5% SI treatment cages.



Fig. 2.5. Daily leaf production on a shoot (A) and areal (B) basis in control and treatment cages.



Table 2.3. Biomass changes in total and individual plant parts as a result of light manipulation in May (initial sampling date) and August 1993 and April and July 1994. No plants survived in the 5% SI treatment cages after November 1993. Values are x ±SE (n=3). nd: no data.



Fig. 2.6. Changes of biomass partitioning of Thalassia testudinum into different plant parts (leaf, short stem, rhizome and roots) as a result of light manipulation between August 1993 and July 1994. Circle area corresponds with total plant biomass listed for each site/date combination.



Fig. 2.7. Carbohydrate carbon concentration in different plant tissues of Thalassia testudinum from control and light treatment cages in August and December 1993 and April 1994.

Control (in situ ambient) Light Manipulation Coarse mesh Fine mesh Average PFD (mol photons m‘ 2 day 1 ) 14.27 4.46 2.37 % ISA 100 31.4 16.1 % SI 46.4 14.3 5.4 H sal (h) 8.5 3.3 0.9 Total irradiance (mol m' 2 yr' 1 ) 5207 1628 864

Table 2.1. Daily average photon flux density (PFD), % of in situ ambient (% ISA), % of surface irradiance (% SI) and the daily period of light saturated photosynthesis (H sat ) in control and light manipulation cages. H sat values based on a saturation irradiance of 140 pmol m' 2 s' 1 for Thalassia testudinum (Dunton, unpub. data)

Sampling Date Chi. a:b ratio Control 14% SI 5% SI July 1993 2.74 ± 0.09 2.51 ± 0.08 2.37 ± 0.08 Sept. 1993 2.72 ± 0.05 2.52 ± 0.06 2.48 ± 0.06 April 1994 2.73 ± 0.07 2.67 ± 0.05 nd July 1994 3.44 ± 0.10 2.10 ± 0.07 nd

Table 2.2. Chlorophyll a.b ratio of Thalassia testudinum leaves from control, 14% SI and 5% SI treatment cages at four different sampling times. Values are x ± SE (n=3). nd: no data

Sampling Date Below/above-ground ratio Control 14% SI 5% SI August 1993 1.3 ± 0.2 3.0 ± 0.6 14.7 ± 6.0 April 1994 5.8 ± 0.9 12.4 ± 2.9 nd July 1994 2.1 ± 0.2 14.0 ± 3.9 nd

Table 2.4. Below- to above-ground ratios of Thalassia testudinum at 46% SI (control), 14% SI and 5% SI in August 1993 and April and July 1994. Values are x ± SE (n=3). nd: no data

Carbohydrate carbon (mg C gdw 1 ) Control cage 14% SI Leaf Rhizome Short stem Root Leaf Rhizome Short stem Root Total 107.0 ±4.8 157.5 ± 4.4 115.2 ±4.4 107.4 ± 2.0 91.0 ±4.5 127.1 ± 3.2 90.4 ± 3.3 117.1 ±2.1 Soluble 49.7 ± 1.1 130.5 ± 6.1 101.6 ± 5.4 70.9 ± 1.4 45.0 ± 1.6 64.7 ± 1.9 56.2 ± 1.8 67.1 ± 1.3 Structural 57.4 ±4.2 31.1 ± 10.6 22.3 ± 35.6 ± 3.5 46.5 ± 3.4 64.7 ± 3.0 36.0 ± 1.2 51.7 ± 2.1 4.8

Table 2.5. Total, soluble and structural carbohydrate carbon content of different plant tissues of Thalassia testudinum from control and 14% SI cages in April 1994.

Discussion

In situ light requirements of Thalassia testudinum

Light reduction resulted in decreases in shoot density and biomass in Thalassia testudinum. Plants in the control cages (46% SI) remained healthy throughout the experiment; in contrast, all plants at 5% SI died within 7 months, and most shoots at 14% SI died after 16 months. Czerny and Dunton (1995) also demonstrated that Thalassia testudinum did not tolerate a light reduction equivalent to 14% SI. This finding is consistent with the minimum light requirements (15-25% SI) reported by Dennison et al. (1993) for Thalassia testudinum from Florida and the Caribbean. Further long-term measurements of in situ PAR in Thalassia testudinum beds at variety of depths is needed to establish the minimum light requirements for Texas plants as has been done for Halodule wrightii (Dunton, 1994).

Thalassia testudinum showed various morphological and physiological adaptations in response to changes in underwater light availability. Seagrasses can respond to light reduction by increasing chlorophyll content and decreasing their chi a : b ratio (Wiginton and McMillan, 1979; Dennison and Alberte, 1982, 1985; Abai et al., 1994). We found agreement with these trends as shown by increases in chlorophyll concentrations and decreases in chi a : b ratios in response to light reduction, although some of these changes were not statistically significant. Wiginton and McMillan (1979) reported that chi a : b ratios were correlated with depth distribution of seagrasses; additionally, they suggested that the chi a : b ratios controlled distributional differences among species. Seagrass occurring in deep areas had low chi a : b ratios, but seagrass occurring at shallow depths had a higher ratio. They suggested that differences in chi a : b ratios were a response to reduced PFD at depth, and not to changes in light quality. However, measurements of underwater spectral irradiance (Weidemann and Bannister, 1986; McPherson and Miller, 1987) indicated that the wavelengths absorbed by chi a decreased more rapidly than the wavelengths absorbed by chi b with increasing water depth. Thus, although plants receiving less light may increase their total chlorophyll concentration to increase light absorption efficiency, rapid increases in chi b relative to chi a would allow more efficient use of the more abundant wavelengths at depth.

Seagrass blade width has been correlated with environmental factors that are ultimately related to underwater light regimes as noted by several investigators (McMillan, 1978; McMillan and Phillips, 1979; Phillips and Lewis, 1983). For example, McMillan (1978) noted that Thalassia populations from turbid bays were characterized by having narrower leaves compared to plants in clear water. Phillips and Lewis (1983) found that Thalassia blade width decreased with increasing depth, and suggested that light attenuation was the causal factor. Our results indicate that decreased light availability has a significant effect on blade width, which decreased about 2 mm (to 4.7 mm) as a function of light reduction and shading duration. Since attainment of the minimum 4.7 mm width was subsequently followed by plant death, decreases in blade width may be a convenient indicator of light stress in Thalassia testudinum.

Changes in biomass and carbon budget

Many studies suggest that whole plant carbon balance is a major factor determining the growth and distribution of seagrasses (Dennison and Alberte, 1982, 1985; Marsh et al., 1986; Zimmerman et al., 1989; Fourqurean and Zieman, 1991; Zimmerman et al., 1991). Carbon balance has often been estimated from rates of respiration and photosynthesis vs irradiance curves constructed for above-ground tissues only (Dennison and Alberte, 1985; Marsh et al., 1986; Zimmerman et al., 1991 ), but there are several other factors that must be considered including a knowledge of carbon and biomass partitioning into different plant parts, carbon metabolism of below-ground tissues, storage of photosynthate and root anoxia.

Leaf biomass from control cages accounted for almost 50% of total biomass of Thalassia testudinum during the warm growing season, and decreased more rapidly than biomass of below-ground tissues as a result of reduced light. Percent leaf biomass as a function of total biomass decreased from 45% to 9.3% in plants at 5% SI during the first four months. Decreased leaf biomass in the shaded cages is probably a product of defoliation and decreased leaf growth. Defoliation is a normal response of terrestrial and submerged plants to reduced light levels (Addicott and Lyon, 1973; Backman and Barilotti, 1976; Neverauskas, 1988), but defoliation during the active growing season may seriously impact seagrass survival through decreases in the production and transport of oxygen to below-ground tissues.

Root biomass decreased more rapidly than rhizome biomass with light reduction. Plants receiving 14% SI for 345 days lost about 90% of their root biomass, while rhizomes still maintained 20-30% of their biomass relative to controls. Results from a litter bag decomposition experiment indicate that Thalassia rhizome is more resistant to decay than root tissues (Kenworthy and Thayer, 1984), which is in agreement with our findings. The increase in the decomposition of below-ground material under low light conditions may also contribute to increases in pore water ammonium and sulfide levels.

The below-ground tissues of seagrasses generally exist in an anoxic environment (Penhale and Wetzel, 1983). In addition to lack of oxygen for aerobic respiration of below-ground tissues, sulfide is produced in anaerobic sediments by bacteria using sulfate as a terminal electron acceptor (Sorensen et al., 1979). Sulfide inhibits respiration, oxygen release and nutrient uptake by plant roots (Bagarinao, 1992). In Florida Bay, pore water sulfide concentrations were considerably higher in die-off areas than in healthy Thalassia beds (Carlson et al., 1994) suggesting that sulfide toxicity may play a role in the loss of seagrass. Photosynthetically produced oxygen is secreted into sediment through roots (Smith et al., 1984), supporting aerobic metabolism and creating an oxidized zone around the roots where pore water sulfide and ammonium oxidation can occur. In this study, the concentration of pore water ammonium and sulfide in shaded cages were significantly higher than that from control cages. This suggests that below-ground tissues in shaded cages were exposed to anoxic conditions more frequently than controls. Thalassia testudinum is more vulnerable to anoxia than other seagrass species because of the relatively high ratio of below-ground biomass to aboveground biomass and its deep rooted growth habit.

Soluble carbohydrate carbon content was highest in rhizome tissues and likely serves as an energy reserve for plant sustenance during the winter period (Dawes and Lawrence, 1980; Durako and Moffler, 1985). In this study levels of soluble carbohydrates decreased significantly in all plant tissues (except roots) with reduced light; however, different plant parts showed distinctive decreasing patterns. Soluble carbohydrate levels in rhizomes and short stems decreased to half that of control plants with light reduction, while levels decreased slightly in blades but remained constant in roots. However, structural carbohydrate levels in plant tissues did not decrease with light reduction. Stored carbohydrate in rhizome tissues can be used to meet the respiratory demands of the plant and can contribute to new growth in above-ground tissues during periods of low photosynthetic production (Dawes and Lawrence, 1979, 1980; Pirc, 1985; Dawes and Guiry, 1992). In addition, when below-ground tissues respire anaerobically, carbon demand increases to meet the metabolic requirements of plants, further decreasing carbohydrate reserves. The demands on stored carbon reserves depleted carbohydrates in rhizome tissues to the levels equivalent to that in the leaves and roots, and consequently the plants were not capable of providing reduced carbon compounds to meet their daily metabolic energy requirements.

In summary, in situ light reduction resulted in a rapid decrease in leaf biomass in Thalassia testudinum though defoliation and low leaf elongation rates. The drop in leaf biomass enhanced anoxia in sediments though decreases in photosynthetic oxygen production and transport to below-ground tissues, ultimately raising the concentration of toxic sulfides and promoting root anaerobic fermentation. Utilization and rapid depletion of stored carbohydrate reserves in rhizome tissues, combined with low productivity and high concentrations of sediment sulfides resulted in plant loss at light levels equivalent to 5 and 14% SI (864 and 1628 mol m' 2 yr 1 , respectively). Our results also indicate that the effects of light reduction in Thalassia testudinum are reflected in a variety of parameters which are conveniently monitored, including shoot density, blade width, leaf growth, chi a : b ratio and chlorophyll content.

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