Citation
Responses of subtropical seagrasses to fluctuations in salinity within an experimental facility

Material Information

Title:
Responses of subtropical seagrasses to fluctuations in salinity within an experimental facility
Creator:
Chesnes, Thomas C., 1973-
Publication Date:
Language:
English
Physical Description:
viii, 207 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Amplitude ( jstor )
Biomass ( jstor )
Fresh water ( jstor )
Leaves ( jstor )
Oxygen ( jstor )
Rhizomes ( jstor )
Salinity ( jstor )
Saltwater ( jstor )
Standard deviation ( jstor )
Statistical significance ( jstor )
Dissertations, Academic -- Environmental Engineering Sciences -- UF ( lcsh )
Environmental Engineering Sciences thesis, Ph.D ( lcsh )
Seagrasses -- Effect of salt on ( lcsh )
Seagrasses -- Physiology ( lcsh )
The Everglades ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Summary:
Thalassia, Halodule, Ruppia.
Thesis:
Thesis (Ph.D.)--University of Florida, 2002.
Bibliography:
Includes bibliographical references (leaves 203-206).
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Thomas C. Chesnes.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
028798970 ( ALEPH )
50512079 ( OCLC )

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' 'By '.


























This manuscript is dedicated to Rose Trivigno.
I know that you would be proud.














ACKNOWLEDGMENTS

Words cannot express my appreciation for those who supported me in this

endeavor. My supervisory committee has been without a doubt instrumental to my

success. I thank Evan Chipouras for his support from the earliest stages of this project, as

well as his ability to help me keep things in perspective. I thank Frank Nordlie for

introducing me to ecological research and mentoring my undergraduate project. Without

him, I certainly would not be in this profession. I thank Stephen Davis, whose

enthusiasm for algae is contagious and inspiring. I especially thank Thomas Crisman

who helped reintroduce me to my ecological roots. His patience and assistance were

essential in getting through a difficult period. Lastly, I thank my chairman Clay

Montague, whose mix of humor and scientific curiosity has inspired me to develop a

strong scientific ethic, an interest in the workings of the universe (on all temporal and

spatial scales), and a never-ending pursuit of truth over accepted ideas.

This study would not be possible without funding and logistical support from the

Everglades National Park Interagency Science Center in Key Largo, Florida and the

South Florida Water Management District, who also provided nutrient analysis. The

Aylesworth Foundation and Greening UF provided additional support. Special thanks to

those who assisted in data collection: Casie Regan, Christos Anastasiou, Joel Dudas,














TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS................................................................................................. iv

ABSTRACT...................................................................................................................... vii

CHAPTERS

1 INTRODUCTION ............................................................................................................ 1

2 DESCRIPTION OF FACILITY DESIGNED FOR SALINITY FLUCTUATION AND
PILOT STUDY ........................................................................................................ 9

Description of Facility.................................................................................................... 9
M materials and M ethods of Pilot Study.......................................................................... 13
Results of Pilot Study ................................................................................................... 16
Discussion of Pilot Study.............................................................................................. 24














Correlations w ith Field Conditions at Collection Sites.............................................. 165

6 PRODUCTIVITY AND LEAF OSMOLALITY-RESULTS OF EXPERIMENT 8 ... 173

Introduction................................................................................................................. 173
M materials and M ethods................................................................................................ 175
D ata Analysis for Experim ent 8.................................................................................. 178
Results......................................................................................................................... 179
D iscussion................................................................................................................... 190

7 DISCU SSION ............................................................................................................... 196

Overall D iscussion...................................................................................................... 196
Conclusions................................................................................................................. 201


REFEREN CES ................................................................................................................ 203

BIOGRAPH ICAL SKETCH ........................................................................................... 207














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
RESPONSES OF SUBTROPICAL SEAGRASSES TO FLUCTUATIONS IN
SALINITY WITHIN AN EXPERIMENTAL FACILITY

By

Thomas C. Chesnes

May 2002

Chairman: Clay L. Montague
Major Department: Environmental Engineering Sciences

The sparsity of seagrass communities within the ponds and bays of northern

Florida Bay may be due to the wide fluctuations of salinity in these habitats. Seven

experiments were performed in a facility designed for salinity fluctuation. Three seagrass

species, Thalassia testudinum, Halodule wrightii, and Ruppia maritima, were

transplanted to the facility and subjected to varying degrees of salinity fluctuation.

Treatments were varied according to salinity wave mean, amplitude, frequency, and

suddenness (slope) of change. Two experiments crossed the effects of salinity fluctuation

with methods of water circulation and reductions in available light. A final experiment,














were negatively correlated with increasing salinity wave amplitudes, frequencies, and

suddenness of change. The effect of salinity fluctuation was dampened when salinity

fluctuated within a range of higher salinities. Salinity fluctuation was more of an

influence on Thalassia survival than the reduction of light.

Halodule condition was impaired by salinity fluctuation, but not to the extent

experienced by Thalassia. Green leaf indices decreased with increases in salinity wave

amplitude, frequency, and slope. The number of turions per sprig correlated negatively

with increasing amplitude and more sudden changes in salinity. Halodule survival was

enhanced by fluctuations within a higher salinity range.

Ruppia was the most resilient of the seagrass species tested. This seagrass was

able to survive all of the salinity fluctuation treatments. Increasing the frequency of

salinity change did have a negative impact on this seagrass, resulting in lower green leaf

indices and number of leaves.

In order to survive fluctuating salinities, seagrasses must regulate their internal

osmotic concentrations in relation to their surrounding waters. Ruppia osmoregulated

more quickly than Thalassia and Halodule and may be the key to its resiliency.














CHAPTER 1
INTRODUCTION



Since 1881, human activities have disrupted the natural flow of freshwater from

the Everglades into Florida Bay (Fourqurean and Robblee 1999). The Everglades

watershed has been engineered and managed for agriculture, flood control, and water

supply for the growing population in South Florida (Light and Dineen 1994). Water

management protocols involving alterations in freshwater flow can change the regime of

salinity fluctuations in the downstream estuary. Sudden releases of flood water may

create rapid drops in salinity, whereas water held back in times of drought may amplify

salinity increases downstream (Montague and Ley 1993). Changes in community

structure will be most noticeable in the estuarine salinity fields closest to land (Estevez

2000).

Salinity related problems arise when estuaries receive too much or too little fresh

water, or water at improper times (Odum 1970). These habitats are characterized as

being harsh due to the salinity changes, especially when compared to the more static

conditions typifying marine or freshwater habitats (Deaton and Greenberg 1986).

Changes in salinity occur rapidly in the shallow basins located within the northern land

margin of Florida Bay. Periods of high freshwater inflow during the onset of the rainy

season, in tropical storms (Chesnes 1999), or water management releases can cause areas








with marine strength salinities to become fresh, in some cases within a matter of days

(Mclvor et al. 1994, Montague and Chipouras 1998).

The meteorologically driven patterns of salinity are clearly seen in the salinity

record of Little Madeira Bay, located within the northern land margin of Florida Bay

(Figure 1-1). During the course of the dry season, salinity steadily rose from 10%o

(December 1998) to above 30%o (May 1999). At the onset of the wet season (May/June

1999), salinity dropped nearly 20%o in the first week, and approximately 30%o within

three.

Acute fluctuations in salinity also occurred during this time period. During July

and August of 1998, salinity fluctuated between 1 and 18%o, with a period of

approximately 4 days between peaks and troughs. The amplitude of the fluctuation

varied over the course of the month (Figure 1-1). Acute fluctuations also occurred in

October and December of 1998, January 1999, and again in the late summer/early

autumn of 1999. The amplitude of salinity change was higher in July and August of 1999

than in 1998, when changes of over 20%o were seen over four day periods.

The pattern of salinity change during transition from high to low salinity in the

ponds and bays of the land margin of Florida Bay is more acute than during the transition

from low to high (Montague and Chipouras 1998). Increases in salinity can be caused by

evaporation during the dry season or by winds pushing saline water from Florida Bay into

the estuary. The transition to saltier conditions is thought to be more gradual, and the

loss of stenohaline freshwater macrophytes may be a more progressive process as the

salinity slowly becomes elevated. Conversely, a sharp decline in the abundance of











stenohaline marine macrophytes follows the acute exposure to low salinity at the onset of

the rainy season (Montague and Chipouras 1998).

Fluctuations in salinity correlate with decreases in submerged macrophytes and

benthic animal density (Montague and Ley 1993, Fears 1993, Jones 1999) as well as

fouling organisms (Chesnes 1999). Each individual has a range of salinity tolerance and

a narrower range of optimal salinity (Remane and Schlieper 1971). Species richness is

especially low in the salinity range between 5 and 8 %o. This paucity in flora and fauna

can be explained by the habitat instability of estuaries (Deaton and Greenberg 1986).

The salinity fluctuation hypothesis suggests that this lack of biota may be the result of

extreme salinity fluctuation (Montague and Ley 1993). Few organisms have evolved the

physiological mechanisms required for life in highly variable environments (Deaton and

Greenberg 1986).

Most studies of salinity and submerged macrophytes have focused on tolerances

to extreme high and low salinities. Few studies have looked at the effects of salinity

fluctuation on submerged aquatic vegetation (Montague and Ley 1993, Fears 1993, Jones

1999, Durako 2000). In some cases, the significance of salinity as a water quality factor

is unknown since its effect is confounded with the presence of other environmental

conditions (Twilley and Barko 1990). For example, a study by Tomasko and Hall (1999)

found that the use of field studies for estimating the lower salinity tolerances of

seagrasses might be inappropriate for those systems where water clarity is positively

correlated with salinity.

Seagrasses are a vital component of estuarine systems. If salinity fluctuates too

much, communities may not become established (Montague and Ley 1993, Montague








1996). Seagrasses provide habitat for many benthic and pelagic organisms (Doering and

Chamberlain 2000), stabilize sediments by slowing water movement and increasing

sedimentation (Ogden 1980) and can form the basis of the plant-based and detrital-based

food chains (Klug 1980). Three seagrass species, Thalassia testudinum, Halodule

(Diplanthera) wrightii, and Ruppia maritima, are commonly found within the northern

land margin of Florida Bay. Little Madeira Bay (Figure 1-1) was included in earlier

macrophytes monitoring studies (Montague and Ley 1993, Montague and Chipouras

1998). All three species inhabited these areas during these studies, though Ruppia was

not as prevalent in the earlier one when salinities were higher and did not fluctuate as

much.

The degree to which rapid and dramatic changes in salinity can influence the

distribution, abundance, and community composition of seagrasses is unknown. Its

importance should be greater at land margins where outwelling freshwater meets

saltwater pushed in by coastal tides and winds. If salinity fluctuation is important, then

changes in water delivery to the coast that is under control of water managers will be

expected to directly influence the distribution and abundance of seagrasses in ways that

could affect habitat for fish and birds. Two other physical factors that are affected by

features of freshwater discharge have received the most attention by water managers and

seagrass scientists, specifically alterations in light and nutrients. In this dissertation

attention will be given to a third and perhaps more influential environmental factor,

salinity fluctuation.

Thalassia testudinum (turtlegrass) is the dominant marine angiosperm of the

Caribbean (Patriquin 1973). It is characterized by a creeping rhizome which has often








been found branched which gives rise to erect branches bearing leaves (Phillips 1960).

This species is considered stenohaline (Jagels 1973), although it was found in habitats

spanning the entire salinity gradient from fresh to marine strength within the northern

land margin of Florida Bay (Montague and Chipouras 1998). Although not thoroughly

explored, Thalassia may have a temperature optimum near 30 C and a salinity optimum

near 30%& (Zieman 1975). Fluctuations in salinity, especially those ranging into fresher

water, may be detrimental to this species.

Halodule wrightii (shoalgrass) had a wider range of salinity tolerance than

Thalassia in experiments by McMillan and Moseley (1967). In salinity tolerance

experiments performed by McMahan (1968), Halodule survived in salinities ranging

from 9 to 52.2%o, but died in salinities of 3.5%0 and in excess of 70%0. This species was

also found in habitats spanning a salinity gradient from nearly fresh to hypersaline within

the northern land margin of Florida Bay (Montague and Chipouras 1998). Based on

these findings, Halodule is expected to be more tolerant to salinity fluctuation than

Thalassia.

Widgeongrass, Ruppia maritima, has a nearly cosmopolitan distribution and

worldwide importance as a waterfowl food (Kantrud 1991). Ruppia has the widest

known range of salinity tolerances of any genus of submerged aquatic vegetation. This

species has been found in waters ranging from 0 to 390%0 with optimum growth between

0.5 and 31%o (Durako 2000). However, a study by McMillan and Moseley (1967) found

Ruppia to be less tolerant of hypersaline conditions than Thalassia and Halodule. In the

field monitoring studies conducted by Montague and Ley (1993) and Montague and

Chipouras (1998) Ruppia was found to be common but ephemeral in ponds located alone





7

streams within the land margin of Florida Bay but rarer in the more open, saline bay

habitats. Salinity fluctuation may be detrimental to this species despite its reported wid

salinity tolerance range.

A facility designed for salinity fluctuation experiments was built in Key Largo,














acclimation rates of seagrasses subjected to salinity fluctuation.













CHAPTER 2
DESCRIPTION OF FACILITY DESIGNED FOR SALINITY FLUCTUATION ANE
PILOT STUDY



Description of Facility


A schematic diagram of the facility used for conducting salinity fluctuation

experiments is provided in Figure 2-1. The facility is located on the grounds of the

National Park Service's Key Largo Ranger Station of Everglades National Park, mile

marker 98.6 bayside Overseas Highway (Figure 2-2). A more complete description of

the facility and discussion of its design criteria, constraints, construction, and testing is

given by Anastasiou (1999). Saltwater for the facility was supplied by a well drilled 13.

meters deep into porous carbonate rock connected to eastern Florida Bay. Salinity in th<

well was approximately 36%o. Chlorinated fresh water was supplied by the Florida Key

Aqueduct Authority. The fresh water was held in a 242 m3 painted concrete reservoir

(formerly a swimming pool), which increased residence time for dechlorination.

The seagrass experiments occurred within twelve 1.1 m3 experimental tanks

(Figure 2-3). Salinities were manipulated by manually adjusting valves on a seawater-

freshwater mixing manifold (center of Figure 2-3). Up to four different salinity regimes

could be delivered simultaneously to randomly chosen replicate experimental tanks by

connection of hoses of 3.81 cm diameter to a distribution manifold. In general, water w-







IU



SActuated solenoid valve (0-60 gpm)
SBall valve, manual @ | From 60,
freshwat
Saltwater pipe fsu ppclt

Freshwater pipe Ir jtw fe
Freswaterpipe pip Freshwater Head \Freshwater Head
____unerb. Tank(2100gal Tank(2100al Water su
Salinity mixing pipe activated
sensors I
Experimental tank fill pipe Pumps tr
level drol
0*0aAa "M ON Saltwater Head Saltwater Headi max
Experimental tank drain pipe Tank (2100 gal) ank (2100 gal)
.4 aa an anaam
Main drain pipe
Experimental tank fill
Conrte sb --(S0-0pn)
0 Experimental tank drain From sa
FW iis
o Stub-up connection to mixing system FW
i---4IIsw
(14or ipe) I I Seawater Freshwater
Mixing System
Sani mix I I I
valves z- rm f '
Random connection with 1.5" hoses.
stub-up *.* ..41.~Q
connctio s
,o,+. fc t l.
to vak-es ... ......t~..


... ............. ..
was


Amm ; 0 1 1. \
A**

mq.n pipe *uuauuugrna
% %

%


Concrete slab
149,
.<~~ ft

EXPERIMENTAL S&
FLUCTUATION FA
3/^-.tS




































IU KM lip -It

















Figure 2-2: Location of collection sites and the Key Largo Ranger Station.
(Map from the USGS)
































Figure 2-3: Photograph of Experimental Facility. Experimental tanks are in the
foreground, storage tanks for fresh and sea water are located on the hill


unless flow was ceased due to experimental design. A constant inflow facilitates water

circulation. Water level was maintained at approximately 50 cm by a stand pipe drain.

Salinity was controlled by varying the quantities of fresh and salt water delivered

through the mixing manifold to the tanks. When fresh water was delivered to a tank

containing water of a higher salinity, an extension was added to the inflow pipe to deliver

water to the bottom of the water column. This enhanced mixing and flushing of the

higher density water. Complete turnover of a 1.1 m3 tank from one extreme salinity to

another occurred within 2 or 3 hours at an inflow rate of 16 m3d"'.








Materials and Methods of Pilot Study


Seagrass Collection and Experiment Preparation

Plants used in this experiment were collected in April 1998. Sprigs of Ruppia

were collected from the northern region of Seven Palm Lake (N 25* 11.69', W 80

43.42'), and Halodule from Terrapin Bay (N 25 09.49', W 80 43.84), both areas

located in north-central Florida Bay (Figure 2-2). Thalassia sprigs were collected from a

floating mat found nearby the Key Largo Ranger Station (Figure 2-2). Twenty-four

polyethylene tubs (Rubbermaid Inc., measuring 57 X 46 cm) were filled with

approximately 22.7 kg of Quikrete Commercial Grade fine sand. Halodule and Ruppia

rhizomes were planted at a density of 40 to 60 percent coverage in separate tubs. Sprigs

were selected that had the presence of green leaves and apical meristems. Thalassia

sprigs were prepared so that each rhizome had only one shoot as well as an apical

meristem. In each Thalassia tub, five rows of three individual seagrasses were planted,

totaling fifteen. Once planted, the tubs of Ruppia, Halodule, and Thalassia were placed

in holding tanks of 1.1 m3 with salinities of 15, 25, and 36%o, respectively. The plants

remained at these salinities for approximately two months.

Eight of the facility's twelve experimental tanks were used in this experiment.

Salinity fluctuation treatments were assigned randomly to the tanks. Four tanks received

a stable salinity treatment (SST1), maintained at 18%o. Two tanks received a four day

period of salinity fluctuation (P4D) in which salinity was alternated between 0 and 36%o

every two days. Two other tanks received an eight day period of salinity fluctuation

between the same extremes (P8D). One tub of each seagrass was randomly selected from








or South) within each of the eight experimental tanks. Treatments were applied during

the period from June 30 to August 1, 1998.

Protocol of Experiment

Salinity, temperature, and amount of ambient light, light just below the water

surface and at the tank bottom were measured daily. The percentage of light reaching the

seagrass was computed using Beers Law. Light measurements were taken within two

hours of solar noon, in an unshadowed area of each tank, using a quantum photometer

(Li-Cor Inc. model LI-185 B). Salinity and temperature were measured using a

refractometer (Leica model TS) and mercury thermometer, respectively. Water inflow

rates were checked three times a week by timing the filling of a bucket of known volume.

Adjustments were made as needed to ensure that each tank was receiving a similar influx


5i.a, uiou .

ush. Notice,

r of scrubbin

Daily m(

lent, evaluati

rated using

ies 1999). T

irs were assu


pJWilu IIuunl, aiiu irom me UoDS anu

rbidity caused by the suspension of el



ag of the seagrasses involved counting

color of the leaves, and noting the pr<

ex (Table 2-1) similar to that used in g

sence of green leaves indicated a heall

indicate a physiological impairment


. wtaAI uy z

ites subsid



number o

,e of new I

Lted field t

hoot, while

ke leaf. W








reliably assessed. A grid was constructed to divide the Halodule and Ruppia seagrass

tubs into nine equal areas of 230 cm2 each. Color index ratings from Table 2-1 were

recorded for each grid region. Color ratings were converted to a percentage, based on the

mean percent within the color rating range (Table 2-1).

Complete shoot counts and color analyses of all three species were performed on

days 1, 2, 3, 15, and 31 of the experiment. On the other days, two grid regions were

randomly selected for coloration surveys and shoot counts in each tub of Ruppia and

Halodule. For Thalassia on these interim days, two of the fifteen shoots were randomly

selected in each tank for coloration analysis.



Table 2-1: Color rating scale used in pilot study.



Index Rating Percent Color Range Mean %

6 100% color 100

5 95% to less than 100% color 97.5

4 66% to less than 95% color 80

3 33% to less than 66% color 50

2 5% to less than 33% color 20

1 Greater than 0% but less than 5% color 2.5








Results of Pilot Study

isical Parameters

Average daily salinity measurements for each treatment are shown in Fig

thin the stable salinity tanks, salinity varied from the desired 18%o. Adjustme

nity of the treatment tanks caused subsequent changes in the flow rates and ho

assure in the other tanks. This adjustment, in turn, caused unintended deviation

nity of the tanks designated for stable salinity treatment. Unexpected salinity

,tuations were also caused by mechanical failure. On Days 9 and 29 the saltw


both the experimental and control tanks for an unknown pe

the total difference in range of average salinity amongst treA

ier thousand (Figure 2-4, middle panel) and the standard de'

considerably higher in the fluctuating treatments as per the dc

el).

temperatures of the treatments were similar, deviating from

C (Figure 2-5, top panel). The fraction of surface light rea

d1 a wider range amongst treatments, however (Figure 2-2, h

ae that were present primarily in the freshwater storage and

ed the turbidity over that in the seawater system. Seagrasse










OI- mbr-^

-n r77 M





0 10 20 30 40
Experiment day


Mean Salinity over Experiment (%o)
18

17-

16 ,
SST1 p8d p4d
Standard Deviation of Salinity over
Experiment (%o)
20

10-


C J











Mean Temperature over Experiment
(oC)

32 -

31

30
SST1 p8d p4d



Intensity of Light at Seagrass Depth
(uE/m2/sec)

1500 -

1300

1100
SST1 p8d p4d


Percent Light Reaching Seagrass Depth

1 -,


0.75


0.5
SST1 p8d p4d



Figure 2-5: Mean temperature (top panel), light intensity at seagrass depth (middle
panel), and percent light reaching seagrass depth (bottom panel) for treatments in pilot
study. Percent is expressed in decimal form.








eagrass Measurements

Figure 2-6 shows the average color ratings obtained from Thalassia. Ra

presented by a stacked bar for each experimental day. Green is given at the b.

ir with the other colors sequentially above from chlorotic to white on top. Sult

iried in coloration for all Thalassia treatments (Figure 2-6). It is evident from

een coloration ratings that Thalassia sprigs used in the SST treatment were in

ndition than those in the other treatments (Figure 2-6).

Daily color ratings were transformed to produce accumulations of color

ter time. At each sample day, color ratings were converted to percent colorati

recentt colorations of each daily measurement were added together and divided

timber of samples. This analysis reduced the noise caused by the subsampling.

cumulations are given for Thalassia in Figure 2-7. Color accumulations are

presented by a stacked bar for each experimental day. Green colorations are 8


nfthe hir with thEp nth.


r colors sequen


accumuiations increase only in me stable salinity treatment (Figure 2-7, top panel). No

green accumulation occurred in the fluctuating treatments over the course of the

experiment. Brown and yellow color accumulations decreased with subsequent samples

in the fluctuation treatments, replaced with increased white coloration.

Starting green coloration ratings of Halodule sprigs were similar (Figure 2-8).

Green coloration ratings oscillated around their initial values in the stable salinity

treatments, but fell (while white ratings increased) in the fluctuation treatments in

samples after ten days of experimentation. Color accumulations were similar over the

course of the stable salinity treatment (Figure 2-9, top panel). In both fluctuation


























P8D Treatment







1 6 1 1 1 6 2 1 2 6 3 1-- * - -




Experiment Day
8

a6 E3wtite
U brown

2 .green


1 6 11 16 21 26 31
Experiment Day


P4D Treatment





U brown
4 -- ^ -


b o yellow
2 2* ------ green
0 1 11 1111 11 11 1111 Hi ll 11 1 11 11 11 [ '', 111-1111 11-1 11 1 1 O~ e



1 6 11 16 21 26 31
Experiment Day

Figure 2-6: Color ratings for Thalassia over pilot study. (* denotes ratings deriv
average of all giants sampled)


6

i4

2

0
1


* brown
D yellow
* green










SST Treatment Color Accumulations

120 -
100- -
~80
|I brown
2 60
~40 N green
20
0
1 6 11 16 21 26 31
F -tt ftBE-!


P4D Treatment Color Accumulations

120 -
100 ---------------------------------------------
100

| 80 - - - - - -Ow te
U brown
~60
.2 60 - - - - - - - -
0 13 yellow
S40 w green
20


1 6 11 16 21 26 31



Figure 2-7: Coloration accumulation for Thalassia in pilot study.


120
100 ----------------------------------

80 - -
S60 - -
o 40
20-
0
l l6 11 16 21 26 31
1 6 11 16 21 26 31


D white
* brown
0 yellow












8






2

0
0 ... ..y -| - - B rv


1 6 11 16 21 26 31
Experiment Day


P8D Treatment


Figure 2-8: Color ratings for Halodule over pilot study. (* denote













SST Treatment Color Accumulations

120
100
80 -o wtite
2
2g 60 -m brown
0
S40 green

20 -

0
1 6 11 16 21 26 31


P8D Treatment Color Accumulations

120 -
100

~80 owit
2

2 60 brown
0
~40
20
0
1 6 11 16 21 26 31


P4D Treatment Color Accumulations

120 -
100

i 80 o *

2 60 U brown
0
U 40 11 1 green
S40
20
0
1 6 11 16 21 26 31


Figure 2-9: Coloration accumulation for Halodule in nilot study.








,atments, green color accumulations in Halodule decreased, while white accur

creased.

Green coloration ratings were highest in Ruppia exposed to the stable sa]

,atment; however; green ratings in the fluctuation treatments persisted through

periment (Figure 2-10). White coloration ratings steadily increased in Ruppia

periment for all treatments, although greatest white color ratings were seen in

situation wave treatments. Ruppia sprigs exposed to the eight day fluctuation


hough green color accumulations remained high in the four day flui

*D) treatment, white color accumulations increased over the experir



Discussion of Pilot Study

The results of the pilot study demonstrate a distinct effect of sa


lent.


salinity, temperature and light) was minor and was unlikely to have measurably

influenced the results. In the three species tested, exposure to fluctuations in salinity

resulted in the replacement of healthy green leaf tissue with dead white tissue. Although

an effect of fluctuation was clear in all three species when compared with stable salinit,

an affect of period of the salinity fluctuation was not evident in the Thalassia and

Halodule experiments. In Ruppia, longer periods were more detrimental.

The method of surveillance did not include the monitoring of individual plants.

Subsampling did not allow the responses of individual sprigs to be evaluated. Repeated

Ramnleq nn the .ame nlnt. wniild hle mnrr. indiretlive nfthp. tff.r.tc nvr time








35T I reatmen


P8D Treatment

8
6 -- ----------

{4 .. -- -


2 4

0
1 6 11 16 21 26 31
Experiment Day


P4D Treatment

8 --------------------


I -


4

2

0
1


6 11 16 21 26 3
Experiment Day


average of all plants sampled) -- -- --- --- -- -
average of all plants sampled)


* uIuwiI
* green


6 11 16 21 26 31
Experiment Day









SST Treatment Color Accumulations

120 -
100

80 white

| 60 brown
40
0 40 green

20 -

0
1 6 11 16 21 26 31



P8D Treatment Color Accumulations

120
100 -
S80 -
SDwhite
.2 60 brown
0
40- E green
* 40

20 -

0
1 6 11 16 21 26 31


P4D Treatment Color Accumulations

120 -

100 i
2 80
Owlut
0 o, IIIIIIIIIAllll//f _.t














amount of starting green coloration may have exacerbated the effects of salinity

fluctuation, giving the plants little chance of survival. Acclimation to appropriate

salinities and stricter criteria for selection in experiments may remedy this problem in

future experiments. Sparse distributions of seagrass communities in northern land

margin of Florida Bay may be influenced by the frequent changes of ambient salinity. If

seagrasses are losing photosynthetic material due to the affects of salinity fluctuation,

energy reserves may not be adequate for vegetative growth and rhizomal elongation, the

main methods of reproduction in both Thalassia and Halodule (McMillan and Mosely

1967, Phillips 1960).

Further experiments are necessary to explore the responses of seagrasses to

varying degrees of salinity fluctuation, including the amplitude, frequency, and

suddenness of change. In addition, the mean about which salinity fluctuates, light, and

nutrient interactions can be examined experimentally. With a more complete and

quantitative examination, the distributions and abundances of seagrasses may be

predicted using models that include salinity fluctuation as well as light, nutrients,

temperature and average salinity.













CHAPTER 3
MATERIALS AND METHODS FOR MAIN STUDY
(EXPERIMENTS 2 THROUGH 7)



Seagrass Collection and Acclimation

All seagrass species used in the salinity fluctuation Experiments 2 through 7 were

collected within Little Madeira Bay (N 25 11.39', W 80 38.34'), a basin located in the

northern land margin of Florida Bay (Figure 2-2). Sprigs of Thalassia testudinum,

Ruppia maritima, and Halodule wrightii were collected that consisted of a length of

rhizome with shoots and roots attached and with healthy leaves. Shoots consist of a

sheath (in Thalassia and Halodule) and at least two leaf blades. Sprigs were carefully

removed from the sediment and placed into coolers half filled with water from the

collection site. Sprigs of Halodule wrightii and Ruppia maritima were selected if they

had a minimum of three shoots and a growing rhizome tip present. Sprigs of Thalassia

testudinum had a minimum of two shoots and a growing rhizome tip. Bottom salinity of

water sampled from seagrass depth was measured at the collection site with a

refractometer. Temperature was measured in the water column with a mercury

thermometer attached to a string. In addition, salinity and temperature data from Little

Madeira Bay were obtained from the South Florida Information Access (SOFIA) website

(Patino and Hittle, unpublished) to determine field conditions in the month prior to

collection.








The seagrasses were transported to the experimental facility in coolers and

allowed to acclimate in the experimental tanks for a minimum of two weeks. During the

acclimation phase, salinities were adjusted from the salinity at the collection site to the

mean salinity of the experiments in increments less than 1 %o per day. Seagrasses were

not monitored during this time. Acclimation periods and ranges for experiments 2

through 7are given in Table 3-1.


Description of Experiments

The effects of different characteristics of salinity fluctuation were tested in a

series of six experiments (numbered 2 through 7). Amplitude, period, suddenness of

change and mean salinity were tested. A general description of the experiments is given

in Table 3-2, and a more detailed description of treatments is given in Table 3-3. The

seven column headings in Table 3-3 describe various aspects of the pattern of salinity

fluctuation. The variables tested in each experiment are designated in the shaded column.

In all but Experiment 4, fluctuating wave patterns treatments were tested against stable
eolinifxr 4"*tr, ,trf,<.Cto









Table 3-1: Acclimation periods and ranges for the facility experiments. The end date of
the acclimation period was the first day of the experiment.



Experiment Start Date End Date Salinity Range Duration at Target Salinity (18%o)


ble3-


ry of Experi


Experiment # Scope of Experiment


2 Effect of Amplitude
3 Suddenness of Change (Square vs. Pyrar
4 Effect of Mean
5 Effect of Period
6 Effect of Water Circulation Method and
Salinity Fluctuation (crossed design)
7 Effect of Light and Amplitude (crossed de,


4 2/6/99 3/3/99 16-18%o
5 4/27/99 5/19/99 30-18%o
6 6/29/99 7/13/99 18%o
7 10/4/99 10/27/99 7-18%o


i

1
If






,fer to m




Numbi
Treatrr


3
3
2
3
4


5


SuayO
days
i days
days





mber of




r of
ents P


Number of
Replicates
-r Treatmer


4
4


licates r










id Wave






ign)


3


2


3


2






















(%.) (da-
-n1


1R 11A A A


18

i around differni
Mean AmI
(NO)


4 32


od Sal. Min. Sal. Max.
(M) (%N) (i
0 18
18 36


ist of Extreme Salinity Fluctuatio
Wave Type Mean Amn


SWp8 square 18 18 0 36 0.13 100

Exp 6: Water Circulation Method and Salinity Fluctuation
Wave Type Mean Amplitude Wave Period Sal. Min. Sal. Max. Inflow Percent Sui
(%0) (%) (days) (%.) (%0) (liter / sec) (%)
SSTt stable 18 0 18 18 IN W 100
SSTb stable 18 0 18 18 100
SWt square 18 8 4 32 100
SWb square 18 8 4 32 100

Exp 7: Low and High Amplitude Fluctuation Crossed with Full and Reduced Sunlight
Wave Type Mean Amplitude Wave Period Sal. Min. Sal. Max. Inflow Percent Sui
(%.) (%.) (days) (%.) (%.) (liter I sec) (%)
SSTs stable 18 0 18 18 0 (air)
SWa7u square 18 8 11 25 0 (air)
SWa7s square 18 8 11 25 0(air)
SWa14u square 18 8 4 32 0 (air)
SWa14s square 18 8 4 32 0 (air)
















salinity max

F amphtude



I mean salinity



salinity min
I
period


Time











Figure 3-1: Characteristics of a salinity fluctuation wave. The square wave is shown in
black, the pyramid wave is gray. Frequency is the reciprocal of period.








throughflow of water versus air bubbling as a means of water circulation in the

experimental tanks was tested along with salinity fluctuation in Experiment 6. The effect

of shading was tested along with salinity fluctuation in Experiment 7.

In Table 3-3, the column titled "inflow" designates the constant inflow of water

into the experimental tanks. For Experiment 6, an air pump was installed to aerate the

saltwater head tanks and selected experimental tanks. Aeration helped reduce sulfides in

the saltwater head tanks and promoted circulation in the experimental tanks (Anastasiou

1999). Water did not flow through the bubbled experimental tanks, rather they were

filled initially with water and aerated. Evaporative losses were replaced daily with

freshwater, a task not required in flow through experimental tanks. When it was time to

change the salinity, a brief period (approximately 60 minutes) of flow through occurred

until the new salinity was achieved.

In Experiment 7, the effects of reduced sunlight were crossed with fluctuation

patterns of low and high amplitude. In this crossed design, interactions between light and

amplitude can be revealed. This was important because the freshwater at times was more

turbid than the saltwater, potentially confounding the results. Randomly selected

experimental tanks were covered by 70 % shade cloths to create reduced light conditions

(30% full sun). The treatments are designated in Table 3-3 under the column labeled

"Percent Sun". Ruppia was not tested in this experiment because there was not adequate

numbers present at the collection site and other areas of northern Florida Bay where

Ruppia has been previously found. Furthermore, an unshaded stable salinity treatment

was not included because of a lack of acclimated sprigs. An unexpected dieoffof

Thalassia and Halodule occurred during the acclimation period. The stable salinity








treatments of the five prior experiments were unshaded, therefore this treatment was not

included to ensure an adequate number of replicates for the remaining treatments tested

in this experiment.

Sprig Preparation and Planting

Immediately prior to each experiment, morphometric measurements were taken

on all seagrass samples. Sprigs with a growing rhizome tip and at least two shoots with

green leaves were randomly selected from the acclimation chamber. The number of

shoots was counted. Rhizome length and mature and immature leaf lengths were

measured to the nearest half millimeter. Sprigs were then planted into polyethylene tubs

(26.5L, Rubbermaid, Inc.), measuring 57 x 46 x 15 cm deep. Fine-grained sand

(Quikcrete brand) was used as sediment in all tubs. Two sprigs of each of the three

species were planted in each tub in random order, for a total of six sprigs per tub. Three

tubs were submerged in each experimental tank, for a total of eighteen sprigs per tank.

Sprig and Tank Monitoring and the Green-Leaf Index

During the course of Experiments 2 through 7, leaves were monitored every two

days. The total numbers of leaves were counted on each of the three youngest shoots on

the sprig, followed by a count of the number of leaves with any green coloration. Finally,

a green-leaf index was assessed for each shoot, based on the number of green leaves

present, prorated visually for partial green coloration. For this index, the number of

leaves with green coloration on a shoot was multiplied by an estimate of the fraction of

all leaves on that shoot that was green. This calculation is performed for the three
























I -- ..__^ -

the seagrass sprigs were dried and weigh





Data and



Statistics were performed using

Associates, Inc.). For each experiment,


responses by each species to each treatr

nrnct'dire. nfwae iiaprl tn i;dpntinfA rlj;flr


'ANUVA 1


IA1 1 -


Pearson product moment corre'


to explore and quantify relationships b

resnnnmfe frnm amnnao fthi optnro Aaf c


frequency. The method of calculation for each descriptor is given in Table 3-5. Mean


TL-


A


_ ? s



















(Cluster of
leaves)

Short-Shool

4-- (Sheath)


Rhizome


Percent Green-Leaf (as a decimal):

0+0.5 +0 0.5 +0.75 1.0 + 0.5 + 1.0

Total per Shoot:

0.5 1.25 2.5

GLI for Sprig (Average of Shoots):

4.25/3 GLI = 1.42





Figure 3-2: Example of Green-Leaf Index calculation. For this example, assume black
colorations on leaves are green.









Table 3-4: Dates and durations of the six facility experiments.


2
3
4
5
6
7


salinity, standard

the salinity wavi

Suddenness giv<

the maximum sl

of this analysis,

determine the m

To aceoi

involving tempe

the stable salinity


10/28/99 11/22/99 26 da


n, and maximum amplitude describe aspects of the ai


inu meL ratC nuiU magIM uuuV Ui MIiiULy unuiiC uy quatuiiyul

occurred in the entire experiment. Due to the exploratory nal

icance threshold was set at p < 0.001 to act as a filter to

tant correlations.

y confounding effects of salinity fluctuation, correlations

ht, and water nutrient concentrations were made with plants
--d- _- 1_-. ,'1L :_L ... ...- Z',l- 1.-. -_. -- _----_ _--.. .. 11 !-


10/25/98 11/25/98 32 di
1/4/99 2/3/99 32 di
I AIQQ MI')QOQ 9 R rit















in Salinity Mean salinity calculated from daily measurements



idard Deviation Standard deviation calculated from daily measurements
alinity


imum 1/2 (maximum minimum salinity measurement)
4litude


denness of Maximum slope of salinity wave
iity Change


iber of Changes Number of salinity changes / Number of days
Day


ificant 1/2 ((number of crossovers -1) / number of days)
juency where "crossover" occurs when salinity crosses designed mean


olute 1/2 (# of peaks + # of troughs / total days)
]uency



























een-le

ippia

ie rest

able s&


h of Taylor River in Little Madeira Bay are plotted and su

and Hittle, unpublished). The daily temperature averages

nts taken at fifteen-minute intervals. Hurricane Georges c


C il ~llli I~ll, L L1 I.IC lil ili Al llI&L1A1ill ~ VT Ol ili 11 C15* A 'l i%. "!-.J AV Ii, 1l.UlI11ilL~l

are coded as follows: 1) SST2- stable salinity treatment, 2) SWa7- square wave treatment

with an amplitude of 7%o and a period of four days, and 3) SWal4- a square wave

treatment with an amplitude of 14%o and a four day period. An indirect hit by Tropical


















































values were measured at 15- minute intervals. Gaps in the salinity record represent w
no data were reported. Temperature values are daily averages. Tabular values bottomo
































.4


Figure 4-2: Satellite Image of Hurricane Georges prior to Florida landfall. (Satellite
image from National Weather Service)









35
30 T
T25-
>20 SST2
k -SWa7
.15 SWa 14
(010-
5
0
O_ !- I-- I_ --

0 10 20 30
Experiment Day


Mean Salinity over Experiment (%@)
(Expanded Scale)
18.5 m

18-

17.5
17ALm
SST2 SWa7 SWa14


Standard Deviation of Salinity over
Experiment %o)
150



0.30
SST2 SWa7 SWa14


Figure 4-3: Salinity patterns for treatments in Experiment 2. (Coding as follows: 1)
SST2- Stable salinity treatment, 2), SWa7- Square wave with amplitude of 14%o, period
of 4 days and 3) SWa14- Square wave with amplitude of 14%o, period of 4 days)










ES was unUKely to nave oeen anectica, owing to me z 10to n

pugh the experimental tanks. Salinity was not measured uni

e constant throughflow of water into the experimental tanks

Le affect of rainfall, however. As intended, mean salinities

one-half part per thousand of 18 %96o. The standard deviation

nity fluctuation) was proportional to the amplitude of the tr

gure 4-3).

i temperatures over the experiment were nearly identical fo

Figure 4-4, top panel). Average light intensity and the fract

rass depth were slightly greater in the stable salinity treatm4

litude treatment, but not compared to the high amplitude tr

d lower panels).

i nutrient concentrations measured over the experiment froi

ment outflow and the freshwater and saltwater supplies are

im the freshwater source had considerably higher concentra

and DIN than water from the seawater source. An equal mi

nflow had intermediate concentrations of the aforemention


the freshwater source and practically none in the seawater supply.

Thalassia Measurements

The biological responses of Thalassia were negatively influenced by the high












Mean Temperature over Experiment
(C)

28
27
26
25
SST2 SWa7 SWa14


Intensity of Light at Seagrass Depth
(uEIm2/sec)
F= 3.89 (p=0.0605)
1500 -
1000-
500
0
SST2 SWa7 SWa14


Percent Light Reaching Seagrass Depth
F= 4.03 (p=0.0562)
1


0.75 -


0.5
SST2 SWa7 SWa14



Figure 4-4: Mean temperature (top panel), light intensity (middle panel) and percent ligh
reaching depth of seagrass (lower panel) for treatments in Experiment 2. Error bars are
intervals derived from Fisher's least significant difference (LSD) procedure. If the means
are not significantly different, the intervals will overlap 95% of the time. F ratios are
given when differences between means are significant. Percent is expressed in decimal
form.




















2.004 I
02.00 0 1:1 2.04
S0 0.00 0 0.01
0%. 18%. 36. 01. 18%. 36/.
Salinity of Source Salinity of Source


Total KJMldahl Nitrogen (TKN) Dissolved Inorganic Nitrogen (DIN)
1 70.00 1 25.1
-0.8 60.000. 20.1
50.00
0.6 I 40.00 0.6 1.1
0.4 E 30o.00o 0. 1 .
20.00
02 10.00 02 5.1
0 0.00 0 0.(
0%. 18%. 36%. 0o. 186%. 36%.
SalMny of Source Salinity of Source


Nitrit (NO2) Nitrate (N032)
1.40 _----0.02 60.00
1.20 0001. 6000
1.00 00.010
0.60 40.00
0.60s .0.01 ~ 30.00-
_U 000oo o
0.40 0.005 20.00
0.20 10.00
0.00 40 0,0M





46

well, but to a lesser extent. Increases in leaf and shoot number were similar between

plants in the stable and low amplitude salinity treatments, but were higher than in plants

exposed to the high amplitude treatment.

Thalassia green-leaf indices (GLI) declined in all treatments over the first 11 days

of the experiment (Figure 4-6, top panel). Plants in the stable salinity treatment however

recovered, showing a net increase in GLI over the course of the experiment (Figure 4-6,

lower panel). An increase in GLI in plants exposed to the low amplitude treatment

occurred during the last seven days of the experiment, but no recovery occurred in plants

in the high amplitude treatment.

The number of Thalassia shoots had increased by the end of the experiment in all

treatments, although this increase was smallest in the high amplitude fluctuation

treatment (Figure 4-7, top panel). Rhizome length decreased for plants in all treatments,

despite the increase in shoot number (Figure 4-7, middle panel). Belowground biomass

in Thalassia was greatest in plants exposed to the high amplitude treatment, although not

statistically different from plants in the other treatments (Figure 4-7, bottom panel).

For all treatments, Thalassia leaves were shorter after the experiment than they

were initially, however plants in either the stable salinity or the low amplitude treatments

had over 30% more leaves on average after the experiment (Figure 4-8). Those that were

in the high amplitude treatment had only 46% of original measurements. Sprigs of






47



1.5-







-U-- SWa7
0 -. -SWa14
0.5




0
0 5 10 15 20 25 30 35
Experiment Day



Before Treatment to After Treatment
Ratio of GLI
F= 15.16 (p =0.0013)
1.5 -


0.5
0"
SST2 SWa7 SWa14


Figure 4-6: Green-leaf indices for Thalassia over Experiment 2 (top panel) and before tc
after treatment ratios (bottom panel). Error bars on time series chart represent standard
error, those on ratio chart are intervals derived from Fisher's least significant difference
(LSD) procedure. If the means are not significantly different, the intervals will overlap
95% of the time.









Before Treatment to After Treatment
Ratio of the Number of Shoot
F =4.89 (p=0.0365)
1.5 -
1

0.5 -
0
SST2 SWa7 SWa14

Before Treatment to After Treatment
Ratio of Rhizome Length

1.5 -
1 __, _
0.5 -
0
SST2 SWa7 SWa14

Belowground Biomass per Rhizome
Length (glcm)

0.05 -
0.04
0.03-T
0.02 J
0.01
0
SST2 SWa7 SWa14





Figure 4-7: Before to after treatment ratios of shoot number (top panel) and rhizome
length (middle panel), and belowground biomass (bottom panel) after treatments for
Thalassia sprigs in Experiment 2. Intervals around means are based on Fisher's LSD
procedure (p < 0.05). F ratios are given when differences between means are significant.








Before Treatment to After Treatment
Ratio of Leaf Length




0.5

0 -
SST2 SWa7 SWa14

Before Treatment to After Treatment
Ratio of Percent Number of Leaves per
Shoot
F= 5.78 (p= 0.0243)
2 -
1.5 -mb

0.5
0.
SST2 SWa7 SWa14

Average Number of Leaves per Shoot
after Treatment
F= 8.09 (p= 0.0098)
2.5
2
1.5
I
0.5


SST2 SWa7 SWa14

Aboveground Biomass per Short Shoot
(g/ss)

0.3 T---------------I
0.21-
0.1
0
SST2 SWa7 SWa14

Figure 4-8: Leaf characteristics for Thalassia after Experiment 2. Intervals around means








rhizome, and leaf measurements, and the standard deviations of the measurements, are

given in Table 4-1.

Halodule Measurements

Halodule exposed to the high amplitude salinity fluctuation treatment had lower

green-leaf indices and a greater reduction in leaf length and number than plants exposed

to the other two treatments. Halodule tolerated the low amplitude treatment, having

similar responses in green-leaf index and leaf length as plants in the stable salinity

treatment.

Halodule green-leaf index declined overall under all three treatments, however

(Figure 4-9). An increase in GLI for plants in all treatments occurred during the final

seven days of the experiment, although the increase was most rapid in the stable salinity

treatment. Those in the low amplitude treatment fared the best, although not statistically

significantly better than those in the stable salinity treatment according to the ANOVA.

As with Thalassia, the high amplitude treatment resulted in the greatest decline in

Halodule green-leaf index (Figure 4-9, bottom panel).

The number of Halodule shoots increased most in the low amplitude (SWa7)

treatment and least in the stable salinity treatment (Figure 4-10, top panel). The increase

in rhizome length in the high amplitude treatment, however, was not statistically different

from that in the two other treatments (Figure 4-10, middle panel). Halodule sprig

biomass was similar among treatments (Figure 4-10, bottom panel).

Average leaf length increased in Halodule sprigs under stable salinity and low

amplitude treatments. A decrease was observed in the high amplitude treatment (Figure










igs prior to and after experimental treatments.

Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. Dev.
Shoot Number
SST2 2.92 0.83 3.67 1.27
SWa7 3.00 0.85 3.70 1.15
SWa14 3.08 0.83 3.38 1.44

Rhizome Length (cm)
SST2 22.73 11.97 19.75 10.68
SWa7 22.16 9.29 19.37 7.82
SWa14 24.64 10.79 17.15 8.93

Leaf length (cm)
SST2 9.78 2.92 2.58 2.00
SWa7 8.73 3.79 2.81 1.77
SWa14 8.47 4.03 1.24 1 A.48

Leaf Number


SWa14 1.32 0.46 0.63 0.65



1). Those in the low amplitude treatment did not change (Fi

The average number of leaves per shoot in Halodule plan

similar to those in high amplitude treatments. The low amp]

st nnmher nf leaves averagino twn leave. ner Qhnnt fPimire


LMl lliiui-sIVI IIVIll, alvc givenl 111 I IDIc +-L.

Ruppia Measurements

In contrast to Thalassia and Halodule, Ruppia fared worst in the stable salinity



























0 5 10 15 20 25 30
Experiment Day



Before Treatment to After Treatment
Ratio of GLI
F= 8.72 (p=0.0078)



0.5

0
SST2 SWa7 SWa14


Figure 4-9: Green-leaf indices for Halodule over Experiment 2 (top panel) and before to
after treatment ratios (bottom panel). Error bars on time series chart represent standard










Before Treatment to After Treatment
Ratio of the Number of Shoots
F= 6.84 (p= 0.0156)
2 -

.5 ---

0
SST2 SWa7 SWa14


Before Treatment to After Treatment
Ratio of Rhizome Length

2 S
1.5

0.5-
0
SST2 SWa7 SWa14


Total Sprig Biomass per Rhizome
Length (glcm)

1.025 -
0.02
1.015
0.01
1.005 -
0
SST2 SWa7 SWa14


are given when differences between means are significant.









Before Treatment to After Treatment
Ratio of Leaf Length
F =5.63 (p= 0.0260)
1.51


0.5 -
0
SST2 SWa7 SWa14

Before Treatment to After Treatment
Ratio of the Number of Leaves per
Shoot
F= 3.36 (p= 0.0814)
1.5
1 ,3
0.5 -
0 -
SST2 SWa7 SWa14

Average Number of Leaves per Shoot
after Treatment
F= 4.30 (p= 0.0490)
2.5-
2-
1.5 ---
I
0
SST2 SWa7 SWa14




Figure 4-11: Leaf characteristics for Halodule after Experiment 2. All percent are
expressed in decimal form. Intervals around means are based on Fisher's LSD procedure
(p < 0.05). F ratios are given when differences between means are significant.










atment (Figure 4-12, bottom panel). Declines were seen in all treatments during the

iddle period of the experiment, but these were far more dramatic in the stable and high

iplitude treatments (Figure 4-12, top panel).

Ruppia sprigs exposed to both the low and high amplitude treatment had more

oots at the conclusion of the experiment, whereas those in the stable salinity treatment

d less (Figure 4-13, top panel). Ruppia rhizome length nearly tripled (from 2.6 to 9.9

i) in the low amplitude treatment (Figure 4-13, middle panel), but no significant

Tferences were observed in total sprig biomass among treatments (Figure 4-13, bottom

nel).


erages and standard deviations of morphometrics measured
and after experimental treatments.

Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. De
mber
SST2 7.39 3.20 8.70 3.18
SWa7 6.58 2.47 10.13 3.53
SWa14 5.38 2.41 7.38 2.75


SWa14 16.99 9.43 10.79 8.44

Leaf length (cm)
SST2 5.36 1.40 6.30 2.22
SWa7 5.86 2.02 6.12 1.81
SWa14 5.42 1.57 4.40 1.66

























0 5 10 15 20 25 30 3
Experiment Day



Before Treatment to After Treatment
Ratio of GLI

1.5 -S aS a
1 -- ^ I -- e m i ---
0.5 -- 1-----------

SST2 SWa7 SWa14


Figure 4-12: Green-leaf indices (GLI) for Ruppia over Experiment 2 (top panel) and
before to after treatment ratios (bottom panel). Error bars on time series chart represent
standard error, those on percent change chart are intervals based on Fisher's least
significant difference (LSD) procedure. If the means are not significantly different, the









Before Treatment to After Treatment
Ratio of the Number of Shoots
F= 8.77 (p =0.0077)
3 ____________...__...... ________________


i ----------------
3

0-
SST2 SWa7 SWa14


Before Treatment to After Treatment
Ratio of Rhizome Length

5 -...
4


3 ---------------------
2 -

0-
SST2 SWa7 SWa14

Total Sprig Biomass per Rhizome
Length (glcm)

0.015 -
0.01

0.005
0
SST2 SWa7 SWa14



Figure 4-13: Before to after treatment ratios of shoot number (top panel) and rhizome
length (middle panel), and total sprig biomass (bottom panel) for Ruppia in Experiment
2. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are
given when differences between means are significant.










ure 4-14, top panel). A slight increase (1%) occurred in 1

e number of Ruppia leaves increased in both the low and

,reas a slight decrease occurred in the stable salinity treat

el). This treatment also had the least numbers of leaves a


lausucC


cant a


were ot


gure 4-14, bottom panel). Average shoot, rhizome, and leal

rd deviations of the measurements, are given in Table 4-3.



it 3: Rate of Change of Salinity Fluctuation (Square vs. Pyr

y fluctuation was detrimental to Thalassia, regardless of the

module was in poor condition after all treatments, although

-x was slightly less in the stable salinity treatment than in tt

Wppia was most negatively affected in the pyramid wave (gr

tive the stable salinity and square wave treatments, but not

r the other two seagrasses.

physical Measurements


month prior to seagrass collection, with a mean around 5%o (Figure 4-15). Mean










f -- -- 1


I ---I

0.5
0



Be
R


1.5 --
1 -

0.5 --
0--


I" Tni


e Tn
o oft







;T2


it to
nber
hoot
2 (p=





SW.


reatr
ves


3
2
1 ------------------


0
SST2 SWa7 SWa14



Figure 4-14: Leaf characteristics for Ruppia after Experiment 2. Intervals around means
are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences
between means are significant.


~1


I MPM









able 4-3: Averages and standard deviations of morphometrics measured on Rupj
prigs prior to and after experimental treatments.


Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. Dev.
Shoot Number
SST2 5.67 1.98 4.38 2.29
SWa7 6.13 1.57 11.04 4.81
SWa14 7.00 2.83 9.48 5.96

Rhizome Length (cm)
SST2 2.06 1.66 3.54 5.21
SWa7 2.60 1.94 9.89 7.05
SWa14 3.18 3.03 5.69 4.87

Leaf length (cm)
SST2 5.75 1.43 4.08 1.37
SWa7 5.88 1.27 5.73 1.47
SWa14 5.64 1.62 4.44 0.68

Leaf Number
SST2 2.37 0.55 2.17 0.60
SWa7 2.31 0.62 2.54 0.50
SWa14 2.32 0.67 2.36 0.54



'i 1/ *-._ A I- .. A .---* A-i_ 1 t *- _l -- ...... *i l 1L .I_ -11 __-" __ -- .. ..r-


ajor deviations from the designed salinity pattern occurred

chanical problems involving the freshwater pump.

neratures among treatments differed less than 0.2 C (Figm


light at bottom) are statistically similar amongst treatments (Figure 4-17, middle and

bottom panels). Total phosphorus concentrations were an order of magnitude higher in

frk









Salinity prior to Collection for Exp3
14 -

12

10

8



ST1117 .8 12I1/ 1




11/17/98 12/1/98 12/16/98


Tabular values (bottom panel) are based on data collected at 15-minute intervals excepti
for data collected at time of collection).


. p +-t-$ p


~~________Salinity (X) Temperatur
Mean 4.8 25.3
Standard Deviation 3.12 1.73
Minimum 0.79 18.92
Maximum 13.2 29.6
measured at Collection 12 21




- cqI-r-I ..._ ---J ... .^--_- / -- 11- _-_ -\ T ;.


5


IZ/U/ao











nM AA^AA SA,, AAk^










1 10 20 30

Experiment Day




Mean Salinity over Experiment (%o)
(Expanded Scale)
19 ,

18.5-
18
17.5
17
SST3 PW SW

Standard Deviation of Salinity over
Experiment (%o)

15_


Figure 4-16: Salinity patterns for treatments in Experiment 3. (Coding as follows: 1)
SST3- Stable salinity treatment, 2) PW- Pyramid wave with amplitude of 14%o, salinity
changing every 12 hours by 1.5%o, and 3) SW- Square wave with amplitude of 14%o,
period of 8 days)









Mean Temperature over Experiment
(oC)
F= 7.89 (p=0.0105)
25 -----------------
24
23
22
SST3 PW SW


Intensity of Light at Seagrass Depth
(uE/m2/sec)

600 -

300

0

SST3 PW SW


Percent Light Reaching Seagrass Depth

1 -


0.75-


0.5 -
SST3 PW SW







Figure 4-17: Mean temperature (top panel), light intensity (middle panel) and percent
light reaching depth of seagrass (bottom panel) for treatments in Experiment 3. Error
bars are intervals based on Fisher's least significant difference (LSD) procedure. If the















4
2
0
0%. 18%. 36%.
SalKinity of Source


Total KJeldahl Nitrogen (TKN)

s o


20

0fl

0%. 18 36%.
Salinity of Source



[4


).25
).2S -
3.15 -
3 .1

).05
0 0
0%. 18%. 36%.
Salinity of Source


Dissolved Inorganic Nitrogen (DIN
1.2 100.00 .-- ...-.-...
1 an nnf mi __


0.8-
0.6

0.4
0.2
0
0%. 18%. 36
SalUnity of Source



Figure 4-18: Mean nutrient concentrations means


0.15 -3
0.1 E 2
0.05 1I l
0 0

0%. 18%. 36%.
Salinity of Source


Ammonium (NH4)
-1.4 20
12 2 1 -M


0.01 50
0.008 j 40 t
0.006 30 -
0.004 20
*0.002 10 -
0 01
a 0%. 18%.
Salinity of Source



hired during Experiment 3.









assia T

Tha,

tinual, J

treatme

ber occ


if length and


ll i Uh 1111stal UsiniU U tr a1 t eAnt11;; 1 IwasUles t1l7). L L UVt1011 eILIU.UU11 111 trLatLeIns

ex in the stable salinity treatment was less than in the other treatments.


'I.1, %I O "II u ILAA I 1U N LI. U 5 '.Ju ,UJL I, I, aI %IA IL A,.%Y" W I

ed in the pyramid wave treatment (Figure 4-20, middle pan

the stable salinity treatment retained the most biomass, sig

Sthe square wave treatment (Figure 4-20, bottom panel).

;e leaf length declined during the experiment in all treatmer

e number of Thalassia leaves per shoot decreased least in t]


0.35 leaves per shoot (approximately one leaf per three shoots) after the pyramid wave

treatment, in contrast to the square wave (0.76) and stable salinity (1.01) treatments

























SIII I
0 5 10 15 20 25 30
Experiment Day





Before Treatment to After Treatment
Ratio of GLI
F= 15.92 (p= 0.0011)
1.5 -
I i-m

0.5 --
0 -" SW....
SST3 PW SW


difference (LSD) procedure. If the means are not significantly different, the intervals will
overlap 95% of the time.










Before Treatment to After Treatment
Ratio of the Number of Shoots

1 I---, I-----,----- ---


0.5

0 -
SST3 PW SW

Before Treatment to After Treatment
Ratio of Rhizome Length
F= 3.17 (p= 0.0905)
1.5 -
1 -
0.5-
0 -
SST3 PW SW

Belowground Biomass per Rhizome
Length (glcm)
F= 4.29 (p= 0.0490)
0.03 -
0.02
0.01
0~
SST3 PW SW






Figure 4-20: Before to after treatment ratios of shoot number (top panel) and rhizoi
length (middle panel), and belowground biomass (bottom panel) for Thalassia in
Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.
ratios are given when differences between means are significant.













0.5
0
SST3

Before Tnr
Ratio of t




0.5
0-
SST3

Average N


After
r of L
it
0.0009

|


w

eavee


Patme
es pe






rSh

er Sh<


0.5
0
SST3 PW SW

Aboveground Biomass per Short Shoot
(g/ss)

0.25-
0.2 -
0.15
0.1
0.05
0 1
SST3 PW SW
Figure 4-21: Leaf characteristics for Thalassia after Experiment 3. Intervals around
means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when
i *rv -f-------------


teTore i reamnenm 0o Aner i reatmeni
Ratio of Leaf Length


I -I


"zip


SIAR


itmer
e Nur
S
= 16.9!


nber


F:



























PW 5.82 2.21 1.44 2.32
SW 6.67 2.49 3.49 3.20

Leaf Number
SST3 1.69 0.66 1.01 0.90
PW 1.67 0.45 0.35 0.52
SW 1.74 0.54 0.76 0.59



tistically significant. Average shoot, rhizome, and leaf measure

viations of the measurements, are given in Table 4-4.

measurements


--- 0 ----- ---o-------~- -^-d -------- ---_ ----- -_---- --_

fluctuation treatments.

A sharp decline in GLI occurred in Halodule by the ninth day of the experiment










2



1.5 T

0I -*- SST3
IW 1 --T-- PW

-----SW

0.5


0 ---
0 5 10 15 20 25 30 35
Experiment Day



Before Treatment to After Treatment
Ratio of GLI

1.5 -
I M
0.5
0-
SST3 PW SW




Figure 4-22: Green-leaf indices for Halodule over Experiment 3 (top panel) and before
to after treatment ratios (bottom panel). Error bars on time series chart represent standard
error, those on percent change chart are intervals based on Fisher's least significant
difference (LSD) procedure. If the means are not significantly different, the intervals will
overlap 95% of the time.










Increased more than in the stable salinity treatment (Figui

crease in rhizome length occurred in the square wave trea

in the others (Figure 4-23, middle panel). The higher Hal

nity treatment was not statistically different from the other

)m panel).

e decline in average leaf length and number of leaves per ,

:nts (Figure 4-24, top and middle panels). In addition, the

shoot after the experiment were very close, with 1.47 leave

atment versus 1.42 leaves counted in both the square and ]

(Figure 4-24, bottom panel). Average shoot, rhizome, anc

ndard deviations of the measurements, are given in Table

measurements


:or Ha

fthee

5). Al


wave treatments, although statistically significant differences occurred only in rhizome

length (top and middle panels of Figure 4-26 and 4-27). Average shoot, rhizome, and











Before Treatment to After Treatment
Ratio of the Number of Shoots
F= 3.66 (p= 0.0687)
2 -
.5 -

5 -
0
SST3 PW SW

Before Treatment to After Treatment
Ratio of Rhizome Length

.5


.5 -
0 -
SST3 PW SW

Total Sprig Biomass per Rhizome
Length (glcm)

0.01 T------------------------
.008 T
,006
.004
.002
0
SST3 PW SW


Figure 4-23: Before to after treatment ratios of shoot number (top panel), and rhizome
length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment
3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are











Before Treatment to After Treatment
Ratio of Leaf Length

1.5 -


0.5 -
0-
SST3 PW SW

Before Treatment to After Treatment
Ratio of Number of Leaves per Shoot

1.5 -


0.5

0 S
SST3 PW SW


1119ra ghrily"1%IftV 0%9 1 fttv M^ lo -









~au -->.;.n. Vsa 4iiIU L4a1uiLLU UcVL.IUI UI IiUipiipumclrL-_ maIU-U1TCu Oun natoaute
rigs prior to and after experimental treatments.

Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. Dev.
Shoot Number
SST3 5.29 2.18 5.87 2.67
PW 4.71 2.29 6.88 2.85
SW 5.00 1.84 7.17 3.06

Rhizome Length (cm)
SST3 9.42 4.02 7.70 4.19
PW 10.29 2.88 9.90 6.15
SW 8.96 4.33 8.67 3.68

Leaf length (cm)
SST3 5.89 1.72 2.68 1.20
PW 6.18 1.74 2.81 1.12
SW 5.73 1.91 2.84 0.75


I












..T ~ ~ ~ A T \k .^ ^ -*1









0 5 10 15 20 25 30
Experiment Day




Before Treatment to After Treatment
Ratio of GLI

'1- ^ -- 4-
1.5

0.5 S--W
0 -
SST3 PW SW


S1J. I. LIJA'.,t, U r.V I ..I LJ.L'WlL X, i|CL '.. LiI IC V. 11, lIlL',. V %0lI UCUV. I .t 1 YAL'O..L 0 iI.MOIX O11ld tJI l C"tI.0 L
difference (LSD) procedure. If the means are not significantly different, the intervals wil
overlap 95% of the time.













Before Treatment to After Treatment
Ratio of Number of Shoots

1.5 -


1 -------------------
0-
SST3 PW SW

Before Treatment to After Treatment
Ratio of Rhizome Length

1.5

0.5 .--.

0 -
SST3 PW SW

Total Sprig Biomass per Length (glcm)

0.015 T----------------
0.015

0.005 -
0.005









Before Treatment to After Treatment


1.5


0.5

SST3

Before Tr
Ratio of Ni

1.5
1
0.5
0 -
SST3


0 Iatm4


Patm(
r Shi



-u


atmei
nber


Aftei
save







N


2-
1
0 -:::a
SST3 PW SW





Figure 4-27: Leaf characteristics for Ruppia after Experiment 3. Intervals around means
are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences
between means are significant.


Before Treatment to After Treatment
J m i m, .=


I


I









)rigs prior to and after experimental treatments.


SW 3.92 0.86 2.49 1.48

Leaf Number
SST3 2.69 0.45 1.94 0.96
PW 2.57 0.49 1.28 0.80
SW 2.57 0.77 1.49 0.92



Experiment 4: Salinity Fluctuation around Different Means


cited at higher salinities. Ruppia had similar responses to b

ses in leaf length and number were greater when salinity fl

range.


treatments is given in Figure 4-29. The treatments are coded as follows: 1) M9- square

wave with an eight day period, an amplitude of 9%o, oscillating around a mean of 9%o

(ranging between 0 and 18%o), and 2) M27- square wave with an eight day period, an


Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. Dev.
Shoot Number
SST3 12.54 6.17 8.96 5.45
PW 9.96 4.13 6.23 5.95
SW 12.13 5.83 6.33 5.83

Rhizome Length (cm)
SST3 13.17 6.18 7.52 5.90
PW 11.08 5.08 3.42 3.91
SW 12.13 4.88 5.79 5.82
















































1113/9 1120/Zw 1/27/W 2



~~___~___Salinity (%.) Temperature (=
Mean 13.12 22.7
Standard Deviation 2.4 2.87
Minimum 1.99 12.84
aximum 17.95 27.35
measured at Collection 16 26


Salinity prior to Collection for Exp 4

8 X








"fA
6M





0 k
4 Al-



116199 1/21/99 2/5/9


Temperature prior to Collection for Exp 4


A A A









40-
35-
30
025 -
20 --- M27
j15
C10
5
0
0 10 20 30
Experiment Day


Mean Salinity over Experiment (%.)

30 ,-,-

210

0


M9 M27


Standard Deviation of Salinity over
Experiment (%o)

12 -
8
4
0
M9 M27

Figure 4-29: Salinity patterns for treatments in Experiment 4. (Coding as follows: 1) T9-
Square wave with amplitude of 9%o, period of eight days, oscillating around 9%0, and 2)
T27- Square wave with amplitude of 9%o, period of eight days, oscillating around 27%o)










Design called for square waves with eight day periods, p


antic


ig bottom, and therefore a significantly greater intensity of

'el (Figure 4-30). The low salinity treatments received high

us, due to elevated concentrations in the freshwater supply,

alinity treatment received slightly higher concentrations of

1).

[easurements

rnity fluctuation affected Thalassia in this experiment, howe

ien salinity fluctuated around a higher mean. The number (

of leaves per shoot increased in the high salinity treatments

-n-leaf indices of Thalassia decreased over the course of th

mnte althoigrh the df.rlintp wua areatfr in the. lPce calinp- tr

Before to after treatment ratios of the number of shoots and rhizome lengths after

the experiment were slight and varied little between the treatments (Figure 4-33, top and

middle panel). The greater belowground biomass in Thalassia in the less saline treatment































Percent Light Reaching Seagrass Depth
F= 314.29 (p=0.0001)
) I
[)75


Figure 4-30: Mean temperature (top panel), light intensity (middle panel) and percent
light reaching depth of seagrass (bottom panel) for treatments in Experiment 4. Error











Total Phosphorus (TP)
0A 10,
-0.3 80.0
S 6,0
0.2
4.0
-0.1 2.0
0 0.0
0%. 180. 36%.
Sanity of Source

Total KJeoldMahl Nitrogen (TKN)
1.2 50.0
-1 40.0+


0 0.0 -
0.4 0.


otal Dissolved Phosphorus (TDP)
0.53 40
0.25
3.0
02
0.15 20
0.1 v10
0.0-L
0.05
0 0.0 -


1%. 0%. 18%. 361
Salinty of Source

Nitrite (NO2)
0.0035 12 .0
S0.003 10.0-
S*00.0025 8.0
| | |:2oSooU Io
0.002 [
0.0015 E 6.0 i1-
*0.001 4.0
0.0005 2.0
0 0.0-


S02 10.0 0.2
0.1 T 0.1
0 .0 0
0%. 18%. 36%.
SaKnitv of Source


1 1__ _ __ _ _ 1 __ _ __ _


Orthophosphate (P04)
-0.14
0.12
0.08
0.04

I. 18.. 036% 2
6 18%. 36%.


)issolved Inoi



I


Am



I


NI



I


E-4- -l


I














H--4--T









I I
0 5 10 15 20 25
Experiment Day





Before Treatment to After Treatment
Ratio of GLI
F= 11.36 (p=0.0071)
-1.5-1



0.5


Figure 4-32: Green-leaf indices for Thalassia over Experiment 4 (top panel) and before
to after treatment ratios (bottom panel). Error bars on time series chart represent standard
error, those on percent change chart are intervals based on Fisher's least significant









Before Treatment to After Treatment
Ratio of Number of Shoots

1.5 -1
1 --

0.5 -
0 -
M9 M27

Before Treatment to After Treatment
Ratio of Rhizome Length

1.5 -
1 --------,--x ----

0.5 -
0 -
M9 M27

Belowground Biomass per Rhizome
Length (g/cm)

0.025
0.02
0.015
0.01
0.005
0
M9 M27




Figure 4-33: Before to after treatment ratios of shoot number (top panel) and rhizome
length (middle panel), and belowground biomass (bottom panel) for Thalassia in
Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F
ratios are given when differences between means are significant.








decreased less in the more saline treatment, and the number of leaves increased by a

average of over 20% (Figure 4-34, top and second panel). Thalassia sprigs in the le:

saline treatment ended with approximately 40% fewer leaves per shoot. Abovegrou

structures in the less saline treatment had more biomass, however (Figure 4-34, botti

panel). Average shoot, rhizome, and leaf measurements, and the standard deviation,

the measurements, are given in Table 4-7.

Halodule Measurements

Overall declines in Halodule GLI were also observed in both treatments (Fig

4-35). In the more saline treatment, Halodule GLI increased during the initial third

experiment. During this time the GLI of sprigs in the less saline treatment sharply

declined. By Day 15, GLI reached its lowest level in the less saline treatment, where

remained for the remainder of the experiment.

The number of Halodule shoots in the more saline treatment decreased less E

treatment than those in the lower salinity treatment (Figure 4-36, top panel). Rhizoi

length increased in the higher salinity treatment as well (Figure 4-36, middle panel).

Sprig biomass was significantly greater in the less saline treatment than those in the

saline treatment, however (Figure 4-36, bottom panel).

Leaf lengths declined by about the same amount in both treatments (Figure 4

top panel). Halodule shoots had an average of 14% more leaves after the more salir

treatment in contrast to the 32% decrease in the less saline treatment (Figure 4-37, n

panel). On average, Thalassia and Halodule had an additional leaf per shoot in the i

saline treatment (Figure 4-34, third panel, Figure 4-37, bottom panel). The average

number of leaves was statistically significant higher for Thalassia and Halodule in t








Before Treatment to After Treatment
Ratio of Leaf Length

1.5 -


0.5 -.

0 -
M9 M27

Before Treatment to After Treatment Ratio
of Number of Leaves per Shoot
F= 7.99 (p= 0.0179)
2
1.5

0.5-
0-
M9 M27

Average Number of Leaves per Shoot
after Treatment
F= 5.25 (p=0.0449)
3-
2
1

0 -
M9 M27

Aboveground Biomass per Short Shoot
(g/ss)

025 -
0.2
0.15
0.1
0.05
0 -
M9 M27

Figure 4-34: Leaf characteristics for Thalassia after Experiment 4. Intervals around
means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when
differences between means are significant.


































lard deviations of the measurements, are given in Table 4-8

surements

ia varied little in GLI between the two treatments, in sharp

ecies (Figure 4-38). Green-leaf index increased in both tre<

The increase in the less saline treatment was slightly large


greater in the less saline treatment (Figure 4-39, bottom panel).

Leaf length changed little in either treatment (Figure 4-40, top panel). The


Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. De
Shoot Number
M9 4.39 1.23 4.03 1.30
M27 3.92 1.23 3.92 1.18
Rhizome Length (cm)
M9 33.11 13.52 33.43 11.21
M27 28.15 8.87 28.06 8.48
Leaf length (cm)
M9 18.27 4.50 8.19 7.42
M27 18.37 4.75 11.34 4.93
Leaf Number
M9 2.39 0.60 1.42 1.20
M27 2.33 0.86 2.53 2.06


i'rivr nnnr in m I Hiitr IPX- ExrwinnIFFItiIHI IT'EHLrInIUIIL


I I


I






























) 5 10 15 20 25
Experiment Day






Before Treatment to After Treatment
Ratio of GLI
F= 38.49 (p= 0.0001)

1.5

-I'





















Before Treatment to After Treatment
Ratio of Rhizome Length
F= 12.67 (p=0.0047)
1.5 -


0.5 -
0 -
M9 M27

Total Sprig Biomass per Rhizome
Length (glcm)
F= 5.50 (p= 0.0410)


Figure 4-36: Before to after treatment ratios of shoot number (top panel) and rhizome
length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment
4. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are


Before Treatment to After Treatment
Ratio of the Number of Shoots


1.5
1-

0.5
0


M9 M27


M9 M2Z


I r









Before Treatment to After Treatment


0.5 I
0


Before 1
Ratio o


4 _____


P1111


eatme


-=11.


Afte
r of I
.t
0.006:
O.O6


atmnei
Ds pei


1-


IVIh7 IVI& I I


after Treatment
F= 14.00 (p= 0.0038)
4
3
2
1


Elm






92


Table 4-8: Averages and standard deviations ofmorphometrics measured on Halodule
sprigs prior to and after experimental treatments.


Pre-Experiment Post-Experiment
Average Std. Dev. Average Std. Dev.
Shoot Number
M9 4.89 1.33 2.42 1.61
M27 4.19 1.09 3.75 1.90

Rhizome Length (cm)
M9 12.03 5.19 9.23 5.32
M27 10.74 4.54 10.77 4.75

Leaf length (cm)
M9 7.24 1.84 3.86 2.09
M27 8.42 2.05 5.61 0.93

Leaf Number
M9 2.25 0.50 1.53 0.97
M27 2.25 0.44 2.56 0.65



8% in the more saline treatment (Figure 4-40, middle panel). The number of Ruppia

leaves per shoot, however, was similar in both treatments (Figure 4-40, bottom panel).

Average shoot, rhizome, and leaf measurements, and the standard deviations of the

measurements, are given in Table 4-9.




Full Text

PAGE 1

RESPONSES OF SUBTROPICAL SEAGRASSES TO FLUCTUATIONS IN SALINITY WITHIN AN EXPERIMENT AL FACILITY By THOMAS C CHESNES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

PAGE 2

Copyright 2002 By Thomas Chesnes

PAGE 3

This manuscript is dedicated to Rose Trivigno. I know that you would be proud.

PAGE 4

ACKNOWLEDGMENTS Words cannot express my appreciation for those who supported me in this endeavor. My supervisory committee has been without a doubt instrumental to my success. I thank Evan Chipouras for his support from the earliest stages of this project, as well as his ability to help me keep things in perspective. I thank Frank Nordlie for introducing me to ecological research and mentoring my undergraduate project. Without him, I certainly would not be in this profession. I thank Stephen Davis, whose enthusiasm for algae is contagious and inspiring. I especially thank Thomas Crisman who helped reintroduce me to my ecological roots. His patience and assistance were essential in getting through a difficult period. Lastly, I thank my chairman Clay Montague, whose mix of humor and scientific curiosity has inspired me to develop a strong scientific ethic, an interest in the workings of the universe ( on all temporal and spatial scales), and a never-ending pursuit of truth over accepted ideas. This study would not be possible without funding and logistical support from the Everglades National Park Interagency Science Center in Key Largo, Florida and the South Florida Water Management District, who also provided nutrient analysis. The Aylesworth Foundation and Greening UF provided additional support. Special thanks to those who assisted in data collection: Casie Regan, Christos Anastasiou, Joel Dudas, Jessica DiEgidio, Ben Loughran, and last, but certainly not least, Bridget Chesnes. Finally, I give thanks to my family and friends who have always supported me. I am truly blessed. lV

PAGE 5

TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iv ABSTRACT ...................................................................................................................... vii CHAPTERS 1 IN'fRODUCTION ............................................................................................................ 1 2 DESCRIPTION OF FACILITY DESIGNED FOR SALINITY FLUCTUATION AND PILOT STUDY ........................................................................................................ 9 Description of Facility .................................................................................................... 9 Materials and Methods of Pilot Study .......................................................................... 13 Results of Pilot Study ................................................................................................... 16 Discussion of Pilot Study .............................................................................................. 24 3 MATERIALS AND METHODS FOR MAIN STUDY .................... ............................ 28 Seagrass Collection and Acclimation ........................................................................... 28 Description and Protocol of Facility Experiments ........................................................ 29 Data and Statistical Analysis ............................................................ ........................... 35 4 RESULTS OF EXPERIMENTS 2 THROUGH 7 IN THE SALINITY FLUCTUATION FACILITY ON KEY LARGO ............................................................................... 39 Experiment 2: Effect of Amplitude of Salinity Fluctuation on Seagrasses .................. 39 Experiment 3: Rate of Change of Salinity Fluctuation (Pyramid vs. Square Wave) .... 58 Experiment 4: Salinity Fluctuation around Different Means ........................................ 78 Experiment 5: Extreme Salinity Fluctuation and Effect of Period ............................... 96 Experiment 6: Salinity Fluctuation and Circulation: Constant Through Flow of Water vs. Air Circulation in Experimental Tanks ............................................................. 114 Experiment 7: Effect of Salinity Fluctuation and Reduction of Light ........................ 134 V

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5 CORRELATIONS BETWEEN BIOLOGICAL MEASUREMENTS AND PHYSICAL VARIABLES IN FACILITY EXPERJMENTS ................................................. .147 Correlations with Salinity ........................................................................................... 147 Correlations with Temperature, Light, and Water Clarity .......................................... 155 Correlations with Water Nutrient Concentrations ...................................................... 160 Correlations with Field Conditions at Collection Sites .............................................. 165 6 PRODUCTIVITY AND LEAF OSMOLALITY-RESULTS OF EXPERJMENT 8 ... 173 Introduction ................................................................................................................. 173 Materials and Methods ................................................................................................ 175 Data Analysis for Experiment 8 .................................................................................. 178 Results ........................................................ .... ......... .. . ................................................ 179 Discussion ................................................................................................................... 190 7 DISCUSSION ............................................................................................................... 196 Overall Discussion ...................................................................................................... 196 Conclusions ......................................................................................... ..... ................... 201 REFERENCES ................................................................................................................ 203 BIOGRAPHICAL SKETCH ........................................................................................... 207 Vl

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RESPONSES OF SUBTROPICAL SEAGRASSES TO FLUCTUATIONS IN SALINITY WITHIN AN EXPERIMENT ALF ACILITY By Thomas C. Chesnes May 2002 Chairman: Clay L. Montague Major Department: Environmental Engineering Sciences The sparsity of seagrass communities within the ponds and bays of northern Florida Bay may be due to the wide fluctuations of salinity in these habitats. Seven experiments were performed in a facility designed for salinity fluctuation. Three seagrass species, Thalassia testudinum, Halodule wrightii, and Ruppia maritima, were transplanted to the facility and subjected to varying degrees of salinity fluctuation. Treatments were varied according to salinity wave mean, amplitude, frequency, and suddenness (slope) of change. Two experiments crossed the effects of salinity fluctuation with methods of water circulation and reductions in available light. A final experiment, performed in buckets, measured the photosynthetic and internal osmotic responses of seagrasses to fluctuations in salinity. To assess the loss of photosynthetic material in the seagrasses during the experiments, a green leaf index (GLI) was developed, based on the percent green leaves Vil

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present per turion, prorated for partial green coloration. Leaf and rhizome morphometrics were measured prior to and following the treatments. Thalassia was the most sensitive to salinity fluctuation. Biological parameters were negatively correlated with increasing salinity wave amplitudes, frequencies, and suddenness of change. The effect of salinity fluctuation was dampened when salinity fluctuated within a range of higher salinities. Salinity fluctuation was more of an influence on Thalassia survival than the reduction of light. Halodule condition was impaired by salinity fluctuation, but not to the extent experienced by Thalassia. Green leaf indices decreased with increases in salinity wave amplitude, frequency, and slope. The number of turions per sprig correlated negatively with increasing amplitude and more sudden changes in salinity. Halodule survival was enhanced by fluctuations within a higher salinity range. Ruppia was the most resilient of the seagrass species tested. This seagrass was able to survive all of the salinity fluctuation treatments. Increasing the frequency of salinity change did have a negative impact on this seagrass, resulting in lower green leaf indices and number of leaves. In order to survive fluctuating salinities, seagrasses must regulate their internal osmotic concentrations in relation to their surrounding waters. Ruppia osmoregulated more quickly than Thalassia and Halodule and may be the key to its resiliency Vlll

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CHAPTER 1 INTRODUCTION Since 1881, human activities have disrupted the natural flow of freshwater from the Everglades into Florida Bay (Fourqurean and Robblee 1999). The Everglades watershed has been engineered and managed for agriculture, flood control, and water supply for the growing population in South Florida (Light and Dineen 1994). Water management protocols involving alterations in freshwater flow can change the regime of salinity fluctuations in the downstream estuary. Sudden releases of flood water may create rapid drops in salinity, whereas water held back in times of drought may amplify salinity increases downstream (Montague and Ley 1993). Changes in community structure will be most noticeable in the estuarine salinity fields closest to land (Estevez 2000). Salinity related problems arise when estuaries receive too much or too little fresh water, or water at improper times (Odum 1970). These habitats are characterized as being harsh due to the salinity changes, especially when compared to the more static conditions typifying marine or freshwater habitats (Deaton and Greenberg 1986). Changes in salinity occur rapidly in the shallow basins located within the northern land margin of Florida Bay. Periods of high freshwater inflow during the onset of the rainy season, in tropical storms (Chesnes 1999), or water management releases can cause areas 1

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2 with marine strength salinities to become fresh, in some cases within a matter of days (Mclvor et al. 1994, Montague and Chipouras 1998). The meteorologically driven patterns of salinity are clearly seen in the salinity record of Little Madeira Bay, located within the northern land margin of Florida Bay (Figure 1-1 ) During the course of the dry season, salinity steadily rose from 10%o (December 1998) to above 30%0 (May 1999). At the onset of the wet season (May / June 1999), salinity dropped nearly 20%0 in the first week, and approximately 30%0 within three. Acute fluctuations in salinity also occurred during this time period. During July and August of 1998, salinity fluctuated between 1 and 18%0, with a period of approximately 4 days between peaks and troughs. The amplitude of the fluctuation varied over the course of the month (Figure 1-1 ). Acute fluctuations also occurred in October and December of 1998, January 1999, and again in the late summer / early autumn of 1999. The amplitude of salinity change was higher in July and August of 1999 than in 1998, when changes of over 20o/oo were seen over four day periods. The pattern of salinity change during transition from high to low salinity in the ponds and bays of the land margin of Florida Bay is more acute than during the transition from low to high (Montague and Chipouras 1998). Increases in salinity can be caused by evaporation during the dry season or by winds pushing saline water from Florida Bay into the estuary. The transition to saltier conditions is thought to be more gradual, and the loss of stenohaline freshwater macrophytes may be a more progressive process as t he salinity slowly becomes elevated. Conversely, a sharp decline in the abundance of

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35 30 25 20 : i cu 15 en 10 5 0 J t t \ .. Wt, ~\... ~I 11 I 't A Ari I I V ,~ J I r \ Jul-98 Aug-98 Sep-98 Oct-98 Nov 98 Dec-98 Jan 99 Feb 99 Mar-99 Apr 99 May 99 Jun-99 Jul 99 Aug-99 Sep-99 Figure 1-1: Daily average salinity measured at the mouth of Taylor River in Little Madeira Bay. (Data from Patino and Hittle unpublished)

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4 stenohaline marine macrophytes follows the acute exposure to low salinity at the onset of the rainy season (Montague and Chipouras 1998). Fluctuations in salinity correlate with decreases in submerged macrophytes and benthic animal density (Montague and Ley 1993, Fears 1993, Jones 1999) as well as fouling organisms (Chesnes 1999). Each individual has a range of salinity tolerance and a narrower range of optimal salinity (Remane and Schlieper 1971 ). Species richness is especially low in the salinity range between 5 and 8 %0. This paucity in flora and fauna can be explained by the habitat instability of estuaries (Deaton and Greenberg 1986). The salinity fluctuation hypothesis suggests that this lack of biota may be the result of extreme salinity fluctuation (Montague and Ley 1993). Few organisms have evolved the physiological mechanisms required for life in highly variable environments (Deaton and Greenberg 1986). Most studies of salinity and submerged macrophytes have focused on tolerances to extreme high and low salinities. Few studies have looked at the effects of salinity fluctuation on submerged aquatic vegetation (Montague and Ley 1993, Fears 1993, Jones 1999, Durako 2000). In some cases, the significance of salinity as a water quality factor is unknown since its effect is confounded with the presence of other environmental conditions {Twilley and Barko 1990). For example, a study by Tomasko and Hall (1999) found that the use of field studies for estimating the lower salinity tolerances of seagrasses might be inappropriate for those systems where water clarity is positively correlated with salinity. Seagrasses are a vital component of estuarine systems. If salinity fluctuates too much, communities may not become established (Montague and Ley 1993, Montague

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5 1996). Seagrasses provide habitat for many benthic and pelagic organisms (Doering and Chamberlain 2000), stabilize sediments by slowing water movement and increasing sedimentation (Ogden 1980) and can form the basis of the plant-based and detrital-based food chains (Klug 1980). Three seagrass species, Thalassia testudinum, Halodule (Diplanthera) wrightii, and Ruppia maritima, are commonly found within the northern land margin of Florida Bay. Little Madeira Bay (Figure 1-1) was included in earlier macrophytes monitoring studies (Montague and Ley 1993, Montague and Chipouras 1998). All three species inhabited these areas during these studies, though Ruppia was not as prevalent in the earlier one when salinities were higher and did not fluctuate as much. The degree to which rapid and dramatic changes in salinity can influence the distribution, abundance, and community composition of seagrasses is unknown Its importance should be greater at land margins where outwelling freshwater meets saltwater pushed in by coastal tides and winds. If salinity fluctuation is important, then changes in water delivery to the coast that is under control of water managers will be expected to directly influence the distribution and abundance of seagrasses in ways that could affect habitat for fish and birds. Two other physical factors that are affected by features of freshwater discharge have received the most attention by water managers and seagrass scientists, specifically alterations in light and nutrients. In this dissertation attention will be given to a third and perhaps more influential environmental factor, salinity fluctuation. Thalassia testudinum (turtlegrass) is the dominant marine angiosperm of the Caribbean (Patriquin 1973). It is characterized by a creeping rhizome which has often

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6 been found branched which gives rise to erect branches bearing leaves (Phillips 1960). This species is considered stenohaline (Jagels 1973), although it was found in habitats spanning the entire salinity gradient from fresh to marine strength within the northern land margin of Florida Bay (Montague and Chipouras 1998). Although not thoroughly explored, Thalassia may have a temperature optimum near 30 C and a salinity optimum near 30%0 (Zieman 1975). Fluctuations in salinity, especially those ranging into fresher water, may be detrimental to this species. Halodule wrightii (shoalgrass) had a wider range of salinity tolerance than Thalassia in experiments by McMillan and Moseley (1967). In salinity tolerance experiments performed by McMahan (1968), Halodule survived in salinities ranging from 9 to 52.2%0, but died in salinities of 3 5%0 and in excess of 70o/oo. This species was also found in habitats spanning a salinity gradient from nearly fresh to hypersaline within the northern land margin of Florida Bay (Montague and Chipouras 1998). Based on these findings, Halodule is expected to be more tolerant to salinity fluctuation than Thalassia. Widgeongrass, Ruppia maritima, has a nearly cosmopolitan distribution and worldwide importance as a waterfowl food (Kantrud 1991). Ruppia has the widest known range of salinity tolerances of any genus of submerged aquatic vegetation This species has been found in waters ranging from Oto 390o/oo with optimum growth between 0.5 and 31 o/oo (Durako 2000). However, a study by McMillan and Moseley (1967) found Ruppia to be less tolerant ofhypersaline conditions than Thalassia and Halodule. In the field monitoring studies conducted by Montague and Ley (1993) and Montague and Chipouras (1998) Ruppia was found to be common but ephemeral in ponds located along

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7 streams within the land margin of Florida Bay but rarer in the more open, saline bay habitats. Salinity fluctuation may be detrimental to this species despite its reported wide salinity tolerance range. A facility designed for salinity fluctuation experiments was built in Key Largo, Florida (Anastasiou 1999). Controlled experiments with replicated treatments are possible in this facility. In addition, doing experiments in such a facility gives a researcher the ability to manage, or at least closely monitor, other environmental factors. Within the facility, seagrasses can be subjected to various salinity treatments, which differ in rate, frequency, and amplitude of salinity change. Salinity change and the rate of this change were shown to directly affect the health and growth of transplanted individuals of these species (Jones 1999). In the facility, the responses of the seagrasses can be closely monitored in terms of changes in morphology, productivity, and osmoregulation. The experimental facility can be used to produce alterations in salinity similar to what was seen in the salinity record during the periods of acute salinity fluctuation (Figure 1-1 ). In addition, the amplitudes and frequencies of salinity change can be modified to expose seagrasses to more or less extreme conditions. If salinity fluctuation is detrimental to the overall health of seagrasses, reductions in biomass, photosynthetic material, and growth should be evident in the plants exposed to these conditions. The impairment of photosynthetic material should result in reduced primary production. If these resources are required by the plants to maintain the functioning of mechanisms used for osmoregulation, there will be less available for building biomass. Consequently, survival and growth will be impaired. In order to test this hypothesis, seven experiments

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8 were performed to explore the responses of the three seagrasses found at the northern land margin of Florida Bay to varying degrees of salinity fluctuation. An eighth experiment was done in Gainesville, Florida to examine the photosynthesis and salinity acclimation rates of seagrasses subjected to salinity fluctuation

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CHAPTER2 DESCRIPTION OF FACILITY DESIGNED FOR SALINITY FLUCTUATION AND PILOT STUDY Description of Facility A schematic diagram of the facility used for conducting salinity fluctuation experiments is provided in Figure 2-1. The facility is located on the grounds of the National Park Service's Key Largo Ranger Station of Everglades National Park, mile marker 98.6 bayside Overseas Highway (Figure 2-2). A more complete description of the facility and discussion of its design criteria, constraints, construction, and testing is given by Anastasiou (1999). Saltwater for the facility was supplied by a well drilled 13.7 meters deep into porous carbonate rock connected to eastern Florida Bay. Salinity in the well was approximately 36%0. Chlorinated fresh water was supplied by the Florida Keys Aqueduct Authority. The fresh water was held in a 242 m 3 painted concrete reservoir (formerly a swimming pool), which increased residence time for dechlorination. The seagrass experiments occurred within twelve 1.1 m 3 experimental tanks (Figure 2-3). Salinities were manipulated by manually adjusting valves on a seawater freshwater mixing manifold (center of Figure 2-3). Up to four different salinity regimes could be delivered simultaneously to randomly chosen replicate experimental tanks by connection of hoses of 3.81 cm diameter to a distribution manifold. In general, water was allowed to flow continuously through each experimental tank at approximately 16 m 3 d1 9

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Actuated solenoid valve 18) Ball valve manual Saltwater p i pe Freshwater pipe Sal i nijy mixing p i pe Dashed l i nes I ndicate pipe under s l ab Exper i mental tank fill pipe Experimental tank drain pipe .......... Ma i n drain pipe l Experimental tank fill Experimental tank drain o Stub-up connection to mixing system Saline mix dislrlbuUo v alves i Dra i n pipe Concrete s l ab $ 149" . . . . : . . 10 ... 0 . . ... . . .. . From 60,000 gal freshwater reservoir fed by city water supply water supply pumps activated by level sensors in head tanks Pumps triggered when level drops 1' from max From saltwater well . . .. . . Dra i n pipe cleanout . . . ..... .... 5 ft EXPERIMENTAL SALINITY FLUCTUATION FACILITY 14 ~, Figure 2-1: Schematic Design of Experimental Facility designed for salinity fluctuation (Anastasiou 1999).

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11 E > 10km Figure 2-2: Location of collection sites and the Key Largo Ranger Station (Map from the USGS)

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12 Figure 2-3: Photograph of Experimental Facility. Experimental tanks are in the foreground, storage tanks for fresh and sea water are located on the hill unless flow was ceased due to experimental design. A constant inflow facilitates water circulation. Water level was maintained at approximately 50 cm by a stand pipe drain. Salinity was controlled by varying the quantities of fresh and salt water delivered through the mixing manifold to the tanks. When fresh water was delivered to a tank containing water of a higher salinity, an extension was added to the inflow pipe to deliver water to the bottom of the water column. This enhanced mixing and flushing of the higher density water Complete turnover of a 1.1 m 3 tank from one extreme salinity to another occurred within 2 or 3 hours at an inflow rate of 16 m 3 d1

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13 Materials and Methods of Pilot Study Seagrass Collection and Experiment Preparation Plants used in this experiment were collected in April 1998. Sprigs of Ruppia were collected from the northern region of Seven Palm Lake (N 25 11.69', W 80 43.42'), and Halodule from Terrapin Bay (N 25 09.49', W 80 43.84), both areas located in north-central Florida Bay (Figure 2-2). Thalassia sprigs were collected from a floating mat found nearby the Key Largo Ranger Station (Figure 2-2). Twenty-four polyethylene tubs (Rubbermaid Inc., measuring 57 X 46 cm) were filled with approximately 22.7 kg of Quikrete Commercial Grade fine sand. Halodule and Ruppia rhizomes were planted at a density of 40 to 60 percent coverage in separate tubs. Sprigs were selected that had the presence of green leaves and apical meristems. Thalassia sprigs were prepared so that each rhizome had only one shoot as well as an apical meristem. In each Thalassia tub, five rows of three individual seagrasses were planted, totaling fifteen. Once planted, the tubs of Ruppia, Halodule, and Thalassia were placed in holding tanks of 1.1 m 3 with salinities of 15, 25, and 36%0, respectively. The plants remained at these salinities for approximately two months. Eight of the facility's twelve experimental tanks were used in this experiment. Salinity fluctuation treatments were assigned randomly to the tanks. Four tanks received a stable salinity treatment (SSTl), maintained at 18o/oo. Two tanks received a four day period of salinity fluctuation (P4D) in which salinity was alternated between 0 and 36o/oo every two days. Two other tanks received an eight day period of salinity fluctuation between the same extremes (P8D). One tub of each seagrass was randomly selected from its respective holding tank and placed into a randomly assigned position (North, Center,

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14 or South) within each of the eight experimental tanks. Treatments were applied during the period from June 30 to August 1, 1998. Protocol of Experiment Salinity, temperature, and amount of ambient light, light just below the water surface and at the tank bottom were measured daily. The percentage oflight reaching the seagrass was computed using Beers Law. Light measurements were taken within two hours of solar noon, in an unshadowed area of each tank, using a quantum photometer (Li-Cor Inc. model LI-185 B). Salinity and temperature were measured using a refractometer (Leica model TS) and mercury thermometer, respectively. Water inflow rates were checked three times a week by timing the filling of a bucket of known volume. Adjustments were made as needed to ensure that each tank was receiving a similar influx of water. Epiphytic algae were removed daily from the seagrass by gently pinching the seagrass blades in an upward motion, and from the tubs and tank walls by scrubbing with a brush. Noticeable turbidity caused by the suspension of epiphytes subsided within an hour of scrubbing. Daily monitoring of the seagrasses involved counting the number of shoots present, evaluating the color of the leaves, and noting the presence of new leaves. Color was rated using an index (Table 2-1) similar to that used in a related field transplant study (Jones 1999). The presence of green leaves indicated a healthy shoot, while the other colors were assumed to indicate a physiological impairment of the leaf. White leaves were assumed to be dead. An overall estimate of amount of green, yellow, brown, and white coloration was made for Thalassia sprigs. Green, brown, and white ratings were made for Halodule and Ruppia because any yellow stage was too short-lived to be

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15 reliably assessed A grid was constructed to divide the Halodule and Ruppia seagrass tubs into nine equal areas of 230 cm 2 each. Color index ratings from Table 2-1 were recorded for each grid region Color ratings were converted to a percentage, based on the mean percent within the color rating range {Table 2-1). Complete shoot counts and color analyses of all three species were performed on days 1, 2, 3, 15, and 31 of the experiment. On the other days, two grid regions were randomly selected for coloration surveys and shoot counts in each tub of Ruppia and Halodule. For Thalassia on these interim days, two of the fifteen shoots were randomly selected in each tank for coloration analysis. Table 2-1: Color rating scale used in pilot study. Index Rating Percent Color Range Mean% 6 100 % color 100 5 95% to less than 100% color 97 5 4 66% to less than 95% color 80 3 33% to less than 66% color 50 2 5% to less than 33% color 20 1 Greater than 0% but less than 5% color 2 5 0 0% color 0

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16 Results of Pilot Study Physical Parameters Average daily salinity measurements for each treatment are shown in Figure 2-4. Within the stable salinity tanks, salinity varied from the desired l 8%0. Adjustments to the salinity of the treatment tanks caused subsequent changes in the flow rates and head pressure in the other tanks. This adjustment, in turn, caused unintended deviations in the salinity of the tanks designated for stable salinity treatment. Unexpected salinity fluctuations were also caused by mechanical failure. On Days 9 and 29 the saltwater pump failed sometime during the previous night, which resulted in lower salinities than desired within both the experimental and control tanks for an unknown period of time. Nevertheless, the total difference in range of average salinity amongst treatments is less than one part per thousand (Figure 2-4, middle panel) and the standard deviation of salinity was considerably higher in the fluctuating treatments as per the design (Figure 24, bottom panel). Mean temperatures of the treatments were similar, deviating from each other by less than 0.5 C (Figure 2-5, top panel). The fraction of surface light reaching seagrass depth exhibited a wider range amongst treatments, however (Figure 2-2, lower panel). Suspended algae that were present primarily in the freshwater storage and delivery system increased the turbidity over that in the seawater system. Seagrasses in the stable salinity treatment (SSTl) received the smallest fraction of available light.

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40 30 0 20 C: ca en 10 0 4 ... ., .. 0 17 ttrfl -~.IJl .,rlYl \ 1h. .. ... --~ --"--'-V ~.J 10 20 Experiment day 30 40 Mean Salinity over Experiment (o/oo) SST1 p8d p4d Standard Deviation of Salinity over Experiment (%0) 10 -t-------t SST1 p8d p4d -+-SST1 --o-p8d -a-p4d Figure 2-4: Salinity patterns for pilot study. Salinity over time (top panel), mean salinity (middle panel), and standard deviation of salinity (bottom panel) are shown. (Coding is as follows: l)SSTlStable salinity treatment, 2) p8dSquare wave with amplitude of l 8%0 period of eight days, and 3)p4dSquare wave with amplitude of l 8%0 period of eight days)

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18 Mean Temperature over Experiment (OC) 32 ...------------------, SST1 p8d p4d Intensity of Light at Seagrass Depth (uE/m 2 /sec) SST1 p8d p4d Percent Light Reaching Seagrass Depth SST1 p8d p4d Figure 2-5: Mean temperature (top panel), light intensity at seagrass depth (middle panel), and percent light reaching seagrass depth (bottom panel) for treatments in pilot study. Percent is expressed in decimal form

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19 Seagrass Measurements Figure 2-6 shows the average color ratings obtained from Thalassia. Ratings are represented by a stacked bar for each experimental day. Green is given at the base of the bar with the other colors sequentially above from chlorotic to white on top. Subsamples varied in coloration for all Thalassia treatments (Figure 2-6). It is evident from the initial green coloration ratings that Thalassia sprigs used in the SST treatment were in better condition than those in the other treatments (Figure 2-6). Daily color ratings were transformed to produce accumulations of color ratings over time. At each sample day, color ratings were converted to percent colorations. The percent colorations of each daily measurement were added together and divided by the number of samples. This analysis reduced the noise caused by the subsampling Color accumulations are given for Thalassia in Figure 2-7. Color accumulations are represented by a stacked bar for each experimental day. Green colorations are given at the base of the bar with the other colors sequentially above. Thalassia green accumulations increased only in the stable salinity treatment (Figure 27, top panel). No green accumulation occurred in the fluctuating treatments over the course of the experiment. Brown and yellow color accumulations decreased with subsequent samples in the fluctuation treatments, replaced with increased white coloration. Starting green coloration ratings of Halodule sprigs were similar (Figure 2-8) Green coloration ratings oscillated around their initial values in the stable salinity treatments, but fell (while white ratings increased) in the fluctuation treatments in samples after ten days of experimentation. Color accumulations were similar over the course of the stable salinity treatment (Figure 2-9, top panel). In both fluctuation

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20 SST Treatment 8 Cl 6 white C brown ... 4 yellow 0 0 u 2 Cl green 0 1 6 11 16 21 26 31 Experiment Day P8D Treatment 8 6 white Cl C ;:i brown /}_ 4 ... yellow 0 0 u 2 green 0 1 6 11 16 21 26 31 Experiment Day P4D Treatment 8 Cl 6 white C brown 4 ... yellow 0 0 u 2 green 0 1 6 11 16 21 26 31 Experiment Day Figure 2-6: Color ratings for Tha/assia over pilot study.(* denotes ratings derived fr om average of all plants sampled)

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21 SST Treatment Color Accumulations 120 100 C white 0 80 ; E brown .2 60 yellow 0 (.) 40 l'!!I green 0 20 0 1 6 11 16 21 26 31 P8D Treatment Color Accumulations 120 100 C white 0 80 ; ca brown ... .2 60 yellow 0 (.) 40 green 0 20 0 1 6 11 16 21 26 31 P4D Treatment Color Accumulations 120 1 00 C white 0 80 ; ca brown ... .2 60 yellow 0 (.) 40 green 0 20 0 1 6 11 16 21 26 31 Figure 2-7 : Coloration accumulation for Tha/assia in pilot study

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22 SST Treatment 8 6 ,,CJI C white 4 brown ... 0 0 green 0 2 0 1 6 11 16 21 26 31 Experiment Day P8D Treatment 8 6 CJI C white 4 brown ... 0 m green 0 0 2 0 1 6 11 16 21 26 31 Experiment Day P4D Treatment 8 6 CJI C white i 4 brown ... 0 0 green 0 2 0 1 6 11 16 21 26 31 Experiment Day Figure 2-8: Color ratings for Halodule over pilot study.(* denotes ratings derived from average of all plants sampled)

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23 SST Treatment Color Accumulations 120 100 C: 0 80 white :;; "' ... .S? 60 brown 0 l?I green (.) 40 0 20 0 1 6 11 16 21 26 31 PSD Treatment Color Accumulations 120 100 C: 0 80 :;; white f! .S? 60 brown 0 (.) 40 green 0 20 0 1 6 11 16 21 26 31 P4D Treatment Color Accumulations 120 100 C: 0 80 :;; white f! .S? 0 60 brown (.) 40 green 0 20 0 1 6 11 16 21 26 31 Figure 2-9: Coloration accumulation for Halodule in pilot study

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24 treatments, green color accumulations in Halodule decreased, while white accumulations increased. Green coloration ratings were highest in Ruppia exposed to the stable salinity treatment; however; green ratings in the fluctuation treatments persisted throughout the experiment (Figure 2-10). White coloration ratings steadily increased in Ruppia over the experiment for all treatments, although greatest white color ratings were seen in the two fluctuation wave treatments. Ruppia sprigs exposed to the eight day fluctuation period (P8D) treatment had the greatest decline in green color accumulation (Figure 2-11 ). Although green color accumulations remained high in the four day fluctuation period (P4D) treatment, white color accumulations increased over the experiment. Discussion of Pilot Study The results of the pilot study demonstrate a distinct effect of salinity fluctuation on seagrass survival and growth. Confounding of environmental variables (mean salinity, temperature and light) was minor and was unlikely to have measurably influenced the results. In the three species tested, exposure to fluctuations in salinity resulted in the replacement of healthy green leaf tissue with dead white tissue. Although an effect of fluctuation was clear in all three species when compared with stable salinity, an affect of period of the salinity fluctuation was not evident in the Thalassia and Halodule experiments In Ruppia, longer periods were more detrimental. The method of surveillance did not include the monitoring of individual plants. Subsampling did not allow the responses of individual sprigs to be evaluated. Repeated samples on the same plants would be more indicative of the effects over time

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25 SST Treatment 8 CJl6 C white 4 brown ... 0 l!I green 0 u 2 0 1 6 11 16 21 26 31 Experiment Day P8D Treatment 8 CJ! 6 C white 4 brown ... 0 0 green u 2 0 1 6 11 16 21 26 31 Experiment Day P4D Treatment 8 CJ! 6 C wh i te 4 brown ... 0 El green 0 u 2 0 1 6 11 16 21 26 31 Experiment Day Figure 2-10 : Color ratings for Ruppia over pilot study (* denotes ratings deri v ed fr om a v erage of all plants sampled)

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26 SST Treatment Color Accumulations 120 100 C 0 80 ; white E .2 60 brown 0 u 40 II green 0 20 0 1 6 11 16 21 26 31 P8D Treatment Color Accumulations 1 20 100 C 0 80 ; white ca ... .2 60 brown 0 u 40 l!I green 0 20 0 1 6 11 16 21 26 31 P4D Treatment Color Accumulations 120 1 00 C 0 80 ; white ca ... .2 60 brown 0 u '$. 40 a green 20 0 1 6 11 16 21 26 31 Fig ur e 2 1 1 : Co l o r ation accumulation for Ruppia in pilot study

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27 The effect of salinity fluctuation on Thalassia was probably obscured in the pilot study due to the poor starting condition of sprigs planted in the fluctuation treatments. Although trends were evident in the color accumulation analysis, the relatively small amount of starting green coloration may have exacerbated the effects of salinity fluctuation, giving the plants little chance of survival. Acclimation to appropriate salinities and stricter criteria for selection in experiments may remedy this problem in future experiments. Sparse distributions of seagrass communities in northern land margin of Florida Bay may be influenced by the frequent changes of ambient salinity. If seagrasses are losing photosynthetic material due to the affects of salinity fluctuation, energy reserves may not be adequate for vegetative growth and rhizomal elongation, the main methods ofreproduction in both Thalassia and Halodule (McMillan and Mosely 1967, Phillips 1960). Further experiments are necessary to explore the responses of seagrasses to varying degrees of salinity fluctuation, including the amplitude, frequency, and suddenness of change. In addition, the mean about which salinity fluctuates, light, and nutrient interactions can be examined experimentally. With a more complete and quantitative examination, the distributions and abundances of seagrasses may be predicted using models that include salinity fluctuation as well as light, nutrients, temperature and average salinity.

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CHAPTER3 MATERIALS AND METHODS FOR MAIN STUDY (EXPERIMENTS2THROUGH7) Seagrass Collection and Acclimation All seagrass species used in the salinity fluctuation Experiments 2 through 7 were collected within Little Madeira Bay (N 25 11.39', W 80 38.34'), a basin located in the northern land margin of Florida Bay (Figure 2-2). Sprigs of Thalassia testudinum, Ruppia maritima, and Halodule wrightii were collected that consisted of a length of rhizome with shoots and roots attached and with healthy leaves. Shoots consist of a sheath (in Thalassia and Ha/odule) and at least two leaf blades. Sprigs were carefully removed from the sediment and placed into coolers half filled with water from the collection site. Sprigs of Halodule wrightii and Ruppia maritima were selected if they had a minimum of three shoots and a growing rhizome tip present. Sprigs of Thalassia testudinum had a minimum of two shoots and a growing rhizome tip. Bottom salinity of water sampled from seagrass depth was measured at the collection site with a refractometer. Temperature was measured in the water column with a mercury thermometer attached to a string. In addition, salinity and temperature data from Little Madeira Bay were obtained from the South Florida Information Access (SOFIA) website (Patino and Hittle, unpublished) to determine field conditions in the month prior to collection. 28

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29 The seagrasses were transported to the experimental facility in coolers and allowed to acclimate in the experimental tanks for a minimum of two weeks. During the acclimation phase, salinities were adjusted from the salinity at the collection site to the mean salinity of the experiments in increments less than 1 o/oo per day. Seagrasses were not monitored during this time. Acclimation periods and ranges for experiments 2 through 7are given in Table 3-1. Description and Protocol of Facility Experiments Description of Experiments The effects of different characteristics of salinity fluctuation were tested in a series of six experiments (numbered 2 through 7). Amplitude, period, suddenness of change and mean salinity were tested. A general description of the experiments is g i ven in Table 3-2, and a more detailed description of treatments is given in Table 3-3. The seven column headings in Table 3-3 describe various aspects of the pattern of salinity fluctuation. The variables tested in each experiment are designated in the shaded column. In all but Experiment 4, fluctuating wave patterns treatments were tested against stable salinity treatments. In Experiment 3, the effect of the large sudden changes of the square wave was compared to more gradual changes by using a pyramid wave of the same period (8 days) and amplitude (14o/oo). An example of a pyramid wave and a square wave are shown in Figure 3-1. Frequent salinity changes are required to simulate a pyramid wave in the salinity fluctuation facility. For the pyramid pattern, salinity was changed in a series of small steps of approximately 1.5 o/oo every twelve hours. The effects of constant

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30 Table 3-1: Acclimation periods and ranges for the facility experiments. The end date of the acclimation period was the first day of the experiment. Exgeriment Start Date End Date Salinit~ Range Duration at Target Salinitv {18o/oo) 2 9/29/98 10/24/98 10-18%o 10 days 3 12/17/98 1/3/99 12-18%o 5 days 4 2/6/99 3/3/99 16 18 %o 22 days 5 4/27/99 5/19/99 30 18 %o 5 days 6 6/29/99 7/13/99 18 %o 15 days 7 10/4/99 10/27/99 7 18 %o 8 da s Table 3-2: Brief Summary of Experiment 27 Replicates refer to number of experimental tanks with identical treatments. Number of Number of Replicates Experiment # Scope of Experiment Treatments Per Treatment 2 Effect of Amplitude 3 4 3 Suddenness of Change (Square vs. Pyramid Wave) 3 4 4 Effect of Mean 2 6 5 Effect of Period 3 3 6 Effect of Water Circulation Method and 4 3 Salinity Fluctuation (crossed design) 7 Effect of Light and Amplitude (crossed design) 5 2

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31 Table 3-3: Detailed Summary of Experiments 2-7. Shaded columns designate salin i ty fluctuation or environmental parameters manipulated in each experiment. Column headings Wave Type, Mean, Amplitude, Period, Salinity Minimum and Maximum describe the salinity fluctuation treatment. Inflow is the rate of which water is delivered to each experimental tank. Percent Sun gives the fraction of sunlight each experimental tank received during an experiment. Exp 2 : Test of the Amplitude of Salinity Fluctuation Wave Type Mean Amplitude Wave Period Sal. Min. Sal. Max. Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter / sec) (% ) SST2 stab l e 18 0 0 18 18 0 13 100 SWa14 square 18 14 4 4 32 0 13 100 SWa7 squa r e 18 7 4 11 25 0 13 1 00 Exp 3: Test of the Rate of Change of Salinity Fluctuation Wave Type Mean Amplitude Wave Period Sal. Min Sal. Max Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter / sec) (% ) SST3 stable 18 0 0 18 18 0 13 1 00 SW square 18 14 8 4 32 0 13 100 PW fil! mid 18 14 8 4 32 0 13 1 00 Exp 4: Test of Fluctuation around differing Mean Salinities Wave Type Mean Amplitude Wave Period Sal. Min Sal. Max Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter/ sec) (%) T9 squa r e 9 9 8 0 18 0 13 1 00 T27 square 27 9 8 18 36 0 13 1 00 Exp 5: Test of Extreme Salinity Fluctuation and Period Wave Type Mean Amplitude Wave Period Sal. Min. Sal. Max Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter/ sec) ( %) SST5 stab l e 18 0 0 18 18 0 13 1 00 SWp4 squa r e 18 18 4 0 36 0 13 1 00 SWp8 square 18 18 8 0 36 0 13 1 00 Exp 6 : Water Circulation Method and Salinity Fluctuation Wave Type Mean Amplitude Wave Period Sal. Min Sal. Max Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter/ sec) ( %) SSTt stab l e 18 0 0 18 18 0.13 1 00 SSTb stable 18 0 0 18 18 0 (air) 1 00 SWt square 18 14 8 4 32 0.13 1 00 SWb square 18 14 8 4 32 0 air 1 00 Exp 7 : Low and High Amplitude Fluctuation Crossed with Full and Reduced Sunlight Wave Type Mean Amplitude Wave Period Sal. Min. Sal. Max Inflow Percent Sun (%o) (%o) (days) (%o) (%o) (liter/ sec) (%) SSTs stable 18 0 0 18 18 0 (air) 30 SWa7u square 18 7 8 11 25 0 (air) 100 SWa7s square 18 7 8 11 25 0 (air) 30 SWa14u square 18 14 8 4 32 0 (air) 100 SWa14s square 18 14 8 4 32 0 (air) 30

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32 salinity max amplitude mean salinity salinity min period Time Figure 3-1 : Characteristics of a salinity fluctuation wave The square wave is shown in black the pyramid wave is gray. Frequency is the reciprocal of period.

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33 throughflow of water versus air bubbling as a means of water circulation in the experimental tanks was tested along with salinity fluctuation in Experiment 6. The effect of shading was tested along with salinity fluctuation in Experiment 7. In Table 3-3, the column titled "inflow" designates the constant inflow of water into the experimental tanks. For Experiment 6, an air pump was installed to aerate the saltwater head tanks and selected experimental tanks. Aeration helped reduce sulfides in the saltwater head tanks and promoted circulation in the experimental tanks (Anastasiou 1999). Water did not flow through the bubbled experimental tanks, rather they were filled initially with water and aerated. Evaporative losses were replaced daily with freshwater, a task not required in flow through experimental tanks. When it was time to change the salinity, a brief period (approximately 60 minutes) of flow through occurred until the new salinity was achieved. In Experiment 7, the effects of reduced sunlight were crossed with fluctuation patterns of low and high amplitude. In this crossed design, interactions between light and amplitude can be revealed. This was important because the freshwater at times was more turbid than the saltwater, potentially confounding the results. Randomly selected experimental tanks were covered by 70 % shade cloths to create reduced light conditions (30% full sun). The treatments are designated in Table 3-3 under the column labeled "Percent Sun". Ruppia was not tested in this experiment because there was not adequate numbers present at the collection site and other areas of northern Florida Bay where Ruppia has been previously found. Furthermore, an unshaded stable salinity treatment was not included because of a lack of acclimated sprigs. An unexpected dieoff of Thalassia and Halodule occurred during the acclimation period. The stable salinity

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34 treatments of the five prior experiments were unshaded, therefore this treatment was not included to ensure an adequate number of replicates for the remaining treatments tested in this experiment. Sprig Preparation and Planting Immediately prior to each experiment, morphometric measurements were taken on all seagrass samples. Sprigs with a growing rhizome tip and at least two shoots with green leaves were randomly selected from the acclimation chamber. The number of shoots was counted. Rhizome length and mature and immature leaf lengths were measured to the nearest half millimeter. Sprigs were then planted into polyethylene tubs (26.5L, Rubbermaid, Inc.), measuring 57 x 46 x 15 cm deep. Fine-grained sand (Quikcrete brand) was used as sediment in all tubs. Two sprigs of each of the three species were planted in each tub in random order, for a total of six sprigs per tub. Three tubs were submerged in each experimental tank, for a total of eighteen sprigs per tank. Sprig and Tank Monitoring and the Green-Leaf Index During the course of Experiments 2 through 7, leaves were monitored every two days. The total numbers ofleaves were counted on each of the three youngest shoots on the sprig, followed by a count of the number ofleaves with any green coloration. Finally, a green-leaf index was assessed for each shoot, based on the number of green leaves present, prorated visually for partial green coloration. For this index, the number of leaves with green coloration on a shoot was multiplied by an estimate of the fraction of all leaves on that shoot that was green. This calculation is performed for the three youngest shoots on a sprig and averaged to give the index value for that sprig. An example of the calculation of the index is illustrated in Figure 3-2.

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35 Daily measurements of salinity, temperature, water inflow, and light were taken in each tank. Epiphytic growths on seagrass leaves were removed daily as in the pilot study. Water samples were collected weekly and sent to the South Florida Water Management District (SFWMD) for analysis of total phosphorus, dissolved inorganic nitrogen, and total Kjeldahl nitrogen. Experiments lasted between 24 and 32 days (Table 3-4) Following an experiment, morphometric measurements were repeated, after which the seagrass sprigs were dried and weighed. Data and Statistical Analysis Statistics were performed using statistical software (Statlets Version l IB, NWP Associates, Inc.). For each experiment, one-way ANOVA tests were run to compare the responses by each species to each treatment. Fisher's least significant difference (LSD) procedure of was used to identify differences among means of statistically significant ANOV As. The significance threshold was set at p < 0.05 Pearson product moment correlations and multiple regression analyses were used to explore and quantify relationships between salinity fluctuation and the seagrass responses from among the entire data set of all experiments. For these analyses, salinity fluctuation was quantified across experiments by using seven wave descriptors: mean salinity, standard deviation of salinity, maximum amplitude, suddenness of salinity change, number of salinity changes per day, significant frequency and absolute frequency The method of calculation for each descriptor is given in Table 3-5. Mean

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Shoot~ (Cluster of leaves) 36 Percent Green-Leaf (as a decimal): 0 + 0.5 + 0 Total per Shoot: 0.5 0 5 + 0.75 1.25 GU for Sprig (Average of Shoots): 4.25 / 3 Rhizome Short-Shoot .__ (Sheath) 1.0 + 0.5 + 1.0 2 5 GLI = 1.42 F igure 3-2: Example of Green-Leaf Index calculation. For this example assume b l ack colorations on l e aves are green

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37 Table 3-4: Dates and durations of the six facility experiments. Experiment # Treatment Start Date Treatment End Date Duration 2 10/25/98 11/25/98 32 days 3 1/4/99 2/3/99 32 days 4 3/4/99 3/29/99 26 days 5 5/20/99 6/13/99 24 days 6 7/6/99 7/31/99 24 days 7 10/28/99 11/22/99 26 days salinity, standard deviation, and maximum amplitude describe aspects of the amplitude of the salinity waves, where the others address frequency characteristics of the waves Suddenness gives insight into the rate and magnitude of salinity change by quantifying the maximum slope that occurred in the entire experiment. Due to the exploratory nature of this analysis, the significance threshold was set at p < 0.001 to act as a filter to determine the most important correlations. To account for any confounding effects of salinity fluctuation, correlations involving temperature, light, and water nutrient concentrations were made with plants in the stable salinity treatments only. The influence of these physical parameters will be masked by the detrimental affects of salinity fluctuation, so seagrasses in the fluctuation treatments were excluded from these analyses. To determine if prior field conditions had any effect on the seagrasses collected, correlations were made between the mean salinities and temperatures measured during the month prior to collection, the salinity at time of sprig collection and the initial green leaf indices and leaf morphometric measurements of all seagrasses used in the experiments. Rhizome length and shoot number were not analyzed since these measurements are influenced more by the collection process than the field conditions.

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38 Table 3-5: Descriptors to quantify characteristics of salinity fluctuation waves. Wave Descriptor Mean Salinity Standard Deviation of Salinity Maximum Amplitude Suddenness of Salinity Change Number of Changes Per Day Significant Frequency Absolute Frequency Method of Calculation Mean salinity calculated from daily measurements Standard deviation calculated from daily measurements 1 /2 (maximum minimum salinity measurement) Maximum slope of salinity wave Number of salinity changes/ Number of days 1/2 ((number of crossovers -1) / number of days) where "crossover" occurs when salinity crosses designed mean 1/2 (# of peaks+# of troughs/ total days)

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CHAPTER4 RESULTS OF EXPERIMENTS 2 THROUGH 7 IN THE SALINITY FLUCTUATION FACILITY ON KEY LARGO Experiment 2: Effect of Amplitude of Salinity Fluctuation on Seagrass Higher amplitude salinity fluctuation was detrimental to both Thalassia and Halodule in this experiment. Ruppia was more resistant. Biological responses suc h as green-leaf index, shoot and leaf number, and rhizome length, were more favorable for Ruppia in the lower amplitude fluctuation treatments than in the stable salinity treatment. The response of Ruppia in the high amplitude treatment was similar to its response i n the stable salinity treatment. Overview of Physical Measurements Salinity and temperature measurements during the month prior to plant collection at the mouth of Taylor River in Little Madeira Bay are plotted and summarized in Figure 4-1 (Patino and Hittle, unpublished). The daily temperature averages are calculated from measurements taken at fifteen-minute intervals Hurricane Georges crossed the collection site on September 23 1998 (Figure 4-2), during a period of already low salinity The plants were collected six days after landfall. The salinity pattern of the three treatments is shown in Figure 4-3. The treatments are coded as follows : 1) SST2stable salinity treatment, 2) SWa7square wave treatment with an amplitude of7%o and a period of four days, and 3) SWal4a square wave treatment with an amplitude of 14%0 and a four day period. An indirect hit by Tropical 39

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25 20 1 5 1 0 5 0 35 30 25 20 15 10 5 8129/98 0 8/29 / 98 40 Salinity prior to Collection for Exp 2 I"'''' HG 9113198 9128/98 Temperature prior to Collection for Exp 2 ..__.... ....... ...........~-~ 9 / 3/98 9/8/98 9 /1319 8 9 / 18/98 9 /23/9 8 9 /2 8/98 Salinity("-) Temperature (C) Mean 7.98 29 5 Standard Deviation 6.74 1 87 Minimum 0 53 25 3 Maximum 19 3 33.56 Measured at Collection 9.5 29 5 Figure 4-1 : Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished). Salinity values were measured at 15minute intervals. Gaps in the salinity record represent where no data were reported. Temperature values are daily averages. Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection). Hurricane Georges crossed the collection site on 23 September (designated by HG on the salinity chart).

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41 Figure 4-2: Satellite Image of Hurricane Georges prior to Florida landfall. (Satellite image from National Weather Service)

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42 35 -,---------------, 30 25 0 20 c: 15 cu u, 10 5 0 -+------"-T"""--------r-------r--~ 0 10 20 30 Experiment Day Mean Salinity over Experiment (o/oo) (Expanded Scale) SST2 SWa7 SWa14 Standard Deviation of Salinity over Experiment (o/oo) 15 ~----------------~ 10 +---------------D 5 -i------------t 0.30 0 -+~----.----'-....... '-------,.-......................... ---! SST2 SWa7 SWa14 ~ssr2 ---swa7 -&-SWa14 Figure 4-3 : Salinity patterns for treatments in Experiment 2. (Coding as follows: 1) SST2Stable salinity treatment, 2), SWa7Square wave with amplitude of 14%0 period of 4 days and 3) SWal4Square wave with amplitude of 14%0, period of 4 days)

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43 Storm Mitch occurred on November 5, 1998, Day 11 of the experiment. The salinity of the treatments was unlikely to have been affected, owing to the 2 to 3 hour turnover time of water through the experimental tanks. Salinity was not measured until the day after landfall. The constant throughflow of water into the experimental tanks may have dampened the affect of rainfall, however. As intended, mean salinities for all treatments were within one-half part per thousand of 18 %0. The standard deviation of salinity ( an index of salinity fluctuation) was proportional to the amplitude of the treatment, as expected (Figure 4-3). Mean temperatures over the experiment were nearly identical for the three treatments (Figure 4-4, top panel). Average light intensity and the fraction of surface light at seagrass depth were slightly greater in the stable salinity treatment, compared to the low amplitude treatment, but not compared to the high amplitude treatment (Figure 44, middle and lower panels). Mean nutrient concentrations measured over the experiment from the stable salinity treatment outflow and the freshwater and saltwater supplies are given in Figure 45. Water from the freshwater source had considerably higher concentrations ofTP, TDP, P04, TKN, and DIN than water from the seawater source. An equal mix of these waters in the l 8%0 inflow had intermediate concentrations of the aforementioned nutrients. Nitrite concentrations, however, were highest in the mixed water, with a lesser amount in the freshwater source and practically none in the seawater supply. Thalassia Measurements The biological responses of Thalassia were negatively influenced by the high amplitude salinity fluctuation treatment. The low amplitude treatment was detrimental as

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44 Mean Temperature over Experiment (OC) 28 -r~ ;:;::;;:;;.::::;:: ;:;----; ;=::..:::: ::;---,~ ~ ;;--i 27 26 25 -1---==~--,---____J~__,::.,a__-r--____i........;.~.J_--f SST2 SWa7 SWa14 Intensity of Light at Seagrass Depth (uE/m 2 /sec) F= 3.89 (p=0 0605) 1500 ..-------------------, 1000 -1--r.-= 500 0 -1-----"-'= SST2 SWa7 SWa14 Percent Light Reaching Seagrass Depth F= 4.03 (p=0.0562) SST2 SWa7 SWa14 Figure 4-4: Mean temperature (top panel), light intensity (middle panel) and percent light reaching depth of seagrass (lower panel) for treatments in Experiment 2. Error bars are intervals derived from Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant. Percent is expressed in decimal form.

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Total Pho5phoru5 (TP) 14 00 ..-------------~ 0 5 12 00 0 4 10 00 i" 8 00 ,:. 6 00 4 00 2 00 0.00 0 3 0 2 !. 0 1 _.._ ___ __ -I0 36 ... Salinity of Source Total Kjeldahl Nitrogen (TKN) 70 00 -r--------------,60 00 50 00 i' 40 00 30 00 20 00 10 00 0 00 o ... Salinity of Source 1 40 1 20 1 00 i' 0 60 0 60 0 40 0 20 0 00 o ... 0 8 0 6 a 0 4 ._ 0 2 0 Nitrite (NO2) 18 ... Salinity of Source Total Dluolved Pho5phoru5 (TOP) 10 00 0 3 i" 6 00 ci, 12 00 lL I 0 4 6 00 0 2 ._ 4 00 0 1 2 00 0 00 -+-_. ___ -+ 0 0 ... 18 ... Salinity of Source Dissolved Inorganic Nitrogen (DIN) 70 00 ..-----------~ 60 00 50 00 i' 40 00 30 00 20 00 10 00 0 00 0 02 0 015 0 01 0 005 0 36 ... o ... :::, ci, !. Salinity of Source 60 00 50 00 40 00 i' 30 00 20 00 10 00 0 00 o ... 0 8 0 6 a 0 4 !. 0 2 0 Nitrate (NO3+2) 18 ... Salinity of Source Figure 4-5: Mean nutrient concentrations measured during Experiment 2. 6 00 i' 4 00 2 00 0 00 Orthopho5phate (PO4) Salinity of Source Ammonium (NH4) 25 00 ..-----------, 20 00 i' 15 00 10 00 5 00 0 00 36 ... 0 8 0 6 0 4 0 2 0 Salin tty of Source =ci, !. 0 25 0 2 0 15 ci, 0 1 !. 0 05 0 0 35 0 3 0 25 0 2 =ci, 0 15 !. 0 1 0 05 0 Vt

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46 well, but to a lesser extent. Increases in leaf and shoot number were similar between plants in the stable and low amplitude salinity treatments, but were higher than in plants exposed to the high amplitude treatment. Thalassia green-leaf indices (GLI) declined in all treatments over the first 11 days of the experiment (Figure 4-6, top panel). Plants in the stable salinity treatment however recovered, showing a net increase in GLI over the course of the experiment (Figure 4-6, lower panel). An increase in GLI in plants exposed to the low amplitude treatment occurred during the last seven days of the experiment, but no recovery occurred in plants in the high amplitude treatment. The number of Thalassia shoots had increased by the end of the experiment in all treatments, although this increase was smallest in the high amplitude fluctuation treatment (Figure 47, top panel). Rhizome length decreased for plants in all treatments, despite the increase in shoot number (Figure 47, middle panel). Belowground biomass in Thalassia was greatest in plants exposed to the high amplitude treatment, although not statistically different from plants in the other treatments (Figure 47, bottom panel) For all treatments, Thalassia leaves were shorter after the experiment than they were initially, however plants in either the stable salinity or the low amplitude treatments had over 30% more leaves on average after the experiment (Figure 4-8). Those that were in the high amplitude treatment had only 46% of original measurements. Sprigs of Thalassia exposed to the higher amplitude salinity fluctuation averaged less than one leaf per shoot (0.63), whereas those in the low amplitude and stable salinity treatments averaged 1. 71 and 1.58 leaves per shoot, respectively (Figure 4-8). No statistically significant difference was observed among aboveground biomass. Average shoot,

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:::::i 1 (!) ,!! II) II) ,!! 0.5 0 5 47 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio of GLI F = 15 16 (p = 0 0013) 35 1.5 -.--------------------, 1 t-___cn;t ~-,----i:!~ r---,---0 5 +--------------0 ...._----------------~ SST2 SWa7 SWa14 -+-SST2 ---swa7 -6-SWa14 Figure 4-6 : Green-leaf indices for Thalassia over Experiment 2 (top panel) and before to after treatment rat i os (bottom panel). Error bars on time series chart represent standard error, those on ratio chart are intervals derived from Fisher's least significant difference (LSD) procedure If the means are not significantly different the intervals will overlap 95% of the time.

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48 Before Treatment to After Treatment Ratio of the Number of Shoot F =4 89 (p = 0 0365) 1.5 --,--------------------, 1 +----' ........ 0 5 +--------------------, 0 -'------------------~ SST2 SWa7 SWa14 Before Treatment to After Treatment Ratio of Rhizome Length 1.5 ...i===================================================i 1 t--r::=:i::::.r-,-,:::.~~::..:i-.---,3~~ ~ r 0.5 -+---------------------; o ~----------------~ SST2 SWa7 SWa14 Belowground Biomass per Rhizome Length (g/cm) 0.05 -r------------------, 0 04 -+---------------,..,,= ---~ ,.:;----< 0 03 -I-__,. 0.02 0.01 0 +-~=:;.&_-,----L-------'----r--'-----..:...&.--i SST2 SWa7 SWa14 Figure 47: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and belowground biomass (bottom panel) after treatments for Tha/assia sprigs in Experiment 2. Intervals around means are based on Fisher's LSD procedure (p < 0.05) F ratios are given when differences between means are significant.

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49 Before Treatment to After Treatment Ratio of Leaf Length SST2 SWa7 SWa14 Before Treatment to After Treatment Ratio of Percent Number of Leaves per Shoot F = 5 78 (p= 0.0243) 2 ....------------------, 1 ~ t===~~ ~ ia===~==:::i ~7::c~ =--1= ==~==~=.~~=~==~ 0.5 -1------------------L ~,-......----i o ..J.._ ______ _ __ ____ __, SST2 SWa7 SWa14 Average Number of Leaves per Shoot after Treatment F= 8.09 (p= 0.0098) 2 5 ~------------------, 2 -1----------~ .------------, 1 5 -t----f!JF};:[ 1 +--l l 0 .5 +----1, 0 +-_ ............... .....__,---___. __ ....__,---__._ __ ..___----i SST2 SWa7 SWa14 Aboveground Biomass per Short Shoot (g/ss) 0 3 -.------------, 0.2 t-~~~ --;:::.~t.-=-. -----r "711T.:':!'1 ----1 0.1 +---il 0 4--_ .i..:....:.,;,;..;..;.:..J.__,--__J ____ .J...__.,----.1.__,~,L__--4 SST2 SWa7 SWa14 Figure 4-8: Leaf characteristics for Thalassia after Experiment 2. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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50 rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 4-1. Halodule Measurements Halodule exposed to the high amplitude salinity fluctuation treatment had lower green-leaf indices and a greater reduction in leaf length and number than plants exposed to the other two treatments. Halodule tolerated the low amplitude treatment, having similar responses in green-leaf index and leaf length as plants in the stable salinity treatment. Halodule green-leaf index declined overall under all three treatments, however (Figure 4-9). An increase in GLI for plants in all treatments occurred during the final seven days of the experiment, although the increase was most rapid in the stable salinity treatment. Those in the low amplitude treatment fared the best, although not statistically significantly better than those in the stable salinity treatment according to the ANOV A. As with Thalassia, the high amplitude treatment resulted in the greatest decline in Halodule green-leaf index (Figure 4-9, bottom panel). The number of Halodule shoots increased most in the low amplitude (SWa7) treatment and least in the stable salinity treatment (Figure 4-10, top panel). The increase in rhizome length in the high amplitude treatment, however, was not statistically different from that in the two other treatments (Figure 4-10, middle panel). Halodule sprig biomass was similar among treatments (Figure 4-10, bottom panel). Average leaf length increased in Halodule sprigs under stable salinity and low amplitude treatments. A decrease was observed in the high amplitude treatment (Figure

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51 Table 4-1: Averages and standard deviations of morphometrics measured on Thal a ssia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev. Average Std. Dev Shoot Number SST2 2 92 0 83 3.67 1 27 SWa7 3 00 0.85 3 70 1 15 SWa14 3 08 0 83 3 38 1.44 Rhizome Length (cm) SST2 22 73 11.97 19.75 10.68 SWa7 22.16 9 29 19 37 7 82 SWa14 24.64 10 79 17 15 8 93 Leaf length ( cm) SST2 9 78 2.92 2 58 2 00 SWa7 8 73 3.79 2.81 1 77 SWa14 8.47 4.03 1 24 1.48 Leaf Number SST2 1 38 0 52 1.58 1.06 SWa7 1.30 0.45 1 74 0.70 SWa14 1 32 0.46 0 63 0 65 4-11, top panel) Those in the low amplitude treatment did not change (Figure 4-11 middle panel). The average number of leaves per shoot in Halodule plants under stable salinity were simi l ar to those in high amplitude treatments. The low amplitude treatment had the greatest number of leaves averaging two leaves per shoot (Figure 4-11, bo tt om panel) Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements are given in Table 4-2 Ruppia Measurements In contrast to Thalassia and Halodu/e Ruppia fared worst in the stable sal i n ity treatment. Increases in shoot number and leaves per shoot occurred in plants exposed to both the low and high amplitude treatments, whereas decreases in these measureme nt s occurred in sprigs in the stable salinity treatment.

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2.5 2 :::i C) 1.5 '5 -2 1 :c: 0.5 0 0 5 52 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio of GLI F= 8.72 (p=0 0078) 35 o ~----------------SST2 SWa7 SWa14 --+--SST2 -swa7 ----o-SWa14 Figure 4-9: Green-leaf indices for Ha/odule over Experiment 2 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant.

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53 Before Treatment to After Treatment Ratio of the Number of Shoots F= 6 84 (p= 0 0156) 2 ~------------------, 1.5 +--------f:;1?.=iTI ------,.-..~ri-------1 1 L _____IE~s_ ~_ li:2:2:.JL ---.----___L ~ru_--1 0.5 +-----------------------1 0 -'----------------~ 2 1.5 1 0.5 0 SST2 SWa7 SWa14 Before Treatment to After Treatment Ratio of Rhizome Length I' ,._ -1 I I SS12 SWa7 SWa14 Total Sprig Biomass per Rhizome Length (g/cm) 0.025 ~-----------------, 0 02 +-----------'f" ----...,,._ -----i 0 015 -l----d =---r::-t";r:1-----r::;~ iill--------1 0.01 0.005 -t-t i 0 +-__.J==...____,_--1_ _;_,__-r-_~ SST2 SWa7 SWa14 Figure 4-10 : Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment 2. Intervals around means are derived from Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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54 Before Treatment to After Treatment Ratio of Leaf Length F = 5 63 {p = 0 0260) 1 5 ~-----------------, 1 +-__J ~= :J.._-,-------1 --=:.;__ ....__r------r --,,,,,.....---------i 0 5 +------------------------i SST2 SWa7 SWa14 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F = 3 36 {p = 0 0814) 1 5 -.--------------------, 1 +-:::;_~~"':... ---.---: :!: t----r---r .=.==;:::..=.. -r----1 0.5 -+--------------------, o _._----------------~ SST2 SWa7 SWa14 Average Number of Leaves per Shoot after Treatment F = 4 30 (p = 0 0490) 2 5 -.--------------------, 2 -t---------, r--.,-:r= ::,-----------, 1 5 +--l 1 +-----f 0 5 -t-------i, 0 +-= = ;;.i__ -,------1---.;.;;..:...,___ r--1,;...;,_;__.,___ --I SST2 SWa7 SWa14 Figure 4-11: Lea f characteristics for Halodule after Experiment 2 All percents are expressed in decimal form. Intervals around means are based on Fisher's LSD procedure (p < 0 05) F ratios are given when differences between means are significant.

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55 The greatest decline in green-leaf index for Ruppia occurred in the stable salinity treatment (Figure 4-12 bottom panel). Declines were seen in all treatments during th e middle period of the experiment but these were far more dramatic in the stable and high amplitude treatments (Figure 4-12, top panel). Ruppia sprigs exposed to both the low and high amplitude treatment had more shoots at the conclusion of the experiment, whereas those in the stable salinity trea tm ent had less (Figure 4-13 top panel) Ruppia rhizome length nearly tripled (from 2.6 to 9.9 cm) in the low amplitude treatment (Figure 4-13, middle panel), but no significant differences were observed in total sprig biomass among treatments (Figure 4-13, bottom panel). Table 4-2: Averages and standard deviations ofmorphometrics measured on Halod u le sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev. Average Std Dev Shoot Number SST2 7 39 3 20 8 70 3 18 SWa7 6.58 2.47 10.13 3 53 SWa14 5 38 2.41 7.38 2 75 Rhizome Length (cm) SST2 17.70 7 96 15.36 12.71 SWa7 18 18 9.87 13.00 9 80 SWa14 16.99 9.43 10 79 8.44 Leaf length (cm) SST2 5.36 1.40 6.30 2.22 SWa7 5.86 2.02 6 12 1 81 SWa14 5.42 1.57 4.40 1 66 Leaf Number SST2 2 07 0 58 1 65 0.43 SWa7 2.17 0 70 2 00 0 68 SWa14 2 19 0.56 1 69 0 54

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2 5 2 :J 1.5 (!) ,!S! 8: :::s 1 a: 0.5 0 0 5 56 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio of GLI 35 1 5 ...-------------------, 1 1 -----n J u i"ft,..F, ,.,; f -r--===f=:=::r---,r--~~ :-,I 0.5 -t---.._.__----------------; o _.__ ________________ ___, SST2 SWa7 SWa14 -+-SST2 -a-SWa7 -1r-SWa14 Figure 4-12: Green-leaf indices (GLI) for Ruppia over Experiment 2 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher s least significant difference (LSD) procedure If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant.

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3 2 1 0 57 Before Treatment to After Treatment Ratio of the Number of Shoots F= 8 77 (p =0 0077) I H T .L SS12 SWa7 SWa14 Before Treatment to After Treatment Ratio of Rhizome Length 5 ~------------------, 4 +-----------=------------, 3 +-----t----............. ---------, 2 -+--------trn~rl------1 1 +-_.__,;;.----'--'-'----,--_.__ __.__ _-r--_ _.__-t__ ---, o ...__-----------------~ SST2 SWa7 SWa14 Total Sprig Biomass per Rhizome Length (g/cm) 0 015 -,-------------------, 0.01 +----..---------------------, 0.005 0 +-__,......_-=......_------. ___.__ ........ _-r-_......_ ____ __,_____, SST2 SWa7 SWa14 Figure 4-13: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Ruppia in Experiment 2. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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58 Leaf length declined in Ruppia plants in the stable salinity and high amplitude treatments (Figure 4-14, top panel). A slight increase (1 %) occurred in the low amplitude treatments. The number of Ruppia leaves increased in both the low and high amplitude treatments whereas a slight decrease occurred in the stable salinity treatment (Figure 414 middle panel). This treatment also had the least numbers ofleaves after the experiment however no statistically significant differences were observed between the treatments (Figure 4-14, bottom panel). Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 4-3. Experiment 3: Rate of Change of Salinity Fluctuation (Sguare vs. Pyramid Wave) Salinity fluctuation was detrimental to Thalassia, regardless of the rate of change of salinity. Halodule was in poor condition after all treatments, although the decline in green-leaf index was slightly less in the stable salinity treatment than in the fluctuation treatments. Ruppia was most negatively affected in the pyramid wave (gradual change) treatments relative the stable salinity and square wave treatments, but not to the extent experienced by the other two seagrasses. Overview of Physical Measurements Salinity at the collection site ranged from nearly fresh to about 13%0 during the month prior to seagrass collection, with a mean around 5%o (Figure 4-15) Mean temperature was approximately 25 C. The salinity pattern of the three treatments is shown in Figure 4-16. The treatments are coded as follows: 1) SST3stable salinity treatment, 2) SWsquare wave with an amplitude of 14%0 (a range from 4 to 32o/oo), and a period of eight days (a salinity

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1 5 1 0.5 0 59 Before Treatment to After Treatment Ratio of Leaf Length T t ~ ,,., I .L .. ,. .J.. .L I SST2 SWa7 SWa14 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F = 9.92 (p= 0.0053) 1 5 -,--------------------, 1 -i-------.----____c~::i_ -------c:::::iiii:::::=.._ --1 0 5 +------------------------t o ...L________________ __. SST2 SWa7 SWa14 Average Number of Leaves per Shoot after Treatment 3 -.---------------------, 2 -1--r;r.i ,,,, tt __ 7"'"'." . J ----l 1 -+-----i o-1-==,;.;;.i_---r--_.1...-=~---.----_.i.;..;;.,;;..;....-J.__~ SST2 SWa7 SWa14 Figure 4-14: Leaf characteristics for Ruppia after Experiment 2. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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60 Table 4-3: Averages and standard deviations of morphometrics measured on Ruppia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev. Average Std. Dev. Shoot Number SST2 5.67 1.98 4.38 2.29 SWa7 6.13 1.57 11 04 4.81 SWa14 7.00 2.83 9.48 5.96 Rhizome Length (cm) SST2 2.06 1.66 3.54 5.21 SWa7 2.60 1.94 9.89 7 05 SWa14 3 18 3 03 5.69 4.87 Leaf length (cm) SST2 5 75 1.43 4.08 1 37 SWa7 5.88 1 27 5 73 1.47 SWa14 5.64 1.62 4.44 0.68 Leaf Number SST2 2.37 0 55 2.17 0 60 SWa7 2.31 0 62 2.54 0.50 SWa14 2.32 0.67 2 36 0.54 32%0, adjusted twice daily in 1.5%0 increments. Although the designed mean salinity for all treatments was l 8%0, the actual means ranged between 18 and 19%0 (Figure 4-16, middle panel). Major deviations from the designed salinity pattern occured on Days 8 and 14, due to mechanical problems involving the freshwater pump. Mean temperatures among treatments differed less than 0 2 C (Figure 4-17, top panel). Light intensities at seagrass depth as well as water clarity (as indicated by percent light at bottom) are statistically similar amongst treatments (Figure 4-17, middle and bottom panels). Total phosphorus concentrations were an order of magnitude higher in the freshwater source than in the seawater source, whereas total Kjeldahl nitrogen and ammonium concentrations were fairly similar (Figure 4-18).

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14 1 2 1 0 8 6 4 2 0 11 / 17 / 98 30 25 2 0 15 1 0 5 ............ 0 11/ 17 / 98 & 61 Sal i n i ty prio r to Collect i on for Exp 3 I 11 I ~w ~Yr l 1 \ ,1 ,I ,,, 'U,.,._.&. l,.J~fv r 'I~ Ni \J 12/1 / 98 12/16 / 98 Temperature prior to Collection for Exp 3 T ..... & & & & .... & & & T . 1 1 / 24 / 98 12/1/98 12/8/98 12/15/98 Salinity (%.) Temperature (C) Mean 4 8 25.3 Standard Deviation 3.12 1 73 Minimum 0.79 18.92 Maximum 13 2 29.6 Measured at Collection 12 21 Figure 4-15: Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished). Salinity values were measu r ed at 15minute intervals. Temperature values are daily averages Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection)

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62 35 ~-----.,...-------------, 30 25 :J e.,.. 20 c: 15 cu en 10 5 0 +------~------,---------.-~ 0 10 20 Experiment Day Mean Salinity over Experiment (o/oo) (Expanded Scale) 30 19 ...-------------;:.;."i:-;, ---, 18.5 -1---------...-------1 '.' 18 -t---1 17.5 17 +----'......;..;;""--'.__--,_ _.__-'-L..._-,-_....__J.--! SST3 PW SW Standard Deviation of Salinity over Experiment (o/oo) 15 ...--------------,.,.,. .,.. ..;;-.::; ---, 10 -t-------,:::..:;;:.:::, ---i .ii' 5 +----,--.,....,------1 ;,; ,. :,;.:~--1 1.46 0 J_ _c~:::::1..-~-1:~:.i_-~_t_:~!..l__ __J SST3 PW SW -+-SST3 -a-PW -lr-$W Figure 4-16: Salinity patterns for treatments in Experiment 3. (Coding as follows: 1) SST3Stable salinity treatment, 2) PWPyramid wave with amplitude of 14%0, salinity changing every 12 hours by 1.5%0, and 3) SWSquare wave with amplitude of 14%0, period of 8 days)

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63 Mean Temperature over Experiment (OC) F = 7 89 (p = 0.0105) 25 -,--------------------, 24 +--f l 23 -t----t 22 -1---'-=~....__--,--____,.___._....__.,--__. ______ ..._ --i SST3 PW SW Intensity of Light at Seagrass Depth (uE/m 2 /sec) SST3 PW SW Percent Light Reaching Seagrass Depth SST3 PW SW Figure 4-17: Mean temperature (top panel), light intensity (middle panel) and percent light reaching depth of seagrass (bottom panel) for treatments in Experiment 3. Error bars are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant. Percent is expressed in decimal form.

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Total Phosphorus (TP) Total Dissolved Phosphorus (TOP) Orthophosphate (PO 12 0 35 8 0 25 5 0 14 10 0 3 0 2 4 0 12 0 25 6 0 1 8 i' 0.2 0, I 0 15 0, i' 3 0 08 0, .:. 6 .. .. ::, .. 0.15 0 1 2 0 06 4 0 .1 0.04 2 2 0 05 0 05 0.02 0 0 0 0 0 0 0%. 18%. 36%, 0%, 18'lo. 36%. 0%, 18%. 36%. Salinity of Source Salinity of Source Salinity of Source Total KJeldahl Nitrogen (TKN) Dissolved Inorganic Nitrogen (DIN) Ammonium (NH4) 80 1 2 100 00 1 4 20 0 3 1 80 00 1 2 0 25 60 15 0 8 C 0 2 =! 40 0, i" 60 00 0 8 0, i' 0, 0 6 10 0 15 .. .:. 40 00 0 6 .. .:. .. 0.4 0.4 0.1 20 5 0 .2 20.00 0 2 0 05 0 0 0.00 0 0 0 0%. 18%. 36%, 0%. 18%. 36%, 0%. 18%. 36%, Salinity of Source Salinity of Source Salinity of Source Nitrite (N02) Nitrate (N03+2) 0 014 70 0 8 0.012 60 0 8 0 01 50 i 0 6 0 008 C i 40 0 6 =0, 0, ::, 0 006 .. 0 4 .:. 30 0.4 .. 0 004 20 0 2 0 002 10 0 2 0 0 0 0 0%, 18%. 36%. 0%, 18%, 36%. Salinity of Source Salinity of Source Figure 4-18: Mean nutrient concentrations measured during Experiment 3.

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65 Thalassia Measurements Thalassia was affected by salinity fluctuation, regardless of the rate of change. Continual, gradual changes were more detrimental to Thalassia than sudden changes. In this treatment, the greatest declines of green-leaf index, rhizome length, leaf length and number occurred. Green-leaf indices (GLI) for Thalassia declined in all treatments over the course of this experiment, however, an increase in GLI occurred in the stable salinity treatment during the final third of the experiment (Figure 4-19). The overall reduction in green-leaf index in the stable salinity treatment was less than in the other treatments. The number of shoots did not change significantly in any treatment (Figure 4-20, top panel). Decreases in rhizome length occurred in all treatments, however. The greatest occurred in the pyramid wave treatment (Figure 4-20, middle panel). Thalassia rhizomes from the stable salinity treatment retained the most biomass, significantly moreso than in the square wave treatment (Figure 4-20, bottom panel). Average leaf length declined during the experiment in all treatments (Figure 4-21, top panel). The number of Thalassia leaves per shoot decreased least in the stable salinity treatment (Figure 4-21, second panel). Plants in the pyramid wave treatment ended the experiment with 20% fewer leaves than initially present. Thalassia averaged 0.35 leaves per shoot (approximately one leaf per three shoots) after the pyramid wave treatment, in contrast to the square wave (0.76) and stable salinity (1.01) treatments (Figure 4-21, third panel) Aboveground biomass was greatest in the stable salinity treatment. The difference in aboveground biomass between the two fluctuation treatments

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:::i 1 (!) .!!:! II) II) ca 0.5 0 5 66 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio ofGLI F= 15.92 (p= 0.0011) 35 1 5 -,--------------------, 1 +---r. 0 5 -+----~---0 -'----------------~ SST3 PW SW ---+-SST3 --PW -ts-SW Figure 4-19: Green-leaf indices for Thalassia over Experiment 3 (top panel) before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals derived from Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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67 Before Treatment to After Treatment Ratio of the Number of Shoots 0 5 -1----------------------1 0 -'------------------~ 1 5 1 0.5 0 SST3 PW SW Before Treatment to After Treatment Ratio of Rhizome Length F= 3 17 (p= 0.0905) SST3 PW SW Belowground Biomass per Rhizome Length (g/cm) F = 4.29 (p= 0.0490) 0 03 -,------------------, 0 02 -t---t ,l 0 01 ;-----;0 -I---' ....... SST3 PW SW Figure 4-20: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and belowground biomass (bottom panel) for Thalassia in Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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68 Before Treatment to After Treatment Ratio of Leaf Length 1.5 ~------------------, 1 ;----,...,,.,.., ,,,,..,...r-----.~~~ ,----r----ri = rr-i-, 0.5 +-=+-=::J-----j 0 -'--------___,;;;;;;..._ _______ __, SST3 PW SW Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F = 16.99 (p = 0.0009) 1 +-r.::-: = -~-----, r-~,:r--r-----r ~~ ,-----, 0.5 -+---~~-----. o ~----------------SST3 PW SW Average Number of Leaves per Shoot after Treatment F= 16.09 (p = 0 0011) 1.5 -,------------------~ 1 +-l"ll'ffl~ --------------1 0 5 +----I 0 -1--~=~----'--..,__ -,-_., __ ..__ ---l SST3 PW SW Aboveground Biomass per Short Shoot (g/ss) 0 25 ~-----------------, o 2 ,------.~n~i.. --------~ r------i 0.15 ...._____, ~~lm: 1----+----r-."~r---l 0 .1 ..J....___J 0 05 +--I 0 +---'----SST3 PW SW Figure 4-21: Lea f characteristics for Thalassia after Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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69 Table 4-4: Averages and standard deviations of morphometrics measured on Thalassia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std. Dev. Shoot Number SST3 3.63 0 92 3 71 1 16 PW 3.46 0.93 3.33 0.82 SW 3 96 1.46 3.96 1 63 Rhizome Length (cm) SST3 28.25 6 22 26.52 7.01 PW 28 29 9 03 24.83 10.79 SW 30.29 10.15 30.63 13.38 Leaf length (cm) SST3 6.07 3 24 2.55 1.98 PW 5 82 2 21 1.44 2 32 SW 6.67 2.49 3.49 3 20 Leaf Number SST3 1.69 0.66 1.01 0 90 PW 1.67 0.45 0 35 0 52 SW 1.74 0.54 0 76 0 59 was not statistically significant. Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 4-4. Halodule Measurements Halodule fared poorly in all treatments, with decreases in green-leaf index, leaf number and length occurring in each. Surprisingly, shoot numbers increased more in the fluctuation treatments. A sharp decline in GLI occurred in Halodule by the ninth day of the experiment in all treatments (Figure 4-22). In the stable salinity treatment, green-leaf indices increased during the remainder of the experiment. Overall, the percent changes of GLI did not differ significantly amongst treatments.

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1 5 :J C) ,S! 1 '5 ,2 cu :z: 0 5 0 5 70 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio of GLI 35 1 11J!3~l, -.---ri 0 5 _j__ _j__ ___ ~~~~t=::::: '----1 o ~----------------~ SST3 PW SW -+-SST3 -a-PW -tr-SW Figure 4-22: Green-leaf indices for Halodule over Experiment 3 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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71 The number of Halodule shoots in both the square wave and pyramid wave treatments increased more than in the stable salinity treatment (Figure 4-23, top panel). A slight increase in rhizome length occurred in the square wave treatment; decreases were seen in the others (Figure 4-23, middle panel). The higher Halodule biomass in the stable salinity treatment was not statistically different from the other treatments (Figure 4-23, bottom panel). The decline in average leaf length and number of leaves per shoot was similar in all treatments (Figure 4-24, top and middle panels). In addition, the average numbers of leaves per shoot after the experiment were very close, with 1.47 leaves per shoot in stable salinity treatment versus 1.42 leaves counted in both the square and pyramid wave treatments (Figure 4-24, bottom panel). Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 4-5. Ruppia Measurements As for Halodule, Ruppia green-leaf index declined in all treatments during the first third of the experiment, but increased afterwards in the stable salinity treatment (Figure 4-25). Although those in the pyramid wave treatment declined most in GLI, differences among the treatments were statistically insignificant. Declines in Ruppia shoot number, rhizome length, leaf length, and leaf number were greatest in the pyramid wave treatments, although statistically significant differences occurred only in rhizome length (top and middle panels of Figure 4-26 and 4-27). Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are in Table 4-6.

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72 Before Treatment to After Treatment Ratio of the Number of Shoots F = 3 66 (p = 0.0687) 2 -,-------------------, 1 5 --1-------------. ....... ~--~~ 't-------l 1 _j____J ==::;jl;:!:!: L_~ _L.:..L.....i.L _-,--__J.:.::Z:~L_-J 0 5 +----------------------. 0 -L-----------------~ 1 5 1 0 5 0 SST3 PW SW Before Treatment to After Treatment Ratio of Rhizome Length T T I .. I .L -'.L SST3 PW SW Total Sprig Biomass per Rhizome Length (g/cm) 0.01 -,----------------, 0 008 +--;:;;;ii;;,,-;., -----------------, 0 .006 -r-----; ''" "' 0 004 -t----1'1 0 002 +--t .!i 0 +___J..::.:;:..;.;=.i.._--,_ ---1.,._....;.;.i_ _---,_ _.L..a.,_..;:,;~ ----l SST3 PW SW Figure 4-23: Before to after treatment ratios of shoot number (top panel), and rhizome length (middle panel), and total sprig biomass (bottom panel) for Ha/odule in Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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73 Before Treatment to After Treatment Ratio of Leaf Length 1 5 ....----------------, 1 -t--.-,. 0 .5 +-----" --0 _._ -----------~ SST3 PW SW Before Treatment to After Treatment Ratio of Number of Leaves per Shoot 1.5 -,----------------, 1 -t-=~ ~,--, ~=.,i----r =~r:::. .--------j 0 5 +-------------------, o _,__ ____ _____ _____ ----, SST3 PW SW Average Number of Leaves per Shoot after Treatment 2 ....----------------, 1.5 --i-;::;;j CMr-~~ ~-------. ~~ ,-----, 1 +---r0 5 -t-1 0 +-__._ ___ SST3 PW SW Figure 4-24 : Leaf characteristics for Halodule after Experiment 3 Intervals around means are based on Fisher s LSD procedure (p < 0.05) F ratios are given when differences between means are significant.

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74 Table 4-5: Averages and standard deviations of morphometrics measured on Halodule sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std Dev Shoot Number SST3 5 29 2 18 5.87 2 67 PW 4 71 2 29 6.88 2.85 SW 5 00 1 84 7.17 3 06 Rhizome Length (cm) SST3 9.42 4.02 7 70 4.19 PW 10.29 2 88 9 90 6.15 SW 8 96 4 33 8 67 3.68 Leaf length (cm) SST3 5.89 1.72 2 68 1 20 PW 6.18 1 74 2.81 1.12 SW 5 73 1.91 2.84 0.75 Leaf Number SST3 2.11 0 52 1.42 0 65 PW 2.04 0 53 1.42 0.58 SW 1.96 0.60 1.42 0.36

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:::; C) ,!! : :::s a: 75 1 5 1 0.5 0 -+----,----..---.-----,r------r----r-----, 0 5 10 15 20 25 30 Experiment Day Before Treatment to After Treatment Ratio of GLI 35 1.5 ~------------------, 1 1---, ~~r---,-77~ 1ir-.---, =1=~ 1 0 5 -+----------1--------------1 o ~----------------~ SST3 PW SW -+-SST3 ---PW -t:s-SW Figure 4-25: Green-leaf indices for Ruppia over Experiment 3 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure If the means are not significantly different, the intervals will overlap 95% of the time.

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76 Before Treatment to After Treatment Ratio of Number of Shoots 1.5 ....-------------------, 1 +-c.~~""":,;_,n--.-----r .--:-~.,--r--r=~r-----j 0 5 +-----------------,----, o _.__----------------~ SST3 PW SW Before Treatment to After Treatment Ratio of Rhizome Length 1 5 ~------------------, 1 +-..,..... ....... ...---.-----,-~.,....--,------r~=,-----1 0 5 -+----,.,,._ ___ __, .......... __. o ...._------------~ 0 015 0 01 0 005 0 SST3 PW SW Total Sprig Biomass per Length {g/cm) I h'"'" I f!, R I SST3 PW SW Figure 4-26: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Ruppia in Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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77 Before Treatment to After Treatment Ratio of Leaf Length 1.5 ~-------------------, 1 ----.:==f:.mJ --r-i:; ~r--.-~~ ~ 1 """ 1s, 0.5 ..l....-------===::f::::"::~ ---------l o _.___ ________________ SST3 PW SW Before Treatment to After Treatment Ratio of Number of Leaves per Shoot 1 5 ....--------------------, 1 -r-. r-::r.::!!llt1?rr-----r-,-;~ 0.5 +----------' """""'.,,...., "'-----.1 ~-----i 0 --L---------------------' SST3 PW SW Average Number of Leaves per Shoot after Treatment 3 -r---------------------, 2 +---r--.....,,...,..._ --------------l 1 +-----t 0 +--.L...;;.;; ___ ....__~ _.__ ___ _.__-,.._ _.__ ....... __.__ -l SST3 PW SW Figure 4-27: Leaf characteristics for Ruppia after Experiment 3. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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78 Table 4-6: Averages and standard deviations of morphometrics measured on Ruppia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std. Dev. Shoot Number SST3 12 54 6.17 8.96 5.45 PW 9.96 4 13 6.23 5.95 SW 12.13 5 83 6.33 5.83 Rhizome Length (cm) SST3 13.17 6 18 7.52 5.90 PW 11.08 5 08 3.42 3 91 SW 12.13 4.88 5 79 5.82 Leaf length (cm) SST3 3.94 1 16 3.32 1.34 PW 3.96 1.08 2 10 1.48 SW 3 92 0.86 2 49 1.48 Leaf Number SST3 2 69 0.45 1.94 0.96 PW 2.57 0.49 1.28 0.80 SW 2.57 0.77 1.49 0.92 Experiment 4: Salinity Fluctuation around Different Means The overall effect of salinity fluctuation was less on Thalassia and Halodule when salinities fluctuated at higher salinities. Ruppia had similar responses to both treatments, although increases in leaf length and number were greater when salinity fluctuated within the less saline range. Overview of Physical Measurements Salinity in Little Madeira Bay during the month prior to collection averaged around 13%0, temperature at 22.7 C (Figure 4-28). The salinity pattern of the two treatments is given in Figure 4-29 The treatments are coded as follows: 1) M9square wave with an eight day period, an amplitude of 9%o, oscillating around a mean of 9%o (ranging between O and 18%0), and 2) M27square wave with an eight day period, an

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1 8 1 6 1 4 12 1 0 8 6 4 2 0 1 / 6 / 99 V \I ........ 30 25 20 15 10 / 5 0 1/6/99 79 Salinity prior to Collection for Exp 4 j >A J\I '1 rM 11 .... 'Y' ... I I I \ I~ r 1/21/99 2/5/99 Temperature prior to Collection for Exp 4 -... T . . ...-T T T T T ....-~ .........., 1/13/99 1/20/99 1/27/99 2/3/99 Salini "Tem erature oc Mean 13.12 22 7 tandard Deviation 2.4 2.87 1.99 12.84 Maximum 17.95 27.35 easured at Collection 16 26 Figure 4-28: Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished). Salinity values were measured at 15minute intervals. Temperature values are daily averages. Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection).

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40 35 30 8 25 20 C: ns 15 en 10 5 0 0 80 10 20 Experiment Day Mean Salinity over Experiment (o/oo) M9 M27 Standard Deviation of Salinity over Experiment ( 0 /oo) 30 12 ~-----------------, 8 -+----4 4 +---0 +---M9 M27 Figure 4-29: Salinity patterns for treatments in Experiment 4. (Coding as follows: 1) T9Square wave with amplitude of 9%o, period of eight days, oscillating around 9%o, and 2) T27Square wave with amplitude of 9o/oo, period of eight days, oscillating around 27%0)

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81 amplitude of 9%o, oscillating around a mean of27%o (ranging between 18 and 36). Although the design called for square waves with eight day periods pump problems made it necessary to run a wave pattern with a ten day, six day, and finally eight day period. Both treatments followed this pattern. The standard deviation of salinity in the T27 treatment was higher than that of the T9 treatment (Figure 4-29, bottom panel) due to an unanticipated salinity drop on Day 15. The more saline water in the T27 treatment was warmer, clearer, had a greater percent of light reaching bottom, and therefore a significantly greater intensity of light at the seagrass level (Figure 4-30). The low salinity treatments received higher concentrations of phosphorus, due to elevated concentrations in the freshwater supply, whereas those in the higher salinity treatment received slightly higher concentrations of total nitrogen (Figure 4-31 ) Thalassia Measurements Salinity fluctuation affected Thalassia in this experiment, however the affect was lessened when salinity fluctuated around a higher mean. The number of shoots as well as the number of leaves per shoot increased in the high salinity treatments. Green-leaf indices of Thalassia decreased over the course of the experiment in both treatments, although the decline was greater in the less saline treatment (Figure 432). An increase in GLI occurred in latter third of the more saline treatment. Before to after treatment ratios of the number of shoots and rhizome lengths after the experiment were slight and varied little between the treatments (Figure 4-33 top and middle panel) The greater belowground biomass in Thalassia in the less saline treatment was not statistically significant (Figure 4-33, bottom panel). Average leaflength

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82 Mean Temperature over Experiment (OC) M9 M27 Intensity of Light at Seagrass Depth (uE/m 2 /sec) F= 98.70 (p=0.0001) 1500 -,---------~ ~~ ,-, 1000 -+--500 -+--0 4_ _Ll :ffi~ :iU......---L.:.:.:.t::~I!.'.J... -___.j M9 M27 Percent Light Reaching Seagrass Depth F = 314 29 (p=0.0001) M9 M27 Figure 4-30 : Mean temperature (top panel), light intensity (middle panel) and percen t light reaching depth of seagrass (bottom panel) for treatments in Experiment 4 Erro r bars are intervals based on Fisher's least significant difference (LSD) procedure If t h e means are not significantly different the intervals will overlap 95% of the time F ra ti os are g i ven when di ff erences between means are significant. Percent i s expressed i n decimal form

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Total Phosphorus (TP) Total Dlssolved Phosphorus (TOP) Orthophosphate (P04) 12 0 0 4 10 0 0 3 4 0 0 14 10 0 8 0 0 25 0 12 0 3 3 0 0 1 8 0 0 2 i" :::i" 6 0 i" 0 08 6 0 0 2 Q 0.15 Q 2 0 Q 2. g 2. 4 0 g 2. 0 06 g 4 0 0 1 0 04 0 1 1 0 2 0 2 0 0 05 0 02 0 0 0 0 0 0 0 0 0 0'18'36'0'18'36'0'18'36'Salinity of Source Salinity of Source Salinlty of Source Total Kjeldahl Nitrogen (TKN) Dluolved Inorganic Nitrogen (DIN) Ammonium (NH4) 80 0 1 2 50 0 0.7 40 0 0 6 40 0 0 6 0 5 60 0 0 5 30 0 0 8 i" 30 0 0 4 i" Q 0 4 Q i" Q 2. 40 0 0 6 g ::, 0.3 g 2. 20 0 0 3 g 20 0 0.4 nd 0 2 0 2 20 0 10 0 0 2 10 0 0 1 0 1 0 0 0 0 0 0 0 0 0 00 0'18'36'0'18'36'0'18'36'\,;..) Salinlty of Source SaNnlty of Source Salinlty of Source Nitrite (N02) Nitrate (ND3+2) 0.25 0 0035 12 0 0 15 0 20 0 003 10 0 0 0025 8 0 0 1 i" 0 15 0 002 Q i" 6 0 Q ::, 0 0015 g 2. g 0 10 0 001 4 0 0 05 0 05 2 0 nd 0 0005 0 00 0 0 0 0 O'18'36'O'18'36'SaNnlty of Source SaHnlty of Source Figure 4-31: Mean nutrient concentrations measured during Experiment 4. (ndno data)

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:J 1 C) -~ II) II) (1:1 0.5 0 5 10 84 15 Experiment Day 20 25 Before Treatment to After Treatment Ratio of GLI F= 11.36 (p=0.0071) 1,5 ....--------------------, 1 +--------.0.5 +---0 --------------------~ M9 M27 7 30 Figure 4-32: Green-leaf indices for Thalassia over Experiment 4 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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1 5 1 0.5 0 1.5 1 0.5 0 85 Before Treatment to After Treatment Ratio of Number of Shoots .... M9 M27 Before Treatment to After Treatment Ratio of Rhizome Length M9 M27 Belowground Biomass per Rhizome Length (g/cm) 0 025 ....---------------0 02 +--t 0.015 +----1 0.01 ---i----t 0.005 +---0 ---1---M9 M27 Figure 4-33: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and belowground biomass (bottom panel) for Thalassia in Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0 05) F ratios are given when differences between means are significant.

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86 decreased less in the more saline treatment, and the number of leaves increased by an average of over 20% (Figure 4-34, top and second panel). Thalassia sprigs in the less saline treatment ended with approximately 40% fewer leaves per shoot. Aboveground structures in the less saline treatment had more biomass, however (Figure 4-34, bottom panel). Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 47. Halodule Measurements Overall declines in Halodule GLI were also observed in both treatments (Figure 4-35). In the more saline treatment, Halodule GLI increased during the initial third of the experiment. During this time the GLI of sprigs in the less saline treatment sharply declined. By Day 15, GLI reached its lowest level in the less saline treatment, where it remained for the remainder of the experiment. The number of Halodule shoots in the more saline treatment decreased less after treatment than those in the lower salinity treatment (Figure 4-36, top panel). Rhizome length increased in the higher salinity treatment as well (Figure 4-36, middle panel). Sprig biomass was significantly greater in the less saline treatment than those in the more saline treatment, however (Figure 4-36, bottom panel). Leaf lengths declined by about the same amount in both treatments (Figure 4-37, top panel). Halodule shoots had an average of 14% more leaves after the more saline treatment in contrast to the 32% decrease in the less saline treatment (Figure 4-37, middle panel). On average, Thalassia and Ha/odule had an additional leaf per shoot in the more saline treatment (Figure 4-34, third panel, Figure 4-37, bottom panel). The average number of leaves was statistically significant higher for Thalassia and Halodule in the

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87 Before Treatment to After Treatment Ratio of Leaf Length 1 5 -,--------------------, 1 +-----.0 5 +------<= 0 -'------------------J M9 M27 Before Treatment to After Treatment Ratio of Number of Leaves per Shoot 2 1 5 1 0 5 0 ..L. M9 F = 7.99 (p = 0.0179) I 1 ...... I "", I M27 Average Number of Leaves per Shoot after Treatment F= 5 25 (p = 0 0449) 3 ~-----------------~ 2 -t------------; : 1 -+---< 0 -+-----'-------------.---...._ ______ -i M9 M27 Aboveground Biomass per Short Shoot (g/ss) 0.25 ~----------------~ 0.2 -+-----------------i 0 .15 ;--------11 0.1 +--I 0 05 +---t'. 0 4----'-".;;.;.:.,;. M9 M27 Figure 4-34: Leaf characteristics for Thalassia after Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0.05) F ratios are given when differences between means are significant.

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88 Table 4-7: Averages and standard deviations ofmorphometrics measured on Thalassia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std. Dev. Shoot Number M9 4.39 1.23 4.03 1.30 M27 3 92 1 23 3 92 1 18 Rhizome Length (cm) M9 33 11 13.52 33.43 11.21 M27 28.15 8 87 28 06 8.48 Leaf length (cm) M9 18 27 4 50 8.19 7.42 M27 18 37 4 75 11.34 4 93 Leaf Number M9 2.39 0.60 1.42 1 20 M27 2 33 0.86 2.53 2.06 higher salinity treatment. Average shoot, rhizome, and leaf measurements for Halodule, and the standard deviations of the measurements, are given in Table 4-8. Ruppia Measurements Ruppia varied little in GLI between the two treatments, in sharp contrast to the other two species (Figure 4-38) Green-leaf index increased in both treatments over the experiment. The increase in the less saline treatment was slightly larger. Increases in Ruppia shoot number were similar between treatments (Figure 4-39, top panel). The increase in Ruppia rhizome length was greater in the more saline treatment but not significantly so (Figure 4-39, middle panel) Total sprig biomass was greater in the less saline treatment (Figure 4-39, bottom panel). Leaf length changed little in either treatment (Figure 4-40, top panel). The number of leaves per shoot increased by 5% in the less saline treatment, but declined by

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:J 1 C) -S? "5 ca l: 0.5 0 5 10 89 15 Experiment Day 20 25 Before Treatment to After Treatment Ratio of GLI F= 38.49 (p= 0 0001) 1 5 -,---------------------, 1 ;--------.-, 0.5 +---I 0 -'--------------------' M9 M27 7 30 Figure 4-35: Green-leaf indices for Halodule over Experiment 4 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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90 Before Treatment to After Treatment Ratio of the Number of Shoots F= 13.07 (p = 0 0047) 1 5 ~------------------, 1 +----.0.5 -t-----L. o ~----------------1.5 1 0.5 0 M9 M27 Before Treatment to After Treatment Ratio of Rhizome Length F= 12.67 (p = 0.0047) .... 1 ~--. :, ,I ..... M9 M27 Total Sprig Biomass per Rhizome Length (g/cm) F = 5 50 (p= 0 0410) 0 008 ~---------------~ 0.006 -t-------= ==i:::::= =-------------, 0.004 -+--0 002 +---0 +----'-......... M9 M27 Figure 4-36: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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91 Before Treatment to After Treatment Ratio of Leaf Length 1 5 ~---------------, 1 -t------.0 .5 -1---------"------------------i o ......_----------------~ 1 5 1 0 5 0 M9 M27 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F= 11 83 (p= 0.0063) T .... : 1 .L M9 M27 Average Number of Leaves per Shoot after Treatment F= 14.00 (p= 0.0038) 4 -.---------------------, 3 -t------------..~---, 2 +--------------t 1 -+----l 0 +--__JL.....::.;==.;::.i._----,----,.__.;....;;.,.;_;;___,___-----1 M9 M27 Figure 4-37: Leaf characteristics for Halodule after Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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92 Table 4-8: Averages and standard deviations ofmorphometrics measured on Halodule sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std. Dev Shoot Number M9 4.89 1 33 2.42 1 61 M27 4.19 1.09 3.75 1 90 Rhizome Length (cm) M9 12.03 5 19 9 23 5 32 M27 10 74 4 54 10 77 4.75 Leaf length (cm) M9 7.24 1.84 3.86 2.09 M27 8.42 2 05 5.61 0.93 Leaf Number M9 2.25 0.50 1 53 0 97 M27 2 25 0.44 2.56 0.65 8% in the more saline treatment (Figure 4-40, middle panel). The number of Ruppia leaves per shoot, however, was similar in both treatments (Figure 4-40, bottom panel) Average shoot, rhizome, and leaf measurements, and the standard deviations of the measurements, are given in Table 4-9.

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::i (!) .!!! 8: ::s a:: 2 5 2 1 5 1 0.5 0 0 5 1 5 1 0.5 0 93 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GLI T T ._,.,. . I M9 M27 30 Figure 4-38: Green-leaf indices for Ruppia over Experiment 4 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error those on percent change chart are intervals based on Fisher s least significant difference (LSD) procedure. If the means are not significantly different the intervals will o v erlap 95% of the time.

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1.5 1 0.5 0 94 Before Treatment to After Treatment Ratio of the Number of Shoots T T I .,. '' M9 M27 Before Treatment to After Treatment Ratio of Rhizome Length 2 5 -,-------------------, 2 -+----------------------, 1 5 +-----=---------r ~ ::-t::: ~ n-----1 1 -l------1--i-----,------L === a_ ----j 0 5 +----------------------, 0 -'-----------------~ M9 M27 Total Sprig Biomass per Rhizome Length {g/cm) 0 008 ~-------------------, 0 006 ;------+--------------; 0 004 +---0.002 -t--0 -1--_,__ M9 M27 Figure 4-39: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Ruppia in Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0.05) F ratios are given when differences between means are significant.

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1.5 1 0.5 0 95 Before Treatment to After Treatment Ratio of Leaf Length T T .L M9 M27 Before Treatment to After Treatment Ratio of the Number of Leaves F= 7.60 (p = 0.0202) 1.5 ~-------------------, ... t +__Ji:3:5it::!!!::o__ _,-----------c~~:::r~ .... 0.5 -!-------------------; 0 ----------------M9 M27 Average Number of Leaves per Shoot after Treatment 4 ~------------------, 3 -t-------;------=~'""'=~ -----1 2 +---1 +---0 +---"--'-..;..a,;,....,._;.___.__ __ __ _,__..;..._ __._ __ -1 M9 M27 Figure 4-40: Leaf characteristics for Ruppia after Experiment 4. Intervals around means are based on Fisher's LSD procedure (p < 0 .0 5). F ratios are given when differences between means are significant.

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96 Table 4-9: Averages and standard deviations of morphometrics measured on Ruppia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev. Average Std. Dev. Shoot Number M9 13.61 5 53 15.33 7 65 M27 15.97 7.44 18.61 12 31 Rhizome Length (cm) M9 11.33 5.13 11.81 6.60 M27 14.28 6.71 21.46 18.68 Leaf length (cm) M9 4 35 0 50 4.59 0.73 M27 4.61 0.77 4.45 1.88 Leaf Number M9 2.97 0.84 3.11 0.46 M27 3.22 0.72 2.97 1.30 Experiment 5: Extreme Salinity Fluctuation and Effect of Period Salinity fluctuation had a profound effect on Thalassia and Halodule in this experiment, regardless of the period (frequency) of the salinity fluctuation treatment pattern Once again, Ruppia was the most resilient, although the high frequency salinity fluctuation treatment was the most detrimental. Overview of Physical Measurements Salinity gradually increased in Little Madeira Bay over the month prior to collection, averaging around 25%0 (Figure 4-41). The salinity pattern for the three treatments is given in Figure 4-42. The three treatments are coded as follows: 1) SST5stable salinity treatment, 2) SWp8square wave with amplitude of l 8%0 (ranging between O and 36%0) and a period of eight days (a salinity change every four days), and 3) SWp4square wave with amplitude of l 8%0 and a period of four days (a salinity change every two days). The square wave period with the eight-day period was plotted

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97 Salinity prior to Collection for Exp 5 35 30 25 20 15 10 5 0 3/26/99 4/9 /9 9 4 / 25/99 Temperature prior to Collection for Exp 5 35 30 25 20 15 1 0 5 0 3/26/99 4/2/99 4 / 9/99 4/16/99 4 / 23/99 Salini %o Tern erature oc Mean 25.6 26 84 tandard Deviation 2.37 2.19 Minimum 20.26 20.86 Maximum 29.4 31.82 Measured at Collection 30 29 Figure 4-41: Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished) Salinity values were measured at 15minute intervals Temperature values are daily averages. Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection).

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98 40 ...-------------------, 35 30 25 20 C: ca 15 (/) 10 5 0 -+-----------1--,-------------.--6--il--------; 0 10 20 30 Experiment Day Mean Salinity over Experiment (o/oo) (E xpand e d Scale) 19 ~-----------------, 18 -+----------------.------, 17 +---------------i 16 4-~ 15 +--rt ,, 14 +_..__ __,__r--__._ __ ._--,_ _.__~ ---1 SST5 SWp8 SWp4 Standard Deviation of Salinity over Experiment (o/oo) 20 ~-----------------, 15 ------f 10 -+------< 5 +---r ~~ l---1 0 +----'...._ ____ ...___r--__.__........,..__--,_ _,._ ___.__ --i SST5 SWp8 SWp4 -+-SST5 ---sWp8 -tr-SWp4 Figure 4-42 : Salinity patterns for treatments in Experiment 5. (Coding as follows: 1) SST5Stable salinity treatment, 2) SWp8Square wave with amplitude of 18%0 and period of eight days, and 3) SWp4Square wave with amplitude of l 8%0 and period of four days)

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99 before the four-day period treatment, because the frequency of the four-day period is higher, and assumed to be more of stress on the seagrasses. Problems with the saltwater pump occurred during the final week of the experiment, lowering the mean salinity of the treatments and increasing the calculated standard deviation of the stable salinity treatment (Figure 4-42, bottom panel). The water in the tanks of the low frequency treatment was clearest: 65% of the light measured at the surface reached seagrass level (Figure 4-43, bottom panel) Seagrass in the low frequency treatment received the greatest intensity of light, followed by those in the high frequency and stable salinity treatments (Figure 4-43, middle panel). Nutrient data are provided in Figure 4-44. Thalassia Measurements The effect of salinity fluctuation on Thalassia was dramatic. Green-leaf index increased in Thalassia in the stable salinity treatment over the course of the experiment (Figure 4-45). Although both frequency treatments resulted in the same percent change of GLI, those in the higher frequency treatment (four-day period) had a steeper decline during the initial third of the experiment than those in the lower frequency treatment (eight-day period). Not until Day 15, did both frequency treatments yield similar GLI values Green-leaf indices for the frequency treatments were 2% of their initial values at the end of the experiment (Figure 4-45, bottom panel) The number of Thalassia shoots decreased in both frequency treatments, whereas in the stable salinity treatment, shoot number increased by 10% (Figure 4-46, top panel). Rhizome length did not change considerably in any treatment (Figure 4-46, middle panel). Rhizome biomass was least in the high frequency (SWp4 ) treatment (Figure 4

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100 Mean Temperature over Experiment (OC) F=l2 33 (p=0.0001) SST5 SWp8 SWp4 Intensity of Light at Seagrass Depth (uE/m 2 /sec) F= 133.58 (p=0 0001) SST5 SWp8 SWp4 Percent Light Reaching Seagrass Depth F= 13.20 (p = 0.0063) SST5 SWp8 SWp4 Figure 4-43: Mean temperature (top panel), light intensity (middle panel), and percent light reaching depth of seagrass (bottom panel) for treatments in Experiment 5. Error bars are intervals based on Fisher's least significant difference (LSD) procedure If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant. Percent is expressed in decimal form.

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Total Phosphorus (TP) Total Dlsoolved Phosphorus (TOP) Orthophosphate (P04) 20 0 5 14 0 4 10 0 3 0 4 12 8 0 25 15 10 0 3 0 2 i" 0 3 a, i" 8 a, i" 6 a, 10 0 2 0 15 .=. 0 2 !. .=. 6 !. .=. 4 !. 4 0 1 5 0 1 0 1 2 2 0 05 0 0 0 0 0 0 o"' 18"' 36"' o"' 18"' 36"' o"' 18"' 36"' Sallnlty of Source Sallnlty of Source Sallnlty of Source Total Kjeldahl Nitrogen (TKN) Dissolved Inorganic Nitrogen (DIN) Ammonium (NH4) 120 2 30 0 0 4 12 0 2 100 25 0 10 1 5 0 3 0 15 80 20 0 8 i" 60 a, i" 15 0 0 2 a, i" 6 0 1 a, .=. !. .=. !. .=. !. 40 10 0 4 nd 0 5 0 1 0 05 20 5 0 2 0 0 0 0 0 0 0 ..... o"' 18"' 36"' o"' 18"' 36"' o"' 18"' 36"' 0 ..... SaUnlty of Source Sallnlty of Source Sa Unity of Source Nitrite (N02) Nitrate (N03+2) 0 014 25 0 35 0 012 20 0 3 0 8 0 01 0 25 i" 0 6 0 008 i" 15 0 2 ci, ::, 0 006 !. .=. 10 0 15 !. 0 4 0 004 0 1 0 2 0 002 5 0 05 0 0 0 0 o"' 18"' 36"' o"' 18"' 36"' Sallnlty of Source Sa Unity of Source Figure 4-44: Mean nutrient concentrations measured during Experiment 5. (ndno data)

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2 5 2 .J (!) 1.5 -~ II) II) -!!! 1 C1' .:: 0 5 0 0 5 102 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GLI F = 119 76 (p= 0.0001) 30 1 5 -.--------------------, 1 L..JM~-----.--o s -t-----------; 0 .J...._ ______ ___, __ ........, _,__ __ __. __ ........ ..._ ___, SSTS SWp8 SWp4 --+--SST5 --swpa -tr-SWp4 Figure 4-45: Green-leaf indices for Thalassia over Experiment 5 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error those on percent change chart are intervals derived from Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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1 5 1 0 5 0 1.5 1 0.5 0 103 Before Treatment to After Treatment Ratio of the Number of Shoots F = 5 53 (p= 0.0435) T -~ ._._ I I 'I I _..,..,. -r-: .L .L SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of Rhizome Length .,.. .... SST5 SWp8 SWp4 Belowground Biomass per Rhizome Length (g/cm) F = 3 62 (p = 0 0930) 0 03 ....------------------, 0.02 0 01 0 +--Ji..:::.-=..J_,------L,----'-L---,-_._..;...;____,__ -1 SST5 SWp8 SWp4 Figure 4-46: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and belowground biomass (bottom panel) for Thalassia in Experiment 5. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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104 Before Treatment to After Treatment Ratio of Leaf Length F= 80.56 (p= 0.0001) 1 -t--,--,-,,.....,..,..--,-----,--rr.'l!~-.-------re 0 .5 -+------------, 0 _L_ _____ ____.! ::::::JE::==!....._ __ ~~:::!__ _J SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F= 36.05 (p= 0.0005) 2 -.-----------------, 1 ~ !==J5~~~1==~= = =~=======~==~::::~=~ 0.5 -+-----------t 0 j_ ______ _1.,,,,,,.,i,,,_;.i__ __ __,!==1=~ __J SST5 SWp8 SWp4 Average Number of Leaves per Shoot after Treatment F = 145.95 (0.0001) 4 --.--------------------, 3 2 -t----1 1 0 +.---1..E~1---,---= =:l:===---,--__J=t::::::]_____j SSTS SWp8 SWp4 Aboveground Biomass per Short Shoot (g/ss) 0 6 --.--------------------, 0.4 ,-------==-----------;;: ;::;:;;~ ------:;::---------i 0 2 _L______f 0 +-__,Ji..;.::,;;;=-'---..---___.-=..._~~___J.-=...;..;...L-~ SSTS SWp8 SWp4 Figure 4-47: Leaf characteristics for Thalassia after Experiment 5. All percents are expressed in decimal form. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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105 Table 4-10: Averages and standard deviations of morphometrics measured on Thalassia sprigs prior to and after experimental treatments Pre-Experiment Post-Experiment Average Std. Dev. Average Std. Dev. Shoot Number SST5 3.89 1.68 4.28 1.67 SWp8 3.56 1 38 2.89 1.13 SWp4 3 89 1 57 3 22 1 56 Rhizome Length (cm) SST5 37.16 20.04 34 75 18.74 SWp8 30 09 13.01 29.92 13.88 SWp4 32 57 10.01 31.61 8 40 Leaf length (cm) SST5 20.33 5.05 14 83 5.31 SWp8 21 12 4.61 1.08 3 25 SWp4 20 54 6.02 0 96 2 79 Leaf Number SST5 2.78 0.73 3.22 0.65 SWp8 2 67 0.49 0.17 0 51 SWp4 2.72 0 67 0.28 0.83 46, bottom panel) The leaf length and number of leaves per shoot decreased considerably, yet increased in the stable salinity treatment (Figure 4-47, top and second panel) On average, Thalassia in this treatment had over three leaves per shoot after the experiment, whereas those in the four-day and eight-day period treatments had 0.28 and 0.17 leaves per shoot, respectively (Figure 4-47, third panel). Averages and standard deviations of shoot, rhizome, and leaf morphometrics are provided in Table 4-10. Halodule Measurements Declines in Halodule green-leaf index occurred in all treatments (Figure 4-48). The magnitude of decline was less in the stable salinity treatment, where Halodule

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106 retained over 50% of its original green-leaf index, versus less than 20% in both frequency treatments The beginning GLI of the Halodule used in the stable salinity treatment was lower as well, as indicated on the top panel of Figure 4-48. In the frequency treatments, the greatest declines occurred midway through the experiment. The number of shoots per Halodule sprig decreased in all treatments, as did the length ofrhizome (Figure 4-49, top and middle panel) Total sprig biomass was least in the high frequency treatment, but was not significantly higher in the other treatments (Figure 4-49, lower panel). The average length of Halodule leaves increased in the stable salinity treatment, although the number of leaves per shoot was essentially the same as that prior to treatment (Figure 4-50, top and middle panel). Similar decreases in leaf length and number occurred for plants in both square wave treatments. At the conclusion of the experiment Halodule in the frequency treatments finished with approximately half of the number ofleaves per shoot than those in the stable salinity treatment (Figure 4-50, bottom panel). Averages and standard deviations ofmorphometrics are given in Table 411. Ruppia Measurements Despite the extreme salinity fluctuation treatments, Ruppia was resilient. A decline in Ruppia green-leaf index occurred only in the high frequency (SWp4) treatment (Figure 4-51). However, Ruppia in this treatment retained over 80% of its initial GLI values. In addition, green-leaf indices increased during the middle segment of the experiment. GLI values of Ruppia in the low frequency (SWp8) treatment increased slightly, 10% more than initial values (Figure 4-51, bottom panel).

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1 5 ::i (!) -5 1 .S2 l: 0.5 0 5 10 107 15 Experiment Day 20 25 Before Treatment to After Treatment Ratio of GLI F= 5 51 (p=0 0438) 1 5 ~----------------1 +---r 0 5 -I-------" 30 0 --'----------==--------==-----' SST5 SWp8 SWp4 --+--SST5 -a-SWp8 -tr-SWp4 Figure 4-48: Green-leaf indices for Halodule over Experiment 5 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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108 Before Treatment to After Treatment Ratio of the Number of Shoots F= 3 63 (p = 0 0928) 1.5 -,--------------------, 1 +----,..,.,.,..._,.,,,..r------,-r-,,--....,----,----.~ 0.5 4-__J ~~,!__ __ ---1 o ....__-------~-----~-SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of Rhizome Length 1 5 ~-----------------, 1 +---. r-........ ~ -. -.--"'!""""'1 ------,---r--:"-:::-:-...,--------, 0.5 +----------------0 ~-----------SST5 SWp8 SWp4 Total Sprig Biomass per Rhizome Length (g/cm) 0.01 -r-----------------0 008 +---------+-------------! 0 006 +----j i------r.::""1-::;:-:J--------r---1 0.004 +---f l 0.002 0 +--'-....;.;..;.;;.;.;;,.;..L__-----,---....i..;,___,;,_._ _.,----L--==---i--1 SST5 SWp8 SWp4 Figure 4-49: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment 5. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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109 Before Treatment to After Treatment Ratio of Leaf Length F= 12.37 (p = 0 0074) 1.5 ...-------------------, 1 +-_E~::.:J.._-----,--~ ~r--,---r:i:r.::::::m:,------1 0 5 4-________ ....__ _______ ---t o _._ ________________ SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of Number of Leaves per Shoot 1.5 ...-------------------, 1 +--+---r------, r:-~,.,------, ~~ ,----1 0 5 -1---------___!= f=:::, e:!._ __ ~ =1~ ='-----l o _._----------------~ SST5 SWp8 SWp4 Average Number of Leaves per Shoot after Treatment F = 11.82 (p = 0 0083) 4 ...--------------------, 3 +---~ -------------; 2 ,---,, 1 +-------i. 0 SST5 SWp8 SWp4 Figure 4-50: Leaf characteristics for Halodule after Experiment 5. Intervals around means are based on Fishe r' s LSD procedure (p < 0 05). F ratios are given when diffe r ences between means are significant.

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110 Table 4-11 : Averages and standard deviations of morphometrics measured on Halodule sprigs prior to and after experimental treatments Pre-Experiment Post-Experiment Average Std. Dev. Average Std. Dev Shoot Number SST5 5 72 3.25 3 06 1 63 SWp8 5 39 3.13 3 28 1 56 SWp4 6.89 2.22 3 06 2 13 Rhizome Length (cm) SST5 16.92 9.68 13 13 10 29 SWp8 15 75 10.42 12 36 7 22 SWp4 22 93 7.85 16.43 8.59 Leaf length (cm) SST5 6 54 1.70 7.62 2 25 SWp8 6.41 1.58 3.94 1.96 SWp4 5.94 0 84 4.12 2.15 Leaf Number SST5 2 72 0.67 2.61 0 78 SWp8 2 61 0 50 1 28 0 75 SWp4 2.72 0 57 1 39 0.85 The greatest decrease in the number of shoots and rhizome length occurred in the high frequency fluctuation treatment (Figure 4-52, top and middle panels). Ruppia in the high frequency treatment had the greatest percent loss of leaves per shoot, as well (F i gure 4-53, middle panel). Leaflength increased in all treatments, most in the stable salini ty treatment (Figure 4-53 top panel) Morphometric values are given in Table 4-12

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2 :::; 1.5 (!) -~ & ::s 1 a:: 0.5 0 0 2 1.5 1 0.5 0 5 111 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GLI F = 6.47 (0.0318) ~ .. T I .1. SST5 SWp8 SWp4 -+-SST5 ---swpa -i:rSWp4 30 Figure 4-51: Green-leaf indices for Ruppia over Experiment 5 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals derived from Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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112 Before Treatment to After Treatment Ratio of the Number of Shoots F = 4.83 (p = 0 0563) 1 5 ~------------------, 1 r~~r-----------c=r~ -r-~~, 0 5 ----------------------1 0 -'---------------------' SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of Rhizome Length F = 5 33 (p = 0.0467) 1.5 ...--------------------, 1 r+-=~,r~~_J ~ ~~-~1 ~I~l: l 0 5 0 -'-----------------------' SST5 SWp8 SWp4 Total Sprig Biomass per Rhizome Length (g/cm) 0,015 ...------------------, 0,01 +---------==----------'T"-----.....-----------1 SST5 SWp8 SWp4 Figure 4-52: Before to after treatment ratios of shoot number (top panel) and rhizom e length (middle pan e l) and total sprig biomass (bottom panel) for Ruppia in Experiment 5 Intervals around means are based on Fisher's LSD procedure (p < 0.05) F ratios are given when differences between means are significant.

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113 Before Treatment to After Treatment Ratio of Leaf Length F= 9 55 (p= 0 0137) 2 5 ~------------------, 2 +--..-----------------, 1 5 +--f ill 1 +---'-' 0.5 +-------------------; o ..L-----------------~ SST5 SWp8 SWp4 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F= 8 36 (p = 0.0184) 1 5 -,--------------------, 1 1 -:f:--.--.=i:=:i-.---, ra im..,----1 0 5 4------------~=;i;==------I o _,__----------------~ SST5 SWp8 SWp4 Average Number of Leaves per Shoot after Treatment F= 14.00 (p=0 0055) 4 -.--------------------, 3 4----o 2 -i--n 1 -i-----1 ,, 0 +--.i..:.;.;;..;.;;;:;:=----r---.L---"'-'---r--..L,.;.,a=..:..a...SST5 SWp8 SWp4 Figure 4-53: Leaf characteristics for Ruppia after Experiment 5. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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114 Table 4 12: Averages and standard deviations of morphometrics measured on Rupp i a sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev. Average Std Dev Shoot Number SST5 11 28 5 20 8 11 2 52 SWp8 11 83 3 60 10.22 4 26 SWp4 12 63 6.27 8.31 4.80 Rh i zome Length (cm) SST5 9 20 5 02 5.06 2 98 SWp8 11 27 5.40 5.59 3.42 SWp4 10 68 8.68 4.41 3.26 Leaf length (cm) SST5 4.46 0.72 7.92 1.79 SWp8 4.65 0 77 5.06 0 73 SWp4 4 37 0.84 4.84 1.71 Leaf Number SST5 3 39 0 61 3 17 0 51 SWp8 3 35 1 09 2.83 0.38 SWp4 3.81 0 91 2.44 0 96 Experiment 6 : Salinity Fluctuation and Circulation : Constant Throughflow of Water vs. Air Circulation in Experimental Tanks Regardless of the method of water circulation, Thalassia was negatively i nfluenced by the salinity fluctuation treatments. Halodule was affected by salinity fluctuation as well, h owever when salinities were stable air circulation lead to greater biological r esponses Ruppia responded well in the air circulated tanks as well desp i te the e x posure to fluctuating salinities.

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115 Overview of Physical Measurements Salinity at the collection site was decreasing in the month prior to collection from nearly 30 to approximately 15%0 during that time (Figure 4-54). Temperature varied a few degrees around 30 C every 5 to 7 days The salinity patterns for the treatments in this experiment are given in Figure 455 The treatments are coded as follows: 1) SSTtstable salinity treatment circulated by constant inflow of water, 2) SSTbstable salinity treatment circulated by constant bubbling, 3) SWtsquare wave with amplitude of 14%0 and eight day period circulated by constant inflow of water, and 4) SWbsquare wave with amplitude of 14o/oo and e i ght day period, circulated by constant bubbling. Problems with the freshwater pump occurred on Days 4 and 16, causing spikes in salinity in the throughflow treatments. Greater control of salinity was achieved in the bubbled treatments Mean salinities were approximately 18o/oo in the aerated treatments; salinity standard deviation in bubbled stable salinity treatment was an order of magnitude less than that in the throughflow stable salinity treatment (Figure 4-55, bottom panel). Temperature was similar in all treatments however light intensity and water clarity was higher in the a i r circulated tanks (Figure 4-56). Nutrient data are provided in Figures 4-57 A noticeable decrease in total phosphorus and total nitrogen concentrations occurred in the freshwater supply relative to that in prior experiments. Thalassia Measurements The effect of fluctuation was far more important than the effect of circulation method in spite of the greater water clarity, water movement, and controllability of salinity in the bubbled tanks. Green-leaf indices of Thalassia in both the stable salinity

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35 30 25 20 1 5 1 0 5 0 35 30 25 20 15 10 5 5/29/99 ~ 0 5/29 /9 9 116 Salinity prior to Collection for Exp 6 6/13/99 Temperature prior to Collection for Exp 6 _.... A --.. ----...., 6 /5/ 99 6/12/99 6/19/99 Salinity ("8) Mean 26.94 Standard Deviation 4 53 Minimum 7.49 Maximum 32.24 Measured at Collection 18 6/29/99 A .... 6/26/99 Temperature (C) 29.78 1.54 26 03 33.3 30 Figure 4-54: Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished). Salinity values were measured at 15minute intervals. Temperature values are daily averages Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection).

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117 40 -.------------------~ 35 30 0 c 25 20 C: ci:i 15 (/) 10 5 0 -+-----------.--------,---------, 0 10 20 Experiment Day Mean Salinity over Experiment (o/oo) SSTt SSTb SWt SWb Standard Deviation of Salinity over Experiment (o/oo) 30 20 -.-------------------, 10 -----------1 SSTt SSTb SWt SWb -+-SSTt ---ssrb -tr-SWt ~SWb Figure 4-55: Salinity patterns for treatments in Experiment 6. (Coding as follows: 1) SSTtStable salinity treatment circulated by constant throughflow of water, 2) SSTb Stable salinity treatment circulated by air bubbling, 3) SWtSquare wave with amplitude of 14%0, period of eight days, and circulated by throughflow of water, and 4) SWb Square wave with amplitude of 14%0, period of eight days, and circulated by air bubbling.)

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118 Mean Temperature over Experiment (OC) 31 ~------------------, 30 t-----;::;:;:;:J=;;;;:,---,----:j~r----m!':f:"'~--;;;~;:..,:;1 29 28 +-~=::u.----,-__,1..;.;....;....;...1.__-r--__a.;;;:.;;.;;..:;;..;;..i_---,---1,......., ___, --f SSTt SSTb SWt SWb Intensity of Light at Seagrass Depth (uE/m 2 /sec) F = 14 00 (p=0.0015) 1200 ~----........,.....,C"T""r ......-....-----rr,-.,_.....,.-...--, 1100 -l-------r-r.,_----~~ 1----t 1000 -+-...------, 900 +-_._ _,__---,-__.__~--r--~-r----"----1 SSTt SSTb SWt SWb Percent Light Reaching Seagrass Depth F = 18.57 (p=0 0006) 1 ...-------------------, SSTt SSTb SWt SWb Figure 4-56: Mean temperature (top panel), light intensity (middle panel) and percent light reaching depth of seagrass (bottom panel) for treatments in Experiment 6. Error bars are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time. F ratios are given when differences between means are significant. Percent is expressed in decimal form.

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Total Phosphorus (TP) Total Dluolved Phosphoru (TDP) Orthophosphate (PO4) 7 0 0 2 7 0 0 2 4 0 0 14 6 0 6 0 0 12 5 0 0 15 5 0 0 15 3 0 0 1 i' 4 0 i' 4 0 ::, i' 0 08 01 0 1 01 0 1 01 2 0 2. 3 0 g 2. 3 0 g 2. 0 06 g 2 0 0 05 2 0 nd 0 05 1.0 0 04 1 0 1 0 0 02 0 0 0 0 0 0 0 0 0 0'1,, 18'1,, 36'1,, 0'1,, 18'1,, 36'1,, 0'1,, 18'1,, 36'1,, Salinity of Source Salinity of Source Salinity of Source Total Kjeldahl Nitrogen (TKN) Dissolved Inorganic Nitrogen (DIN) Ammonium (NH4) 100 0 1.4 35 .0 0 5 12 0 0 2 80 0 1 2 30 0 0 4 10 0 0 15 1 25 0 8 0 i' 60 0 0 8 a, i' 20 0 0 3 a, i' a, 8 0 0 1 2. 40 0 0 6 g 2. 15 0 0 2 g 2. g 0 4 10 0 4 0 0 05 20 0 0 2 5 0 0 1 2 0 0 0 0 0 0 0 0 0 0 0'1,, 18'1,, 36'1,, 0'1,, 18'1,, 36'1,, 0'1,, 18'1,, 36'1,, Sallnlty of Source Sa Unity of Source Saffnlty of Source Nitrite (NO2) Nitrate (NO3+2) 1.0 0 014 20 0 0 .3 0 8 0 012 0 25 0 01 15 0 i' 0 6 0.2 ::, 0 008 a, i' a, 0 006 g 2. 10 0 0 15 g 0 4 0 004 0.1 0 2 5 0 0 002 0 05 0 0 0 0 0 0 0'1,, 18'1,, 36'1,, 0'1,, 18'1,, 36'1,, Sallnlty of Source Sallnlty of Source Figure 4-57: Mean nutrient concentrations measured over Experiment 6. (ndno data)

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120 and fluctuation treatments declined during the first third of the experiment, regardless of the method of circulation (Figure 4-58). Following the initial decline, an increase occurred in the stable salinity throughflow treatment (SSTt), where a gradual decline ensued in the stable air circulated treatment (SSTb ). Both square wave treatments followed a similar trend throughout the experiment, ending in green-leaf indices of about 10% original values (Figure 4-58, bottom panel). Shoot number increased in both stable salinity treatments, whereas slight decreases occurred in the square wave treatments (Figure 4-59, top panel). Rhizome length and belowground biomass varied little among treatments (Figure 4-59, middle and bottom panels). Leaf morphometrics for Tha/assia were affected by salinity treatment but not method of circulation, although those in the air circulated stable salinity treatments decreased less than those in the throughflow treatments (Figure 4-60). Averages and standard deviations of shoot, rhizome, and leaf morphometrics are given in Table 4-13. Halodule Measurements Halodu/e was affected by salinity fluctuation regardless of the circulation method, with declines in GLI after treatment (Figure 4-61) Halodule GLI increased in the bubbled stable salinity treatment, increasing 33% over initial values. This is the first treatment in which an increase in Halodu/e green-leaf index occurred A decline in green-leaf index was measured in the stable throughflow treatment, but not as great as the decline measured in both square wave treatments. The number of Halodu/e shoots and the length of its rhizomes declined in the fluctuation treatments. Halodule in the bubbled stable salinity treatment, however,

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2 5 2 :::i (!) 1 5 -~ II) II) -!!! 1 Ct, t: 0 5 0 0 5 121 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GLI F = 17.75 (p= 0 0007) 30 1.5 ~---------------, 1 -t-r= =,.,.--,---, ~~ ----==~---.-----r= ~ -----1 0.5 +------' ="l'""""'--f 0 ...,__ __________ ......... ___ __._ ___, SSTt SSTb SWt SWb --+---SSTt ---ssTo -tr-SWt ~SWb Figure 4-58: Green-leaf indices for Thalassia over Experiment 6 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher s least significant difference (LSD) procedure. If the means are not significantly different, the intervals will o v erlap 95 % of the time.

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122 Before Treatment to After Treatment Ratio of the Number of Shoots 1.5 ....---------------------, 1 4--1-...,i..;..L__ --,-----l ~,;....a.. -,--,....._,...~ ---,---+~ ------l 0 5 +---------------------, 0 -'------------------1.5 1 0.5 0 SSTt SSTb SWt SWb Before Treatment to After Treatment Ratio of Rhizome Length 'T" -+'T" SSTt SSTb SWt SWb Belowground Biomass per Rhizome Length (g/cm) 0.04 -,-------------------, 0 .03 +-------~----~ 7:l;~ -..,.,.... ........ ---~--1 0.02 0,01 0 +__.__....__--,-__,_......,___,_ ......... ____.'----,-----'------...__---' SSTt SSTb SWt SWb Figure 4-59: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and belowground biomass (bottom panel) for Thalassia in Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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123 Before Treatment to After Treatment Ratio of Leaf Length F = 16 67 (p = 0 0008) 1.5 ~-----------------, 1 +-..,..,,,,.,,..,,.,-----. --,._....,---,------,: .,,,,...,..----,"r"'!"':"--:--,------1 0 5 4-----------1 : 0 -'-------------~ SSTt SSTb SWt SWb Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F = 8.95 (p = 0.0062) 1.5 ~---------------, 1 -t----c-, -:-= ~ ----r---. r,-:-:::-,--,-~~,-----.-----r=-r -----i 0.5 +--......._----; r.; ;;a: ~ --t : 0 -'-------------~ SSTt SSTb SWt SWb Average Number of Leaves per Shoot after Treatment F= 13.58 (p= 0.0017) 4 ~--------------, 3 +------~ -------------, 2 1 0 -------,--~'---r----------.------SSTt SSTb SWt SWb Aboveground Biomass per Short Shoot (g/ss) 0.6 ~----------------~ 0 4 ---..-------1 0 2 0 +--......... ...._-----.__._......__,----_.__....._..___~____.,.........,_._---i SSTt SSTb SWt SWb Figure 4-60: Leaf characteristics for Thalassia after Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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124 Table 4 13: Averages and standard deviations of morphometrics measured on Thalassia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std Dev Shoot Numbe r SSTt 3 28 0.83 3 61 1.46 SSTb 3.11 0 90 3.33 1 37 SWt 3.06 0 94 2 39 0.92 SWb 2 89 1 18 2 56 1.38 Rhizome Length (cm) SSTt 27 06 7 30 27 15 8.72 SSTb 27.09 9 85 29.55 10 04 SWt 28 60 9.62 27.47 10 15 SWb 25.49 8.32 25.78 8 12 Leaf length (cm) SSTt 30.42 5 87 22 30 8.71 SSTb 28 62 6 07 23 98 9.33 SWt 28 59 5 92 8.43 7 34 SWb 27.38 4 98 10 34 9 07 Leaf Number SSTt 3 67 0.84 2.33 0 84 SSTb 3 61 0 78 2 72 0 96 SWt 3.89 0.58 1 22 1.17 SWb 3.61 0 78 1.17 1 04 increased in both shoot number and rhizome length, by 69 and 50%, respectively (F i gure 4-62, top and middle panels). Declines in leaflength and the number ofleaves were s i milar amongst fluctuation treatments for Halodule, although those in the bubbled treatment had less of a decline in leaf length and the greatest percent increase in lea f number (Figure 4-63 top and middle panels). Averages and standard deviations of morphometric measurements are provided in Table 4-14

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1.5 :::i (!) -9! -a 1 0.5 0 5 10 125 15 Experiment Day 20 25 Before Treatment to After Treatment Ratio of GLI F = 15.24 (p = 0.0011) 2 -.---------------------, 1 5 +--------~---------------t 1 +--,-..,...,.,--,-___J ...____._ _,-~=----.---r~-,-----1 0.5 +-----------; o ~----------------~ SSTt SSlb SWt SWb --+-SSTt --ssTo -o-SWt ~SWb 30 Figure 4-61: Green-leaf indices for Halodule over Experiment 6 (top panel) and before to after treatment ratios (bottom panel) Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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126 Before Treatment to After Treatment Ratio of the Number of Shoots Following Treatment F= 34.04 (p = 0.0001) 2 ....-------------, 1.5 -+----------. ,,;;;, 1 +--+----.-___._ __,__ -...,.,..,...~.-.::::::a:=.--; 0 5 +-----------------------, 0 -'---------------~ SSTt SSTb SWt SWb Before Treatment to After Treatment Ratio of Rhizome Length F = 19.16 (p=0 0005) 2 ....--------------------, 1.5 +---------r ""':!:"--i----------; 1 +-----....--..----.--___._ __,__ --r_ ==-r~= .-----1 0.5 +-----------------------, o ~ ------------SSTt SSTb SWt Total Sprig Biomass per Rhizome Length (g/cm) F= 5 25 (p=0 0271) SWb 0 015 -,-----------------, 0.01 -i-----....-_.....-----------------, 0 005 0 -1---=...::...a;.-'---,----'---'----,---'-----...__----r-----'-'-...____, SSTt SSTb SWt SWb Figure 4-62: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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127 Before Treatment to After Treatment Ratio of Leaf Length F= 3 25 (p=0 0809) 1 5 -r-------------------, 1 -t---.-,,,.,.,..., ,..-------,----, r""<:"'!'......,.. -r--rr.i~ ~ --.------,,= ~ ---. 0.5 -1---""'--------_._,_.,......__ _---= ==---l o ~----------------SSTt SSTb SWt SWb Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot 1.5 -r--------------------, 1 +-"""------.-' ==-.---r==,.---.--., ==---, 0 5 +----------------------; o ....__ ________________ ___. SSTt SSTb SWt SWb Average Number of Leaves per Shoot after Treatment F= 5.55 (p=0.0234) 4 -r-------------------, 3 +------=----~------------i 2 1 0 +--'----"'-'----r-_..,_----''----,----'-.;.;.;;...;.....___,_........._ ___ ..__-l SSTt SSTb SWt SWb Figure 4-63: Leaf characteristics for Ha/odule after Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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128 Table 4-14: Averages and standard deviations of morphometrics measured on Halodule sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev Average Std Dev Shoot Number SSTt 4 89 2 05 4 67 2.22 SSTb 5.17 2.20 8.72 3 97 SWt 4 94 2 60 3 67 1.53 SWb 5.53 2.43 4 94 2 61 Rh i zome Length (cm) SSTt 17 86 7.94 15.40 9 18 SSTb 17 53 7 19 25.41 9.51 SWt 17.53 11 79 15.40 8.57 SWb 17.82 9.40 16.04 10 42 Leaf length (cm) SSTt 12.92 2.91 7 68 1.17 SSTb 12 53 3 23 9.14 2 45 SWt 12.49 3 00 6.44 1 96 SWb 11.72 3.32 6 30 1 02 Leaf Number SSTt 2.56 0 51 2 67 0.59 SSTb 2 56 0 51 2.67 0.49 SWt 2.22 0.43 1.94 0 64 SWb 2 65 0.49 2.35 0.49 Ruppia Measurements The method of circulation seemed to be more of a factor than salinity fluctua ti on in determining GLI values for Ruppia. Similar to Halodule, Ruppia green-lea f inde x increased in the bubbled stable salinity treatment (Figure 4-64) A minimal percent decline in GLI occurred in the throughflow stable salinity treatment slightly less than that in the bubbled square wave treatment, and not statistically different than declines in the throughflow square wave treatment.

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3 2 5 2 .J (!) a 1.5 g: a:: 1 0 5 0 0 5 10 129 15 Experiment Day 20 25 Before Treatment to After Treatment Ratio of GLI F = 5.19 (p= 0.0278) 30 1 5 -.--------------------, 1 +-....;..-------._____J ==-,-----,-,.:,--,~ ---.-~ -1 0.5 +----------------------! Q ...L..-------------------' SSTt SSTb SWt SWb -+-SSTt ---ssTo -tr-SWt ~SWb Figure 4-64 : Green-leaf indices for Ruppia over Experiment 6 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals derived from Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time

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130 The largest increases in shoot number and rhizome length were measured on sprigs of Ruppia exposed to the bubbled treatments (Figure 4-65, top and middle panels). The greater whole sprig biomasses measured in these treatments, however, were not statistically significant. Leaf responses were not significantly different regardless of circulation method or salinity fluctuation (Figure 4-66, top and middle panels). Averages and standard deviations of Ruppia shoot, rhizome, and leaf morphometrics are given in Table 4-15. Table 4-15: Averages and standard deviations of morphometrics measured on Rupp i a sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev Average Std. Dev. Shoot Number SSTt 13.35 5 00 13.12 3.37 SSTb 15.94 4.75 29.78 18.53 SWt 13.39 3 60 11.28 5 60 SWb 12.72 4.62 19.00 6 20 Rhizome Length (cm) SSTt 21.29 8.72 18 09 8.12 SSTb 28.18 9.62 47.24 26.17 SWt 23.41 10.24 19.47 10.08 SWb 23.17 6.96 24.88 8.42 Leaf length (cm) SSTt 4 99 0.78 5.74 0.81 SSTb 5.28 0.61 4.93 0 99 SWt 4 87 0 66 4 53 1.20 SWb 5.16 0 71 4 09 0.54 Leaf Number SSTt 3.41 0 51 3.41 0.51 SSTb 3.33 0.49 3.17 0.38 SWt 3.22 0.65 2.83 0.86 SWb 3.28 0.57 3 06 0 24

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131 Before Treatment to After Treatment Ratio of the Number of Shoots F = 5 16 (p= 0 0283) 2 5 .....---------------, 2 +--------, .....+--.---------= ----i 1.5 +--~--1 -+.... -~__J ........_........_ _-r--.--r"""",.-------.--------1,-------1 0.5 -!----------~----------, 0 --L---------------' SSTt SSTb SWt SWb Before Treatment to After Treatment Ratio of Rhizome Length F= 6 75 (p= 0.0139) 2.5 ~------------------, 2 +-----___.,. --------; 1.5 -+--------1 1 +-=:::b:::r---, _J~---L -,--7"'."'.,::-r---.------"=r=-----1 0 5 +----------~--------1 0 ...L.------------------' SSTt SSTb SWt Total Sprig Biomass per Rhizome Length (g/cm) SWb 0.008 -,----------------, 0 006 -+--4-------f"~--------,,-......,. ....... ---l 0 004 0 002 0 -4----L-.;.......-'--~--'----'-~__J'----'---,--'----'---l SSTt SSTb SWt SWb Figure 4-65: Before to after treatment ratios of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Ruppia in Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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132 Before Treatment to After Treatment Ratio of Leaf Length F = 3.43 (p=0 0726) 1 5 ~------------------, 1 -l_._ ___ ....__ --,--, ,...._. -,--__ .... ____ -,---r--=-----1 0 5 +------------------, 0 -'------------------~ 1 5 1 0 5 0 SSTt SSlb SWt SWb Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot .... _._ --xSSTt SSlb SWt SWb Average Number of Leaves per Shoot after Treatment 4 ~-------------------, 3 -1---1 '<"( 2 1 0 -1---L.--L---,--.....__. ___ .___-r--___._ ................. -,r--_._ __ ..____, SSTt SSlb SWt SWb Figure 4-66: Leaf characteristics for Ruppia after Experiment 6. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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133 Experiment 7: Effect of Salinity Fluctuation and Reduction of Light Salinity fluctuation was more of an influence on seagrass survival than light availability. Despite shading to 30% of ambient light, Thalassia and Halodule green-leaf indices were greater in the stable salinity treatment than those exposed even to the low amplitude fluctuations in full sun. Exposure to high amplitude fluctuation treatments resulted in even more reduced GLI, shoot number, and leaves per shoot for both species. Overview of Physical Measurements Salinity at the collection site plummeted approximately 25o/oo ten days prior to collection (Figure 4-67, top panel). Temperatures descended during this month, ranging between 26 and 35C (Figure 4-67, middle panel). Salinity patterns for the five treatments are given in Figure 4-68. The treatments are coded as follows: 1) SSTsstable salinity treatment shaded by 70% shadecloth, 2) SWa7usquare wave treatment with amplitude of 7o/oo, period of eight days, and unshaded, 3) SWa7ssquare wave treatment with amplitude of 7o/oo, period of eight days, and shaded by 70% shadecloth, 4) SWa14usquare wave treatment with amplitude of 14%0, period of eight days, and unshaded and, 5) SWa14usquare wave treatment with amplitude of 14%0, period of eight days, and shaded by 70% shadecloth. Control over salinity from the use of bubble circulation in all treatments is evidenced by the calculated zero standard deviation of salinity in the shaded stable salinity treatment (Figure 4-68, bottom panel). Mean temperatures differed by only 0.13 C amongst tanks, and as designed, light intensities were significantly different between the shaded and unshaded tanks (Figure 4-69). Nutrient data are provided in Figure 470.

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40 35 30 25 20 15 10 5 9/4/99 -~ 0 9/4 / 99 134 Salinity prior to Collection for Exp 7 9/19/99 10/3/99 Temperature prior to Collection for Exp 7 ._. .......... ......... ......... ..,,.....--., 9/11/99 9/18/99 9/25/99 10/2/99 Salinity ("9) Temperature (C) Mean 15 78 29.5 Standard Deviation 9.62 1.91 Minimum 0 8 25.94 Maximum 28.04 35 38 Measured at Collection 7 28 Figure 4-67: Salinity (top panel) and temperature (middle panel) in Little Madeira Bay a month prior to seagrass collection (Data from Patino and Hittle, unpublished). Salinity values were measured at 15-minute intervals. Temperature values are daily averages. Tabular values (bottom panel) are based on data collected at 15-minute intervals ( except for data collected at time of collection).

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135 30 +-----+-----+-----l '--t---+--------1 -+-SSTs C. C. -swa7u 20 -----------41------------------. --trSW a 7 s C: ca "' ~swa14u __._SWa14s 10 ...J_____U~~ -~~!!t------J;~~ ------1 ----1 3 5 7 9 111315 17 19 21 23 25 Experiment Day Mean Salinity over Experiment (%0) (Expanded Scale) SSTs SWa7u SWa7s SWa14u SWa14s Standard Deviation of Salinity over Experiment (%0) 15 ....------------;.. .... ... -~ or..::: ~ -, 10 -r-------------i ,a~, 5 -t-----f ~ m; r---i 0 00 0 +-----r---'--__._..._,..._. __._-,-_.___...___,r-'___ ----i SSTs SWa7u SWa7s SWa14u SWa14s Figure 4-68: Salinity patterns for treatments in Experiment 7. (Coding as follows: 1) SSTsStable salinity treatment shaded by 70% shadecloth, 2) SWa7uSquare wave with amplitude of 7 period of eight days and unshaded, 3) SWa7sSquare wave with amplitude of 7, pe ri od of eight days and shaded by 70%, 4) SWal4u Square wave w i th amplitude of 14, period of eight days and unshaded, and 5) SWal4sSquare wave with amplitude of 14 period of eight days and shaded by 70% )

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136 Mean Temperature over Experiment (OC) 24 -,---------------------, 23 --1--1 ~11:"Tr-----r.:~~ --..----------n~~ -r: 22 21 w:=::1._~e~-,-i:::::::..i~...c:::!!::..1-.,...-1.:E:::r:::l.-l SSTs SWa7u SWa7s SWa14u SWa14s Intensity of Light at Seagrass Depth (uE/m 2 /sec) F=233.08 (p = 0.0001) 1200 -,---------------------, 800 +----400 +----0 ....;.;__;...;.:;.L..,.....i..;.:;=;...a_.,.....L-;.;,...J-,-L..;.;......;.-L....,~i;....~ SSTs SWa7u SWa7s SWa14u SWa14s Percent Light Reaching Seagrass Depth F = 5.34 (p = 0 0475) 1 -,--------------------, 0.75 0 68 0.78 SSTs SWa7u SWa7s SWa14u SWa14s Figure 4-69: Mean temperature (top panel), light intensity (middle panel) and percent light reaching depth of seagrass (bottom panel) for treatments in Experiment 7. Error bars are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time F ratios are given when differences between means are significant. Percent is expressed in decimal form

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Total Phosphorus (TP) Total Dissolved Phosphorus (TOP) Orthophosphate (P04) 10 0 3 12 0 35 10 0 3 8 0 25 1 0 0 3 8 0 25 0 2 8 0 25 0 2 i" 6 DI i" 0 2 DI i" 6 DI 0 15 6 0 15 -=4 g -=0 15 g -=4 g 0 1 4 nd 0 1 0 1 2 0 05 2 0 05 2 0 05 0 0 0 0 0 0 O"18"36"0"18"36"0"18"36"Salinity of Source Salinity of Source Salinity of Source Total Kjekfahl Nitrogen (TKN) Dluolved Inorganic Nitrogen (DIN) Ammonium (NH4) 100 1 2 3 5 0 05 2 0 03 80 3 0 0 04 0 025 2 5 1 5 0.8 0 02 i" 60 i" 2 0 0 03 i' =0 6 DI DI 0 015 DI -=40 g .:!. 1 5 0 02 g -=g 0 4 1 0 0 01 0 5 20 0 2 0 5 0 01 0 005 0 0 0 0 0 0 0 w 0"18"36"0"18"36"0"18"36"--.J SaHnlty of Source Salinity of Source Salinity of Source Nitrite (N02) Nitrate (N03+2) 0 06 0 0008 1 4 0 02 0 05 1 2 0 0006 0 015 0 04 =i' 0 0004 DI i' 0 8 0 01 DI .:!. 0 03 g .:!. 0 6 g 0 02 0 4 0 0002 0 005 0 01 0 2 0 0 0 0 0"18"36"0"18"36"Salinity of Source Salinity of Source Figure 4-70: Mean nutrient concentrations measured during Experiment 7. (ndno data)

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138 Thalassia Measurements Salinity fluctuation was more of factor in Thalassia green-leaf index than light. GLI measured in the high amplitude treatments decreased throughout the experiment (Figure 471 ). Indices in the stable salinity treatment increased throughout the experiment, whereas those in the low amplitude treatments increased only during the final third. Amongst similar amplitude treatments, shaded treatments fared slightly better, although the differences were not statistically significant. The number of shoots per sprig declined on those exposed to the high amplitude salinity treatments (Figure 472, top panel). Declines in rhizome length occurred only in the unshaded high amplitude treatment ( 472, middle panel). The numbers of leaves per shoot increased in Thalassia within the stable salinity treatment (Figure 473, top panel). The lower amount of defoliation in the shaded high amplitude treatment was not significantly different in the unshaded high amplitude treatment. Greatest shoot biomass was measured in the high amplitude square wave plants (Figure 473, bottom panel). Averages and standard deviations of Thalassia morphometric measurements are provided in Table 4-16. Halodule Measurements Like Thalassia Halodule green-leaf indices responded to the salinity fluctuation treatment nearly the same way, regardless oflight (Figure 4-74). All treatments followed a similar pattern in GLI until Day 19, where green-leaf indices in plants in the shaded stable salinity treatment increased.

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:J 1 C) -~ II) II) -!S! (ti 0.5 0 5 139 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GU 30 F = 4.89 = 0.0559 3 ~---------~-----~ 2-----------------1 1 +-_._....__---.----.-t-,------,----,-+-,----,--,.,..,...,---,---,...,...-,,------t 0 ...J.._-------------==i==-----l--.J SSTs SWa7u SWa7s SWa14u SWa14s -+-SSTs ~swa7u -t:r--SWa7s ~SWa14u ---SWa14s Figure 4-71: Green-leaf indices for Thalassia over Experiment 7 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error, those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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140 Before Treatment to After Treatment Ratio of the Number of Shoots F= 3.39 (p= 0.1065) 1.5 -.--------------------, 1 --!---t----. ~ -,...--.---+----. --,...,.,,...,.--,---.:::l=-r--; 0 5 --1-------------&-+-.J--t--; o ..L..-----------------~ SSTs SWa7u SWa7s SWa14u SWa14s Before Treatment to After Treatment Ratio of Rhizome Length 1.5 -.--------------------, 1 --!--~ ---. ----1,----_,.__ --,--_...., __ ---. -. ____ --,-__ ,,,_ --; 0.5 +------------------; 0 -'---------------------' SSTs SWa7u SWa7s SWa14u SWa14s Belowground Biomass per Rhizome Length {g/cm) 0.04 -.---------------------, 0. 03 --!-----,;,.-----T---r ---t----;------i 0.02 0.01 0 --j--'--....L..-,---L.-L-..----'-~_.,____.__,,_.,...._---'-----1 SSTs SWa7u SWa7s SWa14u SWa14s Figure 472: Before to after treatment rati os of shoot number (top panel) and rhizome length (middle panel) and belowground biomass (bottom panel) for Thalassia in Experiment 7. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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141 Before Treatment to After Treatment Ratio of Leaf Length F= 22 30 (p = 0.0022) 1.5 -,------------------~ 1 +-..,..,,..,,.,----,-------,, -..:::::,,..-------y----.----,-------., 0 5 +-----------~ o ..,__ ________________ __, SSTs SWa7u SWa7s SWa14u SWa14s Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F= 6.69 (p= 0.0305) 1.5 -.------------------~ 1 +~c-'----.--..-,.;.;,,....---,-.......... ---.--........ ..---.---.-,.--.-----1 0 5 +-----------==-------1:J-q...._--1c------1 o ~----------------~ SSTs SWa7u SWa7s SWa14u SWa14s Average Number of Leaves per Shoot after Treatment F= 4 27 (p = 0.0717) 3 -.-------------------~ 2 +--1"7'1""::1-:::::::1:::=--=:::i:==---------------i 1 0 -I--_,,____,_~__._ SSTs SWa7u SWa7s SWa14u SWa14s Aboveground Biomass per Short Shoot (g/ss) 1 -.------------------=----, 0.8 -+------------~----+------I 0.6 +------'f'----.------f------i 0.4 -+----+----+-----+----I 0 2 0 +----------~__....=--.__~ ........ ....__~_.___,_----,-__.,_~ SSTs SWa7u SWa7s SWa14u SWa14s Figure 473: Leaf characteristics for Tha/assia after Experiment 7. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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142 Table 4-16 : Ave r ages and standard deviations of morphometrics measured on Thalassia sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std Dev. Average Std Dev. Shoot Number SSTs 2.42 0 51 2.42 0.90 SWa7u 2 92 0.67 2.58 0 90 SWa7s 2 75 0 62 2 75 0 62 SWa14u 2.75 0 75 1 33 1 30 SWa14s 2.92 1.38 1 58 1 38 Rhizome Length (cm) SSTs 27.93 8.47 28 77 8.84 SWa7u 28 48 8.91 28.22 7.99 SWa7s 25 12 9 92 26.82 10.35 SWa14u 27.08 17.51 22.40 8 03 SWa14s 35 16 12 50 35 68 12.02 Leaf length (cm) SSTs 23 55 4 98 17.58 9 50 SWa7u 19 08 3.82 17.68 4.53 SWa7s 20 88 3 12 17 30 5 31 SWa14u 20 26 3 31 7.71 7 19 SWa14s 21.18 3.04 12.42 6.89 Leaf Number SSTs 2 00 0.43 2.08 1 31 SWa7u 2.25 0 87 1 75 0 62 SWa7s 2.50 0 52 1 92 0 79 SWa14u 2.75 1.14 0.83 0 72 SWa14s 2 33 0.49 1 50 1.00 The number of shoots per sprig increased in Halodule in the stable salinity and low amplitude treatments (Figure 475 top panel). Rhizome length also increased in these treatments (Figure 4-75, middle panel). Halodule leaflength and numbers were greater in the low amplitude shaded treatment (SWa7s) (Figure 4-76 top and middle panel) Morphometric values are given in Table 4-17

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2 :J (!) 1 5 J!:? "6 -2 1 ::c: 0 5 0 5 143 10 15 20 25 Experiment Day Before Treatment to After Treatment Ratio of GLI F= 7 84 (p = 0 0222) 30 1.5 ~---------------~ 1 +--.-"l"'"'""" ----r ---,,.....,.. ..--.-,----,-----. ~-..-.....,....--,---; 0.5 +-------!.=,t,o--1 o __.__ ________________ ____. SSTs SWa7u SWa7s SWa14u SWa14s --+--SSTs -a-SWa7u -tr-SWa7s ~SWa14u --SWa14s Figure 4-74: Green-leaf indices for Halodule over Experiment 7 (top panel) and before to after treatment ratios (bottom panel). Error bars on time series chart represent standard error those on percent change chart are intervals based on Fisher's least significant difference (LSD) procedure. If the means are not significantly different, the intervals will overlap 95% of the time.

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144 Before Treatment to After Treatment Ratio of the Number of Shoots F = 6.48 (p = 0 0325) 2 .-------------------, 1.5 +----;;;;;;!=--{ ;, i, J _j 1------------------j 1 +.......... "'"""'--r __.=...___ ---,-----___._._,......_ --r --o -i------,------=:i=t---l 0.5 +-----------------------l o ...__ __ _____________ ___, SSTs SWa7u SWa7s SWa14u SWa14s Before Treatment to After Treatment Ratio of Rhizome Length F= 4.75 (p = 0.0590) 2 -r-----------------, 1 5 ;---------.. ..-------.;;;i;;;..----------------; 1 +-->-.;;;"-J_ ----r ---'-.;.._;..J__ --,-----===,...._ ---r ---....i:...---,----.,,,,,b,,r-----j 0.5 +------------------------l 0 -'---------------------' SSTs SWa7u SWa7s SWa14u SWa14s Total Sprig Biomass per Rhizome Length (g/cm) 0.015 ~----------~ 0.01 +-----+ --=i=----__.,.._ ----1 f----------1 0.005 SSTs SWa7u SWa7s SWa14u SWa14s Figure 4-75: Before to after treatment rati os of shoot number (top panel) and rhizome length (middle panel), and total sprig biomass (bottom panel) for Halodule in Experiment 7. Intervals around means are based on Fisher's LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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145 Before Treatment to After Treatment Ratio of Leaf Length F= 5 70 (p = 0 0419) 1 5 ....---------------------, 1 -t.,.,,...~ ---,----.-=-,-----.-~ =,-----.--,, ~..----.--,-:--.,----i 0 5 +--------------------, 0 ..1.-----------------~ SSTs SWa7u SWa7s SWa14u SWa14s 1 5 1 0 5 0 Before Treatment to After Treatment Ratio of the Number of Leaves per Shoot F = 2.37 (p = 0 1843) .... -=-=-=Jo. SSTs SWa7u SWa7s SWa14u SWa14s Average Number of Leaves per Shoot after Treatment F = 21.00 (p = 0 0025) 4 ~-------------------, 3 +----=-==------=------===-----------1 2 -++ -::;:;:;;1 1--1 +--t.~'.l.ii& J --0 -+_.__ ......... __. SSTs SWa7u SWa7s SWa14u SWa14s Figure 4-76: Leaf characteristics for Halodule after Experiment 7. Intervals around means are based on Fisher s LSD procedure (p < 0.05). F ratios are given when differences between means are significant.

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146 Table 4-17: Averages and standard deviations of morphometrics measured on Halodule sprigs prior to and after experimental treatments. Pre-Experiment Post-Experiment Average Std. Dev. Average Std Dev Shoot Number SSTs 4.67 2 71 6.00 2.92 SWa7u 5.33 2.10 8.17 2.89 SWa7s 4.50 1.51 5.33 1 97 SWa14u 5.00 2.17 4.67 2 71 SWa14s 3.92 0 67 3 50 1.51 Rhizome Length (cm) SSTs 16.86 7 29 20 34 8 08 SWa7u 16 70 9 66 23 20 10 63 SWa7s 18 57 6.93 19.24 6.80 SWa14u 20.33 10.75 18 12 10 31 SWa14s 16 20 5.50 14.84 5 67 Leaf length (cm) SSTs 11.92 2.49 8.52 1.36 SWa7u 12.20 2.28 8.38 1 15 SWa7s 11.38 2.77 8 73 2 01 SWa14u 10.86 2.13 7 92 1 15 SWa14s 11.53 2 37 7.59 1.47 Leaf Number SSTs 3 08 0.29 2.83 0.39 SWa7u 3.17 0.58 2.75 0.45 SWa7s 2.75 0.45 2 83 0.39 SWa14u 2 92 0.67 2.50 0.52 SWa14s 2 75 0.45 2.50 0.52

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CHAPTERS CORRELATIONS BETWEEN BIOLOGICAL MEASUREMENTS AND PHYSICAL VARIABLES INF ACILITY EXPERIMENTS Correlations with Salinity All correlations between Tha/assia green-leaf indices and salinity fluctuation were negative. Changes in green-leaf indices following treatment in Tha/assia correlated significantly with all descriptors except mean salinity and number of changes per day (Table 5-1 ). The strongest correlates were with the amplitude descriptors, standard deviation (r2 = 50.1 %) and maximum amplitude (r2 = 41.9 %). Halodu/e indices were positively correlated with mean salinity, and were negatively correlated with all other parameters. The strongest negative correlation (r2 = 40.5%) was with standard deviation of salinity ( a direct measure of fluctuation). Changes in GLI in Ruppia correlate best with the frequency descriptors, number of changes per day and significant frequency, although not significant at the p < 0.001 level (Table 5-1). Decreases in shoot number in Tha/assia correlated strongly with increasing values of amplitude and suddenness of salinity change (Table 5-2). Decreases in Ha/odu/e shoot number correlated strongly with suddenness of salinity change as well. No statistically significant correlations were observed between changes in Ruppia shoot number and the salinity wave descriptors (Table 5-2). 147

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148 Table 5-1: Pearson product moment correlations between changes in green-leaf index following treatment and salinity wave descriptors. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0 001). Tha/assia Ha/odule Ruppia Mean Salin i ty 0 1566 0.3017 -0 0990 (0.2057) (0.0131) (0.4639) r2= 2.5% r 2 = 7.7% r 2 = 1 0% Standard Deviation -0.7129 -0.6437 -0.0193 of Salinity (0.0000) (0.0000) (0 8434) r2= 50.1% r2 = 40.511/o r2= 0.0% Maximum -0.6543 -0.5810 0 0268 Amplitude (0.0000) (0.0000) (0 8434) r2= 41.9% r2= 32.7% r2= 0.0% Suddenness of -0.5863 -0.5854 0 1380 Salinity Change (0.0000) (0.0000) (0 3061) r2= 33.4% r2= 33.3% r2= 1.9% Number of Changes -0.3056 -0 1262 -0.3195 Per Day (0.0119) (0.3089) (0 0154) r2= 7 9% r 2 = 1 6% r2= 8.6 % Significant -0.3945 -0 3711 -0 3035 Frequency (0.0010) (0 0020) (0 0217) r2= 14.3% r2=12.4% r2= 7.6% Absolute -0.4588 -0.4875 -0 1248 Frequency (0.0001) (0.0000) (0 3549) r2 = 19.8o/o r2 = 22.6o/, r2= 1 6%

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149 Table 5-2: Pearson product moment correlations between changes in number of shoots following treatment and salinity wave descriptors. Pvalues are given in parentheses. R-squared values are given as well Statistically significant correlates are bold (p<0 .0 01) Thalassia Halodule Ruppia Mean Salinity 0.0577 0.2508 -0.0218 (0.6427) (0.0406) (0.8721) r2= 0 0% r2= 4 8% r 2 = 0 1% Standard Deviation -0.5060 -0 3659 -0 1779 of Salinity (0.0000) (0 0023) (0 1854) r2 = 24.s;. r2= 12 1% r2= 1.4% Maximum -0.4469 -0.4033 -0 2559 Amplitude (0.0002) (0.0007) (0 0547) r2= 18.7o/. r2= 15.0o/e r2= 4 9% Suddenness of -0.4518 -0.4801 -0.1202 Salinity Change (0.0001) (0.0000) (0 3730) r2= 19.2% r2 = 21.8o/o r2= 0.0% Number of Changes -0.0434 0 2322 -0.2363 Per Day (0.7272) (0 0587) (0 0768) r2= 0.0% r2= 3 9% r2= 3.8% Significant -0.3119 0 1571 -0 0503 Frequency (0 0102) (0 2044) (0 7104) r2= 8 3% r2= 1 0% r2= 0.0% Absolute -0.0805 -0 1026 0 1372 Frequency (0 5175) (0.4087) (0 3088) r2= 0 0% r2= 0 0% r2= 0.9%

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150 No statistically significant correlations occurred between rhizome length and the salinity fluctuation descriptors for any seagrass (Table 5-3). Thalassia leaflengths were negatively correlated with all salinity wave descriptors except for mean salinity (Table 54) Strongest correlates occurred with absolute frequency and salinity standard deviation Leaf lengths in Halodule were negatively correlated with the amplitude descriptors, standard deviation and maximum amplitude, although not statistically significant. The strongest correlate with Ruppia leaf length was the number of salinity changes per day, which correlated negatively, yet not significant at the p<0.001 level (Table 5-4). All salinity fluctuation descriptors negatively correlated with the change in leaf number in Thalassia except for mean salinity, the strongest being standard deviation of salinity (Table 5-5) Leaf counts in Halodule increased with increasing mean salinity. Changes in leaf number were negatively correlated with the number of salinity changes per day in Ruppia (Table 5-5). Similar to the changes measured by the before to after experiment ratios, the average number of Thalassia leaves per shoot after treatment correlated negatively to all salinity descriptors, except for mean salinity (Table 5-6). Once again, the strongest correlate was standard deviation of salinity (r2 = 27.5%). The average number ofleaves per shoot on Halodule sprigs increased with increasing mean salinity, but was negatively correlated with increasing standard deviation and number of changes per day. Average leaf counts in Ruppia were negatively correlated with the frequency descriptors, number of changes per day and significant frequency (Table 5-6).

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151 Table 5-3: Pearson product moment correlations between changes in rhizome length following treatment and salinity wave descriptors. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0 001) Tha/assia Ha/odu/e Ruppia Mean Salinity 0.0191 0.1610 0 0977 (0.8781) (0.1931) (0.4697) r2= 0.0% r2=1.1% r2= 0.0% Standard Deviation -0.0476 0.0660 -0.3111 of Salinity (0.7023) (0.5912) (0 0185) r2= 0.0% r2= 0 0% r2 = 8.0% Maximum -0 0718 -0 1025 -0.4044 Amplitude (0.5637) (0.4092) (0 0018) r2= 0.5% r 2 = 0 0% r2 = 14 8% Suddenness of 0 0350 -0.0705 -0.2534 Salinity Change (0.7785) (0 5709) (0.0572) r2= 0 0% r 2 = 0.0% r 2 = 4.7% Number of Changes -0 3084 -0.0375 -0.2319 Per Day (0 0111) (0.7631) (0 0826) r2 = 8.1% r2= 0.0% r2= 3 7% Significant -0.2099 -0.0297 -0.0493 Frequency (0.0883) (0.8112) (0.7156) r2= 3.0% r2= 0.0% r2= 0.0% Absolute -0 2934 -0 1403 0 0959 Frequency (0 0160) (0 2575) (0.4781) r2 = 7.2% r2= 0.5% r 2 = 0.0%

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152 Table 5-4 : Pearson product moment correlations between changes in leaf length following treatment and salinity wave descriptors. Pvalues are given in parentheses R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Thalassia Halodule Ruppia Mean Salinity 0.1660 0 0050 -0.1656 (0.3497) (0 9679) (0.2184) r2= 0.0% r 2 = 0 0% r2= 1.0% Standard Deviation -0.4740 -0.3047 -0 0436 of Salinity (0.0001) (0.0122) (0.7476) r2 = 21.3% r2 = 7.9% r2= 0.0% Maximum -0.4170 -0 3226 0 0354 Amplitude (0.0004) (0.0078) (0 7936) r2 = 16.1% r2 = 9 0% r2= 0.0% Suddenness of -0.3294 -0 2345 0.1300 Salinity Change (0 0065) (0.0562) (0 3352) r2 = 9.5% r2= 4.0% r 2 = 0.0% Number of Changes -0.3556 -0 1890 -0 3506 Per Day (0 0031) (0 1256) (0 0075) r2 = 11.3% r2= 2.1% r2 = 10.7% Significant -0.3233 -0.0738 -0.2407 Frequency (0 0076) (0.5526) (0 0713) r2=9.1% r2= 0.0% r2=4.1% Absolute -0.5063 0 0002 -0.1020 Frequency (0.0000) (0.9987) (0.4504) r2 = 24.5% r2= 0 0% r2= 0.0%

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153 Table 5-5: Pearson product moment correlations between changes in number ofleaves per shoot following treatment and salinity wave descriptors. Pvalues are given in parentheses R-squared values are given as well. Statistically significant correlates are in bold (p<0.001). Tha/assia Ha/odu/e Rue_eJa Mean Salinity 0.1904 0.4160 -0.2477 (0.1227) (0.0005) (0.0632) r2= 2.1% r2 = 16.0o/. r2=4.4% Standard Deviation -0.5504 -0.2559 -0.1893 of Salinity (0.0000) (0.0366) (0.1585) r2 = 29.2% r2 = 5.1% r2= 1.8% Maximum -0.4960 -0.1740 -0.2761 Amplitude (0.0000) (0.1592) (0.0376) r2 = 23.4% r 2 = 1.5% r2 = 5.9% Suddenness of -0.3934 -0.1408 -0.0625 Salinity Change (0.0010 (0 2557) (0.6440) r2 = 14.2% r 2 = 0.5% r2= 0.0% Number of Changes -0.3276 -0.1844 -0.4405 Per Day (0.0068) (0.1353) (0.0006) r2 = 9.4% r 2 = 1.9% r2 = 17.9o/e Significant -0.3342 -0.1260 -0.1940 Frequency (0.0057) (0.3096) (0 1482) r2 = 9.8% r2= 0.1% r2= 2.0% Absolute -0.2481 -0.1199 0.0762 Frequency (0.0429) (0.3340) (0.5731) r2 = 4.7% r2= 0.0% r2= 0.0%

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154 Table 5-6: Pearson product moment correlations between number ofleaves per shoot following treatment and salinity wave descriptors. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Tha/assia Halodule Ruppia Mean Salinity 0 1470 0.3672 0.0399 (0.2352) (0.0022) (0.7683} r2= 0.7% r2 = 12.2% r2= 0.0% Standard Deviation -0.5345 -0 2598 -0 0513 of Salinity (0.0000) (0.0338) (0.7044) r2 =27.5% r2 = 5 3% r2= 0.0% Maximum -0.4342 -0 2105 -0.0245 Amplitude (0.0002) (0.0873) (0.8567) r2 = 17.6% r2= 3.0% r2= 0 0% Suddenness of -0.3337 -0.1253 0 1515 Salinity Change (0.0058) (0.3123) (0 2606) r 2 = 9 8% r 2 = 0 6% r2= 0 5% Number of Changes -0.4319 -0 3138 -0.4937 Per Day (0.0003} (0.0097) (0.0001) r2 = 17.4% r2 = 8 5% r2 = 23.0% Significant -0.4709 -0.1197 -0 3752 Frequency (0.0001) (0 3346) (0 0040) r2 = 21.0. r2= 0.0% r2 = 12.5% Absolute -0.4193 -0.2253 -0 0436 Frequency (0.0004) (0 0667) (0.7472) r2 = 16.3% r2= 3 6% r2= 0 0%

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155 Surprisingly, no statistically significant correlations were found between the salinity descriptors and Thalassia aboveground and belowground biomass or Halodule whole sprig biomass (Table 57). The only statistically significant correlate occurred with Ruppia sprig biomass and the number of changes in salinity per day (Table 57). Correlations with Temperature, Light and Water Clarity The percent light reaching seagrass depth was the strongest correlation with green-leaf indices of seagrasses in the stable salinity treatments, although not statistically significant (Table 5-8). This correlation was negative for both Thalassia and Ruppia plants, positive for Halodule. The number of Thalassia shoots increased with increasing light intensities, however (Table 5-9). Shoot number in Halodule increased with higher fractions of light reaching the seagrass depth. None of the physical parameters correlated with changes in rhizome length for Thalassia or Halodule, however the percent light reaching seagrass depth positively correlated with Ruppia rhizome length with a p-value of 0.056 and r2 of 17% (Table 5-9). Leaf lengths of Halodule in the stable salinity treatment correlated with increasing intensities of light at seagrass depth. Leaf lengths in Ruppia correlated negatively with increasing water clarity. Thalassia leaflengths did not correlate with any of the temperature or light measurements (Table 5-10). The changes in leaf length did not correlate with statistical significance for any of the variables for any seagrass (Table 5-10, top panel). Mean temperature and light intensity were strong correlates with increases in Ruppia leaves per shoot and average number of leaves after treatment (Table 5-10, middle and bottom panels).

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156 Table 5-7: Pearson product moment correlations between biomass measurements following treatment and salinity wave descriptors. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Thalassia Tha/assia {shoots) {rhizome} Halodu/e Ruppia Mean Salinity -0 0270 -0 0551 -0.0470 -0 0420 (0.8281) (0.6579) (0.7059) (0.7564) r2= 0.0% r2= 0.0% r2= 0 0% r2= 0 0% Standard Deviation 0 0959 -0 1082 -0.2295 0 0334 of Salinity (0.4402) (0.3834) (0.0617) (0 8050) r2= 0.0% r2= 0 0% r2= 3 8% r2= 0 0% Maximum 0.0099 -0.1876 -0 3176 -0.0679 Amplitude (0.9365) (0 1285) (0.0088) (0.6159) r2= 0 0% r2= 2.0% r2 = 8.7% r2= 0 0% Suddenness of 0 0991 -0.1464 -0.2634 0.1739 Salinity Change (0.4250) (0 2373) (0.0312) (0.1957) r2= 0 0% r2= 0 6% r2 = 5 5% r2= 1 3% Number of Changes -0.2467 -0 0688 -0.0393 -0.4888 Per Day (0.0441) (0.5802) (0.7520) (0.0001) r2 = 4.6% r2= 0.0% r2= 0 0% r2 = 22.5% Significant 0.0789 0 1247 0.1268 -0 1242 Frequency (0.5259) (0 3148) (0 3065) (0 3572) r2= 0.0% r 2 = 0 0% r2= 0.1% r2= 0.0% Absolute -0.1990 0.0636 0.1482 0 0461 Frequency (0 1065) (0 6091) (0 2314) (0 7336) r2= 2.5% r2= 0.0% r2= 070% r2= 0 0%

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157 Table 5-8: Stable salinity treatment Pearson product moment correlations between changes in green-leaf index, temperature and light. Pvalues are given in parentheses. R-squared values are given as well Statistically significant correlates are bold (p<0 001) Tha/assia Halodu/e Rue_e_ia Mean temperature -0 2292 0.1839 0 2001 (0 3451) (0.4512) (0.4414) r2= 0 0% r2= 0 0% r2= 0.0% Intensity of Light at -0.1632 0 1170 -0.0162 Seagrass Depth (0.5043) (0 6334) (0.9506) r2= 0.0% r2= 0.0% r2= 0 0% Percent Light Reaching -0.3854 0.4336 -0 5420 Seagrass Depth (0.1032) (0 0636) (0.0246) r2= 9 8% r2=14.0% r2 = 24 7%

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158 Table 5-9: Stable salinity treatment Pearson product moment correlations between changes in number of shoots, rhizome length, temperature and light. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Number of Shoots Thalassia Halodule Ruppia Mean temperature 0.3978 -0 1121 0.4214 (0 0917) (0.6477) (0 0920) r2= 10 9% r 2 = 0.0% r2=12.3% Intensity of Light at 0.5618 -0 0980 0 2482 Seagrass Depth (0.0123) (0.6899) (0.3367) r2 = 27.5% r2= 0.0% r2= 0.0% Percent Light Reaching 0 2995 0.5769 0 2206 Seagrass Depth (0 2128) (0.0097) (0.3948) r2= 3 6% r2 = 29.4% r2= 0 0% Rhizome Length Tha/assia Halodule Rue_e_ia Mean temperature 0.1914 0.0685 0.2170 (0.4325) (0 7805) (0.4028) r2= 0.0% r 2 = 0.0% r 2 = 0.0% Intensity of Light at 0 0695 -0.0531 0.3347 Seagrass Depth (0 7774) (0.8291) (0.1891) r2= 0.0% r2= 0.0% r2= 5.3% Percent Light Reaching 0 1188 0.2202 0.4719 Seagrass Depth (0.6281) (0.3650) )0.0558) r2= 0.0% r2= 0 0% = 17 1%

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159 Table 5-10: Stable salinity treatment Pearson product moment correlations between changes in leaf length, number of leaves and average number of leaves, temperature and light measurements. Mean temperature Intensity of Light at Seagrass Depth Percent Light Reaching Seagrass Depth Mean temperature Intensity of Light at Seagrass Depth Percent Light Reaching Seagrass Depth Mean temperature Intensity of Light at Seagrass Depth Percent Light Reaching Seagrass Depth Tha/assia 0 2650 (0.2728) r2= 1.6% 0.0404 (0.8695) r2= 0.0% -0.2890 (0.2302) r2= 3 0% Leaf Length Ha/odule 0 3723 (0.1165) r2= 8.8% 0.5369 (0.0178) r2 = 24.6% -0.0589 (0.8108) r2= 0.0% Number of Leaves per Shoot Tha/assia Halodule 0.1004 0.4985 (0.6826) (0.0298) r2= 0.0% r2 = 20.4% 0.2212 0.3771 (0.3627) (0 1115) r2= 0.0% r2= 9.2% -0.1054 (0.0676) (0.6675) r2= 0.0% (0.7833) r2= 0 0% Average Number of Leaves per Shoot Tha/assia Ha/odu/e 0 6257 0.4130 (0 0042) (0.0789) r2 = 35.6% r2=12.2% 0 5137 0 2729 (0.0245) (0.2583) r2 = 22.1% r 2 =2.0% -0.2325 -0.0567 (0.3381) (0.8177) r2= 0 0% r2= 0 0% Ruppia 0.3317 (0.1933) r2 =5.1 % 0 2219 (0.3920) r2= 0.0% -0.6423 (0.0054) r2 = 37.3% Rue,eJa 0.7723 (0.0003) r2 = 60.0% 0.7846 (0.0002) r2 = 59.0o/e 0.3162 ~0.2163) =4 0% Ruppia 0.7937 (0.0001) r2 = 60.5o/. 0.6058 (0.0100) r2 = 32.5% -0.0396 (0 8802) r2= 0.0%

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160 No statistically significant correlates occurred between biomass and temperature and light although Thalassia aboveground biomass correlated strongest with increasing temperature (Table 5-11). In addition to being influenced by temperature, belowground Thalassia biomass correlated with both light intensities and fractions at the seagrass depth Halodule and Ruppia whole sprig biomasses were influenced by light as well, correlating positively with light percent and intensity, respectively (Table 5-11). Correlations with Water Nutrient Concentrations Changes in green-leaf indices of Thalassia in the stable salinity treatment correlated with total phosphorus (r2 = 39.7%), orthophosphate (52 5%) and total dissolved phosphorus (51.0%) concentrations (Table 5-12), although not statistically significant at the p < 0.001 level. Halodule green-leaf indices did not correlate with any of the nutrient concentration measured. A positive relationship existed between Ruppia green-leaf indices and total Kjeldahl nitrogen (TKN) concentrations (Table 5-12) No statistically significant correlates occurred between changes in seagrass shoot number and nutrient concentrations. The strongest correlation in Thalassia was with concentration of total dissolved phosphorus (Table 5-13) Shoot numbers in Halodule correlated negatively with TKN and nitrite concentrations, however a positive correlation existed with nitrate A negative correlation between TKN and Ruppia was the strongest relationship found between changes in rhizome length and nutrient concentrations, although not statistically significant (Table 5-14).

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161 Table 5-11: Stable salinity treatment Pearson product moment correlations between biomass measurements, temperature and light. Pvalues are given in parentheses. squared values are gi v en as well Statistically significant correlates are bold (p<0.00 1 ) Thalassia Thalassia (shoots) (rhizome) Ha/odu/e Ruppia Mean temperature 0.4228 0.4000 0.0754 0.4256 (0 0713) (0.0897) (0 7590) (0 0885) r2=13.0% r2 = 16 0% r2= 0 0% r2=12.7% Intensity of Light at 0 2655 0.4809 0.2062 0 5418 Seagrass Depth (0 2720) (0.0371) (0 3970) (0 0247) r2= 1 6% r2=18 6% r2= 0 0% r2 = 24.7% Percent Light Reach i ng 0.3399 0.5074 0.4669 0 .1 136 Seagrass Depth (0.1545) (0.0266) (0 0439) (0 6641) r2= 6.4% r 2 = 21 4% r2 = 17 2% r2= 0 0%

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162 Table 5-12: Stable salinity treatment Pearson product moment correlations between changes in green-leaf index and nutrient concentrations. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Thalassia Ha/odu/e Ruppia TP 0.6656 -0.4142 0 1220 (0.0094) (0.1410) (0 6777) r2 = 39.7% r2= 10.2% r2= 0 0% PO4 0.6871 -0 3806 0.2514 (0 0195) (0.1794) (0.3860) r 2 = 52.5% r2= 7.4% r2= 0.0% TOP 0.7400 -0.4075 -0.0115 (0 0025) (0.1481) (0.9688) r 2 =51.0% r2=9 7% r2= 0.0% TKN 0 0368 -0 0409 0.7820 (0.9006) (0.8897) (0.0010) r2= 0.0% r2= 0.0% r2 = 57.9% NH4 0.3823 -0.3290 -0.3916 (0.2459) (0 3232) (0.2336) r 2 = 5 1% r2= 0.9% r2= 5.9% NO3+2 -0 2910 -0.1787 -0.0415 (0.3128) (0 5991) (0.9036) r2= 0.8% r2= 0 0% r2= 6.2% NO2 -0 0011 0.1441 0.4656 (0.9970) (0 6231) (0 0934) r 2 = 0.0% r2= 0.0% r2= 15.2% DIN -0 3091 0.0356 -0.5422 (0.2823) (0.9038) (0.0452) r2= 2.0% r2= 0.0% r 2 = 23.5%

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163 Table 5-13 : Stable salinity treatment Pearson product moment correlations betwee n changes in number of shoots following and nutrient concentrations Pvalues are g i ven in parentheses. R-squared values are given as well. Statistically significant correlates are bold (p<0.001) Thalassia Ha/odu/e Ruppia TP 0.4183 -0.2492 -0.4364 (0 1367) (0 3903) (0.1187) r2=10 6% r2= 0.0% r2=12 3% PO4 0.5064 -0.4776 -0 2908 (0.0646) (0.0842) (0 3132) r2= 19.4% r2=16.4% r2= 0.8% TOP 0 6077 -0 1900 -0.3591 (0 0212) (0.5152) (0.2073) r2 = 31 7% r2= 0.0% r2= 5 6% TKN -0.4271 -0 7391 -0.0739 (0.1277) (0.0025) (0 8017) r2 = 11.4% r2 = 50 8% r2= 0.0% NH4 0.2909 0 5454 -0.4549 (0 3856) (0.0827) (0.1597) r2= 0 0% r2=21 9% r2= 11 9% NO3+2 -0 2144 0 5977 -0.2384 (0.5266) (0 0240) (0.4117) r2= 0 0% r2 = 30.4% r2= 0.0% NO2 -0 0931 -0 5935 0 3342 (0 7515) (0 0253) (0 2429) r2= 0 0% r2 = 29 8% r2= 3.8% DIN -0 0650 0.4363 -0.4435 ~0 8494) (0 1797) 1.1719) =0 0% r2=10 0% = 10 7%

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164 Table 5-14: Stable salinity treatment Pearson product moment correlations between changes in rhizome length and nutrient concentrations. Pvalues are given in parentheses R-squared values are given as well. Statistically significant correlates are bold (p<0.001). Thalassia Halodu/e Ruppia TP -0.2779 -0.3416 0.2045 (0 3360) (0.2320) (0.4832) r= 0 0% r= 4.3% r= 0 0% PO4 -0 1531 -0 3244 0.1710 (0.6013) (0.2577) (0.5589) r 2 = 0.0% r= 3 1% r=o.0% TDP -0 2792 -0 3478 0.3787 (0.3337) (0.2231) (0.1818) r 2 = 0 1% r 2 = 4 8% r= 1 2% TKN 0.2045 -0 0126 -0.6662 (0.4830) (0.9659) (0.0093) r= 0 0% r= 0.0% r2 = 39.8% NH4 -0.3980 -0 2455 0 3741 (0.2254) (0.4669) (0 2570) r= 6 4% r=o 0% r2 = 4.4% NO3+2 -0.2590 0.0190 0.0970 (0.4418) (0.9485) (0.7416) r= 0.0% r= 0.0% r=o.0% NO2 0 3553 0.1076 -0 3367 (0 2125) (0.7142) (0 2392) r= 5 3% r= 0 0% r= 3 9% DIN -0.3138 -0.1571 0.0672 (0.3474) (0.6445) (0.8445) r= 0.0% r=o.oo/o r= 0.0%

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165 Halodule leaf lengths had strong positive correlations with orthophosphate and total dissolved phosphorus, with r2 values of 75.7 and 82.3%, respectively {Table 5-15). Ruppia leaf lengths had a statistically significant, positive correlation with total Kjeldahl nitrogen concentration. A strong, yet statistically insignificant, negative correlation occurred with ammonium concentration and Ruppia leaf length {Table 5-15). Although no statistically significant correlates occurred between changes in leaf number and nutrient concentrations, the changes in leaf number in Thalassia were influenced by concentrations ofTP, PO4, and TDP, with r2 values 36.9, 47.4, and 51.0%, respectively {Table 5-16). Average leaf numbers per shoot after treatments were negatively correlated nitrate concentrations for all seagrasses (Table 5-17). Strong, positive correlations occurred between Halodule and Ruppia leaf number and nitrite concentrations, although similar negative relationships occurred with ammonium and dissolved inorganic nitrogen concentrations for both species {Table 5-17). Correlations with Field Conditions at Collection Sites Initial green-leaf indices were higher in Thalassia when conditions in Little Madeira Bay were more saline and experienced less salinity standard deviation {Table 518). Green-leaf indices in Halodule decreased with increasing salinity and increased with higher salinity standard deviations. No correlations were seen with Ruppia starting green-leaf index. Higher salinities also correlated strongly (r2 = 76.9 and 71.1 %)with more Thalassia leaves per shoot {Table 5-19) and longer leaf lengths, although not statistically significant at the p< 0.001 level {Table 5-20). Higher salinities correlated with more

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166 leaves per shoot in Ruppia (Table 5-19), where increased salinity standard deviations led to taller leaves (Table 5-20). No statistically significant relationships were seen involving Halodule leaf numbers or lengths.

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167 Table 5-15: Stable salinity treatment Pearson product moment correlations between changes in leaf length and nutrient concentrations. Pvalues are given in parentheses. R-squared values are given as well Statistically significant correlates are in bold (p<0.001). Tha/assia Halodule Ruppia TP -0.2221 0.7730 0.2920 (0.4453) (0 0012) (0 3111) r2= 0 0% r2 =56.4% r2= 0.9% PO4 0.0230 0.8806 0.5422 (0 9378) (0.0000) (0.0452) r 2 = 0 0% r2 = 75.7% r2 = 23 5% TOP -0.2542 0.9145 0.2343 (0.3805) (0.0000) (0 4200) r2= 0.0% r2 = 82.3% r2= 0.0% TKN 0.5767 -0.1381 0.7874 (0.0308) (0 6377) (0.0008) r2 = 27 7% r2= 0.0% r2 = 58.8% NH4 -0 6663 0.4799 -0.7491 (0.0252) (0.1352) (0.0080) r2 = 38 2% r2= 14 5% r2 = 51 2% NO3+2 -0 5441 -0.3451 -0 6560 (0.0443) (0 2269) (0.0108) r2 = 23 7% r2= 4 6% r2 =38.3% NO2 0 6802 -0.0450 0.6380 (0.0074) (0 8785) (0.0141) r2 = 41 8% r2= 0 0% r2 = 35 8% DIN -0.5203 0 0669 -0.6225 (0.1008) (0.8456) (0.0408) r2=19 0% r2= 0.0% r2 = 32.0%

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168 Table 5-16 : Stab l e salinity treatment Pearson product moment correlations between changes in number of leaves per shoot and nutrient concentrations Pvalues are given in parentheses R-squared values are given as well. Statistically significant correlates are in bold (p<0 001) Thalassia Halodule Ruppia TP 0.6458 -0.1557 0.0211 (0 0126) (0 5950) (0.9429) r2 = 36 9% r2= 0 0% r2= 0 0% PO4 0 7170 0 1203 0 3446 (0.0039) (0 6820) (0.2275) r 2 = 47.4% r2= 0 0% r2= 4.5% TOP 0.7401 -0 0632 0.2554 (0 0025) (0 8302) (0 3782) r2 = 51 0% r2= 0 0% r2= 0.0% TKN -0.0657 0 1967 -0 1889 (0 8234) (0 5002) (0 5177) r2= 0 0% r2= 0 0% r2= 0 0% NH4 0.4057 -0 3734 -0.3550 (0 2157) (0 0640) (0 2841) r2 = 7 2% r2= 25 7% r 2 = 2.9% NO3+2 -0 2520 -0.5889 -0.6867 (0 3847) (0 0267) (0.0067) r2= 0.0% r 2 = 29.2% r2 = 42.8% NO2 -0 0534 0 5878 0.4957 (0 8562) (0.0271) (0.0715) r2 = 0 0% r2=29.1% r2= 18.3% DIN 0 1017 -0 6334 -0 6491 (0 7662) (0 0364) (0 0307) r2= 0 0% r2 = 33.5% r2 = 35.7%

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169 Table 5-17: Stable salinity treatment Pearson product moment correlations between the number of leaves per shoot and nutrient concentrations. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are in bold (p<0 001). Tha/assia Ha/odu/e Ruppia TP 0 2655 -0 1268 -0.2363 (0 3590) (0.6659) (0.4161) r2= 0 0% r2= 0.0% r2= 0.0% PO4 0 6215 0.2767 0.1756 (0 0177) (0.3383) (0.5481) r2 = 33.5% r2= 0 0% r2= 0.0% TOP 0 3379 -0.0302 -0 1397 (0.2374) (0.9184) (0 6339) r2= 4.0% r 2 = 0.0% r 2 = 0 0% TKN 0 5224 0.4505 0.4452 (0.0553) (0.1060) (0.1107) r2=21 2% r2= 13.7% r2= 13.1% NH4 -0.6592 -0.8473 -0.9101 (0.0274) (0.0010) (0.0004) r2= 37 2% r2 = 68.70/o r2 = 80.1% NO3+2 -0.8577 -0.8909 -0.8911 (0.0001) (0.0000) (0.0000) r2 = 71.4% r2 = 77.7% r2 = 77.7% NO2 0.7383 0.8908 0.9300 (0.0026) (0.0000) (0.0000) r2 = 50.7% r2 = 76.7% r2 = 85.4% DIN -0 8205 -0.8970 -0.9288 (0 0020) (0.0002) (0.0000) r 2 = 63.7% r2 = 78.3% r2 = 84.7%

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170 Table 5-18: Pearson product moment correlations between initial green-leaf indices and mean salinity and temperature during the month prior to collection, as well as the salinity at collection Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are in bold (p<0.001). Thalassia Halodu/e Rue_e_ia Mean Sal i n it y 0.8158 -0.7009 0.1198 (0.0000) (0.0008) (0.6469) r2 = 64.6% r2 = 46.1% r2= 0 0% Standard Deviation -0.5386 0 5338 0.3650 of Salinity (0.0174) (0 0186) (0.1497) r2 = 24.8% r2 = 24 3% r2= 7.5% Mean Temperature 0.1637 -0 0399 0.4634 (0.5030) (0 8712) (0 0610) r2= 0.0% r 2 = 0.0% r2=16.2% Salinity Measured 0.7260 -0.8197 -0.3645 at Collection (0.0004) (0.0000) (0 1503) r2 = 52.7% r2 = 65.3% r2= 1 5%

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171 Table 5-19: Pearson product moment correlations between initial number of leaves per shoot and mean salinity and temperature during the month prior to collection, as well as the salinity at collection. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are in bold (p<0.001). Thalassia Ha/odu/e Ruppia Mean Salinity 0.9028 0.6552 0.8914 (0.0137) (0 1578) (0 0423) r2 = 76 9% r 2 = 28 7% r2 = 72 6% Standard Deviation -0.1759 0.4652 -0.6460 of Salinity (0.7389) (0.3525) (0 2389) r2= 0.0% r2= 2.1% r2= 22.3% Mean Temperature 0 1876 0.4228 -0.1168 (0 7219) (0.4036) (0 8516) r2= 0 0% r 2 = 0 0% r2= 0 0% Salinity Measured 0.5022 0.1847 0.8818 at Collection (0 3101) (0 7261) (0.0479) r2= 6.5% r 2 = 0.0% r2 = 70.3%

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172 Table 5-20: Pearson product moment correlations between original leaf lengths and mean salinity and temperature during the month prior to collection, as well as the salinity at collection. Pvalues are given in parentheses. R-squared values are given as well. Statistically significant correlates are in bold (p <0.001 ). Tha/assia Halodule Ruppia Mean Salinity 0.8770 0.5516 0.1071 (0.0218) (0.2565) (0.8639) r2=71.1% r2= 13.0% r2= 0.0% Standard Deviation 0.1781 0.4468 0.8785 of Salinity (0 7357) (0.3743) (0.0499) r2= 0.0% r 2 = 0.0% r2 = 69.6% Mean Temperature 0.4543 0 4477 0.7290 (0 3654) (0 3734) (0.1623) r 2 = 0.8% r2=0.1% r2= 37 5% Salinity Measured 0.3187 -0.1789 -0.0546 at Collection (0.5381) (0.7346) (0.9305) r2= 0 0% r2= 0.0% r2= 0.0%

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CHAPTER6 PRODUCTMTY AND LEAF OSMOLALITYEXPERIMENT 8 Introduction Salinity fluctuation is detrimental to the existence and production of green leaves necessary for photosynthesis, as seen in the previous seven experiments. How primary productivity is affected in the surviving leaves is unknown. Seagrass leaves damaged by fluctuating salinities may have lower rates of productivity due to reductions in photosynthetically viable cells. Many studies have looked at the effect of decreases in salinity on productivity in seagrasses (Barbour 1970, Hammer 1968, and Hellblom and Bjork 1999); however, none address the effects of salinity fluctuation. Lower salinities appear to inhibit photosynthetic activity in seagrasses, not because of reduced salt concentrations, but due to reduced inorganic carbon content. If seawater is diluted with water rich in bicarbonates, assimilation rates are higher (Hammer 1968). Seagrasses exposed to salinity fluctuation treatments should exhibit impaired productivity. By exposing seagrasses to various degrees of light intensity and measuring oxygen evolution, an oxygen evolution vs light curve can be created in order to assess affinity oflight and primary productivity. From what was seen in the facility experiments, it is expected that Thalassia and Halodule will have reduced rates of oxygen evolution in salinity fluctuation treatments with high amplitude and frequency. In 173

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174 addition, Ruppia should have impaired productivity in the high frequency salinity fluctuation treatments. The ability to survive unfavorable salinities depends heavily on the osmotic adjustment within the tissues of the plant (Flowers et al. 1986). Since seagrasses lack salt-secreting glands, they osmoregulate through their epidermal cells (Jagels 1973, 1983). These epidermal cells have highly invaginated plasmalemmas (semipermeable layers of cell protoplasm) with numerous mitochondria situated within, and are closely analogous to the basal cells of salt glands in Spartina and osmoregulatory cells of marine invertebrates (Jagels 1973). Ions are actively transported into channels associated with the plasmalemmas, using an ATP-requiring transport mechanism. In a study of the temperate seagrass Zostera marina, protons (H+ ions) were found to be the major driving ions for this transport mechanism (Fernandez et al. 1999). Other studies of Ruppia (Brock 1981, Durako 2000), found that amino acid accumulation may function as a salt tolerance mechanism. The accumulation of amino acids, such as proline, raises the osmotic potential of the cytoplasm to counterbalance the cell vacuolar solute levels caused by changes in external salinities (Brock 1981 ). The ability for a seagrass to osmoregulate, as well as the energy requirements necessary for ion transport, may be critical for a seagrass to survive fluctuating salinities, especially if salinity change is frequent. The resilience of Ruppia in the facility experiments leads one to expect a high degree of osmoregulation when exposed to fluctuating salinities. Impaired osmoregulation is expected in Thalassia and Halodule, especially in treatments with high amplitudes and frequencies of salinity fluctuation.

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175 Materials and Methods Seagrasses were collected from the same site as the six previous experiments The plants were transported in aerated coolers to Gainesville, Florida where they were acclimated in an approximately 1.1 m 3 round polyethylene tub initially filled with water of the same salinity as the water in which they were collected (14%0). Purified seawater was diluted with aged tap water to achieve a salinity of 14o/oo. The seawater (of approximately 35o/oo) used in Gainesville was collected from the Atlantic Ocean at Marineland, Florida, and subsequently treated in a recirculating seawater purification system consisting of a sand filter, UV sterilizer, and biofilter. Salinity in the tub was gradually adjusted to 18o/oo by the addition of seawater over a 63 day period. Twenty-seven 13 liter white polyethylene buckets (without lids) were used as experimental tanks. Nine treatments were applied that differed in wave amplitude ( 4 to 32 o/oo and 11 to 25 o/oo salinity ranges), frequency (1/4 and 1/8 day), final salinity (low or high) and a stable 18 o/oo salinity (Figure 6-1, bottom panel). Three replicate buckets were used per treatment. Within each bucket, two sprigs each of Thalassia, Halodule, and Ruppia were placedone for oxygen evolution vs. light determination, the other for osmolality measurements. The seagrass sprigs were not planted but were left suspended in the water. All buckets were covered by a 70% shadecloth Thalassia and Halodule sprigs were selected by the same criteria used in the facility experiments. Selected Ruppia sprigs had at least 15 shoots and a growing rhizome tip. The experimental treatments were applied for 9 days, beginning on May 7, 2000. Treatments were divided into two groups and staggered by one day due to the time necessary for data collection. The two groups were 1) high frequency treatments (Codes

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176 C, D, G, and H) and 2) low frequency and stable salinity treatments (Codes A, B, E, F, and SST). A visual survey was not done during the course of the treatments, however a green leaf index was assessed prior to and following the treatments. After the treatment period, seagrasses from the same bucket were randomly selected into groups for oxygen evolution/uptake vs. light and osmolarity measurements. In order to create an oxygen evolution/uptake vs. light curve, selected seagrass sprigs were placed into 300 ml BOD bottles in water of 18o/oo salinity. The BOD bottles were placed in a water bath to keep temperature constant. Treatments for oxygen evolution vs. light included in the absence of light (bottles wrapped in foil) and various light intensities (10%, 30%, and 100% of full sun) using shade cloths placed over the entire water bath. Dissolved oxygen concentrations were measured (YSI 57 DO Meter with a BOD Electrode) initially and approximately every hour until a stable measurement was achieved. Each plant was exposed to every light level for approximately an hour, starting from dark and increasing light incrementally. Following a dissolved oxygen measurement, a layer of shadecloth was gradually folded back over the water bath to expose the seagrasses to increased light intensities. Once all samples in a bath were measured, the shadecloth layer was removed. Blank BOD bottles were measured in all light levels as well so productivity in the incubation water could be estimated and subtracted. Sunlight was measured using a quantum photometer (Li-Cor Inc. model LI190). Fractions of sunlight received in the treatments were estimated by dividing the mean sunlight during the treatment period by the amount of shading provided by the shadecloths

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177 35 A 30 B 0 25 --tr--C 20 :== )( D 15 C: E 10 ns tn 5 F 0 G 0 2 4 6 8 10 -H Experiment Day SST Treatment Amplitude(%.) Period (Days) Mean("-) Final Exposure A 7 8 18 High Salinity (25%o) B 7 8 18 Low Salinity (11%o) C 7 4 18 High Salinity (25%o) D 7 4 18 Low Salinity (1 lo/oo) E 14 8 18 High Salinity (32%o) F 14 8 18 Low Salinity (4%o) G 14 4 18 High Salinity (32 o/oo ) H 14 4 18 Low Salinity (4o/oo) SST 0 0 18 No Step (18o/oo) Figure 6-1: Salinity patterns (top panel) and treatment codes (bottom panel) for Experiment 8

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178 During the first phase of this experiment, the automatic stirrers attached to the BOD electrode ceased to spin. In order to maintain proper circulation of water, the BOD bottles were agitated manually by vigorous shaking. This method was used for the remainder of the experiment. Internal leaf osmolality was determined using a vapor pressure osmometer (Wescor Vapro 5100). Sprigs were taken from their respective treatments and placed in water of 18 %0 salinity. Following at least three but up to six hours at this salinity, the leaves were blotted dry and cut in 6 mm sections to fill the chamber of the osmometer. In most cases, one section of Thalassia, and three sections of Halodule and Ruppia were used to fill the chamber. Osmolality was then measured. Three replicates ( a section from a new seagrass leaf from a replicate bucket) were used for each treatment. Data Analysis for Experiment 8 Oxygen evolution/uptake vs. light curves were created by charting dissolved oxygen measurements normalized by leaf surface area with estimated light intensities received during the period. On the first day, measurements were taken on the low amplitude-high frequency and high amplitude-high frequency treatments. On the second day, measurements were made on the low amplitude-low frequency, high amplitude-low frequency, and stable salinity treatments. Various aspects of the oxygen evolution/uptake vs. light curves were analyzed, including oxygen uptake in the dark, maximum net oxygen evolution, net oxygen evolution at the light intensity of 400 E*m" 2 *sec 1 (a similar light intensity experienced by the seagrasses on both days), gross oxygen evolution (calculated from the dark oxygen

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179 uptake and the maximum net oxygen evolution), and the alpha, or affinity to low light ( estimated as the slope of the oxygen evolution/uptake vs light curve between no light and the lowest light level). One-way ANOV As (Fisher's LSD procedure) were run t o compare the responses by each species to each treatment with regard to the aforementioned aspects of the oxygen evolution vs light curves. The significance threshold was set at p < 0.05. One-way ANOVAs were also used to identify differences among means of osmolalities. Results Salinity patterns for the nine treatments in Experiment 8 are given in Figure 6 -1. Mean temperature for all treatments was approximately 28C. Oxygen Evolution/Uptake vs Light Intensity Oxygen evolution/uptake vs. light curves for Thalassia are given in Figure 6-2. Each line represents a replicate for each treatment. Samples measured on the second day (Treatments A, B, E, F, and SST) received a greater intensity of light, with full sun intensities of approximately 975E*m 2 *sec 1 whereas those on the first day were approximately 407 E*m2 *sec1 Oxygen uptake was greatest in Thalassia in the high amplitude/low frequency treatment ending with high salinity {Treatment E); oxygen uptake rates in the other treatments did not statistically differ from those measured in the stable salinity treatments (Figure 6-3, top panel). Maximum net oxygen evolution and oxygen evolution measured at 400 E*m 2 *sec 1 did not statistically differ amongst treatments (Figure 6-3, second and third panels)

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180 Thalassia gross oxygen evolution was greatest in Treatment E as well (Figure 6-4, top panel). Gross oxygen evolution in this treatment was not statistically greater than the other treatments, except for the low amplitude, low frequency Treatment A. Affinity at low light levels was lowest in the low amplitude, low frequency treatments (Treatments A and B) and the stable salinity treatment (Figure 6-4, middle panel). Oxygen evolution/uptake vs. light curves for Halodule are given in Figure 6-5. Greatest dark oxygen uptakes occurred in the high amplitude, low frequency treatments, Treatments E and F (Figure 6-6, top panel). In general, greatest maximum net oxygen evolutions in Halodule occurred in the high amplitude, high frequency treatments (Treatments G and H) and Treatment F, the high amplitude, low frequency, low final salinity treatment (Figure 6-6, middle panel). Oxygen evolution at 400 E*m. 2 *sec 1 was greatest in treatments with high frequency and final exposure to low salinities, Treatments D and H (Figure 6-6, third panel). Halodule gross oxygen evolution was statistically greater in the high amplitude, low frequency, low final salinity treatment (Treatment F) than that measured in other treatments (Figure 67, top panel). Affinity for low light was least in the stable salinity and low amplitude, low frequency treatments, A and B (Figure 67, middle panel). Oxygen evolution/uptake vs. light curves for Ruppia are given in Figure 6-8. Dark oxygen uptake was greatest in Ruppia in the high amplitude, low frequency, low ending salinity Treatment F (Figure 6-9, top panel). Greatest Ruppia net oxygen evolutions occurred in the low amplitude, low frequency, high ending salinity treatment (Treatment A) and the stable salinity treatment (Figure 6-9, second panel). Net oxygen evolutions at 400 E*m. 2 *sec 1 were similar amongst treatments, except for Treatment B

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SST Treatment 0 002 ~--------------~ 0 0 0 1 -t----~:,,,,_C...~-=:.-c !' ;'~~~.::-.::-_ ~_=.::_ _-_ """ C:::._::~,_ -4~ 1-j 0 +-~ ~.-::==---,----,,----.----I -0 001 ~:..L---------------1 -0 002 =-----------------' 0 20 0 400 600 800 1000 Treatment C (Low Amp HIFreq, HISal) 0 004 0 002 0 -0 002 -0 004 0 100 200 300 400 500 Treatment F (HIAmp, LowFreq, LowSal) 0 002 ~---------------~ 0 00 1 -0 -0 001 .,..--?'7':;...c. ~---------------l -0 002 ....... /'--------------------4 -0 003 -'------------------' 0 00 1 5 0 00 1 0 0005 0 -0 000 5 -0 001 -0 0 0 15 0 0 1 0 005 0 -0 005 -0 0 1 0 003 0 002 0 001 0 Treatment A (Low Amp LowFreq HIS al) ---/ __.-a 1 :r-/"' ...... 0 200 4 0 0 600 800 Treatment D (Low Amp, HIFreq LowSal) 100 200 300 400 Treatment G (HIAmp, HIFreq, HISal) ---... .# --0 -0 001 V -0 002 -0 003 0 200 400 600 800 1000 0 100 200 300 400 1000 500 Treatment B (LowAmp LowFreq, LowSal) 0 004 ----------------= 0 00 2 t= == = = =~;~~---~ ~;:::~:;~~~~;;:~ 0~ -0 002 -0 004 .,__ _______________ _, 0 200 400 600 800 1000 Treatment E (HIAmp LowFreq, HISal) :S k = ;;; I -0 01 .,__ _______________ _.J 0 006 0 004 0 002 0 -0 002 -0 004 -0 006 0 200 400 600 800 1000 Treatment H (HIAmp, HIFreq, LowSal) ----,1,..--7 I/ 500 0 100 200 300 400 500 Figure 6-2 : Oxygen Evolution/Uptake vs Light Curves for Thalassia. X-axis units are light intensity / m 2 /sec), Y-axis units are dissolved oxygen (mg O i/ l*min*cm 2 leaf surface area). Treatments are described by magnitude of amplitude and frequency, as well as fi nal salinity exposure p ri or to 18o/oo ...... 00 ......

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182 Oxygen Uptake In Dark 0 -0 001 0 002 -0 003 -0 004 -0 005 -0 006 A B C D E F G H SST Maximum Net Oxygen Evolution 0 0 1 0.008 0.006 0 004 0 002 0 A B C D E F G H SST Net Oxygen Evolution at 400 uE/m2/sec 0 0 1 0 008 0 006 0.004 0 002 0 A B C D E F G H SST Dark Uptake Max Evolution Evol. At 400 Treatment Amp Freq. Final Sal. ANOVAGp ANOVAGp. ANOVAGp. A 7 %o 1/8 d 25%o B A A B 7 o/oo 1/8 d 11 %o AB A A C 7 %o 1/4 d 25%o B A A D 7 o/ oo 1/4 d 11 %o AB A A E 14 %o 1/8 d 32 %o A A A F 14 %o 1/8 d 4 %o B A A G 14 %o 1/4 d 32 %o B A A H 14 %o 1 / 4 d 4 %o AB A A SST Oo/oo 0d 18 %o B A A F igure 6-3: Thalassia dark oxygen uptake, maximum oxygen evolution, and net oxygen evolution for treatments in Experiment 8. Y axis units are dissolved oxygen (mg 0 2 / l*min*cm 2 leaf surface area). Error bars are standard deviations Overlaps ofletters in ANOV A groups signify no statistical difference at 95% confidence level.

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183 Gross Oxygen Evolution A B C D E F G H SST Alpha Value (Affinity) at Low Light Levels A B C D E F G H SST Gross Evol Affinity Treatment Amp. Freq Final Sal. ANOVAGp ANOVAGp A 7 %o 1/8 d 25 %o A A B 7 %o 1/8 d 11 %o AB A C 7 %o 1/4 d 25 %o AB AB D 7 %o 1/4 d 11 %o AB B E 14 %o 1/8 d 32 %o B AB F 14 %o 1/8 d 4%o AB A G 14 %o 1/4 d 32 %o AB AB H 14 %o 1/4 d 4 %o AB AB SST 0 %o Od 18 %o AB A Figure 6-4 : Tha/a s sia gross oxygen evolution and affinity at low light levels for treatments in Experiment 8. Y axis units for gross oxygen evolution are dissolved oxygen (mg O i/ l*rnin*cm 2 leaf surface area) Y-axis units for affinity are dissolved oxygen per light intensity (mg 02 / l*min*cm 2 leaf surface area I E*m 2 *sec 1 ). Erro r bars are standard deviations. Overlaps of letters in ANOV A groups signify no statistical difference at 95% confidence level.

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SST Treatment 0 004 -.-----------------, 0 00: [: ~ ;~~ ; ==~====~ --0 002 e"">'------------l --0 004 ________ ______ __, 0 200 400 600 800 1000 Treatment C (Low Amp, HIFreq, HiSal) 0 004 -.----------------, 0 002 r~ c====== ~ -----= ~--=::.... ------1 0 +-, ~ ----,~.;; ;;:;::.e~~,,_=----l --0 002 ~ --------------, --0 004 -'------------------' 0 005 0 --0 005 --0 01 --0 015 Treatment A (Low Amp LowFreq HiSal) / 0 200 400 600 800 1000 Treatment D (LowAmp, HIFreq, LowSal) 0 004 -.-----------------, 0 002 +------==-----=-_...::: :......., ,...sa-----i 0 i7'. '{----r--"""= ~ a:::~-=--.---------,------1 --0 002 -+,ff------------< --0 004 ~------------~ Treatment B (Low Amp, LowFreq, LowSal) 0 004 ,...t ========= ;~:. ~ ;;;;~:::::::::~~ ;~ -~ ~ r --0 006 .,_ _______________ ~, 0 200 400 600 800 1000 Treatment E (HIAmp, LowFreq, HISal) 0 00:~ --0 005 ===-------------0 01 ++--------------0 015 ~-------------, R 0 100 200 300 400 500 0 100 200 300 400 500 0 200 400 600 800 1000 0 01 0 005 0 --0 005 --0 01 --0 015 --0 02 Treatment F (HIAmp, LowFreq, LowSal) __.-;:,_ / '/ 0 200 400 600 800 1000 Treatment G (HIAmp, HIFreq, HiSal) 0 006 0 004 0 002 0 --0 002 --0 004 --0 006 0 100 200 300 400 Treatment H (HiAmp, HiFreq, LowSal) 0 006 -.------------------, 0 004 +-----------: ::tt-----1 0 002 +--aa------------, ~~--=------1 o h ~ ::....::;,; ~::::n:::::c:=:= ~;;::;<::,-----.-------, --0 002 +<-i '--~ -=--------1 --0 004 QI--------------; --0 006 -'------------------' 500 0 100 200 300 400 500 Figure 6-5: Oxygen Evolution/Uptake vs. Light Curves for Halodule. X-axis units are light intensity (E/m 2 /sec), Y axis units are dissolved oxygen (mg Oi/l*min*cm 2 leaf surface area) Treatments are described by magnitude of amplitude and frequency as well as fi nal salinity e x posure prior to l 8%0 ..... 00 .i::.

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Treatment A B C D E F G H SST 185 Oxygen Uptake in Dark 0 ~............... .,..,.--,,-.-..--r-.-.-,-..--,--.--,,...,.--i-T....,.......,..-,-,-r-.,-, -0 005 -0 01 +.--:=----------t---, LLJ-------; -0 015 +-----------+--------; -0.02 ......_ _______________ A B C D E F G H SST Maximum Net Oxygen Evolution 0 008 ~------------------, 0.006 -1-----------+--------; 0 004 +---------;;;1;;;;--=---.--------i 0 002 +-rn-----a.l---:=,-----t 0 .+-L-'-......--'--.:.,._,,---1-...L....,--'--'---r--'--'--r........ -,-'--L,--'---,-,..~ A B C D E F G H SST Net Oxygen Evolution at 400 uE/m2/sec 0 005 ....------------------, 0 004 -+-----------------r----, 0.003 +--------+-----+---'l"---1 ~1--------, 0.002 +--t-----lr--t .... i------+------1 .r. t--___, 0.001 -+-+---r n---1f---; J --.......--1 0 ~...._~----.-"-I .......... _._......,_......_......,_ ........ ......,__._..._....,....... ....... ..,.......--, A B C D E F G H SST Dark Uptake Max Evolution Evol. At 400 Amp Freq. Final Sal. ANOVAGp. ANOVAGp. ANOVAGp 7 %o 1/8 d 25 %o B ABC AB 7 %o 1/8 d 11 %o B ABC AB 7 %o 1/4 d 25 %o B AB A 7 %o 1/4 d 11 %o B ABC BC 14 %o 1/8 d 32 %o AB A AB 14 %o 1/8 d 4 %o A C ABC 14 %o 1/4 d 32 o/oo B BC ABC 14 %o 1/4 d 4%o B BC C 0 %o 0d 18 %o B A AB Figure 6-6: Halodule dark oxygen uptake, maximum oxygen evolution, and net oxygen evolution for treatments in Experiment 8. Y-axis units are dissolved oxygen (mg 0 2 /l*min*cm 2 leaf surface area). Error bars are standard deviations. Overlaps ofletters in ANOV A groups signify no statistical difference at 95% confidence level.

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) \ 186 Gross Oxygen Evolution 0.025 -,---------------, 0 02 4-----------+-----------; 0.015 -l------------------------1 0.01 --l--.f-------1-----1 J----------i 0 .005 --+--f 1..~-r:1c:1---=-----.+,,--n : 1~m---1 "~1-------1-:"-t----r---i 0 -4-1,;u_.,.-Lo:.u_.---i::,..i_,--1....L.....,--...&..::."---,...L.;.;.il-.L.,J.-.--' ....... ...,.............., A B C D E F G H SST Alpha Value (Affinity) at Low Light Levels 0 00012 -,------------~ 0 0001 +---------------....-------; 0 00008 -1----------------,t--------t----; 0 .00006 +--,...-----------=i=---+---t-~~-+---mr--------i 0 .00004 -t-=1=------i J---f.1.1--+------i l'iil--~+l--1,...1-----l 0.00002 +-l \ lff----r.t::t-f 1' 0 +-"::.:a_,.=u....,._.__L-r-J.,,;.J..--,-J..&..L~.:.a..-,---1..."-r .......... -=-i A B C D E F G H SST Gross Evol. Affinity Treatment Amp Freq. Final Sal. ANOVAGp. ANOVAGp. A 7 %o 1/8 d 25 %o A ABC B 7 %o 1/8 d 11 %o A AB C 7 %o 1/4 d 25 %o A BC D 7%o 1/4 d 11 %o A BC E 14 %o 1/8 d 32 %o A ABC F 14 %o 1/8 d 4 %o B C G 14 %o 1/4 d 32 %o A BC H 14 %o 1/4 d 4 %o A C SST 0 %o Od 18 %o A A Figure 6-7 : Ha/odule gross oxygen evolution and affinity at low light levels for treatments in Experiment 8. Y-axis units for gross oxygen evolution are dissolved oxygen (mg 02/l*min*cm 2 leaf surface area). Y-axis units for affinity are dissolved oxygen per light intensity (mg 0 2 /l*min*cm 2 leaf surface area/ E*m" 2 *sec 1 ). Error bars are standard deviations. Overlaps ofletters in ANOVA groups signify no statistical difference at 95% confidence level.

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SST Treatment Treatment A (Low Amp, LowFreq HiSal) Treatment B (Low Amp LowFreq, LowSal) 0 0015 0 00 1 5 0 00 1 0 00 1 0 001 -0 0005 .,,.--: ---===== JI 0 0005 0 0005 ./ ,,. 0 0 0 --0 0005 .// --0 000 5 I --0 001 --0 0005 1/ --0 0015 --0 00 1 I --0 001 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Treatment C (LowAmp, HighFreq, HISal) Treatment D (LowAmp, HIFreq, LowSal) Treatment E (HIAmp, Low Freq, HISal) 0 001 0 0008 0 001 ---,JI 0 0006 0 0005 0 0004 ~0 0005 -------0 0002 ----_,.,..,-~ 0 / ./" --....., 0 /I 0 I/ --0 0005 --0 0002 :; --0 0005 I ,, --0 0004 --0 001 --0 0006 --0 001 0 100 200 300 400 500 0 100 200 300 400 500 0 200 400 600 800 1000 Treatment F (HIAmp LowFreq, HISal) Treatment G (HIAmp, HIFreq, HISal) Treatment H (HIAmp, HiFreq, LowSal) 0 001 0 001 0 001 0 0005 0 0005 ----6 ,,,,,-:::.. 0 0005 0 0 --0 0005 _,,,r 0 / .,../ --0 0005 / --0 001 --0.0005 --0 001 --0 0015 --0 001 --0 0015 0 200 400 600 800 1000 0 100 200 300 400 500 0 100 200 300 400 500 Figure 6-8: Oxygen Evolution/Uptake vs. Light Curves for Ruppia. X-axis units are light intensity (E/m 2 /sec), Y-axis units are dissolved oxygen (mg 02/l*min*cm 2 leaf surface area). Treatments are described by magnitude of amplitude and frequency, as well as final salinity exposure prior to l 8%0. 00 -..J

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Treatment A B C D E F G H SST 188 Oxygen Uptake in Dark -0.0005 A B C D E F G H SST Maximum Net Oxygen Evolution 0.0012 ~------------------, 0 001 -t----.ata.--------------.---, 0.0008 0.0006 0 0004 0.0002 0 -+-'"....._.,.............._,_._.........,,......_....._,.___.__......,..__.__._.__,__.._.--,-..._._--.--,----i A B C D E F G H SST Net Oxygen Evolution at 400uE/m2/sec 0.001 ,-------------------, 0 0008 ++--------------------, 0 .0006 +-r -t-t---l .. 1-----=i =------,: =----+ -;;;i;;;; ,----t--=----1 0.0004 0.0002 0 -+-'"_._.,.........--.,_._........., ........ ......_,.. ......... ......,.. __ ,__,_ ........ --,--.--.--,----i A B C D E F G H SST Dark Uptake Max Evolution Evol. At 400 Amp Freq. Final Sal. ANOVAGp. ANOVAGp. ANOVAGp 7 %o 1/8 d 25 %o AB C B 7 %o 1/8 d 11 %o BC A A 7 o/oo 1/4 d 25 o/oo BC A B 7 o/oo 1/4 d 11 %o C A B 14 %o 1/8 d 32 %o BC AB AB 14 o/oo 1/8 d 4%o A A B 14 o/oo 1/4 d 32 %o BC A B 14 %o 1/4 d 4%o ABC A B 0 %o 0d 18 %o AB BC B Figure 6-9: Ruppia dark oxygen uptake, maximum oxygen evolution, and net oxygen evolution for treatments in Experiment 8. Y-axis units are dissolved oxygen (mg 0 2 /l*min*cm 2 leaf surface area). Error bars are standard deviations. Overlaps ofletters in ANOV A groups signify no statistical difference at 95% confidence level.

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0 0025 0 002 0.0015 0.001 0.0005 0 189 Gross Oxygen Evolution T ,ii ~ ~'' r I rf :c ....rt ,- !\ ....i 1 , .j. ,, : A B C D E F G H SST Alpha Value (Affinity) at Low Light Levels 0.00002 ~---------------, 0.000015 +---~ --------r------; 0.00001 +---~ t--.-1-. ----rt, ----j 0 .000005 -t-;;;;-----t-'I ':, 0 4-1-........,..-,.......__,.__,___,'---,-J.._._____.......__,......,_.._,_ ....... .....,..... ___ ___..~ A B C D E F G H SST Gross Evol. Affinity Treatment Amp. Freq. Final Sal. ANOVAGp. ANOVAGp. A 7 loo 1/8 d 25 loo D AB B 7 loo 1/8 d 11 loo ABC AB C 7 loo 1/4 d 25 loo ABCD D D 7 loo 1/4 d 11 loo A CD E 14 loo 1/8 d 32 loo ABC A F 14 loo 1/8 d 4 loo BCD ABC G 14 loo 1/4 d 32 loo A BC H 14 loo 1/4 d 4 loo AB CD SST 0 loo Od 18 loo CD AB Figure 6-10 : Ruppia gross oxygen evolution and affinity at low light levels for treatments in Experiment 8. Y-axis units for gross oxygen evolution are dissolved oxygen (mg O 2 / l*min*cm 2 leaf surface area). Y-axis units for affinity are dissolved oxygen per l ight intensity (mg O 2/ l*min*cm 2 leaf surface area/ E*m 2 *sec1 ). Error bars are standa r d deviations. Overlaps of letters in ANOV A groups signify no statistical difference at 95% confidence level.

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190 which was significantly lower (Figure 6-9, third panel). Affinity to low light in Ruppia was greatest in the treatments with a high frequency of salinity fluctuation (Treatment C,D, G, and H) (Figure 6-10, middle panel). All other treatments had similar affinities. Internal Leaf Osmolality Highest osmotic concentrations were measured in Thalassia sprigs from the stable salinity treatment (Figure 6-6). For each combination of amplitude and frequency, seagrasses with recent exposure to lower salinities had greater leaf osmolalities than those with recent exposure to higher salinities. Internal leaf osmolalities of those exposed to the high amplitude treatment that had recent exposure to high salinities (Treatments E and G) were significantly lower than those with recent exposure to low salinities (F and H). Halodule osmolalities were highest among treatments exposed to low salinities for each amplitude-frequency combination (Figure 6-7). In high frequency treatments, sprigs with recent exposure to low salinities had significantly higher internal leaf osmolalities than those exposed to higher salinities (Treatments C vs. D and G vs. H). No significant differences in Ruppia osmolality occurred amongst treatments (Figure 68), although those in the stable salinity treatment had the greatest mean osmolality. Discussion The increase in oxygen evolution measured in seagrasses exposed to salinity fluctuation treatments could be due in part to two mechanisms. The loss of outside leaves on a seagrass shoot led to increased rates of primary production in the remaining

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Thalassia Leaf Osmolality 1400 1300 1200 C) 1100 ........ 0 1000 ... E .. E 900 800 700 600 AMPLITUDE 7 ppt 7 ppt 7 ppt 7 ppt 14 ppt 14 ppt 14 ppt 14 ppt 0 ppt FREQUENCY 1/8 day 1/8 day 1/4 day 1/4 day 1/8 day 1/8 day 1/4 day 1/4 day Oday FINAL TREATMENT SAL 25 ppt 11 ppt 25 ppt 11 ppt 32 ppt 4 ppt 32 ppt 4 ppt 18 ppt TREATMENT CODE A B C D E F G H SST Figure 6-6 : Internal leaf osmolality of Thalassia in treatments of Experiment 8. Osmotic concentrations of water of salinity of l 8%0 ran g ed between 51 2 and 524 mmol/kg \0

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Halodule Leaf Osmolality 1800 ,. 1600 C) ,. 1400 ........ 0 1200 0 E 1000 E .. 800 600 AMPLITUDE FREQUENCY FINAL TREATMENT SAL TREATMENT CODE Figure 6-7 : Internal leaf osmolality of Halodule in treatments of Experiment 8 Osmotic concentrations of water of salinity of 18%0 range d b etween 5 12 and 5 24 mm o l/kg. N

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Ruppia Leaf Osmolality 1400 1300 ... .. C) 1200 0 0 0 ... ........ 1100 0 0 1000 E 900 E 800 700 600 AMPLITUDE 7 ppt 7 ppt 7 ppt 7 ppt 14 DDt 14 ppt 14 ppt 14 ppt 0 ppt FREQUENCY 1/8 day 1/8 day 1/4 day 1/4 day 1/8 day 1/8 day 1/4 day 1/4 day 0 day FINAL TREATMENT SAL 25 DDt 11 ppt 25 ppt 11 ppt 32 ppt 4 ppt 32 DDt 4 DDt 18 oot TREATMENT CODE A B C D E F G H SST Figure 6 8: Internal leaf osmolality of Ruppia in treatments of Experiment 8. Osmotic concentrations of water of salinity of 18%0 ranged between 512 and 524 mmol/kg

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194 undamaged leaves in a study by Tomasko and Dawes (1989). Remaining leaves, even if undamaged by salinity fluctuation, could be compensating for those lost in the salinity fluctuation treatment. Higher affinities to light in seagrasses exposed to stressful treatments (such as Ruppia in the high frequency treatments) may be due to this compensatory mechanism. Secondly, salinity fluctuation may in fact have damaged these leaves, and impaired their internal lacuna! system, which would allow normally stored oxygen to escape, giving the impression of greater primary production. Normally, as production increases during the day, Thalassia leaves swell to as much as 200-250% their morning volumes, due to the internal production of gases at a much greater rate than can be exchanged through the leaf surface (Zieman 1974). If seagrasses cannot store metabolic gases adequately, oxygen will be released to the water column during light, increasing measured dissolved oxygen concentrations. Osmolality in Ruppia leaf tissues changed within one minute of exposure to a new salinity (Durako 2000). Since osmolalities in Experiment 8 were measured between three to six hours following exposure to a new salinity, the initial change in Ruppia was probably missed. The exact time of exposure to the new salinity was not recorded for seagrasses in this experiment and should be documented in further osmolality studies. All seagrasses tested appear to osmoregulate rather than osmoconform, since internal leaf osmolalities were twice that of the ambient water for all seagrasses exposed to the stable salinity treatment. Osmolalities measured in this study were assumed to be that of the internal extracellular fluids. Seagrasses must adjust the ionic concentrations of their internal fluids when ambient salinity changes. The rate of osmoregulation is also

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195 important. The rapid acclimation rate of osmoregulation in Ruppia may account for its resiliency in the salinity fluctuation experiments. Osmoregulation appears to be much slower in Thalassia and Halodule than in Ruppia. Slower acclimation rates may be costly to seagrass survival, especially when salinity is constantly changing. When salinity fluctuates often, as in the facility experiments or within the northern land margin of Florida Bay, the costs may exceed the gains, causing leaf mortality. With repeated occurrence, fewer and fewer resources will be available for production of new plant structure or propagules, affecting the distribution and abundances of these species. In general, the oxygen evolution/uptake vs. light curves and the osmoregulatory capabilities of the seagrasses reaffirm their distributions in the estuary. Thalassia, with the highest affinity to light and the lowest ability to osmoregulate, prefers deeper waters with more stable salinities. Halodule, with intermediate affinity, is found in more shallow waters than Thalassia. Ruppia, with a lower affinity to light than Halodule and Thalassia and the highest degree of osmoregulation, is commonly found in shallower waters with variable salinity. Furthermore, Ruppia oxygen evolution/uptake vs light curves rarely approached saturation, indicating a preference for high light levels. Ruppia also had the lowest oxygen evolution per unit leaf area. Ruppia has a low investment in rhizomous storage, allocating more resources to leaf and seed production, which may be energetically less expensive.

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CHAPTER 7 DISCUSSION Overall Discussion Salinity fluctuation has a decisively negative effect on seagrass survival and community development. Fluctuations in salinity may be more important than mean salt concentration in determining the distribution of euryhaline macrophytes in estuarine waters (Montague and Ley 1993). The frequent phenomenon of salinity change may cause the sparse seagrass communities within the ponds and bays of the northern land margin of Florida Bay and elsewhere in seagrass dominated estuaries. Seagrass survival may be impaired by the metabolic costs incurred by osmoregulation in response to salinity fluctuation. When submerged macrophytes are exposed to a change in salinity, turgor is maintained by pumping ions through potassium and chloride channels (Fernandez et al. 1999). If the new salinity is higher, ions must be pumped inward to maintain osmotic balance; if the new salinity is lower, ions are pumped outward. In addition, amino acids are used to osmoregulate. The use of organic molecules to achieve osmotic balance diverts production away from growth and reproduction. Active processes, such as pumping ions and amino acid formation, have energetic costs. The loss of photosynthetic tissue as a result of salinity fluctuation further exacerbates the metabolic toll of frequent osmoregulation. Seagrass rhizomes serve as 196

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197 storage sites for carbohydrates and proteins used for leaf regeneration (Valentine et al. 1997). If these storages are frequently tapped in order to make new leaves to replace damaged ones without adequate replenishment, the plant could die. Distributions and abundances of seagrasses are, in turn, influenced by depletion of rhizomatous resources. Sexual reproduction is infrequent in Thalassia and Halodule (McMillan and Mosely 1967). Reproduction is these species is primarily achieved through vegetative growth. For this to occur, viable plants must be present. Starch reserves in rhizomes must not only allow regeneration of leaves, but are necessary for rhizome extension. Ruppia, on the other hand, has very thin rhizomes. High fecundity and rapid growth increase its chances of survival (Kantrud 1991 ), and create its ephemeral boom and bust cycles of abundance. In addition, Ruppia has an advanced seasonal growth cycle compared to Thalassia and Halodule, initiating growth in the winter, prior to the other seagrasses (Lazar and Dawes 1991). Transplantation, like salinity fluctuation, can lead to physiological shock in seagrasses (Bird et al. 1993, Van Tussenbroek 1996). The general defoliation and chlorosis experienced by seagrasses during the first 10 days in all experiments (including stable salinity) may be a result of handling during transplantation. Transplantation may also have made the test sprigs more susceptible to salinity fluctuation, but the recovery in some treatments ( especially in stable salinity) suggests that the stress associated with transplantation would be overcome. The initial leaf loss and chlorosis of all plants in most treatments need not affect the interpretation of the affect of salinity fluctuations

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198 more than would be caused by, perhaps, one or two additional cycles of salinity fluctuation. The range, amplitude, and frequency of salinity fluctuation are major determinants of seagrass survival. The better survival of Thalassia and Halodule in more marine salinities is well documented anecdotally (Phillips 1960, Zieman 1975), and to a lesser degree, experimentally (McMahan 1968). The better survival of these species in fluctuation regimes at higher average salinities is further experimental evidence of marine salinity as a more appropriate environmental condition for these two species. Salinity is non-optimal for seagrass survival and growth more often when the amplitude of fluctuation increases. The loss of photosynthetic material and plant structure in these plants was not as severe when the amplitude of fluctuation waves was smaller, possibly in part because the ambient salinities were closer to their optimal ranges. As shown in simulations of the hypothetical effects of salinity on seagrasses (Fears 1993, Anastasiou 1999), longer lag times in acclimation dampen the amplitude of internal osmolality fluctuations, but also increase the time the plants internal osmolality differs from that in its surrounding water. The resulting greater osmotic potential may directly damage cells through cell bursting or shrinkage. Ruppia has the widest salinity range of any angiosperm (Brock 1981, Kantrud 1991). Despite its tolerance to wide ranges of salinity, the frequency of salinity changes were detrimental to its survival. Defoliation, chlorosis, and higher affinities to light associated with exposures to high frequency salinity fluctuations may provide insight into the osmoregulatory mechanisms of this species. In spite of Ruppia 's ability to

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199 osmoregulate rapidly and possibly to compensate for stress by using more light, it may still be energetically costly if the need for osmoregulation is frequent. Ruppia has little rhizomous storage to affect this process. The susceptibility of Ruppia to salinity fluctuation with high frequency in the facility experiments was contrary to what was found in the pilot study. When the salinity regime of the pilot study was repeated in Experiment 5, Ruppia survival was impaired in the high frequency treatment (p4d), as indicated by decreases in green-leaf index and the morphometric measurements. The seagrasses in the pilot study were subjected to higher temperatures and more light than in Experiment 5. In addition, the pilot study was conducted during July, near the beginning of Ruppia 's die back cycle (Lazar and Dawes 1991). The mechanism responsible for Ruppia 's respondes in the pilot study is not clear, however. Light and nutrient availability are of secondary importance in habitats of extreme salinity fluctuation, especially for species sensitive to salinity fluctuation such as Thalassia. Seagrasses require a high intensity of light for photosynthesis (Zieman and Wetzel 1980). Biweekly light measurements in Little Madeira Bay ranged between 100 and 1600 E / m 2 sec, with a mean intensity of 647 E / m 2 sec (Chesnes 1999). Thalassia was resilient, however, when exposed to severe, periodic light limitations (Kraemer and Hanisak 2000). The survival of Thalassia and Halodule is better under reduced light, but stable salinity treatments versus those in full sun, and a low amplitude salinity fluctuation regime is experimental evidence of the secondary importance oflight in environments exposed to salinity fluctuation.

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200 Seagrasses in northern Florida Bay are limited by phosphorus (Fourqurean and Zieman 1992, Montague and Ley 1993). Phosphorus concentrations in the test facility were an order of magnitude higher than that from the collection site, Little Madeira Bay, during 1996: 14.9g / l (Rudnick et al. 1999). Average total nitrogen concentrations into Little Madeira Bay during 1996 were 1180 g / l (Rudnick et al. 1999), slightly higher than the water used in the test facility. Despite the elevated phosphorus concentrations, the seagrasses in the test facility succumbed to the affects of salinity fluctuation. Whether low nutrient concentrations exacerbate the affects of salinity fluctuation is unknown, but plausible due to the high metabolic demands associated with withstanding highly variable salinity regimes. An odor of sulfide was evident in water pumped from the seawater supply well. Although sulfide concentrations were not measured, sulfide toxicity was probably not a factor in the facility experiments. Seagrasses in the tropics have been thought to be susceptible to sulfide toxicity, since the biogenic carbonate sediments have low iron content, so the production of iron-sulfide compounds, such as pyrite, is relatively limited compared to the iron-rich sediments of much of the U.S. east coast (Erskine and Koch 2000) Short-term ( 48 hour) exposures to root level sulfide concentrations (ranging from 2.0 to 10 mM) failed to produce any visual signs of sulfide toxicity in Thalassia testudinum, although leaf elongation decreased with increased sulfide concentrations (Erskine and Koch 2000). These concentrations are well above the odor threshold for humans (Camp 1968). Halodule wrightii has a wide tolerance to sediment sulfides in comparison to Ruppia maritima (Erskine and Koch 2000). Given the success of Ruppia in the facility experiments, it is assumed that sulfide toxicity was not a factor. The better

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201 growth of Halodule and Ruppia in the bubbled tanks is probably due to better circulation of water around the leaves and higher light rather than reductions in sulfide. Further, the sediments used in the facility experiments were coarser and probably less anaerobic than in the field, therefore sulfide toxicity in the field would seem more likely than in the facility. Conclusions As hypothesized by Montague and Ley (1993), and confirmed by this study, in habitats where salinity fluctuation is common, as in the ponds and bays of the northern land margin of Florida Bay, salinity fluctuation may be the single most important factor dictating the distribution and abundance of submerged macrophytes. Even in cases where nutrient, light, and temperature levels are optimal, seagrasses will be impaired if salinity fluctuates frequently over a wide range. Fluctuations in salinity of sufficient magnitude occur often at the northern land margin of Florida Bay. In estuaries, salinity fluctuations are often associated with the onset of the wet season, tropical storms, drought, or, as seen in the shallow basins of Florida Bay, a simple change in wind direction. The timing and attenuation of water releases is an important consideration in water management for a variety of reasons including BOD, nutrient loading, and fish migration. Salinity fluctuation must also be added to the list. Holding back water in times of drought as well as excessive releases in times of water abundance will exacerbate the spatial range of salinity fluctuation, affecting areas (upstream or downstream from areas of normal salinity fluctuation) whose organisms may not have the ability to adapt.

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202 The position of maximum salinity fluctuation in the estuary is dynamic, especially under various quantities of water discharge. In the northwest fork of the Loxahatchee estuary, the saltwater-freshwater interface can shift approximately 3 to 5 river miles as a result of changes in freshwater inflow, compared to a 0.5 to 1.5 mile shift due to tidal or seasonal influences (Russell and McPherson 1984). For any estuary there is a rate of freshwater flow that will push the band of favorable salinities beyond estuarine boundaries into open waters, eliminating favorable habitat entirely. Likewise, for every estuary there is a freshwater flow so low that the band of favorable salinities retreats upriver where the area of favorable habitat is small. (Browder and Moore 1981, p. 421) For mobile organisms, zones of unfavorable salinity can be dealt with by simply moving to a more favorable area. Immobile organisms must either adapt or die. For immobile organisms such as Thalassia and Halodule that rely on vegetative growth as the main method ofreproduction, the latter option will diminish distributions significantly.

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REFERENCES Anastasiou, C C. 1999. Design and development of a facility to accommodate salinity fluctuation experiments. MS Thesis, University of Florida. 152 pages. Barbour M.G. 1970. Is any angiosperm an obligate halophyte? American M i dland Naturalist 84 : 105-120. Bird K.T., B.R Cody J Jewett-Smith, and M.E. Kane. 1993. Salinity effects on Ruppia maritima cultured In Vitro Botanica marina. 36 : 23-28 Brock, M.A. 1981 Accumulation ofproline in a submerged aquatic halophyte, Ruppia L. Oecologia. 51 : 217-219. Browder, J.A. and D. Moore. 1981. A new approach to determining the quantitative relationship between fishery production and the flow of fresh water to estuaries. In: Proceedings of the National Symposium of Freshwater Inflow to Estuaries. R.D. Cross and D.L. Williams Eds. Fish and Wildlife Service FWS / OBS-81 / 04. Camp T R. 1968 Water and its Impurities Rheinhold Book Corporation, New York, NY. 355 pages. Chesnes T.C 1999. Responses of fouling communities to habitats of varying salinity in the northern land margin of Florida Bay. MS Thesis, University of Florida. 111 pages Deaton, L.E. and M.J Greenberg. 1986 There is no Horohalinicum. Estuaries 9:20-30. Doering, P.H. and R.H. Chamberlain. 2000. Experimental studies on the salinity tolerance of turtle grass, Thalassia Testudinum. In Seagrasses: Monitoring, Ecology Ph y siology and Management S.A. Bortone, Ed. CRC Press, New York, NY, chap 6 Durako, M.J. 2000. Laboratory Microcosm Experiments and Analysis to Test the effect of Salinity Fluctuation on the physiological Health, Growth, and Biochemistry of a Submersed Estuarine Plant common to Northern Florida Bay. Final Report to the South Florida Water Management District. Erskine, J.M. and M.S. Koch. 2000. Sulfide effects on Thalassia testudinum carbon balance and adenylate energy charge. Aquatic Botany 67:275-285 203

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204 Estevez, E.D. 2000. Matching salinity metrics to estuarine seagrasses for freshwater inflow management. In Seagrasses: Monitoring, Ecology, Physiology, and Management, S A. Bortone, Ed. CRC Press, New York, NY, chap 22. Fears S. 1993. The role of salinity fluctuation in determining seagrass distribution and species composition. MS Thesis, University of Florida. 90 pages. Fernandez, F.A., M.J. Garcia-Sanchez, and H.H. Felle. 1999. Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane of the marine angiosperm Zostera marina. Journal of Experimental Botany. 50: 1763-1768. Flowers, T.J., M.A. Hajibagheri, and N.J.W. Clipson. 1986. Halophytes. Quarterl y Review of Biology. 61:313-337. Fourqurean, J.W. and J.C. Zieman. 1992. Phosphorus limitation of primary production in Florida Bay: Evidence from C:N:P ratios of the dominant seagrass Thalassia testudinum. Limnology and Oceanography. 37: 162-171. Fourqurean, J.W. and M.B. Robblee. 1999. Florida Bay: A history ofrecent ecological changes. Estuaries 22:345-357. Hammer, L. 1968. Salzgehalt und photosynthese bei marinen Pflanzen. International Journal on Life in Oceans and Coastal Waters. 1: 185-190. Hellblom, F. and M. Bjork. 1999. Photosynthetic responses in Zostera marina to decreasing salinity, inorganic carbon content and osmolality. Aquatic Botany. 65:97104. J agels, R. 1973. Studies of a marine grass. Thalassia testudinum I. Ultrastructure of the osmoregulatory leaf cells. American Journal of Botany 60(10): 1003-1009. Jagels, R. 1983. Further evidence for osmoregulation in epidermal leaf cells of seagrasses. American Journal of Botany. 70:327-333. Jones, L.M. 1999. Salinity change effects on four transplanted aquatic macrophytes in North-Central Florida Bay. MS Thesis, University of Florida. 79 pages. Kantrud, H.A. 1991. Widgeongrass (Ruppia maritima L.) : A Literature Review. US Fish and Wildlife Service, Fish and Wildlife Research 10. 58 pages. Klug, M.J. 1980. Detritus-decomposition relationships. In: Handbook of Seagrass Biology: An Ecosystem Perspective R.C. Phillips and P.C. McRoy Eds. Garland STPM Press, New York, NY. Chap 12.

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205 Kraemer, G.P. and M.D. Hanisak. 2000. Physiological and growth responses of Thalassia testudinum to environmentally-relevant periods of low irradiance. Aquatic Botany. 67:287-300. Lazar, A.C. and C.J. Dawes. 1991. A seasonal study of the seagrass Ruppia maritima in Tampa Bay, Florida. Organic constituents and tolerances to salinity and temperature. Botanica Marina. 34:265-269. Light, S.S. and J.W. Dineen. 1994. Water control in the Everglades: A historical perspective. In: Everglades: The Ecosystem and its Restoration, S.M. Davis and J.C. Ogden, Eds. St. Lucie Press, Delray Beach, FL, chap 4. Mclvor C.C., J.A. Ley, and R.D. Bjork. 1994. Changes in freshwater inflow from the Everglades to Florida Bay including effects on biota and biotic processes: A review. In Everglades: The Ecosystem and its Restoration, S.M. Davis and J.C. Ogden, Eds. St. Lucie Press, Delray Beach, FL, chap 6. McMahan, C.A. 1968. Biomass and salinity tolerance of shoalgrass and manateegrass in Lower Laguna Madre, Texas. Journal of Wildlife Management 32:501-506. McMillan, C. and F.N. Moseley. 1967. Salinity tolerances of five marine spermatophytes of Redfish Bay, Texas. Ecology 48:503-506. Montague, C.L. 1996. Responses of Submersed Macrophytes to Freshwater Inflow to Florida Bay: Final Work Project Plan to the South Florida Water Management District. Montague C.L. and J.A. Ley. 1993. A possible effect on salinity fluctuation on abundance ofbenthic vegetation and associated fauna in Northeastern Florida Bay. Estuaries 16:703-717. Montague, C.L. and E. Chipouras. 1998. Responses of Submerged Macrophytes to Florida Bay: Final Field Studies Report to the South Florida Water Management District. Odum, W.E. 1970. Insidious alteration of estuarine environment. Transactions American Fisheries Society. 99:836-847. Ogden, J.C. 1980. Fauna! relationships in Caribbean seagrass beds. In: Handbook of Seagrass Biology: An Ecosystem Perspective. R.C. Phillips and P.C. McRoy Eds. Garland STPM Press, New York, NY. Chap 10. Patino, E. and C. Hittle. Unpublished Material. Patflow2Taylor River at Mouth. USGS 251127080382100. Patriquin, D. 1973. Estimation of growth rate, production, and age of the marine angiosperm Thalassia testudinum Konig. Caribbean Journal of Science 13:111-123.

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206 Phillips, R.C. 1960. Observations on the Ecology and Distribution of the Florida Seagrasses. Professional Papers Series, Fla. Board of Conservation. 2: 172. Remane A. and C Schlieper. 1971. Biology of Brackish Water. John Wiley and Sons, New York Russell, G.M. and B.F. McPherson 1984. Freshwater Runoff and Salinity Distribution in the Loxahatchee River Estuary, Southeastern Florida, 1980-82. USGS Water Resources Investigations Report 83-4244. Rudnick, D.T., Chen, Z, Childers, D., Boyer, J. and T.D. Fontaine III. 1999 Phosphorus and nitrogen inputs to Florida Bay: The importance of the Everglades watershed. Estuaries. 22:398-416. Tomasko, D.A. and C.J. Dawes. 1989. Effects of partial defoliation on remaining intact leaves in the seagrass, Thalassia testudinum Banks ex Konig. Botanica Marina. 32:235240. Tomasko, D.A. and M.O. Hall. 1999. Productivity and biomass of the seagrass Thalassia testudinum along a gradient of freshwater influence in Charlotte Harbor, Florida. Estuaries 22:592-602 Twilley, R.R. and J.W. Barko. 1990. The growth of submersed macrophytes under experimental salinity and light conditions. Estuaries 13:311-321. Valentine, J.F ., K.L. Heck, J. Busby, and D. Webb. 1997 Experimental evidence that herbivory increases shoot density and productivity in a subtropical turtlegrass (Thalassia testudinum) meadow. Oecologia. 112:193-200. Van Tussenbroek, B.I. 1996. Integrated growth patterns of Thalassia testudinum banks ex Konig. Aquatic Botany 55:139-144. Zieman, J.C. 1974. Methods for the study of the growth and production of turtle grass, Thalassia testudinum Konig. Aquaculture. 4:139-143. Zieman, J.C. 1975 Seasonal variation ofturtlegrass, Thalassia testudinum Konig, with reference to temperature and salinity effects. Aquatic Botany 1: 107-123. Zieman, J.C. and R.G. Wetzel. 1980. Productivity in seagrasses: Methods and rates In Handbook of seagrass biology: An ecosystem perspective. R.C. Phillips and C.P. McRoy, Eds. Garland, STPM Press, New York, chap 7.

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BIOGRAPHICAL SKETCH Thomas Chesnes, the youngest of six children was born on June 20, 1973, in Palm Beach Gardens, Florida, to Rosemarie and Gerard Chesnes. Growing up in south Florida, he acquired a deep appreciation for the outdoors, especially marine environments. Thomas graduated from Palm Beach Gardens High School in June 1991, and later pursued an undergraduate degree at the University of Florida. Finally deciding on the major of zoology, he received his bachelor's degree with high honors in 1995. Here he had his first exposure to research, studying osmoregulation in estuarine fish for his senior thesis, under the guidance of Dr. Frank Nordlie. Thomas received his master's degree from the Department of Environmental Engineering Sciences in 1999. His research focused on the responses of fouling organisms to habitats of varying salinity in the northern land margin of Florida Bay. He continued his research in this system, studying macrophytes in an experimental facility for his doctoral dissertation. After graduation, he will serve as an Assistant Professor of Biology at Palm Beach Atlantic College in West Palm Beach, Florida. 207

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Engineering Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. a_-~ Thomas L. Crisman Professor of Environmental Engineering Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. Professor Emeritus of Zoology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philoso hy. f( Evan Chipouras 1 Associate Professor of Biology University of Tampa This dissertation was submitted to the Graduate Faculty of the College of Engineering and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May2002 Pramod P Khargonekar Dean, College of Engineering Winfred M. Phillips Dean, Graduate School

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