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Abiotic Stress, Grazing and Disease

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Permanent Link: http://ufdc.ufl.edu/UFE0022281/00001

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Title: Abiotic Stress, Grazing and Disease Implications of Global Change on Zostera Marina Seagrasses
Physical Description: 1 online resource (31 p.)
Language: english
Creator: Blohm, Gabriela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: climate, grazing, labyrinthula, nitrogen, seagrass, zostera
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The effects of anthropogenic activities go beyond the non-living components of ecosystems; humans are directly altering biological communities. Understanding the implications of changing abiotic regimes on the strength of biological interactions and rates of primary production is important for predicting the ecological effects of climate change. Seagrass ecosystems are of particular interest, given their high rates of primary productivity and simultaneous exposure to a number of factors associated with climate change. Ecosystems undergo simultaneous changes in their natural environment. Extrapolating the effects of single stressors from controlled laboratory settings to natural settings has often proven insufficient; changes in abiotic regimes often occur simultaneously in the environment. Seagrass ecosystems are ideal for conducting multi-factor studies at the mesocosm scale through replicated experimental manipulation. We investigated the effects of increased temperature, nutrient enrichment and grazing pressure on Zostera marina disease, biomass and senescence in a replicated mesocosm experiment at the Virginia Institute of Marine Science in Gloucester Point, VA. We found that an average daily temperature increase of 1.7 degrees C results in a reduction of biomass and an increase in senescence. Contrary to previous studies, which showed that increased temperature gives rise to an increase in disease-driven necrosis, we found that nutrient enrichment increased the proportion of grass that exhibited necrosis due to disease. We found no grazer effect and no significant interaction effects, although we caution that the statistical power of the experiments was low. Our results suggest that the grazing, increased temperature and nutrient enrichment operate through different pathways, given that they did not show synergies or antagonisms. In the context of climate change and seagrass disease, our results do not support the previous studies that have reported seagrass diebacks that were associated with disease outbreaks due to warmer water temperatures.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gabriela Blohm.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Osenberg, Craig W.
Local: Co-adviser: Holt, Robert D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022281:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022281/00001

Material Information

Title: Abiotic Stress, Grazing and Disease Implications of Global Change on Zostera Marina Seagrasses
Physical Description: 1 online resource (31 p.)
Language: english
Creator: Blohm, Gabriela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: climate, grazing, labyrinthula, nitrogen, seagrass, zostera
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The effects of anthropogenic activities go beyond the non-living components of ecosystems; humans are directly altering biological communities. Understanding the implications of changing abiotic regimes on the strength of biological interactions and rates of primary production is important for predicting the ecological effects of climate change. Seagrass ecosystems are of particular interest, given their high rates of primary productivity and simultaneous exposure to a number of factors associated with climate change. Ecosystems undergo simultaneous changes in their natural environment. Extrapolating the effects of single stressors from controlled laboratory settings to natural settings has often proven insufficient; changes in abiotic regimes often occur simultaneously in the environment. Seagrass ecosystems are ideal for conducting multi-factor studies at the mesocosm scale through replicated experimental manipulation. We investigated the effects of increased temperature, nutrient enrichment and grazing pressure on Zostera marina disease, biomass and senescence in a replicated mesocosm experiment at the Virginia Institute of Marine Science in Gloucester Point, VA. We found that an average daily temperature increase of 1.7 degrees C results in a reduction of biomass and an increase in senescence. Contrary to previous studies, which showed that increased temperature gives rise to an increase in disease-driven necrosis, we found that nutrient enrichment increased the proportion of grass that exhibited necrosis due to disease. We found no grazer effect and no significant interaction effects, although we caution that the statistical power of the experiments was low. Our results suggest that the grazing, increased temperature and nutrient enrichment operate through different pathways, given that they did not show synergies or antagonisms. In the context of climate change and seagrass disease, our results do not support the previous studies that have reported seagrass diebacks that were associated with disease outbreaks due to warmer water temperatures.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gabriela Blohm.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Osenberg, Craig W.
Local: Co-adviser: Holt, Robert D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022281:00001


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ABIOTIC STRESS, GRAZINTG AND DISEASE: IMPLICATIONS OF GLOBAL CHANGE
ON Zostera marina SEAGRASSES





















By

GABRIELA MAXINE BLOHM


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER INT SCIENCE

UNIVERSITY OF FLORIDA

2008





































O 2008 Gabriela Maxine Blohm

































To the strongest women in my life: my mother, for teaching me how to cultivate my strengths
and to my grandmother, for always reminding me to stop and smell the flowers









ACKNOWLEDGMENTS

I thank Craig W. Osenberg, an inspiring and brilliant mentor and advocate. I thank Bob

Holt for all of his support and positive reinforcement; Marta Wayne for empowering me to push

through and grow from my experience; Benj amin Bolker for his constant and indispensable help;

Michelle Mack for sparking my interest in ecosystem ecology and allowing me to use her lab

space, the SOB lab for all of their helpful feedback on my manuscript; Lindsey Albertson for all

of her help in the field; Brian Silliman for establishing the contact with the Duffy lab and Ondi

Crino for her help in every aspect of these last 3 years.












TABLE OF CONTENTS


page


ACKNOWLEDGMENTS .............. ...............4.....


LIST OF FIGURES .............. ...............6.....


AB S TRAC T ......_ ................. ............_........7


CHAPTER


1 INTRODUCTION ................. ...............9.......... ......


2 METHOD S ................. ...............14.......... .....


Experimental Design .............. ...............14....
Mesocosm Set-up ................. ...............14.................
Treatm ents .............. ...............15....

Sam pling ................ .... ..... .. ...............16.......
Surveys during Experiment ................. ...............16........... ....
Final Survey............... ...............17.
Statistical Analyses ................. ...............18.................


3 RE SULT S ................. ...............19.......... .....


Effectiveness of Treatments .............. .....................19

Temperature ................. ...............19.................
Fertilizer ................ ...............19...

Crustacean Density ................. ...............19.......... ......
Response Variables............... ...............2
Z. marina Biomass .............. ...............21....
Z. marina Necrosis .............. ...............22....


4 DI SCUS SSION ................. ...............25.............


LIST OF REFERENCES ............. ........... ...............28...


BIOGRAPHICAL SKETCH ................. ............... 1..............










LIST OF FIGURES

FiMr page

1-1 Average daily temperature of heated and ambient mesocosms ................ ................ ...23

1-2 Ash-free dry mass of aboveground, belowground and litter of Z. marina in each
treatm ent. ............. ...............23.....

1-3 Proportion of senescent tissue ........... ..... .___ ...............24..

1-4 Proportion of necrotic tissue ........._. ...... .... ...............24..









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ABIOTIC STRESS, GRAZINTG AND DISEASE: IMPLICATIONS OF GLOBAL CHANGE
ON Zostera marina SEAGRASSES

By

Gabriela Maxine Blohm

August 2008

Chair: Craig W. Osenberg
Cochair: Robert D. Holt
Major: Zoology

The effects of anthropogenic activities go beyond the non-living components of

ecosystems; humans are directly altering biological communities. Understanding the implications

of changing abiotic regimes on the strength of biological interactions and rates of primary

production is important for predicting the ecological effects of climate change. Seagrass

ecosystems are of particular interest, given their high rates of primary productivity and

simultaneous exposure to a number of factors associated with climate change. Ecosystems

undergo simultaneous changes in their natural environment. Extrapolating the effects of single

stressors from controlled laboratory settings to natural settings has often proven insufficient;

changes in abiotic regimes often occur simultaneously in the environment. Seagrass ecosystems

are ideal for conducting multi-factor studies at the mesocosm scale through replicated

experimental manipulation.

We investigated the effects of increased temperature, nutrient enrichment and grazing

pressure on Zostera marina disease, biomass and senescence in a replicated mesocosm

experiment at the Virginia Institute of Marine Science in Gloucester Point, VA. We found that an

average daily temperature increase of 1.70C results in a reduction of biomass and an increase in










senescence. Contrary to previous studies, which showed that increased temperature gives rise to

an increase in disease-driven necrosis, we found that nutrient enrichment increased the

proportion of grass that exhibited necrosis due to disease. We found no grazer effect and no

significant interaction effects, although we caution that the statistical power of the experiments

was low. Our results suggest that the grazing, increased temperature and nutrient enrichment

operate through different pathways, given that they did not show synergies or antagonisms. In

the context of climate change and seagrass disease, our results do not support the previous

studies that have reported seagrass diebacks that were associated with disease outbreaks due to

warmer water temperatures.









CHAPTER 1
INTTRODUCTION

The biotic structure of ecosystems and the abiotic context in which they function have

undergone a suite of rapid modifications due to climate change (IPCC 2007, Vitousek 1997,

Harvell 2002). Although the causes of these changes are difficult to identify, global mean

temperatures and N-fixation rates have increased and will continue to do so as human economic

demands increase (IPCC 2007). The effects of anthropogenic activities go beyond the non-living

components of ecosystems; humans are directly altering biological communities. For example, in

many communities the systematic removal of species (mainly predator and large herbivore

species) has cascaded down to alter primary productivity (Deegan 2007, Ripple 2007, Frank

2005, Daskalov 2002).

Biological alterations are not limited to trophic cascade studies that took hold in the 1960s

when Hairston, Smith and Slobodkin (1960) posed the classic question of why the world is

green. "Hidden players," which include pathogens and decomposers, have been recognized as an

important component of community structure. For example, in many ecosystems -from coral

reefs and temperate marshes to tropical montane amphibian communities disease outbreaks

associated with increased UV, drought and increased temperatures greatly altered species

diversity and primary production (Harvell 2002, Silliman 2005 and Pounds 2006).

Understanding the dynamics of hidden players and their response to ecological changes is

important for predicting how communities will respond to climate change.

Forecasting the effects of changing temperature, nutrient regimes and community structure

is a challenging task, especially if they interact non-additively. Multi-factor experiments can

facilitate the identification of key ecological players and possible synergisms, but are limited by

the required spatial scale of the experiments. Mesocosm experiments are useful for predicting










biological responses to climate change (e.g., Kercher 2004, Koch 2007, Christensen 2006). This

approach allows the manipulation of multiple factors using replicated experiments in an

environment that resembles field conditions more closely than a laboratory setting. In this study,

we manipulate three factors related to global change (temperature, nutrients and grazers) in a

seagrass mesocosm array to determine their effects on primary producer biomass, disease and

senescence.

We expect that increased temperature will become an important factor for seagrass survival

as global climate change progresses, having likely effects on Z. marina biomass, allocation and

growth. Stress due to increased herbivory and pathogen loads has been studied in the terrestrial

literature and is expected to increase plant allocation to anti-herbivore chemicals (possibly

changing C and N demands), resulting in tradeoffs on important life history characteristics such

as reproductive allocation (Clay 1996). It has been widely documented in the terrestrial literature

that physical damage due to grazing can increase susceptibility to disease by providing an entry

for pathogens, as well as by altering plant allocation to anti-herbivore chemicals (Clay 1996).

The effects of nitrogen on seagrasses are complex. There are indirect effects due to increased

epiphyte load and reduced light, and direct effects on Z. marina specifically, due to NH4' toxicity

(Van Katwijk 1997, Touchette 2000). Although temperate seagrasses are considered N-limited

during the early growing season, water-column ammonium and nitrate increase as the summer

progresses, giving rise to temporal differences in nutrient limitation. Pulsed agricultural inputs of

NH4' in Z.marina eCOsystems results in the accumulation of NH4' that can become toxic due to

changes in cell membrane potential (Van Katwijk 1997).

Seagrasses serve important roles in the global C and N cycles; thus their feedbacks on

global climate change are of interest. They store an estimated 15% of the ocean' s carbon. Net









production in these systems is approximately 0.6 x 1015 g C/yr (Duarte and Chiscano 1999),

24.3% of which is exported to adj acent terrestrial and marine ecosystems (Duarte and Cebrian

1996). Seagrasses also reduce the flow of fertilizer into coral reefs and other coastal systems that

are highly susceptible to eutrophic conditions (Touchette 2000, Hemminga and Duarte 2000).

Commercially and ecologically important species such as blue crab, shrimp, red drum and

scallops require seagrass habitat during at least part of their life history (Hemminga and Duarte

2000).

The services provided by seagrasses may diminish with the effects of global change.

Indeed, sixty percent of global seagrass habitat loss has been attributed to anthropogenic

activities (Short and Wylie-Escheverria 1996). Additionally, worldwide seagrass declines have

been associated with higher temperatures (Seddon and Cheshire 2000), eutrophication from

agricultural runoff (Short and Wylie-Escheverria 1996) and wasting disease (Muehlstein 1992).

Higher average daily temperatures will increase respiration rates (Short and Neckles 2002), and

proj ected reductions in inorganic C in the water column (Terrados 1999) should reduce

photosynthetic rates. Nutrient inputs increase algal growth, leading to low oxygen conditions

and reduced light availability (Touchette 2000). They also can enhance periphyton production,

leading to shading, and increase grazer populations, possibly enhancing herbivory rates on the

seagrass (Touchette 2000).

These changes are expected to alter distributions and rates of ecological interactions in

seagrasses (Duarte 2002). Interactions among plants and microbes, which include disease and

decomposers, have not been well-described. Previous studies suggest that this group of

interactions can potentially have a large effect on the extent and cover of seagrasses (Renn 1936,

Muehlstein 1997). For example, a series of seagrass diebacks were reported as early as 1930,









coincident with a warm water event (Cottam 1933). The leaves of the seagrass, Z. marina, were

brown and necrotic, suggesting the seagrass had been infected with a wasting disease

(Muehlstein 1992). Although the ecological mechanisms that caused these diebacks were not

experimentally elucidated, Labyrinthula zosterae, a marine protistan wasting disease, was later

found in these necrotic wounds and described as a pathogen using Koch's postulates (Muehlsein

1990). Since then, diebacks associated with wasting disease and warm water temperatures were

documented in the 1980s (Muehlstein 1991) and early 2000s (J. J. Orth, personal

communication). Laboratory experiments have shown that L. zosterae thrives at higher salinities

(Vergeer 1995); however, its responses to higher temperatures and nutrient regimes in the water

column have not been determined experimentally. As a result, it is difficult to predict how L.

zosterae will respond to abiotic factors, and how its possible effect on Z. marina may depend on

other biotic and abiotic stressors that also are expected to occur in these ecosystems.

Studies that investigate the effects of multiple factors related to climate change and

anthropogenic effects can inform conservation efforts while also strengthening our understanding

of how simultaneous changes can affect ecosystems. Seagrass ecosystems are ideal for studying

the effects of multiple factors; they are experiencing coincident changes in temperature, fertilizer

and community structure (Duarte 2002, Hemminga and Duarte 2000); comprise an ecologically

and economically important system (Hemminga and Duarte 2000, Lippson and Lippson 1984);

and can be studied in mesocosm settings.

Few studies have investigated the effects of global climate change (or its various

components) on seagrass biomass or wasting. The goal of this study was to determine the effects

of three putative stressors on seagrass: increased temperature, increased water column fertilizer,

and the presence of herbivorous crustaceans that feed on seagrass. We explore the single and









combined effects of these stressors on seagrass biomass, allocation, wasting disease and

senescence. These results shed light on whether the past documented diebacks were due to

increased temperature, nutrient inputs or increased physical damage due to grazing, and provide

insight about future effects that might be expected with continued climate change.









CHAPTER 2
IVETHOD S

Experimental Design

We applied three putative stressors: increased temperature (by ~1.70C using heaters),

grazers (via the addition ofA. valida) and NH4' (via addition of Osmocote NPK fertilizer) in a 6-

week outdoor mesocosm experiment to determine their effects on 1) two separate types of Z

marina browning: disease-induced necrosis and senescence; and 2) Z. marina biomass. We

conducted a fully factorial experiment (2 levels of each factor; 8 replicates/treatment; yielding 64

mesocosms) at the Virginia Institute of Marine Science in a flow-through system with water

supplied from the York River estuary. Sixty four 5-gallon buckets, were arranged along the

middle of 4 cattle tanks, with two replicates of each treatment per tank (i.e., tanks were treated as

blocks in the design).

Mesocosm Set-up

The river water (salinity approx 15-20 ppt) was pumped through a sand filter and a mesh

filter (500 microns) to remove larvae and other mesofauna. Five three-inch diameter holes

covered in 250 Clm mesh lined the upper edge of each bucket to allow water flow out of the

buckets without the loss of grazers. To prevent backflow into the buckets from the tanks, we

placed a standpipe in each tank that prevented the external water level from exceeding that of the

bottom edge of each mesh hole. We lined the bottom 3 inches of each bucket with a combination

of 4 parts sand to 1 part dried organic matter from the Goodwin Island salt marsh. We allowed

the sediment to settle for 1 day before planting the grass. The tanks were covered with opaque

mesh to reduce UV light penetration into the buckets, which are shallower than most field

conditions.










We collected Z. marina shoots from Goodwin Island. Before transplanting, we counted

sixty four groups of 15 green shoots each and placed them in small mesh bags that were floated

in a tank with flow-through seawater. To ensure that all microcosms were starting with similar

amounts of fouling organisms (e.g., epiphytes), Labyrinthula sp. spores, and tunicate larvae that

could colonize the mesocosms and reach abnormally high densities, we removed the grass from

the mesh bags and exposed each group of shoots to four consecutive 5-minute freshwater

washes. We manually removed all visible fouling organisms such as the invasive tunicates

Botryllus sp. and M~olgula sp and brown shoots. We then removed excess water from the grass

using a salad spinner (20 rotations) and recorded its wet mass, ensuring that each group had

approximately the same mass and the same relative amounts of root and aboveground biomass

(total wet mass averaged 47 g per bucket; range 42-53 g). We haphazardly arranged the grass in

each bucket and planted it so the roots were buried and the blades were floating in the water

column. We allowed the grass to acclimate for 1 week before applying the treatments. We then

populated all buckets with two species of crustaceans (an amphipod, Ganmmarus mucronatus, and

an isopod, Erichsonella attenuate) to control epiphyte growth on the grass throughout the

duration of the experiment.

Treatments

We increased temperature by a mean of 1.70C above ambient levels (using VisiTherm

brand aquarium heaters in the +Heat treatment buckets only). During the first 11 days, we

monitored temperature twice daily using a YSI brand temperature and DO meter. During the

remainder of the experiment, we used 10 HOBO loggers to record temperature at 15-minute

intervals. We rotated the loggers into new buckets 4 times throughout the duration of the

experiment.









To increase water column fertilizer levels (specifically NH4 ), we prepared 25 g of

Osmocote brand fertilizer (19-6-12 NPK) in nylon mesh bags, which we then placed inside 20-

cm PVC pipes that contained holes to allow the slow release of fertilizer into the water column.

Each bucket contained either a PVC pipe with fertilizer or an empty pipe as a control. We

collected water samples from each bucket on days 1, 2 and 3 after the introduction of the nutrient

sticks, filtered the water through 1 Clm filters, and followed the Koroleff method to obtain the

concentration of NH4 -

We introduced an additional species of amphipod (Ampithoe valida) to all buckets in the

"+Grazer" treatment. Unlike the former 2 species, which feed primarily on detritus and

epiphytes that foul Z. marina blades, A. valida feeds directly on Z. marina aboveground tissue

(Lippson and Lippson 1984). All buckets contained the same initial number of individuals (30

crustaceans), but the grazer levels differed in composition. The grazer treatment received 10 A.

valida, 10 E. attenuate and 10 G. mucronatus, while the grazer controls received 15 E. attenuate

and 15 G. mucronatus. A. valida feeds on epiphytes until the grass is exposed; then it switches to

feeding on Z. marina. The initial number of crustaceans was kept the same for all treatments to

ensure that the epiphyte load was controlled similarly across all treatments.

Sampling

We conducted three surveys: week 2 (non-destructive), week 4 (non-destructive) and week

6 (destructive: end of experiment) to measure the abundance of crustaceans and the abundance of

browning.

Surveys during Experiment

For the first two crustacean surveys, we swept the water column and seagrass following

three figure-8 sweeps using a small mesh net, counted all individuals of each species and

returned them to their mesocosms (except for contaminants: A. valida in non-grazer treatments









were noted and removed). For the first two browning surveys, we haphazardly chose five blades

from each bucket and recorded the total length, percent cover of brown (using the Wasting Index

published by Burdick 1997), number of grazer scars and if possible, leaf age. Blade age was

calculated from the location of the seagrass blade on the shoot, where 1 is the innermost blade

and 3 is the outermost blade. Brown tissue was divided into two types based on its morphology:

1) "senesecent" tissue was identified visually as translucent light brown, originating at the tip of

the blade and when cultured in the laboratory (surface-sterilized with peroxide and grown in fetal

bovine serum), it contained mainly fungus and yeast (although some contained Labyrinthula sp.);

and 2) "necrotic" tissue was opaque dark brown, displaying a mottled pattern that originated in

center of the blade and expanded out towards the edges. When observed under the microscope,

the cells in necrotic tissue were partially digested and disfigured and when grown in culture

(surface-sterilized with peroxide and grown in fetal bovine serum), necrotic tissue contained

mainly Labyrinthula sp. and some fungi.

Final Survey

At the end of the experiment (day 42 after initiating the treatments), we siphoned the water

out of each bucket through a 500 Clm sieve. We removed all algae and amphipods from the edge

of the bucket and interstices of the nutrient sticks. We then uprooted the grass and rinsed it in

saltwater to remove excess sediment. We froze the samples of grass and crustaceans in opaque

plastic bags at -100C in the dark for later processing at the University of Florida. We subdivided

each sample of crustaceans 2 or 4 ways using a plankton splitter, identified and counted all

amphipods and isopods in the subsample, and scaled counts up to the entire sample. To minimize

additional browning on the grass due to light-induced breakdown of chlorophyll, we covered the

grass in foil and defrosted it under running water. We then carefully separated each shoot and

recorded the length, percent cover of brown, type of brown, number of scars and leaf age. We









then separated aboveground, litter and belowground biomass, placed each component in

aluminum bags, and dried them at 600C for approximately 5 days. We then weighed the dried

seagrass and placed it in a muffle furnace (at 5000C), reweighed the sample, and obtained ash-

free dry mass as the difference between the two masses.

Statistical Analyses

Crustacean density and total biomass were analyzed using ANOVA. Total Z. marina

biomass was analyzed using ANOVA. Z. marina biomass allocation (above, below and litter)

was analyzed using MANOVA. Litter was included in the analysis as part of total biomass

because all buckets contained no litter initially, and decomposition rates were presumed to be

low given the hypoxic conditions of the sediment and short duration of the experiments. The

proportion of brown tissue was arcsine square root transformed and analyzed using Repeated

Measures ANOVA in two separate models: 1) for senescence and 2) for necrosis. Models were

reduced by sequentially eliminating non-significant terms (P>0.05). All analyses were conducted

in R (C-Ran R Proj ect, 2007).









CHAPTER 3
RESULTS

Effectiveness of Treatments

Temperature

The average daily temperature difference between the ambient and heated treatments was

1.70C (Figure 1-1). This difference persisted during the first three weeks of the experiments. Due

to a decrease in flow rates of seawater into the tanks, however, the mean difference increased to

approximately 50C on June 9-10 and on June 17-19. On both occasions, the heaters were

adjusted to reduce the temperature differential. The sand filter was cleaned to restore normal

water flow rates within 2 days.

Fertilizer

Although we added N, P and K, the target stressor was NH4' because it can be directly

toxic to Z. marina cells. [NH4 ] declined with time and was significantly greater in the nutrient

addition treatments (Fl~s=1 8.72,p=0.0001); there was no effect of heat or grazers on the rate of

nutrient release during the first three days of nutrient addition.

Crustacean Density

A. valida was present in all of the grazer treatments and essentially absent from the non-

grazer treatments (details are summarized below), thus the grazer addition treatment had a

significant effect on A. valida density (Fl~s = 94.42, p=1.3x10-13). G. mucronatus was the most

abundant crustacean, having near 10 times the density of E attenuate, the least abundant species.

The average number of A. valid was greater in the grazer addition treatment than in the

treatments without grazers (F1~s=94.4, p=1.28*10-13) by approximately 120 individuals in each

bucket. A. valida was essentially absent from all non-grazer treatments except for those with

NPK, in which two of the eight replicates were contaminated with more than 30 A. valida










individuals. Exclusion of these replicates had a minimal effect on the results and did not change

the statistical conclusions, so we left these two replicates in the final analyses.

G. mucronatus was the most abundant crustacean and its densities were significantly

affected by fertilizer addition (Fl~s= 35.09, p=2.01*10- ) but not heat (Fl~s = 0.84, p= 0.36). There

were approximately 200 more individuals in the fertilizer addition treatments than all treatments

without fertilizer.

A qualitatively similar pattern was found in the total number of individuals for all species

pooled together (effect of fertilizer: Fl~s= 37.28, P=1.02*10- ), although the effect of heat was

significant (Fl~s= 4.66, p= 0.04). This pattern was driven by G. mucronatus, which was

approximately eight times more abundant than the other two species.

E. attenuate was the least abundant crustacean, which can be explained by the species'

large body size (up to 4-5x the size of the other two species) and slower generation time relative

to that of the other two species. E. attenuate abundances were significantly reduced in the heated

treatment (F1~s=14.46, p=0.0004)suggesting that increased thermal stress might reduce E.

attenuate abundance in Z. marina beds.

Response Variables

Increased temperature, fertilizer and grazing had additive effects on Z. marina

senescence, necrosis and biomass; however each response variable was affected differently by

each of the treatments. Increased temperature best explained the variation in the maj ority of the

results (Z. marina senescence and biomass), followed by NPK, which explained Z~marina

necrosis and allocation to a small extent (0.1l>p>0.05). The predictive power of grazing was

lowest and therefore did not remain in the final models for Z. marina senescence, necrosis and

biomass. We observed some interesting patterns as we monitored the temperature and grazer

treatment data.









Z. marina Biomass

Total seagrass biomass was greatest at ambient temperature, grazer and nutrient levels.

Increased average daily temperatures led to a lower mean total Z. marina biomass of

approximately 30% when compared to all treatments that had ambient temperatures (Fl~s=5.3,

p=0.025). There was no significant effect of grazing or fertilizer addition on total Z. marina

biomass (Fl~s=.0321, p=.86 and Fl~s=0.5, p=0.48, respectively).

Z. marina proportional allocation did not vary significantly among treatments (Grazers: F-

1,54= 0.26, p=0.9; Heat: F1,54= 1.08, p=0.3; Fertilizer: F1,54= 2.25, p=0.093). These results from

the MANOVA suggested that further analysis of proportional allocation might reveal an effect of

fertilizer on proportional allocation. Thus, we conducted individual ANOVAs on the proportion

of total biomass that was allocated to each type of plant material. We found a significant effect of

fertilizer addition on Z. marina belowground allocation (Fl~s=4.18, p=0.046).

Increased temperature led to a significant increase in the proportion of attached tissue

(which is separate from the detached litter that was discussed in the previous section) that was

senescent (F60,18 =4.56, p=0.037) (Figure 1-3). Grazers explained some of the variation in

senescence (F60,18 =2.25, p=0.13) and exhibited some interesting trends in response to heat (see

results for crustaceans). Fertilizer had little effect on the proportion of senescence (F60,18 =0.058,

p=0.8).

At higher temperatures, Z. marina senescence increased steadily through time. At the end

of the experiments, the proportion of senescent tissue in the heated treatments was higher than

those that experienced ambient temperature levels. A sharp increase in the proportion of

senescent tissue during the middle of the experiment, coupled with an increase in the sample

variance for the heated treatments occurred. This could be explained by the temperature spike










that occurred during the same time when the water flow rates decreased, further suggesting a

strong effect of increased temperature on senescence.

Z. marina Necrosis

The best model included only the main effect of fertilizer (Figure 1-4). There were no

significant interactions among the three factors (p>0.3 in all cases) on necrosis, which is the

browning associated with L. zosterae. The addition of fertilizer increased the proportion of

necrotic tissue (F60, 18= 17.06, p=0.0001), suggesting that high levels of NH4+ inCreaSed Z.

marina susceptibility to disease and may provide favorable conditions for the growth ofL.

zosterae. Neither grazers (F60, 18= 0.91, p=0.35) nor heat (F60, 18=0.86, p=0.36) had demonstrable

effects, suggesting that disease outbreaks are better explained by increases in water column

fertilizer than by temperature.



















28







c 20





a,- Ambient
18- .I -0- Heated



5/14/07 5/21/07 5/28/07 6/4/07 6/11/07 6/18/07 6/25/07



Figure 1-1. Average daily temperature of heated and ambient mesocosms. Average daily water
temperature. Readings taken from twice-daily monitoring (maximum and minimum)
using a YSI brand meter between 5/11/107 and 5/21/07. Readings between 5/22/07
and 5/25/07 were recorded using HOBO dataloggers, which monitored temperature at
15-minute intervals.


ct
r
a,

P2
o
a,
5
r
cn
Q
1


C G N GN H GH NH GNH

Treatment



Figure 1-2 Ash-free dry mass of aboveground, belowground and litter of Z. marina in each
treatment. C is the control; G represents A. valida grazerr) addition; H represents

heat; N represents the addition of fertilizer. Plotted are the means +/- 2 SE.


III


































































S2 3 4 5 6 7
Week
-* A and H and G and G+H
-O N and G+N and H+N and G+H+N


0.25 -


0.20 -


D.15 -
-.0


1 2 3 4 5 6 7
Week


Figure 1-3 Proportion of senescent tissue, calculated as (% cover)*(leaf area)/(total leaf area).
Because the effect of N was negligible (p=0.81), the data were pooled (thus, N=1 6).
Plotted are the means +/- 2 SE.


O 20


Figure 1-4 Proportion of necrotic tissue, calculated as (% cover)*(leaf area)/(total leaf area).
Because the effects of Heat and Grazers were negligible (p=0.34 and p=0.3 5,
respectively), the data were pooled for the graph (thus, N=32). Plotted are the means
+/- 2 SE.









CHAPTER 4
DISCUS SION

Temperature, nutrients, and grazers affected the Z. marina community differently.

Increased temperature led to a reduction in Z. marina biomass and an increase in senescence.

The addition of fertilizer increased the proportion of necrosis associated with L. zosterae.

Grazers had no demonstrable effects. The effects of increased temperature, fertilizer and grazing

pressure on Z. marina were additive, although the data are highly variable (leading to low

statistical power), so the absence of interactions should be taken with caution.

The reduction in total Z. marina biomass due to heat, coupled with the positive effect of

heat on senescence suggests that increased temperature led to higher rates of litterfall that were

not compensated by seagrass growth. Given that the proportion of litter was not significantly

affected by increased temperature, it is also possible that heat directly reduced Z. marina growth.

A reduction in growth could have been caused by a reduction in photosynthetic yield, as has

been observed in other temperate seagrass species (Campbell 2006). The upper thermal tolerance

limit for most temperate seagrass species is 350C (Bulthius 1983, Ralph 1998). When

temperatures exceed this limit, carbon production can be reduced because higher temperatures

can increase respiration rates (Bulthius 1983, Ralph 1998) and can denature photosynthetic

enzymes, as was found in some terrestrial plant species (Bruggeman 1992). Studies on the effects

of thermal stress on photosynthesis and productivity in temperate seagrasses highlight that

individual responses to increased temperature depend on the duration of exposure, history of

thermal stress, light levels and leaf age (Bulthuis 1987, Seddon and Cheshire 2000). In tropical

seagrass ecosystems, net primary productivity begins decreasing at 300C (Fong and Harwell

1994). For example, in Thala~ssia testudinum ecosystems, standing crop was reduced at

temperatures between 3 to 40C above ambient levels. In a study of seven temperate seagrasses










(Z. marina was not included), Campbell (2006) found that a reduction on photosynthetic yield at

high temperature pulses (15-30 minute exposures to 35-450C) gave rise to reductions in

photosynthetic yield, explaining the decreased aboveground biomass that was reported with El

Nifio warm water events (Seddon and Cheshire 2000). Although the results of this study do not

show a significant reduction in aboveground biomass due to increased temperature, a reduction

in total biomass was found, suggesting that higher temperatures might affect belowground

growth as well. It is also possible that decomposition rates were greater at higher temperatures,

giving rise to a reduction in the litter that was collected and measured at the end of the

experiment; however the litter that accumulated was relatively in tact and decomposition did not

appear to have been appreciable (Blohm, pers. obs.).

Past studies on L. zosterae suggest that it is an endemic and facultative pathogen found in

decomposer community (Muehlstein 1988, Muehlstein 1990), as opposed to a novel pathogen

that arrived via a specific vector. To understand and predict the future extent ofL. zosterae, it is

important to identify the factors (whether genetic or environmental) that most affect its growth.

At least three large-scale diebacks associated with a wasting disease were attributed to warmer

temperatures (Renn 1936, Muehlstein 1988); however the results of these studies were not

corroborated by experimental evidence. L. zosterae was identified as the causative agent of

seagrass wasting disease using Koch's postulates (Muehlstein 1991); however the results of our

experiments suggest that temperature did not increase necrosis (an indicator of L. zosterae);

instead nutrients increased the incidence of necrosis. Recent unpublished studies have shown

that L. zosteraeis less tolerant of increased temperature than is Z. marina (Erica Smith and

Gabriela Blohm, unpublished data): L zosterae dies at temperatures above 260C, while Z. marina

does well up to 300C. This suggests that past diebacks were not caused by disease outbreaks









related to high temperatures, but rather high nutrient loads or other possible factors that were not

measured.

The results of our experiments suggest that nutrient inputs better explain the abundance of

L. zosterae in seagrasses, although the mechanisms that underlie our results are unclear. Nutrient

addition has a suite of direct and indirect effects on Z. marina growth and survival (Duarte 2002,

Touchette 2000): increased epiphyte growth can give rise to light limitation; increased

respiration in the water column can reduce oxygen availability, especially at night; and NH4+ can

be directly toxic to the leaf tissue. It would follow that if Z. marina is stressed (due to light

limitation, O limitation or NH3+ toxicity), it would be more susceptible to L. zosterae.

Developing a theoretical framework for the study of multiple factors is challenging when

the physiological responses to single and multiple factors is unknown. Scaling up to the

ecological level to explain changes in interaction strengths, community structure and ecosystem

function will require that we understand how stressors affect individual physiology. To

understand how future changes in disease and senescence will affect seagrass ecosystem function

(C sequestration, N provision for adj acent ecosystems, nutrient buffering), we need to

experimentally determine how this ecosystem will respond to increased microbial (disease and

decomposer) activity, with the understanding that the dynamics underlying the cause and

response of each group of decomposers are different. The results of these experiments (coupled

with the documented severity of past diebacks) suggest that previous studies may have reported

both senescence and necrosis as the drivers of seagrass diebacks, and that separating the two

types of leaf loss is important for understanding how to manage seagrass ecosystems in the

context of climate change.










LIST OF REFERENCES


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Bulthuis, D.A., 1983. Effects of temperature on the photosynthesis-irradiance curve of the
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Bulthuis, D.A., 1987. Effects of temperature on the photosynthesis and growth of seagrass.
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Campbell, S. J., L.J. McKenzie and S. P. Kerville. 2006. Photosynthetic responses of seven
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Christensen, M.R., M.D. Graham, R.D. Vinebrooke, D.L. Findlay et al. 2006. Multiple
anthropogenic stressors cause ecological surprises in boreal lakes. Global Change
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Clay, K. 1996. Interactions among fungal endophytes, grasses and herbivores. Researches on
Population Ecology 38, 191-201.

Daskalov, G.M. 2002. Overfishing drives a trophic cascade in the Black Sea. Marine Ecology
Progress Series 225, 53-63.

Deegan, L.A., J.L. Bowen and D. Drake. 2007. Susceptibility of salt marshes to nutrient
enrichment and predator removal. Ecological Applications 17, 542-563

Duarte, C. M. 2002. The future of seagrass meadows. Environmental Conservation 29, 192-206.

Duarte, C.M. & Cebrian, J. 1996. The fate of marine autotrophic production. Limnology and
Oceanography 41, 1758-66.

Fong, P., Harwell, M.A., 1994. Modelling seagrass communities in tropical and subtropical bays
and estuaries: a mathematical model synthesis of current hypotheses. Bulletin of Marine
Science. 54 (3), 757- 781.

Frank, K. T., B. Petrie, J.S. Choi. 2005. Trophic cascades in a formerly cod-dominated
ecosystem. Science 308, 1621-1623.

Hairston, N. G., F. E. Smith, and L. B. Slobodkin. 1960. Community Structure, Population
Control and Competition. American Naturalist 94

Harvell, C.D., C.E. Mitchell, J.R. Ward, S. Altizer, A.P Dobson, R.S. Ostfeld and M.D. Samuel.
2002. Climate Warming and disease risks for terrestrial and marine biota. Science 296,
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Hemminga, M. and Duarte, C.M. 2000. Seagrass Ecology. Cambridge, UK: Cambridge
University Press.

Hoeksema, J.D. and E. Bruna. 2000. Pursuing the big questions about mutualism: a review of
theoretical approaches. Oecologia 125, 321-330

Intergovernmental Panel on Climate Change. 2007.

Koch, M.S., S.A. Schopmeyer, M. Holmer et al. Thala~ssia testudinum response to the interactive
stressors hypersalinity, sulfide and hypoxia. Aquatic Botany 87, 104-110.

Lippson, A. J. and R. L. Lippson. 1984. Life in the Chesepeake Bay. Baltimore, MD. Johns
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Muehlstein, L.K. 1992. The host-pathogen interaction in the wasting disease of eelgrass, Zostera
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Muehlstein, L. K., D. Porter and F. T. Short. 1991. Labyrinthula zosterae: the causative agent of
wasting disease of eelgrass, Zostera marina. Mycologia 83, 180-191.

Muehlstein, L. K., D. Porter and F. T. Short. Labyrinthula sp., a marine slime mold producing
the symptoms of wasting disease in eelgrass, Zostera marina. 1995. Marine Biology 99,
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Pounds J.A., Bustamante M.R., Coloma L.A., Consuegra J.A., Fogden M.P.L., Foster P.N., La
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Ralph, P.J., 1998. Photosynthetic response of laboratory-cultured Halophila ovalis to thermal
stress. Marine Ecology Progress Series 171, 123- 130.

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Ripple, W. J. and R. L. Beschta. 2007. Hardwood tree decline following large carnivore loss on
the Great Plains, USA. Frontiers in Ecology and the Environment 5, 241-246.

Short, F.T. and D. M. Burdick. 1996. Quantifying seagrass habitat loss in relation to housing
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Vergeer, L.H.T., T.L. Aarts and J.D. Degroot. 1995. The wasting disease and the effect of abiotic
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the phenolic content of Zostera marina shoots. Aquatic Botany 52, 3 5-44.

Vitousek, P. M., J. D. Aber, R. W. Howarth, G. E. Likens, P.A. Matson, D.W. Schindler, W. H.
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BIOGRAPHICAL SKETCH

Gabriela Blohm was born in the beautiful country of Venezuela, whose people are kind yet

unaware of the fragility of their nation' s social and ecological stability. She lived with her family

in Caracas until the age of fourteen, when the country's political climate became volatile and the

crime rate escalated to a point where their everyday lives were being affected. She moved to the

United States at the age of fourteen.

During her time in South America, she spent my weekends snorkeling and SCUBA diving

in places that had not suffered some of the impacts of human development. This is when her

aspiration in life became to promote the research and preservation of these quickly and silently

disappearing coasts. She grew up in a home where conversations at the lunch table were about

her grandfather' s program on the reintroduction of the endangered Orinoco crocodile, her

grandmother' s work with the conservation of sea turtles, and the polarized and unstable

socioeconomic divide that their country was experiencing. Her grandparents were founders of

Venezuela' s first environmental NGO, and they always spoke of the challenge of spreading an

environmental awareness throughout Venezuela. There are cultural, political and scientific

challenges to conservation efforts, and all are interwoven.

As an aspiring scientist, she is thrilled by the pursuit of good questions and obj ective

answers. She maj ored in Wildlife Ecology at the University of Florida, and upon graduating with

a BS in 2005, she decided to pursue her MS in Zoology at the University of Florida. She hopes to

continue working as a researcher and educator, with the goal of spreading a sense of ethical

responsibility for natural resource conservation, human rights preservation and education





PAGE 1

1 ABIOTIC STRESS, GRAZING AND DISEASE: IMPLICATIONS OF GLOBAL CHANGE ON Zostera marina SEAGRASSES By GABRIELA MAXINE BLOHM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER IN SCIENCE UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Gabriela Maxine Blohm

PAGE 3

3 To the strongest women in my life: my mother for teaching me how to cultivate my strengths and to my grandmother, for always reminding me to stop and smell the flowers

PAGE 4

4 ACKNOWLEDGMENTS I thank Craig W Osenberg, an inspiring and brilliant mentor and advocate. I thank Bob Holt for all of his support and positive reinfor cement; Marta Wayne for empowering me to push through and grow from my experience; Benjamin Bo lker for his constant and indispensable help; Michelle Mack for sparking my interest in eco system ecology and allowing me to use her lab space, the SOB lab for all of their helpful fee dback on my manuscript; Lindsey Albertson for all of her help in the field; Brian Silliman for es tablishing the contact with the Duffy lab and Ondi Crino for her help in every aspe ct of these last 3 years.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF FIGURES .........................................................................................................................6ABSTRACT ...................................................................................................................... ...............7 CHAP TER 1 INTRODUCTION .................................................................................................................. ..92 METHODS ....................................................................................................................... ......14Experimental Design ........................................................................................................... ...14Mesocosm Set-up ............................................................................................................... .....14Treatments .................................................................................................................... ..........15Sampling ...................................................................................................................... ...........16Surveys during Experiment .............................................................................................16Final Survey .....................................................................................................................17Statistical Analyses .......................................................................................................... .......183 RESULTS ....................................................................................................................... ........19Effectiveness of Treatments ...................................................................................................19Temperature ................................................................................................................... ..19Fertilizer .................................................................................................................... ......19Crustacean Density .......................................................................................................... 19Response Variables .................................................................................................................20Z. marina Biomass ..........................................................................................................21Z. marina Necrosis ..........................................................................................................224 DISCUSSION .................................................................................................................... .....25LIST OF REFERENCES ...............................................................................................................28BIOGRAPHICAL SKETCH .........................................................................................................31

PAGE 6

6 LIST OF FIGURES Figure page 1-1 Average daily temperature of heated and am bient mesocosms. ........................................ 23 1-2 Ash-free dry mass of above ground, belowground and litter of Z. marina in each treatm ent. .................................................................................................................... .......23 1-3 Proportion of senescent tissue ............................................................................................ 24 1-4 Proportion of necrotic tissue ............................................................................................. .24

PAGE 7

7 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ABIOTIC STRESS, GRAZING AND DISEASE: IMPLICATIONS OF GLOBAL CHANGE ON Zostera marina SEAGRASSES By Gabriela Maxine Blohm August 2008 Chair: Craig W. Osenberg Cochair: Robert D. Holt Major: Zoology The effects of anthropogenic activities go beyond the non-living components of ecosystems; humans are directly altering biolog ical communities. Understanding the implications of changing abiotic regimes on the strength of biological interactions and rates of primary production is important for predicting the eco logical effects of climate change. Seagrass ecosystems are of particular interest, give n their high rates of primary productivity and simultaneous exposure to a number of factors as sociated with climate change. Ecosystems undergo simultaneous changes in their natural envi ronment. Extrapolating the effects of single stressors from controlled laborat ory settings to natural settin gs has often proven insufficient; changes in abiotic regimes often occur simultane ously in the environment. Seagrass ecosystems are ideal for conducting multi-factor studies at the mesocosm scale through replicated experimental manipulation. We investigated the effects of increased temperature, nutrient enrichment and grazing pressure on Zostera marina disease, biomass and senescen ce in a replicated mesocosm experiment at the Virginia Institute of Marine Science in Gloucester Poin t, VA. We found that an average daily temperature increase of 1.7C results in a reduction of biom ass and an increase in

PAGE 8

8 senescence. Contrary to previous studies, which showed that incr eased temperature gives rise to an increase in disease-driven necrosis, we found that nutrient enri chment increased the proportion of grass that exhibited necrosis due to disease. We found no grazer effect and no significant interaction effects, al though we caution that the statis tical power of the experiments was low. Our results suggest th at the grazing, increased temperat ure and nutrient enrichment operate through different pathways, given that they did not show synergies or antagonisms. In the context of climate change and seagrass di sease, our results do not support the previous studies that have reported seag rass diebacks that were associated with disease outbreaks due to warmer water temperatures.

PAGE 9

9 CHAPTER 1 INTRODUCTION The biotic structure of ecosystem s and the abiotic context in whic h they function have undergone a suite of rapid modifications due to climate change (IPCC 2007, Vitousek 1997, Harvell 2002). Although the causes of these ch anges are difficult to identify, global mean temperatures and N-fixation rates have increased and will continue to do so as human economic demands increase (IPCC 2007). The effects of anthropogenic activitie s go beyond the non-living components of ecosystems; humans are directly altering biological communities. For example, in many communities the systematic removal of species (mainly predator and large herbivore species) has cascaded down to alter primary productivity (Deegan 2007, Ripple 2007, Frank 2005, Daskalov 2002). Biological alterations are not li mited to trophic cascade studies that took hold in the 1960s when Hairston, Smith and Slobodkin (1960) posed the classic question of why the world is green. Hidden players, which include pathogens and decomposers, have been recognized as an important component of community structure. For example, in many ecosystems from coral reefs and temperate marshes to tropical mont ane amphibian communities disease outbreaks associated with increased UV, drought and incr eased temperatures greatly altered species diversity and primary production (Harve ll 2002, Silliman 2005 and Pounds 2006). Understanding the dynamics of hidden players and their response to ecological changes is important for predicting how communities will respond to climate change. Forecasting the effects of changing temperature, nutrient regimes and community structure is a challenging task, especially if they inte ract non-additively. Multi -factor experiments can facilitate the identification of key ecological players and possible synergisms, but are limited by the required spatial scale of th e experiments. Mesocosm experiments are useful for predicting

PAGE 10

10 biological responses to climate change (e.g., Kercher 2004, Koch 2007, Christensen 2006). This approach allows the manipulation of multiple factors using replicated experiments in an environment that resembles field conditions more closely than a laboratory setting. In this study, we manipulate three factors related to global ch ange (temperature, nutr ients and grazers) in a seagrass mesocosm array to determine their e ffects on primary producer biomass, disease and senescence. We expect that increased temperature will become an important factor for seagrass survival as global climate change progres ses, having likely effects on Z. marina biomass, allocation and growth. Stress due to increased herbivory and pat hogen loads has been studied in the terrestrial literature and is expected to increase plant a llocation to anti-herbivor e chemicals (possibly changing C and N demands), resulting in tradeoffs on important life hist ory characteristics such as reproductive allocation (Clay 1996). It has been widely docu mented in the terrestrial literature that physical damage due to grazing can increase susceptibility to disease by providing an entry for pathogens, as well as by altering plant allo cation to anti-herbivor e chemicals (Clay 1996). The effects of nitrogen on seagrasses are comple x. There are indirect effects due to increased epiphyte load and reduced li ght, and direct effects on Z. marina specifically, due to NH4 + toxicity (Van Katwijk 1997, Touchette 2000). Although temp erate seagrasses are considered N-limited during the early growing season, water-column ammonium and nitrate increase as the summer progresses, giving rise to temporal differences in nutrient limitation. Pulsed agricu ltural inputs of NH4 + in Z. marina ecosystems results in the accumulation of NH4 + that can become toxic due to changes in cell membrane potential (Van Katwijk 1997). Seagrasses serve important role s in the global C and N cycles; thus their feedbacks on global climate change are of interest. They st ore an estimated 15% of the oceans carbon. Net

PAGE 11

11 production in these systems is approximately 0.6 x 1015 g C/yr (Duarte and Chiscano 1999), 24.3% of which is exported to adjacent terrestrial and marine ecosystems (Duarte and Cebrin 1996). Seagrasses also reduce the flow of fertilizer into coral reefs and other coastal systems that are highly susceptible to eu trophic conditions (Touchette 20 00, Hemminga and Duarte 2000). Commercially and ecologically important specie s such as blue crab, shrimp, red drum and scallops require seagrass habitat during at least part of their life history (Hemminga and Duarte 2000). The services provided by seagrasses may dimi nish with the effects of global change. Indeed, sixty percent of global seagrass habita t loss has been attri buted to anthropogenic activities (Short and Wylie-Escheverria 1996). A dditionally, worldwide seagrass declines have been associated with higher temperatures (Seddon and Cheshire 2000), eutrophication from agricultural runoff (Short and Wylie-Escheverria 1996) and wasting dise ase (Muehlstein 1992). Higher average daily temperatures will increase respiration rate s (Short and Neckles 2002), and projected reductions in inorganic C in th e water column (Terrados 1999) should reduce photosynthetic rates. Nutrient inputs increase algal growth, leading to low oxygen conditions and reduced light availability (Touchette 2000). They also can enhance periphyton production, leading to shading, and increase grazer populations, possibly enhancing herbivory rates on the seagrass (Touchette 2000). These changes are expected to alter distributi ons and rates of ecological interactions in seagrasses (Duarte 2002). Intera ctions among plants and microbes, which include disease and decomposers, have not been well-described. Previous studies suggest that this group of interactions can potentially have a large effect on the extent and cover of seagrasses (Renn 1936, Muehlstein 1997). For example, a series of seagrass diebacks were reported as early as 1930,

PAGE 12

12 coincident with a warm water event (Co ttam 1933). The leaves of the seagrass, Z. marina, were brown and necrotic, suggesting the seagrass had been infected with a wasting disease (Muehlstein 1992). Although the eco logical mechanisms that caus ed these diebacks were not experimentally elucidated, Labyrinthula zosterae, a marine protistan wasting disease, was later found in these necrotic wounds and described as a pathogen using Kochs postulates (Muehlsein 1990). Since then, diebacks associated with wast ing disease and warm water temperatures were documented in the 1980s (Muehlstein 1991) and early 2000s (J. J. Orth, personal communication ). Laboratory experiments have shown that L. zosterae thrives at higher salinities (Vergeer 1995); however, its responses to higher temperatures a nd nutrient regimes in the water column have not been determined experimental ly. As a result, it is difficult to predict how L. zosterae will respond to abiotic factors, and how its possible effect on Z. marina may depend on other biotic and abiotic stresso rs that also are expected to occur in these ecosystems. Studies that investigate the effects of multiple factors related to climate change and anthropogenic effects can inform conservation effo rts while also strength ening our understanding of how simultaneous changes can affect ecosystems. Seagrass ecosystems are ideal for studying the effects of multiple factors; they are experienci ng coincident changes in temperature, fertilizer and community structure (Duarte 2002, Hemminga and Duarte 2000); comprise an ecologically and economically important system (Hemmi nga and Duarte 2000, Lippson and Lippson 1984); and can be studied in mesocosm settings. Few studies have investigated the effects of global clim ate change (or its various components) on seagrass biomass or wasting. The goal of this study was to determine the effects of three putative stressors on seagrass: increased temperature, increased water column fertilizer, and the presence of herbivorous crustaceans that feed on seagrass. We explore the single and

PAGE 13

13 combined effects of these stressors on seag rass biomass, allocati on, wasting disease and senescence. These results shed light on whether the past documented diebacks were due to increased temperature, nutrient inputs or increased physical da mage due to grazing, and provide insight about future effects that might be expected with continued climate change.

PAGE 14

14 CHAPTER 2 METHODS Experimental Design We applied three putative stressors: increas ed temperature (by ~1.7C using heaters), graze rs (via th e addition of A. valida ) and NH4 + (via addition of Osmocote NPK fertilizer) in a 6week outdoor mesocosm experiment to determin e their effects on 1) tw o separate types of Z. marina browning: disease-induced necr osis and senescence; and 2) Z. marina biomass. We conducted a fully factorial experiment (2 levels of each factor; 8 replicates/treatment; yielding 64 mesocosms) at the Virginia Institute of Marine Science in a flow-through system with water supplied from the York River estuary. Sixty four 5-gallon buckets, were arranged along the middle of 4 cattle tanks, with two re plicates of each treatment per tank (i.e., tanks were treated as blocks in the design). Mesocosm Set-up The river water (salinity approx 15-20 ppt) wa s pum ped through a sand filter and a mesh filter (500 microns) to remove la rvae and other mesofauna. Five three-inch diameter holes covered in 250 m mesh lined the upper edge of each bucket to allow water flow out of the buckets without the loss of grazers. To prevent backflow into the buckets from the tanks, we placed a standpipe in each tank that prevented the external water level from exceeding that of the bottom edge of each mesh hole. We lined the bott om 3 inches of each bucket with a combination of 4 parts sand to 1 part dried organic matter from the Goodwin Island salt marsh. We allowed the sediment to settle for 1 day before planting the grass. The tanks were covered with opaque mesh to reduce UV light penetration into the buckets, which are shallower than most field conditions.

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15 We collected Z. marina shoots from Goodwin Island. Befo re transplanting, we counted sixty four groups of 15 green shoot s each and placed them in small mesh bags that were floated in a tank with flow-through seawater. To ensure that all microcosms were starting with similar amounts of fouling organisms (e.g., epiphytes), Labyrinthula sp. spores, and tunicate larvae that could colonize the mesocosms and reach abnormally high densities, we removed the grass from the mesh bags and exposed each group of shoots to four consecutive 5-minute freshwater washes. We manually removed all visible foulin g organisms such as th e invasive tunicates Botryllus sp. and Molgula sp and brown shoots. We then removed excess water from the grass using a salad spinner (2 0 rotations) and recorded its wet mass, ensuring that each group had approximately the same mass and the same re lative amounts of root and aboveground biomass (total wet mass averaged 47 g pe r bucket; range 42-53 g). We haphazardly arranged the grass in each bucket and planted it so the roots were burie d and the blades were floating in the water column. We allowed the grass to acclimate for 1 week before applying the treatments. We then populated all buckets with two sp ecies of crustaceans (an amphipod, Gammarus mucronatus and an isopod, Erichsonella attenuata ) to control epiphyte growth on the grass throughout the duration of the experiment. Treatments We increased tem perature by a mean of 1.7 C above ambient levels (using VisiTherm brand aquarium heaters in the +Heat treatment buckets only). During the first 11 days, we monitored temperature twice daily using a YS I brand temperature and DO meter. During the remainder of the experiment, we used 10 HOBO loggers to record temperature at 15-minute intervals. We rotated the l oggers into new buckets 4 times throughout the duration of the experiment.

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16 To increase water column fertilizer levels (specifically NH4 +), we prepared 25 g of Osmocote brand fertilizer (19-6-12 NPK) in nylon mesh bags, which we then placed inside 20cm PVC pipes that contained holes to allow the sl ow release of fertilizer into the water column. Each bucket contained either a PVC pipe with fe rtilizer or an empty pi pe as a control. We collected water samples from each bucket on days 1, 2 and 3 after the introduction of the nutrient sticks, filtered the water through 1 m filters, and followed the Koroleff method to obtain the concentration of NH4 +. We introduced an additi onal species of amphipod ( Ampithoe valida ) to all buckets in the +Grazer treatment. Unlike the former 2 species, which feed primarily on detritus and epiphytes that foul Z. marina blades, A. valida feeds directly on Z. marina aboveground tissue (Lippson and Lippson 1984). All buckets contained the same initial number of individuals (30 crustaceans), but the grazer levels differed in composition. The grazer treatment received 10 A. valida, 10 E. attenuata and 10 G. mucronatus, while the grazer controls received 15 E. attenuata and 15 G. mucronatus. A. valida feeds on epiphytes until the grass is exposed; then it switches to feeding on Z. marina. The initial number of crustaceans was ke pt the same for all treatments to ensure that the epiphyte lo ad was controlled simila rly across all treatments. Sampling We conducted three surveys: week 2 (non-destru ctive), week 4 (non-destructive) and week 6 (destructive: end of experim ent) to measure the abundance of crustaceans and the abundance of browning. Surveys during Experiment For the f irst two crustacean surveys, we swept the water column and seagrass following three figure-8 sweeps using a small mesh net, counted all individuals of each species and returned them to their mesocosms (except for contaminants: A. valida in non-grazer treatments

PAGE 17

17 were noted and removed). For the first two brow ning surveys, we haphazardly chose five blades from each bucket and recorded the total length, pe rcent cover of brown (using the Wasting Index published by Burdick 1997), number of grazer scar s and if possible, leaf age. Blade age was calculated from the location of the seagrass blade on the shoot, where 1 is the innermost blade and 3 is the outermost blade. Brown tissue was divided into two types based on its morphology: 1) senesecent tissue was identified visually as tr anslucent light brown, orig inating at the tip of the blade and when cultured in the laboratory (sur face-sterilized with peroxide and grown in fetal bovine serum), it contained mainly fungus and yeast (although some contained Labyrinthula sp. ); and 2) necrotic tissue was opaque dark brown, di splaying a mottled pattern that originated in center of the blade and expanded out towards th e edges. When observed under the microscope, the cells in necrotic tissue were partially dige sted and disfigured and when grown in culture (surface-sterilized with peroxide and grown in fetal bovine seru m), necrotic tissue contained mainly Labyrinthula sp. and some fungi. Final Survey At the end of the experim ent (day 42 after initiating the tr eatments), we siphoned the water out of each bucket through a 500 m sieve. We removed all algae and amphipods from the edge of the bucket and intersti ces of the nutrient sticks. We then uprooted the grass and rinsed it in saltwater to remove excess sedi ment. We froze the samples of grass and crustaceans in opaque plastic bags at -10C in the dark for later processing at the University of Florida. We subdivided each sample of crustaceans 2 or 4 ways using a plankton splitter, identified and counted all amphipods and isopods in the subsample, and scaled counts up to the entire sample. To minimize additional browning on the grass due to light-induced breakdown of chlorophyll, we covered the grass in foil and defrosted it under running water. We then carefully se parated each shoot and recorded the length, percent cove r of brown, type of brown, numbe r of scars and leaf age. We

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18 then separated aboveground, litter and belowg round biomass, placed each component in aluminum bags, and dried them at 60C for approximately 5 days. We then weighed the dried seagrass and placed it in a muff le furnace (at 500C), reweighed the sample, and obtained ashfree dry mass as the difference between the two masses. Statistical Analyses Crustacean density and total biom a ss were analyzed using ANOVA. Total Z. marina biomass was analyzed using ANOVA. Z. marina biomass allocation (above, below and litter) was analyzed using MANOVA. Litter was included in the analysis as pa rt of total biomass because all buckets contained no litter initially, and decomposition rates were presumed to be low given the hypoxic conditions of the sediment and short duration of the experiments. The proportion of brown tissue was arcsine square ro ot transformed and analyzed using Repeated Measures ANOVA in two separate models: 1) for senescence and 2) for necrosis. Models were reduced by sequentially eliminating non-significant terms (P>0.05). All an alyses were conducted in R (C-Ran R Project, 2007).

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19 CHAPTER 3 RESULTS Effectiveness of Treatments Temperature The average daily tem perature difference betw een the ambient and heated treatments was 1.7C (Figure 1-1). This difference persisted during the first three weeks of the experiments. Due to a decrease in flow rates of seawater into th e tanks, however, the mean difference increased to approximately 5C on June 9-10 and on June 17-19. On both occasions, the heaters were adjusted to reduce the temperature differential The sand filter was cleaned to restore normal water flow rates within 2 days. Fertilizer Although we added N, P and K, the target stressor was NH4 + because it can be directly toxic to Z. marina cells. [NH4 +] declined with time and was si gnificantly greater in the nutrient addition treatments (F1,8=18.72,p=0.0001) ; there was no effect of heat or grazers on the rate of nutrient release during the first th ree days of nutrient addition. Crustacean Density A. valida was present in all of the grazer treatm ents and esse ntially absent from the nongrazer treatments (details are summarized belo w), thus the grazer a ddition treatment had a significant effect on A. valida density (F1,8 = 94.42, p=1.3x10-13). G. mucronatus was the most abundant crustacean, having near 10 times the density of E. attenuata, the least abundant species. The average number of A. valida was greater in the grazer ad dition treatment than in the treatments without grazers (F1,8=94.4, p=1.28*10-13) by approximately 120 individuals in each bucket. A. valida was essentially absent from all non-gr azer treatments except for those with NPK, in which two of the eight replicates were contaminated with more than 30 A. valida

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20 individuals. Exclusion of these replicates had a minimal effect on the results and did not change the statistical conclusions, so we left th ese two replicates in the final analyses. G. mucronatus was the most abundant crustacean a nd its densities were significantly affected by fertilizer addition (F1,8= 35.09, p=2.01*10-7) but not heat (F1,8 = 0.84, p= 0.36). There were approximately 200 more individuals in the fer tilizer addition treatments than all treatments without fertilizer. A qualitatively similar pattern was found in the total number of individuals for all species pooled together (effect of fertilizer: F1,8= 37.28, P=1.02*10-7), although the eff ect of heat was significant (F1,8= 4.66, p= 0.04). This pattern was driven by G. mucronatus which was approximately eight times more abundant than the other two species. E. attenuata was the least abundant crustacean, whic h can be explained by the species large body size (up to 4-5x the size of the other two species) and slower generation time relative to that of the other two species. E. attenuata abundances were significantly reduced in the heated treatment (F1,8=14.46, p=0.0004)suggesting that increa sed thermal stress might reduce E. attenuata abundance in Z. marina beds. Response Variables Increased temperature, f ertilizer and grazing had additive effects on Z. marina senescence, necrosis and biomass; however each response variable was affected differently by each of the treatments. Increased temperature best explained the variation in the majority of the results ( Z. marina senescence and biomass), follo wed by NPK, which explained Z.marina necrosis and allocation to a small extent ( 0.1>p>0.05). The predictive power of grazing was lowest and therefore did not remain in the final models for Z. marina senescence, necrosis and biomass. We observed some inte resting patterns as we monito red the temperature and grazer treatment data.

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21 Z. marina Biomass Total seagrass biom ass was greatest at ambient temperature, grazer a nd nutrient levels. Increased average daily temperatur es led to a lower mean total Z. marina biomass of approximately 30% when compared to all treatments that had ambient temperatures (F1,8=5.3, p=0.025). There was no significant effect of grazing or fertilizer addition on total Z. marina biomass (F1,8=.0321, p=.86 and F1,8=0.5, p=0.48, respectively). Z. marina proportional allocation did not vary sign ificantly among treatments (Grazers: F-1,54= 0.26, p=0.9; Heat: F1,54= 1.08, p=0.3; Fertilizer: F1,54= 2.25, p=0.093). These results from the MANOVA suggested that further analysis of proportional allocation might reveal an effect of fertilizer on proportional allo cation. Thus, we conducted individual ANOVAs on the proportion of total biomass that was allocated to each type of plant material We found a significant effect of fertilizer addition on Z. marina belowground allocation (F1,8=4.18, p=0.046). Increased temperature led to a significant increase in the proportion of attached tissue (which is separate from the det ached litter that was discussed in the previous section) that was senescent (F60,18 =4.56, p=0.037) (Figure 1-3). Grazers explained some of the variation in senescence (F60,18 =2.25, p=0.13) and exhibited some intere sting trends in response to heat (see results for crustaceans). Fert ilizer had little effect on th e proportion of senescence (F60,18 =0.058, p=0.8). At higher temperatures, Z. marina senescence increased steadily through time. At the end of the experiments, the proportion of senescent tiss ue in the heated treatments was higher than those that experienced ambien t temperature levels. A sharp increase in th e proportion of senescent tissue during the middle of the experi ment, coupled with an increase in the sample variance for the heated treatments occurred. This could be explained by the temperature spike

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22 that occurred during the same time when the water flow rates decrea sed, further suggesting a strong effect of increased temperature on senescence. Z. marina Necrosis The best model included only the main effect of fertilizer (Figure 1-4). There were no significant interactions among the three factors (p>0.3 in all cases) on necrosis, which is the browning associated with L. zosterae The addition of fertilizer increased the proportion of necrotic tissue (F60, 18= 17.06, p=0.0001) suggesting that high levels of NH4 + increased Z. marina susceptibility to disease a nd may provide favorable conditions for the growth of L. zosterae. Neither grazers (F60, 18= 0.91, p=0.35) nor heat (F60, 18=0.86, p=0.36) had demonstrable effects, suggesting that diseas e outbreaks are better explained by increases in water column fertilizer than by temperature.

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23 5/14/07 5/21/07 5/28/07 6/4/07 6/11/07 6/18/07 6/25/07 Mean Temperature (degrees C) 16 18 20 22 24 26 28 30 Ambient Heated Figure 1-1. Average daily temperature of heated and ambient mesocosms. Average daily water temperature. Readings taken from twice-daily monitoring (maximum and minimum) using a YSI brand meter between 5/11/07 and 5/21/07. Readings between 5/22/07 and 5/25/07 were recorded using HOBO datal oggers, which monitored temperature at 15-minute intervals. Treatment CGNGNHGHNHGNH Ash-free dry weight (g) 0 1 2 3 4 Figure 1-2 Ash-free dry mass of a boveground, belowground and litter of Z. marina in each treatment. C is the control; G represents A. valida (grazer) addition; H represents heat; N represents the addition of fert ilizer. Plotted are the means +/2 SE.

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24 Figure 1-3 Proportion of senescent tissue, calcula ted as (% cover)*(leaf ar ea)/(total leaf area). Because the effect of N was negligible (p =0.81), the data were pooled (thus, N=16). Plotted are the means +/2 SE. Week 1234567 Proportion necrotic 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 A and H and G and G+H N and G+N and H+N and G+H+N Figure 1-4 Proportion of necrotic tissue, calculate d as (% cover)*(leaf area )/(total leaf area). Because the effects of Heat and Grazers were negligible (p=0.34 and p=0.35, respectively), the data were pooled for the graph (thus, N=32). Plotted are the means +/2 SE.

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25 CHAPTER 4 DISCUSSION Tem perature, nutrients, and grazers affected the Z. marina community differently. Increased temperature le d to a reduction in Z. marina biomass and an increase in senescence. The addition of fertilizer increased the proportion of necrosis associated with L. zosterae. Grazers had no demonstrable effects. The effects of increased temperature, fertilizer and grazing pressure on Z. marina were additive, although the data are highly variable (leading to low statistical power), so the absence of interactions should be taken with caution. The reduction in total Z. marina biomass due to heat, coupled with the positive effect of heat on senescence suggests that increased temper ature led to higher rates of litterfall that were not compensated by seagrass growth. Given that the proportion of litter was not significantly affected by increased temperature, it is also possible that heat directly reduced Z. marina growth. A reduction in growth could have been caused by a reduction in photosynthetic yield, as has been observed in other temperate seagrass sp ecies (Campbell 2006). The upper thermal tolerance limit for most temperate seagrass specie s is 35C (Bulthius 1983, Ralph 1998). When temperatures exceed this limit, carbon production can be reduced because higher temperatures can increase respiration rates (Bulthius 1983, Ralph 1998) and can denature photosynthetic enzymes, as was found in some terrestrial plant species (Bruggeman 1992). Studies on the effects of thermal stress on photosynthesis and productiv ity in temperate seagrasses highlight that individual responses to increas ed temperature depend on the duration of exposure, history of thermal stress, light levels and leaf age (B ulthuis 1987, Seddon and Cheshire 2000). In tropical seagrass ecosystems, net primary productivity begins decreasing at 30C (Fong and Harwell 1994). For example, in Thalassia testudinum ecosystems, standing crop was reduced at temperatures between 3 to 4C above ambient le vels. In a study of se ven temperate seagrasses

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26 ( Z. marina was not included), Campbell (2006) found that a reduction on photos ynthetic yield at high temperature pulses (15-30 minute exposures to 35-45C) gave rise to reductions in photosynthetic yield, explaining th e decreased aboveground biomass that was reported with El Nio warm water events (Seddon an d Cheshire 2000). Although the results of this study do not show a significant reduction in aboveground biomass due to increased te mperature, a reduction in total biomass was found, suggesting that hi gher temperatures might affect belowground growth as well. It is also po ssible that decomposition rates were greater at higher temperatures, giving rise to a reduction in the litter that was collected and measured at the end of the experiment; however the litter that accumulated was relatively in tact and decomposition did not appear to have been appreci able (Blohm, pers. obs.). Past studies on L. zosterae suggest that it is an endemic and facultative pathogen found in decomposer community (Muehlstein 1988, Muehls tein 1990), as opposed to a novel pathogen that arrived via a specific vector. To unde rstand and predict the future extent of L. zosterae, it is important to identify the factors (whether genetic or environmental) that most affect its growth. At least three large-scale diebacks associated with a wasting disease were attributed to warmer temperatures (Renn 1936, Muehlstein 1988); howeve r the results of thes e studies were not corroborated by experimental evidence. L. zosterae was identified as the causative agent of seagrass wasting disease using Kochs postulates (Muehlstein 1991); however the results of our experiments suggest that temperat ure did not increase necrosis (an indicator of L. zosterae); instead nutrients increased the incidence of necrosis. Recent unpublished studies have shown that L. zosterae is less tolerant of increased temperature than is Z. marina (Erica Smith and Gabriela Blohm, unpublished data ): L zosterae dies at temperatures above 26C, while Z. marina does well up to 30C. This suggests that past diebacks were not caused by disease outbreaks

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27 related to high temperatures, but ra ther high nutrient loads or othe r possible factors that were not measured. The results of our experiments suggest that nutrient inputs better e xplain the abundance of L. zosterae in seagrasses, although the mechanisms that underlie our results are unclear. Nutrient addition has a suite of direct and indirect effects on Z. marina growth and survival (Duarte 2002, Touchette 2000): increased epiphy te growth can give rise to light limitation; increased respiration in the water column can reduce oxyg en availability, especially at night; and NH4 + can be directly toxic to the leaf tissue It would follow that if Z. marina is stressed (due to light limitation, O limitation or NH3 + toxicity), it would be more susceptible to L. zosterae. Developing a theoretical framework for the study of multiple factors is challenging when the physiological responses to single and mu ltiple factors is unknown. Scaling up to the ecological level to explain change s in interaction strengths, co mmunity structure and ecosystem function will require that we understand how stressors aff ect individual physiology. To understand how future changes in disease and se nescence will affect seagrass ecosystem function (C sequestration, N provision for adjacent ecosystems, nutrient buffering), we need to experimentally determine how this ecosyste m will respond to increased microbial (disease and decomposer) activity, with the understanding th at the dynamics underlying the cause and response of each group of decomposers are differe nt. The results of these experiments (coupled with the documented severity of past diebacks) suggest that previous studies may have reported both senescence and necrosis as the drivers of seagrass diebacks, and th at separating the two types of leaf loss is importan t for understanding how to manage seagrass ecosystems in the context of climate change.

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28 LIST OF REFERENCES Bruggem an, W., Van Der Kooij, T.A.W., Van Hasselt, P.R., 1992. Long-Term chilling of young tomato plants under low light and subseque nt recovery: II. Chlorophyll fluorescence, carbon metabolismand activity of ribulo se-1,5-bisphosphate carboxylase/oxygenase. Plants 186, 179 187. Bulthuis, D.A., 1983. Effects of temperature on the photosynthesis-irra diance curve of the Australian seagrass, Heterozostera tasmanica Marine Biology Letters 4, 47 57. Bulthuis, D.A., 1987. Effects of temperature on the photosynthesis and growth of seagrass. Aquatic Botany 27, 27 40. Campbell, S. J., L.J. McKenzie and S. P. Kerville. 2006. Photosynthetic responses of seven tropical seagrasses to elevated seawater temperature. Jour nal of Experimental Marine Biology and Ecology 330, 455-468. Christensen, M.R., M.D. Graham, R.D. Vinebrooke, D.L. Findlay et al. 2006. Multiple anthropogenic stressors cause ecological su rprises in boreal lakes. Global Change Biology 12, 2316-2322. Clay, K. 1996. Interactions am ong fungal endophytes, grasses and herbivores. Researches on Population Ecology 38, 191-201. Daskalov, G.M. 2002. Overfishing drives a trophic cascade in the Black Sea. Marine Ecology Progress Series 225, 53-63. Deegan, L.A., J.L. Bowen and D. Drake. 2007. Susceptibility of salt marshes to nutrient enrichment and predator removal. Ecological Applications 17, 542-563 Duarte, C. M. 2002. The future of seagrass meadows. Environmental Conservation 29, 192-206. Duarte, C.M. & Cebrin, J. 1996. The fate of marine autotrophic production. Limnology and Oceanography 41, 1758. Fong, P., Harwell, M.A., 1994. Modelling seagrass communities in tropical and subtropical bays and estuaries: a mathematical model synthesis of current hypotheses. Bulletin of Marine Science. 54 (3), 757 781. Frank, K. T., B. Petrie, J.S. Choi. 2005. Tr ophic cascades in a formerly cod-dominated ecosystem. Science 308, 1621-1623. Hairston, N. G., F. E. Smith, and L. B. Slobodkin. 1960. Community Structure, Population Control and Competition. American Naturalist 94 Harvell, C.D., C.E. Mitchell, J.R. Ward, S. A ltizer, A.P Dobson, R.S. Ostfeld and M.D. Samuel. 2002. Climate Warming and disease risks for te rrestrial and marine biota. Science 296, 2158-2162.

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29 Hemminga, M. and Duarte, C.M. 2000. S eagrass Ecology. Cambridge, UK: Cambridge University Press. Hoeksema, J.D. and E. Bruna. 2000. Pursuing the bi g questions about mutualism: a review of theoretical approaches Oecologia 125, 321-330 Intergovernmental Panel on Climate Change. 2007. Koch, M.S., S.A. Schopmeyer, M. Holmer et al. Thalassia testudinum response to th e interactive stressors hypersalinity, sulfide and hypoxia. Aquatic Botany 87, 104-110. Lippson, A. J. and R. L. Lippson. 1984. Life in the Chesepeake Bay. Baltimore, MD. Johns Hopkins University Press. Muehlstein, L.K. 1992. The host-pa thogen interaction in the wasting disease of eelgrass, Zostera marina. Canadian Journal of Botany 70, 2081-2088. Muehlstein, L. K., D. Port er and F. T. Short. 1991. Labyrinthula zosterae: the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia 83, 180-191. Muehlstein, L. K., D. Po rter and F. T. Short. Labyrinthula sp., a marine slime mold producing the symptoms of wasting disease in eelgrass, Zostera marina. 1995. Marine Biology 99, 465-472. Pounds J.A., Bustamante M.R., Coloma L.A., Co nsuegra J.A., Fogden M.P.L., Foster P.N., La Marca E., Masters K.L., Merino-Viteri A., Puschendorf R., Ron S.R., Santiago-Azofeifa G.A., Still C.J., Young B.E. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature. 439, 161. Ralph, P.J., 1998. Photosynthetic response of laboratory-cultured Halophila ovalis to thermal stress. Marine Ecology Pr ogress Series 171, 123 130. Renn, C. E. 1936. The wasting disease of Zostera marina. A phytological investigation of the diseased plant. Biological Bulletin. 70, 148-158. Ripple, W. J. and R. L. Beschta. 2007. Hardw ood tree decline following large carnivore loss on the Great Plains, USA. Frontiers in Ecology and the Environment 5, 241-246. Short, F.T. and D. M. Burdick. 1996. Quantifying seagrass habitat loss in relation to housing development and nitrogen loading in Wa quoit Bay, Massachusetts. Estuaries 19, 730 739. Silliman, B.R., J. van de Koppel and M.D. Bertness. 2005. Drought, snails, and large-scale dieoff of southern U.S. salt marshes. Science 310, 1803-1806. Terrados, J., Duarte, C.M., Kamp-Nielsen, L., Boru m, J., Agawin, N.S.R., Fortes, M.D., Gacia, E., Lacap, D., Lubanski, M. and Greve, T. (1999) Are seagrass growth and survival affected by reducing conditions in the sediment? Aquatic Botany 65, 175.

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30 Touchette, B. W. and J. M. Burkholder. 2000. Review of nitrogen and phosphorous metabolism in seagrasses. Journal of Experime ntal Marine Biology and Ecology 250, 133-167. Short, F.T. and H. A. Neckles. 1999. The effects of global climate change on seagrasses. Aquatic Botany 63, 169. Short, F.T. and S. Wyllie-Echeverria. 1996. Natural and human-induced disturbance of seagrasses. Environmental Conservation 23, 17. Seddon, S., Cheshire, A., 2000. Photosynthetic responses of Amphibolis antartica and Posidonia australis to temperature and desiccation using chlorophyll fluorescence. Marine Ecology Progress Series 220, 119 130. Van Katwijk, M.M., Vergeer, L.H.T., Schmitz, G.H.W. & Roelofs, J.G.M. 1997. Ammonium toxicity in eelgrass Zostera marina. Marine Ecology Progress Series 157, 159. Vergeer, L.H.T., T.L. Aarts and J.D. Degroot. 1995. The wasting disease and the effect of abiotic factors (light-intensity, temperature, salinity) and infection with Labyrinthula zosterae on the phenolic content of Zostera marina shoots. Aquatic Botany 52, 35-44. Vitousek, P. M., J. D. Aber, R. W. Howarth, G. E. Likens, P.A. Matson, D.W. Schindler, W. H. Schlesinger, D.G. Tilman 1997. Human alterati on of the global nitrogen cycle: sources and consequences. Ecological Applications 7, 737-750.

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31 BIOGRAPHICAL SKETCH Gabriela Blohm was born in the beautiful c ountry of Venezuela, whose people are kind yet unaware of the fragility of their nations social and ecol ogical stability. She lived with her family in Caracas until the age of fourteen, when the co untrys political climate became volatile and the crime rate escalated to a point where their everyd ay lives were being affected. She moved to the United States at the age of fourteen. During her time in South America, she spen t my weekends snorkeling and SCUBA diving in places that had not suffered some of the im pacts of human development. This is when her aspiration in life became to promote the research and preservation of these quickly and silently disappearing coasts. She grew up in a home wher e conversations at the lunch table were about her grandfathers program on the reintroduction of the endangered Orinoco crocodile, her grandmothers work with the conservation of sea turtles, and the polarized and unstable socioeconomic divide that their country was ex periencing. Her grandparents were founders of Venezuelas first environmental NGO, and they always spoke of the challenge of spreading an environmental awareness throughout Venezuela. Th ere are cultural, poli tical and scientific challenges to conservation efforts, and all are interwoven. As an aspiring scientist, she is thrilled by the pursuit of good questions and objective answers. She majored in Wildlif e Ecology at the University of Florida, and upon graduating with a BS in 2005, she decided to pursue her MS in Zool ogy at the University of Florida. She hopes to continue working as a researcher and educator, with the goal of spread ing a sense of ethical responsibility for natural resource conserva tion, human rights preser vation and education


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