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Graduate Theses and Dissertations Graduate School

2004

Effects of hydrodynamic regime on photosynthesisin the green alga CaulerpaMark D. DriscollUniversity of South Florida

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Scholar Commons CitationDriscoll, Mark D., "Effects of hydrodynamic regime on photosynthesis in the green alga Caulerpa" (2004). Graduate Theses andDissertations.http://scholarcommons.usf.edu/etd/1018

Effects of hydrodynamic regime on photosynthesis in the green alga Caulerpa.

by

Mark D. Driscoll

A thesis submitted in partial fulfillment

of the requirements for the degree of Master of Science

Department of Biology College of Arts and Science University of South Florida

Major Professor: Florence I.M. Thomas, Ph.D.

Kevin S. Beach, Ph.D. Clinton J. Dawes, Ph.D.

Date of Approval: March 19, 2004

Keywords: PAM fluorescence, hydrodynamics, primary production, boundary layers,

photosystem II kinematics.

© Copyright 2004, Mark D. Driscoll

Dedication

This thesis is dedicated to my wife, Jodie Corsetti Driscoll, for her patience,

encouragement and support.

Acknowledgements

I would like to thank Dr. Flo Thomas for her friendship, support and guidance

over the past few years, Dr. Kevin Beach for introducing me to the world of marine

algae, and for encouraging me to pursue a graduate degree, and Dr. Clinton Dawes for

thoughtful review of my thesis. This research was made possible by a National Science

Foundation PECASE award (OCE-9996361) to Florence I. M. Thomas and a SIGMA XI

grants in aid of research award to Mark D. Driscoll. I would also like to thank Chris

Cornelisen and Sean Kinane for field assistance, Jodie Corsetti Driscoll and Dan Huber

for laboratory assistance and Dan Huber, Justin Bowels, Dr. Bruce Cowell, and Dr. Gary

Huxel for assistance with statistical analysis. I would also like to thank my parents, John

and Karen Driscoll, for their un-wavering support throughout my educational career.

i

Table of Contents

List of Tables ii List of Figures iii Abstract v Introduction 1 Limits of Algal Productivty 2 How Hydrodynamics affects algal productivity 6 Objectives 12 Background information on Caulerpa spp. 13 Accoustic Doppler Velocimenter 16 The PAM Fluorescence measuring technique 17 Materials and Methods 22 Caulerpa 24 Measurement of PSII kinematics 25 Field Experiments 28 Time in Flow 30 Results 33 Does the magnetic sample holder affect values of Pmax? 33 Laboratory measurements of PSII kinematics in C. mexicana after a short eposure to hydrodynamic regime. 33 In situ field measurements of PSII kinematics on C. sertularioides. 34 Laboratory measurements of PSII kinematics in C. racemosa after long term exposure to hydrodynamic regime. 34 Discussion 36 References 67

ii

List of Tables

Table 1. Values of Pmax, a and Ik measured on C. sertularioides in situ. 62 Table 2. Values of Pmax, a, Ik, qP and NPQ measured on C. racemosa in laboratory flumes over 7 hours. 63 Table 3. Values of NPQ at each measurement hour. 64 Table 4. Values of Pmax, a, Ik, qP and NPQ measured on C. racemosa Over 5 hours in a laboratory flume. 65 Table 5. Values of NPQ after 3 hours of immersion. 66

iii

List of Figures

Figure 1. Diagram of anti-herbivory metabolite caulerpenyne 42 Figure 2. The three main energy pathways for photosystem II 43 Figure 3. Skematic diagram of the pulse amplitude modulated fluorescence measuring principle in both light and dark adapted samples. 44 Figure 4. Representative curve that would be analyzed using the Platt inhibition model. 45 Figure 5. Representative curve that would be analyzed using the hyperbolic tangent model. 46 Figure 6. Drawing of Caulerpa sertularioides. 47 Figure 7. Drawing of Caulerpa racemosa. 48 Figure 8. Drawing of Caulerpa mexicana. 49 Figure 9. Collection site for C. racemosa. 50 Figure 10. 110 Litre racetrack flume. 51 Figure 11. Maps of study sites in the Florida Keys. 52 Figure 12. Orientation of the acoustic doppler velocimeter during field measurements. 53 Figure 13. Ratio of Pmax in fast flow to Pmax in slow flow at each measuring interval. 54 Figure 14. Results of dark acclimation trial using magnetic leaf clips under full Illumination and a dark room. 55 Figure 15. Values of Pmax for C. mexicana run at high and low velocities in a laboratory flume. 56 Figure 16. In situ measures of Pmax in C. sertularioides. 57

iv

Figure 17. In situ measurements of saturation irradiance in C. sertularioides. 58 Figure 18. Laboratory measurements of NPQ in C. racemosa. 59 Figure 19. Laboratory measurements of Pmax in C. racemosa. 60 Figure 20. Laboratory measurements of NPQ in C. racemosa. 61

v

Effects of hydrodynamics on photosynthesis in the green alga Caulerpa.

Mark D. Driscoll

ABSTRACT

The delivery of nutrients to the surface of marine algae can be controlled by the

local hydrodynamic regime: in higher flow velocities, the Diffusive Boundary Layer

(DBL) at the uptake surface is thinner, which can increase the flux of dissolved chemicals

to the algal surface. If the primary productivity of an alga is controlled by the availability

of a dissolved chemical, increased water flow should result in greater primary

productivity due to increased chemical flux. To test the hypothesis that increased water

flow will increase Photosystem II kinematics (PSII) in the green alga Caulerpa we used a

Diving Pam Fluorometer to measure the maximum relative electron transport rate

(Pmax), Saturation Irradiance (Ik), Non-photochemical quenching (NPQ), the light

limited slope of photosynthesis vs. irradiance curve (α) and photo-chemical quenching

(qP) and compared these measured values among treatments of varying flow speeds in a

portable laboratory flume. We also measured the influence of water flow on values of

Pmax, Ik, α , qP and NPQ in the field. Results showed that in C. racemosa collected from

Tampa bay, and tested in a laboratory flume, values of Pmax and Ik were positively

correlated to increase water flow, possibly indicating mass-transfer limitation. C.

mexicana, collected from the Florida Keys, showed a decrease in values of Pmax, and Ik

vi

with increasing water velocity in flume experiments, indicating that the increased flow

was resulting in physiological stress. This result was supported with field measurements

for C. sertularioides, which showed a negative correlation between Pmax and flow

velocity and Ik and flow velocity.

1

Introduction

Marine macro-algal communities represent a highly productive and often

overlooked component of marine systems (Hader 1999, Hurd 2000). As primary

producers, providers of food and shelter to a wide variety of marine life and as sediment

stabilizers (Williams 1990), macroalgae play an integral role in the health and

development of many coastal marine ecosystems (Wheeler and Björnsäter 1992, Gacia et

al. 1996, Campbell 2001). Rates of algal productivity have been shown to be some of the

highest on the planet (Littler 1980, Hurd 2000) and in near shore marine communities,

macroalgae play an important role in overall production (Wing and Patterson 1993,

Hader et al. 2001). While the vast majority of macroalgae are a healthy component of

marine ecosystems, some are dangerous invaders, destroying native eco-systems and

remaining unresponsive to eradication efforts (Hill et al. 1998, Argyrou et al. 1999,

Aussem and Hill 1999, Ceccherelli et al. 2000, Uchimura et al. 2000, Stimson et al. 2001,

Harris and Tyrell 2001).

Members of the genus Caulerpa (Bryiopsidacea) are important components of

tropical marine ecosystems worldwide (Collado-Vides and Robledo 1999, Collado-Vides

2002). With approximately 70 species within the genus, Caulerpa is often one of the

dominant macro-algal species within tropical shallow water habitats (Gacia et al. 1996).

Recently coastal marine systems in California and the Mediterranean Sea have been

2

invaded by a rogue strain of C. taxifolia (Vahl) C. Agardh, which has overgrown native

seagrass beds and detrimentally affected the native marine ecosystems (Ceccherelli and

Cinelli 1999, Cecherelli et al. 2000, Meinesz et al. 2001, Ceccherelli and Sechi 2002,

Ceccherelli and Campo 2002, Boudouresque et al. 2002). It is clear that macroalgae are

important components of healthy marine systems, as well as devastating components of

some disturbed systems. Due to the substantial impact that macro-algae can have on

coastal marine systems it is important that we understand the factors that control algal

production.

Limits of Algal Productivity:

Marine macrophyte physiology shows an incredible amount of diversity (Littler

1980, Littler and Arnold 1982, Saffo 1987, Koehl and Alberte 1988, Jensen et al 1985,

Hader et al 1996, Beach et al 1997, Stevens and Hurd 1997, Larned 1998, Williams and

Carpenter 1998, Hurd 2000, McCook et al 2000, Stimson and Larned 2000, Hillis 2001,).

This diversity allows algal communities to flourish in a vast range of environmental

conditions. Researchers have shown that macroalgae are able to change their overall

morphology (Koehl and Alberte 1988), pigment quality and quantity (Beach and Smith

1996a) and photosystem structure (Fork and Satoh 1986, Beach and Smith 1996b) in

response to varying environmental conditions. However, even with the highly plastic

nature of these organisms, light and nutrients can severely limit photo-production

3

(Hatcher and Larkum 1983, Duke et al. 1989, Wheeler and Björnsäter 1992, Fong et al.

2001, Harrison and Hurd 2001).

The availability of saturating irradiance is a controlling factor for photosynthesis

in marine macroalgae (Engleman 1883, Beach and Smith 1997a, Beach and Smith 1997b

Beach et al. 1997, Gomez et al. 1998, Cloern 1999, Yentsch et al. 2002). The availability

of light also contributes to the vertical zonation of marine algae (Engleman 1883). While

it is well known that not enough light can limit the primary production of algae, it must

also be considered that an overabundance of light can be detrimental given certain

circumstances (Jensen et al 1985, Beach et al 1995, Beach and Smith 1996, Hader et al

1996, Beach and Smith 1997a, Beach and Smith 1997b, Beach et al 1997, Hader et al.

1999, Hader et al. 2000, Gorbunov et al. 2000). Species that are low light acclimated can

see a drastic drop in photosynthetic activity when brought into drastically higher light

regimes (Hader et al. 1996). It is believed that this decrease is caused by damage to the

D1 protein complex within photosystem II of the electron transport chain (Greene et al.

1994, Hader 1996, Hader 1999). If damage to the D1 protein occurs, the protein must be

replaced before normal functions resume. Light inhibition can often be overcome if the

algae are acclimated in small steps to the desired level of irradiance (Hader et al 1996).

Researchers have shown that given enough time to acclimate to a new light regime,

macroalgae are able to adjust pigment levels and pigment ratios, allowing the organism to

more efficiently utilize the available light regime (Riechert and Dawes 1986).

When light levels are saturating, nutrient availability becomes the major limiting

factor to macroalgal production (Chapman 1974, Duke et al. 1989, Björnsäter and

4

Wheeler 1990, Wheeler and Björnsäter 1992, Greene et al. 1994, Martin et al. 1994,

Jones et al. 1996, Pedersen and Borum 1996, Landry et al. 1997, Ceccherelli and Cinelli

1999, Schwartz et al. 2000). This is especially evident in tropical environments where

coastal waters are known to be nutrient deplete (Hatcher and Larkum 1983, Carpenter et

al. 1991, Stimson and Larned 2000, Fong et al. 2001). The major limiting nutrients in the

marine environment are nitrogen (N) (Ryther and Dunstan 1971, Kinsley 1974,

Björnsäter and Wheeler 1990, Wheeler and Björnsäter 1992, Duke et al. 1989), phosphate

(P) (Kinsey and Domm 1974, Björnsäter and Wheeler 1990, Wheeler and Björnsäter

1992, Atkinson and Smith 1983), carbon (C) (Björnsäter and Wheeler 1990, Wheeler and

Björnsäter 1992) which is most often in the form of bi-carbonate in marine systems, and

micronutrients such as iron (Fe) (Greene et al. 1994, Martin et al. 1994).

Many studies have experimentally examined the effect of nutrient limitation in

algae; however, due to the metabolic requirements of individual species, nutrient

limitation varies by species and study site. For example increased productivity was seen

when samples of the coral reef algae Chlorodesmis fastigiata were artificially fertilized

with nitrate and phosphate (Kinsey and Domnm 1974). Similarly, chlorophyll content

and overall biomass was shown to increase after N and N+P additions to the water

column (Menendez et al. 2002). Greene et al. (1994) proposed that in areas where C, N

and P are not limiting, Fe is the main limiting nutrient to phytoplankton growth. By

seeding the open ocean with Fe Green et al. (1994) showed that plankton growth and

productivity was shown to drastically increase.

5

Nutrient enrichment is not always beneficial to marine vegetative assemblages.

Taylor et al. (1995) showed that while nutrients may positively affect the growth of one

component of an ecosystem, the same nutrient pulse could be detrimental to other

components. Their research showed that the biomass of Nannochloropsis spp. increased

as a result of N fertilization as well as N+P fertilization, the nutrient pulse resulted in a

loss of below ground biomass for the associated seagrass community.

Certain species of algae are known to display mechanisms that allow them to

obtain nutrients from sources other than the water column. The thallus of Dictyospheria

cavernosa grows in such a manner as to isolate a parcel of water between it and the

benthos (Larned and Stimpson 1996). This “chamber” provides two sources of nutrients

other than what is available from the water column. The first source is metabolic waste

products from invertebrates that colonize the inside of the chamber, and the second

source is the efflux of nutrients from the sediment that is trapped under the algal thallus.

The nutrient concentrations inside these chambers were found to be ten times higher than

the surrounding water column (Larned and Stimpson 1996). This mechanism was

hypothesized to be the factor that allows D. cavernosa to thrive in waters where the

ambient water column nutrients are not high enough for algal growth (Larned and

Stimpson 1996). Transport of nutrients from the sediment to the vegetative tissue of

algae has also been seen . Uptake of labeled 15N by the rhizoids of the alga Caulerpa

cupressoides was shown by Williams (1984), demonstrating that in some species of

Caulerpa sediment nutrients can play a large role in the productivity of the algae.

6

In terms of nutrient availability, organisms can be considered to be either

physically limited, biologically limited or in between the two. In a physically limited

system, the organism is not experiencing a high enough nutrient flux to saturate its

physiological processes (e.g. uptake potential, enzyme loads). Biologically limited

systems receive more than enough nutrient flux to saturate their processes; however,

these organisms do not have the capability to process all of the available nutrients (e.g.

not enough uptake sites, insufficient enzyme stores).

How hydrodynamics affects algal productivity:

Many researchers have proposed that the hydrodynamic regime experienced by

an organism can have a large effect on its metabolic processes (Odum 1956, Wheeler

1980, Riber and Wetzel 1987, Fonseca and Kenworthy 1987, Koehl and Alberte 1988,

Patterson et al. 1991, Koch 1993, Williams and Carpenter 1998, Kuffner 2001). Studies

have shown that relative irradiance levels (Dromgoole 1987, Koehl and Alberte 1988,

Patterson 1992, Wing and Patterson 1993, Wing et al. 1993) and nutrient delivery (Koehl

and Alberte 1988, Bilger and Atkinson 1992) can be drastically changed depending on

the ambient hydrodynamic regime of a given area. Changes in the local hydrodynamic

regime can affect an organism in two major ways. The three dimensional orientation of

the organism can be changed, affecting the relative light levels and the structural reaction

to flow (Koehl and Alberte 1988). Secondly, the nutrient delivery to the organism’s

7

surface can be affected by disruptions to the boundary layers surrounding the organism

(Gerard 1982, Thomas and Cornelisen 2003)

The flow of water past a submerged organism results in drag forces acting on that

particular organism, which can have a major impact on its physiology. Organisms

located in high-energy areas generally experience more drag than those found in sheltered

areas. Temperate kelps are able to protect themselves from this increased drag by

changing the morphology of their blades so as to reduce form drag (Koehl and Alberte

1988). In high velocity areas the blades of Nereocystis leutkiana take on a smooth and

narrow form that allows the blades to clump together into a streamlined cone, reducing

drag. In low velocity areas the same blades take on a more ruffled, wider form (Koehl

and Alberte 1988). This clumping in high velocity reduces the cumulative amount of

light that reaches the entire plant due to self-shading. In low velocity areas the blades do

not clump together, resulting in a higher surface area exposed to irradiance.

The clumping of blades into a more streamlined shape is not the only condition

related to hydrodynamics regimes that can be detrimental to plants. Dromgoole (1987),

Wing and Patterson (1993) and Wing et al. (1993) showed that hydrodynamic regimes

can drastically alter the light climate reaching a benthic community. Light flecks are

periods of intense light caused by canopy movement or surface waves. Surface waves in

particular can cause a short term, intense focusing of light, up to five times the ambient

levels onto the benthos (Sagert and Schubert 2000, Schubert et al. 2001). It is possible

that under certain conditions, these periods of intense light are strong enough to cause

8

permanent damage to the photosystems of aquatic primary producers, resulting in photo-

inhibition.

The local hydrodynamic regime can affect the nutrient delivery to an algal thallus

by increasing advection of nutrients over the surface and by decreasing the thickness of

the diffusive boundary layer (DBL (a gradient in concentration where chemical transport

is controlled only by molecular diffusion, not momentum.)). Hydrodynamics controls

the thickness of the DBL and therefore greatly affects nutrient delivery (Kohel and

Alberte 1988, Bilger and Atkinson 1992). If nutrient delivery is influenced by

hydrodynamics, then metabolic processes dependent on that delivery are then linked to

hydrodynamics as well.

The momentum boundary layer (MBL) is usually a completely turbulent region,

where the rates of chemical transport are dominated by inertial forces. The turbulence

in this layer is a function of water velocity and the properties of the benthic roughness

elements. Due to the presence of turbulence in the water column, the rates of chemical

transport in this layer are much higher than would be expected from molecular diffusion

alone (Kays and Crawford 1993). Eddies vertically mix dissolved chemicals through this

layer to the surface of the viscous sub layer, transporting them through the water column

faster than molecular convection or molecular diffusion.

The viscous sub layer (VSL) is a thin region immediately next to the surface of a

solid. In this layer, the vertical transport of momentum due to turbulence is no longer

effective, often resulting in a region of laminar flow. Chemical transport in this region is

governed by viscous transport, or molecular conduction between the layers (Kays and

9

Crawford 1993). The concentration of solutes within the viscous sub layer is controlled

by the delivery of chemicals from the momentum boundary layer. In areas of low

turbulence, the transport of these solutes may be slower, resulting in a lower

concentration within the viscous sub layer.

Below the viscous sub layer is the diffusive boundary layer (DBL). In this

region, the transport of solutes to and from a solid is completely reliant on molecular

diffusion. If an organism uses a chemical faster than it can be delivered through this

layer, it will become physically limited. Similarly, if an organism excretes a metabolic bi-

product into this layer faster than it can diffuse out, then an elevated concentration of that

bi-product will occur in the water next to the tissue surface. For plants, boundary layers

can cause a major problem. All photosysnthesis results in the production of oxygen,

which can compete with carbon for binding sites on the enzyme Ribulose 1-5

Bisphosphate Carboxylase Oxygenase (RUBISCO). When this happens, primary

productivity may be reduced due to the oxygen build up in the DBL.

Hydrodynamic regimes may greatly affect the nutrient limited metabolic

processes such as photosynthesis under some circumstances. As the boundary layers

surrounding a submerged solid are broken up by turbulent energy, or made thinner by

increased velocity, molecular diffusion to that solid is increased. In this manner, even in

low nutrient waters, the relative nutrient flux experienced by an organism can be quite

high if the area is swept by high velocity water flow. Conversely, if there is little

turbulent energy or velocity in the water column, an organism in a nutrient rich

environment may experience nutrient limitation due to the thickness of the diffusive

10

boundary layer. Two branches of research have developed models of how water flow

affects aquatic organisms. The first branch focuses on the effects of water flow on

photosynthesis and gross morphology, and the second branch has demonstrated the

dependence of nutrient uptake on water velocity.

Wheeler (1980) demonstrated that increased water velocity influenced the

photosynthetic rates of Macrocystis pyrifera during laboratory experiments using 3.0 cm

tissue discs. Oxygen evolution was shown to increase from a level of 0.3µmol O2 cm-2 in

stagnant water to a level of 1.3 µmol O2 cm-2 in water moving at the equivalent of 7.0 cm

s-1. Similar results were reported by Gerard (1982), who determined that small tissue

samples of M. pyrifera is mass transfer limited in flows less than 3.0 to 4.0 cm s-1 . These

studies were followed by Koehl and Alberte (1988) who demonstrated increased

photosynthetic activity in tissue discs of Nereocystis leutkiana as a result of increased

flow regimes in laboratory experiments. Koehl and Alberte (1988) also demonstrated

that the morphology of N. leutkiana is highly variable depending upon environmental

conditions. In high flows, blades were found to be smooth and without marginal spines,

allowing the blades to clump together and reduce form drag, where as in low flow, blades

were found to have large marginal spines and ruggose morphology, increasing turbulence

around the blade. This increased turbulence acts to disrupt the viscous sub-layer and

increase the nutrient flux to the edge of the diffusive boundary layer. In several studies

that utilized whole plants, instead of dissected tissue disks and blades, rates of primary

production ([µgO2 {µg Chl a}-1 h-1]) and acetylene reduction, an indicator of nitrogen

fixation, in tropical algal turfs have been positively correlated to water velocity (0-22cm

11

s-1) (Carpenter et al. 1991, Williams and Carpenter 1998). The effect of water velocity on

photo-production in marine algae is not uniform, Koch (1993) demonstrated that

increasing friction velocities (u* (a measure of the intensity of the turbulence in a

system)) from 0.0 cm s -1 to 0.3 cm s -1 significantly increased the photosynthetic rate of

Ulva lactuca. However, increasing µ* beyond 0.3 cm s -1 had no effect on the algae, and it

was postulated that under less than saturating irradiance increased water flow could be

detrimental to photosynthesis. Koch postulated that this decreased performance in higher

flows may have been due to the algae being accustomed to the flow regime in the area

where it was collected. By changing this regime, the plants may have become stressed,

reducing their efficiency (Koch 1993).

In addition to measuring primary productivity in response to varying

hydrodynamic regimes, several studies have investigated the affects of water flow on

nutrient uptake rates. Increased N and P uptake rates as a function of velocity have been

widely reported in the literature (Atkinson and Bilger 1992, Bilger and Atkinson 1992,

Bilger and Atkinson 1995, Thomas and Atkinson 1997, Thomas et al. 2000, Thomas and

Cornelisen 2003). Atkinson and Bilger have mainly worked on organisms in flume

systems. These studies noted dependence of the first order rate constant for the uptake of

important nutrients by coral reef assemblages on hydrodynamic regime. Along the same

lines, Thomas and Cornelisen have focused on field based experiments using a large field

flume capable of encompassing 3.7 m2 of the benthos. These experiments have

demonstrated that nutrient uptake of an entire community in situ is directly correlated to

water velocity. Additionally, these studies showed that the uptake rates of individuals

12

within the community are correlated to water flow and to whole community uptake

(Cornelisen and Thomas 2002).

Objectives:

To clarify the role of hydrodynamics in photo-production new technology that

allows for measurements of hydrodynamic regimes and direct measurements of

photosystem II kinematics will be used in situ. By measuring PSII kinematics in

laboratory experiments, I intend to show that a Diving PAM Fluorometer is capable of

measuring changes in PSII kinematics due to varying hydrodynamic regimes. I also plan

on utilizing field measurements to directly measure PSII kinematics without removing

the plants from their natural environmental conditions, which has been suggested as a

stressor to aquatic vegetation (Koch 1993). These studies will focus on the genus

Caulerpa due to its extensive presence in near-shore tropical and sub-tropical marine

systems. The detailed goals of this thesis are:

1. To determine if hydrodynamic regime affects photosystem II kinematics

in situ and in a controlled laboratory flume.

2. To determine if the effects of hydrodynamic regime on photosystem II

kinematics can be measured by integrating data collected with an Acoustic

Doppler Velocimeter and a Diving Pam Flourometer.

13

3. To determine the magnitude of the dependence of photosystem II

kinematics on hydrodynamic regime within the genus Caulerpa.

Background information on Caulerpa spp.

Research has shown that members of the genus Caulerpa are important

components of tropical marine ecosystems around the world (Collado-Vides and Robledo

1999, Collado-Vides 2002). The morphology of the genus Caulerpa is highly diverse,

and within species the morphology is amazingly plastic (Gacia et al. 1996, Benzie et al.

2000, Collado-Vides 2002), changing drastically in accordance to environmental

conditions. As early as 1906 water motion and ambient light levels were being

considered as important factors affecting the morphology of Caulerpa (Svedelius 1906).

Barilotti (1970) showed that the morphology of C. prolifera (FØrsskål) Lamouroux was

determined by available light levels; Peterson (1972) then showed a similar dependence

on ambient light levels in C. racemosa.

The genus Caulerpa has drawn special attention in the Mediterranean Sea, where

two species, Caulerpa taxifolia and C. racemosa (Forsskål) J. Agardh, have become

dominant invaders (Ceccherelli and Cinelli 1999, Cecherelli et al. 2000, Ceccherelli and

Piazzi 2001, Meinesz et al. 2001, Ceccherelli and Sechi 2002, Ceccherelli et al. 2002,

Ceccherelli and Campo 2002, Verlaque et al. 2002). Since its introduction off the coast

of Monaco in 1984, C. taxifolia has been shown to out-compete the seagrasses Posidonia

oceanica (Ceccherelli and Cinelli 1999, Cecherelli et al. 2000) and Cymodocea nodosa

14

(Ceccerelli and Sechi 2002). Caulerpa racemosa, which is an immigrant species from

the red sea, has shown a similar pattern as C. taxifolia in the Southern Mediterranean Sea,

out-competing C. nodosa, Zostera noltii (Ceccherelli et al. 2002) and Posidonia oceanica

(Ceccherelli et al. 2000). The negative effect of these algae has not been limited to the

overgrowth of submerged aquatic vegetation. Arigoni et al (2002) showed that native

labrid fishes underwent a drastic color change in C. taxifolia bed as compared to normal

color morphs in P. oceanica beds. Decreases in invertebrate abundance (Bellan-Santini

et al. 1996), species richness, and fish biomass (Francour et al. 1995, Relini et al. 2000)

were also seen within C. taxifolia beds as compared to natural seagrass assemblages. The

high levels of secondary metabolites present in Caulerpa spp may explain this decline in

species diversity. The acetylenic sesquiterpene metabolite caulerpenyne (Figure 1), which

is the main anti-herbivory defense in Caulerpa spp. has been shown to compromise a

substantial (0.2-13.0%) component of the frond weight depending on the season (Dumay

et al. 2002). The high levels of secondary metabolites in Caulerpa is not surprising,

given that the genus is mainly found in tropical waters which are known for high rates of

herbivory (Pennings and Paul 1992, Cronin and Hay 1996).

Data collected on Caulerpa spp. has demonstrated in many systems its ability to

colonize a wide range of substrate types, ranging from muddy unconsolidated sediments

to rocky intertidal areas (Meinesz et al. 1995, Chisholm and Jaubert 1997, Ceccherelli

and Cinelli 1998, Arigoni et al. 2002). Caulerpa has been reported at depths of 50

meters, well below the photic zone of most green algae (Arigoni et al. 2002). This

flexibility allows Caulerpa a vast range of options when settlement of fragments or

15

zygotes occurs, giving Caulerpa an advantage over species that are more selective in

their substratum choices. Ecologically, Caulerpa has shown that it is an important

component in the succession of tropical seagrass beds (Williams 1990). By consolidating

loose sediments, Caulerpa makes it possible for climax species, such as seagrasses to

colonize an area.

The growth strategy of Caulerpa is based on an extremely fast rate of vegetative

expansion (Chisholm and Jaubert 1997, Ceccherelli and Piazzi 2001). This rapid growth

allows the alga to quickly extend new tissue into a large area immediately surrounding

the initial settlement plot. Caulerpa is known to grow at night, as is seen by the white

growing tips visible in the early morning (Dawes and Barilotti 1969). As light levels

increase, cytoplasm replaces the “meristemplasm” that occupies the white areas of the

cell and Chloroplasts re-position themselves in this newly available area (Dawes and

Barilotti 1969). Like many members of the Bryopsidales, Caulerpa has the ability to

reproduce both sexually via holocarpy (Zuljevic and Antolic 2000) and asexually through

vegetative fragmentation (Smith and Walters 1999, Ceccherelli and Piazzi 2001, Walters

et al. 2002). Vegetative fragmentation is an extremely effective mechanism of

reproduction in Caulerpa, fragments as small as 2.0 mm have been shown to successfully

settle, attach and grown into full size plants (Goddard and Dawes 1983, Smith and

Walters 1999, Ceccherelli and Piazzi 2001). The effectiveness of this growth strategy is

seen in C. taxifolia, which, in the Mediterranean has spread solely through vegetative

fragmentation, as only male gametes have been observed (Zuljevic and Antolic 2000).

The same is true for C. racemosa in the southern Mediterranean; no gamete production

16

has been seen in the alga, meaning that while sexual reproduction of C. racemosa is

important in tropical waters, vegetative growth and fragmentation are solely responsible

for the alga’s success in the Mediterranean Sea (Ceccherelli and Piazzi 2001).

Acoustic Doppler Velocimeter

Early studies utilized methods such as hot film anemometers, plaster of paris

dissolution, and “life saver” dissolution to measure bulk flow velocities (Wheeler 1980,

Koehl and Alberte 1988) Plaster of paris and life saver dissolution give a general idea of

water velocity by measuring the amount of mass lost over a given time. Identical

samples can be calibrated at a known water velocity and compared to samples tested

(Wheeler 1980, Koehl and Alberte 1988). Hot film anemometers measure the advection

of heat from the probe tip and correlate it with velocity (Koch 1993). These techniques,

while informative, do not measure the turbulent energy in a system, which affects the

DBL. Acoustic Doppler Velocimeters measure water flow in three vectors

simultaneously; “x” which is parallel to the bulk flow, “y” which is perpendicular to the

bulk flow, and “z” which is the vertical perpendicular to the bulk flow. The amount of

variation between these measures allows researchers to collect data on the amount of

energy, or turbulence in the system.

17

The PAM Fluorescence Measuring Technique

Traditional methods of measuring primary production have relied on gas

exchange or increased biomass as the primary indicating factor. While looking at an

increase of biomass or gas exchange over time can give a gross estimate of primary

productivity, these estimates may not give accurate insights into the efficiency of algal

photosystems. The advances seen in chlorophyll fluorescence technology in the past

decade have drastically changed the ability of eco-physiologists to discern photosystem II

(PSII) kinematics in the laboratory and in situ.

Pulse amplitude modulated fluorescence technology is based on exposing

photosynthetic tissue to a 655nm light source that is rapidly switched on and off at a rate

of 20 kHz. A photodiode then detects only the fluorescence at wavelengths greater than

700 nm to determine the fluorescent output of the sample (Walz manual 1998, Maxwell

and Johnson 2000). The ability to collect a non-invasive, real time fluorescence reading

from samples in situ has expanded the scope of the questions that can be addressed

concerning plant physiology.

The basics of chlorophyll fluorescence are relatively uncomplicated. Energy that

enters the photosynthetic apparatus of a given plant can follow three different pathways

(Figure 2). These pathways control the photochemistry of the system or the actual

conversion of light energy to glucose, and the dissipation of excess energy before it can

cause irreparable damage to the photosystem. Energy entering the first such pathway is

used to drive photosynthesis (production of ATP and NADPH) and is known as

18

photochemical quenching (qP). Energy that enters the second such pathway is dissipated

as heat; this pathway is linked to photo-protection, the dissipation of excess energy before

it can cause permanent damage to PSII (NPQ). The third option is for the energy to be

re-emitted from the PSII antennae complex at a higher wavelength (fluorescence).

Fluorescence is thought to be responsible for the dissipation of approximately

5% of excess the energy present in a given PSII antennae complex. The three energy

pathways of PSII are competing circuits, if the amount of energy entering one increases,

it will in turn decrease the amount entering the other two (Maxwell and Johnson 2000,

Krouse and Weis 1991). By looking at the relative amounts of energy in each pathway, it

is possible to look at the overall efficiency of PSII.

The performance of PSII can be determined from data collected from the changes

in fluorescent levels between times of no, low and high light (Figure 3). PAM

fluorescence can be used with the plants either in a dark acclimated or light acclimated

state. In dark acclimated conditions, the vegetative tissue is kept in complete darkness

for a varying length of time; this darkness ensures that all of the photo-receptors are in a

non-oxidized or open state. A small measuring light (0.15µ mol photon per m2) is

applied to the sample to determine the background fluorescence level (Fo). A pulse of

light with intensity high enough to oxidize (close) all available photo-receptors is then

applied to obtain the maximum fluorescent yield (Fm) for the sample. The difference

between the values of Fm, and Fo is known as the variable fluorescence or Fv. After the

saturating light pulse, a photosynthesis driving light is applied to the sample for a

prescribed length of time. A second saturating pulse is then switched on and the samples

19

maximum fluorescence in the light (Fm’) is measured. Immediately before the second

saturation pulse is applied a measure of the samples residual fluorescence (Ft) is taken.

The comparison of these values allows one to investigate the efficiency of the

photosystem.

The effective quantum yield of energy conversion in a given sample is determined

from the following equation Yield = (Fm’-Ft)/Fm’ (Maxwell and Johnson 2000, Waltz

Manual 1998). This value can be used to interpret how much energy is being consumed

in photochemistry. The potential quantum yield of PSII (ΦPSII) can be determined by

dividing Fv/Fm = (Fm-Fo)/Fm = PSII/qP. This measure can be used to determine the

possible yield for a sample if all of its reaction centers are “open” (Maxwell and Johnson

2000). Photochemical quenching (qP) is an indication of how many reaction centers are

open, or able to accept solar energy. Calculated as (Fm’-Ft)/ (Fm’-Fo’), qP allows one to

look at the actual processes affecting yield (Maxwell and Johnson 2000). Non-

photochemical quenching or NPQ, measures the amount in heat dissipation in dark

acclimated samples.

Fm cannot be measured without the presence of non-photochemical quenching

(Maxwell and Johnson 2000). Due to this fact if an accurate measure of Fm is desired,

samples must be dark acclimated prior to the initial reading, and a relative measure taken

from this dark adapted state. Dark acclimation ensures that all available photo-centers

are in the “open” state, meaning that they are capable of accepting photo-chemical

energy. qN can also affect the level of Fo in some situations, which in turn leads to a

reduction in the yield of Fo`. When qN reaches a point above 0.4, Fo can be quenched,

20

reducing the yield of Fo`. This quenching of Fo` can be addressed only by the use of a far

red light pulse to enhance Qa-reoxidation. This light source however, is not available in

the diving PAM. The diving PAM does however give a second measure of non-

photochemical quenching (NPQ) that is not as sensitive to Fo` quenching. Where qN is

affected by this quenching at 0.4, NPQ is affected by quenching at 4.0 (Maxwell and

Johnson 2000). Due to this increased tolerance, the value of NPQ has become the

measure of choice for heat dissipation analysis.

Field studies using PAM fluorometery have demonstrated the usefulness of this

technology in measuring the parameters surrounding marine primary productivity. Much

of the present work has focused on the effects of increased irradiance on PSII (Hader et

al. 1996. Hader et al. 1999). This work has indicated that the diving PAM fluorometry

system is able to measure in situ changes in quantum yield and qN in PSII as well

recovery time due to photo-inhibition. A correlation between increased photokinetics and

irradiance levels was also shown in seagrasses (Ralph et al. 1998), sponges (Beer and Ilan

1998a) and corals (Beer et al. 1998b). This work has set the base for continued work

using PAM technology to look at PSII kinematics as a function of various other

environmental fluctuations (e.g. water flow and salinity).

The development of chlorophyll fluorescence technology along with advances in

acoustic doppler velocimeters has made research questions that were not measurable a

short time ago valid research avenues. The ability to take detailed in situ measurements

of hydrodynamic regime and tie them in with direct measures of photosystem kinematics

may allow eco-physiologists, physical biologists, and oceanographers to produce more

21

accurate models of aquatic productivity. This thesis investigates the hypothesis that

increased water flow will increase PSII kinematics in Caulerpa spp. This hypothesis is

based on the fact that in higher water flow; the boundary layer is reduced, allowing for a

higher rate of nutrient transport to the algal thallus, or a higher rate of O2 removal from

the surface. This study is the first to use a direct measure photosystem activity to look at

the effects of flowing water on aquatic vegetation.

22

Materials and Methods

To determine the effects of water flow on the photoproduction of the genus

Caulerpa. I preformed three sets of experiments. These experiments were conducted

with specific goals in mind. The first set of experiments was conducted to determine

whether photosystem efficiency was dependent on changes in water velocity in a

laboratory flume. The second set of experiments was conducted to determine the effects

of water flow on photosynthesis in a natural setting, and the third set of experiments was

conducted in order to determine the effects of water flow on photosynthesis over varying

lengths of time.

Measurements on several species of macroalgae within the genus Caulerpa

from the Florida Keys and Tampa Bay were used to characterize the effects of

hydrodynamic regime on photosystem II (PSII) kinematics. Laboratory measurements

were collected in order to determine if photosystem kinematics are a function of

hydrodynamic regime and field measurements were collected in order to elucidate the

role of hydrodynamics in algal productivity in situ. All photosynthetic parameters

analyzed in this study were measured using rapid light curves produced by a Diving PAM

Fluorometer. PSII kinematics measurements were taken approximately half way up the

frond from the rhizome, and in no case was new tissue (complete lack of pigment) used

for measurement of photosynthetic activity. Unlike past studies on productivity in

flowing waters, the development of the Diving PAM Fluorometer allows for non-

23

invasive, direct measurements of PSII kinematics. In all experiments, values of

maximum electron transport rate (Pmax), the initial slope of the photosynthetic curve (α)

and saturation irradiance (Ik) were analyzed. In several experiments photochemical

quenching (qP) and non-photochemical quenching (NPQ) were also analyzed.

In all of the experiments conducted, rapid light curves were collected and analyzed

with two different non-linear models using the sigma plot software suite. The Platt

inhibition model, (f=px(1-exp((-axx)/p))x(exp(-bxx/p)) (Henely 1993) was used when light

curves showed a marked inhibition of photosystem structure (Figure 4) and the

hyperbolic tangent model (f=Pmax*tanh((alphaxx)/Pmax)+Rd) (Henely 1993) was used

when there was no inhibition in the light curve (Figure 5). Only curves where the model

fit with a r2 value of 0.90 or better were used in the data analysis.

The recent advances in technology used to measure hydrodynamic regimes have also

drastically changed the scope of the questions that can be raised concerning the

interactions of biological processes and water flow. In all flume experiments, the water

velocity was measured by taking a velocity profile (measures of velocity were collected

at 1.0 cm intervals with an ADV) through the water column and calculating a depth

average velocity. Field measurements of velocity were bulk flow measures taken at the

approximate midpoint of the water column. Care was taken when near a surface, to

ensure that the ADV signal was not being intercepted by the substrate (Finelli 1999).

24

Caulerpa

Caulerpa racemosa, C. sertularioides and C. mexicana were chosen for this study

due to their abundance in Florida’s coastal eco-systems. The unifying characteristics

Caulerpa spp. is a coenocytic thallus, a rhizomatous mass used to anchor the algae to the

substrate and trabeculae supporting the exterior cell walls. Caulerpa spp. often grow in

an intertwined matt of rhizomes, acting to stabilize unconsolidated sediments and

allowing the settlement of climax species such as seagrasses which require solid

sediments.

Caulerpa sertularioides (Figure 6) is characterized by an erect featherlike frond

up to 20.0 cm in length and 1.0-2.0 cm in width. The branchlets or pinnules are

oppositely branched and approximately 3.0-11.0 cm long and pointed at the tip. The

rhizome is up to 2.5 mm in diameter and can grow up to 2.0 m in length. C.

sertularioides is most often found in shallow waters on sandy substrates. C.

sertularioides is found from the shallow subtidal down to a depth of 10.0m. The range of

C. sertularioides is from Florida in the North throughout the Southern Caribbean (Littler

and Littler 2000).

Caulerpa racemosa (Figure 7) is characterized by a creeping or erect frond that is

1.0-5.0 cm tall. The pinnules, of C. racemosa are highly diversified and may include

rounded, mushroom capped, or oval shaped apices. The stolon of C. racemosa is thick

(2.0-3.0 mm) and creeping with a large number of branched rhizoids. C. racemosa is

often found attached to rocks in areas of considerable surf as well as sandy sediments that

25

experience little to no water flow, such as in thick seagrass beds. C. racemosa is found

from the intertidal zone to a depth of 50.0 m. The range of C. racemosa is from Florida

in the North throughout the Southern Caribbean (Littler and Littler 2000).

Caulerpa mexicana (Figure 8) is characterized by an erect frond that can range

from 2.0 cm in height in high-energy areas to 25.0 cm in height in low-energy

environments. Pinnules are opposite of each other and resemble the fronds of a fern. The

stolon of C. mexicana can be relatively thin ranging from 0.6 mm to 1.5 mm in diameter.

The rhizoids are thin and numerous, allowing the algae to anchor in very soft sediment.

The depth range of C. mexicana is reported as the shallow subtidal through a depth of

15.0 m. The species range of C. mexicana is the same as the other two species

mentioned, with a northern limit of Florida throughout the Southern Caribbean (Littler

and Littler 2000).

Measurement of PSII kinematics

Before the start of each experiment, samples were dark acclimated in order to

normalize the light history of individual plants. This dark acclimation involved securing

the sample inside the magnetic leaf clip of the Diving PAM Fluorometer for a period of

ten minutes. The acclimation period ensured that all of the reaction centers within PSII

were oxidized, or open for the capture of photochemical energy. The sample clips

required by the Diving PAM Fluorometer to dark acclimate samples present an

immediate problem when studying the effects of hydrodynamic regime on photosystem

26

function. Samples must be held in the clips for at least 11.5 minutes (10 minutes for dark

acclimation and at least 1.5 minutes for a rapid light curve) during which time the

boundary layer around the thallus is increased due to the added size of the leaf clip. This

increased boundary layer may act to slow the acquisition of nutrients by the algae. To

ensure that this increased boundary layer around the plants due to the presence of the

magnetic leaf clip was not a source of nutrient (e.g. C, N, P) limitation a small flume

experiment was conducted to determine if there was a difference in Pmax when samples

were acclimated using the clips vs. when they were acclimated in a dark room. Samples

of Caulerpa racemosa were collected from the South East jetty of the Sunshine Skyway

Bridge at the mouth of Tampa Bay (Figure 9). All samples were transported in aerated

seawater back to the lab within one hour, cleaned of all macro-epiphytes and kept for 24

hours in aerated seawater. The samples were then placed into a 110L racetrack flume

filled with artificial seawater with a salinity of 35ppt (Figure 10). Samples were held in

place using zip ties attached to a false floor in the flume and allowed to acclimate to the

hydrodynamic regime for 30 minutes before measurements were taken. A light bank of

10,000 K Coralife© full spectrum grow lights suspended above the flume provided

illumination. Samples were dark acclimated using either the magnetic leaf clip (Waltz,

Germany) under full artificial illumination or with the room in complete darkness. Water

flow in the flume was controlled using a small trolling motor and kept at approximately

15 cm s-1. After dark acclimation, rapid light curves using light levels of 0, 8, 12, 22, 30,

48, 66, 104, 155 µmol quanta m-2 s-1 were conducted. To determine if the magnetic leaf

27

clip was negatively affecting Pmax between the two treatments, an ANOVA (SYSTAT)

was run after assessing the normality of the data (SYSTAT).

An initial, small scale experiment was conducted in order to determine whether or

not a diving pam fluorometer could be used to measure short-term changes in

photosystem kinematics due to changes in water velocity. A series of small flume

experiments were conducted at the Keys Marine Lab (KML), on Long Key Florida

(Figure 11). Samples of Caulerpa mexicana were collected from the Thalassia

testudinum bed behind KML over five days of December of 2002. Samples were

transferred within five minutes to a re-circulating seawater table and stored for 24 hours

under natural light conditions before experiments were conducted. Samples were

cleaned of all macro-epiphytes and placed into a 110L racetrack flume filled with un-

filtered seawater (Figure 10), where the water velocity was controlled using a small

trolling motor. High velocity trials were run at 25.0-30.0 cm s-1, and low velocity trials

were run at 5.0-10.0cm s-1. Water velocity profiles were measured using a Sontek

Acoustic Doppler Velocimeter (ADV) at a sample rate of (25 hz) and a depth average

velocity was calculated. The samples were held in place using zip ties attached to a false

floor in the flume. The plants were allowed to acclimate to the flow conditions for one

hour before measurements were taken. A bank of overhead fluorescent lights placed

approximately 30.0 cm above the flume provided irradiance. Samples of C. mexicana

were placed in a magnetic leaf clip (Waltz, Germany) so that no pinnules were

overlapping and dark acclimated for 10 minutes. Rapid light curves using a Diving PAM

Fluorometer were taken using light levels of 0, 48, 66, 99, 138, 212, 311, 463, 734 µmol

28

quanta m-2 s-1. Light curves were analyzed for maximum electron transport rate (Pmax),

saturation irradiance (Ik) and the initial ascending slope of the curve (α) by fitting the data

to either the hyperbolic tangent model or the Platt inhibition model (Henely 1993). Data

were tested for normality using SYSTAT 10 statistical packages and values of Ik, were

normalized to the square root of the data (SIGMA STAT). An ANOVA (SYSTAT) was

used to assess the affect of hydrodynamic regime on Pmax, Ik and α.

Field Experiments

Unlike laboratory experiments that use constant uni-directional flow, organisms

living in natural conditions experience constant changes in the magnitude and direction of

water flow due to tidal changes and wind driven waves (Hearn et al. 2001, Wing and

Patterson 1993). To assess the role of hydrodynamics in algal photo-physiology in situ a

series of field measurements were taken on samples of Caulerpa sertularioides growing

on a carbonate shoal approximately 2.0 km North-West of the Long Key Channel Bridge.

Rapid light curves were measured on samples of Caulerpa sertularioides growing on Old

Sweat Bank, on the Florida Bay side of Long Key, Florida (Figure 11) between June 6,

2002 and June 9, 2002. Much of the water from Florida Bay, including water originating

from the Everglades empties into the Atlantic Ocean through the Long Key Channel

(Wang 1998). Water velocities on the shoal range from near 0.0 cm s-1 at slack tides to

greater than 1.0 m s-1 during flood tides. Old Sweat Bank is characterized as a carbonate

shoal composed mainly of Halimeda spp. segments and crushed coral. Vegetative

29

growth on the shoal is comprised of Thallassia testudinum and Syringonia filiforme,

extensive Halimeda spp. and Penicillus spp. beds, and intermittent patches of Caulerpa

sertularioides. Caulerpa patches were approximately 1.0 m in diameter and were located

along the midline of the shoal. Large patches of Porites porites are scattered over the

shoal along with various species of sponges. The bank is surrounded on all sides by

dense T. testudinum beds, and cut into three sections by two tidal channels (Figure 11).

Water depth over the shoal ranges from approximately 1.0 m at flood tide to <10cm at

neap tide. All light curves were run between 7:30 am and 8:00 pm to ensure that a

variety of water flow velocities (>1.0 ms-1 <0.05 cms-1) were encountered at all times of

photosynthetic activity for the algae.

Samples of Caulerpa sertularioides were placed into the magnetic leaf clip in

such a manner that no pinnules overlapped and dark acclimated in situ for ten minutes

(Waltz, Germany). Rapid light curves were then run on all samples using a diving pam

fluorometer at light levels of 0, 48, 66, 99, 138, 212, 311, 463, 734 µmol quanta m-2 s-1.

Light curves were analyzed for maximum electron transport rate (Pmax), saturation

irradiance (Ik) and the initial ascending slope of the curve (α) by fitting the data to either

the hyperbolic tangent model or the Platt inhibition model (Henely 1993). Ambient light

levels were measured using the external light sensor on the Diving PAM Fluorometer.

Light levels at the algal thallus ranged from 40-1167 µmol quanta m-2 s-1. The 2π

quantum sensor was mounted onto the top of fluorometer, which was the approximate

height of the algae off the substrate. Simultaneous acoustic doppler velocimeter (ADV,

Nortek) readings were taken adjacent to the algae. To measure bulk flow over the shoal

30

at mid water depth, the ADV sensor head was mounted perpendicular to the substrate

when water height above the bottom was >15cm (Figure 12). When water height was

<15cm the sensor was mounted parallel to the substrate (Figure 12). ADV samples were

taken at 25hertz with burst intervals of 5 minutes. Using the internal clocks features of

the fluorometer and the ADV, individual light curves were matched by time to the

corresponding bulk flow measurement over the shoal. Water velocities over the shoal

were separated into three discreet categories, <20cm s-1, 20-30cm s-1, and >30cm s-1. To

determine if photosystem kinematics were dependent on hydrodynamic regime a two way

ANOVA (SYSTAT) was used to test for differences in Pmax, Ik, and α, with time of day

as a covariate. Data was tested for normality (SYSTAT) and subsequently normalized

(SIGMA STAT) to the square root of the data. All outliers were removed according to

Grubbs outliers test (Rolph and Sokal 1981) before analysis was preformed.

Time in flow

In an attempt to isolate the effects of hydrodynamics on the photosystem

kinematics, and to determine the effects of “time in flow” on the PSII kinematics of C.

racemosa. a series of small flume experiments was conducted in a controlled laboratory

setting. In this manner the amount of light, nutrient levels and salinity can be controlled

with hydrodynamic regime as the only fluctuating component.

Samples of Caulerpa racemosa were collected from the South East jetty of the

Sunshine Skyway Bridge at the mouth of Tampa Bay (Figure 9) between July 2, 2002

31

and August 5, 2002. Samples were collected just after sunrise and were returned to the

lab within one hour. Samples were cleaned of macro-epiphytes and kept in aerated

seawater for 24 hrs before experiments were conducted. Only samples that had not been

damaged in the collection process were used for experiments. Three individual plants

were then placed into each of two identical 110L racetrack flume (Figure 10) filled with

artificial seawater (35 ppt). The sea water was fertilized with 0.0161g l-1 PO4, 0.031 g l-1

NH4, and 0.005 g l-1 Nitrate. These nutrient levels correspond to a ten year average

during the summer months at the Sunshine Skyway Bridge

(http://www.floridamarine.org, 2003). Water velocity within the flumes was controlled

by a small trolling motor. Velocity profiles within the flume were taken using an ADV

(SONTEK) and a depth average velocity was calculated for use as a bulk flow

measurement. Illumination was provided by an overhead bank of 10,000 K Coralife©

grow lights suspended over the flumes. Samples were allowed to acclimate to the

hydrodynamic regime for a period of one hour before Rapid Light Curves were collected

using light levels of 0, 8, 12, 22, 30, 48, 66, 104, 155 µmol quanta m-2 s-1. Rapid light

curves were taken every hour for 7 hours. Light curves were analyzed for maximum

electron transport rate (Pmax), saturation irradiance (Ik) and the initial ascending slope of

the curve (α) by fitting the data to either the hyperbolic tangent model or the Platt

inhibition model (Henely 1993). Photochemical quenching (qP) and non-photochemical

quenching (NPQ) values were obtained for each sample by selecting the set of data

within each curve nearest the saturation irradiance for the sample. To assess the role of

hydrodynamic regime on the photosystem kinematics of C. racemosa a two way repeated

32

measures ANOVA (SIGMA STAT) was preformed with water flow as one factor and the

amount of time in the flow as the second factor. The measurements from each of the

three plants in each flume were averaged to form one set of data per trial. The data was

then assessed for normality (SYSTAT) and transformed (SIGMA STAT) to the square

root of the data for Pmax, Ik, and NPQ. Data was transformed to the ln of the data for α,

and qP. All outliers were removed using Grubbs outliers test (Rohlf and Sokal 1981).

The ratio of Pmax in high flow to Pmax in low flow was plotted for each of the

seven time periods (FIGURE 13). A distinct drop in the ratios was seen after two hours

of immersion. The data for measurements three through seven was then assessed for

normality (SYSTAT) and transformed (SIGMA STAT) to the square root of the data for

Pmax, Ik, and NPQ. Data was transformed to the ln of the data for α, and qP. All outliers

were removed using Grubbs outliers test (Rohlf and Sokal 1981). A two way repeated

measures ANOVA was then used to assess the role of hydrodynamics after two hours of

immersion.

33

Results

Does the magnetic sample holder affect values of Pmax?

The differences in values of Pmax between trials using a magnetic leaf clip to

dark acclimate a sample and trials using a dark room to dark acclimate samples were not

significantly different (P=0.790, F=0.073, Figure 14). Samples acclimate with clips had a

mean Pmax of 8.6 (SE = 1.4, n=9) and samples acclimated in a darkened room had a

mean Pmax of 8.9 (SE=3.1, n=9).

Laboratory measurements of PSII kinematics on C. mexicana after a short exposure

to hydrodynamic regime.

The effect of water velocity on values of Pmax from C. mexicana was significant

(P<0.05, F=6.811, Figure 15). Samples run at high (25-30cm s-1) and low (5-10 cm s-1)

velocities had mean values of Pmax of 26.5 (SE=7.0, n=4) and 52.3 (SE=7.0, n=4)

respectively. Values of α from C. mexicana in the two different hydrodynamic regimes

were not significantly different. The effect of water velocity on saturation irradiance was

very close to being significant, (P=0.055, F=5.66). The mean of Ik in high flow was 16.2

(SE=1.7 n=4) and the mean of Ik in low flow was 22.0 (SE=1.7, n=4).

34

In situ field measurements of PSII kinematics on C. sertularioides

Water velocity had a significant affect on the values of Pmax and Ik in Caulerpa

sertularioides measured in situ (P<0.001, F=9.387, Figure 16, Table 1 and P<0.05,

F=3.754, Figure 17, Table 1 respectively). Water flow did not affect values of α

(P=0.570, F=5.67) Table 1). There was no significant interaction of time on the values of

Pmax, α or Ik (Table 1).

Laboratory measurements of PSII kinematics in C. racemosa after long term

exposure to hydrodynamic regime.

Values of Pmax (P=0.067, F=3.865), α (P=0.100, F=3.044,), Ik (P=0.893,

F=0.0188), qP (P=0.198, F=1.800) and NPQ (P=0.490, F=0.497) for C. racemosa run in

laboratory flumes at different water flow velocities over a period of seven hours were not

significantly different (Table 2). There was no significant difference in Values of Pmax

(P=0.343, F=1.143), α (P=0.104, F=1.818), Ik (P= 0.674, F=0.669) and qP (P=.135,

F=1.677) due to the time spent in the flow (Table 2). There was a significant effect of

time in flow for values of NPQ (P<.005, F=4.825, Figure 18, Table 2, Table 3). The

interaction of time spent in water flow and water velocity for Pmax (P=0.266, F=1.297),

α (P=0.409, F=1.032), Ik (P=0.078, F=1.972), qP (P=0.164, F=1.570) and NPQ (P=0.286,

F=1.259) was not significant (Table 2).

The ratio of Pmax in fast flow to Pmax in slow flow was calculated to determine

if this value changed over time (Figure 13). The ratio was found to be relatively constant

for the first two hours, then the ratio dropped, indicating that there may be a delay in

35

response to changes in water flow. If the data is analyzed for the time after two hours

there is a significant difference between Pmax in fast flow and Pmax in slow flow. A

second repeated measures ANOVA was run for measurements three through seven.

Water velocity significantly affected values of Pmax values at times three through seven

(P<0.05, F=4.816, Figure 19, Table 4). The mean value of Pmax in low flow velocity

was 3.5 (SE=0.201, n=45), the mean value of Pmax in high flow was 4.7 (SE=0.278,

n=45). Water velocity did not affect the values of α, Ik, qP and NPQ (P=0.098, F=3.096,

P=0.971, F=0.00138, P=0.098, F=3.096 and P=0.321, F=1.039 respectively Table 4). The

amount of time spent in the hydrodynamic regime did not affect the values of Pmax, α,

Ik, and qP(P=0.264, F=1.342, P=0.167, F=1.65, P=0.665, F=0.618 and P=0.167, F=1.675

respectively, Table 3). The amount of time in the hydrodynamic regime did affect values

of NPQ significantly (P=.001, F=4.197, Figure 20, Table 4, Table 5). The interaction of

water velocity and time spent in flow did not affect the values of Pmax, α, Ik , qP and

NPQ (P=0.273, F=1.318, P=0.458, F=0.920, P=0.139, F=1.810, P=0.403, F=1.023 and

P=0.374, F=0.826 respectively, Table 3).

36

Discussion The objectives of this thesis were to determine if hydrodynamic regime affects

photosystem II kinematics in three species of the green algae Caulerpa, and to determine

the extent of the dependence of PSII kinematics on hydrodynamic regime. Experiments

also showed that the effect of hydrodynamics on members of the genus Caulerpa may be

species, or location specific.

The results of these experiments indicate that photosystem II kinematics of

Caulerpa sertularioides, C. mexicana, and C. racemosa are influenced by hydrodynamic

regime in lab experiments and in situ. Lab experiments on C. racemosa (Figure 19)

collected from Tampa Bay, support the findings of Wheeler (1980), Gerard (1982), Koehl

and Alberte (1988), Carpenter et al. (1991) and Williams and Carpenter (1998) that

increased water flow positively correlates with Pmax (figure b) and Ik (figure c) in marine

algae. These results support our hypothesis that increased water flow would positively

influence PSII kinematics. A possible explanation is that in Tampa Bay, C. racemosa is

physically limited. As water velocity was artificially increased more nutrients were

delivered to the plant surface, allowing for an increase in the electron transport rate.

Field measurements of C. sertularioides from the Florida Keys and lab

measurements of C. mexicana did not support our hypothesis. Results showed a drastic

decrease in Pmax as a function of water velocity (Figure 13, Figure 15). These findings

are supported by several studies on the effects of water flow on marine and freshwater

37

algae. Gerard and Mann (1979) noted that the growth of Laminaria longicrursis in

exposed sites was significantly slower than the growth of L. longicrursis in protected

sites. More recently, Borchardt (1994) demonstrated that in freshwater plants which are N

and P limited, flows greater than 30 cm s-1 were physiologically costly. Several

mechanisms may be responsible for these results, including nutrient limitation due to the

high water velocity, light flecking, and self shading.

Nutrient limitation is often thought to occur only during periods of low flow,

when nutrient delivery is impeded by the thickness of the diffusive boundary layer.

However, nutrient limitation can occur during periods of relatively high water flow if

there are competing mechanisms within an organism that are triggered by the increase in

water velocity. Gerard and Mann (1979) concluded that in high flowing waters N was

more limiting than in slow flows in L. longicrursis because of specialized structures the

algae needed to produce and maintain in order to survive in high velocity environments.

If the plants need N to build or maintain these structures, then less is available for the

cells photosynthetic pathways. Borchardt (1994) proposed that the actual nutrient

requirements of nutrient limited Spirogyra fluviatilis changed with changes in

hydrodynamic regime; the plants needed more of a limiting nutrient when flow was

increased. This conclusion supports the conclusions of Gerard and Mann (1979), that

while nutrient delivery is increased in higher flows, the actual cellular or organismal

quota is also increased. Carbon limitation may actually increase with flowing waters

depending on the form of C that a plant is capable of using (Borchardt 1994). If a plant

utilizes an extra cellular bi-carbonate converting mechanism to convert bi-carbonate to

38

CO2, increased water flow may actually increase C limitation. These conversion

mechanisms often rely on the algae actively pumping a high gradient of protons (H+) into

the diffusive boundary layer next to the thallus surface. These H+ are needed to convert

bi-carbonate to CO2. If water velocity increased and these H+ were transported away

from the thallus, then higher levels of N and P may be needed to increase H+ transport

across the cell membranes.

The light field experienced by any photosynthetic organism can drastically change

its rate of photosynthesis. Sun flecks are intense burst of irradiance that result from the

movement of canopy structures and surface waves that can account for significant

differences in photosynthetic capacity of marine plants (Dromgoole 1987, Wing and

Patterson 1993, Wing et al. 1993). Due to their focusing effect, surface waves result in

high-frequency light fluctuations (millisecond range ), while the movement of canopy

structures results in slower frequency and longer duration fluctuations (seconds to

minutes ) (Green and Gerard 1990, Wing and Patterson 1993, Sagert and Schubet 2000).

These fluctuations in light levels and intensity are known to be both beneficial and costly

to algae. Under high frequencies of fluctuating light, the photosynthetic rates of many

species have been shown to increase (Dromgoole 1987, Dromgoole 1988, Green and

Gerard 1990). Conversely, other studies have demonstrated a negative correlation

between light flecks and photosynthetic rates (Wellnitz and Rinne 1999). For a short

period of time (milliseconds) focusing of irradiance by surface waves can increase the

amount of light reaching the benthos by as much as five times the surface irradiance

(Sagert and Schubert 2000, Schubert et al. 2001). During periods of high water flow on

39

Old Sweat Bank, small surface waves ran over the shoal with a very high frequency

(Driscoll personal observations). The light regime resulting from this intense focusing of

light may cause photo-inhibition in C. sertularioides due to temporarily damage the

antennae complexes of PSII.

Caulerpa, like several other genera of the siphonous green algae have the ability

to translocate their chloroplasts in times of stress and growth (Dawes and Barilotti 1969,

Drew and Abel 1990, Chisholm and Jaubert 1997). This chloroplast movement was

witnessed in C. sertularioides and C. racemosa during periods of high water flow. This

chloroplast movement may have induced self-shading (of plastids) effects which could

have resulted in an underestimation of PSII kinematics in high flow conditions.

In high velocity flows marine algae have been shown to “clump” together or bend

to reduce the effects of form drag (Koehl and Alberte 1988). This clumping, while

reducing drag can also decrease the photosynthetic activity of a plant by limiting the

amount of light reaching the individual photosynthetic surfaces of the plant. The frond

axis of C. sertularioides in the Florida Keys remained upright at all water velocities,

however, observations showed the pinnules bending with the bulk flow so that they were

almost parallel to the main velocity vector. This bending of the pinnules, while not

quantified, may have caused self-shading during times of high water flow, decreasing

values of Pmax.

Comparison between sites in the Florida Keys and Tampa Bay are made difficult

by the differences in light regime and ambient nutrient concentration. The collection site

in Tampa Bay is often extremely turbid due to terrestrial run-off and storm activity,

40

limiting the amount of light penetrating the water column. Nutrient levels are also high

due to extensive anthropogenic inputs. Conversely, Old Sweat Bank has very little

turbidity in the water column, allowing high levels of irradiance to penetrate the water.

Like most tropical systems the nutrient levels of carbonate shoals in the keys are deplete

(Lapointe and Clark 1992). These factors make comparisons between locations

equivocal, continued experimentation at both sites will be needed to draw conclusions on

a wider scale than species level.

These experiments are the first to use a direct measure of PSII kinematics to look

at the role of hydrodynamics in photo-production. Previous studies have relied on

oxygen evolution as the measure of photosynthetic activity (Raven et. al. 1979, Carpenter

et. al. 1991, Gacia et al. 1996). The measure of oxygen evolution as a function of water

velocity creates a situation that may drastically affect the results of experiments. Just as

Nitrogen, Phosphate, and Carbon are mass transfer limited to photosynthetic surface,

oxygen may be mass transfer limited from the same surface. This limitation would mean

that in low flows a gradient of oxygen would form within the boundary layer with higher

levels near the tissue surface. By increasing water velocity this oxygen would be carried

into the water column as the boundary layer thickness is decreased, erroneously

increasing the measured rates of oxygen evolution. By measuring chlorophyll

fluorescence this artifact is removed since fluorescence originates in the PSII antennae

complex and the measure itself is not affected by associated boundary layers.

This research is the first set of data that looks at direct measures of photo-

production as a function of hydrodynamic regime. Our findings support the hypothesis

41

that hydrodynamic regimes can influence physiological processes in marine algae. The

method of this influence and the degree of the dependence of PSII on water flow

conditions are still unclear, however, the results of these experiments and observations

suggest that the use of PAM Fluorescence could be applied to elucidate the answers to

these questions. Future directions for this project may involve altering the concentrations

of nutrients in a variety of hydrodynamic regimes in an attempt to isolate the limiting

factor, if any, in the photosynthetic pathways of Caulerpa spp. In situ measurements on

several more species of Caulerpa would also be beneficial in determining the role of

hydrodynamics in photosystem function.

42

Figure 1. Diagram of the anti-herbivory metabolite caulerpenyne. This compound is on

of the main factors allowing for the success of Caulerpa spp. In certain cases

caulerpenyne may compose up to 13% of the wet frond weight of C. taxifolia, making the

algae in-edible to any known herbivore.

43

Figure 2. The three main energy pathways for photosystem II. (1) Energy is

shunted off to the electron transport chain, (2) energy is dissipated as heat before it

damages the photosystem and (3) energy is re-emitted from photosystem II at a higher

wavelength (Fluorescence).

44

Figure 3. Skematic diagram of the pulse amplitude modulated fluorescence

measuring principle in both light and dark adapted samples. (Walz 2001).

45

0

2

4

6

8

0 200 400 600 800

µmol photons m2s-1

Pmax

µ

mol

pho

tons

m2 s-1

Figure 4. Representative curve that would be analyzed using the Platt inhibition

model. A distinct decrease in levels of Pmax (photo-inhibition) is seen. µmol

photons m2s-1

46

05

10152025

0 200 400 600 800

µmol photons m2s-1

Pmax

µ

mol

pho

tons

m2 s-1

Figure 5. Representative curve that would be analyzed using the hyperbolic

tangent model. No photo-inhibition is seen in the curve.

47

Figure 6. Drawing of Caulerpa sertularioides from Caribbean Reef Plants (Littler and Littler 2000). The pinnules are clearly much finer than those found in C. mexicana and C. racemosa.

48

Figure 7. Drawing of C. racemosa from Caribbean Reef Plants (Littler and Littler 2000). This diagram depicts the variety in pinnule shapes and formations encountered in Tampa Bay, Florida.

49

Figure 8. Drawing of Caulerpa mexicana from Caribbean reef plants (Littler and Litter 2000). The pinnules of C. mexicana grown in a very flat broad shape, which is very similar to that of C. taxifolia.

50

Figure 9. Collection site for C. racemosa at the Sunshine Skyway Bridge south causeway. Specimens were collected from a depth of approximately 0.5m and returned to the lab within one hour of collection.

51

Figure 10. 110L racetrack flume. Water velocity within the flume is created by a small trolling motor and augmented using a diode control. Turbulence within the flume is dampened using three sets of flow straighteners.

52

Figure 11. Maps of Study sites in the Florida Keys. Upper image shows the Florida Keys and Florida Bay. Lower cutout diagrams Old Sweat Bank. C. sertularioides collection sites are indicated as dark shapes in each section of the shoal.

53

Figure 12. Orientation of the acoustic doppler velocimeters during field measurements. The ADV was attached to a large ring stand device with cable ties and hose clamps, and height was adjusted using several ring stand clamps. The sensor head was placed perpendicular to the substrate when water depth was greater than 15 cm and it was oriented parallel to the substrate when the water depth was less than 15 cm.

54

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8Time in flow (hours)

Pm

ax fa

st fl

ow /

Pmax

slo

w fl

ow

Figure 13. Ratio of Pmax in fast flow to Pmax in slow flow at each measuring interval. A clear drop in the ratio of Pmax fast/ Pmax slow is seen between times two and three indicating a possible lag time for the effects of hydrodynamics to be seen in PSII.

55

0

5

10

15

Clip Dark room

Pmax

(µm

ol e

lect

ron

m2

s-1)

Figure 14. Results of dark acclimation trial using magnetic leaf clips under full illumination and a dark room. There was no significant difference (P value = 0.790) between trials utilizing the magnetic leaf clips and a dark room to dark acclimate tissue samples. These results indicate that the samples used were not nutrient limited by the presence of the magnetic leaf clip.

56

0 10

20 30

40 50

60 70

5-10 cm s-120-25 cm s-1

Pmax

(µm

ol e

lect

ron

m2

s-1)

Figure 15. Values of Pmax for C. mexicana run at high and low velocities in a laboratory flume. Water velocity was shown to significantly affect values of Pmax (P value = 0.04). High flow was 20 – 25 cm s -1 and low flow was 5-10 cm s-1 . Values of Pmax were significantly higher in slow hydrodynamic regimes.

57

0 2 4 6 8

10 12 14 16

<20 cm s-1 20-30 cm s-1 >30 cm s-1

Pm

ax

(µm

ol e

lect

ron

m2 s-1

)

Figure 16. In situ measures of Pmax in C. sertularioides. PSII kinematics parameters were measured without removing the plant from its natural environment. Water velocity was shown to significantly affect values of Pmax measured in the field using a Diving PAM Fluorometer (P value <0.001). Low flow was less than 20 cm s -1 , medium flow was 20 – 30 cm s -1 and high flow was above 30 cm s -1 . The highest values of Ik were seen when velocity was less than 20 cm s-1. The lowest values of Pmax were measured when water velocity was greater than 30 cm s-1.

A

B

C

58

0 10 20 30 40 50 60 70

<20 cm s -1 20-30 cm s -1 >30 cm s -1

I k (µ

mol

qua

nta

m-1

s-1)

Figure 17. In situ measurements of Saturation irradiance in C. sertularioides. Saturation irradiances measured on C. sertularioides measured in situ were significantly different as a function of water velocity (P value = 0.018). Low flow was less than 20 cm s -1 , medium flow was 20 – 30 cm s -1 and high flow was above 30 cm s -1 . The highest values of Ik were seen when velocity was less than 20 cm s-1.

A

B

C

59

0 0.020.040.060.080.1

0.120.14

1 2 3 4 5 6 7 Time in flow

NPQ

Figure 18. Laboratory measurements of NPQ in C. racemosa. Samples were collected from Tampa Bay, and PSII kinematics parameters were measured in a laboratory flume every hour for seven consecutive hours. Values of NPQ, measured from hours three through seven were shown to be significantly different as a function the amount of time spent in the water flow (P value = 0.002).

AA A

AA

BB

60

3.4 3.6

3.8 4

4.2 4.4

4.6 4.8

>15 cm s -1<15 cm s -1

Pm

ax

(µm

ol e

lect

ron

m2

s-1)

Figure 19. Laboratory measurements of Pmax in C. racemosa. C. racemosa was collected from the Sunshine Skyway jettie, and exposed to different water flow regimes in a small laboratory flume. After three hours in a hydrodynamic regime, values of Pmax from C. racemosa were shown to be significantly different as a function of water velocity (P value = 0.022). High flow was considered velocities above 15 cm s-1 and low flow was considered velocities lower than 15 cm s-1. Higher values of Pmax were measured in velocities greater than 15 cm s-1.

61

Figure 20. Laboratory measurements of NPQ in C. racemosa. Samples were collected from Tampa Bay, and PSII kinematics parameters were measured in a laboratory flume. Values of NPQ, measured from hours three through seven were shown to be significantly different as a function the amount of time spent in the water flow (P value = 0.002). Hours labeled “A” are significantly different than hours labeled "B".

0 0.02 0.04 0.06

0.08 0.1

0.12 0.14

3 4 5 6 7 Measurement hours

NPQ

A A

BB

B

62

Flow Speed

Low Medium High

P value

Mean

Mean Mean Velocity time

Pmax (µmol electron m2s-1)

12.8

(0.794)

9.4 (0.759)

7.2 (1.127)

<0.001 0.47

α (µmol electron m2s-1)

0.224 (0.017)

0.217 (0.019)

0.257 (0.031)

0.57 0.26

Ik (µmol quanta m2s-1)

56.8

(5.632)

46.4 (5.245)

30.3 (7.534)

<0.05 0.64

Table 1. Values of Pmax, α, and Ik measured on C. sertularioides in situ. Measurements were taken on plants that had not been removed from their natural habitats, and that were exposed to natural water flow and irradiance conditions. Values of Pmax, measured in situ were found to be significantly different as a function of water velocity (P value <0.001). Values of α and Ik were not found to be significantly different in different water flows when measured in situ.

63

Flow Speed Flow Speed High Low

P value

Mean Mean velocity time Velocity x time Pmax

(µmol electron m2s-1) 4.4

(0.195) 4.0 0.07 0.34 0.26

α (µmol electron m2s-1)

0.390 (0.01)

0.378 0.10 0.10 0.41

Ik (µmol quanta m2s-1)

27.9 (1.256)

28.4 0.89 0.67 0.08

QP 0.238 (0.00973

)

0.226 0.19 0.13 0.16

NPQ 0.083 (0.00547

)

0.098 0.49 0.002 0.73

Table 2. Values of Pmax, α, Ik, qP, and NPQ measured on C. racemosa in a laboratory flume over seven hours. Samples were collected from Tampa Bay and measurements were taken in a laboratory flume. Measured values of Pmax, α, Ik, qP and NPQ were not found to be significantly different as a function of water velocity. The values of Pmax, α, Ik, and qP were not found to be significantly different as a function of the amount of time spent in a given hydrodynamic regime. Values of non-photochemical quenching were found to be significantly different as a function of the amount of time spent in a given flow regime (P value <0.005). There was no significant interaction between velocity and time.

64

Time NPQ Sample Size

Mean SE N 1 0.075 0.012 17 2 0.089 0.011 18 3 0.069 0.007 14 4 0.101 0.008 18 5 0.103 0.007 16 6 0.115 0.014 17 7 0.084 0.013 13

Table 3. Values of NPQ at each measurement hour. Measurements were taken on samples in a laboratory flume. NPQ values are significantly different as a function of time immersed in flow. Values of NPQ at measurement intervals of 3 and seven hours were significantly different than at the other five measurement times.

65

Flow Speed Flow Speed High Low

Pvalue

Mean Mean Velocity time Velocity x timePmax

(µmol electron m2s-1) 4.7

(0.278) 3.5

(0.201) <0.05 0.26 0.27

α (µmol electron m2s-1)

0.168 (0.00755)

0.141 (0.00904)

0.98 0.17 0.36

Ik (µmol quanta m2s-1)

27.8 (1.150)

27.7 (1.426)

0.97 0.67 0.14

qP 1.967 (0.0595)

1.772 (0.0617)

0.09 0.17 1.12

NPQ 0.096 (0.00716)

0.090 (0.00586)

0.32 <0.002 0.83

Table 4. Values of Pmax, α, Ik, qP, and NPQ measured on C. racemosa over five hours in a laboratory flume. Samples were collected from Tampa Bay, and measurements were taken while the samples were immersed in a given water flow regime in a laboratory flume. Measurements were taken once every hour for seven hours. Measured values of Pmax for times three through seven were found to be significantly different as a function of water velocity (P value <0.05). There was a higher electron transport rate in samples experiencing a faster hydrodynamic regime. Values of α, Ik, qP and NPQ at times three through seven in fast and slow hydrodynamic regimes were shown to not be significantly different. Measured values of NPQ were shown to be significantly different as a function of the amount of time spent in a given hydrodynamic regime (P value = 0.002). Values of Pmax, α, Ik and qP were not significantly different in at different measuring periods. There was no interaction of time and velocity.

66

Time NPQ Sample Size

Mean SE N 3 0.069 0.007 14 4 0.101 0.008 18 5 0.103 0.007 16 6 0.115 0.014 17 7 0.084 0.013 13

Table 5. Values of NPQ after 2 hours of immersion. PSII kinematic parameters were measured on samples collected from Tampa Bay were measured once an hour for seven hours. Values of NPQ are significantly different as a function of time immersed in flow (P value = 0.002).

67

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