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Adaptation and acclimation of populations of Ludwigia repens to growth in high- and lower-CO2 springs

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Title:
Adaptation and acclimation of populations of Ludwigia repens to growth in high- and lower-CO2 springs
Creator:
Lytle, Steven Todd ( Dissertant )
Bowes, George E. ( Thesis advisor )
Allen, Hartwell ( Reviewer )
Fox, Alison ( Reviewer )
Mulkey, Stephen ( Reviewer )
Harmon, Alic ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
2003
Language:
English

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Subjects / Keywords:
Carbon ( jstor )
Carbon dioxide ( jstor )
Cells ( jstor )
Leaves ( jstor )
Photosynthesis ( jstor )
Plant growth ( jstor )
Plant roots ( jstor )
Plants ( jstor )
Population growth ( jstor )
Species ( jstor )
Botany thesis, Ph.D
Dissertations, Academic -- UF -- Botany

Notes

Abstract:
Characteristics of an amphibious plant, Ludwigia repens, growing in high- and lower-CO2 natural springs were examined to test the hypothesis that the populations are genetically adapted to their respective CO2 environments. The two springs exhibited stable temperature, pH, and inorganic carbon concentrations over at least a 55-year period, and similar characteristics except that the high-CO2 spring had greater alkalinity, inorganic carbon, P, and NO3- concentrations, and lower pH than the lower-CO2 spring. Plants from the high-CO2 spring had terrestrial-like anatomy, with leaves possessing a cuticle and stomates, and stems having a cambium. Aerial leaves had a better-developed palisade and 168 and 287% greater starch grain and stomatal density, respectively, than submersed leaves. The two populations showed genetic divergence; with 11% genetic distance and 69% fragment diversity. The high-CO2 population reproduced sexually and asexually, but the lower-CO2 population reproduced only asexually. Submersed leaf photosynthesis rates of both populations were 31% inhibited by atmospheric [O2], indicating C3-like photosynthesis, and they did not use HCO3-. Photosynthesis rates and Rubisco activities were greater for the high- than the lower-CO2 plants. High-CO2 plants had twice the number of axillary shoots as lower-CO2 plants, whereas lower-CO2 plants had greater weight, density, leaf area and number of leaves. Shoots from both populations were grown submersed and emergent in a common garden experiment at different [CO2]. Relative growth rates (dry weight and leaf area) and number of axillary shoots responded more to elevated-CO2 in plants from the high- than the lower-CO2 population, showing that the populations are adapted to the spring [CO2]. Plants from both populations showed greater RGR, stem length, and number of axillary shoots in elevated CO2 when emergent. At ambient [CO2], neither population grew when submersed, and high-CO2 plants senesced even after emergence. This is the first CO2 spring adaptation study to demonstrate a genetic difference between populations growing in differing [CO2] and to use aquatic plants in a common garden experiment. It shows that plants may be adapted to their natural growth [CO2] and emergent plants will likely respond more than submersed plants as atmospheric CO2 rises.
Subject:
adaptation, aquatic, co2, emergent, ludwigia, photosynthesis, springs, submersed
General Note:
Title from title page of source document.
General Note:
Includes vita.
Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
General Note:
Text (Electronic thesis) in PDF format.

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University of Florida
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University of Florida
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Copyright Lytle, Steven Todd. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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9/9/1999

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Full Text
ADAPTATION AND ACCLIMATION OF POPULATIONS OF LUDWIGIA REPENS
TO GROWTH IN HIGH- AND LOWER-CO2 SPRINGS
By
STEVEN TODD LYTLE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003




This dissertation is dedicated to my wife, Stephanie Sullivan Lytle.




ACKNOWLEDGMENTS
I would like to acknowledge the members of my supervisory committee, Drs. George Bowes, Hartwell Allen, Alison Fox, Stephen Mulkey, and Alice Harmon, for contributing their time and effort in providing constructive criticism and advice throughout my degree program. I am especially indebted to Dr. George Bowes, for showing me what is required of a researcher at a large university, from the hypothesis to the final paper stage. Although not on my committee, Drs. Terry Lucansky and Karou Kitajima selflessly supplied valuable behind-the-scenes advice on many topics as well as supplying lab space and equipment.
I thank Drs. Julia Reiskind and Srinath Rao, and Gonzalo Estavillo for supplying daily motivation, generosity, and good humor in my work environment. I express gratitude to Drs. Mark Whitten and Matt Gitzendanner for their assistance with genetic fingerprinting, the staff at Fanning Springs State Recreation Area and Rainbow Springs State Park for support in research collections, John McKay for aid in flower and seed studies, and Sarah Bouchard for help doing the fiber analyses. I thank Dave Nolletti for performing the C: N analysis, Nate Bazinet, Sandra Diaz, Mike Durham, and Matt Young for their assistance in maintaining plant colonies, and Andy Freenock and Wayne Wynn for their engineering advice.
I would like to praise my father, Brian Lytle, for instilling the desire to pursue
higher education to its fullest, and my mother, Linda Lytle, for all her support along the way. My final acknowledgement is the most important; my wife Stephanie provided iii




endless encouragement and patience throughout the pursuit of this degree and I share this achievement with her.
iv




TABLE OF CONTENTS
pM.ge
ACKNOWLEDGMENTS ...................................................................... iii
LIST OF TABLES .............................................................................. vii
LIST OF FIGURES ............................................................................ viii
ABSTRACT ..................................................................................... x
CHAPTER
1 TERRESTRIAL AND AQUATIC PLANT RESPONSES TO ELEVATED CO2 1..
Rising Atmospheric CO2 ..................................................................1.
Terrestrial Plant Responses to Elevated CO2............................................1.
Aquatic Plant Biology and Responses to Elevated-CO2 ................................ 24
L udw igia R epens .....................................................36
Summary Statement ........................................................................ 37
2 ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS .............. 40
Intro du ction . . . . . . . . . . . . . . . . . . . . . . . . . . . ..4 0
Materials and Methods..................................................................... 43
Results ..................................................................................... 44
Discussion................................................................................. 47
3 CHARACTERISTICS OF SUBMERSED LUIDWIGIA REPENS PLANTS
FROM TWO POPULATIONS GROWING TN SPRINGS WITH DIFFERING
CO2 CONCENTRATIONS .............................................60
Introduction ................................................................................ 60
Materials and Methods..................................................................... 64
Results ..................................................................................... 71
Discussion .................................................................................. 78
4 THlE EFFECT OF CO2 AND EMERGENCE ON THlE GROWTH OF
LUDWIGIA REPENS PLANTS FROM HIGH AND LOWER-CO2 SPRING
POPULATIONS............................................................................ 95
Introduction ................................................................................ 95
v




M aterials and M ethods ............................................................................................. 100
R e su lts ....................................................................................................................... 1 0 4
D isc u ssio n ................................................................................................................. 1 0 9
5 CON CLU D IN G REM ARK S .................................................................................... 124
LIST OF REFEREN CES ................................................................................................. 127
BIOGRAPH ICAL SKETCH ........................................................................................... 145
vi




LIST OF TABLES
Table
2-1 Characteristics of fully-expanded aerial and submersed leaves of L. repens ..... 54 3-1 Water chemistry of the high- and lower-CO2 springs ........................................ 85
3-2 Weights, densities, and morphological characteristics of L. repens plants
growing in high- and low er-CO 2 springs ............................................................ 87
3-3 Individual leaf characteristics of L. repens plants growing in high- and
low er-C O 2 spring s ............................................................................................. . 88
3-4 Stem and root characteristics of L. repens plants growing in high- and
low er-C O 2 spring s ............................................................................................. . 90
3-5 Reproductive characteristics of L. repens plants collected from high- and lowerCO2 populations and grown in a growth chamber .............................................. 91
4-1 Leaf area (LA) and specific leaf area (SLA) for individual, fully-expanded
submersed leaves of L. repens plants taken from high- and lower-CO2
populations and grown at 350, 750, and 1500 gmol CO2 mo1-1 in SPAR
c h a m b e rs ................................................................................................................ 1 2 0
4-2 Number of axillary shoots per plant and shoot: root dry weight ratio for
L. repens plants taken from high- and lower-CO2 populations and grown at
350, 750, and 1500 gmol CO2 mol1 in SPAR chambers ....................................... 122
vii




LIST OF FIGURES
Figure p
1-2 Acclimated and non-acclimated assimilation/intercellular CO2 (A/Ci) curves
for a typical C3 plant at high light and 21% 02 .................................................... 39
2-1 Ludwigia repens growing emergent in Fanning Springs, Florida ....................... 52
2-2 Micrograph cross-sections of aerial (A) and submersed (B) L. repens leaves ......... 53
2-3 Micrograph cross-section of the major vascular bundle of a submersed
L rep en s leaf .................................................................................................... . 5 5
2-4 Micrograph cross-sections of the epidermis and cortex of aerial (A) and
subm ersed (B ) L. repens stem s .......................................................................... 56
2-5 Micrograph cross-sections of the inner cortex, vascular cylinder, and pith of
aerial (A) and submersed (B) L. repens stems ................................................... 57
2-6 Micrograph cross-sections of submersed water (A), and hydrosoil (B) L. repens
r o o t s ... ..... ................................................................................................................ 5 8
2-7 Micrograph cross-section of the endodermis, pericycle, and stele of a hydrosoil
L rep en s ro ot .................................................................................................... . 5 9
3-2 Leaf cross sections for L. repens plants growing in (a) high- and (b) lower-CO2
sp rin g s ..... ................................................................................................................ 8 9
3-3 Oxygen inhibition of net photosynthesis rates on a leaf area basis for L. repens
plants growing in high- and lower-CO2 springs ................................................. 92
3-4 Net photosynthesis rates on a leaf area basis for L. repens plants growing
in high- and lower-CO2 springs to test for bicarbonate use ................................ 93
3-5 Net photosynthesis rates and Rubisco activities on a leaf area basis for
submersed leaves of L. repens plants collected from high-CO2 and
lo w er-C O 2 sp rin g s .................................................................................................... 9 4
4-1 Increase in dry weight per plant (% of initial) over time for L. repens plants
taken from high- (filled symbols) and lower-CO2 (open symbols) populations
and grown at 375, 750, and 1500 tmol CO2 mo1-1 in SPAR chambers ................. 115
viii




4-2 Relative growth rates (RGR) for L. repens plants taken from high- and
lower-CO2 populations and grown at 375, 750, and 1500 tmol CO2 mo11 in
S P A R ch am b ers ...................................................................................................... 1 16
4-3 Ludwigia repens plants from the high-CO2 population after 79-d growth at 375,
750, and 1500 tmol CO2 mol1 in SPAR chambers ............................................... 117
4-4 Increase in leaf area per plant (% of initial) over time for L. repens plants taken
from high- (filled symbols) and lower-CO2 (open symbols) populations and
grown at 375, 750, and 1500 tmol CO2 mol1 in SPAR chambers ........................ 118
4-5 Leaf area relative growth rate (LARGR) for L. repens plants taken from highand lower-CO2 populations and grown at 375, 750, and 1500 tmol CO2 mol1
in S P A R ch am b ers ................................................................................................ 1 19
4-6 Total (main and axillary) stem length per plant collapsed across the high- and
lower-CO2 populations for L. repens plants grown at 350, 750, and 1500 tmol
C O 2 m ol1 in SP A R cham bers ................................................................................ 12 1
4-7 Final (day 79) C: N ratios on a dry weight basis for organs of L. repens plants
taken from high- and lower-CO2 populations and grown at different [C02] in
S P A R ch am b ers ...................................................................................................... 12 3
ix




Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
ADAPTATION AND ACCLIMATION OF POPULATIONS OF LUDWIGIA REPENS,
TO GROWTH IN HIGH- AND LOWER-CO2 SPRINGS By
Steven Todd Lytle
August 2003
Chair: Dr. George Bowes
Major Department: Botany
Characteristics of an amphibious plant, Ludwigia repens, growing in high- and
lower-CO2 natural springs were examined to test the hypothesis that the populations are genetically adapted to their respective CO2 environments. The two springs exhibited stable temperature, pH, and inorganic carbon concentrations over at least a 55-year period, and similar characteristics except that the high-CO2 spring had greater alkalinity, inorganic carbon, P, and N03- concentrations, and lower pH than the lower-CO2 spring.
Plants from the high-CO2 spring had terrestrial-like anatomy, with leaves
possessing a cuticle and stomates, and stems having a cambium. Aerial leaves had a better-developed palisade and 168 and 287% greater starch grain and stomatal density, respectively, than submersed leaves.
The two populations showed genetic divergence; with 11% genetic distance and 69% fragment diversity. The high-CO2 population reproduced sexually and asexually, but the lower-CO2 population reproduced only asexually. Submersed leaf photosynthesis
x




rates of both populations were 31% inhibited by atmospheric [02], indicating C3-like photosynthesis, and they did not use HC03-. Photosynthesis rates and Rubisco activities were greater for the high- than the lower-CO2 plants. High-CO2 plants had twice the number of axillary shoots as lower-CO2 plants, whereas lower-CO2 plants had greater weight, density, leaf area and number of leaves.
Shoots from both populations were grown submersed and emergent in a common garden experiment at different [C02]. Relative growth rates (dry weight and leaf area) and number of axillary shoots responded more to elevated-CO2 in plants from the highthan the lower-CO2 population, showing that the populations are adapted to the spring [C02]. Plants from both populations showed greater RGR, stem length, and number of axillary shoots in elevated CO2 when emergent. At ambient [C02], neither population grew when submersed, and high-CO2 plants senesced even after emergence.
This is the first CO2 spring adaptation study to demonstrate a genetic difference between populations growing in differing [C02] and to use aquatic plants in a common garden experiment. It shows that plants may be adapted to their natural growth [C02] and emergent plants will likely respond more than submersed plants as atmospheric CO2 rises.
xi




CHAPTER 1
TERRESTRIAL AND AQUATIC PLANT RESPONSES TO ELEVATED CO2 Rising Atmospheric CO2
Fluctuations in the [C02] of the Earth's atmosphere have occurred over geologic time. The atmosphere 420 million years ago may have contained over 4,000 gmol CO2 mo1-1, but the last 160,000 years have seen glacial lows of 180 gmol CO2 mo1-1 and interglacial highs of only 250-300 gmol CO2 mol1- (Post et al. 1990). Atmospheric [C02] recorded at Mauna Loa, Hawaii, since the 1950s indicate a steady increase in C02, with our current [C02] estimated at 370 gmol mol-1 (Keeling & Whorf 2002). This value is expected to double in this century as anthropogenic sources of CO2 continue to increase.
Terrestrial Plant Responses to Elevated CO2 For the past 20-30 million years the atmosphere has had a relatively low [C02], thus plants have presumably adapted to a low-CO2 environment. However, within the last 150 years, [C02] has been gradually rising; therefore plants may be re-adapting to the higher values (Bowes 1996). Rising CO2 has probably already enhanced the photosynthesis, water use efficiency, and growth of many plants, especially those that perform C3 photosynthesis (Amthor 1995; Bowes 1996; Drake et al. 1996a). The trend with the current CO2 increase is that it is more rapid than in past periods so adaptation may lag behind. How plants adapt to rising [C02] and other global climate changes will ultimately impact primary productivity, global gas exchange patterns, and vegetation boundaries of the world (Cook et al. 1997).




2
The primary reason that rising [CO2] affects the physiology of plants is that CO2 is the activator and substrate for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). Rubisco functions as a carboxylase of ribulose-1,5bisphosphate (RuBP) using CO2 in the photosynthetic carbon reduction (PCR) cycle, and as an oxygenase of RuBP with 02 in the photosynthetic carbon oxidation (PCO) cycle (i.e. photorespiration). These gases compete for the active sites of Rubisco. Operation of the PCO cycle indirectly reduces photosynthesis since both processes require RuBP as a substrate and photorespiration releases previously fixed carbon. As [CO2] rises, Rubisco's carboxylase activity increases at the expense of its oxygenase activity, and more CO2 molecules are assimilated in photosynthesis.
Responses of plants to elevated-[CO2] are partly dependent on their photosynthesis category [C3, C4, or Crassulacean acid metabolism (CAM)]. Species in the C3 category operate only the PCR cycle, whereas C4 and CAM species also utilize a C4 acid cycle to concentrate CO2 at the site of Rubisco. The C4 and CAM CO2 concentrating mechanisms (CCM) shift Rubisco activity away from oxygenation toward its carboxylation function. In effect, [CO2] is already elevated at the carboxylation site in C4 and CAM plants, lessening the effect that rising [CO2] has on plants in these categories.
Approximately 95% of known plant species are in the C3 category with examples including soybean, wheat, and most woody plants. Under present atmospheric conditions, Rubisco of C3 plants functions below its Km, meaning its active sites are not saturated with CO2, and thus enables oxygenation (i.e. photorespiration) to occur. Photorespiration reduces photosynthesis of C3 species by about 35% at 25C, with higher temperatures increasing this inhibition (Jordan & Ogren 1984; Bowes 1996).




3
Stimulation of carboxylation at elevated-[C02] is usually greatest for C3 species. Bowes (1996) estimated that doubling atmospheric [C02] should more than halve photorespiration by C3 plants. Initially, the increase in photosynthesis is quite large, but usually there is a decline or acclimation that occurs when other resources limit photosynthesis (Sage et a!. 1989). Regardless, acclimation is rarely great enough to completely negate the positive stimulation Of C3 photosynthesis that occurs at elevatedCO2 (Drake et a!. 1996a). A survey Of C3 crops by Cure and Acock (1986) showed that doubling [C02] increased photosynthesis by 5200 but this enhancement dropped to 29%o following acclimation.
Acclimation to C02
Acclimation is a phenotypic adjustment that occurs in an individual organism as a response to declining performance following exposure to unfavorable levels of one or more environmental factors (Schmid et a!. 1996; Lambers et a!. 1998). This adjustment involves a redistribution of resources toward the most limiting processes, resulting in a re-optimization for growth and reproduction under the prevailing conditions. It is a relatively short-term phenomenon that occurs at the level of gene expression or enzyme regulation and may be contrasted with adaptation, a process that requires multiple generations to cause changes in the gene pool of a population. The shortest acclimation responses include changes in the capacity of enzyme function and occur in a timeframe from minutes to hours. Lengthier acclimation responses invoke gene effectors that modify gene expression to affect the type and amount of proteins produced. Changes in gene effectors re-establish a balance within the photosynthetic apparatus, rather than just changing the in vivo turnover rate (Sage 1994) and take hours to months to occur.




4
Photosynthesis response to elevated-CO2
Photosynthetic acclimation to elevated-CO2 can result in modifications to plant anatomy, morphology, physiology and/or biochemistry (Bowes 1991). Photosynthesis can be measured directly by quantifying CO2 consumption and/or 02 evolution, or can be indirectly assessed by analyzing the activity and amount of individual enzymes (e.g., Rubisco) or the amount of metabolites (e.g., RuBP) involved in photosynthesis. The elevated-CO2 acclimation response "down-regulates" photosynthesis.
Photosynthetic acclimation can be assessed by comparing the net CO2 assimilation rate as a function of intercellular CO2 (A/Ci curves) of plants before and after growth at elevated [C02]. Figure 1-2 shows an acclimated and non-acclimated CO2 A/Ci curves for a typical C3 terrestrial plant at high light and 21% 02 (Sage 1994). The shape of response of A to Ci is usually that of a rectangular hyperbola and can be divided into two phases: an initial linear phase that is limited by the amount of active Rubisco present (which provides a measure of carboxylation efficiency or Rubisco capacity); and a phase that is limited by the rate at which RuBP can be regenerated from triose phosphates (e.g., ATP) in the PCR cycle (Sage 1994; Bowes 1996). Triose phosphate is limited by non-cyclic electron transport and by the rate that inorganic phosphate (Pi) is recycled. In this phase, A continues to increase as CO2 rises because photorespiration is reduced and a greater proportion of RuBP is used in carboxylation. However, as the photorespiration effect eventually subsides, the curve levels off at the maximum assimilation rate (Amax), which is an indicator of photosynthetic or Pi regeneration capacity.
Changes in the shape of the curve are indicative of a re-allocation of resources within the plant. A key premise of the A/Ci acclimation function is that the photosynthetic biochemistry in the steady state is regulated so the rate at which Rubisco




5
consumes RuBP equals the rate at which RuBP is regenerated (Sage et al. 1990). The slope of the initial linear portion of the curve can decrease and/or there can be an increase in Amax. The decrease in the slope of an A/Ci curve is an indication that the photosynthetic efficiency has decreased. At elevated CO2, the plant consumes all of the RuBP available so Rubisco content may be reduced to enhance resource (e.g., nitrogen) use efficiency. Long and Drake (1992) calculated that Rubisco could be down-regulated by 35% in a C3 leaf when [CO2] is doubled before Rubisco would limit photosynthesis. The increase in Amax is due to an increase in Pi regeneration in the PCR reactions that occurs at elevated-CO2, which leads to greater RuBP concentrations, and is an indicator of an increase in photosynthetic capacity.
Sage (1994) conducted a survey of 34 published and unpublished A/Ci studies, and as expected, the most common response was reduced A at low to moderate Ci, and increased A at high Ci. No pattern existed between the type of A/Ci response and the life form or the ecological requirements of the species studied.
Reduction in Rubisco amount, activity, and gene expression are all good indicators that a plant is acclimating to elevated-CO2. The activity and amount of Rubisco decreased on a leaf area (LA) basis when Oryza sativa was exposed to a range of [CO2] (Rowland-Bamford et al. 1991). Rubisco content of Lycopersicon esculentum decreased by 50% when plants were grown at 1000 pmol CO2 mo1-1 (Besford et al. 1990). As with the gas exchange data, Rubisco acclimation exceptions do occur in the literature. Although Chenopodium album and Brassica oleracea had lower Rubisco amount at 9001000 pmol CO2 mo1-1, no differences were present for Solanum tuberosum, Solanum melongena, and Phaseolus vulgaris (Sage et al. 1989). Glycine max Rubisco activity and




6
amount showed minimal response to manipulation of [C02] (Campbell et al. 1988). Hydroponically-grown Lolium temulentum, showed no change in maximum Rubisco activity, but did have greater Rubisco protein at double-[C02] (Lewis et al. 1999).
Decreased expression of mRNA genes that encode Rubisco can also serve as an
indicator of acclimation to enriched-CO2. Long-term exposure of Arabidopsis thaliana to high-CO2 caused the mRNA for the large and the small subunits of Rubisco to decrease by 35-40 and 60%, respectively, with a subsequent reduction in the expression of Rubisco protein (Cheng et al. 1998). The correlation between mRNA transcripts and protein levels of Rubisco is not always tightly coupled. Gesch et al. (1998) observed a decrease in the small subunit transcript within 24 hours after switching 0. sativa plants from 350 to 700 tmol CO2 mol-1; however, Rubisco amount and total activity showed no response, even eight days after the switch. The decrease in Rubisco protein found in 12 out of 16 crop species grown at 1000 mol CO2 mol1 showed no specific association with changes in transcript levels for the Rubisco small subunit (Moore et al. 1998). Causes of the acclimation response
A variety of hypotheses for what causes photosynthetic acclimation have been
proposed, and all are related to either the photosynthetic machinery or other physiological processes not being able to keep pace with the increasing C availability. Acclimation can occur due to reallocation of resources from non-limiting C acquisition processes to those that may be more limiting, such as light harvesting, electron transport, and carbohydrate handling (Sage et al. 1987; Bowes 1991; Sage 1994). Acclimation may occur because the additional C cannot be transported to sinks or storage rapidly enough. Therefore, the response should be to either down-regulate photosynthesis or to up-regulate carbohydrate metabolism and N assimilation.




7
Explanations for the effect that carbohydrates have on down-regulation of photosynthesis have focused on the increased flux as well as accumulation of carbohydrates. Current research in this area points toward the increased flux of carbohydrates as being the trigger for the signal that leads to the down-regulation of photosynthesis. Specifically, hexokinase has been identified as being important in the acclimation signaling process (Jang & Sheen, 1997; Moore et al. 1998; Moore et al. 2003). Hexose sugars generated in photosynthesis are phosphorylated by hexokinase, so that its activity is increased as sugar flux is increased at elevated-CO2. A plant's capacity to export photosynthate and its sink size may be insufficient at elevated [C02] so that increasing carbohydrate pools lead to feedback inhibition of photosynthesis (Stitt 1991; Sheen 1994). Also, extreme cases of starch accumulation may distort chloroplasts (Madsen 1968; Woodward et al. 1991).
Empirical data show a variety of non-structural carbohydrates may be involved in the down-regulation of photosynthesis. Elevated CO2 (700 gmol CO2 mo1-) enhanced non-structural carbohydrate accumulation in leaves of five Trticum aestivum cultivars showing photosynthetic down-regulation (Barnes et al. 1995). Both Flaveria and Panicum species showed photosynthetic acclimation at 700 gmol CO2 mol1, but the species differed in carbohydrate accumulation; Flaveria accumulated high levels of starch, while Panicum built up stores of sugars (Leonardos & Grodzinski 2000). However, others have deduced that higher total non-structural carbohydrate and starch content is not related with the photosynthetic down-regulation (Moore et al. 1998; Van der Kooij et al. 1999).




8
The C and N metabolism pathways must be coordinated precisely to achieve an
appropriate balance between supply and demand so that a plant can build the appropriate macromolecules. Carbon and N metabolism compete for ATP, reducing power, and C skeletons. Elevated CO2 normally modifies the C: N balance of plant tissues (especially leaves) toward C. Oryza sativa, Gossypium hirsutum, Zea mays, and T aestivum grown at higher-[C02] had lower leaf [N] (Rowland-Bamford et al. 1991; Wong 1979; Hocking & Meyer 1991a, b). Exceptions do exist, with C. album, S. tuberosum, S. melongena, and B. oleracea indicating no difference in leaf [N] at enriched-CO2 (Sage et al. 1989).
The extremity of the elevated-CO2 acclimation effect is partly dependent on whether there is sufficient N available in the plant, especially if other resources are adequate. Gossypium hirsutum, Z. mays, and hydroponic 0. sativa showed greater photosynthetic down-regulation when supplied with lower nitrogen in elevated CO2 (Wong 1979; Nakano et al. 1997). To avoid the negative effects of the C: N ratio shifting too far toward C, the acclimation response should be to favor N assimilation, export, and re-mobilization over C assimilation. The initial regulation response should be to lower the activity of Rubisco, which then might be followed by a reduction in the amount of Rubisco (Bowes 1996). In low-[N] treatments grown at high-C02, G. hirsutum and Z mays both had lower Rubisco activity (Wong 1979), while tobacco had decreased Rubisco content (Walch-Liu et al. 2001).
Dark Respiration
The effect that elevated [C02] has on dark respiration has received less attention than photosynthesis, even though these processes are closely linked. Half or more of the C assimilated during photosynthesis may be lost as CO2 during subsequent respiration (Allen & Amthor 1995). Respiration serves as the primary means for supplying a plant's




9
cells with C skeletons (intermediates), usable energy (ATP), and reductant (NADH) required for growth, maintenance, transport, uptake, and nutrient assimilation processes.
It is essential to distinguish between direct and indirect effects of CO2 on
respiration. Direct effects are indicated by immediate responses that can be rapidly relieved by reducing [C02] to its original level (Amthor 1991; Bowes 1996). Indirect effects occur after growth in elevated [C02] for extended periods (Amthor 1991), and are acclimation effects that cannot be reversed quickly.
Direct effects of elevated-CO2 on respiration generally involve a decrease in
respiratory CO2 release (Drake et al. 1999). A survey across a variety of species and tissues (leaves, roots, and shoots) by Drake et al. (1996b) concluded that specific respiration was reduced by about 20% at twice ambient-CO2. Doubling the [C02] in which G. max cotyledons were grown decreased their mitochondrial 02 uptake (Gonzdilez-Meler et al. 1996), and inhibition of CO2 evolution by Rumex crispus leaves and Oryza sativa canopy was observed at high-[C02] (Amthor et al. 1992; Baker et al. 2000). However respiration does not always decrease at elevated-C02, and there are recent reports that the effects can be attributed to experimental artifacts (Jahnke 2001; Jahnke & Krewitt 2002).
The mechanism responsible for the direct inhibition of respiration has not been clearly defined (Allen & Amthor 1995), although it may be related to a decrease in the activity of two key enzymes of the mitochondrial electron transport chain, cytochrome c oxidase and succinate dehydrogenase (Drake et al. 1996b). The activities of these enzymes were inhibited at elevated [C02] for mitochondria isolated from G. max cotyledons (Gonzdilez-Meler et al. 1996).




10
Indirect respiration effects are dependent on the impact CO2 has on physiological mechanisms that affect growth and maintenance. Determining the causes of indirect effects on respiration can be complicated since many factors are involved. Respiration effects are mediated through the influence of CO2 on processes that supply substrates of respiration and use respiration intermediates and end products (Amthor 1994). Respiration slows when respiratory products accumulate in cells, and increases when the products are consumed, through a series of feedback mechanisms (Amthor 1995).
Several authors indicate that rising CO2 indirectly reduces respiration through the changes it causes in plant tissue composition (Amthor 1995; Drake et al. 1996a). As CO2 rises, the C: N ratio of plants usually shifts toward C. A decrease in N (and therefore, protein) concentration results in a decrease in specific maintenance respiration since tissues lower in N are less costly to synthesize and maintain (Amthor 1995; Drake et al. 1996a). Respiration rates were decreased at elevated [CO2] in stems of Scirpus olneyi and leaves of Lindera benzoin, while Spartinapatens leaves were unaffected (Azc6nBieto et al. 1994). Greater specific respiration rates in subambient-[CO2] treatments were associated with higher plant tissue [N] for 0. sativa (Baker et al. 1992). Growth and morphology of C3 species
Generally, growth rates of plants are stimulated at elevated [CO2]. The stimulation may be due to greater tillering or branching, leaf area (due to greater area per leaf and more leaves per plant), and leaf thickness which all result in greater total plant photosynthesis (Acock & Allen 1985; Smith et al. 1987; Allen 1990). Besides providing more of a limiting resource, additional CO2 has the potential to improve the use of other resources (Bowes 1993). Nitrogen use efficiency (NUE) and water use efficiency (WUE)




11
may both increase at elevated CO2. Therefore, even when other resources are low and limit photosynthesis, the additional CO2 may still enhance growth (Bowes 1996).
Growth and photosynthetic responses to elevated [C02] are partly dependent on the other resources to which the plant is exposed. Kimball et al. (1990) showed that growth of cultivated plants in well-tilled soil with abundant nutrients will respond positively to rising-CO2 over their entire life span. There is much less research published on how natural plant communities respond to enriched-C02, but increased competition for other resources may limit growth enhancement (Fordham et al. 1997a).
Most researchers have concluded that growth of C3 species increases with CO2
enrichment. Yields of a combination of crop and non-crop C3 species were enhanced by an average of 33% when ambient [C02] was doubled (Kimball 1983). A survey by Poorter (1993) reported that average dry weight for vegetative plants of both cultivated and wild C3 species increased by 41%, with wild species showing a 35% increase. Examples in the literature of CO2 not stimulating the growth of C3 plant species are rare, however, the annual C3 species, Cardamin hursuta, Poa annua, and Spergula arvensis showed no response to CO2 (Leishman et al. 1999).
Morphological changes caused by elevated [C02] show common trends in C3
species, including initiation of new organ growth, a shift in the partitioning of biomass toward roots, greater leaf area, and a decrease in specific leaf area (SLA). Increased branching and tillering in plants grown at enriched-CO2 is common since increases in photosynthesis lead to accumulation of carbohydrates that are either used in new growth or stored. The grasses Bromus tectorum, Oryzopsis hymenoides, and Agropyron smithii all had more basal stems when grown at high-CO2 (Smith et al. 1987). Whether grown in




12
a phytotron or hydroponically, T aestivum cultivars grown at elevated CO2 showed a stimulated tillering response (Barnes et al. 1995; Monje & Bugbee 1998). Not all experiments verify the increase in tillering though; Leishman et al. (1999) grew C. hursuta, P. annua, S. vulgaris, S. arvensis at 550 pmol CO2 mo1-1 and found no tiller effects.
Since leaves and shoots provide the apparatus for photosynthesis, which is likely saturated at elevated-CO2, a plant might benefit by investing more in root tissues to increase access to resources other than CO2. The root: shoot ratio of C3 plants generally increases at enriched-CO2. Two cultivars of Oryza sativa had their root: shoot ratios increased by 24 and 92% when they developed under high-CO2 (Ziska & Teramura 1992).
Leaf area of C3 plants is often enhanced when CO2 is increased, although the effect is not usually a major one. The grasses B. tectorum, O. hymenoides, and A. smithii all had greater LA when grown at elevated-CO2 (Smith et al. 1987). Tobacco had more and larger leaves at 700 pmol CO2 mo1-1 than at ambient-CO2 (Backhausen & Scheibe 1999). In contrast, C. hursuta, P. annua, S. arvensis, and L. esculentum showed no LA enhancement in response to increased CO2 (Leishman et al. 1999; Besford et al. 1990).
An aspect of leaf growth that is more sensitive to CO2 is SLA. Specific leaf area on an individual and total leaf basis typically decreases because leaves get thicker at enriched-CO2. Ten Acacia tree species grown at high-CO2 had lower SLA and this was attributed to increased thickness of the leaves (Atkin et al. 1999). Sage et al. (1989) noted that C. album and B. oleracea had lower SLA in elevated-CO2 treatments, while S. tuberosum, S. melongena, and P. vulgaris did not. Finally, the SLA response of three




13
Oryza sativa cultivars to high-CO2 was the opposite expected; SLA increased (RowlandBamford et al. 1991; Ziska & Teramura 1992). Greater leaf thickness is due to the rapid development of leaves during lamina differentiation and cell enlargement (Acock & Allen 1985). Leaf thickness increased in Panicum tricanthum exposed to 900 pmol CO2 mol-1 (Tipping & Murray 1999) and G. max grown at 800 pmol CO2 mol-1 had 37% greater leaf thickness due to an increase in the number of palisade cells (Vu et al. 1989). Stomates
As atmospheric CO2 increases, more CO2 diffuses into leaves, therefore the degree of stomatal opening and the density of stomates should decrease (Woodward 1987; Bowes 1996). Plants normally show partial stomatal closure at higher than present atmospheric CO2 (Morison 1985), however, there are reports of enormous variation between species (Jarvis & Mansfield 1999). When exposed to high CO2, abaxial stomatal density decreased by 22% for P. tricanthum (C3) (Tipping & Murray 1999). Stomatal density increased in Oryza sativa plants as [CO2] was increased at intervals from 160 to 330 pmol CO2 mol-1, but then showed no further increase at intervals from 330 to 900 pmol CO2 mo11 (Rowland-Bamford et al. 1990).
A decrease in stomatal opening results in a decline in stomatal conductance. Stomatal conductance is often correlated with changes in photosynthesis, so that as photosynthesis is down-regulated, conductance also decreases (Drake et al. 1996a). Conductance is usually regulated to maintain Ci within narrow limits (Morison 1985). Using 41 observations covering 28 species, the average reduction in stomatal conductance due to a rise in CO2 was 20% (Drake et al. 1996a). All the grass species [B. tectorum (C3), O. hymenoides (C3) A. smithii (C3) E. orcuttiana (C4)] tested by Smith et




14
al. (1987) showed a decrease in stomatal conductance when grown at 680 gmol CO2 mol 1, rather than ambient-CO2.
Anatomy
Minimal research has been done to determine the effects of elevated CO2 on anatomical features of plants. Most studies thus far have tried to elucidate what role anatomy plays in the increased growth and photosynthetic responses when plants are grown at high-CO2. Changes in leaf anatomy may influence a plant's capacity to assimilate carbon, and thus be associated with the photosynthetic down-regulation that occurs at high-CO2 (Pritchard et al. 1999). The magnitude of anatomical changes hinges upon plant genetic plasticity, nutrient availability, temperature, and phenology (Pritchard et al. 1999).
Of all the plant organs, leaves exhibit the greatest structural plasticity in response to environmental conditions (Esau 1977). Although variable, leaves typically have greater rates of expansion and therefore produce larger leaves in high-CO2 environments. Greater leaf size can be reflected in lamina thickness, which is often correlated with changes in internal anatomy. No single process has been identified as being responsible for producing greater leaf size, however, cell expansion plays a greater role than enhanced cell division (Pritchard et al. 1999). Increased leaf growth may be due to increased cell expansion, resulting in larger cells (Populus clones, Radoglou & Jarvis 1990; Plantago media, Taylor et al. 1994; Sanguisorba minor, Lotus corniculatus, Anthyllis vulneraria, Ferris & Taylor 1994). Increased leaf expansion rate and final leaf size were correlated with greater epidermal cell size and number in Populus nigra andP. euramericana, while P. alba only had increased epidermal cell number (Ferris et al. 2001). Other researchers single out an increase in cell number as being the factor




15
contributing to increased leaf growth in high-CO2. Glycine max grown at 800 tmol CO2 mo1-1 had an increased number of palisade cells and leaf thickness (Vu et al. 1989).
Other studies have focused on the proportion of the different tissue layers produced at enriched-CO2. Mesophyll and vascular tissue cross-sectional areas and volumes typically increase, with the mesophyll becoming more clearly defined. Also at high-C02, leaves tend to have more dense tissues with smaller cells and less internal spaces. When grown at enriched-C02, the second trifoliate leaf of P. vulgaris had reduced epidermal and intercellular space volume and cell area (Bray & Reid 2002). Also, leaf thickness, palisade cell length and volume density of the spongy mesophyll and palisade mesophyll were greater for P. vulgaris plants grown at high versus ambient [C02]. Other studies also report increased mesophyll cross-sectional area in leaves (Populus trichocarpa, Radoglou & Jarvis 1990). In addition to a decrease in intercellular spaces, an extra layer of palisade cells was observed in G. max grown in high CO2 (Vu et al. 1989). However, reductions in mesophyll have also been reported (Pinusponderosa, Pushnik et al. 1995). Vascular tissue area increased in P. ponderosa and L. platyglosa leaves exposed to highCO2 (Pushnik et al. 1995; St. Omer & Horvath 1984).
Even though stems are important in mediating the flux of resources acquired above and below the ground, studies relating stem anatomical characteristics with rising CO2 is scarce. Elevated-CO2 increases stem growth primarily by stimulating cell division within shoot apices (Pritchard et al. 1999). Increased stem diameter has been reported for many species growing under elevated-CO2. Pinus radiata had greater stem diameter when grown at high CO2 (Conroy et al. 1990) and Sigurdsson et al. (2001) concluded that P. trichocarpa showed at increase in stem diameter at high-CO2 and nutrient availability.




16
Still Pushnik et a!. (1995) found no increase in secondary growth of P. ponderosa stems. A review by Pritchard et a!. (1999) showed that stem secondary growth normally increases elevated CO2.
Although roots often exhibit the greatest relative increase in biomass of all plant organs when grown in elevated C02, there are few studies on root anatomical responses (Pritchard et a!. 1999). Increase in girth of roots has been found in some experiments. There was a 2700 increase in root diameter in the root hair zone, a 230% rise in stele diameter, and a 280% escalation in cortex width in G. max grown in elevated CO2 (Rogers et a!. 1992). Pinus taeda also had larger root diameters in high-CO2 treatments (Larigauderie et a!. 1994). These results notwithstanding, St. Omer and Horvath (1984) found no difference in stele or tracheary element diameter of Layiaplayglossa roots grown at higher than ambient-CO2 levels.
C02 Adaptation
In contrast to acclimation, adaptation is a change in a population's gene pool
induced by an environmental change over multiple generations due to drift, migration, or selection. Research on long-term effects Of CO2 on plant adaptation is lacking. There is great urgency to understand the implications of our future CO2 atmosphere, but even for short-lived species, periods of ten or more years are often required for genetic shifts in the population (Miglietta et a!. 1993). Elevated-CO2 research, which is aimed at predicting responses expected to occur over decades to centuries, is often conducted over periods that are considerably shorter than may be required for full expression or equilibrium of the responses (Koch 1993). The general rule is the longer the experiment, the better, however, empirical studies of relatively short duration will continue to be used to support models that simulate processes over the longer time-scales of relevance.




17
Several long-term experiments have been initiated, but they are still in their infancy (Field et al. 1996). Different species and cultivars vary widely in their response to elevated C02, yet little is known about whether variation also occurs within natural populations (Curtis et al. 1994).
There are three main categories of elevated-CO2 adaptation research. In the first type, herbarium and fossil specimens are examined for differences that may be related to the [C02] in which they grew. In the second method, plants of the same species, but with different genotypes (often established cultivars), are exposed to similar environments and their responses to elevated [C02] are monitored. The third strategy utilizes natural settings (e.g., geothermal gas vents) in which high [C02] have prevailed over evolutionary time. Plants growing near natural CO2 sources may be adapted to elevated [C02] relative to populations of the same species growing outside the CO2 plumes (Sage 1994). A criticism of the natural experiments is that the researchers do not verify that genetic differences exist between the populations being compared, and the differences that show up may not reflect adaptation. Just because a species exhibits certain traits in a particular environment, does not mean that these traits are beneficial and resulted from natural selection.
Comparisons of herbarium and fossil specimens versus present-day species have generally shown a decrease in the number of stomates and stomatal density over time as [C02] has increased (Wagner et al. 1999). Using four collection dates over the last 3000 years, Beerling and Chaloner (1993) deduced that ancient leaf material of Olea europaea showed a decrease in stomatal density as CO2 increased. Parallel to an increase in CO2 from AD 1720 to present, there were decreases in stomatal and epidermal cell density for




18
14 species of trees, shrubs, and herbs collected and stored as dried herbarium specimens, while stomatal index was unchanged (Pefiuelas & Matamala 1990). However, there are also instances reported where CO2 does not influence stomatal characteristics. Korner (1988) looked at over 200 plant species and found no significant difference in overall stomatal density between 1918 and 1985.
Different cultivars of the same species often diverge in their CO2 response. When five cultivars of T aestivum were grown at elevated C02, net assimilation was enhanced by an average of 45% across cultivars, however, this enhanced rate was eventually downregulated by 15% in only two of the five cultivars (Barnes et al. 1995). Wulff and Alexander (1985) found differences in the germination and growth of progeny derived from five maternal families of Plantago lancelolata when grown at 675 gmol CO2 mo1-1. Some families responded much more to CO2 than others, suggesting that there may be genetic variation in response to CO2 during seed maturation and seedling development. Progeny from five families of wild radish, Raphanus raphanistrum, were grown at double [CO2] and showed stimulation in net assimilation rates, and flower and seed production across paternal families (Curtis et al. 1994). However, in three families there were no significant CO2 effects, while in one family lifetime fecundity increased by 50%. These genotype-specific effects altered fitness rankings of the five paternal families providing evidence for heritable responses to elevated CO2.
Little is known about variation across natural plant populations (Curtis et al. 1994). Perhaps the only situation in which terrestrial vegetation chronically and naturally experiences greatly increased ambient-CO2 levels is in areas close to natural discharges of CO2 from geological sources (Koch 1993). Geothermal gas vents such as springs,




19
geysers, and volcanoes emit C02, with the CO2 from natural spring vents approaching 99% purity (Cook et al. 1997; van Gardingen et al. 1997). The [C02] in the air around geothermal gas vents can be as high as 10,000 gmol CO2 mo1-1 (Miglietta & Raschi 1993). These high-CO2 environments allow researchers to examine plant responses over time-scales that are much greater than possible with human-designed experimental systems (Koch 1993). The environmental conditions may be assumed to have occurred for hundreds of years, with vegetation around these vents subjected to a [C02] gradient, with a decreasing concentration as the distance from the vents increases (Miglietta et al. 1993).
Research testing the effect that the elevated-CO2 has on plants growing in these environments has only been instituted for about a decade. The most positive aspect of this research is that the CO2 effects on the plants are long-term, therefore fully acclimated physiological responses, which may be adaptations, can be examined. Another significant characteristic is that the observations are made in a natural setting. Also, there is no need to set up expensive, large-scale Free Air CO2 Enrichment (FACE) experiments. A negative characteristic of CO2 vent experiments is that they cannot be replicated. Also, there is temporal and spatial heterogeneity of the [C02] in the air surrounding the vent due to diffusive, convective, and turbulent mixing. Additionally, when comparing plants growing in elevated-CO2 near the vents versus plants in nearby lower-CO2 sites, there is often diversity in the two environments other than [C02]. Heterogeneity of background environmental factors around a natural-CO2 source may complicate comparisons to ambient-CO2 sites and make it difficult to isolate CO2 effects on vegetation characteristics. Finally, phytotoxic gases are often present in these sites.




20
Most natural CO2 source experiments take place in spring environments in which toxic gases are not released and that have moderate temperatures as opposed to the high temperatures found near volcanoes and geysers. A natural spring is created when water is discharged as leakage or overflow from an aquifer through an opening in the ground. Carbon dioxide from the atmosphere and from plant and microbial respiration combines with water as it percolates through the ground to form carbonic acid (H2C03). Other acids from organic matter may also combine with the solution (Nordlie 1990). This H2C03 solution reacts with the bases of rocks and can dissolve tunnels deep into bedrock (Ferguson et a!. 1947). Limestone of calcium-enriched rock is especially solubilized by H2C03 to produce calcium bicarbonate (Ca(HCO3)2), thus increasing Ca 2 and HC03ions in the water (Wetzel 1983). Bicarbonate may then be converted to free CO2 depending on the pH of the water the spring effluent enters. Water confined in the dissolved cavities by overlying sediments creates a pressure head that cracks open the confining rock bed further down-slope, releasing it to the surface and creating springs (Nordlie 1990). Carbon dioxide and other gases are released steadily from mineral springs as gas-laden waters ascend and effervesce upon reaching atmospheric pressure (Koch 1993).
In its history, Florida was repeatedly inundated by marine water, thereby layering it with limestone produced as marine sediments were eventually covered by clay and sand (Nordlie 1990). Since the Florida aquifer is mainly composed of limestone it is easily dissolved by carbonic acid solution. The high solubility of limestone rock by natural waters is probably the most important factor responsible for the large number of springs (over 300) in Florida (Ferguson et a!. 1947; Stamm 1994). Surveys indicate that the




21
physical and chemical makeup of many of Florida's freshwater springs have changed little over a thirty-year period encompassing the 1940s to 1970s (Ferguson et a. 1947; Rosenau et a!. 1977).
There are two main types of experiments using elevated-CO2 springs. The first compares a population of plants growing near a high-CO2 vent with a population in the same ecosystem but further from the vent where [C02] is near ambient. These experiments attempt to uncover adaptation phenomena, but all experiments thus far have disregarded testing to determine if the populations differ genetically. Often the populations are in close proximity so genetic exchange is likely, therefore lessening the likelihood of genetic differences. Correlations between physiology and environment in the field provide a basis for interesting physiological hypotheses, but these hypotheses can rarely be tested without complementary approaches such as growth experiments or phylogenetic analyses. The second kind of test involves collecting individual seed (preferably) or seedlings from the high- and ambient-CO2 springs, and growing them in a common garden (Clausen et a!. 1940). Any differences in CO2 responses that arise between the populations are assumed to be due to adaptations. The problem is that the plants may differ due to initial non-genetic variability in their "mother material" created by growth in different environments.
Many experiments have been conducted in which plants growing near to and further from natural CO2 springs are compared, but there is still great uncertainty in drawing conclusions from the data. However, typical high-CO2 acclimation responses seem less prevalent in the terrestrial plant populations growing near the high-CO2 springs, thereby pointing to adaptation of these populations to high [C02]. Thus far it has




22
not yet been demonstrated that exposure to long-term elevated-CO2 from springs produces large differences in biomass (Miglietta & Raschi 1993; Miglietta et al. 1993; Korner & Miglietta 1994). The photosynthesis rate of Scirpus lacustris did not differ between high- and ambient-CO2 sites (Miglietta et al. 1993; Bettarini et al. 1997). Scirpus lacustris responded to growth in elevated-CO2 with a decrease in stomatal density but no effect on Ci: Ca ratio or N content (Bettarini et al. 1997). Photosynthesis was not reduced in Quercuspubescens growing closer to high-CO2 springs (van Gardingen et al. 1997), but this contradicted earlier work by Miglietta and Raschi (1993) in which the photosynthetic capacity of Q. pubescens leaves was down-regulated. Miglietta and Raschi (1993) also compared leaf anatomy of Q. pubescens, and found little evidence of acclimation in the high-CO2 population. Stomatal and epidermal cell numbers were the same at the two sites, but size of guard cells was reduced in leaves of plants grown in the enriched-CO2 atmosphere. Bettarini et al. (1998) concluded that long-term exposure to elevated-CO2 in the spring did not cause adaptive modification in stomatal number and distribution for 17 species.
Other experiments indicate that terrestrial plant populations growing near spring
vents are not fully able to utilize the high levels of CO2 and shows acclimation responses. When compared to an ambient population, Nardus stricta growing near a spring with 790 pmol CO2 mo1-1 showed reduced photosynthetic capacity, Rubisco content and activation state, and chlorophyll content (Cook et al. 1997). Spring plants also had lower leaf [N] and greater starch in some of the years sampled, both of which could have contributed to acclimation. Earlier senescence, greater leaf area index, and lower SLA were also traits of the plants growing in high-CO2. Leaf photosynthetic capacity, stomatal density, and




23
conductance were down-regulated in Phragmites australis plants growing nearest to spring vents (van Gardingen et al. 1997). Korner and Miglietta (1994) compared multiple grassland herbs and forest trees growing around a spring at 500-1000 tmol CO2 mo1-1 with others growing in ambient-CO2. Although they found no evidence that plants in the spring area grew larger or flowered earlier, TNC accumulation (especially starch) and N depletion occurred in most herbaceous and tree leaves growing under elevatedCO2.
There has been only one elevated-CO2 spring experiment that used an aquatic plant. Koch (1993) compared populations of Ludwigia uruguayensis, an emergent aquatic species, growing at various distances from a CO2 spring, but even this study focused on the aerial parts, rather than the underwater portions, of the plant. Ludwigia uruguayensis plants growing nearest to the spring showed no evidence of photosynthetic downregulation, even though more starch accumulated. Primarily as a result of the increased starch levels, there was also a decrease in SLA for plants growing closer to the vent.
True adaptation experiments involve collecting seed or small seedlings from highand ambient-CO2 sites with otherwise similar environmental conditions, and growing them under a common [C02] in what is known as a common garden experiment. By using only a portion of a plant or its offspring in these experiments the environmental effects are kept to a minimum while the genetic effects are maximized. A criticism of these studies is that there have been no genotypic analyses of the populations to go with the physiological comparisons. The populations must be genetically distinct to imply adaptation has occurred.




24
The majority of the common garden experiments using terrestrial populations
suggest that the high-CO2 plants may be adapted to their high-CO2 environment. Scirpus lacustris grown in the laboratory from rhizomes collected in CO2 springs did not have reduced photosynthetic capacity when compared with those collected from control sites (Bettarini et al. 1997). Fordham et al. (1997a) selected Agrostis canina seed from a spring with [C02] ranging from 451-610 gmol CO2 mo1-1 and grew them in the lab at ambient and 700 gmol CO2 mol1. Elevated-CO2 stimulated the growth of all of the populations, and the seeds taken from closer to the spring produced plants with greater initial relative growth rate (RGR) irrespective of chamber [C02]. The weight of the seeds was positively correlated with the [C02] where they were collected, but these differences only accounted for a portion of the variation in RGR. Plantago major plants originating from a high-CO2 spring had intrinsically greater biomass and RGR than two populations growing at ambient-CO2 when both were grown in 350 and 700 gmol CO2 mol1 treatments (Fordham et al. 1997b). Differences in original seed weight only explained a small portion of the variation in RGR between populations of P. major. Seed from spring populations of Boehmeria cylindrica growing at varying [C02] from 350-550 gmol CO2 mol1 were grown in controlled environments at 350, 525, and 675 gmol CO2 mol1 (Woodward 1987). Differences between populations in plant growth were noted only at the highest CO2 treatment; at 675 gmol CO2 mol1, the high-CO2 population had the greatest growth and height.
Aquatic Plant Biology and Responses to Elevated-CO2
Aquatic plants have lower dry to fresh weight ratios than terrestrial plants, and those that are completely submersed have the lowest ratios (Spencer & Bowes 1990). Freshwater is 775-times denser than air, and provides almost 1000-times greater




25
buoyancy, which renders extensive structural tissue in submersed plants superfluous (Scuithorpe 1967).
Another major difference from terrestrial plants is that the shoots of submersed plants contribute to the overall nutrition by absorbing minerals from the surrounding aquatic medium. Although aquatic plant roots were regarded only as anchorage devices, it has since been shown that they are involved in nutrient uptake. Barko et a!. (1991 a) concluded that N, P, Fe, Mn, and micronutrients are primarily taken up from the sediments, while Ca, Mg, Na, K, SO4, and Cl are mainly absorbed from the surrounding water.
Terrestrial and emergent plants are generally more productive than submersed
species, with production by floating plants intermediate (Spencer & Bowes 1990). Per unit area, some emergent aquatic plant communities are among the most productive of the world's vegetation types due to ample provision of water and nutrients, two factors that commonly limit plant growth on land (Wade 1990).
Although seeds are of paramount importance to the reproductive phase of terrestrial species, aquatic plants tend to have greater reliance on vegetative reproduction as their principle mode of population growth (Wade 1990). Vegetative reproduction occurs via fragmentation, creeping stems (layers, runners, stolons, rhizomes, and stem tubers), modified shoot bases (bulbs, corms), root suckers (creeping roots, tap roots, root tubers), and pseudovivipary (Grace 1993). The buoyant and protective nature of water makes the aquatic environment extremely favorable for the dispersal of clonal propagules (Grace 1993). Sexual reproduction produces offspring with variability, with the possibility that some will be able to survive when conditions change or when the species invades a




26
different environment. But for a plant that is well adapted to prevailing conditions (such as those found in aquatic waters), vegetative reproduction with minimal variety in the offspring ensures they will be similarly suited and successful in a consistent environment (Spencer & Bowes 1990).
Photosynthesis in the Aquatic Environment
The photosynthetic mechanisms that aquatic plants employ are based partly on their habit (e.g., submersed, emergent, or floating). Obviously each of these habits allows different access to the resources present in air and water. Light, nutrients, and dissolved inorganic carbon (DIC) can all limit the photosynthesis rates of aquatic plants in the field. The photosynthesis rates of submersed species are typically low when compared to terrestrial and aerial species, and this trend is maintained even at light- and DICsaturation (Van et a!. 1976; Salvucci & Bowes 1982; Spencer & Bowes 1990).
The light environment in aquatic ecosystems is highly variable. Attenuation of
light with water depth is common (Wetzel 1983), so that both the quality and quantity of light vary with depth (Spencer & Bowes 1990). Low irradiance often limits the photosynthesis and growth of submersed plants. Submersed plants invariably can be categorized as shade plants, because leaf photosynthesis is saturated at an irradiance of less than half full sunlight, even for those species that inhabit shallow waters (Salvucci & Bowes 1982; Wade 1990).
In terms of nutrients, comparisons of tissue and freshwater nutrient concentrations suggest that N and P are most likely to limit photosynthesis and growth under natural conditions (Raven 1984). Low tissue [N] has been correlated with a reduction in photosynthetic capacity, carbon affinity, chlorophyll content, and growth of submersed macrophytes (Gerloff & Krombholz 1966; Madsen & Sand-Jensen 1987; Van Wijk




27
1989). Photosynthetic capacity, HC03- uptake capacity, and Rubisco activity of Elodea canadensis were all enhanced when N was increased in hydroponic cultures (Madsen & Baattrup-Pedersen 1995).
Inorganic Carbon in the Aquatic Environment
There is great spatial and temporal variability in the concentration of inorganic carbon species in aquatic ecosystems due to shifts in the carbonate equilibria:
Air [C02] <-> Dissolved [C02] + H20 <-> H2CO3 <-> H+ + HC03- <-> H+ + C032CO2 is the preferred form of DIC for photosynthesis by plants, but HC03- can also be used by some species (Spence & Maberly 1985).
Fluctuations in the carbonate equilibrium occur for a variety of reasons. The
equilibrium is very sensitive to pH fluctuations, with CO2 most prevalent in acidic waters (pH 5 and below), HC03- common from pH 7 to 9, and C032- abundant in alkaline waters (above pH 9.5) (Wetzel 1983). When vegetation is dense, shifts may be related to the amount of photosynthesis and respiration taking place in the ecosystem, resulting in a decoupling of normal water-air mixing. High photosynthesis rates during the day can cause [C02] to approach zero and raise the pH. In contrast, [C02] may far exceed air equilibrium when respiration exceeds photosynthesis at night. Diel changes in pH of more than two units occur in freshwaters and can result in over a hundred-fold change in free-CO2 (Bowes 1996). Carbon dioxide concentration is usually highest before dawn and after plants have been respiring in the evening, and lowest in the late afternoon after plants have been photosynthesizing all day (Sand-Jensen & Frost-Christensen 1998). Seasonal cycles in the carbonate equilibria may also occur as CO2 is depleted when plant abundance and photosynthesis are highest and lowest during the summer and winter,




28
respectively. In productive sites, [C02] may approach zero for several months (Talling 1976; Madsen & Maberly 1991).
Water in equilibrium with ambient air contains approximately 10 mmol CO2 m-3 at 25C; however, most freshwater systems are not in equilibrium with the atmosphere (Cole et al. 1994; Sand-Jensen & Frost-Christensen 1998). Free-CO2 values in freshwaters range from zero to over 350 mmol CO2 m-3 (Spencer & Bowes 1990). Among other things, the [C02] of a water system depends on the type of system (e.g., lake, river, spring) and the amount of mixing within the system. The [C02] of freshwater systems may be lower than air-equilibrium values because water has a massive boundary layer resistance (which results in low exchange rates between air and water), low gas diffusion (approximately 104 times lower in water than air), and rapid CO2 depletion due to photosynthesis of aquatic organisms (Raven 1970; Van et al. 1976; Maberly & Spence 1989; Madsen 1991). Inorganic carbon is most often limiting to plants due to the diffusion constraints, rather than low concentrations available in the water. Aquatic Plant Photosynthesis and CO2
The mechanisms that aquatic plants have evolved to assimilate carbon can vary from the functional groups described for terrestrial plants (e.g., C3, C4, CAM) as a consequence of the physical and chemical differences between the two environments. Most submersed plants fit best in the C3 terrestrial category, however, they show a variety of modifications to the typical C3 pattern. Submersed species require higher [C02] to reach the equivalent photosynthetic rates of terrestrial plants. Submersed C3 species require 30-times more free-CO2 to saturate photosynthesis than their emergent counterparts and exhibit much higher apparent K1 2(CO2) values (Maberly & Spence




29
1983; Bowes & Salvucci 1989; Madsen & Sand-Jensen 1991). Moreover, coinciding with their lower dry weight to fresh weight ratio, submersed plants have less metabolic machinery than emergent or terrestrial species, as demonstrated by low chlorophyll and Rubisco activities (Van et al. 1976; Holaday et al. 1983).
Depleted [DIC] is common in aquatic ecosystems and can severely restrict
photosynthesis and growth by submersed aquatic macrophytes (Van et al. 1976; Bowes & Salvucci 1989; Titus 1992; Vadstrup & Madsen 1995). Submersed aquatic macrophytes show physiological/biochemical and anatomical/morphological adaptation strategies to increase the availability of CO2 at the Rubisco carboxylation site. There are two major physiological/biochemical adaptations. Submersed plants may utilize HC03- as a carbon source for photosynthesis, which is the dominant carbon form in most aquatic habitats (Wetzel 1983). Also, there are photosynthetic systems employed by some species in which additional biochemical pathways are added, including C4- and CAM-like metabolism (Bowes & Salvucci 1984; Casati et al. 2000; Madsen & Sand-Jensen 1991). Anatomical and morphological adaptations to low aqueous DIC include development of gas lacunae and an aerial growth habit (Spencer & Bowes 1990).
Although all plants utilize CO2 as the preferred form for photosynthesis, about 50% of submersed species also use HC03- as the initial C source (Spence & Maberly 1985). The capacity for HC03- use varies with the growth conditions and the photorespiratory state of the plant (Salvucci & Bowes 1983a, b; Bowes 1987; Sand-Jensen & Gordon 1984). Plants that use HC03- may concentrate CO2 around Rubisco resulting in reduced photorespiration rates, thereby increasing photosynthetic efficiency (Maberly & Madsen 1998).




30
Whereas many submersed species can use HCO3-, amphibious species are generally unable to utilize it (Sand-Jensen et al. 1992). Maberly and Madsen (1998) concluded that aquatic species that are restricted to CO2-use only (e.g., Myriophyllum verticillata, Callitriche cophocarpa, Sparganium emersum), had higher photosynthetic affinities for CO2 than species that were also able to use HCO3- (e.g., Myriophyllum spicatum, Elodea canadensis, Potamogeton crispus, Vallisneria spiralis). Spence and Maberly (1985) reviewed the distribution of aquatic species in relation to alkalinity and DIC composition of the water in which they grew and concluded that most CO2-only species were restricted to waters where CO2 was the dominate form of DIC and that HCO3-users were more prevalent in waters with high alkalinity.
Sediments are usually hypoxic or anoxic so the underground organs of aquatic
plants must be adapted to such conditions in order to survive (Waisel & Agami 1996). A major adaptation is the formation of aerenchyma by the development of larger gas-spaces than typically found between cells in ground parenchyma tissues (Jackson & Armstrong 1999). Aerenchyma can account for up to 60% of some tissues in submersed plants (Wade 1990). Aerenchyma can substantially reduce internal impedance to gas transport, especially between roots and shoots (Jackson & Armstrong 1999). The primary function of aerenchyma is to increase 02 diffusion from the atmosphere or photosynthesis to roots and the rhizophere, thereby enabling aerobic respiration to continue (Allen 1996). Aerenchyma can also facilitate the movement of CO2, whether it originates from the atmosphere, from the sediment, or as a respiratory product in plant tissues. The placement of biomass at the water surface in some aquatic plants, and use of hydrosoil CO2 for photosynthesis seem mutually exclusive, as the path length from roots to leaves




31
is generally too long for transfer of sufficient CO2 to support photosynthesis (Spencer & Bowes 1990). Several groups have assessed the contribution of aerenchymal CO2 to photosynthesis of emergent macrophytes. For completely submersed Phragmites australis, S. lacustris, and C. papyrus, sediment-, root-, and rhizome-derived CO2 played major roles in photosynthesis, but these roles were diminished when plants became emergent (Brix 1990; Singer et al. 1994; Jackson & Armstrong 1999); so inorganic carbon was only limiting below, and not above the water in these experiments. The most often cited examples of hydrosoil-CO2 use involve isoetid species. In some, a thick cuticle inhibits the escape of CO2, while growth close to the hydrosoil minimizes the CO2 diffusion pathway (Spencer & Bowes 1990).
Perhaps the simplest adaptation of aquatic plants to increase access to greater CO2 is to produce aerial shoots. In many species, the morphology of aerial leaves is different from submersed leaves. Unlike submersed leaves, aerial leaves utilize free CO2 as their only inorganic carbon source. Therefore, aerial leaves are not usually faced with the potentially wide fluctuations in DIC, 02, and pH that submersed leave encounter (Spencer & Bowes 1990). Some aquatic plants have developed flotation devices that keep their photosynthesizing organs in contact with the atmosphere. An example would be the use of aerenchyma in the shoots of some Sagittaria species to maintain buoyancy at the water surface.
A variety of other anatomical and morphological adaptations to increase access to DIC exist in leaves. Submersed leaves often have chloroplasts in their epidermis, no or non-functional stomates, and little cuticle (Spencer & Bowes 1990). Also, to maximize




32
exposure to the water and access to DIC, they are frequently thin, sometimes finely dissected, and only contain a few cell layers (Wade 1990; Spencer & Bowes 1990). Elevated-CO2 and HC03- Effects on Aquatic Plants
The response of aquatic plants to rising CO2 may be quite different than that of
terrestrial plants. Research on aquatic plants lags behind that of terrestrial plants, thus the C assimilation mechanisms of aquatic plants are less well understood and categorized, thereby making it difficult to make generalizations about their potential responses to rising [C02]. Also, it is difficult to predict how rising CO2 will influence water systems, since they already vary greatly in pH and concentration of free CO2 and HC03- (Bowes 1991; Raven 1994). Huge changes in free [C02] can occur in water bodies, so submersed aquatic species are already exposed to greater CO2 fluctuations than terrestrial species. With this in mind, it has been stated that the rise in atmospheric-CO2 will impact the submersed vegetation of aquatic ecosystems less than those of terrestrial ecosystems (Raven 1994; Bowes 1996). Furthermore, the probability of aquatic plants showing increased growth responses under CO2 enrichment has been suggested to be greater for emergent than for submersed macrophytes (Wetzel & Grace 1983).
Down-regulation of photosynthetic capacity in response to high DIC occurs in aquatic macrophytes and is often coupled to reduced CO2 affinity and suppression of HC03--use, and C4 and CAM metabolism (Holaday et al. 1983; Sand-Jensen & Gordon 1986; Jones et al. 1993). Suppression of these mechanisms is consistent with these processes having costs as well as benefits related to their increased energy expenditure. Thus, these systems may be up-regulated when the plants are DIC-limited, but absent when DIC-replete. The decline in photosynthetic capacity and CO2 affinity seems to be most pronounced in species with the ability to use HC03- (Madsen 1991).




33
Spencer et al. (1994) identified acclimation of photosynthetic phenotypes as a response to environmental heterogeneity within an ecosystem. Hydrilla verticillata forms a vegetation mat with the environmental conditions being very different within and on the edge of the mat, due to the photosynthesis and respiration effects. They found that [DIC], pH, and dissolved oxygen were 0.1 mol m-3, 10.2, and 0.48 mol m-3, respectively, in the mat, while the edge values were 0.7 mol m-3, pH 7.1, and 0.13 mol m-3, respectively. Hydrilla verticillata growing in the mat had greater biomass density, but lower net photosynthesis, daily C gain, and RGR than edge plants. CO2 compensation points were positively correlated with CO2 and HC03- and negatively associated with pH, dissolved oxygen, and biomass, so that low and high CO2 compensation point photosynthetic phenotypes were associated with mat and edge habitats, respectively.
Submersed species have shown enhanced photosynthesis and growth at enrichedCO2 and HC03- in laboratory experiments. Nielsen and Sand-Jensen (1989) concluded that the photosynthesis rates of 14 submersed macrophytes were still limited at four times greater than air equilibrium [CO2] (45 [LM), but had rates three times higher when their CO2 supply was increased to 1600 [tM. Van den Berg et al. (2002) grew two HCO3-users, Chara aspera and Potamogetonpectinatus, at pH greater than 9.5 so that little CO2 was present and they could test the growth response of these species to HCO3- only. Chara aspera was a more efficient HCO3- user based on it having greater photosynthetic rates at low [HCO3-] than P. pectinatus. Net biomass increased for both species at the higher HCO3- treatment, but the ash-free dry weight fraction only increased for C. aspera. Vallisneria americana grown at elevated [CO2] and [DIC] with fertile soils in a greenhouse had stimulated growth rates when compared to those grown at air-equilibrium




34
[CO2] and [DIC] (Titus et al. 1990), but no elevated-CO2 response was identified for V. americana by Barko et al. (1991b). Barko et al. (1991b) did find that CO2 had strong positive effect on biomass of H. verticillata under high irradiance, and that the CO2 response of both populations was greater when fertility was sufficient.
Callitriche cophocarpa and E. canadensis are submersed macrophytes that have received substantial attention in comparing their response to varying [DIC]. Callitriche cophocarpa uses only CO2 for photosynthesis (Madsen 1991) and is heterophyllous with apical rosettes of floating leaves, while Elodea is homophyllous and uses HCO3 in addition to CO2 (Madsen & Sand-Jensen 1987). These species had down-regulated photosynthesis but increased growth at high [DIC] (Madsen et al. 1996). As DIC (CO2 and HCO3-) increased, both species responded with a decrease in maximum photosynthesis rate, initial slope of A/Ci curves, Rubisco activity, protein content, and chlorophyll content, whereas CO2 compensation points increased. In addition, the rate of HCO3--dependent photosynthesis decreased for the HCO3 user, E. canadensis. For both species, the growth response to increased CO2 was greater than that to increased HCO3, and the root: shoot ratio increased with increasing [CO2], but was unaffected by HCO3-. Specific leaf area declined with C availability in the heterophyllous species, C. cophocarpa, whereas no change was observed in E. canadensis. The in situ growth of both species responded positively to elevated-CO2, while enrichment with HCO3 affected E. canadensis only (Vadstrup & Madsen 1995)
Emergence of amphibious plants may play a role in their response to elevated-CO2. The growth rate of C. cophocarpa shoots in contact with air was about three times faster than the rate of fully submerged shoots when both were grown in air-equilibrated water




35
(16 mmol m-3) (Madsen & Breinholt 1995). This difference decreased as dissolved freeCO2 in the water was increased, and the two shoot types grew at the same rate when the submersed shoots received greater than 350 mmol m-3 free-CO2.
Very little research has been done to correlate the acclimation responses of aquatic plants to elevated CO2 with nutrient and carbohydrate levels as is common in the terrestrial literature. Nutrients limited the growth enhancement of H. verticillata and V americana to elevated-CO2 (Barko et al. 1991b). Titus (1992) determined that V americana had greater biomass at enriched-CO2 on all sediments tested and that there was no C02-sediment interaction for biomass, however, allocation of biomass differed with sediment type. Plants grown on the less fertile sediments showed greater relative allocation to horizontal versus vertical growth than plants grown on relatively fertile sediment. Tissue N and P concentrations were consistently lower at high- than airequilibrium C02; however, sediment effects on V. americana growth could not be attributed to either of these nutrients. Madsen et al. (1998) took a different approach by investigating the effect of DIC on the nitrogen requirement of E. canadensis and C. cophocarpa, two species that assimilate N through their leaves (Madsen et al., unpublished data). At elevated C02, growth rates increased, while tissue [N] decreased for both species. Thus the efficiency of N use was improved at high DIC availability and the tissue [N] needed to sustain maximum growth was reduced. The reduced tissue [N] at high CO2 was not caused by an increased accumulation of starch and other non-structural carbohydrates; rather, it was a result of reduced net N uptake. These results suggested that depending on species and relative differences in [DIC], higher growth rates can be expected in systems with higher [DIC], even without a concomitant increase in N load.




36
Ludwigia Repens
Ludwigia repens (Forster), red ludwigia, is a perennial eudicot that is native to Florida. Ludwigia repens belongs in the Onagraceae (evening primrose) family, tribe Jussiaeae, and section Dantia (Godfrey & Wooten 1981; Peng 1989). It has been identified on the East Coast of the United States from South Carolina to South Florida and then West along the Gulf Coast to Texas (Godfrey & Wooten 1981). Red ludwigia is commonly sold as an aquarium plant (Tarver et al. 1979). In Florida, this plant has been collected growing in high-[C02] natural springs, as well as lower-[C02] spring environments (University of Florida, Herbarium), which makes it useful for elevated-CO2 research purposes.
Ludwigia repens is an amphibious plant producing both submersed and aerial
leaves, and can grow in completely terrestrial conditions (Godfrey & Wooten 1981). The term creeping emergent was suggested by Rejmdnkovd (1992) to describe the growth form of a similar species, Ludwigiapeploides. Ludwigia repens is rooted in the substrate, but also produces roots along the stem in the water column. Often, it grows in soils along the border between the shoreline and shallow waters (Hoyer et al. 1996). Mats can be formed as the stems creep along the sediment, sending up flaccid stems in the water (Hoyer et al. 1996). The plant varies from dark red/purple to green. Underwater portions of the plant usually have more red pigmentation, but they shift to a green color as they become aerial. Leaves grow opposite and are typically 7 x 2 cm (Hoyer et al. 1996).
Both sexual and asexual reproduction occurs in L. repens. Flowers are yellow and are usually found on the aerial stems from the late spring through early fall. A sessile fruit or capsule (2.5-3.0 x 2.0 mm) contains a cluster of very small seeds that provide the means of sexual reproduction (Tarver et al. 1979). A related species, L. peploides was




37
found to germinate readily on soil and in water, floating or when maintained submerged (Yen & Myerscough 1989). Ludwigia repens also reproduces with fragments breaking off from the whole plant. Regeneration is possible from fragmented stem pieces with only a few nodes (Lytle, unpublished results). Sculthorpe (1967) noted that species of Ludwigia growing entirely submersed in deep water do not reproduce sexually.
Very little physiological research has been performed with L repens, however,
research has been done using similar species within the genus. Ludwigia uruguayensis was used to assess the effect of a plant species growing in a high-CO2 and resource spring (Koch 1993). There was no evidence of down-regulation of photosynthesis in the plants near the high-CO2 spring vent. Prins et al. (1980) determined that Ludwigia natans used only CO2 in photosynthesis, and not HCO3-.
Summary Statement
The literature regarding the short-term responses of terrestrial plants to elevatedCO2 is well established, with most, but not all, C3 plants showing down-regulation of photosynthesis and increased growth. Studies of more long-term plant responses to elevated-CO2 have been undertaken mainly in natural springs with high [C02]. Although this area of research is in its infancy, these studies indicate that terrestrial plant acclimation responses to elevated-CO2 found in the short-term studies are less prevalent after plants have been exposed to elevated [C02] over many generations. Thus far, CO2 spring experiments have implied that adaptation differences exist between the plant populations growing in high- and ambient-[C02] around the springs, however, researchers have yet to establish genetic differences between the populations to back up this claim. Natural CO2 spring research is an important tool in order to predict how plants will respond to a future high-CO2 atmosphere. The effect of elevated-CO2 on




38
aquatic plants has received far less attention than terrestrial plants, and submersed plants have been completely neglected in regard to natural C02 spring experiments. Submersed plants differ from their terrestrial counterparts in many respects, so that their responses to elevated-C02 cannot be extrapolated from terrestrial results. Freshwater and freshwater ecosystems are vital resources, and will become increasingly scarce and valuable commodities as population and agricultural pressures increase. In this context, aquatic plants are an integral component of any "healthy" aquatic ecosystem and should not be overlooked in elevated-C02 research.




39
Net CO2 assimilation
rate ([tmol CO2 m-2 S-1) Limiting factors in the non-acclimated curve 50 < -------Rubisco-------> < ----------------------RuBP------------------------->
40 Increased Amax due to increased Acclimated
Pi and RuBP regeneration .......................................
30 Non-acclimated
20
10 Decreased slope due to decreased Rubisco content
0
0 I I I
C 200 400 600 800 1000
Intercellular CO2 (pmol mol1) Figure 1-2. Acclimated and non-acclimated assimilation/intercellular CO2 (A/C) curves
for a typical C3 plant at high light and 21% 02.




CHAPTER 2
ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS Introduction
Ludwigia repens (Forster), red ludwigia, is a perennial eudicot that is native to
Florida. It belongs in the order Myrtales, family Onagraceae, tribe Jussiaeae, and section Dantia (Godfrey & Wooten 1981; Peng 1989). It is thought to have originated in South America (Keating 1982) and has been identified on the East Coast of the United States from South Carolina to South Florida and then West along the Gulf Coast to Texas (Godfrey & Wooten 1981). Commercially, this species is commonly sold as an aquarium plant (Tarver et al. 1979).
Ludwigia repens is an amphibious plant that produces both submersed and aerial shoots, and can grow entirely terrestrial (Godfrey & Wooten 1981). It is rooted in the substrate, but also produces roots along the stem in the water column. Often, plants grow in soils along the border between the shoreline and shallow waters, and mats can be formed as the stems creep horizontally along the sediment, sending up stems vertically in the water (Hoyer et al. 1996). The underwater portions of the plants usually have more red and purple pigmentation, but their color shifts to green when they become aerial. Leaves grow opposite and are typically about 7 x 2 cm (Hoyer et al. 1996).
Both sexual and asexual reproduction occurs in L. repens. Flowers are yellow and are usually found on the aerial stems from the late spring through early fall. A sessile fruit or capsule (2-3 x 2 mm) contains a cluster of very small seeds (Tarver et al. 1979). This species also reproduces vegetatively by fragmentation and clonal ramets separating
40




41
from the genet and surviving as individuals. Regeneration is possible from fragmented stem pieces with only a few nodes (Lytle, unpublished data).
There are no anatomical descriptions of L. repens, although members within the genus have been characterized. Summarizing results from observations of the leaves of 25 Onagraceae species, Keating (1982) noted that Ludwigia represents a phylogenetic line separate from all other Onagraceae, and that a distinguishing characteristic of Onagraceae leaves is the presence of abundant raphide crystals in the vegetative tissues.
Aerial and submersed leaf anatomy in some species of Ludwigia differed even to the extent that the plants are considered heterophyllous (Kuwabara et al. 2001). Differences in aerial versus submersed leaf anatomy and morphology for an amphibious plant may represent acclimation to the conditions in their respective air and water environments (Sculthorpe 1967). More so than in the aerial environment, light and inorganic carbon can limit photosynthesis of leaves that are underwater (Spencer & Bowes 1990). Submersed leaves that are thin, finely dissected, and only contain only a few cell layers optimize access to dissolved inorganic carbon and light (Wade 1990). Features that reduce evapotranspiration and provide support in air, such as a thick cuticle, several photosynthetic cell layers, functional stomates, and lignin lose their value underwater (Maberly & Madsen 2002). Thus submersed leaves of Ludwigia arcuata are narrower, with a higher leaf length to width ratio, and a lower stomatal density than the aerial counterparts (Kuwabara et al. 2001). Narrower and longer submersed leaves as compared to emergent leaves are also found in L. arcuata, L. repens, and L. palustris (Petch 1928; Fassett 1957).




42
Stems of submersed plants often contain aerenchyma and have less support tissues such as lignin when compared to emergent stems, which is likely an adaptation to the buoyancy of water (Jackson & Armstrong 1999; Maberly & Madsen 2002). Stem aerenchyma produced by a phellogen was common in the semi-aquatic members of the Onagraceae (Schenck 1889). Ludwigia species are predominantly herbaceous; the most familiar species are herbs of very wet habitats such as ponds, ditches, and streams, therefore, it is somewhat surprising that some woody species exist. The periderm of L. octovalvis plants swelled when they were grown in flooded as opposed to non-flooded environments (Angeles 1992). Carlquist (1987) using Ludwigia anastomosans, L. peduncularis, and L. torulosa determined that their aquatic habit was reflected in the lowest degree of vessel grouping in the Onagraceae family.
Root anatomy of Ludwigia has received attention, with most of the interest directed toward root dimorphism in certain species. Ludwigiaperuviana and L. peploides produce both upward- and downward-growing roots on the same plant (Schenck 1889; Ellmore 1981a, b). In transverse section, the stele of upward-growing roots of L. peruviana roots was surrounded by a wide zone of aerenchyma produced by a phellogen, whereas this zone is absent in downward-growing roots (Schenck 1889; Ellmore 1981 a, b).
The purpose of the present study is to characterize the vegetative anatomy of L. repens, including both aerial and submersed organs. In Florida, this plant has been collected from natural springs (University of Florida Herbarium, FLAS). Anatomical information for this species may contribute to a better physiological understanding as it relates to the plant's function in the both aerial and submersed habits.




43
Materials and Methods
Random collections of twenty mature, emergent L. repens plants were made from Fanning Springs, Florida (29 0 35' 15" N, 82 0 56' 08" W) on 19 February 1999. A voucher specimen was deposited at the University of Florida Herbarium (FLAS). Tissue from ten of the plants was fixed into a formalin-acetic acid-alcohol (FAA) solution at the field site. Mature leaves, stems, and roots were separated from the fixed material to produce slides. Submersed and aerial leaves and stems were also separated, while roots were separated into those growing in the water column or the hydrosoil. Tissue was dehydrated in a tertiary-butyl alcohol series, and embedded in paraffin at 56"5C (Johansen 1940). Cross- and longitudinal-sections (8-20 [tm) of all organs were stained with a safranin (1% w/v in 50% v/v EtOH)-fast green (1% w/v in 95% v/v EtOH) series. Whole leaves were cleared in a 5-10% (w/v) NaOH solution and stained with a safranin (1% w/v in 50% v/v EtOH) solution. Leaf thickness and the density of starch grains and crystals (e.g., raphides and druses) were determined for mature submersed and aerial leaves from micrographs at 100x magnification.
In order to compare stomatal density of the submersed and aerial leaves, dried
leaves were flooded with acetone and compressed with cellulose acetate sheets. The dry cellulose acetate was stripped away leaving a negative replica of the leaf surface. An
2
area of 1.8 mm at 100x magnification was used to count the stomates, with three replicates per leaf




44
Results
Plants
Figure 2-1 shows L. repens growing emergent at the collection site. Leaves
Micrograph cross-sections of aerial and submersed Ludwigia repens leaves are
shown in Figure 2-2, while quantitative characteristics of these leaves are documented in Table 2-1. The leaves are homophyllous since they showed only slight anatomical differences. There was no difference in the thickness of aerial and submersed leaves (Table 2-1). A thin cuticular layer was present on the adaxial and abaxial surfaces of both leaf types. The epidermal cells were thin-walled, with some tangential thickening (Fig. 2-2). There was little difference between the adaxial and abaxial epidermal cells, although the adaxial cells of aerial leaves were somewhat smaller and had thinner cell walls than the abaxial cells (Fig. 2-2). No chloroplasts were observed in the epidermal cells, even of the submersed leaves. Stomates were present on the adaxial and abaxial surfaces of aerial and submersed leaves, making them amphistomatous. The stomates exhibited guard cells, but no subsidiary cells, i.e., they anomyocytic. Aerial leaves had two- and seven-times greater stomatal densities than submersed leaves on the adaxial and abaxial surfaces, respectively (Table 2-1). Stomatal density did not differ between the adaxial and abaxial surfaces of aerial leaves, whereas submersed leaves had about fourfold greater stomatal density on the adaxial as compared to the abaxial surface (Table 21).
The mesophyll tissue was typically differentiated into one layer of palisade and
four to five layers of spongy mesophyll (Fig. 2-2). However, the palisade mesophyll was more defined in aerial as compared to submersed leaves, and there were instances of




45
submersed leaves near the base of the stem having no palisade mesophyll layer. The palisade mesophyll in aerial leaves was tightly packed, whereas the spongy mesophyll and the entire mesophyll of submersed leaves were loosely arranged with abundant intercellular air spaces; however, no lacunae were present (Fig. 2-2). The mesophyll was made up of chlorenchyma cells, with both the palisade and spongy mesophyll containing starch grains, but more were present in the palisade layer (Fig. 2-2). Aerial leaves had 168% greater starch grain density than submersed leaves, but the leaf types contained similar numbers of raphide and druse crystals (Table 2-1).
The vasculature of L. repens leaves consisted of bicollateral bundles in the midrib and major veins (Fig. 2-3), and collateral bundles in the minor veins. There were a large number of vascular bundles in the leaf margin, with four to five on each side of the midvein. The bundle sheaths were indistinct with ground parenchyma tissue surrounding a small amount of primary xylem and phloem (Fig. 2-3). The xylem tissue of the bundles consisted of rows of small vessel members interspersed with xylem parenchyma cells that were few and small in number (Fig. 2-3). The elongate vessel members had transverse or oblique end walls. There were small clusters of phloem on both sides of the xylem (i.e., bicollateral phloem).
Stems
Figures 2-4 and 2-5 show cross-sections of aerial and submersed L. repens stems. No cuticle was present on the stems of L. repens and epidermal cells were thick-walled with mucilage present (Fig. 2-4). The cortex was differentiated into three zones; all made up of thin-walled parenchyma cells that contained starch grains, raphides, and mucilage (Fig. 2-4). Mucilage was less prevalent in all the layers of aerial as compared to submersed stems (Fig. 2-4). The outer layer of cortex was composed of cells of small




46
size with the largest quantity of mucilage (Fig. 2-4). The middle layers contained cells that were larger than those in the other zones, and was evenly interspersed with large air lacunae (Fig. 2-4). These air lacunae were smaller and less prevalent in aerial as opposed to submersed stems (Fig. 2-4). The cells in the inner cortical layers exhibited the largest number of raphides, and contained styloid and cuboidal crystals as well.
The stem endodermis was composed of thick-walled parenchyma cells that
contained mucilage and starch grains (Fig. 2-5). The endodermis and stele of the aerial stem had less mucilage than their submersed counterparts (Fig. 2-5). The individual vascular bundles present in younger stems were bicollateral, with the primary phloem clustered in two distinct internal and external zones around the xylem poles, while the primary xylem showed endarch maturation. Although it was not well defined, L. repens produced a cambium that generated infrequent secondary xylem with spiral thickenings, but no secondary phloem. The xylem formed a continuous cylinder in older stems (Fig. 2-5). Xylem tissues were composed of vessel members with alternate pits and some parenchyma (Fig. 2-5). The secondary vessel members had reticulately thickened walls. The stem pith was made up of thin-walled parenchyma cells with small intercellular spaces (Fig. 2-5). Many of these cells contained mucilage, starch grains, and/or raphides. Roots
Figure 2-6 shows cross-sections of submersed water-column and hydrosoil roots of L. repens. These root types did not differ anatomically (Fig. 2-6). In some of the roots the epidermis was crushed or compressed. Root epidermal cells were parenchymatous with their walls tangentially and radially thickened (Fig. 2-6).
The cortex of roots was either divided into two zones, with an outer layer
composed of about two layers of larger cells (Fig. 2-6A), or was undifferentiated (Fig. 2-




47
6B). This distinction was not a water-column versus hydrosoil root phenomenon as it occurred in both root types. The cortex of both root types was made up of thin-walled parenchyma cells containing starch grains (Fig. 2-6). Lacunae were not present in the roots; however, roots did have many intercellular spaces of various sizes that were larger than the isodiametric cells in which they were embedded (Fig. 2-6).
The endodermis of the roots of L repens was distinct. No Casparian strip was
evident, but the endodermis did contain mucilage (Fig. 2-7). The pericycle of the stele was composed of one to two layers of thin-walled parenchyma cells (Fig. 2-7), and lateral roots were generated from the pericycle. The stele exhibited a protostele pattern and there was no vascular cambium in the roots (Fig. 2-7). Vessel member and parenchyma cells made up the xylem tissues, and these cells were often filled with mucilage (Fig. 27). Maturation of the xylem was exarch, with the smaller xylem cells on the periphery. The smaller protoxylem cells had spiral thickenings, and the metaxylem cells had alternate pits. Phloem was restricted to isolated bundles between xylem poles and was composed of sieve tube members and parenchyma cells.
Discussion
Ludwigia repens exhibited terrestrial-like anatomical characteristics even though it grows more frequently submersed or emergent than fully terrestrial. Similar to terrestrial plants, aerial and submersed leaves possessed a cuticle and stomates, and stems contained a cambium. Traits that were more aquatic included the possession of aerenchyma in the stems and roots, and a lack of sub-stomatal cavities in leaves.
A thin layer of cuticle was present on both the adaxial and abaxial sides of all
leaves examined. Most submersed plant leaves have little cuticle since water loss is not a problem (Spencer & Bowes 1990), and its absence can enhance CO2 diffusion. Keating




48
(1982) examined 24 species of Ludwigia (not L. repens) and found no cuticle on the leaves, so L. repens presents an anomaly in this respect. Mucilage was also present in all the L. repens organs examined. The cuticle and mucilage present on submersed leaves should retard water loss if the submersed leaves become aerial, as water levels fluctuate in the natural habitat.
Stomates of L. repens in this study were anomyocytic, unlike other species in the Onagraceae, which are surrounded by three or more subsidiary cells (Metcalfe & Chalk 1950). Stomates were present on both the aerial and submersed leaves, with greater density on the aerial leaves. Submersed aquatic leaves typically have few or nonfunctional stomates (Sculthorpe 1967; Spencer & Bowes 1990). It is possible that the submersed leaves on a plant growing in the sample site could become aerial due to water level fluctuations. Stomates are developed while leaves are still in the bud (Costantin 1886), but presumably function only when they are exposed to the atmosphere (Sculthorpe 1967). Ludwigia arcuata, another amphibious species but with heterophyllous leaves, is similar in regard to stomates in that they are found on leaves of both terrestrial and submersed plants, with about three-fold greater density on terrestrial leaves (Kuwabara et al. 2001).
The stomatal density of submersed leaves ofL. repens was greater on the adaxial than on the abaxial side; but there was no difference for the aerial leaves. Terrestrial eudicots commonly contain more stomates on the abaxial leaf surface, but this pattern tends to be reversed in submersed species (Sculthorpe 1967). Ludwigia arcuata, had greater stomatal density on the adaxial surfaces of both terrestrial and submersed leaves




49
(Kuwabara et al. 2001), while stomates were equally common on both leaf surfaces of 24 Ludwigia species (Keating 1982).
A cambium was present in both the aerial and submersed L. repens stems, and this has been reported for other Ludwigia species (Carlquist 1987; Angeles 1992). Wood and other structural materials are typically minimized in submersed plant parts since they are supported by the buoyancy of water (Westlake 1965; Sculthorpe 1967). Thus, the Ludwigia genus, which primarily contains aquatic species, is somewhat unusual in having a cambium present.
Stems of Ludwigia repens contained lacunae, while leaves and roots had small and large intercellular spaces, respectively. Extensive aerenchyma is common in aquatic plants as it facilitates more rapid diffusion of CO2 and 02 in the gas phase for photosynthesis or respiration, or to discharge volatiles (Jackson & Armstrong 1999). Aerial stems had fewer lacunae than submersed stems in accordance with results in Sculthorpe (1967) and reports that flooding triggers the formation of aerenchyma (Jackson & Armstrong 1999). It is interesting to note that in his classic study of gasfilled tissue in marsh plants in which the term aerenchyma originated, Schenck (1889) used stems of Jussiaea, now in the genus Ludwigia, to describe the development of schizogenous spaces between cells derived from a phellogen.
The abundant raphide and druse crystals found in all the organs ofL. repens was in agreement with results showing that raphide crystals in vegetative tissues are a distinguishing characteristic of the Onagraceae (Keating 1982). Calcium oxalate crystals were present in all 24 species of Ludwigia examined by Keating (1982), with raphides present in nearly all specimens. Fanning Springs, where L. repens was collected, is




50
designated a calcium carbonate spring (Woodruff 1993), therefore it is possible that calcium accumulation was a factor in the calcium oxalate crystal formation.
Although the bicollateral vascular bundles, i.e., bundles with the phloem in primary in two distinct zones (internal and external) found in the leaves and stems of L. repens is a rare anatomical feature, other genera in the Onagraceae also possess this trait (Keating 1982).
The aerial and submersed leaves showed differences in anatomy and morphology, but they are too minor to consider the leaves heterophyllous. Aerial leaves had a betterdeveloped palisade layer and had greater starch grain and stomatal density. These traits may reflect the greater reliance on the aerial leaves for photosynthesis by the plant and could be related to enhanced CO2 diffusion. Aerial leaves of amphibious plants tend to have higher photosynthesis rates and light-saturation points than submersed leaves (Bowes 1987; Salvucci & Bowes 1982). Other Ludwigia species have a welldifferentiated mesophyll with one adaxial layer of palisade occupying about half of the mesophyll, however they were not necessarily aquatic species (Keating 1982). Submersed leaves often show an increase in palisade at the expense of spongy mesophyll or lack palisade in their mesophyll tissues (Sculthorpe 1967; Ronzhina & P'yankov 2001). Similar to L. repens, aerial leaves of L. arcuata had greater stomatal density than submersed leaves, but unlike L. repens the leaf types were different enough in morphology and anatomy that they were considered heterophyllous (Kuwabara et al. 2001).
Roots growing in the water column and hydrosoil showed no differences. This is in contrast to L. peploides, which although it has similar growth habit to L. repens, being a




51
prostrate amphibious plant anchored in waterlogged soil, shows root dimorphism (Ellmore 1981a, b).
Leaf anatomy of L. repens reflected a physiology better suited to an emergent,
rather than a submersed existence, at least with respect to photosynthesis. The stomates and cuticle serve no purpose and hinder CO2 diffusion in submersed leaves, whereas they are vital in reducing water loss during aerial leaf photosynthesis. These traits, plus the better-developed palisade layer in aerial leaves provide evidence that the aerial leaves serve as the primary photosynthetic tissues of emergent L. repens plants.




52
Figure 2-1. Ludwigia repens growing emergent in Fanning Springs, Florida.




53
ad
-.0 sg
~ab 0-1 mm A. Aerial leaf
ad
'No
sg
ab
0-1 mm B. Submersed leaf Figure 2-2.Micrograph cross-sections of aerial (A) and submersed (B) L. repens leaves.
Labels denote adaxial epidermis (ad), palisade mesophyll (p), spongy
mesophyll (s), starch grain (sg), and abaxial epidermis (ab).




54
Table 2-1. Characteristics of fully-expanded aerial and submersed leaves of L. repens.
Parameter Aerial leaves Submersed leaves
Thickness leaf (mm) 0-16 + 0-02 0-18 + 0-03
Stomatal density (no. stomates mm-2)
Adaxial epidermis 163 + 35** 95 + 22
Abaxial epidermis 181 + 48** 25 + 11
Starch grain density
(no. mm-2) 5200 + 900** 3100 + 400
Crystal density (no. mm-2)
Raphides 8 + 5 6 + 2
Druses 7 + 4 7 + 7
Mean SD (n = 4-8). P-values are from t-tests and significant differences between populations atP < 0-01 are noted by **




55
p
x
ph
0.05 mm Figure 2-3. Micrograph cross-section of the maj or vascular bundle of a submersed L.
repens leaf. Labels denote parenchyma (p), xylem (x), and phloem (ph) cells.




56
0.25 mm A. Aerial stem
~m
e
fk r
Sg
0.25 mm B. Submersed stem Figure 2-4 Micrograph cross-sections of the epidermis and cortex of aerial (A) and
submersed (B) L. reopens stems. Labels denote epidermis (e), parenchyma cell
(p), lacunae (1) starch grain (sg), and mucilage (m).




57
K Aph
x
_4 4 Ifi, sg Tm
e
0-3 mm A. Aerial stem
i 7f ;
ph
e, m
0.2 mm B. Submersed stem Figure 2-5.Micrograph cross-sections of the inner cortex, vascular cylinder, and pith of
aerial (A) and submersed (B) L. repens stems. Labels denote parenchyma cell
(p), xylem (x), phloem (ph), endodermis (e), starch grain (sg), and mucilage
(m).




58
e
x
en, m
p
0-2 mm A. VAter-colufnn root
i
p
en, m
x
T
e
0- 14 mm B. Hydrosoil root Figure 2-6.Micrograph cross-sections of submersed water (A), and hydrosoil (B) L.
repens roots. Labels denote epidermis (e), parenchyma cell (p), intercellular
space (i), endodermis (en), xylem (x), and mucilage (m).




59
e, m
p
ph
x
0-05 mm Figure 2-7.Micrograph cross-section of the endodermis, pericycle, and stele of a
hydrosoil L. repens root. Labels include endodermis (e), pericycle (p), xylem
(x), phloem (ph), and mucilage (m).




CHAPTER 3
CHARACTERISTICS OF SUBMERSED LUDWIGIA REPENS PLANTS
FROM TWO POPULATIONS GROWING IN SPRINGS WITH DIFFERING CO2 CONCENTRATIONS
Introduction
Atmospheric [C02] is currently over 371 gmol CO2 mo1-1 (Keeling & Whorf 2002), and expected to double in this century. Much research on the short-term (days to months) acclimation responses of terrestrial plants to elevated CO2 exists, with the responses differing among species (Drake et al. 1996a; Pritchard et al. 1999). Because Rubisco is the initial C-fixing enzyme in C3 plants and subject to competitive inhibition by oxygen (Bowes & Ogren 1972), C3 plants generally show greater responses to elevated-CO2 than C4 and CAM species that are able to concentrate CO2 at the site of fixation. Although photosynthesis rates of C3 plants respond favorably to enriched-C02, this initial enhancement is often reduced as rates are down-regulated over longer time frames (Lewis eta. 1999). Directly correlated with the down-regulation of photosynthesis are reduced Rubisco activity and protein amount (Besford et al. 1990; Rowland-Bamford et al. 1991). In addition, water use efficiency may increase in elevated-CO2 because of reduced stomatal conductance through decreases in stomatal density or partial closure of stomates (Amthor 1995). Both direct and indirect effects of elevated-CO2 have been reported to decrease respiration (Drake et al. 1999), although the response is still debatable, with effects at least in part attributable to measurement artifacts (Jahnke 2001; Jahnke & Krewitt 2002).
60




61
Even though photosynthesis may be down-regulated, C3 plants grown in elevatedCO2 usually have enhanced growth (Poorter 1993). Explanations for why acclimation responses occur are related to other physiological mechanisms not keeping pace with increased C-fixation. As C-fixation and source output increase, sink reservoirs (Stitt 1991; Sheen 1994) and N assimilation (Stitt & Krapp 1999) may fail to meet demand. Acclimation responses to high-CO2 are less evident when sink (e.g., root) growth is not restricted (Arp 1991) and nutrient requirements are met (Geiger et al. 1999).
Terrestrial C3 plants grown in elevated-CO2 usually are larger (Poorter 1993), with greater leaf area (Smith et al. 1987). Sink tissues and tiller production are favored, and root: shoot ratios become greater (Smith et al. 1987; Lewis et al. 1999). Tissue density of plants growing in high-CO2 is normally increased, with an associated decrease in specific leaf area (SLA) (Vu et al. 1989) as leaves become thicker (Tipping & Murray 1999). Along with the morphological changes, plant anatomy is affected with increases in cell size, cell number, and proportion of mesophyll tissue in leaves (Vu et al. 1989; Radoglou & Jarvis 1990; Ferris et al. 2001).
Although less studied, submersed plant acclimation responses to high-CO2 tend to be similar to their terrestrial counterparts, with some novel differences (Bowes 1991; Bowes 1993). Because of the diffusion resistance of water, submersed species require higher [C02] to saturate their photosynthesis rates and generally have much lower maximum rates than terrestrial plants (Maberly & Spence 1983; Bowes & Salvucci 1989). Also, coinciding with their lower dry weight to fresh weight ratio, submersed plants have less metabolic machinery than terrestrial species, as demonstrated by low chlorophyll and Rubisco activities per unit mass (Van et al. 1976; Holaday et al. 1983).




62
Based on these characteristics, submersed species should respond favorably to CO2 enrichment. However, some submersed species are able to use bicarbonate ions in photosynthesis, and this may reduce the dependency on free-CO2 and lead to a decreased response to elevated-CO2 (Madsen et al. 1996). Reported responses of submersed aquatic plants to enriched-CO2 include increased growth and root: shoot ratio, decreased SLA, down-regulation of photosynthesis, and decreased Rubisco activity and amount (Sand-Jensen & Gordon 1986; Madsen & Sand-Jensen 1994; Madsen et al. 1996). The degree of CO2 enhancement is also influenced by the nutrient status of the medium in which the plants are grown (Barko et al. 1991b).
In contrast to the abundance of literature regarding short-term responses to
elevated-C02, there are few experiments that extend past a single growing period (Field et al. 1996; Korner et al. 1996). Long-term population-level experiments are crucial to make predictions regarding how plants may genetically adapt to rising-CO2. Different genotypes within a species may differ in CO2 responses (Wulff & Alexander 1985), so the potential for genetic selection to elevated-CO2 exists. However, periods of ten or more years are often required for genetic shifts in populations (Miglietta et al. 1993).
One technique to study long-term elevated-CO2 effects has been to take advantage of natural environments in which plants have been growing in high [C02] for many generations (Raschi et al. 1997). Geothermal gas vents such as springs, geysers, and volcanoes can emit CO2 and expose terrestrial plants in the vicinity to higher than ambient concentrations (van Gardingen et al. 1997). In particular, freshwater springs may produce high [C02] without the toxic components found in some other C02-emitting sources. Using these natural systems, intraspecific populations of terrestrial plants




63
growing at different distances from a natural CO2 source, with [C02] ranging from ambient to 1000 gmol CO2 mol-1 have been investigated (Cook et al. 1998). Adaptation is often implied from these experiments, but acclimation responses cannot be ruled out.
An interesting difference from short-term studies is that terrestrial plant populations growing for long periods near to high-CO2 spring sources do not necessarily produce greater biomass than populations growing further away (Korner & Miglietta 1994; Cook et al. 1998). Down-regulation of photosynthesis is less widespread for plants growing near high-CO2 sources (Koch 1993; Bettarini et al. 1997), but the plants do exhibit decreased stomatal conductance, although the decrease is not always due to modifications in stomatal density (Bettarini et al. 1998). Unlike in short-term studies, plants growing near enriched-CO2 springs do not always show carbohydrate buildup or decreased shoot N content (Korner & Miglietta 1994; Bettarini et al. 1997; Cook et al. 1998) or greater tissue density. Specific leaf area of plants growing close to CO2 vents does not appear to be greater than that of plants growing at ambient [C02] (Koch 1993; Cook et al. 1998).
An underlying assumption in natural CO2 source experiments is that genetic differences exist among the populations being compared, but to our knowledge this assumption has not been tested (Miglietta et al. 1993; Fordham et al. 1997a). Also, in many instances research has compared plant populations that are in close geographic proximity (e.g. 100 m) so that genetic exchange cannot be ruled out. This is an important factor in any study of potential genetic adaptation. Furthermore, fully submersed plants have been neglected in high-CO2 spring research. This is surprising considering the environmental parameters of natural freshwater springs can be remarkably stable over long periods (Rosenau et al. 1977).




64
This study is the first elevated-CO2 spring experiment to examine potential adaptation using submersed plants and to perform a genetic analysis. This project characterizes two submersed populations of a perennial angiosperm native to Florida, Ludwigia repens, that has been growing in two springs with greatly differing [C02] for many generations. The springs are 85 km apart and not connected, therefore, genetic exchange between the two populations is unlikely. The hypothesis being examined is that the two populations should exhibit characteristics consistent with genotypic adaptation to their respective environmental [C02]. The specific objectives were to 1) establish if the high- and lower-CO2 populations differ genetically; 2) determine whether the submersed population of L. repens exposed to high-CO2 for many generations shows down-regulation of photosynthesis and Rubisco activity when compared to the population from a lower-CO2 spring; and 3) resolve if plants from the high-CO2 spring differ morphologically and anatomically from their lower-CO2 counterparts.
Materials and Methods
Collection Sites
Two populations of Ludwigia repens L. were selected for the study, one in a high[C02] spring, Fanning Springs, FL (29 0 35' 15" N, 82 0 56' 08" W) and the other in a spring with a much lower [C02], Rainbow Springs, FL (29 0 06' 08" N, 82 0 26' 16" W). Fanning Springs is approximately 85 km northwest of Rainbow Springs, and the two springs are not connected. Both springs reside on the Ocala geological uplift and fit into the calcium bicarbonate category of spring classification system developed by Woodruff (1993). At both springs, the Ludwigia populations grow both partially emergent and completely submersed near the shore by the boils and occupy approximately 16 M2. All data were collected between the springs and summers of 2001 and 2002.




65
Water from the high- and lower-CO2 springs was analyzed for nutrients, minerals, and conductivity at the Analytical Research Laboratory (University of Florida, Gainesville, Florida, USA). To eliminate air bubbles, containers were sealed below the water surface and placed on ice during transport. Temperature, irradiance, alkalinity, and pH were measured at each field site on the day of collection. Irradiance and the [C02] in the air 3 cm above the water surface were measured with a LI-250 light meter and a LI6200 (LI-COR Inc., Lincoln, NE).
Inorganic carbon species were calculated using the FWcarb computer program
(SC Maberly, Acme Liquid Software Company, 1991). FWcarb requires alkalinity, pH, temperature, and ionic strength as input data and assumes equilibrium conditions to calculate total dissolved inorganic carbon (DIC), C02, and HC03- (Mackereth et al. 1978). Ionic strength (I) was calculated using the formula: I = 2"5 x 10.5 (Sd),
where Sd is total inorganic dissolved solids in mg L-1 (Loewenthal & Marais 1976). The Sd was determined from values obtained from the Analytical Research Laboratory. Morphology and Anatomy
The morphological characteristics of submersed plants from both populations were compared. An individual plant was defined as a single vertical stem with its associated leaves, roots, and axillary stems. The lengths of roots, internodes, and main and axillary stems; and the number of leaves and axillary shoots were determined for eight plants randomly selected from each population. Plants cleaned of epiphytes were separated into leaves, stems, and roots and their fresh weights and density were determined. Leaf areas were measured with an area meter (LI-COR Inc., Lincoln, NE). For dry weight determinations, plant organs were dried at 65 'C for 3 d. Individual leaf characteristics




66
were also measured for a further six plants using four fully-expanded leaves taken from nodes four and five counting down from the shoot apex.
For anatomical comparisons, six plants from each population were fixed in a formalin-acetic acid-ethanol solution (Johansen 1940) at the field sites. For section preparations, fully-expanded leaves from nodes four and five, stem tissue 10 cm from the shoot apex, and hydrosoil roots at the widest diameter, were selected. The tissues were dehydrated in a tertiary-butyl alcohol series, and embedded in paraffin at 56.5 'C (Johansen 1940). Sections (8-20 .m) of leaves, stems, and roots were stained with a safranin (1% w/v in 50% v/v ethanol)-fast green (1% w/v in 95% v/v ethanol) series. Whole leaves were cleared in an 8% (w/v) NaOH solution and stained with a safranin (1% w/v in 50% v/v ethanol) solution. The sections were examined to determine the number of cell layers in each tissue, the frequency of crystals, and the amount of aerenchyma. Leaf thickness was measured 5 mm from the main vein of leaves. The percent aerenchyma per area of stem cross-section was established by passing photographs of stem sections with and without the aerenchyma removed through an area meter.
To determine stomatal frequency, fully-expanded leaves from eight plants were
dried, flooded with 100% acetone, and compressed with cellulose acetate sheets. The dry cellulose acetate was stripped away. This negative replica was used for three replicate counts per leaf of stomates in a 1.8 mm2 surface area. Sexual Reproduction
Flower and seed capsule production at each site was noted from September 1998 to March 2002. The average number of flowers per plant from the high-CO2 spring was determined using ten plants. The ability to flower and produce viable seed over a 56-d




67
period was further studied in a growth chamber with a photon irradiance of 300 mol m-2 s1 (400-700 nm), 12 h photoperiod, 24 'C, and atmospheric CO2. Plants from both populations were collected and grown in 250-mL sand-filled styrofoam cups, supplied with a 1.2 g stick of slow-release fertilizer (13% N; 4% P; 5% K by weight), in aquaria containing deionized water that was changed every 3-d. Flowering of each population was monitored using four plants with each of the following treatments: submersed, emergent, and emergent with gibberellic acid (GA3) applied. For the submersed treatment the water level was maintained above the shoot apices. For the emergent treatments, it was set so that the shoot apices were at the water surface at the start of the experiment, and subsequent growth was emergent. For the GA3 treatment, 200 tL of 50mmol m-3 GA3 immobilized in lanolin was applied to the apical meristem every week for four weeks.
Mature seed capsules were collected from flowering plants just prior to being shed, and the seeds stored at room temperature. To monitor germination, seeds were placed in petri dishes on filter paper moistened with deionized water, in growth chambers set at 24 'C, a photon irradiance of 120 tmol m-2 S- (400-700 nm), and a 12-h photoperiod. Gas Exchange
Gas exchange measurements were performed with an 02 electrode system
(Hansatech Instruments, Norfolk, England). One day prior to use, submersed plants were collected and placed in 20-L aquaria in a growth chamber equilibrated with atmosphericCO2 at an irradiance of 400 mol m-2 S- (400-700 nm), and 23 'C. All measurements were made in triplicate on fully-expanded, detached leaves at 23 'C in a 2-mL chamber filled with buffer set at the appropriate pH and dissolved [02], and a saturating irradiance of 700 mol m-2 S- (400-700 nm). NaHCO3 was injected to initiate photosynthesis. To




68
exhaust intercellular C02, the leaves were incubated prior to measurement in DIC-free buffer at the assay pH and an irradiance of 400 [tmol m-2 s-1. Leaf fresh weight and area were determined for use in rate calculations.
The net photosynthesis (NPS) rates of leaves from plants of both populations were determined at the free [C02] in their natural environments (460 and 50 mmol CO2 m-3 for the high- and lower-CO2 spring, respectively), and also at a saturating [C02] of 1200 mmol m-3. The degree to which 02 inhibited the photosynthesis of plants from both populations was determined by measuring NPS rates at 21% and 1% 02 (equivalent to 270 and 13 mmol m-3 in solution, respectively) with 10 mmol m-3 [C02] (air-equilibrium) in 20 mol m3 WES buffer at pH 5. To assess the ability of the leaves to use bicarbonate ions, NPS was compared for leaves at pH 5 and 9 using 20 mol m3 WES or CHES buffer, respectively, with 200 mmol m3 free [C02] at each pH.
Dark respiration (Rd) was also measured at 23 'C and 21% 02 with detached leaves from both populations at the [C02] and pH of the collection site. For these measurements the chambers were darkened and contained 20 mol m3 HEPES at pH 7.2 and 7.8; the pH values of the high- and lower-CO2 sites, respectively. Rubisco Analysis
A minimum of 100 mg of fully expanded leaves (approximately 5 leaves) was collected from each of four plants during a full-sun period (12:00-14:00) and was immediately frozen in LN2 at the high- and lower-CO2 spring sites. Total Rubisco activities of these leaves were assayed as described by Vu et al. (1997), with the following modifications. Approximately 100-150 mg leaf material was ground with 1 mL of extraction medium per 50 mg of leaf material. The extraction and assay medium




69
contained Tricine rather than Bicine buffer, and no isoascorbate was added. Rubisco activities were expressed on a fresh weight basis. Carbohydrates, Fiber and Ash
For carbohydrate analyses, six plants from the high- and the lower-CO2 populations were separated into their respective leaves, stems, and roots and were quick-frozen in LN2. Samples of approximately 150 mg fresh weight were collected at dusk to ensure that carbohydrates were near their maximum amount. Soluble carbohydrates were extracted using 80% (v/v) ethanol at 85 'C. Starch and total soluble sugars were quantified using the microtiter method of Hendrix (1993). Carbohydrate assays were performed in triplicate on six and four separate extractions for starch and total soluble sugars, respectively.
Fiber was measured using the ANKOM Filter Bag System (Fairport, NY). Four plants were separated into their respective parts and dried at 65 'C for 3-d. Approximately 500 mg dry weight of material was then passed through a 1 mm screen in a Wiley mill. Fiber components were separated using an ANKOM 200 Fiber Analyzer (Fairport, NY). Percent fiber was measured as ash-free neutral-detergent fiber (NDF) (Goering & Van Soest 1970) with decalin and sodium sulfite omitted (Golding et al. 1985). Removal of hemicellulose with the addition of acid to the NDF resulted in acidfree detergent fiber (ADF). Washing ADF with 72% (v/v) H2SO4 removed the cellulose portion, leaving lignin. There was not enough root material to perform a fiber analysis on individual high-CO2 plants; thus, the four replicates were pooled for analysis.
To determine ash content, five plants from the high-and lower-CO2 populations were collected, separated into leaves, stems and roots, and a minimum of 100 mg was




70
dried at 500 oC for 16 h before being ground with a mortar and pestle and subjected to analysis (Horwitz 2000).
DNA Fingerprinting
DNA fingerprinting of the two Ludwigia populations was performed using amplified fragment length polymorphisms (AFLP) (Zabeau & Vos 1993; Vos et al. 1995). Ten plants were randomly collected from each population and dried in silica gel. DNA was extracted according to Doyle & Doyle (1987). The DNA samples were cleaned using the Qiaquick PCR Purification Kit (Qiagen Inc., Santa Clarita, CA).
AFLP was performed using the Plant Mapping Protocol (PE Applied Biosystems Inc., Foster City, CA) unless otherwise noted. The restriction enzymes, EcoRI and Msel, and the T4 DNA ligase and buffer came from New England Biolabs (Beverly, MA). PCR products were generated using a Hybaid PCR Express Thermal Cycler (Ashford, Middlesex, UK). Specific selective primers tested were EcoRI ACT and ACA labeled with D4 (Genset, Paris, France) and Msel CAC, CTC, CTT, CTA, and CTG (Integrated DNA Technologies, Coralville, IA), resulting in ten different primer combinations. Selective amplification reactions contained 200 mmol m-3 each dNTP (BoehringerMannheim, Mannheim, Germany) and 1 U AmpliTaq Gold (PE Applied Biosystems, Foster City, CA). Amplification products were run on a Beckman-Coulter Inc., CEQ 8000 Capillary Genetic Analysis System (Fullerton, CA). Primers chosen for further analysis were EcoRI ACT and Msel CAC.
Only dense bands with more than 70 base pairs were included in the fragment analyses. The analysis and selective amplification were performed five times for each sample. Thirty-six loci were analyzed for all individuals resulting in a binary matrix that was analyzed using Tools for Population Genetics software (Miller 1997). This




71
software was used to calculate Wright's F-statistic ((IDt) (Weir & Cockerham 1984), a 95% confidence interval (using bootstrapping and 5000 replications), and genetic distance and identity (Nei 1972) for diploid dominant alleles. Statistics
Equality of variances between the high- and lower-CO2 populations was tested for each response variable. All variables that were deemed to have equal variances were analyzed using a 2-sample t-test with pooled variances. Variables with unequal variances were analyzed using Sattherthwaite' s t-test for unequal variances. All statistical calculations were carried out using SAS release 8.2 (SAS Inst. Inc., Cary, NC, USA) and alpha > 0.05 and 0.01 levels of significance.
Results
Spring Characteristics
Table 3-1 shows the water chemistry of the two springs measured in 2001
compared with data compiled from the literature going back in most cases over half a century. Both springs showed very little change in the concentrations of inorganic carbon species, in temperature, and pH over a fifty-five year period (Table 3-1). Several CO2 measurements for the high-CO2 spring over this time period show that it oscillated around 410 mmol m3 with no general trend over time (Rosenau et al. 1977; Hornsby et al. 2001; Lytle, data not shown). Likewise, most of the nutrients remained relatively constant over time, although there were exceptions. Most notably, the concentration of nitrate-N in both springs increased ten-fold from 1946 to the present, although NH4-N and total P did not change. The concentrations of K, Na, and Cl increased in the highCO2 spring over time, as did Ca in the lower-CO2 water (Table 3-1).




72
The irradiance characteristics of the two springs were similar, as both were
potentially exposed to full sun at midday, but were shaded in early morning and late afternoon (data not shown). Irradiance (400-700 nm) measurements taken at midday in both springs averaged 2000 mol m-2 s-1 just above the water surface and 1100 mol m-2 s-1 underwater at the top of the submersed Ludwigia plants. Water depth was similar, with the high- and lower-CO2 springs measuring 0.83 0.31 and 1.2 0.21 m deep, respectively.
The two springs differed in several key respects. Fanning Springs had a lower pH with almost ten-fold higher free [C02], and over two-fold higher [HCO3-], total Ci and alkalinity. It also had approximately four-fold greater [N03-N] and higher total [P] than the lower-CO2 Rainbow Spring. The free [CO2] in both springs was greater than the airequilibrium value of 13 mmol m-3 at 23 'C, being 35 and 4 times greater for the high- and lower-CO2 spring, respectively. The high-CO2 spring also had 130% greater [C02] in the air just above the water surface, with values for the high- and lower-CO2 springs of 521 + 58 and 402 14 gmol CO2 mo1-1, respectively; n = 9. Morphology, Anatomy and Chemical Constituents
Submersed plants from the two populations showed morphological differences.
Unexpectedly, submersed plants from the high-CO2 population appeared to be less robust than their lower-CO2 counterparts (Fig. 3-1). High-CO2 plants had fewer leaves, more axillary stems, and were greener than the darker, more brownish-red lower-CO2 population plants (Fig. 3-1).
These observations were borne out by quantitative morphological measurements (Table 3-2). The weight and density of individual plants from the high-CO2 population




73
were significantly less than those from the lower-CO2 spring, and the dry weight content was also lower (11-4 versus 14-8%). High-CO2 plants also invested less in leaves. Thus, the total leaf fresh and dry weights per plant were about half of those found in the lowerCO2 plants. This was due in part to the fact that each high-CO2 plant had only 6500 of the number of leaves found on lower-CO2 plants, and only 63%o of the leaf area (Table 3-2). Total stem dry weight was less for individual plants from the high-CO2 population, even though the high-CO2 plants produced more axillary stems, which resulted in a 22000 greater total axillary stem length (Table 3-2). The average main stem length was similar between the populations, but the internodes were 2-4-times longer on the high-CO2 plants, which is in accord with the finding that plants from this population produced fewer leaves (Table 3-2). As with stem and leaves, root weights were significantly lower for the high-CO2 population plants. However, the difference in total root length was not significant (Table 3-2). Similarly, although the high-CO2 plants appeared to have higher shoot: root ratios (12-5 versus 7-7 on a dry weight basis) the difference was not significantly different.
Individual, fully-expanded leaves on submersed plants in the two populations did not differ significantly in terms of fresh and dry weights, or density (Table 3-3). However, individual leaves from high-CO2 plants were significantly larger in leaf area, but they were also thinner and averaged fewer spongy mesophyll layers, and crystals (Table 3-3). Not unexpectedly, leaf starch content was greater for plants growing in high-CO2. In contrast, leaf fiber and cellulose were greater for plants in the lower-CO2 spring (Table 3-3).




74
Figure 3-2 shows anatomical cross sections of submersed leaves from the two plant populations. The leaf from the lower-CO2 plant was slightly thicker (Fig 3-2b). In both, a single layer of epidermis and a cuticle of similar thickness were present. There was a single layer of palisade mesophyll, but it was more clearly defined and the cells were more elongated in leaves from the high-CO2 population (Fig. 3-2). Aerenchyma was absent from Ludwigia leaves; although the cells were loosely arranged and similar amounts of small, intercellular air spaces were evident in the leaves from both populations.
Stomates were present on the leaves, even though the plants were submersed. The high-CO2 plants exhibited a significantly greater stomatal density, up to ten-fold higher than the lower-CO2 plants (Table 3-3). The distribution was amphistomatous, with the adaxial surfaces of the high- and lower-CO2 plants containing, respectively, three and sixteen times more stomates per unit leaf area than the abaxial surfaces (Table 3-3).
The major stem and root characteristics are shown in Table 3-4. Stem and root diameters and densities were less in plants from the high-CO2 population, and for the stem the latter coincided with the lower dry to fresh weight value of 10 versus 16% (Table 3-2). In terms of anatomy, a single epidermal layer surrounded the stems of plants from both population, but it is notable that the high-CO2 plants contained 61% more aerenchyma and fewer cortical cell layers in the main stem cortex than plants from the lower-CO2 population (Table 3-4). Cells in the stems of the high-CO2 plants produced far fewer crystals. Unlike the leaves where starch was high, the stems of the high-CO2 plant contained significantly less starch than those of the lower-CO2 plants, although sugars, cellulose, lignin, and ash contents were greater (Table 3-4).




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In contrast to the stems, the roots did not contain aerenchyma; however, they did have a large number of small, intercellular air spaces. Concomitant with their smaller diameter, the roots of high-CO2 plants exhibited significantly fewer cell layers in the cortex and inside the cortex, and crystals within the cortical cells (Table 3-4). In terms of chemical constituents, roots from both populations were similar in their contents of TNC, starch, sugar, cellulose, and hemicellulose, but differed in total fiber, lignin and ash with the high-CO2 plant roots containing less than their lower-CO2 counterparts (Table 3-4). Reproduction
Monthly visits to the springs in 2001 revealed that flowering in the high-CO2 spring occurred from May through September, while seed capsules were present from July through November. Flower production by plants in the high-CO2 spring was abundant, while the lower-CO2 population produced almost no flowers. Virtually all the plants in the high-CO2 population that grew emergent or near the water surface produced flowers, but only about 1000 of the fully submersed plants flowered. Flowers from the high-CO2 population usually developed on tissue that was at the air-water interface or completely emergent. The number of flowers per plant was determined for the high-CO2 population in the middle of the flowering period (July), and it averaged 8-6 3-9 (n = 10). In contrast, only three plants with flowers were found in the lower-CO2 population from 1997 through 2002, and each had only a single seed capsule that was submersed and undeveloped. Plants of both populations reproduced asexually in the field by fragmentation and ramet formation.
Growth chamber data confirmed the field data regarding flower production. When the plants were maintained submersed they failed to flower (Table 3-5). When emergent,




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only plants from the high-CO2 population flowered and the number of flowers per plant were similar to values measured in the field. Increasing or decreasing the photoperiod failed to induce flowering of plants from the lower-CO2 spring (data not shown). However, the application of GA3 did induce flowering, and almost doubled the number of flowers per high-CO2 plant (Table 3-5). Plants treated with GA3 elongated more rapidly and had longer internodes than those without exposure to GA3 (data not shown).
Seed capsules produced by plants treated with GA3 were about 50% longer than those produced without GA3 (data not shown). For the high-CO2 plants, GA3 treatment did not change the number of seeds produced per capsule, which averaged 60, nor did it modify the number of seeds that subsequently germinated (90%). Flowers of the GA3induced low-CO2 plants produced very few capsules, each with only about ten seeds, none of which germinated.
Photosynthetic Characteristics
Figure 3-3 shows the effects of [02] on the net photosynthesis (NPS) rates of
submersed leaves of plants from the two Ludwigia populations. The measurements were performed at pH 5.0 with an air-equivalent [C02] of 10 mmol m-3. The NPS rates at 21% (air-equivalent) 02 were lower than at 1% 02. Thus 02 inhibited the photosynthesis of plants from both populations, with similar inhibition values of 31 and 29% for the highand lower-CO2 populations, respectively. Figure 3-4 illustrates the effects of HC03- on the NPS rates, with measurements at pH 5.0 and 9.0, but at the same [C02] of 200 mmol m3. The rates were not significantly different at the two pH values, even though the [HCO3-] was 8000-fold higher at pH 9.0.
Figure 3-5 compares the NPS rates of submersed leaves measured at the pH and
[C02] of their natural spring locations, as well as at saturating [C02]. It also includes the




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values for total Rubisco activities in submersed leaves harvested from the two populations in full sun irradiance and quick-frozen in liquid N2. When measured at their respective natural spring [C02], leaves from the high-CO2 spring had significantly higher NPS rates, by almost six-fold, than their lower-CO2 counterparts. In contrast, when measured at saturating [C02], the difference in NPS rates was much less (Fig. 3-5). Total Rubisco activities were sufficient to account for the NPS values measured at saturating [C02]. However, submersed leaves from the high-CO2 plants exhibited almost twice the Rubisco activity of those from the lower-CO2 spring (Fig. 3-5). The dark respiration rates were similar for leaves from both populations, being 13 3 and 12 2 [mol 02 m-2 leaf area h-1 for the high- and lower-CO2 plants, respectively. However, when compared to the NPS rates at the spring pH and [C02], dark respiration was equivalent to only 11% of the high-CO2 plant NPS rate, whereas it was 55% of the lower-CO2 leaf NPS rate. DNA Fingerprinting
The phenotypic differences observed between the two plant populations were reflected in the results of the AFLP analysis. The populations showed clear genetic differences with a high level of separation. Neis' genetic identity (I) and distance (D) between the populations were 0.89 and 0.11, respectively. Similarly, Wright's F-statistic (PDt) was 0.69 with a 95% C. I. of 0.48-0.87, showing that fragment variation was much greater between than within the populations. The high- and lower-CO2 populations differed at eight of the 36 loci examined. When the within-population variation was examined, the high-CO2 population differed at two loci, whereas the lower-CO2 population showed fragment diversity at five loci.




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Discussion
Both freshwater springs have been very stable in terms of temperature, pH,
alkalinity, and for this study particularly [C02] and [HCO3]1, over long periods of time, probably far exceeding the past fifty-five years for which data are available. Thus, if it is possible to adapt to the local [C02], plant populations in these springs have likely had time to do so, especially sexually reproducing populations. An effect of nitrate cannot be excluded as the concentration of this nutrient differs in the two springs, and has risen substantially in both. However, this troubling rise is relatively recent, as Fanning Springs shows the greatest increase only since the 1970s (Hornsby & Ceryak 1998). In the case of Rainbow Springs the increase has been attributed to fertilizer run-off from pastureland (Jones et a!. 1996).
Previous research using natural CO2 sources to assess the long-term responses of plants to elevated [C02] has focused on populations growing at different distances from a single CO2 source, but generally less than 1 km apart (Cook et a! 1998; Polle et a!. 200 1). As a consequence, gene flow among the populations usually cannot be excluded, and any such exchange would lessen the possibility that the population close to the source is genotypically adapted to elevated CO2. By contrast, the high- and lower-CO2 populations of L. repens in the present study were separated by 85 km. Furthermore, previous studies have not included an analysis to determine if in fact the populations differ genetically, whereas in this work they were subjected to DNA fingerprinting. The diversity index suggested appreciable genetic differentiation; greater than that found with other species (Wright 1978, Smulders et a!. 2000). Likewise, the 1100 genetic distance also indicates the two populations were genetically distinct. In fact, genetic distances of 10o or higher can be used to support categorizing two plant groups into distinct species (Nei 1987).




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Consequently, this degree of genetic separation is great enough to propose that the two populations of L. repens are adapted, rather than just acclimated, to their particular environments, however it does not exclude the possibilities that the differences were due to drift or environmental factors in addition to [C02]. The phenotypic differences that were observed in this study in reproductive, morphological, and photosynthetic characteristics are also consistent with genotypic adaptation of the two populations.
In regard to sexual reproduction, only plants from the high-CO2 population
flowered in their natural spring environment or in the growth chamber, and set viable seed. Altering the photoperiod did not induce flowering in plants from the lower-CO2 population. They only flowered when treated with GA3, and even then the few seeds formed were not viable.
Flowering and seed set was typically confined to plants with emergent shoots, not plants that remained fully submersed. Sculthorpe (1967) also noted that Ludwigia species growing entirely submersed in deep water tended to be sterile. In most instances, aquatic angiosperms produce their flowers above the water surface, as pollen is essentially a terrestrial adaptation (Sculthorpe 1967; Spencer & Bowes 1990). Both populations could grow emergent, but the water level in the high-CO2 spring fluctuated more than in the lower-CO2 spring, allowing shoots of the high-CO2 plants to become emergent more often. This may partially explain the prolific flowering in the high-CO2 population, but it does not explain the lack of flower production by the lower-CO2 population in the growth chamber experiments. The three flowers that were found on the lower-CO2 plants in Rainbow Springs, and the GA3-induced flowering, suggest that this population had some potential to reproduce sexually, but the inability of the seeds to




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germinate makes it unlikely that it did so. The constant environmental conditions of natural springs may favor asexual reproduction, and both populations were observed to reproduce by fragmentation and ramet formation. Cloning is a common phenomenon in submersed species (Sculthorpe 1967; Grace 1993). The two populations appear to differ in reproductive strategies. The high-CO2 population, with greater access to CO2 and N, devoted resources to flowering, fragmentation, and ramet formation, while the lower-CO2 plants appear to be solely dependent on fragmentation and ramet formation. It is interesting that even though the lower-CO2 population only reproduced asexually it showed greater within-population variation than the high-CO2 population, which tends to rule out a founder effect. However, more extensive genetic analysis is needed to confirm it.
Plants from both populations were C3 based on the observation that 02 inhibited their photosynthesis rates by an average of 31%, which is similar to that found with terrestrial C3 species (Bowes 1993). Furthermore, they appeared to be C02-only users, since there was no evidence that the presence of bicarbonate ions enhanced photosynthesis. Another submersed species in the genus, L. natans, was also found to be a C02-only user (Prins et al. 1980). Based on these gas exchange characteristics, it does not appear that the plants possessed any CO2 concentrating mechanism. With these photosynthesis characteristics, the growth of plants in both populations should benefit from a C02-enriched environment.
From acclimation studies with other C3 species one might expect the high-CO2
plants to exhibit lower Rubisco activity, but this was not the case. Total Rubisco activity in leaves from the high-CO2 population, and the light- and C02-saturated photosynthesis




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rates, were not down-regulated relative to values found for the lower-CO2 plants. Rubisco constitutes a large proportion of the [N] in plants therefore higher nitrate concentrations in Fanning Springs may be one of the factors maintaining the Rubisco activity in these high-CO2 plants. Because photosynthesis was not down-regulated, the naturally ten-fold higher [C02] enabled the high-CO2 leaves to exhibit much greater photosynthesis rates than their lower-CO2 counterparts when both were measured at the respective pH and [C02] of their spring environments. In fact the high-CO2 leaves probably operated at close to C02-saturated rates in their natural habitat; in contrast the leaves of lower-CO2 plants were probably at less than 20% of capacity. Similarly, a terrestrial species, L. uruguayensis, exposed to naturally high [C02] also exhibited higher photosynthesis rates than plants further away from the CO2 source (Koch 1993).
Individual leaves of high-CO2 L. repens plants were larger in area than lower-CO2 leaves, and, together with the higher photosynthesis rates, they were more effective at carbon gain in their natural habitat. However, the greater carbon gain of individual leaves has to be balanced against the fact that high-CO2 plants had 13500 longer stem internodes that resulted in fewer leaves per plant. In contrast, the lower-CO2 population had more leaves, resulting in more leaf dry weight and a 600% greater investment in total leaf area per plant. This greater emphasis on leaf area may be an adaptation to enhance CO2 capture in the Rainbow Spring habitat where CO2 appears to be a major limiting resource, and should partially compensate for the lower photosynthetic capacity per unit leaf area.
On a per unit leaf area basis dark respiration rates were similar for plants from the two populations measured under conditions akin to those in their respective habitats.




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There was no evidence for lower rates in the high-CO2 plants as has been reported for terrestrial species (Azc6n-Bieto et a!. 1994, Drake et al. 1999). It has recently been suggested that elevated CO2 has little effect on dark respiration (Jahnke 2001), and the present data are consistent with this proposal. However, when the greater photosynthesis rates and reduced leaf area are considered, dark respiration probably consumes a smaller fraction of the fixed carbon of the high-CO2 plants in their natural habitat.
The greater photosynthesis rates and Rubisco activities of the high-CO2 leaves did not translate into greater mass for individual plants. Thus far, plants growing near highCO2 springs have not shown large biomass differences compared to populations growing in ambient [C02] (Cook et a!. 1998). Plants and organs from the high-CO2 population generally exhibited lower weight and density than those from the lower-CO2 population. Thus, on a total plant basis, high-CO2 plants had a lower dry to fresh weight ratio, and all of the dry weight components measured (TNC, starch, fiber, lignin, and ash) were lower for them, only sugars ran counter to this trend. A further factor contributing to the lower density of the high-CO2 plants was the degree of aerenchyma in the stems; they had 6000 more than the lower-CO2 plant stems. Westlake (1965) has pointed out that the density of most freshwater macrophytes is largely a function of the proportion of internal air spaces.
When the plants were subdivided into their component organs, leaves made up the majority of the dry weight for both populations, although as noted earlier, there was less total leaf dry weight in high-CO2 plants. Although the weight of individual leaves from the two populations was similar, leaves from the high-CO2 population were not as thick, and had less fiber and cellulose, fewer mesophyll layers, and fewer crystals than those from the lower-CO2 population.




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The palisade mesophyll layer in the leaves of high-CO2 plants was more clearly
defined than in the lower-CO2 plants, with more elongated and tightly packed cells. Most submersed leaves have a homogeneous mesophyll (Sculthorpe 1967), but they are often in conditions where CO2 is the major limitation to photosynthesis (Bowes & Salvucci 1989; Madsen & Vadstrup 1995). In the case of the high-CO2 leaves, irradiance was likely to be co-limiting with CO2 and the organized palisade layer may enhance absorption of light.
Despite the fact that high-CO2 plants contained less TNC overall, by dusk their leaves accumulated more starch than those of the lower-CO2 plants, which is consistent with higher photosynthesis rates. Carbohydrate accumulation has been observed in some other investigations with plants growing for long periods near elevated-CO2 sources (Korner & Miglietta 1994; Cook et al. 1997; Stylinski et al. 2000). Excessive carbohydrate accumulation in leaves grown at elevated [C02] is often associated with photosynthetic acclimation (Stitt 1991; Sheen 1994), but this did not occur in the present study. Korner & Miglietta (1994) have hypothesized that such carbohydrate buildup is not due to a short-term imbalance of carbon, but rather an intrinsic inability of plants to dissipate the carbohydrate pools.
Even though the high-CO2 plants had a lower total stem dry weight, they produced far more axillary shoots, and thus had a greater total stem length, than plants from the lower-CO2 population. With more aerenchyma and lesser amounts of starch, lignin, mucilage, cortical cell layers, and crystals, the density of the stems from the high-CO2 plants was lower than those from the lower-CO2 population. Furthermore, the diameter of the axillary stems was much smaller than that of the main stems. It is possible that the




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high-CO2 population invested the additional carbon in producing axillary stems, rather than leaves, as a means of increasing asexual reproduction through fragmentation and ramet formation.
Lacuna in the plants from both populations was restricted to the stems, while the
leaves and roots contained large intercellular air spaces. It is not clear if the difference in amount of air space was related to the [C02] to which the tissues were exposed. As with the stems, the roots of high-CO2 plants exhibited less dry weight and density than those of the lower-CO2 plants.
Populations of L. repens collected in the high- and lower-CO2 springs exhibited characteristics consistent with genotypic adaptation to their respective environmental [C02]. The level of genetic difference between the populations was great enough to suggest that they have diverged with little or no gene flow between them. Plants from the high-CO2 population did not show down-regulation of photosynthesis and Rubisco activity when compared to the lower-CO2 population. It is also possible that in the highCO2 population the investment in sexual and asexual reproductive methods may be facilitated by the greater availability of resources, including CO2. By contrast, plants in the lower-CO2 spring may invest in greater leaf surface area to compensate for the limited availability Of C02, and put more resources into existing plants, rather than into prolific reproduction. It should be noted that plants in the high-CO2 springs are unlikely to show further adaptation as atmospheric [C02] rise because they already grow in an environment with [C 02] far higher than in equilibrium with the atmosphere.




Table 3-1. Water chemistry of the high- and lower-CO2 springs. The 2001 data are reported as mean SD (n = 4), while all other data
are single values obtained from the literature. The 1946 data are from Ferguson et al. (1946). Missing data from 1946 are indicated with the year the supplemental data was taken in parentheses. Data for 1956 and 1974 were from Rosenau et al.
(1977), while the 1995 data came from the Suwannee River Water Management District (Hornsby et al. 2001)
High-CO2 (Fanning) Spring Lower-CO2 (Rainbow) Spring
Parameter 1946 2001 1946 2001
Temperature (oC) 23 23 + 1 24 23 + 1
pH 7-3 72 + 0-1 7-9 7-8 + 0-1
Total alkalinity (meq L-1) 3-4 (1956) 3-7 + 0-2 1-1 (1974) 1-5 + 0-2
CO2 (mmol m-3) 390 460 + 20 45 50 + 5
HCO3- (mmol m-3) 3400 3700 + 200 1300 1500 + 180
Ct (mmol m-3) 3800 4200 + 200 1400 1600 + 200
NH4-N (mmol m-3) 0-71 (1995) 1-4 + 0-7 1-4 (1974) 0-71 + 0-35
NO3-N (mmol m-3) 31 351 + 8 13 82 + 4
Total P (mmol m-3) 2-3 (1995) 2-6 + 0-3 0-97 0-97 + 0-32
K (mmol m-3) 15 59 + 3 10 5-1 + 2-6
Ca (mmol m-3) 1700 2100 + 25 530 1500 + 25
Mg (mmol m-3) 200 230 + 4 170 240 + 4
Na (mmol m-3) 110 200 + 17 130 120 + 9
Cl (mmol m-3) 110 320 + 49 100 140 + 6
Fe (mmol m-3) 1-4 0-89 + 0-36 1-4 0-36 + 0-18




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High-CO2 plant Lower-CO2 plant
Figure 3-1. Submersed L. repens plants from the high- and lower-CO2 populations.




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Table 3-2. Weights, densities, and morphological characteristics of L. repens plants
growing in high- and lower-CO2 springs.
Parameter High-CO2 population Lower-CO2 population
Fresh weight (g plant-1) 2-31 + 0-.68** 4-42 + 1-01
Dry weight (g plant-1) 0-26 + 0-09** 0-65 + 0-15
Density (g cm-3 plant-1) 0-68 + 0-15** 0-89 + 0-15
Leaf fresh weight (g plant-1) 0-96 + 0-33** 2-27 + 0-42
Leaf dry weight (g plant-) 0-13 + 0-06** 0-35 + 0-07
Leaves (no. plant-1) 39 + 15** 60 + 11
Leaf area (cm2 plant-1) 52 + 18** 83 + 15
Total stem fresh weight (g plant-1) 1-1 + 0-4 1-5 + 0-5
Total stem dry weight (g plant-1) 0-12 + 0-04** 0-23 + 0-08
Main stem length (cm plant-1) 23 + 3 21 + 5
Main stem internode length 1-60 + 0-45** 0-68 + 0-12
(cm plant-1)
Axillary stems (no. plant-1) 9-1 + 3.4** 2-1 + 1-1
Axillary stem length (cm plant-1) 12-8 + 10-1** 4-0 + 3-2
Root fresh weight (g plant-) 0-22 + 0.06* 0-62 + 0-38
Root dry weight (g plant-) 0-020 + 0-004** 0-075 + 0-043
Total root length (cm plant-) 107 + 32 161 + 97
Mean SD (n = 8). P-values are from t-tests and significant differences between populations atP < 0-05 and 0-01 are noted by and **, respectively.




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Table 3-3. Individual leaf characteristics of L. repens plants growing in high- and lowerCO2 springs.
Parameter High-CO2 population Lower-CO2 population
Fresh weight (mg leaf 1) 52 + 17 42 + 18
Dry weight (mg leaf 1) 6-8 + 2-0 8-0 &+ 1-3
Density (g cm-3) 0-68 + 0-18 0-82 + 0-14
Area (cm2 leaf1) 2-1 + 0.6* 1-4 + 0-1
Thickness (mm leaf 1) 0-16 + 0.01* 0.18 + 0.01
TNC (mg g-1 dry weight) 115 + 59 47 + 10
Starch 85 + 41* 30 + 13
Sugars 30 + 22 17 + 10
Fiber (mg g1 dry weight) 195 + 25* 262 + 46
Lignin 39 + 22 84 + 39
Cellulose 89 + 7** 119 + 10
Hemicellulose 66 + 7 59 + 4
Ash (mg g-1 dry weight) 125 + 17 130 + 37
Spongy mesophyll cell layers 3-3 + 0.3** 4-1 + 0-5
(no. leaf 1)
Crystals (no. mm-2) 5-1 + 1-0** 23-9 + 9-1
Adaxial stomates (no. mm-2) 103 + 26** 47 + 9
Abaxial stomates (no. mm-2) 33 + 11** 3 + 2
TNC, total non-structural carbohydrate. Mean SD (n = 4-8). P-values are from t-tests and significant differences between populations at P < 0-05 and 0-01 are noted by and
**, respectively.




89
(a) (b)
30 m 30
Il* '4.
pp
t S
e epidenrmal cell
p palisade mesophyll cell s spongy mesophyll cell
c crystal
Figure 3-2.Leaf cross sections for L. repens plants growing in (a) high- and (b) lowerCO2 springs.




Full Text

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ADAPTATION AND ACCLIMATION OF POPULATIONS OF LUDWIGIA REPENS TO GROWTH IN HIGHAND LOWER-CO 2 SPRINGS By STEVEN TODD LYTLE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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This dissertation is dedicated to my wife, Stephanie Sullivan Lytle.

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ACKNOWLEDGMENTS I would like to acknowledge the members of my supervisory committee, Drs. George Bowes, Hartwell Allen, Alison Fox, Stephen Mulkey, and Alice Harmon, for contributing their time and effort in providing constructive criticism and advice throughout my degree program. I am especially indebted to Dr. George Bowes, for showing me what is required of a researcher at a large university, from the hypothesis to the final paper stage. Although not on my committee, Drs. Terry Lucansky and Karou Kitajima selflessly supplied valuable behind-the-scenes advice on many topics as well as supplying lab space and equipment. I thank Drs. Julia Reiskind and Srinath Rao, and Gonzalo Estavillo for supplying daily motivation, generosity, and good humor in my work environment. I express gratitude to Drs. Mark Whitten and Matt Gitzendanner for their assistance with genetic fingerprinting, the staff at Fanning Springs State Recreation Area and Rainbow Springs State Park for support in research collections, John McKay for aid in flower and seed studies, and Sarah Bouchard for help doing the fiber analyses. I thank Dave Nolletti for performing the C: N analysis, Nate Bazinet, Sandra Diaz, Mike Durham, and Matt Young for their assistance in maintaining plant colonies, and Andy Freenock and Wayne Wynn for their engineering advice. I would like to praise my father, Brian Lytle, for instilling the desire to pursue higher education to its fullest, and my mother, Linda Lytle, for all her support along the way. My final acknowledgement is the most important; my wife Stephanie provided iii

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endless encouragement and patience throughout the pursuit of this degree and I share this achievement with her. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 TERRESTRIAL AND AQUATIC PLANT RESPONSES TO ELEVATED CO 2 ......1 Rising Atmospheric CO 2 ..............................................................................................1 Terrestrial Plant Responses to Elevated CO 2 ................................................................1 Aquatic Plant Biology and Responses to Elevated-CO 2 .............................................24 Ludwigia Repens........................................................................................................36 Summary Statement....................................................................................................37 2 ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS.....................40 Introduction.................................................................................................................40 Materials and Methods...............................................................................................43 Results.........................................................................................................................44 Discussion...................................................................................................................47 3 CHARACTERISTICS OF SUBMERSED LUDWIGIA REPENS PLANTS FROM TWO POPULATIONS GROWING IN SPRINGS WITH DIFFERING CO 2 CONCENTRATIONS........................................................................................60 Introduction.................................................................................................................60 Materials and Methods...............................................................................................64 Results.........................................................................................................................71 Discussion...................................................................................................................78 4 THE EFFECT OF CO 2 AND EMERGENCE ON THE GROWTH OF LUDWIGIA REPENS PLANTS FROM HIGH AND LOWER-CO 2 SPRING POPULATIONS.........................................................................................................95 Introduction.................................................................................................................95 v

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Materials and Methods.............................................................................................100 Results.......................................................................................................................104 Discussion.................................................................................................................109 5 CONCLUDING REMARKS....................................................................................124 LIST OF REFERENCES.................................................................................................127 BIOGRAPHICAL SKETCH...........................................................................................145 vi

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LIST OF TABLES Table page 2-1 Characteristics of fully-expanded aerial and submersed leaves of L. repens...........54 3-1 Water chemistry of the highand lower-CO 2 springs..............................................85 3-2 Weights, densities, and morphological characteristics of L. repens plants growing in highand lower-CO 2 springs.................................................................87 3-3 Individual leaf characteristics of L. repens plants growing in highand lower-CO 2 springs....................................................................................................88 3-4 Stem and root characteristics of L. repens plants growing in highand lower-CO 2 springs....................................................................................................90 3-5 Reproductive characteristics of L. repens plants collected from highand lower-CO 2 populations and grown in a growth chamber...................................................91 4-1 Leaf area (LA) and specific leaf area (SLA) for individual, fully-expanded submersed leaves of L. repens plants taken from highand lower-CO 2 populations and grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers.............................................................................................................120 4-2 Number of axillary shoots per plant and shoot: root dry weight ratio for L. repens plants taken from highand lower-CO 2 populations and grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers.......................................122 vii

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LIST OF FIGURES Figure page 1-2 Acclimated and non-acclimated assimilation/intercellular CO 2 (A/C i ) curves for a typical C 3 plant at high light and 21% O 2 ........................................................39 2-1 Ludwigia repens growing emergent in Fanning Springs, Florida............................52 2-2 Micrograph cross-sections of aerial (A) and submersed (B) L. repens leaves.........53 2-3 Micrograph cross-section of the major vascular bundle of a submersed L. repens leaf............................................................................................................55 2-4 Micrograph cross-sections of the epidermis and cortex of aerial (A) and submersed (B) L. repens stems................................................................................56 2-5 Micrograph cross-sections of the inner cortex, vascular cylinder, and pith of aerial (A) and submersed (B) L. repens stems.........................................................57 2-6 Micrograph cross-sections of submersed water (A), and hydrosoil (B) L. repens roots..................................................................................................................58 2-7 Micrograph cross-section of the endodermis, pericycle, and stele of a hydrosoil L. repens root............................................................................................................59 3-2 Leaf cross sections for L. repens plants growing in (a) highand (b) lower-CO 2 springs..................................................................................................................89 3-3 Oxygen inhibition of net photosynthesis rates on a leaf area basis for L. repens plants growing in highand lower-CO 2 springs.......................................................92 3-4 Net photosynthesis rates on a leaf area basis for L. repens plants growing in highand lower-CO 2 springs to test for bicarbonate use.....................................93 3-5 Net photosynthesis rates and Rubisco activities on a leaf area basis for submersed leaves of L. repens plants collected from high-CO 2 and lower-CO 2 springs....................................................................................................94 4-1 Increase in dry weight per plant (% of initial) over time for L. repens plants taken from high(filled symbols) and lower-CO 2 (open symbols) populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers.................115 viii

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4-2 Relative growth rates (RGR) for L. repens plants taken from highand lower-CO 2 populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers......................................................................................................116 4-3 Ludwigia repens plants from the high-CO 2 population after 79-d growth at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers...............................................117 4-4 Increase in leaf area per plant (% of initial) over time for L. repens plants taken from high(filled symbols) and lower-CO 2 (open symbols) populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers........................118 4-5 Leaf area relative growth rate (LARGR) for L. repens plants taken from highand lower-CO 2 populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers.................................................................................................119 4-6 Total (main and axillary) stem length per plant collapsed across the highand lower-CO 2 populations for L. repens plants grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers................................................................................121 4-7 Final (day 79) C: N ratios on a dry weight basis for organs of L. repens plants taken from highand lower-CO 2 populations and grown at different [CO 2 ] in SPAR chambers......................................................................................................123 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ADAPTATION AND ACCLIMATION OF POPULATIONS OF LUDWIGIA REPENS, TO GROWTH IN HIGHAND LOWER-CO 2 SPRINGS By Steven Todd Lytle August 2003 Chair: Dr. George Bowes Major Department: Botany Characteristics of an amphibious plant, Ludwigia repens, growing in highand lower-CO 2 natural springs were examined to test the hypothesis that the populations are genetically adapted to their respective CO 2 environments. The two springs exhibited stable temperature, pH, and inorganic carbon concentrations over at least a 55-year period, and similar characteristics except that the high-CO 2 spring had greater alkalinity, inorganic carbon, P, and NO 3 concentrations, and lower pH than the lower-CO 2 spring. Plants from the high-CO 2 spring had terrestrial-like anatomy, with leaves possessing a cuticle and stomates, and stems having a cambium. Aerial leaves had a better-developed palisade and 168 and 287% greater starch grain and stomatal density, respectively, than submersed leaves. The two populations showed genetic divergence; with 11% genetic distance and 69% fragment diversity. The high-CO 2 population reproduced sexually and asexually, but the lower-CO 2 population reproduced only asexually. Submersed leaf photosynthesis x

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rates of both populations were 31% inhibited by atmospheric [O 2 ], indicating C 3 -like photosynthesis, and they did not use HCO 3 Photosynthesis rates and Rubisco activities were greater for the highthan the lower-CO 2 plants. High-CO 2 plants had twice the number of axillary shoots as lower-CO 2 plants, whereas lower-CO 2 plants had greater weight, density, leaf area and number of leaves. Shoots from both populations were grown submersed and emergent in a common garden experiment at different [CO 2 ]. Relative growth rates (dry weight and leaf area) and number of axillary shoots responded more to elevated-CO 2 in plants from the highthan the lower-CO 2 population, showing that the populations are adapted to the spring [CO 2 ]. Plants from both populations showed greater RGR, stem length, and number of axillary shoots in elevated CO 2 when emergent. At ambient [CO 2 ], neither population grew when submersed, and high-CO 2 plants senesced even after emergence. This is the first CO 2 spring adaptation study to demonstrate a genetic difference between populations growing in differing [CO 2 ] and to use aquatic plants in a common garden experiment. It shows that plants may be adapted to their natural growth [CO 2 ] and emergent plants will likely respond more than submersed plants as atmospheric CO 2 rises. xi

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CHAPTER 1 TERRESTRIAL AND AQUATIC PLANT RESPONSES TO ELEVATED CO 2 Rising Atmospheric CO 2 Fluctuations in the [CO 2 ] of the Earths atmosphere have occurred over geologic time. The atmosphere 420 million years ago may have contained over 4,000 mol CO 2 mol -1 but the last 160,000 years have seen glacial lows of 180 mol CO 2 mol -1 and interglacial highs of only 250-300 mol CO 2 mol -1 (Post et al. 1990). Atmospheric [CO 2 ] recorded at Mauna Loa, Hawaii, since the 1950s indicate a steady increase in CO 2 with our current [CO 2 ] estimated at 370 mol mol -1 (Keeling & Whorf 2002). This value is expected to double in this century as anthropogenic sources of CO 2 continue to increase. Terrestrial Plant Responses to Elevated CO 2 For the past 20-30 million years the atmosphere has had a relatively low [CO 2 ], thus plants have presumably adapted to a low-CO 2 environment. However, within the last 150 years, [CO 2 ] has been gradually rising; therefore plants may be re-adapting to the higher values (Bowes 1996). Rising CO 2 has probably already enhanced the photosynthesis, water use efficiency, and growth of many plants, especially those that perform C 3 photosynthesis (Amthor 1995; Bowes 1996; Drake et al. 1996a). The trend with the current CO 2 increase is that it is more rapid than in past periods so adaptation may lag behind. How plants adapt to rising [CO 2 ] and other global climate changes will ultimately impact primary productivity, global gas exchange patterns, and vegetation boundaries of the world (Cook et al. 1997). 1

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2 The primary reason that rising [CO 2 ] affects the physiology of plants is that CO 2 is the activator and substrate for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). Rubisco functions as a carboxylase of ribulose-1,5-bisphosphate (RuBP) using CO 2 in the photosynthetic carbon reduction (PCR) cycle, and as an oxygenase of RuBP with O 2 in the photosynthetic carbon oxidation (PCO) cycle (i.e. photorespiration). These gases compete for the active sites of Rubisco. Operation of the PCO cycle indirectly reduces photosynthesis since both processes require RuBP as a substrate and photorespiration releases previously fixed carbon. As [CO 2 ] rises, Rubiscos carboxylase activity increases at the expense of its oxygenase activity, and more CO 2 molecules are assimilated in photosynthesis. Responses of plants to elevated-[CO 2 ] are partly dependent on their photosynthesis category [C 3 C 4 or Crassulacean acid metabolism (CAM)]. Species in the C 3 category operate only the PCR cycle, whereas C 4 and CAM species also utilize a C 4 acid cycle to concentrate CO 2 at the site of Rubisco. The C 4 and CAM CO 2 concentrating mechanisms (CCM) shift Rubisco activity away from oxygenation toward its carboxylation function. In effect, [CO 2 ] is already elevated at the carboxylation site in C 4 and CAM plants, lessening the effect that rising [CO 2 ] has on plants in these categories. Approximately 95% of known plant species are in the C 3 category with examples including soybean, wheat, and most woody plants. Under present atmospheric conditions, Rubisco of C 3 plants functions below its K m meaning its active sites are not saturated with CO 2 and thus enables oxygenation (i.e. photorespiration) to occur. Photorespiration reduces photosynthesis of C 3 species by about 35% at 25C, with higher temperatures increasing this inhibition (Jordan & Ogren 1984; Bowes 1996).

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3 Stimulation of carboxylation at elevated-[CO 2 ] is usually greatest for C 3 species. Bowes (1996) estimated that doubling atmospheric [CO 2 ] should more than halve photorespiration by C 3 plants. Initially, the increase in photosynthesis is quite large, but usually there is a decline or acclimation that occurs when other resources limit photosynthesis (Sage et al. 1989). Regardless, acclimation is rarely great enough to completely negate the positive stimulation of C 3 photosynthesis that occurs at elevated-CO 2 (Drake et al. 1996a). A survey of C 3 crops by Cure and Acock (1986) showed that doubling [CO 2 ] increased photosynthesis by 52% but this enhancement dropped to 29% following acclimation. Acclimation to CO 2 Acclimation is a phenotypic adjustment that occurs in an individual organism as a response to declining performance following exposure to unfavorable levels of one or more environmental factors (Schmid et al. 1996; Lambers et al. 1998). This adjustment involves a redistribution of resources toward the most limiting processes, resulting in a re-optimization for growth and reproduction under the prevailing conditions. It is a relatively short-term phenomenon that occurs at the level of gene expression or enzyme regulation and may be contrasted with adaptation, a process that requires multiple generations to cause changes in the gene pool of a population. The shortest acclimation responses include changes in the capacity of enzyme function and occur in a timeframe from minutes to hours. Lengthier acclimation responses invoke gene effectors that modify gene expression to affect the type and amount of proteins produced. Changes in gene effectors re-establish a balance within the photosynthetic apparatus, rather than just changing the in vivo turnover rate (Sage 1994) and take hours to months to occur.

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4 Photosynthesis response to elevated-CO 2 Photosynthetic acclimation to elevated-CO 2 can result in modifications to plant anatomy, morphology, physiology and/or biochemistry (Bowes 1991). Photosynthesis can be measured directly by quantifying CO 2 consumption and/or O 2 evolution, or can be indirectly assessed by analyzing the activity and amount of individual enzymes (e.g., Rubisco) or the amount of metabolites (e.g., RuBP) involved in photosynthesis. The elevated-CO 2 acclimation response down-regulates photosynthesis. Photosynthetic acclimation can be assessed by comparing the net CO 2 assimilation rate as a function of intercellular CO 2 (A/C i curves) of plants before and after growth at elevated [CO 2 ]. Figure 1-2 shows an acclimated and non-acclimated CO 2 A/C i curves for a typical C 3 terrestrial plant at high light and 21% O 2 (Sage 1994). The shape of response of A to C i is usually that of a rectangular hyperbola and can be divided into two phases: an initial linear phase that is limited by the amount of active Rubisco present (which provides a measure of carboxylation efficiency or Rubisco capacity); and a phase that is limited by the rate at which RuBP can be regenerated from triose phosphates (e.g., ATP) in the PCR cycle (Sage 1994; Bowes 1996). Triose phosphate is limited by non-cyclic electron transport and by the rate that inorganic phosphate (P i ) is recycled. In this phase, A continues to increase as CO 2 rises because photorespiration is reduced and a greater proportion of RuBP is used in carboxylation. However, as the photorespiration effect eventually subsides, the curve levels off at the maximum assimilation rate (A max ), which is an indicator of photosynthetic or P i regeneration capacity. Changes in the shape of the curve are indicative of a re-allocation of resources within the plant. A key premise of the A/C i acclimation function is that the photosynthetic biochemistry in the steady state is regulated so the rate at which Rubisco

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5 consumes RuBP equals the rate at which RuBP is regenerated (Sage et al. 1990). The slope of the initial linear portion of the curve can decrease and/or there can be an increase in A max The decrease in the slope of an A/C i curve is an indication that the photosynthetic efficiency has decreased. At elevated CO 2 the plant consumes all of the RuBP available so Rubisco content may be reduced to enhance resource (e.g., nitrogen) use efficiency. Long and Drake (1992) calculated that Rubisco could be down-regulated by 35% in a C 3 leaf when [CO 2 ] is doubled before Rubisco would limit photosynthesis. The increase in A max is due to an increase in P i regeneration in the PCR reactions that occurs at elevated-CO 2 which leads to greater RuBP concentrations, and is an indicator of an increase in photosynthetic capacity. Sage (1994) conducted a survey of 34 published and unpublished A/C i studies, and as expected, the most common response was reduced A at low to moderate C i and increased A at high C i No pattern existed between the type of A/C i response and the life form or the ecological requirements of the species studied. Reduction in Rubisco amount, activity, and gene expression are all good indicators that a plant is acclimating to elevated-CO 2 The activity and amount of Rubisco decreased on a leaf area (LA) basis when Oryza sativa was exposed to a range of [CO 2 ] (Rowland-Bamford et al. 1991). Rubisco content of Lycopersicon esculentum decreased by 50% when plants were grown at 1000 mol CO 2 mol -1 (Besford et al. 1990). As with the gas exchange data, Rubisco acclimation exceptions do occur in the literature. Although Chenopodium album and Brassica oleracea had lower Rubisco amount at 900-1000 mol CO 2 mol -1 no differences were present for Solanum tuberosum, Solanum melongena, and Phaseolus vulgaris (Sage et al. 1989). Glycine max Rubisco activity and

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6 amount showed minimal response to manipulation of [CO 2 ] (Campbell et al. 1988). Hydroponically-grown Lolium temulentum, showed no change in maximum Rubisco activity, but did have greater Rubisco protein at double-[CO 2 ] (Lewis et al. 1999). Decreased expression of mRNA genes that encode Rubisco can also serve as an indicator of acclimation to enriched-CO 2 Long-term exposure of Arabidopsis thaliana to high-CO 2 caused the mRNA for the large and the small subunits of Rubisco to decrease by 35-40 and 60%, respectively, with a subsequent reduction in the expression of Rubisco protein (Cheng et al. 1998). The correlation between mRNA transcripts and protein levels of Rubisco is not always tightly coupled. Gesch et al. (1998) observed a decrease in the small subunit transcript within 24 hours after switching O. sativa plants from 350 to 700 mol CO 2 mol -1 ; however, Rubisco amount and total activity showed no response, even eight days after the switch. The decrease in Rubisco protein found in 12 out of 16 crop species grown at 1000 mol CO 2 mol -1 showed no specific association with changes in transcript levels for the Rubisco small subunit (Moore et al. 1998). Causes of the acclimation response A variety of hypotheses for what causes photosynthetic acclimation have been proposed, and all are related to either the photosynthetic machinery or other physiological processes not being able to keep pace with the increasing C availability. Acclimation can occur due to reallocation of resources from non-limiting C acquisition processes to those that may be more limiting, such as light harvesting, electron transport, and carbohydrate handling (Sage et al. 1987; Bowes 1991; Sage 1994). Acclimation may occur because the additional C cannot be transported to sinks or storage rapidly enough. Therefore, the response should be to either down-regulate photosynthesis or to up-regulate carbohydrate metabolism and N assimilation.

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7 Explanations for the effect that carbohydrates have on down-regulation of photosynthesis have focused on the increased flux as well as accumulation of carbohydrates. Current research in this area points toward the increased flux of carbohydrates as being the trigger for the signal that leads to the down-regulation of photosynthesis. Specifically, hexokinase has been identified as being important in the acclimation signaling process (Jang & Sheen, 1997; Moore et al. 1998; Moore et al. 2003). Hexose sugars generated in photosynthesis are phosphorylated by hexokinase, so that its activity is increased as sugar flux is increased at elevated-CO 2 A plant's capacity to export photosynthate and its sink size may be insufficient at elevated [CO 2 ] so that increasing carbohydrate pools lead to feedback inhibition of photosynthesis (Stitt 1991; Sheen 1994). Also, extreme cases of starch accumulation may distort chloroplasts (Madsen 1968; Woodward et al. 1991). Empirical data show a variety of non-structural carbohydrates may be involved in the down-regulation of photosynthesis. Elevated CO 2 (700 mol CO 2 mol -1 ) enhanced non-structural carbohydrate accumulation in leaves of five Trticum aestivum cultivars showing photosynthetic down-regulation (Barnes et al. 1995). Both Flaveria and Panicum species showed photosynthetic acclimation at 700 mol CO 2 mol -1 but the species differed in carbohydrate accumulation; Flaveria accumulated high levels of starch, while Panicum built up stores of sugars (Leonardos & Grodzinski 2000). However, others have deduced that higher total non-structural carbohydrate and starch content is not related with the photosynthetic down-regulation (Moore et al. 1998; Van der Kooij et al. 1999).

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8 The C and N metabolism pathways must be coordinated precisely to achieve an appropriate balance between supply and demand so that a plant can build the appropriate macromolecules. Carbon and N metabolism compete for ATP, reducing power, and C skeletons. Elevated CO 2 normally modifies the C: N balance of plant tissues (especially leaves) toward C. Oryza sativa, Gossypium hirsutum, Zea mays, and T. aestivum grown at higher-[CO 2 ] had lower leaf [N] (Rowland-Bamford et al. 1991; Wong 1979; Hocking & Meyer 1991a, b). Exceptions do exist, with C. album, S. tuberosum, S. melongena, and B. oleracea indicating no difference in leaf [N] at enriched-CO 2 (Sage et al. 1989). The extremity of the elevated-CO 2 acclimation effect is partly dependent on whether there is sufficient N available in the plant, especially if other resources are adequate. Gossypium hirsutum, Z. mays, and hydroponic O. sativa showed greater photosynthetic down-regulation when supplied with lower nitrogen in elevated CO 2 (Wong 1979; Nakano et al. 1997). To avoid the negative effects of the C: N ratio shifting too far toward C, the acclimation response should be to favor N assimilation, export, and re-mobilization over C assimilation. The initial regulation response should be to lower the activity of Rubisco, which then might be followed by a reduction in the amount of Rubisco (Bowes 1996). In low-[N] treatments grown at high-CO 2 G. hirsutum and Z. mays both had lower Rubisco activity (Wong 1979), while tobacco had decreased Rubisco content (Walch-Liu et al. 2001). Dark Respiration The effect that elevated [CO 2 ] has on dark respiration has received less attention than photosynthesis, even though these processes are closely linked. Half or more of the C assimilated during photosynthesis may be lost as CO 2 during subsequent respiration (Allen & Amthor 1995). Respiration serves as the primary means for supplying a plant's

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9 cells with C skeletons (intermediates), usable energy (ATP), and reductant (NADH) required for growth, maintenance, transport, uptake, and nutrient assimilation processes. It is essential to distinguish between direct and indirect effects of CO 2 on respiration. Direct effects are indicated by immediate responses that can be rapidly relieved by reducing [CO 2 ] to its original level (Amthor 1991; Bowes 1996). Indirect effects occur after growth in elevated [CO 2 ] for extended periods (Amthor 1991), and are acclimation effects that cannot be reversed quickly. Direct effects of elevated-CO 2 on respiration generally involve a decrease in respiratory CO 2 release (Drake et al. 1999). A survey across a variety of species and tissues (leaves, roots, and shoots) by Drake et al. (1996b) concluded that specific respiration was reduced by about 20% at twice ambient-CO 2 Doubling the [CO 2 ] in which G. max cotyledons were grown decreased their mitochondrial O 2 uptake (Gonzlez-Meler et al. 1996), and inhibition of CO 2 evolution by Rumex crispus leaves and Oryza sativa canopy was observed at high-[CO 2 ] (Amthor et al. 1992; Baker et al. 2000). However respiration does not always decrease at elevated-CO 2 and there are recent reports that the effects can be attributed to experimental artifacts (Jahnke 2001; Jahnke & Krewitt 2002). The mechanism responsible for the direct inhibition of respiration has not been clearly defined (Allen & Amthor 1995), although it may be related to a decrease in the activity of two key enzymes of the mitochondrial electron transport chain, cytochrome c oxidase and succinate dehydrogenase (Drake et al. 1996b). The activities of these enzymes were inhibited at elevated [CO 2 ] for mitochondria isolated from G. max cotyledons (Gonzlez-Meler et al. 1996).

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10 Indirect respiration effects are dependent on the impact CO 2 has on physiological mechanisms that affect growth and maintenance. Determining the causes of indirect effects on respiration can be complicated since many factors are involved. Respiration effects are mediated through the influence of CO 2 on processes that supply substrates of respiration and use respiration intermediates and end products (Amthor 1994). Respiration slows when respiratory products accumulate in cells, and increases when the products are consumed, through a series of feedback mechanisms (Amthor 1995). Several authors indicate that rising CO 2 indirectly reduces respiration through the changes it causes in plant tissue composition (Amthor 1995; Drake et al. 1996a). As CO 2 rises, the C: N ratio of plants usually shifts toward C. A decrease in N (and therefore, protein) concentration results in a decrease in specific maintenance respiration since tissues lower in N are less costly to synthesize and maintain (Amthor 1995; Drake et al. 1996a). Respiration rates were decreased at elevated [CO 2 ] in stems of Scirpus olneyi and leaves of Lindera benzoin, while Spartina patens leaves were unaffected (Azcn-Bieto et al. 1994). Greater specific respiration rates in subambient-[CO 2 ] treatments were associated with higher plant tissue [N] for O. sativa (Baker et al. 1992). Growth and morphology of C 3 species Generally, growth rates of plants are stimulated at elevated [CO 2 ]. The stimulation may be due to greater tillering or branching, leaf area (due to greater area per leaf and more leaves per plant), and leaf thickness which all result in greater total plant photosynthesis (Acock & Allen 1985; Smith et al. 1987; Allen 1990). Besides providing more of a limiting resource, additional CO 2 has the potential to improve the use of other resources (Bowes 1993). Nitrogen use efficiency (NUE) and water use efficiency (WUE)

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11 may both increase at elevated CO 2 Therefore, even when other resources are low and limit photosynthesis, the additional CO 2 may still enhance growth (Bowes 1996). Growth and photosynthetic responses to elevated [CO 2 ] are partly dependent on the other resources to which the plant is exposed. Kimball et al. (1990) showed that growth of cultivated plants in well-tilled soil with abundant nutrients will respond positively to rising-CO 2 over their entire life span. There is much less research published on how natural plant communities respond to enriched-CO 2 but increased competition for other resources may limit growth enhancement (Fordham et al. 1997a). Most researchers have concluded that growth of C 3 species increases with CO 2 enrichment. Yields of a combination of crop and non-crop C 3 species were enhanced by an average of 33% when ambient [CO 2 ] was doubled (Kimball 1983). A survey by Poorter (1993) reported that average dry weight for vegetative plants of both cultivated and wild C 3 species increased by 41%, with wild species showing a 35% increase. Examples in the literature of CO 2 not stimulating the growth of C 3 plant species are rare, however, the annual C 3 species, Cardamin hursuta, Poa annua, and Spergula arvensis showed no response to CO 2 (Leishman et al. 1999). Morphological changes caused by elevated [CO 2 ] show common trends in C 3 species, including initiation of new organ growth, a shift in the partitioning of biomass toward roots, greater leaf area, and a decrease in specific leaf area (SLA). Increased branching and tillering in plants grown at enriched-CO 2 is common since increases in photosynthesis lead to accumulation of carbohydrates that are either used in new growth or stored. The grasses Bromus tectorum, Oryzopsis hymenoides, and Agropyron smithii all had more basal stems when grown at high-CO 2 (Smith et al. 1987). Whether grown in

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12 a phytotron or hydroponically, T. aestivum cultivars grown at elevated CO 2 showed a stimulated tillering response (Barnes et al. 1995; Monje & Bugbee 1998). Not all experiments verify the increase in tillering though; Leishman et al. (1999) grew C. hursuta, P. annua, S. vulgaris, S. arvensis at 550 mol CO 2 mol -1 and found no tiller effects. Since leaves and shoots provide the apparatus for photosynthesis, which is likely saturated at elevated-CO 2 a plant might benefit by investing more in root tissues to increase access to resources other than CO 2 The root: shoot ratio of C 3 plants generally increases at enriched-CO 2 Two cultivars of Oryza sativa had their root: shoot ratios increased by 24 and 92% when they developed under high-CO 2 (Ziska & Teramura 1992). Leaf area of C 3 plants is often enhanced when CO 2 is increased, although the effect is not usually a major one. The grasses B. tectorum, O. hymenoides, and A. smithii all had greater LA when grown at elevated-CO 2 (Smith et al. 1987). Tobacco had more and larger leaves at 700 mol CO 2 mol -1 than at ambient-CO 2 (Backhausen & Scheibe 1999). In contrast, C. hursuta, P. annua, S. arvensis, and L. esculentum showed no LA enhancement in response to increased CO 2 (Leishman et al. 1999; Besford et al. 1990). An aspect of leaf growth that is more sensitive to CO 2 is SLA. Specific leaf area on an individual and total leaf basis typically decreases because leaves get thicker at enriched-CO 2 Ten Acacia tree species grown at high-CO 2 had lower SLA and this was attributed to increased thickness of the leaves (Atkin et al. 1999). Sage et al. (1989) noted that C. album and B. oleracea had lower SLA in elevated-CO 2 treatments, while S. tuberosum, S. melongena, and P. vulgaris did not. Finally, the SLA response of three

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13 Oryza sativa cultivars to high-CO 2 was the opposite expected; SLA increased (Rowland-Bamford et al. 1991; Ziska & Teramura 1992). Greater leaf thickness is due to the rapid development of leaves during lamina differentiation and cell enlargement (Acock & Allen 1985). Leaf thickness increased in Panicum tricanthum exposed to 900 mol CO 2 mol -1 (Tipping & Murray 1999) and G. max grown at 800 mol CO 2 mol -1 had 37% greater leaf thickness due to an increase in the number of palisade cells (Vu et al. 1989). Stomates As atmospheric CO 2 increases, more CO 2 diffuses into leaves, therefore the degree of stomatal opening and the density of stomates should decrease (Woodward 1987; Bowes 1996). Plants normally show partial stomatal closure at higher than present atmospheric CO 2 (Morison 1985), however, there are reports of enormous variation between species (Jarvis & Mansfield 1999). When exposed to high CO 2 abaxial stomatal density decreased by 22% for P. tricanthum (C 3 ) (Tipping & Murray 1999). Stomatal density increased in Oryza sativa plants as [CO 2 ] was increased at intervals from 160 to 330 mol CO 2 mol -1 but then showed no further increase at intervals from 330 to 900 mol CO 2 mol -1 (Rowland-Bamford et al. 1990). A decrease in stomatal opening results in a decline in stomatal conductance. Stomatal conductance is often correlated with changes in photosynthesis, so that as photosynthesis is down-regulated, conductance also decreases (Drake et al. 1996a). Conductance is usually regulated to maintain C i within narrow limits (Morison 1985). Using 41 observations covering 28 species, the average reduction in stomatal conductance due to a rise in CO 2 was 20% (Drake et al. 1996a). All the grass species [B. tectorum (C 3 ), O. hymenoides (C 3 ) A. smithii (C 3 ) E. orcuttiana (C 4 )] tested by Smith et

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14 al. (1987) showed a decrease in stomatal conductance when grown at 680 mol CO 2 mol -1 rather than ambient-CO 2 Anatomy Minimal research has been done to determine the effects of elevated CO 2 on anatomical features of plants. Most studies thus far have tried to elucidate what role anatomy plays in the increased growth and photosynthetic responses when plants are grown at high-CO 2 Changes in leaf anatomy may influence a plants capacity to assimilate carbon, and thus be associated with the photosynthetic down-regulation that occurs at high-CO 2 (Pritchard et al. 1999). The magnitude of anatomical changes hinges upon plant genetic plasticity, nutrient availability, temperature, and phenology (Pritchard et al. 1999). Of all the plant organs, leaves exhibit the greatest structural plasticity in response to environmental conditions (Esau 1977). Although variable, leaves typically have greater rates of expansion and therefore produce larger leaves in high-CO 2 environments. Greater leaf size can be reflected in lamina thickness, which is often correlated with changes in internal anatomy. No single process has been identified as being responsible for producing greater leaf size, however, cell expansion plays a greater role than enhanced cell division (Pritchard et al. 1999). Increased leaf growth may be due to increased cell expansion, resulting in larger cells (Populus clones, Radoglou & Jarvis 1990; Plantago media, Taylor et al. 1994; Sanguisorba minor, Lotus corniculatus, Anthyllis vulneraria, Ferris & Taylor 1994). Increased leaf expansion rate and final leaf size were correlated with greater epidermal cell size and number in Populus nigra and P. euramericana, while P. alba only had increased epidermal cell number (Ferris et al. 2001). Other researchers single out an increase in cell number as being the factor

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15 contributing to increased leaf growth in high-CO 2 Glycine max grown at 800 mol CO 2 mol -1 had an increased number of palisade cells and leaf thickness (Vu et al. 1989). Other studies have focused on the proportion of the different tissue layers produced at enriched-CO 2 Mesophyll and vascular tissue cross-sectional areas and volumes typically increase, with the mesophyll becoming more clearly defined. Also at high-CO 2 leaves tend to have more dense tissues with smaller cells and less internal spaces. When grown at enriched-CO 2 the second trifoliate leaf of P. vulgaris had reduced epidermal and intercellular space volume and cell area (Bray & Reid 2002). Also, leaf thickness, palisade cell length and volume density of the spongy mesophyll and palisade mesophyll were greater for P. vulgaris plants grown at high versus ambient [CO 2 ]. Other studies also report increased mesophyll cross-sectional area in leaves (Populus trichocarpa, Radoglou & Jarvis 1990). In addition to a decrease in intercellular spaces, an extra layer of palisade cells was observed in G. max grown in high CO 2 (Vu et al. 1989). However, reductions in mesophyll have also been reported (Pinus ponderosa, Pushnik et al. 1995). Vascular tissue area increased in P. ponderosa and L. platyglosa leaves exposed to high-CO 2 (Pushnik et al. 1995; St. Omer & Horvath 1984). Even though stems are important in mediating the flux of resources acquired above and below the ground, studies relating stem anatomical characteristics with rising CO 2 is scarce. Elevated-CO 2 increases stem growth primarily by stimulating cell division within shoot apices (Pritchard et al. 1999). Increased stem diameter has been reported for many species growing under elevated-CO 2 Pinus radiata had greater stem diameter when grown at high CO 2 (Conroy et al. 1990) and Sigurdsson et al. (2001) concluded that P. trichocarpa showed at increase in stem diameter at high-CO 2 and nutrient availability.

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16 Still Pushnik et al. (1995) found no increase in secondary growth of P. ponderosa stems. A review by Pritchard et al. (1999) showed that stem secondary growth normally increases elevated CO 2 Although roots often exhibit the greatest relative increase in biomass of all plant organs when grown in elevated CO 2 there are few studies on root anatomical responses (Pritchard et al. 1999). Increase in girth of roots has been found in some experiments. There was a 27% increase in root diameter in the root hair zone, a 23% rise in stele diameter, and a 28% escalation in cortex width in G. max grown in elevated CO 2 (Rogers et al. 1992). Pinus taeda also had larger root diameters in high-CO 2 treatments (Larigauderie et al. 1994). These results notwithstanding, St. Omer and Horvath (1984) found no difference in stele or tracheary element diameter of Layia platyglossa roots grown at higher than ambient-CO 2 levels. CO 2 Adaptation In contrast to acclimation, adaptation is a change in a populations gene pool induced by an environmental change over multiple generations due to drift, migration, or selection. Research on long-term effects of CO 2 on plant adaptation is lacking. There is great urgency to understand the implications of our future CO 2 atmosphere, but even for short-lived species, periods of ten or more years are often required for genetic shifts in the population (Miglietta et al. 1993). Elevated-CO 2 research, which is aimed at predicting responses expected to occur over decades to centuries, is often conducted over periods that are considerably shorter than may be required for full expression or equilibrium of the responses (Koch 1993). The general rule is the longer the experiment, the better, however, empirical studies of relatively short duration will continue to be used to support models that simulate processes over the longer time-scales of relevance.

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17 Several long-term experiments have been initiated, but they are still in their infancy (Field et al. 1996). Different species and cultivars vary widely in their response to elevated CO 2 yet little is known about whether variation also occurs within natural populations (Curtis et al. 1994). There are three main categories of elevated-CO 2 adaptation research. In the first type, herbarium and fossil specimens are examined for differences that may be related to the [CO 2 ] in which they grew. In the second method, plants of the same species, but with different genotypes (often established cultivars), are exposed to similar environments and their responses to elevated [CO 2 ] are monitored. The third strategy utilizes natural settings (e.g., geothermal gas vents) in which high [CO 2 ] have prevailed over evolutionary time. Plants growing near natural CO 2 sources may be adapted to elevated [CO 2 ] relative to populations of the same species growing outside the CO 2 plumes (Sage 1994). A criticism of the natural experiments is that the researchers do not verify that genetic differences exist between the populations being compared, and the differences that show up may not reflect adaptation. Just because a species exhibits certain traits in a particular environment, does not mean that these traits are beneficial and resulted from natural selection. Comparisons of herbarium and fossil specimens versus present-day species have generally shown a decrease in the number of stomates and stomatal density over time as [CO 2 ] has increased (Wagner et al. 1999). Using four collection dates over the last 3000 years, Beerling and Chaloner (1993) deduced that ancient leaf material of Olea europaea showed a decrease in stomatal density as CO 2 increased. Parallel to an increase in CO 2 from AD 1720 to present, there were decreases in stomatal and epidermal cell density for

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18 14 species of trees, shrubs, and herbs collected and stored as dried herbarium specimens, while stomatal index was unchanged (Peuelas & Matamala 1990). However, there are also instances reported where CO 2 does not influence stomatal characteristics. Krner (1988) looked at over 200 plant species and found no significant difference in overall stomatal density between 1918 and 1985. Different cultivars of the same species often diverge in their CO 2 response. When five cultivars of T. aestivum were grown at elevated CO 2 net assimilation was enhanced by an average of 45% across cultivars, however, this enhanced rate was eventually down-regulated by 15% in only two of the five cultivars (Barnes et al. 1995). Wulff and Alexander (1985) found differences in the germination and growth of progeny derived from five maternal families of Plantago lancelolata when grown at 675 mol CO 2 mol -1 Some families responded much more to CO 2 than others, suggesting that there may be genetic variation in response to CO 2 during seed maturation and seedling development. Progeny from five families of wild radish, Raphanus raphanistrum, were grown at double [CO 2 ] and showed stimulation in net assimilation rates, and flower and seed production across paternal families (Curtis et al. 1994). However, in three families there were no significant CO 2 effects, while in one family lifetime fecundity increased by 50%. These genotype-specific effects altered fitness rankings of the five paternal families providing evidence for heritable responses to elevated CO 2 Little is known about variation across natural plant populations (Curtis et al. 1994). Perhaps the only situation in which terrestrial vegetation chronically and naturally experiences greatly increased ambient-CO 2 levels is in areas close to natural discharges of CO 2 from geological sources (Koch 1993). Geothermal gas vents such as springs,

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19 geysers, and volcanoes emit CO 2 with the CO 2 from natural spring vents approaching 99% purity (Cook et al. 1997; van Gardingen et al. 1997). The [CO 2 ] in the air around geothermal gas vents can be as high as 10,000 mol CO 2 mol -1 (Miglietta & Raschi 1993). These high-CO 2 environments allow researchers to examine plant responses over time-scales that are much greater than possible with human-designed experimental systems (Koch 1993). The environmental conditions may be assumed to have occurred for hundreds of years, with vegetation around these vents subjected to a [CO 2 ] gradient, with a decreasing concentration as the distance from the vents increases (Miglietta et al. 1993). Research testing the effect that the elevated-CO 2 has on plants growing in these environments has only been instituted for about a decade. The most positive aspect of this research is that the CO 2 effects on the plants are long-term, therefore fully acclimated physiological responses, which may be adaptations, can be examined. Another significant characteristic is that the observations are made in a natural setting. Also, there is no need to set up expensive, large-scale Free Air CO 2 Enrichment (FACE) experiments. A negative characteristic of CO 2 vent experiments is that they cannot be replicated. Also, there is temporal and spatial heterogeneity of the [CO 2 ] in the air surrounding the vent due to diffusive, convective, and turbulent mixing. Additionally, when comparing plants growing in elevated-CO 2 near the vents versus plants in nearby lower-CO 2 sites, there is often diversity in the two environments other than [CO 2 ]. Heterogeneity of background environmental factors around a natural-CO 2 source may complicate comparisons to ambient-CO 2 sites and make it difficult to isolate CO 2 effects on vegetation characteristics. Finally, phytotoxic gases are often present in these sites.

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20 Most natural CO 2 source experiments take place in spring environments in which toxic gases are not released and that have moderate temperatures as opposed to the high temperatures found near volcanoes and geysers. A natural spring is created when water is discharged as leakage or overflow from an aquifer through an opening in the ground. Carbon dioxide from the atmosphere and from plant and microbial respiration combines with water as it percolates through the ground to form carbonic acid (H 2 CO 3 ). Other acids from organic matter may also combine with the solution (Nordlie 1990). This H 2 CO 3 solution reacts with the bases of rocks and can dissolve tunnels deep into bedrock (Ferguson et al. 1947). Limestone of calcium-enriched rock is especially solubilized by H 2 CO 3 to produce calcium bicarbonate (Ca(HCO 3 ) 2 ), thus increasing Ca 2+ and HCO 3 ions in the water (Wetzel 1983). Bicarbonate may then be converted to free CO 2 depending on the pH of the water the spring effluent enters. Water confined in the dissolved cavities by overlying sediments creates a pressure head that cracks open the confining rock bed further down-slope, releasing it to the surface and creating springs (Nordlie 1990). Carbon dioxide and other gases are released steadily from mineral springs as gas-laden waters ascend and effervesce upon reaching atmospheric pressure (Koch 1993). In its history, Florida was repeatedly inundated by marine water, thereby layering it with limestone produced as marine sediments were eventually covered by clay and sand (Nordlie 1990). Since the Florida aquifer is mainly composed of limestone it is easily dissolved by carbonic acid solution. The high solubility of limestone rock by natural waters is probably the most important factor responsible for the large number of springs (over 300) in Florida (Ferguson et al. 1947; Stamm 1994). Surveys indicate that the

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21 physical and chemical makeup of many of Florida's freshwater springs have changed little over a thirty-year period encompassing the 1940s to 1970s (Ferguson et al. 1947; Rosenau et al. 1977). There are two main types of experiments using elevated-CO 2 springs. The first compares a population of plants growing near a high-CO 2 vent with a population in the same ecosystem but further from the vent where [CO 2 ] is near ambient. These experiments attempt to uncover adaptation phenomena, but all experiments thus far have disregarded testing to determine if the populations differ genetically. Often the populations are in close proximity so genetic exchange is likely, therefore lessening the likelihood of genetic differences. Correlations between physiology and environment in the field provide a basis for interesting physiological hypotheses, but these hypotheses can rarely be tested without complementary approaches such as growth experiments or phylogenetic analyses. The second kind of test involves collecting individual seed (preferably) or seedlings from the highand ambient-CO 2 springs, and growing them in a common garden (Clausen et al. 1940). Any differences in CO 2 responses that arise between the populations are assumed to be due to adaptations. The problem is that the plants may differ due to initial non-genetic variability in their mother material created by growth in different environments. Many experiments have been conducted in which plants growing near to and further from natural CO 2 springs are compared, but there is still great uncertainty in drawing conclusions from the data. However, typical high-CO 2 acclimation responses seem less prevalent in the terrestrial plant populations growing near the high-CO 2 springs, thereby pointing to adaptation of these populations to high [CO 2 ]. Thus far it has

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22 not yet been demonstrated that exposure to long-term elevated-CO 2 from springs produces large differences in biomass (Miglietta & Raschi 1993; Miglietta et al. 1993; Krner & Miglietta 1994). The photosynthesis rate of Scirpus lacustris did not differ between highand ambient-CO 2 sites (Miglietta et al. 1993; Bettarini et al. 1997). Scirpus lacustris responded to growth in elevated-CO 2 with a decrease in stomatal density but no effect on C i : C a ratio or N content (Bettarini et al. 1997). Photosynthesis was not reduced in Quercus pubescens growing closer to high-CO 2 springs (van Gardingen et al. 1997), but this contradicted earlier work by Miglietta and Raschi (1993) in which the photosynthetic capacity of Q. pubescens leaves was down-regulated. Miglietta and Raschi (1993) also compared leaf anatomy of Q. pubescens, and found little evidence of acclimation in the high-CO 2 population. Stomatal and epidermal cell numbers were the same at the two sites, but size of guard cells was reduced in leaves of plants grown in the enriched-CO 2 atmosphere. Bettarini et al. (1998) concluded that long-term exposure to elevated-CO 2 in the spring did not cause adaptive modification in stomatal number and distribution for 17 species. Other experiments indicate that terrestrial plant populations growing near spring vents are not fully able to utilize the high levels of CO 2 and shows acclimation responses. When compared to an ambient population, Nardus stricta growing near a spring with 790 mol CO 2 mol -1 showed reduced photosynthetic capacity, Rubisco content and activation state, and chlorophyll content (Cook et al. 1997). Spring plants also had lower leaf [N] and greater starch in some of the years sampled, both of which could have contributed to acclimation. Earlier senescence, greater leaf area index, and lower SLA were also traits of the plants growing in high-CO 2 Leaf photosynthetic capacity, stomatal density, and

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23 conductance were down-regulated in Phragmites australis plants growing nearest to spring vents (van Gardingen et al. 1997). Krner and Miglietta (1994) compared multiple grassland herbs and forest trees growing around a spring at 500-1000 mol CO 2 mol -1 with others growing in ambient-CO 2 Although they found no evidence that plants in the spring area grew larger or flowered earlier, TNC accumulation (especially starch) and N depletion occurred in most herbaceous and tree leaves growing under elevated-CO 2 There has been only one elevated-CO 2 spring experiment that used an aquatic plant. Koch (1993) compared populations of Ludwigia uruguayensis, an emergent aquatic species, growing at various distances from a CO 2 spring, but even this study focused on the aerial parts, rather than the underwater portions, of the plant. Ludwigia uruguayensis plants growing nearest to the spring showed no evidence of photosynthetic down-regulation, even though more starch accumulated. Primarily as a result of the increased starch levels, there was also a decrease in SLA for plants growing closer to the vent. True adaptation experiments involve collecting seed or small seedlings from highand ambient-CO 2 sites with otherwise similar environmental conditions, and growing them under a common [CO 2 ] in what is known as a common garden experiment. By using only a portion of a plant or its offspring in these experiments the environmental effects are kept to a minimum while the genetic effects are maximized. A criticism of these studies is that there have been no genotypic analyses of the populations to go with the physiological comparisons. The populations must be genetically distinct to imply adaptation has occurred.

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24 The majority of the common garden experiments using terrestrial populations suggest that the high-CO 2 plants may be adapted to their high-CO 2 environment. Scirpus lacustris grown in the laboratory from rhizomes collected in CO 2 springs did not have reduced photosynthetic capacity when compared with those collected from control sites (Bettarini et al. 1997). Fordham et al. (1997a) selected Agrostis canina seed from a spring with [CO 2 ] ranging from 451-610 mol CO 2 mol -1 and grew them in the lab at ambient and 700 mol CO 2 mol -1 Elevated-CO 2 stimulated the growth of all of the populations, and the seeds taken from closer to the spring produced plants with greater initial relative growth rate (RGR) irrespective of chamber [CO 2 ]. The weight of the seeds was positively correlated with the [CO 2 ] where they were collected, but these differences only accounted for a portion of the variation in RGR. Plantago major plants originating from a high-CO 2 spring had intrinsically greater biomass and RGR than two populations growing at ambient-CO 2 when both were grown in 350 and 700 mol CO 2 mol -1 treatments (Fordham et al. 1997b). Differences in original seed weight only explained a small portion of the variation in RGR between populations of P. major. Seed from spring populations of Boehmeria cylindrica growing at varying [CO 2 ] from 350-550 mol CO 2 mol -1 were grown in controlled environments at 350, 525, and 675 mol CO 2 mol -1 (Woodward 1987). Differences between populations in plant growth were noted only at the highest CO 2 treatment; at 675 mol CO 2 mol -1 the high-CO 2 population had the greatest growth and height. Aquatic Plant Biology and Responses to Elevated-CO 2 Aquatic plants have lower dry to fresh weight ratios than terrestrial plants, and those that are completely submersed have the lowest ratios (Spencer & Bowes 1990). Freshwater is 775-times denser than air, and provides almost 1000-times greater

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25 buoyancy, which renders extensive structural tissue in submersed plants superfluous (Sculthorpe 1967). Another major difference from terrestrial plants is that the shoots of submersed plants contribute to the overall nutrition by absorbing minerals from the surrounding aquatic medium. Although aquatic plant roots were regarded only as anchorage devices, it has since been shown that they are involved in nutrient uptake. Barko et al. (1991a) concluded that N, P, Fe, Mn, and micronutrients are primarily taken up from the sediments, while Ca, Mg, Na, K, SO 4 and Cl are mainly absorbed from the surrounding water. Terrestrial and emergent plants are generally more productive than submersed species, with production by floating plants intermediate (Spencer & Bowes 1990). Per unit area, some emergent aquatic plant communities are among the most productive of the worlds vegetation types due to ample provision of water and nutrients, two factors that commonly limit plant growth on land (Wade 1990). Although seeds are of paramount importance to the reproductive phase of terrestrial species, aquatic plants tend to have greater reliance on vegetative reproduction as their principle mode of population growth (Wade 1990). Vegetative reproduction occurs via fragmentation, creeping stems (layers, runners, stolons, rhizomes, and stem tubers), modified shoot bases (bulbs, corms), root suckers (creeping roots, tap roots, root tubers), and pseudovivipary (Grace 1993). The buoyant and protective nature of water makes the aquatic environment extremely favorable for the dispersal of clonal propagules (Grace 1993). Sexual reproduction produces offspring with variability, with the possibility that some will be able to survive when conditions change or when the species invades a

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26 different environment. But for a plant that is well adapted to prevailing conditions (such as those found in aquatic waters), vegetative reproduction with minimal variety in the offspring ensures they will be similarly suited and successful in a consistent environment (Spencer & Bowes 1990). Photosynthesis in the Aquatic Environment The photosynthetic mechanisms that aquatic plants employ are based partly on their habit (e.g., submersed, emergent, or floating). Obviously each of these habits allows different access to the resources present in air and water. Light, nutrients, and dissolved inorganic carbon (DIC) can all limit the photosynthesis rates of aquatic plants in the field. The photosynthesis rates of submersed species are typically low when compared to terrestrial and aerial species, and this trend is maintained even at lightand DIC-saturation (Van et al. 1976; Salvucci & Bowes 1982; Spencer & Bowes 1990). The light environment in aquatic ecosystems is highly variable. Attenuation of light with water depth is common (Wetzel 1983), so that both the quality and quantity of light vary with depth (Spencer & Bowes 1990). Low irradiance often limits the photosynthesis and growth of submersed plants. Submersed plants invariably can be categorized as shade plants, because leaf photosynthesis is saturated at an irradiance of less than half full sunlight, even for those species that inhabit shallow waters (Salvucci & Bowes 1982; Wade 1990). In terms of nutrients, comparisons of tissue and freshwater nutrient concentrations suggest that N and P are most likely to limit photosynthesis and growth under natural conditions (Raven 1984). Low tissue [N] has been correlated with a reduction in photosynthetic capacity, carbon affinity, chlorophyll content, and growth of submersed macrophytes (Gerloff & Krombholz 1966; Madsen & Sand-Jensen 1987; Van Wijk

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27 1989). Photosynthetic capacity, HCO 3 uptake capacity, and Rubisco activity of Elodea canadensis were all enhanced when N was increased in hydroponic cultures (Madsen & Baattrup-Pedersen 1995). Inorganic Carbon in the Aquatic Environment There is great spatial and temporal variability in the concentration of inorganic carbon species in aquatic ecosystems due to shifts in the carbonate equilibria: Air [CO 2 ] Dissolved [CO 2 ] + H 2 O H 2 CO 3 H + + HCO 3 H + + CO 3 2CO 2 is the preferred form of DIC for photosynthesis by plants, but HCO 3 can also be used by some species (Spence & Maberly 1985). Fluctuations in the carbonate equilibrium occur for a variety of reasons. The equilibrium is very sensitive to pH fluctuations, with CO 2 most prevalent in acidic waters (pH 5 and below), HCO 3 common from pH 7 to 9, and CO 3 2abundant in alkaline waters (above pH 9.5) (Wetzel 1983). When vegetation is dense, shifts may be related to the amount of photosynthesis and respiration taking place in the ecosystem, resulting in a decoupling of normal water-air mixing. High photosynthesis rates during the day can cause [CO 2 ] to approach zero and raise the pH. In contrast, [CO 2 ] may far exceed air equilibrium when respiration exceeds photosynthesis at night. Diel changes in pH of more than two units occur in freshwaters and can result in over a hundred-fold change in free-CO 2 (Bowes 1996). Carbon dioxide concentration is usually highest before dawn and after plants have been respiring in the evening, and lowest in the late afternoon after plants have been photosynthesizing all day (Sand-Jensen & Frost-Christensen 1998). Seasonal cycles in the carbonate equilibria may also occur as CO 2 is depleted when plant abundance and photosynthesis are highest and lowest during the summer and winter,

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28 respectively. In productive sites, [CO 2 ] may approach zero for several months (Talling 1976; Madsen & Maberly 1991). Water in equilibrium with ambient air contains approximately 10 mmol CO 2 m -3 at 25C; however, most freshwater systems are not in equilibrium with the atmosphere (Cole et al. 1994; Sand-Jensen & Frost-Christensen 1998). Free-CO 2 values in freshwaters range from zero to over 350 mmol CO 2 m -3 (Spencer & Bowes 1990). Among other things, the [CO 2 ] of a water system depends on the type of system (e.g., lake, river, spring) and the amount of mixing within the system. The [CO 2 ] of freshwater systems may be lower than air-equilibrium values because water has a massive boundary layer resistance (which results in low exchange rates between air and water), low gas diffusion (approximately 10 4 times lower in water than air), and rapid CO 2 depletion due to photosynthesis of aquatic organisms (Raven 1970; Van et al. 1976; Maberly & Spence 1989; Madsen 1991). Inorganic carbon is most often limiting to plants due to the diffusion constraints, rather than low concentrations available in the water. Aquatic Plant Photosynthesis and CO 2 The mechanisms that aquatic plants have evolved to assimilate carbon can vary from the functional groups described for terrestrial plants (e.g., C 3 C 4 CAM) as a consequence of the physical and chemical differences between the two environments. Most submersed plants fit best in the C 3 terrestrial category, however, they show a variety of modifications to the typical C 3 pattern. Submersed species require higher [CO 2 ] to reach the equivalent photosynthetic rates of terrestrial plants. Submersed C 3 species require 30-times more free-CO 2 to saturate photosynthesis than their emergent counterparts and exhibit much higher apparent K 1/2 (CO 2 ) values (Maberly & Spence

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29 1983; Bowes & Salvucci 1989; Madsen & Sand-Jensen 1991). Moreover, coinciding with their lower dry weight to fresh weight ratio, submersed plants have less metabolic machinery than emergent or terrestrial species, as demonstrated by low chlorophyll and Rubisco activities (Van et al. 1976; Holaday et al. 1983). Depleted [DIC] is common in aquatic ecosystems and can severely restrict photosynthesis and growth by submersed aquatic macrophytes (Van et al. 1976; Bowes & Salvucci 1989; Titus 1992; Vadstrup & Madsen 1995). Submersed aquatic macrophytes show physiological/biochemical and anatomical/morphological adaptation strategies to increase the availability of CO 2 at the Rubisco carboxylation site. There are two major physiological/biochemical adaptations. Submersed plants may utilize HCO 3 as a carbon source for photosynthesis, which is the dominant carbon form in most aquatic habitats (Wetzel 1983). Also, there are photosynthetic systems employed by some species in which additional biochemical pathways are added, including C 4 and CAM-like metabolism (Bowes & Salvucci 1984; Casati et al. 2000; Madsen & Sand-Jensen 1991). Anatomical and morphological adaptations to low aqueous DIC include development of gas lacunae and an aerial growth habit (Spencer & Bowes 1990). Although all plants utilize CO 2 as the preferred form for photosynthesis, about 50% of submersed species also use HCO 3 as the initial C source (Spence & Maberly 1985). The capacity for HCO 3 use varies with the growth conditions and the photorespiratory state of the plant (Salvucci & Bowes 1983a, b; Bowes 1987; Sand-Jensen & Gordon 1984). Plants that use HCO 3 may concentrate CO 2 around Rubisco resulting in reduced photorespiration rates, thereby increasing photosynthetic efficiency (Maberly & Madsen 1998).

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30 Whereas many submersed species can use HCO 3 amphibious species are generally unable to utilize it (Sand-Jensen et al. 1992). Maberly and Madsen (1998) concluded that aquatic species that are restricted to CO 2 -use only (e.g., Myriophyllum verticillata, Callitriche cophocarpa, Sparganium emersum), had higher photosynthetic affinities for CO 2 than species that were also able to use HCO 3 (e.g., Myriophyllum spicatum, Elodea canadensis, Potamogeton crispus, Vallisneria spiralis). Spence and Maberly (1985) reviewed the distribution of aquatic species in relation to alkalinity and DIC composition of the water in which they grew and concluded that most CO 2 -only species were restricted to waters where CO 2 was the dominate form of DIC and that HCO 3 -users were more prevalent in waters with high alkalinity. Sediments are usually hypoxic or anoxic so the underground organs of aquatic plants must be adapted to such conditions in order to survive (Waisel & Agami 1996). A major adaptation is the formation of aerenchyma by the development of larger gas-spaces than typically found between cells in ground parenchyma tissues (Jackson & Armstrong 1999). Aerenchyma can account for up to 60% of some tissues in submersed plants (Wade 1990). Aerenchyma can substantially reduce internal impedance to gas transport, especially between roots and shoots (Jackson & Armstrong 1999). The primary function of aerenchyma is to increase O 2 diffusion from the atmosphere or photosynthesis to roots and the rhizophere, thereby enabling aerobic respiration to continue (Allen 1996). Aerenchyma can also facilitate the movement of CO 2 whether it originates from the atmosphere, from the sediment, or as a respiratory product in plant tissues. The placement of biomass at the water surface in some aquatic plants, and use of hydrosoil CO 2 for photosynthesis seem mutually exclusive, as the path length from roots to leaves

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31 is generally too long for transfer of sufficient CO 2 to support photosynthesis (Spencer & Bowes 1990). Several groups have assessed the contribution of aerenchymal CO 2 to photosynthesis of emergent macrophytes. For completely submersed Phragmites australis, S. lacustris, and C. papyrus, sediment-, root-, and rhizome-derived CO 2 played major roles in photosynthesis, but these roles were diminished when plants became emergent (Brix 1990; Singer et al. 1994; Jackson & Armstrong 1999); so inorganic carbon was only limiting below, and not above the water in these experiments. The most often cited examples of hydrosoil-CO 2 use involve isoetid species. In some, a thick cuticle inhibits the escape of CO 2 while growth close to the hydrosoil minimizes the CO 2 diffusion pathway (Spencer & Bowes 1990). Perhaps the simplest adaptation of aquatic plants to increase access to greater CO 2 is to produce aerial shoots. In many species, the morphology of aerial leaves is different from submersed leaves. Unlike submersed leaves, aerial leaves utilize free CO 2 as their only inorganic carbon source. Therefore, aerial leaves are not usually faced with the potentially wide fluctuations in DIC, O 2 and pH that submersed leave encounter (Spencer & Bowes 1990). Some aquatic plants have developed flotation devices that keep their photosynthesizing organs in contact with the atmosphere. An example would be the use of aerenchyma in the shoots of some Sagittaria species to maintain buoyancy at the water surface. A variety of other anatomical and morphological adaptations to increase access to DIC exist in leaves. Submersed leaves often have chloroplasts in their epidermis, no or non-functional stomates, and little cuticle (Spencer & Bowes 1990). Also, to maximize

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32 exposure to the water and access to DIC, they are frequently thin, sometimes finely dissected, and only contain a few cell layers (Wade 1990; Spencer & Bowes 1990). Elevated-CO 2 and HCO 3 Effects on Aquatic Plants The response of aquatic plants to rising CO 2 may be quite different than that of terrestrial plants. Research on aquatic plants lags behind that of terrestrial plants, thus the C assimilation mechanisms of aquatic plants are less well understood and categorized, thereby making it difficult to make generalizations about their potential responses to rising [CO 2 ]. Also, it is difficult to predict how rising CO 2 will influence water systems, since they already vary greatly in pH and concentration of free CO 2 and HCO 3 (Bowes 1991; Raven 1994). Huge changes in free [CO 2 ] can occur in water bodies, so submersed aquatic species are already exposed to greater CO 2 fluctuations than terrestrial species. With this in mind, it has been stated that the rise in atmospheric-CO 2 will impact the submersed vegetation of aquatic ecosystems less than those of terrestrial ecosystems (Raven 1994; Bowes 1996). Furthermore, the probability of aquatic plants showing increased growth responses under CO 2 enrichment has been suggested to be greater for emergent than for submersed macrophytes (Wetzel & Grace 1983). Down-regulation of photosynthetic capacity in response to high DIC occurs in aquatic macrophytes and is often coupled to reduced CO 2 affinity and suppression of HCO 3 -use, and C 4 and CAM metabolism (Holaday et al. 1983; Sand-Jensen & Gordon 1986; Jones et al. 1993). Suppression of these mechanisms is consistent with these processes having costs as well as benefits related to their increased energy expenditure. Thus, these systems may be up-regulated when the plants are DIC-limited, but absent when DIC-replete. The decline in photosynthetic capacity and CO 2 affinity seems to be most pronounced in species with the ability to use HCO 3 (Madsen 1991).

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33 Spencer et al. (1994) identified acclimation of photosynthetic phenotypes as a response to environmental heterogeneity within an ecosystem. Hydrilla verticillata forms a vegetation mat with the environmental conditions being very different within and on the edge of the mat, due to the photosynthesis and respiration effects. They found that [DIC], pH, and dissolved oxygen were 0 mol m -3 10, and 0 mol m -3 respectively, in the mat, while the edge values were 0 mol m -3 pH 7, and 0 mol m -3 respectively. Hydrilla verticillata growing in the mat had greater biomass density, but lower net photosynthesis, daily C gain, and RGR than edge plants. CO 2 compensation points were positively correlated with CO 2 and HCO 3 and negatively associated with pH, dissolved oxygen, and biomass, so that low and high CO 2 compensation point photosynthetic phenotypes were associated with mat and edge habitats, respectively. Submersed species have shown enhanced photosynthesis and growth at enriched-CO 2 and HCO 3 in laboratory experiments. Nielsen and Sand-Jensen (1989) concluded that the photosynthesis rates of 14 submersed macrophytes were still limited at four times greater than air equilibrium [CO 2 ] (45 M), but had rates three times higher when their CO 2 supply was increased to 1600 M. Van den Berg et al. (2002) grew two HCO 3 users, Chara aspera and Potamogeton pectinatus, at pH greater than 9.5 so that little CO 2 was present and they could test the growth response of these species to HCO 3 only. Chara aspera was a more efficient HCO 3 user based on it having greater photosynthetic rates at low [HCO 3 ] than P. pectinatus. Net biomass increased for both species at the higher HCO 3 treatment, but the ash-free dry weight fraction only increased for C. aspera. Vallisneria americana grown at elevated [CO 2 ] and [DIC] with fertile soils in a greenhouse had stimulated growth rates when compared to those grown at air-equilibrium

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34 [CO 2 ] and [DIC] (Titus et al. 1990), but no elevated-CO 2 response was identified for V. americana by Barko et al. (1991b). Barko et al. (1991b) did find that CO 2 had strong positive effect on biomass of H. verticillata under high irradiance, and that the CO 2 response of both populations was greater when fertility was sufficient. Callitriche cophocarpa and E. canadensis are submersed macrophytes that have received substantial attention in comparing their response to varying [DIC]. Callitriche cophocarpa uses only CO 2 for photosynthesis (Madsen 1991) and is heterophyllous with apical rosettes of floating leaves, while Elodea is homophyllous and uses HCO 3 in addition to CO 2 (Madsen & Sand-Jensen 1987). These species had down-regulated photosynthesis but increased growth at high [DIC] (Madsen et al. 1996). As DIC (CO 2 and HCO 3 ) increased, both species responded with a decrease in maximum photosynthesis rate, initial slope of A/C i curves, Rubisco activity, protein content, and chlorophyll content, whereas CO 2 compensation points increased. In addition, the rate of HCO 3 -dependent photosynthesis decreased for the HCO 3 user, E. canadensis. For both species, the growth response to increased CO 2 was greater than that to increased HCO 3 and the root: shoot ratio increased with increasing [CO 2 ], but was unaffected by HCO 3 Specific leaf area declined with C availability in the heterophyllous species, C. cophocarpa, whereas no change was observed in E. canadensis. The in situ growth of both species responded positively to elevated-CO 2 while enrichment with HCO 3 affected E. canadensis only (Vadstrup & Madsen 1995) Emergence of amphibious plants may play a role in their response to elevated-CO 2 The growth rate of C. cophocarpa shoots in contact with air was about three times faster than the rate of fully submerged shoots when both were grown in air-equilibrated water

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35 (16 mmol m -3 ) (Madsen & Breinholt 1995). This difference decreased as dissolved free-CO 2 in the water was increased, and the two shoot types grew at the same rate when the submersed shoots received greater than 350 mmol m -3 free-CO 2 Very little research has been done to correlate the acclimation responses of aquatic plants to elevated CO 2 with nutrient and carbohydrate levels as is common in the terrestrial literature. Nutrients limited the growth enhancement of H. verticillata and V. americana to elevated-CO 2 (Barko et al. 1991b). Titus (1992) determined that V. americana had greater biomass at enriched-CO 2 on all sediments tested and that there was no CO 2 -sediment interaction for biomass, however, allocation of biomass differed with sediment type. Plants grown on the less fertile sediments showed greater relative allocation to horizontal versus vertical growth than plants grown on relatively fertile sediment. Tissue N and P concentrations were consistently lower at highthan air-equilibrium CO 2 ; however, sediment effects on V. americana growth could not be attributed to either of these nutrients. Madsen et al. (1998) took a different approach by investigating the effect of DIC on the nitrogen requirement of E. canadensis and C. cophocarpa, two species that assimilate N through their leaves (Madsen et al., unpublished data). At elevated CO 2 growth rates increased, while tissue [N] decreased for both species. Thus the efficiency of N use was improved at high DIC availability and the tissue [N] needed to sustain maximum growth was reduced. The reduced tissue [N] at high CO 2 was not caused by an increased accumulation of starch and other non-structural carbohydrates; rather, it was a result of reduced net N uptake. These results suggested that depending on species and relative differences in [DIC], higher growth rates can be expected in systems with higher [DIC], even without a concomitant increase in N load.

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36 Ludwigia Repens Ludwigia repens (Forster), red ludwigia, is a perennial eudicot that is native to Florida. Ludwigia repens belongs in the Onagraceae (evening primrose) family, tribe Jussiaeae, and section Dantia (Godfrey & Wooten 1981; Peng 1989). It has been identified on the East Coast of the United States from South Carolina to South Florida and then West along the Gulf Coast to Texas (Godfrey & Wooten 1981). Red ludwigia is commonly sold as an aquarium plant (Tarver et al. 1979). In Florida, this plant has been collected growing in high-[CO 2 ] natural springs, as well as lower-[CO 2 ] spring environments (University of Florida, Herbarium), which makes it useful for elevated-CO 2 research purposes. Ludwigia repens is an amphibious plant producing both submersed and aerial leaves, and can grow in completely terrestrial conditions (Godfrey & Wooten 1981). The term creeping emergent was suggested by Rejmnkov (1992) to describe the growth form of a similar species, Ludwigia peploides. Ludwigia repens is rooted in the substrate, but also produces roots along the stem in the water column. Often, it grows in soils along the border between the shoreline and shallow waters (Hoyer et al. 1996). Mats can be formed as the stems creep along the sediment, sending up flaccid stems in the water (Hoyer et al. 1996). The plant varies from dark red/purple to green. Underwater portions of the plant usually have more red pigmentation, but they shift to a green color as they become aerial. Leaves grow opposite and are typically 7 x 2 cm (Hoyer et al. 1996). Both sexual and asexual reproduction occurs in L. repens. Flowers are yellow and are usually found on the aerial stems from the late spring through early fall. A sessile fruit or capsule (2.5-3.0 x 2.0 mm) contains a cluster of very small seeds that provide the means of sexual reproduction (Tarver et al. 1979). A related species, L. peploides was

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37 found to germinate readily on soil and in water, floating or when maintained submerged (Yen & Myerscough 1989). Ludwigia repens also reproduces with fragments breaking off from the whole plant. Regeneration is possible from fragmented stem pieces with only a few nodes (Lytle, unpublished results). Sculthorpe (1967) noted that species of Ludwigia growing entirely submersed in deep water do not reproduce sexually. Very little physiological research has been performed with L repens, however, research has been done using similar species within the genus. Ludwigia uruguayensis was used to assess the effect of a plant species growing in a high-CO 2 and resource spring (Koch 1993). There was no evidence of down-regulation of photosynthesis in the plants near the high-CO 2 spring vent. Prins et al. (1980) determined that Ludwigia natans used only CO 2 in photosynthesis, and not HCO 3 Summary Statement The literature regarding the short-term responses of terrestrial plants to elevated-CO 2 is well established, with most, but not all, C 3 plants showing down-regulation of photosynthesis and increased growth. Studies of more long-term plant responses to elevated-CO 2 have been undertaken mainly in natural springs with high [CO 2 ]. Although this area of research is in its infancy, these studies indicate that terrestrial plant acclimation responses to elevated-CO 2 found in the short-term studies are less prevalent after plants have been exposed to elevated [CO 2 ] over many generations. Thus far, CO 2 spring experiments have implied that adaptation differences exist between the plant populations growing in highand ambient-[CO 2 ] around the springs, however, researchers have yet to establish genetic differences between the populations to back up this claim. Natural CO 2 spring research is an important tool in order to predict how plants will respond to a future high-CO 2 atmosphere. The effect of elevated-CO 2 on

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38 aquatic plants has received far less attention than terrestrial plants, and submersed plants have been completely neglected in regard to natural CO 2 spring experiments. Submersed plants differ from their terrestrial counterparts in many respects, so that their responses to elevated-CO 2 cannot be extrapolated from terrestrial results. Freshwater and freshwater ecosystems are vital resources, and will become increasingly scarce and valuable commodities as population and agricultural pressures increase. In this context, aquatic plants are an integral component of any healthy aquatic ecosystem and should not be overlooked in elevated-CO 2 research.

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39 Net CO 2 assimilation rate (mol CO 2 m -2 s -1 ) Limiting factors in the non-acclimated curve 50 <-------Rubisco-------> <----------------------RuBP-------------------------> 40 Increased A max due to increased Acclimated P i and RuBP regeneration 30 Non-acclimated 20 10 Decreased slope due to decreased Rubisco content 0 0 200 400 600 800 1000 Intercellular CO 2 (mol mol -1 ) Figure 1-2. Acclimated and non-acclimated assimilation/intercellular CO 2 (A/C i ) curves for a typical C 3 plant at high light and 21% O 2

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CHAPTER 2 ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS Introduction Ludwigia repens (Forster), red ludwigia, is a perennial eudicot that is native to Florida. It belongs in the order Myrtales, family Onagraceae, tribe Jussiaeae, and section Dantia (Godfrey & Wooten 1981; Peng 1989). It is thought to have originated in South America (Keating 1982) and has been identified on the East Coast of the United States from South Carolina to South Florida and then West along the Gulf Coast to Texas (Godfrey & Wooten 1981). Commercially, this species is commonly sold as an aquarium plant (Tarver et al. 1979). Ludwigia repens is an amphibious plant that produces both submersed and aerial shoots, and can grow entirely terrestrial (Godfrey & Wooten 1981). It is rooted in the substrate, but also produces roots along the stem in the water column. Often, plants grow in soils along the border between the shoreline and shallow waters, and mats can be formed as the stems creep horizontally along the sediment, sending up stems vertically in the water (Hoyer et al. 1996). The underwater portions of the plants usually have more red and purple pigmentation, but their color shifts to green when they become aerial. Leaves grow opposite and are typically about 7 x 2 cm (Hoyer et al. 1996). Both sexual and asexual reproduction occurs in L. repens. Flowers are yellow and are usually found on the aerial stems from the late spring through early fall. A sessile fruit or capsule (2-3 x 2 mm) contains a cluster of very small seeds (Tarver et al. 1979). This species also reproduces vegetatively by fragmentation and clonal ramets separating 40

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41 from the genet and surviving as individuals. Regeneration is possible from fragmented stem pieces with only a few nodes (Lytle, unpublished data). There are no anatomical descriptions of L. repens, although members within the genus have been characterized. Summarizing results from observations of the leaves of 25 Onagraceae species, Keating (1982) noted that Ludwigia represents a phylogenetic line separate from all other Onagraceae, and that a distinguishing characteristic of Onagraceae leaves is the presence of abundant raphide crystals in the vegetative tissues. Aerial and submersed leaf anatomy in some species of Ludwigia differed even to the extent that the plants are considered heterophyllous (Kuwabara et al. 2001). Differences in aerial versus submersed leaf anatomy and morphology for an amphibious plant may represent acclimation to the conditions in their respective air and water environments (Sculthorpe 1967). More so than in the aerial environment, light and inorganic carbon can limit photosynthesis of leaves that are underwater (Spencer & Bowes 1990). Submersed leaves that are thin, finely dissected, and only contain only a few cell layers optimize access to dissolved inorganic carbon and light (Wade 1990). Features that reduce evapotranspiration and provide support in air, such as a thick cuticle, several photosynthetic cell layers, functional stomates, and lignin lose their value underwater (Maberly & Madsen 2002). Thus submersed leaves of Ludwigia arcuata are narrower, with a higher leaf length to width ratio, and a lower stomatal density than the aerial counterparts (Kuwabara et al. 2001). Narrower and longer submersed leaves as compared to emergent leaves are also found in L. arcuata, L. repens, and L. palustris (Petch 1928; Fassett 1957).

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42 Stems of submersed plants often contain aerenchyma and have less support tissues such as lignin when compared to emergent stems, which is likely an adaptation to the buoyancy of water (Jackson & Armstrong 1999; Maberly & Madsen 2002). Stem aerenchyma produced by a phellogen was common in the semi-aquatic members of the Onagraceae (Schenck 1889). Ludwigia species are predominantly herbaceous; the most familiar species are herbs of very wet habitats such as ponds, ditches, and streams, therefore, it is somewhat surprising that some woody species exist. The periderm of L. octovalvis plants swelled when they were grown in flooded as opposed to non-flooded environments (Angeles 1992). Carlquist (1987) using Ludwigia anastomosans, L. peduncularis, and L. torulosa determined that their aquatic habit was reflected in the lowest degree of vessel grouping in the Onagraceae family. Root anatomy of Ludwigia has received attention, with most of the interest directed toward root dimorphism in certain species. Ludwigia peruviana and L. peploides produce both upwardand downward-growing roots on the same plant (Schenck 1889; Ellmore 1981a, b). In transverse section, the stele of upward-growing roots of L. peruviana roots was surrounded by a wide zone of aerenchyma produced by a phellogen, whereas this zone is absent in downward-growing roots (Schenck 1889; Ellmore 1981a, b). The purpose of the present study is to characterize the vegetative anatomy of L. repens, including both aerial and submersed organs. In Florida, this plant has been collected from natural springs (University of Florida Herbarium, FLAS). Anatomical information for this species may contribute to a better physiological understanding as it relates to the plants function in the both aerial and submersed habits.

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43 Materials and Methods Random collections of twenty mature, emergent L. repens plants were made from Fanning Springs, Florida (29 35' 15" N, 82 56' 08" W) on 19 February 1999. A voucher specimen was deposited at the University of Florida Herbarium (FLAS). Tissue from ten of the plants was fixed into a formalin-acetic acid-alcohol (FAA) solution at the field site. Mature leaves, stems, and roots were separated from the fixed material to produce slides. Submersed and aerial leaves and stems were also separated, while roots were separated into those growing in the water column or the hydrosoil. Tissue was dehydrated in a tertiary-butyl alcohol series, and embedded in paraffin at 56C (Johansen 1940). Crossand longitudinal-sections (8-20 m) of all organs were stained with a safranin (1% w/v in 50% v/v EtOH)-fast green (1% w/v in 95% v/v EtOH) series. Whole leaves were cleared in a 5-10% (w/v) NaOH solution and stained with a safranin (1% w/v in 50% v/v EtOH) solution. Leaf thickness and the density of starch grains and crystals (e.g., raphides and druses) were determined for mature submersed and aerial leaves from micrographs at 100x magnification. In order to compare stomatal density of the submersed and aerial leaves, dried leaves were flooded with acetone and compressed with cellulose acetate sheets. The dry cellulose acetate was stripped away leaving a negative replica of the leaf surface. An area of 1 mm 2 at 100x magnification was used to count the stomates, with three replicates per leaf.

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44 Results Plants Figure 2-1 shows L. repens growing emergent at the collection site. Leaves Micrograph cross-sections of aerial and submersed Ludwigia repens leaves are shown in Figure 2-2, while quantitative characteristics of these leaves are documented in Table 2-1. The leaves are homophyllous since they showed only slight anatomical differences. There was no difference in the thickness of aerial and submersed leaves (Table 2-1). A thin cuticular layer was present on the adaxial and abaxial surfaces of both leaf types. The epidermal cells were thin-walled, with some tangential thickening (Fig. 2-2). There was little difference between the adaxial and abaxial epidermal cells, although the adaxial cells of aerial leaves were somewhat smaller and had thinner cell walls than the abaxial cells (Fig. 2-2). No chloroplasts were observed in the epidermal cells, even of the submersed leaves. Stomates were present on the adaxial and abaxial surfaces of aerial and submersed leaves, making them amphistomatous. The stomates exhibited guard cells, but no subsidiary cells, i.e., they anomyocytic. Aerial leaves had twoand seven-times greater stomatal densities than submersed leaves on the adaxial and abaxial surfaces, respectively (Table 2-1). Stomatal density did not differ between the adaxial and abaxial surfaces of aerial leaves, whereas submersed leaves had about four-fold greater stomatal density on the adaxial as compared to the abaxial surface (Table 2-1). The mesophyll tissue was typically differentiated into one layer of palisade and four to five layers of spongy mesophyll (Fig. 2-2). However, the palisade mesophyll was more defined in aerial as compared to submersed leaves, and there were instances of

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45 submersed leaves near the base of the stem having no palisade mesophyll layer. The palisade mesophyll in aerial leaves was tightly packed, whereas the spongy mesophyll and the entire mesophyll of submersed leaves were loosely arranged with abundant intercellular air spaces; however, no lacunae were present (Fig. 2-2). The mesophyll was made up of chlorenchyma cells, with both the palisade and spongy mesophyll containing starch grains, but more were present in the palisade layer (Fig. 2-2). Aerial leaves had 168% greater starch grain density than submersed leaves, but the leaf types contained similar numbers of raphide and druse crystals (Table 2-1). The vasculature of L. repens leaves consisted of bicollateral bundles in the midrib and major veins (Fig. 2-3), and collateral bundles in the minor veins. There were a large number of vascular bundles in the leaf margin, with four to five on each side of the midvein. The bundle sheaths were indistinct with ground parenchyma tissue surrounding a small amount of primary xylem and phloem (Fig. 2-3). The xylem tissue of the bundles consisted of rows of small vessel members interspersed with xylem parenchyma cells that were few and small in number (Fig. 2-3). The elongate vessel members had transverse or oblique end walls. There were small clusters of phloem on both sides of the xylem (i.e., bicollateral phloem). Stems Figures 2-4 and 2-5 show cross-sections of aerial and submersed L. repens stems. No cuticle was present on the stems of L. repens and epidermal cells were thick-walled with mucilage present (Fig. 2-4). The cortex was differentiated into three zones; all made up of thin-walled parenchyma cells that contained starch grains, raphides, and mucilage (Fig. 2-4). Mucilage was less prevalent in all the layers of aerial as compared to submersed stems (Fig. 2-4). The outer layer of cortex was composed of cells of small

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46 size with the largest quantity of mucilage (Fig. 2-4). The middle layers contained cells that were larger than those in the other zones, and was evenly interspersed with large air lacunae (Fig. 2-4). These air lacunae were smaller and less prevalent in aerial as opposed to submersed stems (Fig. 2-4). The cells in the inner cortical layers exhibited the largest number of raphides, and contained styloid and cuboidal crystals as well. The stem endodermis was composed of thick-walled parenchyma cells that contained mucilage and starch grains (Fig. 2-5). The endodermis and stele of the aerial stem had less mucilage than their submersed counterparts (Fig. 2-5). The individual vascular bundles present in younger stems were bicollateral, with the primary phloem clustered in two distinct internal and external zones around the xylem poles, while the primary xylem showed endarch maturation. Although it was not well defined, L. repens produced a cambium that generated infrequent secondary xylem with spiral thickenings, but no secondary phloem. The xylem formed a continuous cylinder in older stems (Fig. 2-5). Xylem tissues were composed of vessel members with alternate pits and some parenchyma (Fig. 2-5). The secondary vessel members had reticulately thickened walls. The stem pith was made up of thin-walled parenchyma cells with small intercellular spaces (Fig. 2-5). Many of these cells contained mucilage, starch grains, and/or raphides. Roots Figure 2-6 shows cross-sections of submersed water-column and hydrosoil roots of L. repens. These root types did not differ anatomically (Fig. 2-6). In some of the roots the epidermis was crushed or compressed. Root epidermal cells were parenchymatous with their walls tangentially and radially thickened (Fig. 2-6). The cortex of roots was either divided into two zones, with an outer layer composed of about two layers of larger cells (Fig. 2-6A), or was undifferentiated (Fig. 2

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47 6B). This distinction was not a water-column versus hydrosoil root phenomenon as it occurred in both root types. The cortex of both root types was made up of thin-walled parenchyma cells containing starch grains (Fig. 2-6). Lacunae were not present in the roots; however, roots did have many intercellular spaces of various sizes that were larger than the isodiametric cells in which they were embedded (Fig. 2-6). The endodermis of the roots of L repens was distinct. No Casparian strip was evident, but the endodermis did contain mucilage (Fig. 2-7). The pericycle of the stele was composed of one to two layers of thin-walled parenchyma cells (Fig. 2-7), and lateral roots were generated from the pericycle. The stele exhibited a protostele pattern and there was no vascular cambium in the roots (Fig. 2-7). Vessel member and parenchyma cells made up the xylem tissues, and these cells were often filled with mucilage (Fig. 2-7). Maturation of the xylem was exarch, with the smaller xylem cells on the periphery. The smaller protoxylem cells had spiral thickenings, and the metaxylem cells had alternate pits. Phloem was restricted to isolated bundles between xylem poles and was composed of sieve tube members and parenchyma cells. Discussion Ludwigia repens exhibited terrestrial-like anatomical characteristics even though it grows more frequently submersed or emergent than fully terrestrial. Similar to terrestrial plants, aerial and submersed leaves possessed a cuticle and stomates, and stems contained a cambium. Traits that were more aquatic included the possession of aerenchyma in the stems and roots, and a lack of sub-stomatal cavities in leaves. A thin layer of cuticle was present on both the adaxial and abaxial sides of all leaves examined. Most submersed plant leaves have little cuticle since water loss is not a problem (Spencer & Bowes 1990), and its absence can enhance CO 2 diffusion. Keating

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48 (1982) examined 24 species of Ludwigia (not L. repens) and found no cuticle on the leaves, so L. repens presents an anomaly in this respect. Mucilage was also present in all the L. repens organs examined. The cuticle and mucilage present on submersed leaves should retard water loss if the submersed leaves become aerial, as water levels fluctuate in the natural habitat. Stomates of L. repens in this study were anomyocytic, unlike other species in the Onagraceae, which are surrounded by three or more subsidiary cells (Metcalfe & Chalk 1950). Stomates were present on both the aerial and submersed leaves, with greater density on the aerial leaves. Submersed aquatic leaves typically have few or non-functional stomates (Sculthorpe 1967; Spencer & Bowes 1990). It is possible that the submersed leaves on a plant growing in the sample site could become aerial due to water level fluctuations. Stomates are developed while leaves are still in the bud (Costantin 1886), but presumably function only when they are exposed to the atmosphere (Sculthorpe 1967). Ludwigia arcuata, another amphibious species but with heterophyllous leaves, is similar in regard to stomates in that they are found on leaves of both terrestrial and submersed plants, with about three-fold greater density on terrestrial leaves (Kuwabara et al. 2001). The stomatal density of submersed leaves of L. repens was greater on the adaxial than on the abaxial side; but there was no difference for the aerial leaves. Terrestrial eudicots commonly contain more stomates on the abaxial leaf surface, but this pattern tends to be reversed in submersed species (Sculthorpe 1967). Ludwigia arcuata, had greater stomatal density on the adaxial surfaces of both terrestrial and submersed leaves

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49 (Kuwabara et al. 2001), while stomates were equally common on both leaf surfaces of 24 Ludwigia species (Keating 1982). A cambium was present in both the aerial and submersed L. repens stems, and this has been reported for other Ludwigia species (Carlquist 1987; Angeles 1992). Wood and other structural materials are typically minimized in submersed plant parts since they are supported by the buoyancy of water (Westlake 1965; Sculthorpe 1967). Thus, the Ludwigia genus, which primarily contains aquatic species, is somewhat unusual in having a cambium present. Stems of Ludwigia repens contained lacunae, while leaves and roots had small and large intercellular spaces, respectively. Extensive aerenchyma is common in aquatic plants as it facilitates more rapid diffusion of CO 2 and O 2 in the gas phase for photosynthesis or respiration, or to discharge volatiles (Jackson & Armstrong 1999). Aerial stems had fewer lacunae than submersed stems in accordance with results in Sculthorpe (1967) and reports that flooding triggers the formation of aerenchyma (Jackson & Armstrong 1999). It is interesting to note that in his classic study of gas-filled tissue in marsh plants in which the term aerenchyma originated, Schenck (1889) used stems of Jussiaea, now in the genus Ludwigia, to describe the development of schizogenous spaces between cells derived from a phellogen. The abundant raphide and druse crystals found in all the organs of L. repens was in agreement with results showing that raphide crystals in vegetative tissues are a distinguishing characteristic of the Onagraceae (Keating 1982). Calcium oxalate crystals were present in all 24 species of Ludwigia examined by Keating (1982), with raphides present in nearly all specimens. Fanning Springs, where L. repens was collected, is

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50 designated a calcium carbonate spring (Woodruff 1993), therefore it is possible that calcium accumulation was a factor in the calcium oxalate crystal formation. Although the bicollateral vascular bundles, i.e., bundles with the phloem in primary in two distinct zones (internal and external) found in the leaves and stems of L. repens is a rare anatomical feature, other genera in the Onagraceae also possess this trait (Keating 1982). The aerial and submersed leaves showed differences in anatomy and morphology, but they are too minor to consider the leaves heterophyllous. Aerial leaves had a better-developed palisade layer and had greater starch grain and stomatal density. These traits may reflect the greater reliance on the aerial leaves for photosynthesis by the plant and could be related to enhanced CO 2 diffusion. Aerial leaves of amphibious plants tend to have higher photosynthesis rates and light-saturation points than submersed leaves (Bowes 1987; Salvucci & Bowes 1982). Other Ludwigia species have a well-differentiated mesophyll with one adaxial layer of palisade occupying about half of the mesophyll, however they were not necessarily aquatic species (Keating 1982). Submersed leaves often show an increase in palisade at the expense of spongy mesophyll or lack palisade in their mesophyll tissues (Sculthorpe 1967; Ronzhina & P'yankov 2001). Similar to L. repens, aerial leaves of L. arcuata had greater stomatal density than submersed leaves, but unlike L. repens the leaf types were different enough in morphology and anatomy that they were considered heterophyllous (Kuwabara et al. 2001). Roots growing in the water column and hydrosoil showed no differences. This is in contrast to L. peploides, which although it has similar growth habit to L. repens, being a

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51 prostrate amphibious plant anchored in waterlogged soil, shows root dimorphism (Ellmore 1981a, b). Leaf anatomy of L. repens reflected a physiology better suited to an emergent, rather than a submersed existence, at least with respect to photosynthesis. The stomates and cuticle serve no purpose and hinder CO 2 diffusion in submersed leaves, whereas they are vital in reducing water loss during aerial leaf photosynthesis. These traits, plus the better-developed palisade layer in aerial leaves provide evidence that the aerial leaves serve as the primary photosynthetic tissues of emergent L. repens plants.

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52 Figure 2-1. Ludwigia repens growing emergent in Fanning Springs, Florida.

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53 ad p s sg ab 0 1 mm 0 1 mm p s ad sg ab A. Aerial leaf A. Submersed leaf B. Submersed leaf Figure 2-2. Micrograph cross-sections of aerial (A) and submersed (B) L. repens leaves. Labels denote adaxial epidermis (ad), palisade mesophyll (p), spongy mesophyll (s), starch grain (sg), and abaxial epidermis (ab).

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54 Table 2-1. Characteristics of fully-expanded aerial and submersed leaves of L. repens. Mean SD (n = 4-8). P-values are from t-tests and significant differences between populations at P 0 are noted by **. Parameter Aerial leaves Submersed leaves Thickness leaf -1 (mm) 0 0 0 0 Stomatal density (no. stomates mm -2 ) Adaxial epidermis Abaxial epidermis 163 35** 181 48** 95 22 25 11 Starch grain density (no. mm -2 ) 5200 900** 3100 400 Crystal density (no. mm -2 ) Raphides Druses 8 5 7 4 6 2 7 7

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55 x p ph 005 mm Figure 2-3. Micrograph cross-section of the major vascular bundle of a submersed L. repens leaf. Labels denote parenchyma (p), xylem (x), and phloem (ph) cells.

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56 e 025 mm 025 mm p m l p le m A. Aerial stem B. Submersed stem sg sg Figure 2-4 Micrograph cross-sections of the epidermis and cortex of aerial (A) and submersed (B) L. repens stems. Labels denote epidermis (e), parenchyma cell (p), lacunae (l) starch grain (sg), and mucilage (m).

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57 p x sg ph m ph x sg e, m p 0 3 mm 0.2 mm e A. Aerial stem B. Submersed stem Figure 2-5. Micrograph cross-sections of the inner cortex, vascular cylinder, and pith of aerial (A) and submersed (B) L. repens stems. Labels denote parenchyma cell (p), xylem (x), phloem (ph), endodermis (e), starch grain (sg), and mucilage (m).

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58 02 m m 014 m m e p ixen, m i p en, m xe B. H y drosoil root. Wateroot Br A. Water-column root Figure 2-6. Micrograph cross-sections of submersed water (A), and hydrosoil (B) L. repens roots. Labels denote epidermis (e), parenchyma cell (p), intercellular space (i), endodermis (en), xylem (x), and mucilage (m).

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59 e, m p ph x 005 mm Figure 2-7. Micrograph cross-section of the endodermis, pericycle, and stele of a hydrosoil L. repens root. Labels include endodermis (e), pericycle (p), xylem (x), phloem (ph), and mucilage (m).

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CHAPTER 3 CHARACTERISTICS OF SUBMERSED LUDWIGIA REPENS PLANTS FROM TWO POPULATIONS GROWING IN SPRINGS WITH DIFFERING CO 2 CONCENTRATIONS Introduction Atmospheric [CO 2 ] is currently over 371 mol CO 2 mol -1 (Keeling & Whorf 2002), and expected to double in this century. Much research on the short-term (days to months) acclimation responses of terrestrial plants to elevated CO 2 exists, with the responses differing among species (Drake et al. 1996a; Pritchard et al. 1999). Because Rubisco is the initial C-fixing enzyme in C 3 plants and subject to competitive inhibition by oxygen (Bowes & Ogren 1972), C 3 plants generally show greater responses to elevated-CO 2 than C 4 and CAM species that are able to concentrate CO 2 at the site of fixation. Although photosynthesis rates of C 3 plants respond favorably to enriched-CO 2 this initial enhancement is often reduced as rates are down-regulated over longer time frames (Lewis et al. 1999). Directly correlated with the down-regulation of photosynthesis are reduced Rubisco activity and protein amount (Besford et al. 1990; Rowland-Bamford et al. 1991). In addition, water use efficiency may increase in elevated-CO 2 because of reduced stomatal conductance through decreases in stomatal density or partial closure of stomates (Amthor 1995). Both direct and indirect effects of elevated-CO 2 have been reported to decrease respiration (Drake et al. 1999), although the response is still debatable, with effects at least in part attributable to measurement artifacts (Jahnke 2001; Jahnke & Krewitt 2002). 60

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61 Even though photosynthesis may be down-regulated, C 3 plants grown in elevated-CO 2 usually have enhanced growth (Poorter 1993). Explanations for why acclimation responses occur are related to other physiological mechanisms not keeping pace with increased C-fixation. As C-fixation and source output increase, sink reservoirs (Stitt 1991; Sheen 1994) and N assimilation (Stitt & Krapp 1999) may fail to meet demand. Acclimation responses to high-CO 2 are less evident when sink (e.g., root) growth is not restricted (Arp 1991) and nutrient requirements are met (Geiger et al. 1999). Terrestrial C 3 plants grown in elevated-CO 2 usually are larger (Poorter 1993), with greater leaf area (Smith et al. 1987). Sink tissues and tiller production are favored, and root: shoot ratios become greater (Smith et al. 1987; Lewis et al. 1999). Tissue density of plants growing in high-CO 2 is normally increased, with an associated decrease in specific leaf area (SLA) (Vu et al. 1989) as leaves become thicker (Tipping & Murray 1999). Along with the morphological changes, plant anatomy is affected with increases in cell size, cell number, and proportion of mesophyll tissue in leaves (Vu et al. 1989; Radoglou & Jarvis 1990; Ferris et al. 2001). Although less studied, submersed plant acclimation responses to high-CO 2 tend to be similar to their terrestrial counterparts, with some novel differences (Bowes 1991; Bowes 1993). Because of the diffusion resistance of water, submersed species require higher [CO 2 ] to saturate their photosynthesis rates and generally have much lower maximum rates than terrestrial plants (Maberly & Spence 1983; Bowes & Salvucci 1989). Also, coinciding with their lower dry weight to fresh weight ratio, submersed plants have less metabolic machinery than terrestrial species, as demonstrated by low chlorophyll and Rubisco activities per unit mass (Van et al. 1976; Holaday et al. 1983).

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62 Based on these characteristics, submersed species should respond favorably to CO 2 enrichment. However, some submersed species are able to use bicarbonate ions in photosynthesis, and this may reduce the dependency on free-CO 2 and lead to a decreased response to elevated-CO 2 (Madsen et al. 1996). Reported responses of submersed aquatic plants to enriched-CO 2 include increased growth and root: shoot ratio, decreased SLA, down-regulation of photosynthesis, and decreased Rubisco activity and amount (Sand-Jensen & Gordon 1986; Madsen & Sand-Jensen 1994; Madsen et al. 1996). The degree of CO 2 enhancement is also influenced by the nutrient status of the medium in which the plants are grown (Barko et al. 1991b). In contrast to the abundance of literature regarding short-term responses to elevated-CO 2 there are few experiments that extend past a single growing period (Field et al. 1996; Krner et al. 1996). Long-term population-level experiments are crucial to make predictions regarding how plants may genetically adapt to rising-CO 2 Different genotypes within a species may differ in CO 2 responses (Wulff & Alexander 1985), so the potential for genetic selection to elevated-CO 2 exists. However, periods of ten or more years are often required for genetic shifts in populations (Miglietta et al. 1993). One technique to study long-term elevated-CO 2 effects has been to take advantage of natural environments in which plants have been growing in high [CO 2 ] for many generations (Raschi et al. 1997). Geothermal gas vents such as springs, geysers, and volcanoes can emit CO 2 and expose terrestrial plants in the vicinity to higher than ambient concentrations (van Gardingen et al. 1997). In particular, freshwater springs may produce high [CO 2 ] without the toxic components found in some other CO 2 -emitting sources. Using these natural systems, intraspecific populations of terrestrial plants

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63 growing at different distances from a natural CO 2 source, with [CO 2 ] ranging from ambient to 1000 mol CO 2 mol -1 have been investigated (Cook et al. 1998). Adaptation is often implied from these experiments, but acclimation responses cannot be ruled out. An interesting difference from short-term studies is that terrestrial plant populations growing for long periods near to high-CO 2 spring sources do not necessarily produce greater biomass than populations growing further away (Krner & Miglietta 1994; Cook et al. 1998). Down-regulation of photosynthesis is less widespread for plants growing near high-CO 2 sources (Koch 1993; Bettarini et al. 1997), but the plants do exhibit decreased stomatal conductance, although the decrease is not always due to modifications in stomatal density (Bettarini et al. 1998). Unlike in short-term studies, plants growing near enriched-CO 2 springs do not always show carbohydrate buildup or decreased shoot N content (Krner & Miglietta 1994; Bettarini et al. 1997; Cook et al. 1998) or greater tissue density. Specific leaf area of plants growing close to CO 2 vents does not appear to be greater than that of plants growing at ambient [CO 2 ] (Koch 1993; Cook et al. 1998). An underlying assumption in natural CO 2 source experiments is that genetic differences exist among the populations being compared, but to our knowledge this assumption has not been tested (Miglietta et al. 1993; Fordham et al. 1997a). Also, in many instances research has compared plant populations that are in close geographic proximity (e.g. 100 m) so that genetic exchange cannot be ruled out. This is an important factor in any study of potential genetic adaptation. Furthermore, fully submersed plants have been neglected in high-CO 2 spring research. This is surprising considering the environmental parameters of natural freshwater springs can be remarkably stable over long periods (Rosenau et al. 1977).

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64 This study is the first elevated-CO 2 spring experiment to examine potential adaptation using submersed plants and to perform a genetic analysis. This project characterizes two submersed populations of a perennial angiosperm native to Florida, Ludwigia repens, that has been growing in two springs with greatly differing [CO 2 ] for many generations. The springs are 85 km apart and not connected, therefore, genetic exchange between the two populations is unlikely. The hypothesis being examined is that the two populations should exhibit characteristics consistent with genotypic adaptation to their respective environmental [CO 2 ]. The specific objectives were to 1) establish if the highand lower-CO 2 populations differ genetically; 2) determine whether the submersed population of L. repens exposed to high-CO 2 for many generations shows down-regulation of photosynthesis and Rubisco activity when compared to the population from a lower-CO 2 spring; and 3) resolve if plants from the high-CO 2 spring differ morphologically and anatomically from their lower-CO 2 counterparts. Materials and Methods Collection Sites Two populations of Ludwigia repens L. were selected for the study, one in a high-[CO 2 ] spring, Fanning Springs, FL (29 35' 15" N, 82 56' 08" W) and the other in a spring with a much lower [CO 2 ], Rainbow Springs, FL (29 06' 08" N, 82 26' 16" W). Fanning Springs is approximately 85 km northwest of Rainbow Springs, and the two springs are not connected. Both springs reside on the Ocala geological uplift and fit into the calcium bicarbonate category of spring classification system developed by Woodruff (1993). At both springs, the Ludwigia populations grow both partially emergent and completely submersed near the shore by the boils and occupy approximately 16 m 2 All data were collected between the springs and summers of 2001 and 2002.

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65 Water from the highand lower-CO 2 springs was analyzed for nutrients, minerals, and conductivity at the Analytical Research Laboratory (University of Florida, Gainesville, Florida, USA). To eliminate air bubbles, containers were sealed below the water surface and placed on ice during transport. Temperature, irradiance, alkalinity, and pH were measured at each field site on the day of collection. Irradiance and the [CO 2 ] in the air 3 cm above the water surface were measured with a LI-250 light meter and a LI-6200 (LI-COR Inc., Lincoln, NE). Inorganic carbon species were calculated using the FWcarb computer program (SC Maberly, Acme Liquid Software Company, 1991). FWcarb requires alkalinity, pH, temperature, and ionic strength as input data and assumes equilibrium conditions to calculate total dissolved inorganic carbon (DIC), CO 2 and HCO 3 (Mackereth et al. 1978). Ionic strength (I) was calculated using the formula: I = 25 x 10 -5 (s d ), where s d is total inorganic dissolved solids in mg L -1 (Loewenthal & Marais 1976). The s d was determined from values obtained from the Analytical Research Laboratory. Morphology and Anatomy The morphological characteristics of submersed plants from both populations were compared. An individual plant was defined as a single vertical stem with its associated leaves, roots, and axillary stems. The lengths of roots, internodes, and main and axillary stems; and the number of leaves and axillary shoots were determined for eight plants randomly selected from each population. Plants cleaned of epiphytes were separated into leaves, stems, and roots and their fresh weights and density were determined. Leaf areas were measured with an area meter (LI-COR Inc., Lincoln, NE). For dry weight determinations, plant organs were dried at 65 C for 3 d. Individual leaf characteristics

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66 were also measured for a further six plants using four fully-expanded leaves taken from nodes four and five counting down from the shoot apex. For anatomical comparisons, six plants from each population were fixed in a formalin-acetic acid-ethanol solution (Johansen 1940) at the field sites. For section preparations, fully-expanded leaves from nodes four and five, stem tissue 10 cm from the shoot apex, and hydrosoil roots at the widest diameter, were selected. The tissues were dehydrated in a tertiary-butyl alcohol series, and embedded in paraffin at 565 C (Johansen 1940). Sections (8 m) of leaves, stems, and roots were stained with a safranin (1% w/v in 50% v/v ethanol)-fast green (1% w/v in 95% v/v ethanol) series. Whole leaves were cleared in an 8% (w/v) NaOH solution and stained with a safranin (1% w/v in 50% v/v ethanol) solution. The sections were examined to determine the number of cell layers in each tissue, the frequency of crystals, and the amount of aerenchyma. Leaf thickness was measured 5 mm from the main vein of leaves. The percent aerenchyma per area of stem cross-section was established by passing photographs of stem sections with and without the aerenchyma removed through an area meter. To determine stomatal frequency, fully-expanded leaves from eight plants were dried, flooded with 100% acetone, and compressed with cellulose acetate sheets. The dry cellulose acetate was stripped away. This negative replica was used for three replicate counts per leaf of stomates in a 18 mm 2 surface area. Sexual Reproduction Flower and seed capsule production at each site was noted from September 1998 to March 2002. The average number of flowers per plant from the high-CO 2 spring was determined using ten plants. The ability to flower and produce viable seed over a 56-d

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67 period was further studied in a growth chamber with a photon irradiance of 300 mol m -2 s -1 (400-700 nm), 12 h photoperiod, 24 C, and atmospheric CO 2 Plants from both populations were collected and grown in 250-mL sand-filled styrofoam cups, supplied with a 1 g stick of slow-release fertilizer (13% N; 4% P; 5% K by weight), in aquaria containing deionized water that was changed every 3-d. Flowering of each population was monitored using four plants with each of the following treatments: submersed, emergent, and emergent with gibberellic acid (GA 3 ) applied. For the submersed treatment the water level was maintained above the shoot apices. For the emergent treatments, it was set so that the shoot apices were at the water surface at the start of the experiment, and subsequent growth was emergent. For the GA 3 treatment, 200 L of 50-mmol m -3 GA 3 immobilized in lanolin was applied to the apical meristem every week for four weeks. Mature seed capsules were collected from flowering plants just prior to being shed, and the seeds stored at room temperature. To monitor germination, seeds were placed in petri dishes on filter paper moistened with deionized water, in growth chambers set at 24 C, a photon irradiance of 120 mol m -2 s -1 (400-700 nm), and a 12-h photoperiod. Gas Exchange Gas exchange measurements were performed with an O 2 electrode system (Hansatech Instruments, Norfolk, England). One day prior to use, submersed plants were collected and placed in 20-L aquaria in a growth chamber equilibrated with atmospheric-CO 2 at an irradiance of 400 mol m -2 s -1 (400-700 nm), and 23 C. All measurements were made in triplicate on fully-expanded, detached leaves at 23 C in a 2-mL chamber filled with buffer set at the appropriate pH and dissolved [O 2 ], and a saturating irradiance of 700 mol m -2 s -1 (400 nm). NaHCO 3 was injected to initiate photosynthesis. To

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68 exhaust intercellular CO 2 the leaves were incubated prior to measurement in DIC-free buffer at the assay pH and an irradiance of 400 mol m -2 s -1 Leaf fresh weight and area were determined for use in rate calculations. The net photosynthesis (NPS) rates of leaves from plants of both populations were determined at the free [CO 2 ] in their natural environments (460 and 50 mmol CO 2 m -3 for the highand lower-CO 2 spring, respectively), and also at a saturating [CO 2 ] of 1200 mmol m -3 The degree to which O 2 inhibited the photosynthesis of plants from both populations was determined by measuring NPS rates at 21% and 1% O 2 (equivalent to 270 and 13 mmol m -3 in solution, respectively) with 10 mmol m -3 [CO 2 ] (air-equilibrium) in 20 mol m -3 MES buffer at pH 5. To assess the ability of the leaves to use bicarbonate ions, NPS was compared for leaves at pH 5 and 9 using 20 mol m -3 MES or CHES buffer, respectively, with 200 mmol m -3 free [CO 2 ] at each pH. Dark respiration (R d ) was also measured at 23 C and 21% O 2 with detached leaves from both populations at the [CO 2 ] and pH of the collection site. For these measurements the chambers were darkened and contained 20 mol m -3 HEPES at pH 72 and 78; the pH values of the highand lower-CO 2 sites, respectively. Rubisco Analysis A minimum of 100 mg of fully expanded leaves (approximately 5 leaves) was collected from each of four plants during a full-sun period (12:00:00) and was immediately frozen in LN 2 at the highand lower-CO 2 spring sites. Total Rubisco activities of these leaves were assayed as described by Vu et al. (1997), with the following modifications. Approximately 100-150 mg leaf material was ground with 1 mL of extraction medium per 50 mg of leaf material. The extraction and assay medium

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69 contained Tricine rather than Bicine buffer, and no isoascorbate was added. Rubisco activities were expressed on a fresh weight basis. Carbohydrates, Fiber and Ash For carbohydrate analyses, six plants from the highand the lower-CO 2 populations were separated into their respective leaves, stems, and roots and were quick-frozen in LN 2 Samples of approximately 150 mg fresh weight were collected at dusk to ensure that carbohydrates were near their maximum amount. Soluble carbohydrates were extracted using 80% (v/v) ethanol at 85 C. Starch and total soluble sugars were quantified using the microtiter method of Hendrix (1993). Carbohydrate assays were performed in triplicate on six and four separate extractions for starch and total soluble sugars, respectively. Fiber was measured using the ANKOM Filter Bag System (Fairport, NY). Four plants were separated into their respective parts and dried at 65 C for 3-d. Approximately 500 mg dry weight of material was then passed through a 1 mm screen in a Wiley mill. Fiber components were separated using an ANKOM 200 Fiber Analyzer (Fairport, NY). Percent fiber was measured as ash-free neutral-detergent fiber (NDF) (Goering & Van Soest 1970) with decalin and sodium sulfite omitted (Golding et al. 1985). Removal of hemicellulose with the addition of acid to the NDF resulted in acid-free detergent fiber (ADF). Washing ADF with 72% (v/v) H 2 SO 4 removed the cellulose portion, leaving lignin. There was not enough root material to perform a fiber analysis on individual high-CO 2 plants; thus, the four replicates were pooled for analysis. To determine ash content, five plants from the high-and lower-CO 2 populations were collected, separated into leaves, stems and roots, and a minimum of 100 mg was

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70 dried at 500 C for 16 h before being ground with a mortar and pestle and subjected to analysis (Horwitz 2000). DNA Fingerprinting DNA fingerprinting of the two Ludwigia populations was performed using amplified fragment length polymorphisms (AFLP) (Zabeau & Vos 1993; Vos et al. 1995). Ten plants were randomly collected from each population and dried in silica gel. DNA was extracted according to Doyle & Doyle (1987). The DNA samples were cleaned using the Qiaquick PCR Purification Kit (Qiagen Inc., Santa Clarita, CA). AFLP was performed using the Plant Mapping Protocol (PE Applied Biosystems Inc., Foster City, CA) unless otherwise noted. The restriction enzymes, EcoRI and MseI, and the T4 DNA ligase and buffer came from New England Biolabs (Beverly, MA). PCR products were generated using a Hybaid PCR Express Thermal Cycler (Ashford, Middlesex, UK). Specific selective primers tested were EcoRI ACT and ACA labeled with D4 (Genset, Paris, France) and MseI CAC, CTC, CTT, CTA, and CTG (Integrated DNA Technologies, Coralville, IA), resulting in ten different primer combinations. Selective amplification reactions contained 200 mmol m -3 each dNTP (Boehringer-Mannheim, Mannheim, Germany) and 1 U AmpliTaq Gold (PE Applied Biosystems, Foster City, CA). Amplification products were run on a Beckman-Coulter Inc., CEQ 8000 Capillary Genetic Analysis System (Fullerton, CA). Primers chosen for further analysis were EcoRI ACT and MseI CAC. Only dense bands with more than 70 base pairs were included in the fragment analyses. The analysis and selective amplification were performed five times for each sample. Thirty-six loci were analyzed for all individuals resulting in a binary matrix that was analyzed using Tools for Population Genetics software (Miller 1997). This

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71 software was used to calculate Wrights F-statistic ( st ) (Weir & Cockerham 1984), a 95% confidence interval (using bootstrapping and 5000 replications), and genetic distance and identity (Nei 1972) for diploid dominant alleles. Statistics Equality of variances between the highand lower-CO 2 populations was tested for each response variable. All variables that were deemed to have equal variances were analyzed using a 2-sample t-test with pooled variances. Variables with unequal variances were analyzed using Sattherthwaites t-test for unequal variances. All statistical calculations were carried out using SAS release 8 (SAS Inst. Inc., Cary, NC, USA) and alpha 005 and 001 levels of significance. Results Spring Characteristics Table 3-1 shows the water chemistry of the two springs measured in 2001 compared with data compiled from the literature going back in most cases over half a century. Both springs showed very little change in the concentrations of inorganic carbon species, in temperature, and pH over a fifty-five year period (Table 3-1). Several CO 2 measurements for the high-CO 2 spring over this time period show that it oscillated around 410 mmol m -3 with no general trend over time (Rosenau et al. 1977; Hornsby et al. 2001; Lytle, data not shown). Likewise, most of the nutrients remained relatively constant over time, although there were exceptions. Most notably, the concentration of nitrate-N in both springs increased ten-fold from 1946 to the present, although NH 4 -N and total P did not change. The concentrations of K, Na, and Cl increased in the high-CO 2 spring over time, as did Ca in the lower-CO 2 water (Table 3-1).

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72 The irradiance characteristics of the two springs were similar, as both were potentially exposed to full sun at midday, but were shaded in early morning and late afternoon (data not shown). Irradiance (400-700 nm) measurements taken at midday in both springs averaged 2000 mol m -2 s -1 just above the water surface and 1100 mol m -2 s -1 underwater at the top of the submersed Ludwigia plants. Water depth was similar, with the highand lower-CO 2 springs measuring 083 031 and 12 021 m deep, respectively. The two springs differed in several key respects. Fanning Springs had a lower pH with almost ten-fold higher free [CO 2 ], and over two-fold higher [HCO 3 ], total C i and alkalinity. It also had approximately four-fold greater [NO 3 -N] and higher total [P] than the lower-CO 2 Rainbow Spring. The free [CO 2 ] in both springs was greater than the air-equilibrium value of 13 mmol m -3 at 23 C, being 35 and 4 times greater for the highand lower-CO 2 spring, respectively. The high-CO 2 spring also had 130% greater [CO 2 ] in the air just above the water surface, with values for the highand lower-CO 2 springs of 521 58 and 402 14 mol CO 2 mol -1 respectively; n = 9. Morphology, Anatomy and Chemical Constituents Submersed plants from the two populations showed morphological differences. Unexpectedly, submersed plants from the high-CO 2 population appeared to be less robust than their lower-CO 2 counterparts (Fig. 3-1). High-CO 2 plants had fewer leaves, more axillary stems, and were greener than the darker, more brownish-red lower-CO 2 population plants (Fig. 3-1). These observations were borne out by quantitative morphological measurements (Table 3-2). The weight and density of individual plants from the high-CO 2 population

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73 were significantly less than those from the lower-CO 2 spring, and the dry weight content was also lower (114 versus 148%). High-CO 2 plants also invested less in leaves. Thus, the total leaf fresh and dry weights per plant were about half of those found in the lower-CO 2 plants. This was due in part to the fact that each high-CO 2 plant had only 65% of the number of leaves found on lower-CO 2 plants, and only 63% of the leaf area (Table 3-2). Total stem dry weight was less for individual plants from the high-CO 2 population, even though the high-CO 2 plants produced more axillary stems, which resulted in a 220% greater total axillary stem length (Table 3-2). The average main stem length was similar between the populations, but the internodes were 24-times longer on the high-CO 2 plants, which is in accord with the finding that plants from this population produced fewer leaves (Table 3-2). As with stem and leaves, root weights were significantly lower for the high-CO 2 population plants. However, the difference in total root length was not significant (Table 3-2). Similarly, although the high-CO 2 plants appeared to have higher shoot: root ratios (125 versus 77 on a dry weight basis) the difference was not significantly different. Individual, fully-expanded leaves on submersed plants in the two populations did not differ significantly in terms of fresh and dry weights, or density (Table 3-3). However, individual leaves from high-CO 2 plants were significantly larger in leaf area, but they were also thinner and averaged fewer spongy mesophyll layers, and crystals (Table 3-3). Not unexpectedly, leaf starch content was greater for plants growing in high-CO 2 In contrast, leaf fiber and cellulose were greater for plants in the lower-CO 2 spring (Table 3-3).

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74 Figure 3-2 shows anatomical cross sections of submersed leaves from the two plant populations. The leaf from the lower-CO 2 plant was slightly thicker (Fig 3-2b). In both, a single layer of epidermis and a cuticle of similar thickness were present. There was a single layer of palisade mesophyll, but it was more clearly defined and the cells were more elongated in leaves from the high-CO 2 population (Fig. 3-2). Aerenchyma was absent from Ludwigia leaves; although the cells were loosely arranged and similar amounts of small, intercellular air spaces were evident in the leaves from both populations. Stomates were present on the leaves, even though the plants were submersed. The high-CO 2 plants exhibited a significantly greater stomatal density, up to ten-fold higher than the lower-CO 2 plants (Table 3-3). The distribution was amphistomatous, with the adaxial surfaces of the highand lower-CO 2 plants containing, respectively, three and sixteen times more stomates per unit leaf area than the abaxial surfaces (Table 3-3). The major stem and root characteristics are shown in Table 3-4. Stem and root diameters and densities were less in plants from the high-CO 2 population, and for the stem the latter coincided with the lower dry to fresh weight value of 10 versus 16% (Table 3-2). In terms of anatomy, a single epidermal layer surrounded the stems of plants from both population, but it is notable that the high-CO 2 plants contained 61% more aerenchyma and fewer cortical cell layers in the main stem cortex than plants from the lower-CO 2 population (Table 3-4). Cells in the stems of the high-CO 2 plants produced far fewer crystals. Unlike the leaves where starch was high, the stems of the high-CO 2 plant contained significantly less starch than those of the lower-CO 2 plants, although sugars, cellulose, lignin, and ash contents were greater (Table 3-4).

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75 In contrast to the stems, the roots did not contain aerenchyma; however, they did have a large number of small, intercellular air spaces. Concomitant with their smaller diameter, the roots of high-CO 2 plants exhibited significantly fewer cell layers in the cortex and inside the cortex, and crystals within the cortical cells (Table 3-4). In terms of chemical constituents, roots from both populations were similar in their contents of TNC, starch, sugar, cellulose, and hemicellulose, but differed in total fiber, lignin and ash with the high-CO 2 plant roots containing less than their lower-CO 2 counterparts (Table 3-4). Reproduction Monthly visits to the springs in 2001 revealed that flowering in the high-CO 2 spring occurred from May through September, while seed capsules were present from July through November. Flower production by plants in the high-CO 2 spring was abundant, while the lower-CO 2 population produced almost no flowers. Virtually all the plants in the high-CO 2 population that grew emergent or near the water surface produced flowers, but only about 10% of the fully submersed plants flowered. Flowers from the high-CO 2 population usually developed on tissue that was at the air-water interface or completely emergent. The number of flowers per plant was determined for the high-CO 2 population in the middle of the flowering period (July), and it averaged 86 39 (n = 10). In contrast, only three plants with flowers were found in the lower-CO 2 population from 1997 through 2002, and each had only a single seed capsule that was submersed and undeveloped. Plants of both populations reproduced asexually in the field by fragmentation and ramet formation. Growth chamber data confirmed the field data regarding flower production. When the plants were maintained submersed they failed to flower (Table 3-5). When emergent,

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76 only plants from the high-CO 2 population flowered and the number of flowers per plant were similar to values measured in the field. Increasing or decreasing the photoperiod failed to induce flowering of plants from the lower-CO 2 spring (data not shown). However, the application of GA 3 did induce flowering, and almost doubled the number of flowers per high-CO 2 plant (Table 3-5). Plants treated with GA 3 elongated more rapidly and had longer internodes than those without exposure to GA 3 (data not shown). Seed capsules produced by plants treated with GA 3 were about 50% longer than those produced without GA 3 (data not shown). For the high-CO 2 plants, GA 3 treatment did not change the number of seeds produced per capsule, which averaged 60, nor did it modify the number of seeds that subsequently germinated (90%). Flowers of the GA 3 -induced low-CO 2 plants produced very few capsules, each with only about ten seeds, none of which germinated. Photosynthetic Characteristics Figure 3-3 shows the effects of [O 2 ] on the net photosynthesis (NPS) rates of submersed leaves of plants from the two Ludwigia populations. The measurements were performed at pH 50 with an air-equivalent [CO 2 ] of 10 mmol m -3 The NPS rates at 21% (air-equivalent) O 2 were lower than at 1% O 2 Thus O 2 inhibited the photosynthesis of plants from both populations, with similar inhibition values of 31 and 29% for the highand lower-CO 2 populations, respectively. Figure 3-4 illustrates the effects of HCO 3 on the NPS rates, with measurements at pH 50 and 90, but at the same [CO 2 ] of 200 mmol m -3 The rates were not significantly different at the two pH values, even though the [HCO 3 ] was 8000-fold higher at pH 90. Figure 3-5 compares the NPS rates of submersed leaves measured at the pH and [CO 2 ] of their natural spring locations, as well as at saturating [CO 2 ]. It also includes the

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77 values for total Rubisco activities in submersed leaves harvested from the two populations in full sun irradiance and quick-frozen in liquid N 2 When measured at their respective natural spring [CO 2 ], leaves from the high-CO 2 spring had significantly higher NPS rates, by almost six-fold, than their lower-CO 2 counterparts. In contrast, when measured at saturating [CO 2 ], the difference in NPS rates was much less (Fig. 3-5). Total Rubisco activities were sufficient to account for the NPS values measured at saturating [CO 2 ]. However, submersed leaves from the high-CO 2 plants exhibited almost twice the Rubisco activity of those from the lower-CO 2 spring (Fig. 3-5). The dark respiration rates were similar for leaves from both populations, being 13 3 and 12 2 mol O 2 m -2 leaf area h -1 for the highand lower-CO 2 plants, respectively. However, when compared to the NPS rates at the spring pH and [CO 2 ], dark respiration was equivalent to only 11% of the high-CO 2 plant NPS rate, whereas it was 55% of the lower-CO 2 leaf NPS rate. DNA Fingerprinting The phenotypic differences observed between the two plant populations were reflected in the results of the AFLP analysis. The populations showed clear genetic differences with a high level of separation. Neis genetic identity (I) and distance (D) between the populations were 089 and 011, respectively. Similarly, Wright's F-statistic ( st ) was 069 with a 95% C. I. of 04887, showing that fragment variation was much greater between than within the populations. The highand lower-CO 2 populations differed at eight of the 36 loci examined. When the within-population variation was examined, the high-CO 2 population differed at two loci, whereas the lower-CO 2 population showed fragment diversity at five loci.

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78 Discussion Both freshwater springs have been very stable in terms of temperature, pH, alkalinity, and for this study particularly [CO 2 ] and [HCO 3 ], over long periods of time, probably far exceeding the past fifty-five years for which data are available. Thus, if it is possible to adapt to the local [CO 2 ], plant populations in these springs have likely had time to do so, especially sexually reproducing populations. An effect of nitrate cannot be excluded as the concentration of this nutrient differs in the two springs, and has risen substantially in both. However, this troubling rise is relatively recent, as Fanning Springs shows the greatest increase only since the 1970s (Hornsby & Ceryak 1998). In the case of Rainbow Springs the increase has been attributed to fertilizer run-off from pastureland (Jones et al. 1996). Previous research using natural CO 2 sources to assess the long-term responses of plants to elevated [CO 2 ] has focused on populations growing at different distances from a single CO 2 source, but generally less than 1 km apart (Cook et al 1998; Polle et al. 2001). As a consequence, gene flow among the populations usually cannot be excluded, and any such exchange would lessen the possibility that the population close to the source is genotypically adapted to elevated CO 2 By contrast, the highand lower-CO 2 populations of L. repens in the present study were separated by 85 km. Furthermore, previous studies have not included an analysis to determine if in fact the populations differ genetically, whereas in this work they were subjected to DNA fingerprinting. The diversity index suggested appreciable genetic differentiation; greater than that found with other species (Wright 1978, Smulders et al. 2000). Likewise, the 11% genetic distance also indicates the two populations were genetically distinct. In fact, genetic distances of 10% or higher can be used to support categorizing two plant groups into distinct species (Nei 1987).

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79 Consequently, this degree of genetic separation is great enough to propose that the two populations of L. repens are adapted, rather than just acclimated, to their particular environments, however it does not exclude the possibilities that the differences were due to drift or environmental factors in addition to [CO 2 ]. The phenotypic differences that were observed in this study in reproductive, morphological, and photosynthetic characteristics are also consistent with genotypic adaptation of the two populations. In regard to sexual reproduction, only plants from the high-CO 2 population flowered in their natural spring environment or in the growth chamber, and set viable seed. Altering the photoperiod did not induce flowering in plants from the lower-CO 2 population. They only flowered when treated with GA 3 and even then the few seeds formed were not viable. Flowering and seed set was typically confined to plants with emergent shoots, not plants that remained fully submersed. Sculthorpe (1967) also noted that Ludwigia species growing entirely submersed in deep water tended to be sterile. In most instances, aquatic angiosperms produce their flowers above the water surface, as pollen is essentially a terrestrial adaptation (Sculthorpe 1967; Spencer & Bowes 1990). Both populations could grow emergent, but the water level in the high-CO 2 spring fluctuated more than in the lower-CO 2 spring, allowing shoots of the high-CO 2 plants to become emergent more often. This may partially explain the prolific flowering in the high-CO 2 population, but it does not explain the lack of flower production by the lower-CO 2 population in the growth chamber experiments. The three flowers that were found on the lower-CO 2 plants in Rainbow Springs, and the GA 3 -induced flowering, suggest that this population had some potential to reproduce sexually, but the inability of the seeds to

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80 germinate makes it unlikely that it did so. The constant environmental conditions of natural springs may favor asexual reproduction, and both populations were observed to reproduce by fragmentation and ramet formation. Cloning is a common phenomenon in submersed species (Sculthorpe 1967; Grace 1993). The two populations appear to differ in reproductive strategies. The high-CO 2 population, with greater access to CO 2 and N, devoted resources to flowering, fragmentation, and ramet formation, while the lower-CO 2 plants appear to be solely dependent on fragmentation and ramet formation. It is interesting that even though the lower-CO 2 population only reproduced asexually it showed greater within-population variation than the high-CO 2 population, which tends to rule out a founder effect. However, more extensive genetic analysis is needed to confirm it. Plants from both populations were C 3 based on the observation that O 2 inhibited their photosynthesis rates by an average of 31%, which is similar to that found with terrestrial C 3 species (Bowes 1993). Furthermore, they appeared to be CO 2 -only users, since there was no evidence that the presence of bicarbonate ions enhanced photosynthesis. Another submersed species in the genus, L. natans, was also found to be a CO 2 -only user (Prins et al. 1980). Based on these gas exchange characteristics, it does not appear that the plants possessed any CO 2 concentrating mechanism. With these photosynthesis characteristics, the growth of plants in both populations should benefit from a CO 2 -enriched environment. From acclimation studies with other C 3 species one might expect the high-CO 2 plants to exhibit lower Rubisco activity, but this was not the case. Total Rubisco activity in leaves from the high-CO 2 population, and the lightand CO 2 -saturated photosynthesis

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81 rates, were not down-regulated relative to values found for the lower-CO 2 plants. Rubisco constitutes a large proportion of the [N] in plants therefore higher nitrate concentrations in Fanning Springs may be one of the factors maintaining the Rubisco activity in these high-CO 2 plants. Because photosynthesis was not down-regulated, the naturally tenfold higher [CO 2 ] enabled the high-CO 2 leaves to exhibit much greater photosynthesis rates than their lower-CO 2 counterparts when both were measured at the respective pH and [CO 2 ] of their spring environments. In fact the high-CO 2 leaves probably operated at close to CO 2 -saturated rates in their natural habitat; in contrast the leaves of lower-CO 2 plants were probably at less than 20% of capacity. Similarly, a terrestrial species, L. uruguayensis, exposed to naturally high [CO 2 ] also exhibited higher photosynthesis rates than plants further away from the CO 2 source (Koch 1993). Individual leaves of high-CO 2 L. repens plants were larger in area than lower-CO 2 leaves, and, together with the higher photosynthesis rates, they were more effective at carbon gain in their natural habitat. However, the greater carbon gain of individual leaves has to be balanced against the fact that high-CO 2 plants had 135% longer stem internodes that resulted in fewer leaves per plant. In contrast, the lower-CO 2 population had more leaves, resulting in more leaf dry weight and a 60% greater investment in total leaf area per plant. This greater emphasis on leaf area may be an adaptation to enhance CO 2 capture in the Rainbow Spring habitat where CO 2 appears to be a major limiting resource, and should partially compensate for the lower photosynthetic capacity per unit leaf area. On a per unit leaf area basis dark respiration rates were similar for plants from the two populations measured under conditions akin to those in their respective habitats.

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82 There was no evidence for lower rates in the high-CO 2 plants as has been reported for terrestrial species (Azcn-Bieto et al. 1994, Drake et al. 1999). It has recently been suggested that elevated CO 2 has little effect on dark respiration (Jahnke 2001), and the present data are consistent with this proposal. However, when the greater photosynthesis rates and reduced leaf area are considered, dark respiration probably consumes a smaller fraction of the fixed carbon of the high-CO 2 plants in their natural habitat. The greater photosynthesis rates and Rubisco activities of the high-CO 2 leaves did not translate into greater mass for individual plants. Thus far, plants growing near high-CO 2 springs have not shown large biomass differences compared to populations growing in ambient [CO 2 ] (Cook et al. 1998). Plants and organs from the high-CO 2 population generally exhibited lower weight and density than those from the lower-CO 2 population. Thus, on a total plant basis, high-CO 2 plants had a lower dry to fresh weight ratio, and all of the dry weight components measured (TNC, starch, fiber, lignin, and ash) were lower for them, only sugars ran counter to this trend. A further factor contributing to the lower density of the high-CO 2 plants was the degree of aerenchyma in the stems; they had 60% more than the lower-CO 2 plant stems. Westlake (1965) has pointed out that the density of most freshwater macrophytes is largely a function of the proportion of internal air spaces. When the plants were subdivided into their component organs, leaves made up the majority of the dry weight for both populations, although as noted earlier, there was less total leaf dry weight in high-CO 2 plants. Although the weight of individual leaves from the two populations was similar, leaves from the high-CO 2 population were not as thick, and had less fiber and cellulose, fewer mesophyll layers, and fewer crystals than those from the lower-CO 2 population.

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83 The palisade mesophyll layer in the leaves of high-CO 2 plants was more clearly defined than in the lower-CO 2 plants, with more elongated and tightly packed cells. Most submersed leaves have a homogeneous mesophyll (Sculthorpe 1967), but they are often in conditions where CO 2 is the major limitation to photosynthesis (Bowes & Salvucci 1989; Madsen & Vadstrup 1995). In the case of the high-CO 2 leaves, irradiance was likely to be co-limiting with CO 2 and the organized palisade layer may enhance absorption of light. Despite the fact that high-CO 2 plants contained less TNC overall, by dusk their leaves accumulated more starch than those of the lower-CO 2 plants, which is consistent with higher photosynthesis rates. Carbohydrate accumulation has been observed in some other investigations with plants growing for long periods near elevated-CO 2 sources (Krner & Miglietta 1994; Cook et al. 1997; Stylinski et al. 2000). Excessive carbohydrate accumulation in leaves grown at elevated [CO 2 ] is often associated with photosynthetic acclimation (Stitt 1991; Sheen 1994), but this did not occur in the present study. Krner & Miglietta (1994) have hypothesized that such carbohydrate buildup is not due to a short-term imbalance of carbon, but rather an intrinsic inability of plants to dissipate the carbohydrate pools. Even though the high-CO 2 plants had a lower total stem dry weight, they produced far more axillary shoots, and thus had a greater total stem length, than plants from the lower-CO 2 population. With more aerenchyma and lesser amounts of starch, lignin, mucilage, cortical cell layers, and crystals, the density of the stems from the high-CO 2 plants was lower than those from the lower-CO 2 population. Furthermore, the diameter of the axillary stems was much smaller than that of the main stems. It is possible that the

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84 high-CO 2 population invested the additional carbon in producing axillary stems, rather than leaves, as a means of increasing asexual reproduction through fragmentation and ramet formation. Lacuna in the plants from both populations was restricted to the stems, while the leaves and roots contained large intercellular air spaces. It is not clear if the difference in amount of air space was related to the [CO 2 ] to which the tissues were exposed. As with the stems, the roots of high-CO 2 plants exhibited less dry weight and density than those of the lower-CO 2 plants. Populations of L. repens collected in the highand lower-CO 2 springs exhibited characteristics consistent with genotypic adaptation to their respective environmental [CO 2 ]. The level of genetic difference between the populations was great enough to suggest that they have diverged with little or no gene flow between them. Plants from the high-CO 2 population did not show down-regulation of photosynthesis and Rubisco activity when compared to the lower-CO 2 population. It is also possible that in the high-CO 2 population the investment in sexual and asexual reproductive methods may be facilitated by the greater availability of resources, including CO 2 By contrast, plants in the lower-CO 2 spring may invest in greater leaf surface area to compensate for the limited availability of CO 2 and put more resources into existing plants, rather than into prolific reproduction. It should be noted that plants in the high-CO 2 springs are unlikely to show further adaptation as atmospheric [CO 2 ] rise because they already grow in an environment with [CO 2 ] far higher than in equilibrium with the atmosphere.

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Table 3-1. Water chemistry of the highand lower-CO 2 springs. The 2001 data are reported as mean SD (n = 4), while all other data are single values obtained from the literature. The 1946 data are from Ferguson et al. (1946). Missing data from 1946 are indicated with the year the supplemental data was taken in parentheses. Data for 1956 and 1974 were from Rosenau et al. (1977), while the 1995 data came from the Suwannee River Water Management District (Hornsby et al. 2001) 85 High-CO 2 (Fanning) Spring Lower-CO 2 (Rainbow) Spring Parameter 1946 2001 1946 2001 Temperature (C) 23 23 1 24 23 1 pH 7 72 01 7 7 01 Total alkalinity (meq L -1 ) 34 (1956) 37 02 11 (1974) 1 02 CO 2 (mmol m -3 ) 390 460 20 45 50 5 HCO 3 (mmol m -3 ) 3400 3700 200 1300 1500 180 C t (mmol m -3 ) 3800 4200 200 1400 1600 200 NH 4 -N (mmol m -3 ) 071 (1995) 14 07 14 (1974) 071 035 NO 3 -N (mmol m -3 ) 31 351 8 13 82 4 Total P (mmol m -3 ) 23 (1995) 26 03 0 097 032 K (mmol m -3 ) 15 59 3 10 5 26 Ca (mmol m -3 ) 1700 2100 25 530 1500 25 Mg (mmol m -3 ) 200 230 4 170 240 4 Na (mmol m -3 ) 110 200 17 130 120 9 Cl (mmol m -3 ) 110 320 49 100 140 6 Fe (mmol m -3 ) 1 089 036 1 036 018

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86 Lower-CO2 plantHigh-CO2 plant Figure 3-1. Submersed L. repens plants from the highand lower-CO 2 populations.

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87 Table 3-2. Weights, densities, and morphological characteristics of L. repens plants growing in highand lower-CO 2 springs. Parameter High-CO 2 population Lower-CO 2 population Fresh weight (g plant -1 ) 2 068** 4 101 Dry weight (g plant -1 ) 0 009** 0 015 Density (g cm -3 plant -1 ) 0 015** 0 015 Leaf fresh weight (g plant -1 ) 0 033** 2 042 Leaf dry weight (g plant -1 ) 0 006** 0 007 Leaves (no. plant -1 ) 39 15** 60 11 Leaf area (cm 2 plant -1 ) 52 18** 83 15 Total stem fresh weight (g plant -1 ) 1 04 1 05 Total stem dry weight (g plant -1 ) 0 004** 0 008 Main stem length (cm plant -1 ) 23 3 21 5 Main stem internode length (cm plant -1 ) 1 045** 0 012 Axillary stems (no. plant -1 ) 9 34** 2 11 Axillary stem length (cm plant -1 ) 12 101** 4 32 Root fresh weight (g plant -1 ) 0 006* 0 038 Root dry weight (g plant -1 ) 0020 0004** 0075 0043 Total root length (cm plant -1 ) 107 32 161 97 Mean SD (n = 8). P-values are from t-tests and significant differences between populations at P 005 and 001 are noted by and **, respectively.

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88 Table 3-3. Individual leaf characteristics of L. repens plants growing in highand lower-CO 2 springs. Parameter High-CO 2 population Lower-CO 2 population Fresh weight (mg leaf -1 ) 52 17 42 18 Dry weight (mg leaf -1 ) 6 20 8 13 Density (g cm -3 ) 0 018 0 014 Area (cm 2 leaf -1 ) 2 06* 1 01 Thickness (mm leaf -1 ) 0 001* 0 001 TNC (mg g -1 dry weight) 115 59 47 10 Starch 85 41* 30 13 Sugars 30 22 17 10 Fiber (mg g -1 dry weight) 195 25* 262 46 Lignin 39 22 84 39 Cellulose 89 7** 119 10 Hemicellulose 66 7 59 4 Ash (mg g -1 dry weight) 125 17 130 37 Spongy mesophyll cell layers (no. leaf -1 ) 3 03** 4 05 Crystals (no. mm -2 ) 5 10** 23 91 Adaxial stomates (no. mm -2 ) 103 26** 47 9 Abaxial stomates (no. mm -2 ) 33 11** 3 2 TNC, total non-structural carbohydrate. Mean SD (n = 4-8). P-values are from t-tests and significant differences between populations at P 005 and 001 are noted by and **, respectively.

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89 (a)(b)e epidermal cellp palisade mesophyll cells spongy mesophyll cellc crystal Figure 3-2. Leaf cross sections for L. repens plants growing in (a) highand (b) lower-CO 2 springs.

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90 Table 3-4. Stem and root characteristics of L. repens plants growing in highand lower-CO 2 springs. High-CO 2 population Lower-CO 2 population Parameter Stem Root Stem Root Aerenchyma (% area) 29 3** 18 4 Cortex cell layers (no.) 14 2* 95 14** 16 1 133 12 Cell layers inside cortex (no.) 17 1 70 07** 19 3 84 05 Crystals (no. mm -2 ) 0 097** 11 27** 10 2 21 94 Density (g cm -3 ) 073 013* 062 018* 116 048 092 032 Diameter (mm) 21 02** 057 009 2 01 061 011 TNC (mg g -1 dry weight) 73 18 29 8 128 45 27 9 Starch 43 15* 24 7 118 49 31 16 Sugars 30 3** 28 05 11 6 29 09 Fiber (mg g -1 dry weight) 324 19 456 a 302 27 539 22 Lignin 40 3** 132 a 68 23 233 24 Cellulose 193 15** 196 a 124 23 203 14 Hemicellulose 91 8 128 a 110 15 104 7 Ash (mg g -1 dry weight) 137 13** 118 27** 99 12 171 17 TNC, total non-structural carbohydrate. a Pooled the low-CO 2 population replicates since there was not enough root material per plant to analyze individual replicates, therefore, no statistical analysis was done. Mean SD (n = 4-8). Using t-tests, significant differences between populations and within organ at P 005 and 001 are noted by and **, respectively.

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Table 3-5. Reproductive characteristics of L. repens plants collected from highand lower-CO 2 populations and grown in a growth chamber. For the gibberellic acid (GA 3 ) treatments, 200 L of 50-mmol m 3 GA 3 immobilized in lanolin was applied to the apical meristem every week for four weeks. Plants Producing Flowers (%) Flowers (no. plant -1 ) Habit GA 3 (mmol m -3 ) High-CO 2 Lower-CO 2 High-CO 2 Lower-CO 2 Submersed 0 0 0 0 c 0 c Emergent 0 100 0 55 19 b 0 c Emergent 50 100 100 100 16 a 63 32 ab Mean SD (n = 3-4). T-tests were performed between the flowering treatments and significant differences at P 005 are noted by a b and c 91

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92 NPS rate (mol O2 m-2 LA h-1) 0102030 High-CO2 population 1% O2 21% O2 O2 inhibition:Lower-CO2 population Figure 3-3. Oxygen inhibition of net photosynthesis rates on a leaf area basis for L. repens plants growing in highand lower-CO 2 springs. Rates were measured at [CO 2 ] = 10 mmol m -3 and pH 5 and can be converted to a fresh weight basis by dividing the values shown for the highand lower-CO 2 populations by 17 and 23, respectively. Mean SD (n = 4).

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93 NPS rate (mol O2 m-2 LA h-1) 04080120160 High-CO2 populationpH 9 pH 5 HCO3use: Lower-CO2 population Figure 3-4. Net photosynthesis rates on a leaf area basis for L. repens plants growing in highand lower-CO 2 springs to test for bicarbonate use. Measured at [CO 2 ] = 200 mmol m -3 Net photosynthesis rates can be converted to a fresh weight basis by dividing the values shown for the highand lower-CO 2 populations by 17 and 23, respectively. Mean SD (n = 4).

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94 NPS rate (mol O2 m-2 LA h-1) 060120180240300360Total Rubisco activity (mol CO2 m-2 LA h-1) 060120180240300360 NPS rate at spring [CO2] NPS rate at saturating [CO2] Total Rubisco activity PopulationHigh-CO2Lower-CO2 a a a b b b Figure 3-5. Net photosynthesis rates and Rubisco activities on a leaf area basis for submersed leaves of L. repens plants collected from high-CO 2 and lower-CO 2 springs. To measure net photosynthesis (NPS) rates, [CO 2 ] used for the highand lower-CO 2 springs were 460 and 50 mmol CO 2 m -3 respectively, while saturating [CO 2 ] was 1200 mmol CO 2 m -3 for both springs. Rates can be converted to a fresh weight basis by dividing the values shown for the highand lower-CO 2 populations by 17 and 23, respectively. Mean SD (n = 4-8). P-values are from t-tests and significant differences between populations at P 005 are noted by a and b.

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CHAPTER 4 THE EFFECT OF CO 2 AND EMERGENCE ON THE GROWTH OF LUDWIGIA REPENS PLANTS FROM HIGH AND LOWER-CO 2 SPRING POPULATIONS Introduction Carbon dioxide in the atmosphere has been steadily rising (Keeling & Whorf 2002) with a doubling possible in the present century (Cias 1999). This increase has generated considerable research investigating its effects on plants. Short-term responses of terrestrial plants to elevated-CO 2 have been well documented, and are especially dependent on the mode of photosynthetic CO 2 fixation (Bowes 1993; Drake et al. 1996a; Pritchard et al. 1999). Thus, C 3 plants usually show greater responses than their C 4 and CAM counterparts, which already concentrate CO 2 spatially or temporally at the site of fixation by Rubisco (Bowes 1993). When grown at elevated-CO 2 C 3 plants have enhanced carboxylation and decreased oxygenation of Rubisco, leading to higher photosynthesis rates (Drake et al. 1996a). Over longer time periods this stimulation in photosynthesis is often down-regulated as the plant undergoes acclimation to the elevated CO 2 Acclimation may occur because of the increase in carbohydrate concentration or nutrient and sink strength limitations (Drake et al. 1996a). Hexokinase is the most likely candidate for initiating the signaling process that leads to down-regulation of photosynthesis (Koch 1996; Jang & Sheen 1997; Moore et al. 1998). Even with down-regulation effects, C 3 plants generally have greater productivity due to the initial growth enhancement that occurs at high-CO 2 Poorter (1993) from a survey of the literature 95

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96 reported that on average the dry weight of both cultivated and wild C 3 plant species increased by 41% when the [CO 2 ] was doubled. Research on aquatic plants lags far behind terrestrial plant research. The CO 2 assimilation mechanisms of submersed plant species are less well documented and are less easily classified into the terrestrial categories of C 3 C 4 and CAM photosynthesis, thereby making it more difficult to derive generalizations about their responses. Great plasticity exists in aquatic plants with the photosynthetic characteristics of many species being modified by their environment (Bowes & Salvucci 1989; Bowes 1993). Also, there are additional forms of inorganic carbon available in water and, along with CO 2 about 50% of submersed species are able to use bicarbonate ions for photosynthesis (Spence & Maberly 1985). Similar to terrestrial plants, there is evidence that submersed plants show enhanced growth at elevated CO 2 but may also be subject to down-regulation of photosynthesis (Sand-Jensen & Gordon 1986; Madsen & Sand-Jensen 1994; Madsen et al. 1996). Current thinking is that rising atmospheric [CO 2 ] should impact terrestrial and emergent flora more than submersed species (Bowes 1993, 1996; Raven 1994). This is because CO 2 diffuses 10 4 times slower in water than in air (Raven 1970), which greatly limits the photosynthesis of submersed leaves, far more so than the diffusion limitations for terrestrial species. The use of HCO 3 reduces the dependence of some submersed species on CO 2 and also lessens their potential response to elevated [CO 2 ] (Madsen et al. 1996). Amphibious plants produce both submersed and aerial photosynthetic tissues. The aboveand below-water environments differ considerably; therefore the aerial and submersed parts of a given plant experience very different limitations on photosynthesis

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97 and growth. It has long been recognized that the emergent leaves of amphibious plants have higher net photosynthesis rates than their submersed counterparts (Lloyd et al. 1977; Salvucci & Bowes 1982; Spencer & Bowes 1985). Consequently, Bowes (1987) proposed that the aerial shoots of amphibious plants improved access to CO 2 when it is severely limiting in water. Prins and Guia (1987) concluded that Stratiotes aloides was CO 2 -limited during the early, fully submersed stages, but this limitation was reduced after the plants became emergent. Madsen and Breinholt (1995) tested the importance of air contact on growth of an amphibious species, C. cophocarpha. They reported that the growth rate of plants with aerial leaves was three-times higher than that of totally submersed plants when both were grown in water at an air-equilibrium [CO 2 ]. However, when [CO 2 ] in the water was increased, the difference in growth rate between the submersed and emergent plants was reduced. While short-term acclimation studies have provided valuable insight as to how plants respond to elevated-CO 2 in order to predict how plants will adapt to the rising [CO 2 ] in the atmosphere it is crucial that experiments are extended over longer time periods. Currently, research investigating plant adaptation to high-CO 2 is sparse because of the long time frames necessary to elicit genetic adaptation. Natural systems in which CO 2 has been elevated for many generations have been identified with the hope that they may provide answers regarding CO 2 adaptation of plants (Raschi et al. 1997). Geothermic gas vents such as volcanoes, geysers, and natural springs constantly emit [CO 2 ] at greater than ambient values. Natural springs have proven the most fruitful of these sites, since they do not produce toxic gases and extremely high temperatures.

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98 Historically, changes in water chemistry are typically small in the spring environments (Rosenau et al. 1977) so selective pressures may be consistent over many decades. Research using natural springs to investigate high-CO 2 responses of terrestrial plants began over a decade ago. Thus far, the evidence suggests that many spring-CO 2 exposed terrestrial plants tend to show less down-regulation than is found in short-term studies, and thus may have adapted to their high-CO 2 environment. Two major types of experiments have been conducted. The first involves the collection of plant populations in the same community but growing at varying distances from the spring, and thus exposed to differing [CO 2 ]. Commonly, plants growing nearest to high-CO 2 springs do not produce greater biomass than plants further from the springs (Krner & Miglietta 1994; Cook et al. 1998) and show little evidence of photosynthetic down-regulation (Koch 1993; Bettarini et al. 1997). These experiments suffer from the fact that the populations being compared are close so that gene flow cannot be ruled out. Furthermore no genetic analyses of the populations have been performed, to determine if they differ. The second general category of spring experiments is common garden experiments. These involve the collection of seeds or seedlings from populations at different [CO 2 ] within the same spring environment, which are then grown in common, controlled environments, but at different [CO 2 ]. By using only a portion of a plant or its offspring in these experiments prior environmental effects are minimized. Again, there has been no genetic analysis of the plant populations, and as they are often collected less than 100 m apart that genetic exchange cannot be ruled out. Despite these drawbacks, the data for terrestrial species are consistent with adaptation to a high-CO 2 environment. In the case of Agrostis canina and Plantago major populations near a CO 2 spring, exposure to

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99 elevated-CO 2 in a common environment stimulated the growth of all the plants, but those collected near the spring exhibited the greater initial relative growth rate (RGR) (Fordham et al. 1997a, b). Seed from spring populations of Boehmeria cylindrica were grown at 350, 525, and 675 mol CO 2 mol -1 (Woodward 1987). Differences in the growth of the populations were noted only at the highest CO 2 treatment, with the plants that developed from the seed collected where [CO 2 ] was the highest showing the greatest growth and height. In the previous chapter submersed populations of a native plant, Ludwigia repens, growing in highand lower-CO 2 springs (460 and 50 mmol m -3 respectively) were characterized. This was the first such study that compared submersed populations. The springs where the plants were growing are not connected by water and are 85 km apart, but they share similar environmental characteristics. The data showed that Ludwigia repens performs C 3 -like photosynthesis and is unable to use HCO 3 Genetic fingerprinting of the populations using amplified fragment length polymorphisms (AFLP) showed substantial genetic diversity between the populations. Plants from the high-CO 2 population had greater photosynthesis rates, and number of axillary shoots, but lower dry weight and leaf area than those from the lower-CO 2 population. The high-CO 2 population reproduced both sexually and asexually. In contrast the lower-CO 2 population did not flower and seemed entirely dependent on asexual reproduction. While this study addressed the lack of a genetic analysis in CO 2 spring studies, it did not eliminate the other major drawback that the differences between the populations may be due to environmental acclimation, rather than genetic adaptation differences.

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100 In the present study, plants from the highand lower-CO 2 spring populations were used to perform a common garden experiment. The hypothesis being tested is that if the plants from the two populations are inherently CO 2 -adapted they will differ in their responses to [CO 2 ] when grown in a common environment. In addition, because of the massive CO 2 diffusion limitations in water, it is anticipated that the plants will show greater responses to elevated [CO 2 ] when their shoots become emergent. The major experimental objectives to test these two hypotheses were to 1) determine if RGR and morphology of highand lower-CO 2 population plants differ when exposed to elevated [CO 2 ] under the same growth conditions; and 2) establish if emergence of these plants affects their RGR and morphology responses to elevated [CO 2 ]. Materials and Methods Plant Material and Collection Site Two populations of Ludwigia repens L. were selected for the study, one in a high-[CO 2 ] spring, Fanning Springs, FL (29 35' 15" N, 82 56' 08" W) and the other in a spring with much lower [CO 2 ], Rainbow Springs, FL (29 06' 08" N, 82 26' 16" W). Both springs reside on the Ocala geological uplift and fit into the calcium bicarbonate category of spring classification system developed by Woodruff (1993). Analyses of the spring conditions were described in the previous chapter. The free [CO 2 ] in both springs was greater than air-equilibrium, being 35 (460 mmol m -3 ) and four times (50 mmol m -3 ) more for the highand lower-CO 2 spring, respectively. The high-CO 2 Fanning Springs had a lower pH with almost ten-fold higher free [CO 2 ], and over two-fold higher [HCO 3 ], total DIC and alkalinity than Rainbow Springs. There was also approximately four-fold greater [NO 3 -N] and higher total [P] in the lower-CO 2 spring. Both springs were about 1-m deep and potentially exposed to full sun at midday, but were shaded in

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101 early morning and late afternoon. They showed very little change in the concentrations of inorganic carbon species, in temperature, and pH over a fifty-five year period. Likewise, most of the nutrients remained relatively constant over time, although there were exceptions. Most notably, the concentration of nitrate-N in both springs increased ten-fold from 1946 to the present, although NH 4 -N and total P did not change. At both springs, the L. repens populations grow both partially emergent and completely submersed along the shore near the boils and occupy approximately 16 m 2 Submersed 12-cm long apical pieces of L. repens, comprised of the main stem and leaves, were collected from the highand lower-CO 2 springs and thoroughly cleaned of epiphytes prior to use. Growth Conditions This experiment was conducted from September through December of 1999. Stem pieces were planted in 400-mL styrofoam cups filled with sand. Five cups from each population were placed randomly in 40-L aquaria filled with water from the headsprings of Rainbow Springs, because it has lower DIC and nutrient concentrations than Fanning Springs. Three aquaria each were placed in six Soil-Plant-Atmospheric-Research (SPAR) plant growth chambers located in Gainesville, Florida, USA. These chambers were similar to those described in Jones et al. (1984). Aboveground chamber dimensions were 20 10 m in cross-section and 15 m in height. The chambers were constructed of an aluminum frame covered with transparent polyethylene telephthalate (Sixlight, Taiyo Kogyo Co., Tokyo, Japan), which decreased incident PAR at the top of the chamber by only about 10%. To mimic the shading that occurs in the field due to surrounding vegetation the sides of the chambers were covered with shade cloth. This decreased the

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102 incident PAR by about 60% where the plants were growing in the early morning and late afternoon. All stem pieces were grown at air-equilibrium [CO 2 ] (375 mol mol -1 ) for 21 d to acclimate them to the SPAR chamber environment and to allow roots to become established. After 21 d at ambient-air, the SPAR chambers were randomly assigned one of three [CO 2 ] treatments: ambient (375 mol mol -1 ), twice ambient (750 mol mol -1 ) and four-times ambient (1500 mol mol -1 ), with two SPAR chambers per treatment. The data for microclimate conditions and canopy carbon-exchange rate were collected and monitored according to Pickering et al. (1994) and Gesch et al. (1998), with aquarium water temperature and air [CO 2 ] measurements taken every 5 min. Carbon dioxide concentrations in the air of the SPAR chambers were similar within each [CO 2 ] treatment. The ambient treatment actually averaged slightly higher than ambient at 430 46, while the elevated-CO 2 treatments averaged 740 52, and 1500 68 mol CO 2 mol -1 Water in the aquaria was continually aerated to equilibrate with these air-CO 2 mixtures. Following Henry's Law of solubility the dissolved [CO 2 ] in equilibrium with the air-CO 2 mixtures was 13, 25, and 51 mmol m -3 Shifts in the carbonate equilibrium can alter pH; therefore to ensure there was no drastic variation in [CO 2 ] in the water during the day and between aquaria, the pH was monitored. The aquarium water temperature was also monitored in all the SPAR chambers and averaged 23 2 C and 21 1 C during the day and night, respectively, which is similar to the natural spring temperature. To ensure that nutrient limitation did not occur, a 04 g Jobes slow-release fertilizer stick (N:P:K = 13:4:5) was inserted into the sand in each cup on days 1 and 41.

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103 Plants were maintained completely submersed until day 40, and then they were allowed to become emergent. On day 40 the water level was set below the youngest pair of leaves on the apical meristem and the water level marked on each plant. The water level was maintained at this point on each plant so that all growth from the apical meristem for the remainder of the experiment was emergent. Growth and Morphology Total stem length, number of axillary shoots, leaf area (LA), shoot dry weight, and root dry weight were determined on a per plant basis on days 0, 10, 20, 38, 60, and 79. Plants were then separated into their organs. Leaf area was measured with an area meter (LI-COR Inc., Lincoln, NE). Shoots and roots were dried in an oven at 65 C for 3 d to determine dry weight. Shoot: root ratios were determined from the dry weights. Throughout the experiment, dead material from each plant was collected, dried, and weighed for addition into total dry weight. Relative growth rate (RGR) and relative leaf area growth rate (LARGR) per plant were calculated using linear regression for plots of total dry weight and leaf area, respectively, over time. Regression lines for each submersed and emergent treatment were constructed using 12 and nine data points, respectively. Individual LA and SLA for fully expanded submersed leaves were also determined on days 36 and 79. C: N Ratio Analysis On day 79, the dried roots and submersed and emergent shoots from the growth and morphology analysis were used to determine the C: N ratios. Material was ground with a mortar and pestle and the percentage C and N in homogenous samples of approximately 6 mg were determined in a Carlo-Erba NCS 2500 Elemental Analyzer.

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104 Experimental Design and Statistical Analysis There were three replicates for each measurement and plants were sampled randomly. Population and CO 2 treatments were greatly influenced by habit; so submersed and emergent data were separated for comparison. The RGR and LARGR were calculated over the 0 to 38 and 38 to 79 day periods while other data used the day 38 and day 79 values for the submersed and emergent habits, respectively. The experimental design for all variables, except the C: N ratio, was a factorial fixed model with crossed fixed factors of population (highand lower-CO 2 ), habit (submersed and emersed), and CO (375, 750, and 1500 mol CO 2 mol -1 ). Plants from each population were planted in cups, which were put into aquaria placed into one of the six SPAR chambers, with two chambers per CO 2 treatment. Aquarium and SPAR chamber effects were not tested due to an inadequate number of degrees of freedom. Normality and homogeneity of variances were tested using the Shapiro test (Neter et al. 1990). The model for C: N ratio was similar, except that organ substituted for habit. 2 All data except RGR and LARGR were analyzed using a general linear model (GLM), and mean separations were conducted using the Bonferonni t-test. For RGR and LARGR confidence intervals were used to test for significant differences between treatments and separate means. All tests were conducted at the 5% level of significance. Results Figure 4-1 shows the increase in percent dry weight per plant as a function of time for plants from the two populations during growth at different [CO 2 ]. The plants remained submersed until day 40 at which time their shoots were allowed to become emergent. While submersed, plants from the high-CO 2 population in the two elevated [CO 2 ] treatments began to gain weight after day 20, but plants from the lower-CO 2

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105 population showed little weight gain while submersed. When the shoots emerged from the water, the dry weight gain for plants from both populations was enhanced, especially in the elevated CO 2 treatments. There was a clear difference in response between the two populations. Plants from the high-CO 2 population showed a much greater percentage increase in dry weight at elevated [CO 2 ] than plants from the lower-CO 2 population. Over time plants in all the treatments gained in dry weight, except for the high-CO 2 population plants grown at ambient CO 2 They had no gain through day 60, after which they actually lost weight (Fig. 4-1). Plants from the high-CO 2 population grown in the elevated-CO 2 SPAR units produced flowers after emergence (data not shown). The flowers formed on shoots above the water surface and went on to develop seed capsules. Plants from the lower-CO 2 population did not flower; neither did the high-CO 2 population plants in the ambient-CO 2 treatment. Figure 4-2 shows the overall Relative Growth Rates at the three growth [CO 2 ] while the plants were submersed, and after they became emergent. The data were calculated on a dry weight basis. While the plants were submersed the overall RGR values (calculated for days 0 through 38) were not significantly different among the [CO 2 ] treatments, although the values for plants from the high-CO 2 population appeared greater than those of their lower-CO 2 counterparts. The response of both populations to elevated-CO 2 became much more pronounced following emergence (days 38 to 79). Both populations responded positively to the elevated-CO 2 treatments, but the RGR of those from the high-CO 2 spring exhibited the most stimulation, with the 1500 mol CO 2 mol -1 growth treatment producing the greatest enhancement (Fig. 4-2). In contrast, at

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106 ambient-CO 2 the high-CO 2 population plants lost dry weight and thus showed a significant negative RGR, while growth of plants from the lower-CO 2 spring was essentially zero. High-CO 2 population plants from the three growth-CO 2 treatments on the final day of the experiment are shown in Figure 4-3. Enhanced shoot and root mass under the elevated CO 2 treatments are evident, while the plant at ambient-CO 2 was barely surviving. The percent increase over time in total leaf area per plant of the highand lower-CO 2 populations exposed to different [CO 2 ] is shown in Figure 4-4. As with plant dry weight, the leaf area per plant responded positively to elevated-CO 2 with the high-CO 2 population plants showing the greatest percent increase. However, unlike the dry weight data, the percent change in leaf area for plants of both populations showed a substantial elevated-CO 2 effect while they were still completely submersed (Fig. 4-4). For the high-CO 2 population, the plants grown at 1500 mol mol -1 maintained a consistent linear increase in leaf area while submersed and after emergence, but that of the other elevatedand ambient-CO 2 treatments reached a plateau or decreased following emergence (Fig. 4-4). Plants grown at ambient CO 2 did poorly, with the high-CO 2 population losing leaves after day 20, and those of the lower-CO 2 population increasing in leaf area until emergence (day 40), after which they too showed leaf abscission (Fig. 4-4). The leaf area responses differed in part from the dry weight observations because abscised leaves were removed from the leaf category, but were included in the total dry weight category. Following emergence, plants tended to lose older submersed, rather than emergent leaves.

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107 The RGR for leaf area is shown in Figure 4-5. Unlike the dry weight RGR, the leaf area RGR was greatest when the plants were submersed, and declined after they became emergent. While submersed, the leaf area RGR of the high-CO 2 population plants showed a significant enhancement in the elevated-CO 2 treatments, whereas [CO 2 ] had no significant effect on the leaf area RGR values for plants from the lower-CO 2 population (Fig. 4-5). Following emergence, only the highest [CO 2 ] was able to maintain a positive leaf area RGR, for plants from either population (Fig. 4-5). At ambient CO 2 emergent plants from both populations lost leaves and had negative leaf area RGR values (Fig. 4-5). Table 4-1 shows the mean area of individual submersed leaves and specific leaf area (SLA) for plants of both populations as a function of CO 2 treatment. The leaves were fully expanded at the time of measurement. The population-by-habit-by-CO 2 interaction was significant (P = 005); therefore mean separations were performed for each factor within the other factor levels. The high-CO 2 population plants had substantially lower area and greater SLA of individual submersed leaves than plants from the lower-CO 2 population. The leaves from both populations were generally larger in area when the plants were grown in the elevated-CO 2 conditions. This CO 2 -enhanced increase in the submersed leaves occurred irrespective of whether the plants were totally submersed (through day 38) or had been emergent for 39 days (day 79), and had thus also developed aerial leaves. In contrast to the area data, there was little effect of [CO 2 ] on the SLA of the submersed leaves (Table 4-1). Figure 4-6 shows the total (main and axillary) stem length of the L. repens plants grown at ambient and elevated [CO 2 ] after 38 days, during which period they were totally

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108 submersed, and after 79 days when they had been emergent for 39 days. Population had no effect on stem length (P = 0), thus the analysis was collapsed across the populations. For shoot length, the population-by-habit-by-CO 2 interaction was not significant (P = 098), however, habit-by-CO 2 was significant (P < 001), and therefore mean separations were performed for habit within CO 2 and CO 2 within habit. The mean SD (n = 3) for stem length of the high and lower-CO 2 populations across CO 2 and habit were 22 10 and 24 10 cm plant -1 respectively. Even though the plants from both populations started at the same length (12 cm) and the high-CO 2 population plants had a higher RGR, it was not reflected in a greater total stem length. Plants grown at ambient CO 2 showed little increase in shoot length over the course of the experiment (Fig. 4-6). Until they grew out of water, there was no response to elevated-CO 2 but following emergence the plants grown at 750 and 1500 mol CO 2 mol -1 had 70 and 123% greater stem lengths than the ambient-CO 2 treatments (Fig. 4-6). For both populations at elevated-CO 2 the main stems continued to lengthen throughout the experiment, and in addition submersed and emergent axillary shoots were produced. The number of axillary shoots per plant and the shoot: root ratio for plants from the highand lower-CO 2 populations are shown in Table 4-2. For the number of axillary shoots and also the shoot: root ratio, the population-by-habit-by-CO 2 interaction was significant (P = 005; P < 0), therefore mean separations were performed for each factor within the other factor levels. Even though the high-CO 2 population plants started with about half the number of nodes on their main stems (data not shown) they ended the experiment with twice the number of axillary shoots in each of the treatments (Table 4-2), although they tended to be shorter than those of the lower-CO 2 population. While

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109 submersed the plants produced less than two axillary shoots per plant, however following emergence, both populations exhibited a substantial increase in the number of axillary shoots in the elevated-CO 2 treatments (Table 4-2). Plants grown at ambient CO 2 had no increase in axillary shoot production over the course of the experiment. The majority of the roots were produced in the sand substrate, but some grew from nodes on the stem above the sand. There seemed to be no discernable trends in the ratio in response to [CO 2 ] for the submersed plants (Table 4-2). However, following emergence, the shoot: root ratios for plants from both populations tended to be lower in the elevated-CO 2 treatments. Plants from the highand lower-CO 2 populations were harvested on the final day of the experiment to determine the C: N ratio on a dry weight basis for the submersed shoots, emergent shoots, and the roots, and the data are displayed in Figure 4-7. The population-by-organ-by-CO 2 interaction was not significant (P = 020). However, the population-by-organ interaction was significant (P < 001), therefore mean separations were performed for habit within CO 2 and CO 2 within habit. The plant C: N ratios showed no response to CO 2 (P = 017). In terms of the individual organs, the C: N ratios of the submersed shoots and roots from the high-CO 2 population were similar, and higher than that of the emergent shoots (Fig. 4-7). The lower-CO 2 population also had the highest ratios in the submersed organs. Discussion Elevated CO 2 Response of the Highand Lower-CO 2 Populations When shoots from both populations were grown under the same conditions those originally from the high-CO 2 Fanning Springs exhibited a much greater enhancement of growth in the elevated [CO 2 ] treatments than their lower-CO 2 Rainbow Springs

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110 counterparts. This difference in dry weight enhancement as a function of [CO 2 ] is good evidence to support the hypothesis that the two populations are genotypically adapted to the [CO 2 ] of their respective environments, rather than just phenotypically acclimated. This is especially so, given that the two populations show considerable genetic difference (Chapter 3). The stimulation by elevated CO 2 of leaf area and number of axillary shoots from the high-CO 2 spring plants are also consistent with this population being better adapted to utilize high [CO 2 ]. Even though the high-CO 2 plants produced more axillary shoots, there was little difference in stem length since axillary shoots produced by plants from the high-CO 2 population were shorter than those from the lower-CO 2 population. This response in greater number of axillary shoots was also observed for the high-CO 2 population in its natural habitat. Despite their genetic differences, both populations of L. repens retained C 3 photosynthetic characteristics and neither showed any propensity for HCO 3 use (Chapter 3). Terrestrial C 3 species also have been reported to be differentially adapted to elevated CO 2 in both natural species (Fordham et al. 1997a, b; Woodward 1987) and cultivars of the crop plant Oryza sativa (Ziska & Teramura 1992). However, to our knowledge this is the first demonstration of the phenomenon for an aquatic plant. The increase in growth of the plants at elevated-CO 2 led to individual submersed leaves having greater LA, a response that has been documented in the terrestrial C 3 plant literature as well (Backhausen & Scheibe 1999). Specific leaf area normally decreases in terrestrial C 3 plants grown at elevated-CO 2 as carbohydrates accumulate in leaves (Madsen 1968; Woodward et al. 1991), thereby increasing leaf dry weight. Woodward et

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111 al. (1991) reported that SLA of Boehmeria cylindrica decreased for plants grown at elevated-CO 2 from seed collected near a high-CO 2 source, however, Fordham et al. (1997a) found no SLA response in a similar experiment using Agrostis canina. In the SPAR chamber experiment, SLA was unaffected by elevated-CO 2 since the dry weight and LA of individual submersed leaves grown at elevated-CO 2 increased in a similar manner. Shoot: root dry weight ratios were lower when plants were grown at elevatedthan at ambient CO 2 for both populations. Storage of the extra photosynthate generated at elevated-CO 2 into roots is often favored (Bowes 1993); this phenomenon has been demonstrated for terrestrial plant populations (Woodward et al. 1991; Fordham et al. 1997a). The C: N dry weight ratio of the shoots and roots of plants from both populations was unaffected by elevated-CO 2 even though most terrestrial species show an increase in the C: N ratio when they are grown at enriched-CO 2 (Drake et al. 1996a), including plants growing in natural springs (Fordham et al. 1997a). An appropriate balance between C and N is necessary for normal plant growth (Garnier & Roy 1994) so plants with little change in the C: N ratio at elevated-CO 2 may show greater growth responses. Rubisco can constitute 25% of leaf [N] in a C 3 leaf (Bainbridge et al. 1995); so adequate N must be present for Rubisco synthesis and photosynthesis. The C: N ratio of the roots and submersed shoots was lower for plants from the high-CO 2 population than their lower-CO 2 counterparts. This difference may partially explain why the high-CO 2 plants had greater growth than the lower-CO 2 plants in the elevated-CO 2 treatments and is consistent with their adaptation to high-CO 2 In contrast, spring adaptation studies with

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112 terrestrial species have revealed no difference between populations in the C: N ratio or N content (Bettarini et al. 1997; Fordham et al. 1997a). Both populations maintained lower C: N ratios in emergent shoots compared to submersed shoots and roots, which may be important in maintaining the aerial leaves as the primary photosynthetic organ. Plants from the high-CO 2 population grown at elevated-CO 2 produced flowers and seed capsules, whereas high-CO 2 plants grown at ambient-CO 2 and lower-CO 2 plants did not. These observations confirm those from field and laboratory studies in the previous chapter that showed the high-CO 2 population was able to reproduce sexually and asexually, whereas the lower-CO 2 population only reproduced asexually. This data also coincides with a survey by Jablonski et al. (2002) that indicated terrestrial C 3 plants have enhanced sexual reproduction at enriched-CO 2 Ideally, seed collected from plants growing in the springs would have been used in this experiment, but flowering and viable seed could not be obtained from the lower-CO 2 population (Chapter 3). The lower-CO 2 population produced flowers in the laboratory with gibberellic acid application, but the seed capsules that developed had very few seeds and these seeds failed to germinate. Although small shoots from highand lower-CO 2 plants were used to begin this experiment, each plant started with material that was produced in the spring environments, but to minimize residual phenotypic traits they were exposed to the same 21-d acclimation period in the SPAR chambers. Effect of Habit on the CO 2 Response of the Spring Populations Plants after emergence were able to respond to a much greater extent to the same elevated [CO 2 ] than when they were completely submersed. Aerial shoots from both populations exhibited greater RGR, stem length, and number of axillary shoots than submersed shoots. However, this again was especially true for those originally from the

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113 high-CO 2 spring. Thus, whether photosynthesis was occurring under water or in air the high-CO 2 plants appeared better adapted to make use of the high CO 2 It was noteworthy that neither of the plant populations performed well at ambient-CO 2 even after emergence, but plants from the high-CO 2 population were dying, while the lower-CO 2 plants at least maintained their biomass. Emergence may have increased the response of the plants because it allowed the plants to derive CO 2 directly from the gas phase, and thus reduced the CO 2 diffusion resistance, especially since these plants were unable to use HCO 3 A greater stomatal density of the high-CO 2 populations plants (Chapter 3) may have further facilitated access to air-CO 2 Madsen and Breinholt (1995) determined that air contact stimulated growth of the amphibious plant, Callitriche cophocarpa, because emergence enhanced the plants access to inorganic carbon, rather than improved nutrient acquisition. Aerial leaves are more effective at photosynthesis than submersed leaves when [CO 2 ] are limiting because they have lower K 1/2 (CO 2 ) values (Salvucci & Bowes 1982; Spencer & Bowes 1985). Emergent Berula erecta and Mentha aquatica plants also showed greater RGR when plants were emergent as opposed to submersed (Sand-Jensen & Frost-Christensen 1999). Of all the growth response variables, only LARGR was larger when the plants were submersed as opposed to emergent. While the plants were submersed, a greater surface area could reduce the CO 2 diffusion limitations in water. After the plants became emergent, the majority of the older, submersed leaves abscised, which increased the reliance on aerial leaves and their enhanced access to CO 2

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114 Shoot: root dry weight ratios only differed between habits for plants grown at ambient CO 2 For these treatments, shoot: root dry ratio increased when the plants became emergent because their roots died. This was the first time an aquatic plant has been used in a common garden experiment comparing plant populations collected from natural CO 2 springs. These results may be useful in predicting how aquatic vegetation will respond to the rising [CO 2 ] in our atmosphere. This study supports that contention that emergent or amphibious aquatic species should respond more to rising atmospheric [CO 2 ] than submersed species (Bowes 1993, 1996; Raven 1994). It was surprising that even after emergence the plants from the high-CO 2 population were unable to survive at ambient [CO 2 ] despite the greater access to CO 2 that the aerial environment provides. It should be noted that submersed species growing in waters enriched in CO 2 are unlikely to respond to increases in atmospheric [CO 2 ], since such waters are a source of, not a sink for, CO 2 and can far exceed air-equilibrium values.

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115 Time (d) 020406080 100 Increase in dry weight plant-1 (% of initial) -2000200400600 375 750 1500 EmergenceCO2 (micromol mol-1) CO2 (mol mol -1 ) Figure 4-1. Increase in dry weight per plant (% of initial) over time for L. repens plants taken from high(filled symbols) and lower-CO 2 (open symbols) populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Mean (n = 3).

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116 RGR (mg g-1 d-1) -20-10010203040 375 750 1500 High-CO2 population Lower-CO2 populationa a ac b aa a ab a aSubmersedCO2 (mol mol-1) Submersed Emergent Submersed Emergent Figure 4-2. Relative growth rates (RGR) for L. repens plants taken from highand lower-CO 2 populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Mean SE. Submersed (open bars; n = 12) and emergent (lined bars; n = 9) data are reported for days 0-38 and 38-79, respectively. Differences in RGR between CO 2 treatments within population and habit using 95% confidence intervals are noted by a, b, and c.

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117 Figure 4-3. Ludwigia repens plants from the high-CO 2 population after 79-d growth at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers.

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118 Time (d) 020406080 100 Increase in leaf area (% of initial) -1000100200300400 375 750 1500 Emergence CO2 ( mol mol-1 ) Figure 4-4. Increase in leaf area per plant (% of initial) over time for L. repens plants taken from high(filled symbols) and lower-CO 2 (open symbols) populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Mean (n = 3).

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119 LARGR (mm m-1d-1) -20-10010203040 375 750 1500 High-CO2 population Lower-CO2 populationb a ab b aa a ac b aCO2 (mol mol-1) Submersed Emergent Submersed Emergent Figure 4-5. Leaf area relative growth rate (LARGR) for L. repens plants taken from highand lower-CO 2 populations and grown at 375, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Mean SE. Submersed (open bars; n = 12) and emergent (lined bars; n = 9) data are reported for days 0-38 and 38-79, respectively. Differences in RGR between CO 2 treatments within population and habit using 95% confidence intervals are noted by a, b, and c.

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120 Table 4-1 Leaf area (LA) and specific leaf area (SLA) for individual, fully-expanded submersed leaves of L. repens plants taken from highand lower-CO 2 populations and grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Growth [CO 2 ] LA SLA Population Time (d) (mol mol -1 ) (mm 2 leaf -1 ) (m 2 g -1 ) High-CO 2 36 375 38 07 a 64 03 a 750 60 26 a 58 08 a 1500 64 17 a 65 08 a 79 375 32 14 b 57 14 a 750 62 22 ab 84 26 a 1500 99 24 a 60 12 a Lower-CO 2 36 375 65 04 b 36 07 ab 750 78 24 b 41 05 a 1500 183 50 a 28 03 b 79 375 53 22 b 33 07 a 750 142 28 a 34 03 a 1500 161 49 a 32 01 a Mean SD (n = 3). According to the General Linear Model for LA and SLA, the population-by-time-by-CO 2 interaction was significant at P 0 therefore mean separations were performed for each factor within the other factor levels. Significant differences between the CO 2 treatments within population and habit using the Bonferonni t-test at P 005 are noted by a and b

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121 Total stem length (cm plant-1) 0204060 375 750 1500 HabitbmersedEmergent a a a b a aCO2 (mol mol-1) Su Submersed Habit Emergent Habit Figure 4-6. Total (main and axillary) stem length per plant collapsed across the highand lower-CO 2 populations for L. repens plants grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Submersed and emergent shoot lengths are reported for days 38 and 79, respectively. Mean SD (n = 6). According to the General Linear Model for stem length, the population-by-habit-by-CO 2 interaction was not significant at P < 005, however, the habit-by-CO 2 interaction was significant, therefore mean separations were performed for habit within CO 2 and CO 2 within habit. Population was not significant (P = 052). Significant differences between CO 2 treatments within habit using the Bonferonni t-test at P < 005 are noted by a and b.

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122 Table 4-2 Number of axillary shoots per plant and shoot: root dry weight ratio for L. repens plants taken from highand lower-CO 2 populations and grown at 350, 750, and 1500 mol CO 2 mol -1 in SPAR chambers. Plants were grown submersed until day 40, after which they became emergent. Submersed and emergent values are reported days 38 and 79, respectively. Growth [CO 2 ] Axillary shoots Shoot: root ratio plant -1 Population Habit (mol mol -1 ) (no. plant -1 ) High-CO 2 Submersed 375 07 06 14 4 750 13 15 32 13 1500 10 10 13 3 Emergent 375 20 26 b 43 20 a 750 193 47 a 14 6 b 1500 227 40 a 13 5 b Lower-CO 2 Submersed 375 03 06 8 4 b 750 03 06 54 1 a 1500 07 06 25 8 b Emergent 375 03 06 c 51 18 a 750 77 25 b 46 19 a 1500 137 32 a 9 1 b Mean SD (n = 3). According to the General Linear Model for no. axillary shoots and shoot: root ratio, the population-by-habit-by-CO 2 interaction was significant at P 0 therefore mean separations were performed for each factor within the other factor levels. Significant differences between the CO 2 treatments within population and habit using the Bonferonni t-test at P 005 are noted by a b and c

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123 C: N ratio on a dry weight basis 0102030 Emergent shoot Submersed shoot Root PopulationHigh-CO2Lower-CO2b a ac a bPlant organ Figure 4-7 Final (day 79) C: N ratios on a dry weight basis for organs of L. repens plants taken from highand lower-CO 2 populations and grown at different [CO 2 ] in SPAR chambers. Mean SD (n = 9). According to the General Linear Model for C: N ratio, the population-by-time-by-CO 2 interaction was not significant at P < 005, however the population-by-organ interaction was significant (P < 001), therefore mean separations were performed for population within organ and organ within population. CO 2 was not significant (P = 017). Significant differences between organs within population using the Bonferonni t-test at P < 005 are noted by a, b, and c.

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CHAPTER 5 CONCLUDING REMARKS This study demonstrates that a natural population of aquatic plants can genetically adapt to the [CO 2 ] to which it is exposed, given sufficient time and isolation from other populations. A plant population growing in an elevated-CO 2 spring can maintain high photosynthesis rates and Rubisco activities without down-regulation providing there is sufficient N in the environment. Plants from high-CO 2 spring are unlikely to show further adaptation as atmospheric CO 2 rises since they are already in an environment with [CO 2 ] far greater than in equilibrium, whereas the population adapted to low [CO 2 ] may not be able to take full advantage of rising [CO 2 ] unless it becomes adapted to the new concentration. This is first elevated-CO 2 spring research to provide a genetic analysis of the plant populations being compared. It showed that the highand lower-CO 2 populations are distinct populations with little or no gene flow between them. This is also the first research to compare populations in spring environments and to find differential CO 2 adaptation of an aquatic plant. It was unexpected that even though the high-CO 2 population had the greater photosynthesis rates it produced plants in the springs with less weight and density than the lower-CO 2 population. Measurements throughout the growing season on both aerial and submersed leaves might help to explain this phenomenon. Time of year would undoubtedly affect growth of the high-CO 2 population since part of the year it will be investing in sexual reproduction. In addition to the common garden experiment 124

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125 performed here, reciprocal planting would further test how each population responds in the alternate CO 2 -spring environment. Further determinations of photosynthetic and Rubisco properties of the populations would be valuable to explain the mechanism behind the greater growth response of the high-CO 2 population to elevated [CO 2 ] than the lower-CO 2 population and of the emergent compared to the submersed plants in the SPAR experiment. Specifically, down-regulation of photosynthesis and Rubisco activity of both the aerial and submersed leaves could be compared for the two populations grown at a variety of [CO 2 ] and the same [N] in SPAR chambers. The nature and magnitude of genetic variation in plant responses to CO 2 are poorly known (Curtis et al. 1996). Although a founder effect is unlikely, because of the within-population differences observed, this possibility could be eliminated by increasing the number of individuals sampled from each population. To establish that this is not an isolated ecotypic occurrence, it would be useful to investigate Ludwigia repens populations growing in other springs which differ in [CO 2 ] to determine if they show similar traits to the two populations studied in the present work. Evidence was provided in this work that emergence has a major impact on the CO 2 response of amphibious plants. Anatomically, aerial leaves of the high-CO 2 population had a better-developed palisade layer and greater starch grain and stomatal density than submersed leaves. Both populations also had lower C: N ratios in aerial than submersed leaves. These traits may have reflected greater reliance on aerial leaves for photosynthesis by the plant and could be related to enhanced CO 2 diffusion.

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126 The greater response of the emergent compared to the submersed plants is an interesting area for additional study. It would be worthwhile to compare the response of plants from the highand lower-CO 2 populations that were grown emergent or submersed throughout the entire experiment and for longer periods rather than growing them submersed for a portion of the experiment prior to emergence. This would also indicate whether the plants from the high-CO 2 population growing emergent at ambient CO 2 have the capacity to recover. This study uncovered a variety of interesting phenomenon regarding how plants respond to long-term exposure to elevated-CO 2 and may provide the groundwork for future experiments utilizing this system and elevated-CO 2 spring research.

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LIST OF REFERENCES Acock B. & Allen L.H., Jr. (1985) Crop responses to elevated carbon dioxide. In Direct effects of increasing carbon dioxide in vegetation (ed E. Lemon), pp. 286. U.S. Department of Energy, Washington, DC. Allen E.D. & Amthor J.S. (1995) Plant physiological responses to elevated CO 2 temperature, air pollution, and UV-B radiation. In Biotic feedbacks in the global climatic system: will the warming feed the warming? (eds G.M. Woodwell & F.T. Mackenzie), pp. 51-84. Oxford University Press, New York. Allen L.H., Jr. (1990) Plant responses to rising carbon dioxide and potential interactions with air-pollutants. Journal of Environmental Quality 19, 15-34. Allen L.H., Jr. (1996) Mechanisms and rates of O 2 transfer to and through submerged rhizomes and roots via aerenchyma. Soil and Crop Sciences Society of Florida Proceedings 56, 41-54. Amthor J.S. (1991) Respiration in a future, higher-CO 2 world. Plant, Cell and Environment 14, 13-20. Amthor J.S. (1994) Plant respiratory responses to the environment and their effects on the carbon balance. In Plant-environment interactions (ed R.E. Wilkinson), pp. 501-554. Marcel Dekker, New York. Amthor J.S. (1995) Terrestrial higher-plant response to increasing atmospheric [CO 2 ] in relation to the global carbon-cycle. Global Change Biology 1, 243-274. Amthor J.S., Koch G.W. & Bloom A.J. (1992) CO 2 inhibits respiration in leaves of Rumex crispus L. Plant Physiology 98, 757-760. Angeles G. (1992) The periderm of flooded and non-flooded Ludwigia octovalvis (Onagraceae). IAWA Bulletin 13, 195-200. Arp W.J. (1991) Effects of source-sink relations on photosynthetic acclimation to elevated CO 2 Plant, Cell and Environment 14, 869-875. Atkin O.K., Schortemeyer M., McFarlane N. & Evans J.R. (1999) The response of fastand slow-growing Acacia species to elevated CO 2 : an analysis of the underlying components of relative growth rate. Oecologia 120 544-554. 127

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BIOGRAPHICAL SKETCH Steven Todd Lytle was born in Chesterton, Indiana, on June 9, 1970. He was raised there receiving great support from his parents Brian and Linda, brother David, and sister Denise. He graduated from Chesterton High School in 1988 with a desire to study veterinary medicine after working as a veterinarians assistant for five years. In January 1999, he entered Purdue University (North Central Branch) in Westville, Indiana, with a major of biology. Life at home was a struggle and his parents eventually made it possible for him to travel to Purdues main campus in West Lafayette, Indiana, to continue his biological studies. It was here that he was reunited with a high school friend, Mike Scharf, that hired him as his research assistant in the Entomology Department. This taught him the nuts and bolts of doing biological research leading to his interest in veterinary medicine waning. During this stint, his life took a surprising change for the better as he met his future wife, Stephanie Sullivan, while playing cards with friends. He graduated in 1993 with a bachelors degree in biology focusing on ecology, and immediately entered the Entomology Department at Purdue to pursue his masters degree. Along the way he learned about industrial pest control and insect behavior before settling on host plant resistance as a research area. He received an extention assistantship at the Diagnostic Training Center and thoroughly enjoyed working on the farm at the Center. He obtained his masters degree in entomology in the summer of 1996. He wanted to understand the plant side of host-plant interactions and decided to continue his graduate studies in plant biology so he moved to Gainesville, Florida to pursue a 145

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146 doctorate degree in the Botany Department at the University of Florida. He is proud to have married Stephanie Sullivan and learned to surf during his many days in Florida. Upon completion of the degree requirements, he will gladly pursue a career teaching college students.