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ADAPTATION AND ACCLIMATION OF POPULATIONS OF LUDWIGIA REPENS
TO GROWTH IN HIGH- AND LOWER-CO2 SPRINGS
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
This dissertation is dedicated to my wife, Stephanie Sullivan Lytle.
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
endless encouragement and patience throughout the pursuit of this degree and I share this
achievement with her.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S .................................................................... ......... .............. iii
LIST OF TABLES ......... ... ........... ... ............. ......... .............. .. vii
LIST OF FIGURES ..................... .......... .................................. viii
A B STR A C T ................................................. ..................................... .. x
1 TERRESTRIAL AND AQUATIC PLANT RESPONSES TO ELEVATED CO2......1
R isin g A tm o sp h eric C O 2 .............................................................................................. 1
Terrestrial Plant Responses to Elevated CO2........................................... ............... 1
Aquatic Plant Biology and Responses to Elevated-CO2...................................... 24
Ludwigia Repens .................... .................... ........ .......36
Sum m ary Statem ent .................. .......................................... ........ .... 37
2 ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS...................40
In tro du ctio n ...................................... ................................................ 4 0
M materials and M methods ....................................................................... ..................43
R e su lts ...................................... .................................................... 4 4
D iscu ssio n ...................................... ................................................. 4 7
3 CHARACTERISTICS OF SUBMERSED LUDWIGIA REPENS PLANTS
FROM TWO POPULATIONS GROWING IN SPRINGS WITH DIFFERING
C O 2 C O N C E N TR A T IO N S ............................................................. .....................60
In tro du ctio n ..................................... ................... ............................ 6 0
M materials and M methods ....................................................................... ..................64
R e su lts ........................................................................................... 7 1
D iscu ssio n ........................................................................................7 8
4 THE EFFECT OF CO2 AND EMERGENCE ON THE GROWTH OF
LUDWIGIA REPENS PLANTS FROM HIGH AND LOWER-CO2 SPRING
P O P U L A T IO N S .............................................................................. ................. .. 9 5
In tro d u ctio n ...................................... ................................................ 9 5
M materials and M methods ............................................ ....................................... 100
R e su lts ..........................................................................................1 0 4
D isc u ssio n ........................................................................................1 0 9
5 CONCLUDING REMARKS ......................................................... ............. 124
L IST O F R E FE R E N C E S ......................................................................... ................... 127
BIOGRAPHICAL SKETCH ..............................................................145
LIST OF TABLES
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 ofL. repens plants
growing in high- and lower-CO2 springs. ..................................... ............... 87
3-3 Individual leaf characteristics of L. repens plants growing in high- and
low er-C O 2 springs. .......................... .... ............ ...........................88
3-4 Stem and root characteristics ofL. repens plants growing in high- and
low er-C O 2 springs. .......................... .... ............ ...........................90
3-5 Reproductive characteristics ofL. repens plants collected from high- and lower-
CO2 populations and grown in a growth chamber. ...............................................91
4-1 Leaf area (LA) and specific leaf area (SLA) for individual, fully-expanded
submersed leaves ofL. repens plants taken from high- and lower-CO2
populations and grown at 350, 750, and 1500 imol CO2 mo1-1 in SPAR
cham bers ......... ........... ......... .............................. ............................120
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 imol CO2 mo1-1 in SPAR chambers................ ...............122
LIST OF FIGURES
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 ens leaf. ..........................................................................55
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
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) 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 ofL. repens plants collected from high-CO2 and
low er-C O 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-CO2 (open symbols) populations
and grown at 375, 750, and 1500 Lmol CO2 mo1-1 in SPAR chambers.................115
4-2 Relative growth rates (RGR) for L. repens plants taken from high- and
lower-CO2 populations and grown at 375, 750, and 1500 Ctmol CO2 mo-1 in
SP A R ch am b ers ..... .... .... .. ................................................ .................. 116
4-3 Ludwigia repens plants from the high-CO2 population after 79-d growth at 375,
750, and 1500 itmol CO2 mol11 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 itmol CO2 mol11 in SPAR chambers...................118
4-5 Leaf area relative growth rate (LARGR) for L. repens plants taken from high-
and lower-CO2 populations and grown at 375, 750, and 1500 Ctmol CO2 mol11
in SPA R cham bers. ......... ... ................ ........ ... ....... .. ........ .. .. 119
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 itmol
CO2 mol11 in SPAR chambers. ............................................................................ 121
4-7 Final (day 79) C: N ratios on a dry weight basis for organs ofL. repens plants
taken from high- and lower-CO2 populations and grown at different [CO2] in
SP A R ch am b ers ..... .... .... .. ................................................ .................. 12 3
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
Steven Todd Lytle
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
rates of both populations were 31% inhibited by atmospheric , 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
[C02]. 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 [C02] 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
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 imol CO2
mol-1, but the last 160,000 years have seen glacial lows of 180 imol CO2 mol-1 and
interglacial highs of only 250-300 imol CO2 mol11 (Post et al. 1990). Atmospheric [CO2]
recorded at Mauna Loa, Hawaii, since the 1950s indicate a steady increase in C02, with
our current [C02] estimated at 370 imol 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).
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-1,5-
bisphosphate (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 [C02] 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, [C02] is already elevated at the carboxylation site in C4 and CAM plants,
lessening the effect that rising [C02] 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 C02, and thus enables oxygenation (i.e. photorespiration) to occur.
Photorespiration reduces photosynthesis of C3 species by about 35% at 25 C, with higher
temperatures increasing this inhibition (Jordan & Ogren 1984; Bowes 1996).
Stimulation of carboxylation at elevated-[C02] is usually greatest for C3 species.
Bowes (1996) estimated that doubling atmospheric [CO2] 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 al. 1989). Regardless, acclimation is rarely great enough to
completely negate the positive stimulation of C3 photosynthesis that occurs at elevated-
CO2 (Drake et al. 1996a). A survey of C3 crops by Cure and Acock (1986) showed that
doubling [CO2] increased photosynthesis by 52% but this enhancement dropped to 29%
Acclimation to CO2
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.
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 [CO2]. 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
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 C02, 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 imol CO2 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 pmol CO2 mol11, no differences were present for Solanum tuberosum, Solanum
melongena, and Phaseolus vulgaris (Sage et al. 1989). Glycine max Rubisco activity and
amount showed minimal response to manipulation of [CO2] (Campbell et al. 1988).
Hydroponically-grown Lolium temulentum, showed no change in maximum Rubisco
activity, but did have greater Rubisco protein at double-[CO2] (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 ofArabidopsis 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 MImol 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 imol CO2 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.
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 [CO2] 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 Mmol CO2 mol11) 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 imol CO2 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).
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-[CO2] 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-CO2, G. hirsutum and Z
mays both had lower Rubisco activity (Wong 1979), while tobacco had decreased
Rubisco content (Walch-Liu et al. 2001).
The effect that elevated [CO2] 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
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 [CO2] to its original level (Amthor 1991; Bowes 1996). Indirect
effects occur after growth in elevated [CO2] 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 [CO2] in
which G. max cotyledons were grown decreased their mitochondrial 02 uptake
(Gonzalez-Meler et al. 1996), and inhibition of CO2 evolution by Rumex crispus leaves
and Oryza sativa canopy was observed at high-[CO2] (Amthor et al. 1992; Baker et al.
2000). However respiration does not always decrease at elevated-CO2, 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 [CO2] for mitochondria isolated from G. max
cotyledons (Gonzalez-Meler et al. 1996).
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 (Azc6n-
Bieto 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)
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 [CO2] 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-CO2, 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 [CO2] 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 [CO2] 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
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 imol CO2 mol-1 and found no tiller
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
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, 0. 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 imol 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
Oryza sativa cultivars to high-CO2 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 n it tiluhitnl exposed to 900 imol CO2
mol-1 (Tipping & Murray 1999) and G. max grown at 800 imol CO2 mol-1 had 37%
greater leaf thickness due to an increase in the number of palisade cells (Vu et al. 1989).
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. I/ ikni/nn (C3) (Tipping & Murray 1999).
Stomatal density increased in Oryza sativa plants as [CO2] was increased at intervals
from 160 to 330 Imol CO2 mol-1, but then showed no further increase at intervals from
330 to 900 Mmol CO2 mo1-1 (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 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), 0. hymenoides (C3) A. smithii (C3) E. orcuttiana (C4)] tested by Smith et
al. (1987) showed a decrease in stomatal conductance when grown at 680 Mmol CO2 mol
1, rather than ambient-CO2.
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,
Ainhiy)lli, 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
contributing to increased leaf growth in high-CO2. Glycine max grown at 800 Ctmol CO2
molP1 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-CO2,
leaves tend to have more dense tissues with smaller cells and less internal spaces. When
grown at enriched-CO2, the second trifoliate leaf ofP. 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 [CO2]. 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 high-
CO2 (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.
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 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 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 CO2 (Rogers
et al. 1992). Pinus taeda also had larger root diameters in high-CO2 treatments
(Larigauderie et al. 1994). These results notwithstanding, St. Omer and Horvath (1984)
found no difference in stele or tracheary element diameter of Layiaplatyglossa roots
grown at higher than ambient-CO2 levels.
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 al. 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.
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 [CO2] 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 [CO2] are monitored. The third strategy utilizes natural
settings (e.g., geothermal gas vents) in which high [CO2] 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
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
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 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 imol CO2 mo-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,
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 [CO2] in the air around
geothermal gas vents can be as high as 10,000 imol 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 [CO2] gradient,
with a decreasing concentration as the distance from the vents increases (Miglietta et al.
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 [CO2] 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.
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 (H2CO3). Other
acids from organic matter may also combine with the solution (Nordlie 1990). This
H2CO3 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
H2CO3 to produce calcium bicarbonate (Ca(HCO3)2), thus increasing Ca2+ and HCO3
ions 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
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
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 etal. 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 [CO2] 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 al. 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 [CO2]. Thus far it has
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 strict 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
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 itmol CO2
mol-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 elevated-
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 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 high-
and ambient-CO2 sites with otherwise similar environmental conditions, and growing
them under a common [CO2] 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.
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 [CO2] ranging from 451-610 imol CO2 mol-1 and grew them in the lab at
ambient and 700 imol CO2 mol-1. 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 [CO2]. The weight of the seeds
was positively correlated with the [CO2] 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 imol CO2 mo1-1
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 ofBoehmeria cylindrica growing at varying [CO2] from 350-550 imol CO2
mol-1 were grown in controlled environments at 350, 525, and 675 imol CO2 mol11
(Woodward 1987). Differences between populations in plant growth were noted only at
the highest CO2 treatment; at 675 imol CO2 mol-1, 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
buoyancy, which renders extensive structural tissue in submersed plants superfluous
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, SO4, and Cl are mainly absorbed from the surrounding
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
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 DIC-
saturation (Van etal. 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
1989). Photosynthetic capacity, HCO3- uptake capacity, and Rubisco activity of Elodea
canadensis were all enhanced when N was increased in hydroponic cultures (Madsen &
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 [CO2] + H20 <- H2CO3 < H+ + HCO3- < H+ + CO32-
CO2 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 CO32- 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 [CO2] to approach zero and raise the pH. In contrast, [CO2] 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,
respectively. In productive sites, [CO2] 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
250C; 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 [CO2] of a water system depends on the type of system (e.g.,
lake, river, spring) and the amount of mixing within the system. The [CO2] 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 [CO2] 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(C02) values (Maberly & Spence
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 HCO3- may concentrate CO2 around Rubisco resulting in reduced
photorespiration rates, thereby increasing photosynthetic efficiency (Maberly & Madsen
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 C02, 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
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
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 [CO2]. 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 HCO3- (Bowes
1991; Raven 1994). Huge changes in free [CO2] 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
HCO3-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 HCO3- (Madsen 1991).
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 HCO3- 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 enriched-
CO2 and HCO3- 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
[C02] 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 ofH. 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 HC03- 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 HC03-) 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 HC03- 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 HC03-.
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 HC03-
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
(16 mmol m-3) (Madsen & Breinholt 1995). This difference decreased as dissolved free-
CO2 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 ofH. 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 CO2-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 air-
equilibrium 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.
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-[CO2] natural springs, as well as lower-[C02] spring
environments (University of Florida, Herbarium), which makes it useful for elevated-CO2
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 Rejmankova (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
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-.
The literature regarding the short-term responses of terrestrial plants to elevated-
CO2 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 [CO2]. 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 [CO2] 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
aquatic plants has received far less attention than terrestrial plants, and submersed plants
have been completely neglected in regard to natural CO2 spring experiments. Submersed
plants differ from their terrestrial counterparts in many respects, so that their responses to
elevated-CO2 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-CO2 research.
Net CO2 assimilation
rate (tmol CO2 m2 m21)
Limiting factors in the non-acclimated curve
<-------Rubisco-------> <----------------------RuBP---------- -- >
Increased Amax due to increased Acclimated
Pi and RuBP regeneration ..............
Intercellular CO2 (tmol mo1-1)
Figure 1-2. Acclimated and non-acclimated assimilation/intercellular CO2 (A/Ci) curves
for a typical C3 plant at high light and 21% 02.
** Decreased slope due to decreased Rubisco content
ANATOMICAL CHARACTERIZATION OF LUDWIGIA REPENS
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
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 ofL. 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).
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 ofL.
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 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 plant's function in the both aerial and submersed habits.
Materials and Methods
Random collections of twenty mature, emergent L. repens plants were made from
Fanning Springs, Florida (29 o 35' 15" N, 82 o 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 itm) 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 1-8 mm2 at 100x magnification was used to count the stomates, with three
replicates per leaf.
Figure 2-1 shows L. repens growing emergent at the collection site.
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 four-
fold greater stomatal density on the adaxial as compared to the abaxial surface (Table 2-
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
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.,
Figures 2-4 and 2-5 show cross-sections of aerial and submersed L. repens stems.
No cuticle was present on the stems ofL. 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
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.
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-
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.
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
(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 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
(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 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 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
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 ofL. repens is
a rare anatomical feature, other genera in the Onagraceae also possess this trait (Keating
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 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 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.
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
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.
Figure 2-1. Ludwigia repens growing emergent in Fanning Springs, Florida.
A. Aerial 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).
Table 2-1. Characteristics of fully-expanded aerial and submersed leaves of L. repens.
Thickness leaf (mm)
Stomatal density (no. stomates mm-2)
Starch grain density
0-16 + 002
163 + 35**
181 + 48**
5200 + 900**
0-18 + 003
Crystal density (no. mm-2)
Mean SD (n = 4-8). P-values are from t-tests and significant differences between
populations at P 0-01 are noted by **
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.
__criW-: .- i .- N
A '. A erial stem
t *, o I Z ,
-' .Li --r"-"
SubmA. Aerseial stem
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 (1) starch grain (sg), and mucilage (m).
S '" 'e ---
', .* ... ,
... ./ *. *ff i 'st
^ ,.. .^ .
,,* '* :. ; l .,^.^
,* -'^;^t : l~v*^d
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
A. AVter-colufnn root
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).
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).
CHARACTERISTICS OF SUBMERSED LUDWIGIA REPENS PLANTS
FROM TWO POPULATIONS GROWING IN SPRINGS WITH
DIFFERING CO2 CONCENTRATIONS
Atmospheric [C02] is currently over 371 imol 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-CO2, 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-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 &
Even though photosynthesis may be down-regulated, C3 plants grown in elevated-
CO2 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 [CO2] 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).
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-CO2, 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 often 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 [CO2] 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
growing at different distances from a natural CO2 source, with [CO2] ranging from
ambient to 1000 imol 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 [CO2] (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).
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 [CO2] 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 [CO2]. 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
Two populations of Ludwigia repens L. were selected for the study, one in a high-
[C02] spring, Fanning Springs, FL (29 o 35' 15" N, 82 o 56' 08" W) and the other in a
spring with a much lower [CO2], Rainbow Springs, FL (29 o 06' 08" N, 82 o 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.
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 LI-
6200 (LI-COR Inc., Lincoln, NE).
Inorganic carbon species were calculated using the FWcarbc computer program
(SC Maberly, Acme Liquid Software Company, 1991). FWcarbc 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 = 25 x 105 (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
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 [im) 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
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.
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
period was further studied in a growth chamber with a photon irradiance of 300 imol m-2
s- (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 [iL of 50-
mmol m-3 GA3 immobilized in lanolin was applied to the apical meristem every week for
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 itmol m-2 S-1 (400-700 nm), and a 12-h photoperiod.
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 atmospheric-
CO2 at an irradiance of 400 imol 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 , and a saturating irradiance
of 700 mrol m-2 S-1 (400-700 nm). NaHCO3 was injected to initiate photosynthesis. To
exhaust intercellular C02, the leaves were incubated prior to measurement in DIC-free
buffer at the assay pH and an irradiance of 400 itmol 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 [CO2] in their natural environments (460 and 50 mmol CO2 m-3 for
the high- and lower-CO2 spring, respectively), and also at a saturating [CO2] 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 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 [CO2] 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 m-3 HEPES at pH 7-2 and 7-8; the pH
values of the high- and lower-CO2 sites, respectively.
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
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
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) 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
dried at 500 oC for 16 h before being ground with a mortar and pestle and subjected to
analysis (Horwitz 2000).
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 (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 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 Geneticsc software (Miller 1997). This
software was used to calculate Wright's F-statistic (Dst) (Weir & Cockerham 1984), a
95% confidence interval (using bootstrapping and 5000 replications), and genetic
distance and identity (Nei 1972) for diploid dominant alleles.
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 SASc release 8-2 (SAS Inst. Inc., Cary, NC, USA)
and alpha > 0-05 and 0-01 levels of significance.
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 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 NH4-N
and total P did not change. The concentrations of K, Na, and Cl increased in the high-
CO2 spring over time, as did Ca in the lower-CO2 water (Table 3-1).
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 imol m-2 s-1 just above the water surface and 1100 imol 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,
The two springs differed in several key respects. Fanning Springs had a lower pH
with almost ten-fold higher free [CO2], 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 air-
equilibrium 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 [CO2] in the
air just above the water surface, with values for the high- and lower-CO2 springs of 521 +
58 and 402 14 imol CO2 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-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
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 lower-
CO2 plants. This was due in part to the fact that each high-CO2 plant had only 65% of the
number of leaves found on lower-CO2 plants, and only 63% 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 220%
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
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).
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
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).
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).
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 10% 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,
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 GA3-
induced low-CO2 plants produced very few capsules, each with only about ten seeds,
none of which germinated.
Figure 3-3 shows the effects of  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 [CO2] 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 high-
and 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 [CO2] 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
[CO2] of their natural spring locations, as well as at saturating [CO2]. It also includes the
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 [CO2], 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 [CO2], 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
[CO2]. 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 [CO2], 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.
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
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.
Both freshwater springs have been very stable in terms of temperature, pH,
alkalinity, and for this study particularly [CO2] and [HCO3-], 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 [CO2], 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 CO2 sources to assess the long-term responses of
plants to elevated [CO2] has focused on populations growing at different distances from a
single CO2 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 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 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).
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 [CO2]. 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
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
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 CO2-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 CO2-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 CO2-saturated photosynthesis
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 [CO2] 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 CO2-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 135% 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 60% 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
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.
There was no evidence for lower rates in the high-CO2 plants as has been reported for
terrestrial species (Azc6n-Bieto et al. 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 high-
CO2 springs have not shown large biomass differences compared to populations growing
in ambient [CO2] (Cook et al. 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 60%
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.
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
high-CO2 population invested the additional carbon in producing axillary stems, rather
than leaves, as a means of increasing asexual reproduction through fragmentation and
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 [CO2] 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 ofL. repens collected in the high- and lower-CO2 springs exhibited
characteristics consistent with genotypic adaptation to their respective environmental
[CO2]. 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 high-
CO2 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 [CO2] 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
Total alkalinity (meq L-1)
CO2 (mmol m-3)
HCO3- (mmol m-3)
Ct (mmol m-3)
NH4-N (mmol m-3)
NO3-N (mmol m-3)
Total P (mmol m-3)
K (mmol m-3)
Ca (mmol m-3)
Mg (mmol m-3)
Na (mmol m-3)
Cl (mmol m-3)
Fe (mmol m-3)
0-89 + 036
1500 + 180
1600 + 200
0-71 + 035
0-97 + 032
1500 + 25
240 + 4
0-36 + 018
High-CO2 plant Lower-CO2 plant
Submersed L. repens plants from the high- and lower-CO2 populations.
Table 3-2. Weights, densities, and morphological characteristics ofL. repens plants
growing in high- and lower-CO2 springs.
Fresh weight (g plant-1)
Dry weight (g plant-1)
Density (g cm-3 plant-1)
Leaf fresh weight (g plant-1)
Leaf dry weight (g plant-1)
Leaves (no. plant-1)
Leaf area (cm2 plant-1)
Total stem fresh weight (g plant-1)
Total stem dry weight (g plant-1)
Main stem length (cm plant-1)
Main stem internode length
Axillary stems (no. plant-1)
Axillary stem length (cm plant-1)
Root fresh weight (g plant-1)
Root dry weight (g plant-1)
Total root length (cm plant1)
2-31 + 0-68**
0-26 + 0-09**
0-68 + 0-15**
0-96 + 0-33**
0-13 + 0-06**
39 + 15**
52 + 18**
0-12 + 0-04**
1-60 + 0-45**
9-1 + 3-4**
12-8 + 10-1**
0-22 + 0-06*
0-020 + 0-004**
107 + 32
4-42 + 1-01
0-65 + 015
0-89 + 015
2-27 + 0-42
0-35 + 007
60 + 11
83 + 15
0-23 + 0-08
0-68 + 012
0-62 + 038
0-075 + 0-043
161 + 97
Mean SD (n = 8). P-values are from t-tests and significant differences between
populations at P 0-05 and 0-01 are noted by and **, respectively.
Table 3-3. Individual leaf characteristics of L. repens plants growing in high- and lower-
Fresh weight (mg leaf1)
Dry weight (mg leaf1)
Density (g cm-3)
Area (cm2 leaf1)
Thickness (mm leaf1)
TNC (mg g-1 dry weight)
Fiber (mg g'1 dry weight)
Ash (mg g-1 dry weight)
Spongy mesophyll cell layers
Crystals (no. mm-2)
Adaxial stomates (no. mm-2)
Abaxial stomates (no. mm-2)
52 + 17
0-68 + 018
2-1 + 0-6*
0-16 + 001*
85 + 41*
195 + 25*
89 + 7**
125 + 17
3-3 + 0-3**
5.1 + 10**
103 + 26**
33 + 11**
8-0 + 13
0-82 + 014
0-18 + 001
30 + 13
262 + 46
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
-30 tm 30m
e epidermal cell
c c stal
Figure 3-2.Leaf cross sections for L. repens plants growing in (a) high- and (b) lower-
p palisade mesophyll cell
s spongy mesophyll cell
Figure 3-2.Leaf cross sections forL. repens plants growing in (a) high- and (b) lower-