Aquatic Botany 89 (2008) 379–384
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Aquatic Botany
journal homepage: www.elsevier.com/locate/aquabot
Why the free ﬂoating macrophyte Stratiotes aloides mainly grows in
highly CO2-supersaturated waters
Lasse Tor Nielsen, Jens Borum *
Freshwater Biological Laboratory, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 11 December 2007
Received in revised form 31 March 2008
Accepted 7 April 2008
Available online 23 April 2008
We examined how the freely ﬂoating macrophyte, Stratiotes aloides L., sampled from a CO2supersaturated pond, changes leaf morphology, photosynthesis and inorganic carbon acquisition during
its different submerged and emerged life stages in order to evaluate whether S. aloides requires
consistently supersaturated CO2 conditions to grow and complete its life cycle. Submerged rosettes
formed from over-wintering turions had typical traits of submerged plants with high speciﬁc leaf area
and low chlorophyll a concentrations. Emergent leaf parts of mature, ﬂoating specimens had typical
terrestrial traits with stomata, low speciﬁc leaf area and high chlorophyll a content, while offsets formed
vegetatively and basal, submerged parts of mature plants showed traits in between. All submerged leaf
types exhibited some ability to use HCO3 but only rosettes formed from turions had efﬁcient HCO3 use.
Rosettes also had the highest CO2 afﬁnity and maximum CO2-saturated photosynthesis in water. Halfsaturation constants for CO2 (21–74 mM CO2) were for all submerged leaf parts 5–140 times lower than
the concentrations of free CO2 in the pond (350–2800 mM CO2). Emergent leaves were less efﬁcient in
water but had signiﬁcantly higher photosynthesis than submerged, mature leaf parts in air, and rates of
photosynthesis of emergent leaves in air were three to ﬁve times higher than rates of CO2-saturated
photosynthesis of the three submerged leaf types in water. Underwater photosynthetic rates estimated at
CO2 concentrations corresponding to air equilibrium were not sufﬁciently high to support any noticeable
growth except for rosettes, in which bicarbonate utilization combined with high CO2 afﬁnity resulted in
photosynthetic rates corresponding to almost 34% of maximum rates at high free CO2. We conclude that
S. aloides requires consistently high CO2-supersaturation to support high growth and to complete its life
cycle, and we infer that this requirement explains why S. aloides mainly grows in ponds, ditches and reed
zones that are characterized by strong CO2-supersaturation.
ß 2008 Elsevier B.V. All rights reserved.
Keywords:
Stratiotes aloides
Photosynthesis
Inorganic carbon acquisition
Bicarbonate utilization
CO2-supersaturation
1. Introduction
The free ﬂoating amphibious plant Stratiotes aloides L. (Water
Soldier) grows in ponds and ditches and in reed zones of rivers and
larger lakes in temperate areas (Sculthorpe, 1967; Cook and UrmiKo¨nig, 1983). When present in small water bodies, the species is
often dominant and completely covers the water surface with a
dense and highly productive stand of up to 50 cm high ﬂoating
rosettes (Sculthorpe, 1967). However – and maybe luckily – the
species seems less able to establish and spread on open surfaces of
larger lakes. Free ﬂoating plants are traditionally thought to occur
in small, sheltered water bodies because they are vulnerable to
high physical exposure and require relatively high nutrient
richness due to lack of sediment nutrient access (Sculthorpe,
* Corresponding author. Tel.: +453 532 1904.
E-mail address: [email protected] (J. Borum).
0304-3770/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquabot.2008.04.008
1967). However, in addition to shelter, the typical growth locality
of S. aloides is water of high total alkalinity but with relatively low
pH, and therefore consistent CO2-supersaturation (Prins and
Deguia, 1986). Here we wished to evaluate whether the distribution of this species could also be determined by its need for a richer
CO2-supply than normally offered in large alkaline lakes with high
pH and low free CO2 availability.
S. aloides L. is a dioecious, perennial species with a life cycle of
alternating submerged and emergent stages. In northern temperate areas, including Denmark, mostly female specimens of S.
aloides occur, and the plant only reproduces vegetatively (Cook and
Urmi-Ko¨nig, 1983; Moeslund et al., 1990). Populations in nutrient
poor, transparent waters have been reported to consist only of
submerged individuals that never rise to the surface (Erixon, 1979;
Renman, 1989), but most populations shift from a completely
submerged life form during winter to a predominantly free ﬂoating
form from spring through autumn. In late autumn S. aloides sinks to
the bottom where it resides during winter, all the time carrying
380
L.T. Nielsen, J. Borum / Aquatic Botany 89 (2008) 379–384
green leaves but no roots (Erixon, 1979). In spring, plants rise to the
surface and again form vigorously growing, ﬂoating rosettes of
partly emergent leaves with stomata for efﬁcient atmospheric CO2
uptake (Diannalidis, 1950). New plants are produced from overwintering turions formed by mature plants in the fall. Initially,
leaves of the turions are thin, ﬂaccid and fully submerged. Later,
emergent leaves are produced. During summer, offsets connected
by a stolon to the mother plant are produced by mature plants. At
ﬁrst, the offsets may rely on resource allocation from mother
plants, but later they must build up their own capacity for
underwater photosynthesis. Accordingly, S. aloides has different
life cycle stages exposed to very different CO2-availabilities. S.
aloides may partly meet the challenge of inorganic carbon
acquisition in different life stages by acclimating leaf morphology
and physiology, but we also hypothesized that either the species
must have efﬁcient bicarbonate use or it requires consistently high
availability of free CO2 to complete its life cycle.
Submerged leaves of amphibious rooted macrophytes are most
often restricted to uptake of free CO2, and they have low
photosynthetic rates at free CO2 concentrations in equilibrium with
the atmosphere (Maberly and Spence, 1989; Sand-Jensen et al.,
1992). The CO2 extraction capacity is somewhat higher in
heterophyllous amphibious plants than in homophyllous due to
thinner or ﬁnely dissected leaves, but maximum photosynthetic
rates are still relatively low at equilibrium CO2 (Beer et al., 1991), and
dense stands of amphibious macrophytes are primarily found in
environments rich in free CO2, such as in streams and rivers (SandJensen et al., 1992). A bit surprising, submerged leaves of S. aloides
seem able to utilize bicarbonate in contrast to other amphibious
macrophytes (Prins and Deguia, 1986). Although the bicarbonate
utilization efﬁciency was relatively low compared to that of an
efﬁcient bicarbonate user, Myriophyllum spicatum, and although
Prins and Deguia (1986) assumed that the relative contribution from
bicarbonate utilization in S. aloides was low in CO2-supersaturated
ponds, bicarbonate utilization in submerged life stages may be
sufﬁcient to ensure life cycle completion in S. aloides.
The main purpose of this study was to test the hypothesis, that
S. aloides requires consistently CO2-supersaturated water to
complete its life cycle. We assumed that if submerged leaves of
S. aloides are efﬁcient bicarbonate users or are able to obtain high
photosynthetic rates at concentrations of free CO2 close to air
equilibrium, other factors than inorganic carbon availability would
be responsible for its preference for ponds, ditches and reed zones.
We, therefore, examined the efﬁciency of bicarbonate utilization of
the different submerged leaf types and the photosynthetic rates as
functions of manipulated concentrations of free CO2. Finally, we
compared rates of photosynthesis in air of emergent leaves with
underwater photosynthesis.
2. Materials and methods
Plants were collected from a small (0.5 ha), 5 m deep pond,
Lyngsø (558580 N, 128250 E), in Northern Sealand, Denmark, during
mid-summer 2006. The plants were brought submerged or semisubmerged to the laboratory and kept in aerated water at ambient
temperature and dim light for no longer than 48 h before being
used in experiments. The plants were divided into four morphologically distinct leaf types following the developmental stages and
life forms outlined earlier: (1) underwater rosettes originating
from turions, (2) offsets still attached by a stolon to the mother
plant, (3) basal, submerged parts of leaves of mature, emergent
individuals and (4) emerged parts of leaves of emergent
individuals identiﬁed by the presence of stomata.
Water samples (n = 3) were collected from the pond several
times during summer and on one occasion in winter. The water
was kept cold in completely ﬁlled glass bottles with airtight
closing and analysed for pH, conductivity and alkalinity immediately after return to the laboratory. The pH was measured with a
combination electrode connected to a pH-M82 standard pHmeter (Radiometer, Denmark) and conductivity with a YSI 30M/
10FT multimeter (YSI Incorporated, Yellow Springs, OH 45387,
USA). Alkalinity was determined by end-point titration with HCl,
and the concentration of free CO2 was calculated based on
alkalinity, pH, conductivity and temperature according to
Mackereth et al. (1978).
pH-drift experiments were conducted in order to evaluate
whether each of the four leaf types were able to utilize HCO3 as
an inorganic carbon source. Experiments were carried out in
50 ml glass bottles using a 1:1 mixture of water from the
sampling site and demineralised water to reduce the buffer
capacity. All four leaf types were examined along with control
bottles without plant material. Each leaf type was represented by
ﬁve replicate bottles. Three to four 3 cm long leaf sections of S.
aloides were placed in each bottle. Initial pH was 7.80 0.01
(mean S.D.), initial alkalinity 0.98 0.08 meq l1 and initial
conductivity 119 1 mS cm1. All bottles were mounted on a
rotating wheel placed in a water-ﬁlled chamber cooled to 15 8C. The
light source was a 500 W mercury lamp delivering 300–
400 mmol photons m2 s1 (PAR) inside the bottles. The bottles
were incubated for 24 h after which pH, conductivity and alkalinity
in the bottles were recorded and applied in calculations of terminal
CO2-concentrations.
Submerged photosynthesis at varying pH corresponding to
different concentrations of free CO2 was measured with the purpose
of determining (1) the rate of maximum photosynthesis (Pmax), (2)
the CO2 compensation point (CO2comp), (3) the half-saturation
constant for CO2 uptake (K0.5) and (4) the initial slope (a) of
photosynthesis versus free CO2 concentration relationships for the
four leaf types. The experiments were carried out by mounting two
to four 7 cm long leaf sections of S. aloides in a closed, water ﬁlled
Plexiglas chamber (167 ml) cooled to 15 8C. Before mounting, the
leaves were gently wiped with paper cloth under water to remove
epiphytes. The water was from the sampling site, and a magnetic
stirrer homogenised the water. Irradiance was supplied by a halogen
spot light (600 mmol photons m2 s1). The oxygen concentration
in the chamber was continuously recorded with an oxygen
minielectrode (OX500, Unisense, Denmark) connected to a Unisense
PA2000 picoameter and data were logged through an AD-converter
(ADC16, PicoLog, UK) connected to a computer. A pH-electrode was
also mounted in the chamber and connected to a pH-M82 pH-meter.
Initially pH was reduced to 6.5 by adding HCl and the chamber was
closed. After an acclimation period of 30 min, changes in oxygen
concentrations were recorded. The availability of free CO2 was
gradually reduced by raising pH with NaOH to 10 in steps of 0.5 0.1
pH units (mean S.D.; n = 4). Rate of change in oxygen concentration
was measured at each pH-level and associated to CO2 concentration.
Recording periods of 3–5 min with linear increase in oxygen
concentration were required to calculate photosynthetic rates but
without raising pH by more than 0.01 unit during the incubation. The
free CO2 concentrations were calculated from pH assuming constant
DIC in the chamber (Mackereth et al., 1978).
For each experiment, leaf area was measured with a LI-COR LI3000 (Lambda Instruments Corporation, USA). Emergent leaves
were cut longitudinally in advance to allow correct area
measurements. Leaves were freeze-dried and weighed to determine dry mass. For determination of chlorophyll a content, ca.
5 mg dry material was ground and extracted in 96% ethanol for
24 h in darkness. After ﬁltration, absorbance was measured on a
spectrophotometer and chlorophyll a content was calculated
according to Wintermans and DeMots (1965).
L.T. Nielsen, J. Borum / Aquatic Botany 89 (2008) 379–384
Aerial photosynthesis was measured in a 4.9 l Plexiglas chamber
containing atmospheric air and placed in a 14 8C thermostatically
controlled room. Two to four leaves were mounted on a net in the
middle of the chamber. A small pump and water-soaked tissue paper
was placed in the bottom to ensure well-mixed, humid air. The
chamber was closed by a Plexiglas lid and sealed with vaseline.
Irradiance was 250 mmol photons m2 s1 and full light saturation
may not have been achieved. Hence, differences in photosynthetic
rates might have been conservatively estimated. The experiments
were initiated by taking out two 2 ml samples with syringes through
a serum stopper placed in the lid. Samples were analysed in an
ADC225 MK3 infrared gas analyzer (Analytical Development Co.,
UK), and the CO2 concentrations were determined using 100 ml
aliquots of an acidiﬁed 1.00 mM HCO3 solution as standard. Sets of
two samples were taken from the chamber at 6–10 min intervals
and the photosynthetic rate was calculated from the linear decrease
in CO2 observed over periods of 20–50 min. A photosynthetic
quotient of 1.0 (mol/mol) was used to convert photosynthetic rates
measured as CO2 to O2. Aerial photosynthesis was determined only
for the submerged and emerged part of emergent leaves, each of
which was represented by three replicates. Leaf area, dry weight and
chlorophyll content were determined as described above.
2.1. Calculations and statistical methods
Final pH and CO2 concentration of the four leaf types in the pHdrift experiment were compared by analysis of variance (one-way
ANOVA) and a Tukey test for all pairwise multiple comparisons.
Rates of aerial photosynthesis were compared by Student t-test.
Rates of carbon uptake of submerged leaves were for each replicate
series ﬁtted by non-linear regression to a Hill–Whittingham
equation (Hill and Whittingham, 1955), modiﬁed by adding a
parameter (P CO2 ¼0 ) representing carbon uptake at ‘‘zero’’ free CO2:
1
P ¼ 0:5ðð½CO2 R
1
þ K 0:5 R
þ Pmax Þ
2
0:5
ðð½CO2 R1 þ K 0:5 R1 þ P max Þ 4½CO2 R1 P max Þ
Þ þ PCO2 ¼0
where P is the net photosynthesis, [CO2] is the concentration of free
CO2 and R is the CO2 transport resistance. The P CO2 ¼0 , the
maximum net photosynthesis (Pmax), the half-saturation constant
(K0.5) and the CO2 compensation point (CO2comp) were extracted or
calculated from the individual ﬁts, while initial slopes of the
photosynthesis versus CO2 curves (a) were determined by linear
regression using the 9–12 data points with the lowest CO2
concentration. Photosynthetic parameters of the four leaf types
were compared by one-way ANOVA and a Tukey test for all
pairwise multiple comparisons. K0.5 values were log transformed
before statistical analysis in order to meet the requirement of
normality. The signiﬁcance level of all statistical analyses was set
at 0.05.
3. Results
3.1. Inorganic carbon availability in the pond
With a relatively constant alkalinity of around 2.2 meq l1 in
the pond, equilibrium pH should be approximately 8.3 but varied
between a minimum of 6.2 in August and 7.3 in December
reﬂecting that the pond was consistently supersaturated with free
CO2. Total dissolved inorganic carbon varied between 2.5 and
5.0 mM and free CO2 ranged from 350 in December to 2800 mM in
summer versus the less than 20 mM at equilibrium with atmospheric air.
381
Table 1
End pH and CO2 concentration of the pH-drift experiments with four Stratiotes
aloides leaf types (mean S.D.; n = 4–5)
CO2 (mM)
pH
Rosettes
Offsets
Submerged
Emergent
a
10.05 0.05
9.56 0.21b
9.35 0.24b
9.59 0.33b
0.11 0.02 a
0.59 0.28b
0.99 0.56b
0.45 0.32b
Initial pH was 7.80 and the concentration of free CO2 was 41 mM. Letters (a and b)
indicate statistical differences (Tukey test).
3.2. Chlorophyll content and speciﬁc leaf area
Rosettes, offsets and submerged leaf parts had chlorophyll a
contents ranging from 22.6 to 45.7 mg chl. a m2, respectively
(Table 2). Emerged leaf parts had signiﬁcantly more chlorophyll
per area with a mean chlorophyll content of 125 mg chl. a m2. For
comparison, 14 species of submerged macrophytes had a mean
chlorophyll content of 70 mg chl. a m2 (Nielsen and Sand-Jensen,
1989), and 22 terrestrial species had a mean chlorophyll content of
228 mg chl. a m2 (Murchie and Horton, 1997, values for both sun
and shade leaves).
Speciﬁc leaf area decreased in the order rosettes > offsets >
submerged > emerged from 0.13 to 0.04 m2 g1 DW, showing that
rosette leaves were the thinnest of the four and emerged leaves the
thickest. The speciﬁc leaf area of the 14 submerged macrophyte
species from the reference above ranged from 0.05 to 0.35 m2 g1
DW (average 0.17). The range of speciﬁc leaf area in 2548
terrestrial species was 0.000667–0.0714 m2 g1 DW (Wright et al.,
2004).
3.3. HCO3 use examined by pH-drift experiments
All four S. aloides leaf types raised pH and reduced the
concentration of free CO2 signiﬁcantly from the initial values of
7.80 and 41 mM. Final pH ranged from 10.05 for rosettes to 9.35 for
submerged leaf parts with offsets and emergent leaves lying in
between (Table 1). Correspondingly, rosettes had the lowest mean
CO2 compensation point of 0.11 mM and submerged the highest of
0.99 mM, while offsets and emergent depleted CO2 concentrations
to 0.59 and 0.45 mM. End pH was signiﬁcantly higher and CO2
compensation point signiﬁcantly lower for rosettes than for the
other three leaf groups, which did not have signiﬁcantly different
end pH or compensation point.
3.4. Rates of photosynthesis versus free CO2 availability
Rates of net photosynthesis varied substantially with changing
concentrations of free CO2 for all four leaf types (Fig. 1). For three of
the four, the Hill–Whittingham ﬁt described data satisfactorily
(r2 > 0.84), while high variability in the photosynthetic capacity of
emergent leaves made the ﬁt poor (r2 = 0.69). There were
systematic differences in the photosynthetic parameters derived
from the Hill–Whittingham ﬁt for the four leaf types (Table 2).
Rates of maximum under-water photosynthesis at high free CO2
varied from 59 mg O2 m2 h1 for offsets to 207 mg O2 m2 h1 for
rosettes, while K0.5 ranged from 20.6 mM CO2 for offsets to 326 mM
for emerged leaf parts. The estimated photosynthetic rate of
rosettes at zero free CO2 was signiﬁcantly above zero and higher
than for the other leaf types. Rates at zero CO2 were positive for
offsets and submerged leaves but not signiﬁcantly different from
zero, while the rate for emergent leaves was negative, and
emergent leaves had a CO2 compensation point of 28.7 mM
(Table 2). Initial slopes of the photosynthesis versus CO2 curves
decreased in the order rosettes > offsets > submerged > emergent
L.T. Nielsen, J. Borum / Aquatic Botany 89 (2008) 379–384
382
Fig. 1. Net photosynthesis of the four Stratiotes aloides leaf types in water as a function of the free CO2 concentration. The combined data set for each leaf type was ﬁtted to a
modiﬁed Hill–Whittingham equation by non-linear regression.
Table 2
Photosynthetic parameters and leaf characteristics of different Stratiotes aloides leaf types
K0.5a
Rosettes
Offsets
Submerged
Emergent
ac
Pmax b
b
68.7 21.8
20.6 5.3a
74.4 19.3b
326 209c
c
207 32
59 14a
150 11b
113 73b
PCO2 ¼0 d
c
13.0 3.4
7.1 1.6b
4.6 3.8ab
0.47 0.14a
CO2compe
c
31.9 6.4
2.2 2.5b
3.3 3.6b
13.5 7.8a
–
–
–
28.7
Chl. af
Areasg
ab
28.5 3.4
45.7 6.7b
22.6 10.7 a
125 27c
0.129 0.011a
0.104 0.007ab
0.075 0.031b
0.038 0.006c
Photosynthetic parameters attained from the photosynthesis experiments in water (mean S.D.; n = 4). Letters (a–c) indicate statistical differences (Tukey test).
a
Half-saturation constant (K0.5) in mM CO2.
b
Maximum net photosynthesis (Pmax) in mg O2 m2 h1.
c
Initial slope at free CO2 light (a) in mg O2 m2 h1 mM1 CO2.
d
Net photosynthesis at zero CO2 (P CO2 ¼0 ) in mg O2 m2 h1.
e
CO2 compensation point (CO2comp) in mM CO2.
f
Chlorophyll a content (Chl. a) in mg chl. a m2 leaf surface.
g
Speciﬁc leaf area (Areas) in m2 g1 DW.
from 13.0 to 0.47 mg O2 m2 h1 mM1 CO2 reﬂecting that rosettes
had the most efﬁcient CO2 extraction and emergent leaves the least
efﬁcient.
Photosynthetic rates at free CO2 concentrations in equilibrium
with air (16 mM at 15 8C) were estimated from Hill–Whittingham
ﬁts for the individual leaf types. Rates were relatively high for
rosettes (71 mg O2 m2 h1) but low for offsets and submerged leaf
parts (28 and 30 mg O2 m2 h1, respectively) and negative for
emergent leaves (8.2 mg O2 m2 h1). On a dry weight basis,
rates were 9.1, 2.9, 2.0 and 0.30 mg O2 g1 DW h1 for rosettes,
offsets, submerged and emergent, respectively.
Rates of aerial photosynthesis were determined only for
submerged and emerged leaf parts of emergent mature leaves
Table 3
Rates of net photosynthesis in air of submerged and emerged leaf parts of mature,
ﬂoating Stratiotes aloides plants (mean S.D.; n = 3)
Photosynthetic rates (mg O2 m2 h1)
Submerged
Emerged
75 35
711 118
Means were signiﬁcantly different (Student t-test, p < 0.05).
(Table 3). Emergent leaves had a mean photosynthetic rate in air of
711 mg O2 m2 h1, which was signiﬁcantly higher than the rate of
75 mg O2 m2 h1 displayed by the submerged leaf parts and
about ﬁve times higher than the maximum photosynthetic rate in
water (Table 2).
4. Discussion
Our results showed that S. aloides copes with the different
environments experienced during its life stages by exhibiting
variable morphology and physiology towards that of submerged or
emergent characteristics. Emergent leaf parts of mature rosettes
had clear traits of terrestrial leaves characterized by well
developed stomata, higher chlorophyll content than submerged
leaves and a low speciﬁc leaf area. These traits allowed emergent
leaves in air to obtain three to ﬁve times higher photosynthetic
rates on a leaf area basis than submerged leaf parts. Submerged
leaf parts of mature leaves and offsets had characteristics in
between terrestrial and submerged leaves, but closest to the latter,
while the rosettes originating from over-wintering turions had
typical submerged traits with the high speciﬁc leaf area and low
chlorophyll content that allow efﬁcient light harvesting at low
L.T. Nielsen, J. Borum / Aquatic Botany 89 (2008) 379–384
light intensities and extraction of inorganic carbon via liquid
phase diffusion (Nielsen and Sand-Jensen, 1989). Many amphibious rooted macrophytes are heterophyllous with ‘‘terrestrial’’
leaves above water and thinner and more dissected leaves below
water resulting in a much higher relative surface area (Maberly
and Spence, 1989; Sand-Jensen et al., 1992). S. aloides does not
have dissected, submerged leaves but the different leaf stages
exhibit distinct morphological and functional adaptations to the
ambient growth conditions with respect to both light and
inorganic carbon.
The pH-drift experiment and the measurement of photosynthetic rates at low concentrations of free CO2 conﬁrmed the results
reported by Prins and Deguia (1986), that at least rosettes formed
from turions are able to use bicarbonate. As a general trend,
amphibious plants seem, in contrast to consistently submerged
rooted macrophytes, not able to use bicarbonate for photosynthesis (Spence and Maberly, 1985; Madsen and Sand-Jensen, 1991),
but, clearly, S. aloides rosettes exhibited substantial capacity for
bicarbonate utilization. In the pH-drift experiment, leaves formed
from turions raised pH to above 10, and when manipulating the
availability of free CO2, photosynthesis was signiﬁcantly positive
when extrapolating to zero free CO2. The bicarbonate extraction
was not as efﬁcient and prominent as in many truly submerged,
primary water plants (Sand-Jensen et al., 1992) but sufﬁciently
high to support rates of around 15% of maximum net photosynthesis at CO2 saturation. The ﬁnding of relatively efﬁcient bicarbonate utilization was also surprising considering the very high
concentrations of free CO2 (350–2800 mM) measured in Lyngsø,
because at least some water plants reduce investment in enzyme
capacity for bicarbonate utilization when living in a CO2-rich
environment (Maberly and Spence, 1983; Madsen and SandJensen, 1987). While the capacity of bicarbonate utilization was
relatively high in leaves from over-wintering turions, the other
three leaf types had reduced or lost the capacity, and the
bicarbonate utilization found in the present investigation would
not be sufﬁcient to allow successful completion of the S. aloides life
cycle and reproduction in alkaline lakes with low concentrations of
free CO2.
The estimated photosynthetic rates of the different S. aloides
leaf types obtained at CO2 concentrations corresponding to air
equilibrium (16 mM at 15 8C) was only substantial for rosettes
formed from over-wintering turions. The rate for rosettes was
71 mg O2 m2 h1 and corresponded to as much as 34% of the
maximum photosynthetic rate at high free CO2 concentrations.
This high rate was due to both more efﬁcient bicarbonate use and
higher CO2 afﬁnity than for the other leaf types. Accordingly,
rosettes would likely be able to grow also under conditions
prevailing in lakes with low availability of free CO2. We speculate
that the good performance of rosettes in CO2 poor waters may
explain the occurrence of only submerged S. aloides populations in
a shallow lake characterized by isoetids (Erixon, 1979; Renman,
1989). The phenomenon was attributed to low nutrient availability
(Renman, 1989), but low CO2 availability might have allowed
rosettes to survive and grow but not form emergent rosettes. In
contrast to rosettes (9.1 mg O2 g1 DW h1), the other leaf types
were estimated to obtain rates of net photosynthesis in light of less
than 2.9 mg O2 g1 DW h1, which is close to the dark respiration
rates of 0.4–1.5 mg O2 g1 DW h1 measured for whole shoots of
different aquatic plants by Nielsen and Sand-Jensen (1989). When
taking root respiration in light into account, these low photosynthetic rates most likely would not be sufﬁcient to support
substantial growth and successful completion of the S. aloides life
cycle in larger lakes despite the fact that larger lakes in general also
seem to be at least slightly over-saturated with free CO2 (Cole et al.,
1994).
383
Even without bicarbonate utilization, all submerged leaf
types seemed potentially able to saturate photosynthesis under
the CO2-supersaturated conditions in Lyngsø. Values of K0.5 (CO2)
for submerged leaves were between 21 and 74 mM or 5–140
times lower than the actual concentrations of CO2 found in the
pond. Even in winter, when CO2 concentrations were the lowest
(350 mM), there seemed to be sufﬁcient free CO2 to saturate
photosynthesis. Strong supersaturation with CO2 is known from
other aquatic systems, with lowland streams as suitable sites for
growth of submerged and amphibious rooted macrophytes
under CO2-rich conditions (Sand-Jensen and Frost-Christensen,
1999). Some of these streams may be permanently supersaturated (Kelly et al., 1983) while others exhibit marked diel
CO2 ﬂuctuations with photosynthetic carbon limitation occurring during part of the day (Sand-Jensen and Frost-Christensen,
1999). Although we did not measure changes in pH or CO2 in
Lyngsø over diel cycles, we assume that such changes were small
because of the very high concentrations of free CO2, and, hence,
photosynthesis and growth of submerged parts of S. aloides
should not be limited by inorganic carbon availability in spite of
the likely lower stirring in dense stands of S. aloides than that
used in the experimental setup. Hence, competition for space,
light and perhaps nutrients among individuals of S. aloides in
Lyngsø should be much more severe than competition for
inorganic carbon.
As expected, the aerial leaf parts of S. aloides tended to have
lower photosynthetic rates and signiﬁcantly higher K0.5 (CO2)
when submerged than the submerged parts of the same leaves.
This may be due to the cuticle or wax layers on leaf surfaces
exposed to air to prevent desiccation (Frost-Christensen and Floto,
2007). On the other hand, the formation of stomata on aerial leaf
parts resulted in three to ﬁve times higher photosynthetic rates at
ambient atmospheric CO2 than maximum rates obtained for any
submerged leaf type. While formation of aerial leaves may not
inevitably lead to higher productivity of amphibious plants due to
restrictions in growth season and increasing costs to produce
supportive tissues (Madsen and Sand-Jensen, 1991), we infer that
S. aloides greatly beneﬁts from the presence of aerial leaves because
the photosynthetic rates of these leaves were markedly higher
than that of any other leaf type. It should also be noted, that actual
aerial rates may be even higher than estimated here, because the
CO2 availability immediately above the water surface should be
elevated compared to the atmosphere in general due to the outﬂux of CO2 from the supersaturated pond. In addition light
availability is also much higher for emergent than for submerged
leaves in the rather turbid water of Lyngsø. Finally, the S. aloides
population beneﬁts further from the emergent leaves, because
carbon ﬁxed in air contributes to maintain high CO2-supersaturation in the water column, when ﬂoating rosettes sink to the
bottom and decompose.
We conclude that S. aloides exhibits marked acclimations in leaf
morphology and inorganic carbon uptake that allow the species to
efﬁciently cope with the challenges experienced as an amphibious
plant with both submerged and emergent leaves and life stages.
The ﬂexible and efﬁcient ability to utilize inorganic carbon from
different sources allows this species to form dense stands in CO2supersaturated, temperate ponds, ditches and shallow reed zones
of larger lakes, while the species seems much less well adapted to
complete its life cycle in open waters of larger lakes with free CO2
concentrations closer to air equilibrium. Although physical shelter
and high nutrient availability may still be important factors for the
success of S. aloides (Sculthorpe, 1967), consistent CO2-supersaturation is likely as important for completion of the S. aloides life
cycle as it is for the growth of amphibious plants in streams (SandJensen et al., 1992).
384
L.T. Nielsen, J. Borum / Aquatic Botany 89 (2008) 379–384
Acknowledgements
This work was partly supported by the CLEAR-project. We
thank the Lyngsø family for allowing access to the pond and for
pleasant company. We also thank the students Anders Winkel,
Helle Wilken-Jensen and Lasse Bust Hansen for their efforts with a
pilot project and Elmir Maric for technical assistance. Finally, we
thank Kaj Sand-Jensen and two anonymous reviewers for thorough
comments and constructive suggestions.
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