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The metabolic cost of bicarbonate use in the submerged plant Elodea nuttallii.
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J. Iwan Jones1,2
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Kingdom.
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Dorchester, DT2 8ZD, United Kingdom.
School of Biological Sciences, University of Liverpool, Liverpool, L69 3GS, United
Present address: CEH Dorset, Winfrith Technology Centre, Winfrith Newburgh,
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Author for correspondence:
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Iwan Jones
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Tel: +44 1305 213559
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Fax: +44 1305 213600
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Email: IWAJ@CEH.ac.uk
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Abstract
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The aquatic plant Elodea nuttallii(Planch.) St. John has been shown to express plasticity in
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the source of inorganic carbon it uses for photosynthesis. An investigation was undertaken
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to determine what effect the switch from CO2 to HCO3– use had on the growth of E.
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nuttallii. Plants were grown under reduced CO2 availability that favoured the switch,
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together with control plants (CO2 at equilibrium with air) that continued to use CO2 only.
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The extent to which both sets of plants could utilise HCO3– was determined (as the ratio of
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oxygen evolution at pH 9 and pH 6.5), and several measures of growth were made.
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Although reduced CO2 availability produced an increase in HCO3– utilisation, no
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differences were found in the measured growth of the plants. Therefore, it was possible to
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estimate, from the difference between the estimated rate of photosynthesis of the plants
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utilising HCO3– and those using CO2 only, the approximate cost of constructing,
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maintaining and running the bicarbonate utilisation mechanism in this species as 69 µmol
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photons m-2 s-1. This value can be used to estimate an irradiance of circa 80 µmol m-2 s-1
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below which HCO3– use would not be expected in this species, an irradiance commonly
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experienced by submerged macrophytes in the field.
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Keywords:
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photosynthesis; polar leaf; plasticity.
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aquatic plant; bicarbonate use; carbon dioxide; light; growth; macrophyte;
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Introduction
Dissolved inorganic carbon (DIC) is present in natural waters in different,
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interconvertible forms. These consist of CO2* (free CO2 = H2CO3 + CO2 dissolved),
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HCO3– and CO32-, in proportions largely determined by pH, with the equilibrium shifting
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towards CO32- with increasing pH. In many productive waters, concentrations of CO2* are
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low and can be limiting as a photosynthetic carbon source, a problem exacerbated by the
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low flow conditions found in plant stands (Losee and Wetzel, 1993) and by the slow
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diffusion of dissolved gases in the aquatic environment (Raven, 1970; Smith and Walker,
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1980; Black et al., 1981; Jones et al., 2000a).
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The photosynthesis of plants growing in such waters raises oxygen levels and
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reduces CO2* to very low concentrations, severely restricting further photosynthetic
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assimilation (Jones et al., 1996) and inducing conditions for increased photorespiration
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(Hough, 1974; Van et al., 1976; Ondoket al., 1984). These adverse conditions can occur
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both as part of rapidly changing diel cycles (Goulder, 1970; Unni, 1972; Brown et al.,
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1974; Van et al., 1976; Ondok et al., 1984; Frodge et al., 1990; Madsen and Maberly, 1991;
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Jones et al., 1996) and as much longer-term seasonal changes (Bindloss, 1976; Talling,
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1976; Howard et al., 1984; Frodge et al., 1990). Furthermore, dissolved inorganic carbon
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can also be the basis for resource competition between submerged plants and the periphytic
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algae which grow over their surfaces (Jones et al., 2000a; Jones et al., 2002). Under such
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stress conditions those plant species able to maintain the most net photosynthesis and
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growth will gain a competitive advantage over those with less net photosynthesis and
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growth. One potentially advantageous mechanism is the ability to use bicarbonate as a
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photosynthetic carbon source. This ability has been described for many species common in
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eutrophic and hard waters (Prins et al., 1982; Maberly and Spence, 1983; Raven et al.,
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1985; Spence and Maberly, 1985; Madsen and Sand-Jensen, 1991 Maberly and Madsen,
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2002) and the possession of this ability (or lack of it) has been used to explain the field
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distribution of some species (Kadono, 1982; Adams, 1985).
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Because bicarbonate utilisation involves additional energy usage to gain carbon,
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when compared with the uptake of CO2* by simple diffusion (Raven and Lucas, 1985), it
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would be beneficial for the plant to use HCO3– by active uptake only when photosynthesis
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is restricted by CO2* supply. Acclimation to such conditions would also provide a benefit
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in terms of a reduction in the photorespiratory costs involved in the use of CO2*, since
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enhanced carbon concentrations at the RUBISCO site will suppress the oxygenase activity
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of this enzyme (Raven and Lucas, 1985). Such an acclimatory capability has been reported
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previously for Elodea canadensis Michx. and Elodea nuttallii (Sand-Jensen and Gordon,
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1986; Jones et al., 1993). This ability could be crucial to the success of submerged plants in
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the changeable conditions of lentic eutrophic waters. Although the utilisation of HCO3–
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must involve some energetic cost to the plant [It is apparent that species capable of utilising
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HCO3– may incur an additional cost due to a lower affinity for CO2* (Maberly and Madsen,
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1998), which, if plastic, will be included in the energetic costs of construction and
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maintenance], given sufficient light a vast reserve of carbon will be opened up to
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exploitation by the plant and this, coupled with reduced photorespiratory costs, could ease
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the restriction on growth. The following study was undertaken to compare the growth of E.
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nuttalli plants utilising HCO3– with that of plants using CO2* only. This was achieved by
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growing plants under controlled, light saturated conditions whilst manipulating the
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proportions of the various forms of inorganic carbon available to them.
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Materials and Methods
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Growth
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Six cleaned, healthy, unbranched, 10 cm long shoots of E. nuttallii, collected from
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the Leeds and Liverpool Canal (53°29' N, 2°56' W), were planted into each of 16 covered,
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plastic beakers, filled with nutrient rich sediment collected from the same site, and each
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grown in glass jar containing 2 l of filtered water collected from the same site (2.4 mM
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DIC, further details of the carbonate system given in Howard et al. (1984) and Jones et al.
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(1996)), for 21 days at 20 ± 1 °C, illuminated with 150 µmol m-2 s-1 PAR in a 16 hr
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photoperiod. Each of the jars was bubbled with either untreated air or air that had passed
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through soda lime to reduce the CO2 content, with the treatments arranged in a 4 x 4 Latin
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square, giving a total of eight replicate jars per treatment.
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At the start of the experiment, the ability to utilise bicarbonate of eight, randomly
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selected plants from the stock collection was assessed by measuring photosynthesis at pH
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6.5 and pH 9 as below. A further twenty shoots were cut to 10 cm length and used to
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measure the mean length of the first 10 discernable internodes, blotted wet weight and,
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after drying to constant mass at 60 °C, dry weight.
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At intervals during the growing period, the pH of the water within all the jars was
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measured (CD66, WPA Scientific Instruments, Saffron Walden, Essex). At the same time
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approximately 100 ml of water was removed using a narrow-necked, screw-top bottle from
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three randomly selected jars per treatment, and the concentrations of CO2* calculated from
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pH and total DIC calculated after dual Gran titration (Mackereth et al., 1978). To
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compensate for this removal, and for evaporation, the jars were regularly topped up with
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canal water. On day three a small quantity of 0.1 M HCl was added to the jars bubbled with
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unadjusted air in order to bring the water to pH 7.7, which resulted in an increase in the
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concentration of CO2* and reduction in the total DIC.
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After 21 days one plant was removed from each replicate jar and its ability to utilise
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bicarbonate assessed as below. The following day the remaining five plants in each jar were
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harvested. Each plant was carefully brushed clean of any periphyton with a soft paint brush
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and the following variables measured: length of the main shoot, total length of the main
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shoot and all the side branches, number of side branches, mean length of the first 10
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discernable internodes, blotted wet weight and, after drying to constant mass at 60 °C, dry
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weight. Results were analysed with a nested, mixed model design using SAS.
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Physiological determinations
The extent to which the plants were capable of utilising bicarbonate was determined
by measuring the rate of oxygen evolution at 20 °C in media of pH 6.5 and pH 9
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successively, using a Clarke-type oxygen electrode (Hansatech, King's Lynn, UK),.
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Forsberg II medium was used (Forsberg 1965) modified by omission of Na2SiO3, buffer,
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carbon sources and modified to the appropriate pH (unbuffered-pH adjusted to be pH 6.5
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and pH 9 after the addition of NaHCO3). Photosynthesis was initiated by the injection of
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0.1 ml of NaHCO3 solution into the reaction chamber to give a final concentration of
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2.4mM. Bicarbonate utilisation was expressed as the ratio of photosynthesis in these two
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media (9.0:6.5). At pH 6.5, CO2* is plentiful, 980 µM, and any limited change in pH
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produces little change in photosynthetic rate. At pH 9, CO2* is only 5 µM, and the plant
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can therefore only carry out significant photosynthesis if it can utilise HCO3–. Use of the
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ratio removes effects due to variations in absolute rates between plants and gives an
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estimate of the ability to utilise bicarbonate as a photosynthetic source.
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Determinations were made on one complete whorl of three leaves, excised from the
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stem 3cm from the shoot apex then gently brushed to remove periphyton. These separate
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leaves were placed so that they were freely moving in the electrode reaction chamber
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containing 1.5 cm3 of modified Forsberg medium. The chamber was illuminated with 290
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µmol m-2 s-1 incident light, or darkened with close fitting black plastic for respiration. In
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each case photosynthesis was initiated by the injection of 0.1 cm3 of NaHCO3 into the
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reaction chamber to give a final concentration of 2.4 mM DIC. To reduce the effects of
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photorespiration on photosynthesis, all photosynthetic rates were determined within 1 mg
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O2 l-1 amplitude change, in solutions containing 9 mg O2 l-1 (100% saturation), being
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sparged with N2 and then O2 gas to achieve this concentration before measurements began.
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This process also removed any dissolved CO2 from the medium, such that the added
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NaHCO3 was the only carbon source available. After the two determinations were made,
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the length of the leaves was measured. Rates of oxygen evolution and uptake were
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standardised to the chlorophyll content of the leaves, determined by actone extraction
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(Arnon, 1949).
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Results
By bubbling the jars with untreated air, or air that had passed through self-
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indicating soda lime, two concentrations of CO2* were produced (Table 1), namely full
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CO2* and reduced CO2*. The pH was higher and the concentration of CO2* lower, in the
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reduced CO2* treatment at all times (Table 1). This had a marked effect on the physiology
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of the plants, with almost all those under reduced CO2* capable of HCO3– utilisation after
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21 days (Table 2). However, the plant sampled from one reduced CO2* jar did not appear
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to be using HCO3– to any great extent (photosynthesis HCO3–/CO2 = 0.076), perhaps due
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to inadequate aeration. As the object of this study was to determine the differences between
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plants utilising HCO3– and those relying on CO2 alone, this replicate was removed from all
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subsequent calculations, with the statistical tests adjusted accordingly. This did not affect
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the outcome of any of the statistical tests. The plants which had been subjected to the
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higher CO2* concentration showed a significant decrease in HCO3– utilisation, when
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compared with the starting population (p < 0.05, Table 2). After 21 days of incubation, the
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full CO2* plants were unable to use HCO3– as a carbon source for photosynthesis, whereas
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the reduced CO2* plants were able to use HCO3– in photosynthesis at a rate equivalent to
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about one third of that when CO2* was plentiful (Table 2).
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Together with the changes in HCO3– utilisation, other changes occurred during the
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growth period. Both sets of plants showed a significant reduction in the length of internodes
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and in the total chlorophyll content of the leaves, probably through a reduction in leaf size,
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during the incubation period (Table 2). The reduction in internode length led to an increase
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in dry weight per unit length of stem, as the leaves became more densely packed on the
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stem. This, together with an increase in the number of side shoots, gave the plants a short
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bushy appearance when compared with the starting population.
There was also an increase in the rate of photosynthesis at pH 6.5 in the plants from
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the reduced CO2* treatment, i.e. in the plants utilising HCO3– (Table 2), but not in the
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plants from the full CO2* treatment.
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However, the most notable result was that no significant differences were found
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between the plants from the two experimental treatments for any of the growth variables
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measured, namely length of main shoot, total length, number of branches, internode length,
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leaf length, wet weight and dry weight (Table 2).
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Discussion
Large changes in the physiology of the plants grown under low CO2* were evident.
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The most important of the changes was the extent to which the plants could utilise HCO3–
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as a carbon source for photosynthesis. At the end of the incubation period, the plants under
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the reduced CO2* treatment were using HCO3– extensively, whilst those under the full
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CO2* treatment had lost any ability which they had previously. The plants utilising HCO3–
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also showed an increased chlorophyll specific rate of photosynthesis when utilizing CO2*.
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It is possible that this increased capacity for CO2* use may result from changes in
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RUBISCO as has been shown in other aquatic plants including E. canadensis (Madsen et
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al., 1996). Together with variations between waterbodies in the ability of individual species
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to use HCO3– (Maberly and Spence, 1983; Sand-Jensen and Gordon, 1986; Madsen and
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Sand-Jensen, 1987), seasonal changes within a waterbody have been reported, with plants
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collected from the field having a tendency to show increased affinity in summer (Maberly
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and Spence, 1983; Sand-Jensen and Gordon, 1986). This may be the result of periods when
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productivity and temperature are high and CO2* correspondingly low. These conditions are
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often associated with periods of low water movement and high light intensity, which
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accentuate the problem of carbon supply. However, it has been noted that HCO3– uptake is
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unlikely to occur under conditions of light limitation (Sand-Jensen and Gordon, 1986;
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Madsen and Maberly, 1991), and as low light conditions are more likely to predominate in
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winter, the affinity for HCO3– may be reduced at this time (Sand-Jensen and Gordon,
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1986).
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Here the E. nuttallii plants responded clearly to the reduced availability of CO2* by
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increasing their capacity to utilise HCO3–. At the same time it was noted that there was a
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reduction in the total chlorophyll content of the leaves, leaf length and internode length of
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plants from both experimental treatments during culture compared to field collected
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material. These changes, together with the production of side shoots, are associated with
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increased light availability (Spence and Dale, 1978; Pizarro and Montecino, 1992; Maberly,
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1993; Janes et al., 1996), indicating that the plants were not light limited. The fact that the
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plants from the reduced CO2* treatment switched to HCO3– use would support this
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suggestion.
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Since the physiology of the plants from the two treatments was markedly different,
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it was surprising that no significant differences were found between the growth of the two
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sets of plants. The similarities in growth are remarkable considering that CO2* was only
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available to the full CO2* plants at concentrations that are low relative to saturation of
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photosynthesis (40 µM cf. 1.2 mM; Jones et al., 2000b) and would have been severely
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restricting to photosynthesis, whilst the concentration of HCO3– was 30 fold higher. If we
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accept that the switch to HCO3– utilisation does not occur under conditions of light
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limitation (Sand-Jensen and Gordon, 1986; Madsen and Maberly, 1991), and that reduced
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internode length (Maberly, 1993; Janes et al., 1996) and a decline in chlorophyll
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concentration without a decline in activity (Pizarro and Montecino, 1992; Barko and Filbin,
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1983) indicates an increase in the availability of incident light, then light limitation of
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growth can be ruled out. There are, therefore, only two possible explanations for the lack of
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difference in growth between the two sets of plants. Either, even with increased
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photosynthesis at high pH, the overall cost involved in the utilisation of HCO3– is large, or
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the growth of the plants was limited by some factor other than carbon.
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Since rooted macrophytes take up the majority of their nutrients via their roots
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(Carignan and Kalff, 1980; Barko et al., 1991), and the experimental plants were provided
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with sediment from a site where E. nuttallii grows plentifully into which they rooted well, it
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is very unlikely that they experienced nutrient limitation.
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If limited access to carbon as a photosynthetic substrate was the major constraint on
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growth, then the lack of difference in the growth between the plants must be an indication
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of the metabolic cost involved in the construction, maintenance and running of the polar-
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leaf mechanism in E. nuttallii. These costs are even greater if the loss to photorespiration in
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the full air plants, caused by the low CO2:O2, is taken into account (Raven and Lucas,
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1985). There are two lines of evidence indicating that HCO3– utilisation is metabolically
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costly for aquatic macrophytes.
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i)
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of submerged macrophytes taking up HCO3– is far below that when relying solely on CO2*,
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typically about 50% for species possessing a polar-leaf mechanism (Maberly and Spence,
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1983; Raven and Lucas, 1985; Spence and Maberly, 1985; Sand-Jensen and Gordon, 1986;
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Madsen and Sand-Jensen, 1987, 1991) [This may not be true of microphytes (Maberly and
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Spence, 1983) which apparently use far more energetically efficient mechanisms to
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accumulate dissolved carbon (Raven, 1970; Olofsson, 1980; Lucas, 1983; Shapiro, 1990)].
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ii)
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to HCO3– use (Sand-Jensen and Gordon, 1986; Adamec and Ondok, 1992; Jones et al.,
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1993). In teleological terms, why should a plant wait until HCO3– concentrations are so
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many times greater than CO2* before tapping it as a resource, when photosynthesis is quite
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clearly restricted at such low CO2* concentrations.
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When supplied with saturating DIC and light, the maximum rate of photosynthesis
The very low concentration of CO2* required before a plant will switch from CO2*
If we assume that when utilising HCO3–, carbon is being fixed at the maximum rate,
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with photorespiration reduced to a minimum (Raven and Lucas, 1985), then any energetic
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cost due to the polar-leaf mechanism can be estimated as equivalent to a reduction in
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photosynthesis from this maximum value (assuming the costs of growth, other than those
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involved in HCO3– use, are equal). As the growth of the plants utilising HCO3– was
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equivalent to those using CO2* only, we can assume that the net gains from photosynthesis
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are equivalent. The cost of constructing, maintaining and running the bicarbonate utilisation
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mechanism can, therefore, be estimated as the difference between the maximum rate of
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carbon fixation of the plants utilising HCO3– (without photorespiration) and the rate of
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carbon fixation of the plants using CO2* only.
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Photosynthesis of E. nuttallii collected from the same site in the previous year
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without photorespiration (PmaxFree) was 41 % greater than that at equilibrium with air (See
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Fig. 1. Measured under comparable conditions of 2.4 mM DIC and pH 6.5, Jones et al.,
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1
2000b). Using the mean value of photosynthesis at pH 6.5 and ambient O2 for the full CO2*
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plants (762 µmol O2 g-1 chl min-1, Table 2), PmaxFree can be estimated as 1076 µmol O2 g-1
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chl min-1. An estimate of the rate of photosynthesis of E. nuttallii (collected from the same
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site in the previous year) at the CO2* concentration found in the full CO2* treatment (40
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µM) and ambient concentration of O2 (Pair), is given as 148 µmol O2 g-1 chl min-1 by Jones
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et al. (2000b). The overall cost of the polar-leaf mechanism in terms of reduction of
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photosynthesis is therefore,
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Overall Cost = PmaxFree – Pair = 928 µmol O2 g-1 chl min-1
Using a photosynthetic quotient (O2 released to CO2* fixed) of 1.18, as found in other
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members of the Hydrocharitaceae (Pokorny et al., 1989), and assuming 1 mole of CO2*
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fixed to carbohydrate equates to 25 moles of photons (Raven and Lucas, 1985):
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= 328 µmol photons g-1 chl s-1
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Since chlorophyll was found to be present in the leaves of E. nuttallii plants collected from
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the Leeds and Liverpool Canal at a concentration of 21.18 µg cm-2 (Jones, 1994), this can
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be converted to incident light,
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= 69 µmol photons m-2 s-1
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This value, an irradiance commonly experienced by submerged macrophytes in the field,
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can be used to estimate the value below which HCO3– use would not be expected to occur.
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The compensation point of E. nuttallii and related species has been recorded as between 3
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and 15 µmol photons m-2 s-1 (Simpson et al. 1980; Birch 1990; Sand-Jensen & Madsen
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1991). As HCO3– use at irradiances below 69 µmol photons m-2 s-1 will be energetically
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inefficient, plants will be restricted solely to the use of CO2* at these low irradiances.
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Hence, the compensation point for plants using HCO3– will include this additional cost and
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occur at an irradiance 69 µmol photons m-2 s-1 higher, i.e. 72 to 84 µmol photons m-2 s-1.
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Bicarbonate use would not be expected to occur in plants experiencing an irradiance lower
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than this, though it is possible that the return switch to use of CO2* may not occur under the
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same conditions.
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This method of calculating the costs of this mechanism does, obviously, include the
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errors involved in comparing populations of plants taken at different times, and relies on
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several large assumptions. The HCO3– use by the polar-leaf mechanism works through
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external conversion to CO2* (Prins et al., 1982), hence the rate of CO2* production, and
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photosynthesis, will depend on the capacity of the water to buffer pH changes at the abaxial
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leaf surface. This effect will also incur a cost that is changeable, dependent on the
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composition of the water and its pH (Prins et al., 1982; Prins and Zanstra, 1985). Also, as
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there is no active uptake of carbon in darkness, there will be no benefit of HCO3– use
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during darkness. However, the costs of production (a once only cost) and maintenance of
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the apparatus required for HCO3– utilisation (proteins, plasmalemma, cell structures, etc.)
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will be incurred whether the apparatus is being used or not, i.e. both in the dark and the
14
light, and it might be appropriate to take into account the proportion of time spent in
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darkness each day (8/24) when determining the cost. Nevertheless, the running costs of
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HCO3– utilisation will be incurred only whilst the mechanism is operating, i.e. in the light.
17
The polar leaf mechanism is only switched on during light (Prins and Zanstra, 1985), and it
18
is assumed here that the energy required to pump H+ across the plasmalemma of the cells,
19
sufficient to maintain a pH gradient across the leaf blade of circa 6 pH units (Jones, 1994),
20
is far in excess of that required to produce the structures required to create this pH gradient.
21
Hence, no adjustments have been made to take into account any imbalance between cost
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(potentially light and dark) and benefit (light).
23
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Nevertheless, the method used does give a realistic value for the overall cost of the
mechanism of 69 µmol photons m-2 s-1, which is testable by laboratory growth experiments.
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1
The most important conclusion to be drawn from this study is that HCO3– use in
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E.nuttallii is costly and only used under conditions of severe CO2* limitation, where
3
photosynthesis would otherwise be virtually arrested. Furthermore, there is an obvious pay-
4
off between light and carbon, with HCO3– use confined to areas where sufficient light is
5
available. It is not an easy way to success, with the plant gaining ready access to plentiful
6
supplies of carbon, but a method by which it can persist where other species are excluded.
7
8
Acknowledgements
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This work was funded by a Natural Environment Research Council postgraduate
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studentship, for which I am grateful. I also thank my supervisors Dr. J.W. Eaton and Dr. K.
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Hardwick, and Prof. B. Moss for helpful comments on the manuscript.
12
13
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