Limnol. Oceanogr., 31(6), 1986, 1322-1334 0 1986, by the American Society of Limnology and Oceanography, Inc.

Comparison of methods for measuring production by the submersed macrophyte, Potamogeton perfoliatus L. l p2

W. Michael Kemp, Marlon R. Lewis, 3 and Thomas W. Jones4 University of Maryland, Horn Point Environmental Laboratories, P.O. Box 775, Cambridge 2 16 13

Abstract

Six conventional methods for measuring primary production of submersed vascular plants were compared to test for previously suggested inherent shortcomings in the O,-exchange techniques. Production was measured for experimental populations of Potamogeton perfoliatus L., from upper Chesapeake Bay in five comparative studies, each including an O,-exchange method. All techniques tested (14C incorporation and 0, evolution for both bottle incubations and undisturbed populations; biomass accumulation and inorganic carbon consumption for intact populations) compared fa- vorably, with mean (molar) ratios of oxygen to carbon production ranging from 0.9 to 1.6. In addition, direct measurements indicated that rhizosphere release of 0, would introduce only small errors (< 5%) in 0, production estimates. Although changes in lacuna1 storage of 0, can result in brief (5-25 min) delays between 0, production and evolution to the external water, simple meth- odological precautions can overcome such problems. We conclude that all available methods for measuring productivity of this and related species are potentially useful, each having its particular strengths and weaknesses, and the use of more than one method is recommended.

Submersed vascular plants are a conspic- uous feature of many aquatic ecosystems. Organic production by submersed angio- sperms often dominates the annual carbon budgets of lakes and estuaries (Sondergaard and Sand-Jensen 1978; Thayer et al. 1975). Thus, dependable measurement of primary production for these plants is of fundamen- tal importance in the study of shallow-water ecosystems. Various techniques have been applied over the past several decades for measuring production of these macrophytes (see Wetzel 1964, 1974).

One of the earliest methods involved di- rect harvest of plant biomass (Rickett 1922; Westlake 1965), an approach later modified with measurements for elongation of marked leaves (e.g. Zieman and Wetzel 1980) and analysis of population age-structure to cor- rect for mortality (Mathews and Westlake 1969; Carpenter 1980). Observations on the exchange of metabolic gases (CO, and, es- pecially, 0,) between macrophytes and the surrounding medium have also been used

I Research supported in part by the U.S. EPA grants R805932010 and X003248010.

2 University of Maryland Center for Environmental and Estuarine Studies Contribution 1678.

3 Present address: Dept. Oceanography, Dalhousie University, Halifax, Nova Scotia, B3H 451.

4 Present address: Biology Dept., Salisbury State Col- lege, Salisbury, Maryland 2 180 1.

for plants in bottles or under bell jars (e.g. Schemer 1934; Odum 1956). The intro- duction of 14C isotopes for measuring phy- toplankton photosynthesis led to subse- quent development of 14C methodologies for submersed macrophytes (Wetzel 1964).

In their review and critique of various macrophyte production methods, Zieman and Wetzel (1980) suggested that, with the exception of leaf-marking techniques, all available methods are equivocal and should be used only with caution. In particular, they emphasized the shortcomings of oxygen (0,) techniques, which have been in disfavor since Wetzel’s (1964) earlier review. Zie- man and Wetzel (1980, p. 110) concluded that, “[aIdequate evidence exists on both physiological and environmental bases that the oxygen light and dark enclosure method should bc abandoned as applied to sub- mersed angiosperms. ” Results of several re- cent studies indicate that these caveats may, however, deserve reconsideration (e.g. Westlake 1978; Capone et al. 1979; Kelly et al. 198 1; Sand-Jensen et al. 1982; Lin- dcboom and DeBree 1982).

Several specific problems with O2 meth- ods have been cited, including 0, storage in plant lacuna1 systems, recycling of O2 through coupled photosynthesis and respi- ration internally within the plant, and O2 transport through the lacuna1 system (from leaves to roots) and release from roots to

1322

Macrophyte production 1323

the external rhizosphere. Some evidence of these processes has been provided for se- lected plant species (e.g. Hartman and Brown 1967; Oremland and Taylor 1977), al- though quantitative data had been generally lacking until recently (Westlake 1978; Kelly et al. 198 1; Sand-Jensen et al. 1982). A few direct comparisons of rates obtained with different methods are available; however, results often have been conflicting, and studies have been limited generally to con- temporaneous measurements with two techniques only (Wetzel 1964; Bittaker and Iverson 1976; Capone et al. 1979; Titus et al. 1979; Lindcboom and DeBree 1982).

Here we compare six methods for mea- suring primary production in submersed macrophytes to test for possible inherent shortcomings in the oxygen-exchange tech- niques. Methods investigated include 0, evolution and 14C incorporation for indi- vidual plants incubated in bottles, and 0, evolution, 14C incorporation, total inorgan- ic carbon uptake, and biomass accumula- tion for undisturbed populations. We did five comparative studies using Potamoge- ton perfoliatus L., an historically abundant species in upper (0-157~ salinity) Chesa- peake Bay (Kemp et al. 1984). Each study involved at least one O2 method, to provide a point of reference for interrelating all re- sults. We also obtained quantitative esti- mates of internal storage, transport, and release (to the rhizosphere) of photosyn- thetically produced O2 for this species. We are indebted to our colleagues and students including W. Boynton, J. Cunningham, W. Goldsborough, L. Murray, C. Peters, K. Staver, C. Stevenson, and R. Twilley. Other individuals provided comments in reviews of earlier versions of this paper: D. Patri- quin, P. Penhale, M. Kelly, K. Mann, D. Westlake, R. L. Wetzel, J. Zieman, and an anonymous reviewer.

Methods Experimental systems -Experiments

were conducted between July and October of 1978-1980 with individual plants or whole populations of P. perfoliatus grown in 50- and 700-liter glass aquaria under con- trolled temperature and light conditions. The larger aquaria, used for time-course studies

where substantial removal of plants was re- quired, were 1.8 m long, 0.6 m wide, and 0.8 m high. The smaller aquaria, used for measurements on whole intact macrophyte communities, were 0.6 x 0.3 x 0.4 m. These systems are described in more detail else- where (Cunningham et al. 1984; Kemp et al. 1985).

For b,oth types of aquaria environmental conditions were identical. Plants and sedi- ments (76% sand, 24% silt-clay, 1% organ- its) were collected in June and July by hand from a Chesapeake Bay tributary site having a mean depth of 0.9 m and typically ca. 90% attenuation of irradiance over the water col- umn depth (Kemp et al. 1984). Plants were handled with special care to minimize dam- age during collection and transport. Exper- imental populations were established with healthy appearing plants in aquaria sedi- ments (depth, ca. 15 cm) at least 30 d before comparative studies. Potential macrophyte herbivores (e.g. grass shrimp) were excluded from these aquaria. Salinities in experimen- tal systems were similar to those at the source areas (9-l 1%0), and aquarium temperatures were kept at 20°-23°C. Light was provided by “cool-white” fluorescent bulbs on a 14-h photoperiod, producing 120-l 40 PEinst me2 s-l photosynthetically active radiation (PAR) at the water surface. Aquarium water was continually recirculated by submersible pumps with outlet diffuser systems.

Bottle incubations: O2 production and 14C incorporation -Productivity experiments comparing 14C incorporation and 0, pro- duction were conducted on several occa- sions using five replicate individual P. per- foliatus plants with intact stem plus leaves (20-30 cm) and a rhizome segment (6-8 cm) plus associated roots (experiment A). These plants were gently removed by hand from sediments, cleaned of attached epiphytes and sediment (by scraping with a fingernail in a stream of water), and placed into transpar- ent BOD bottles. The bottles were filled with filtered (GF/C, 1.2 pm) water from the aquarium, and five bottles without plants were also filled and treated identically as controls. To each bottle 2 PCi H14C03- was added, and duplicate samples of the labeled substrate were taken for determination of initial activity. Initial oxygen concentra-

1324 Kemp et al.

tions were measured in all bottles by po- larographic O2 electrodes air-calibrated at the beginning and end of each incubation. On each of six dates, stoppered bottles were suspended at plant height in front of the diffuser outlet to provide gentle continuous mixing, and each bottle was shaken every 10-l 5 min to further reduce the effects of boundary-layer stratifications (Westlake 1967). No bubble formation was observed, and final O2 concentrations never exceeded 125% saturation.

After l-4 h of incubation, oxygen con- centrations were determined in all bottles to obtain “net apparent O2 production” (daytime net O2 evolution), and plants were removed, fumed with HCl, and dried at 70°C until constant mass was achieved (ca. 48 h). Contents of the bottles were filtered, and filters were fumed with HCl, dried as above, and placed directly into scintillation vials containing Aquasol (New England Nucle- ar). To measure organic excretion, we acid- ified (pH = 1.5) a 1 O-ml subsample of fil- trate, bubbled it with N2 for 30 min, and transferred 1 .O ml of this dilution to Aqua- sol for subsequent counting. Dried plant material was ground to a fine powder, sol- ubilized in nitric acid (Lewis et al. 1982), and the digest was placed directly into buff- ered Aquasol. This technique for recovery of 14C incorporated by aquatic macrophytes compares well (&2%) with combustion methods (Lewis et al. 1982). All samples were counted in a Packard Tri-Carb (model 460C) with quench correction by the exter- nal standard ratios method.

To extend the range of production rates, we compared O2 and 14C methods (experi- ment B) for individual P. perfoliatus shoots incubated in BOD bottles with filtered water containing 10 and 100 pg liter-’ of a pho- tosynthetic inhibitor (2-chloro-4-ethylami- no-6-isopropylamino-s-triazine). Five rep- licates were exposed to each of the two treatments, and five replicates receiving only the carrier, methanol, served as controls. Production of O2 and incorporation of H14C03- were measured as described above.

Intact populations: Biomass accumula- tion - Accumulation of “shoot” (stem plus leaf) biomass was estimated for plant pop- ulations in the larger aquaria by counting

and measuring lengths of each shoot in the experimental system at weekly intervals over a 6-week period (experiment C). Each week, 5-l 0 individual shoots were removed at random (~2% of the total population), cleaned of epiphytes and sediments, mea- sured, and dried to constant mass at 70°C (ca. 48 h). Significant regressions of indi- vidual shoot length vs. dry mass (r2 = 0.76, P < 0.01) were used to calculate total bio- mass from the length measurements (Cun- ningham et al. 1984). Initial biomass values for “roots” (including rhizomes) of P. per- foliatus planted in the aquarium were esti- mated from measurements of rhizome lengths before insertion of plants into sed- iments and length-mass regressions (r2 = 0.87) for a subsample of 25 rhizome-root sections. At the end of this experiment all root and shoot material for P. perfoliatus in the aquarium was harvested, sorted, cleaned, and dried to constant mass. Root biomass was estimated for the four intermediate dates by linear interpolation. The direct harvest measurement of total biomass was similar (+6%) to that calculated from the final length-mass regressions. Rates of biomass accumulation here were compared to diel net O2 production as estimated once per week from incubations of five replicate plants in clear and opaque bottles, where dark respiration (integrated over the noc- turnal hours) was subtracted from net ap- parent daytime production (integrated over the light hours).

Intact populations: Production and res- piration-Areal rates of production (P) and respiration (R) for whole aquarium com- munities were measured as temporal changes (2-h intervals) in concentration (c) of 0, or total dissolved inorganic carbon (DIC) in experimental water columns of depth z (ex- periment D). The time (t) rate of change in concentration can be described as (dC/dt)z = k[(C,-C)IC,] + (P - R). Corrections for O2 exchange across the air-water interface were made by applying a gas-transfer coefficient (Copeland and Duffer 1964), k = 0.15 g O2 rnw2 h-l per atmosphere (equivalent piston velocity = 0.5 x 1O-3 cm s-l: Johnson et al. 1979). This coefficient was measured di- rectly by observing the rate of O2 diffusion (in the dark) into aquaria containing filtered

Macrophyte production 1325

(GF/F, 0.45 pm) water (but no plant or sed- iments) after removal of 0, by bubbling with N2 gas (Kemp et al. 1985). Corrections for respiration effects (< 10%) were made with parallel incubations starting at O2 satura- tion. Diffusion of CO2 across the air-water interface was estimated with the approaches of Odum (1956) and Hornberger and Kelly (1975). Rates of 0, and CO, diffusion were always < 10% and < 2% of total O2 and DIC concentration changes. Dissolved oxygen concentrations were measured polarograph- ically as described above, and concentra- tions of dissolved inorganic carbon were measured using infrared gas analysis (Titus et al. 1979).

Production of intact P. perfoliatus pop- ulations was also measured in the smaller (50 liter) experimental systems (triplicate) by adding 0.3 mCi H14C03- to aquarium water columns (experiment E). Rates of O2 production in the open water, in light and dark BOD bottles containing whole water, and in clear and opaque acrylic chambers (63 cm2, 600 ml) randomly placed over un- vegetated sediment surface were deter- mined at the same time. At the end of a 6-h incubation, plants were harvested. Repre- sentative leaf, stem, root, and rhizome por- tions of P. perfoliatus were selected from the total plant material collected (8-14 g DW), and this was treated as described above. Ra- dioactivity associated with epiphytic ma- terial was determined after its removal from host plant leaves (Penhale 1977), and that associated with periphyton growth on aquarium walls was estimated after scraping randomly selected areas (ca. 25 cm2) of walls with a razor blade. Both epiphytes and wall periphyton were collected on filters and put in buffered Aquasol for scintillation count- ing. Community O2 production was moni- tored at 2-h intervals throughout the incu- bation. Macrophyte (plus epiphyte) O2 production was estimated by subtracting measurements of planktonic and benthic contributions from the total community rate as above. Dark respiration of these com- munities was measured as O2 decreases in the water during the night before this ex- periment. “Net apparent production” is de- fined here as O2 evolution in excess of res- piration (P - R) during the day, and “gross

apparent production” is operationally de- fined as the sum of hourly rates of net ap- parent production plus dark respiration. . O2 transport and release to rhizosphere- Oxygen transport through I? perfoliatus la- cunae (from leaves to roots and subsequent release from roots to the external rhizo- sphere) was measured with a split com- partment acrylic chamber (experiment F) similar to those described by Iizumi et al. (1980) and Sand-Jensen et al. (1982). For each experiment four plants (20-50-cm shoot, 5-lo-cm rhizome) were collected in August from experimental ponds (Twilley et al. 1985) and held overnight in aerated estuarine water. Plants, which were care- fully removed from sediments so as not to damage root systems, were washed clean of loosely attached epiphytes and sediments and inserted into the “root chamber” in split serum stoppers through holes in the lid. Broken ends of rhizomes were sealed with vacuum grease, as were ports connecting root and shoot chambers. A stirred polarograph- ic O2 electrode was inserted through another port in the chamber’s lid. The root chamber was filled with filtered water (GF/F, 0.45 pm) in which 0, concentrations had been previously reduced to ca. 1 O-l 5% satura- tion by bubbling with N2 gas.

The entire root chamber (with plants and 0, electrode) was placed in the “shoot chamber,” whose diameter allowed 2 cm of clearance on all sides, on a pedestal over a magnetic stir-bar, and filtered water (GF/F, 0.45 pm) in the shoot chamber was contin- uously circulated during the incubation. O2 was measured at intervals of 15-60 min with an electrode inserted into the top of the shoot chamber. The root chamber water was left unstirred except briefly (ca. 1 min) during O2 measurements. Irradiance (PAR) of 150 PEinst m-2 s-’ was provided by “cool-white” fluorescent lamps, and temperature was kept at 20°-23°C.

Results and discussion Method comparisons: Bottle incuba-

tions- Rates of photosynthesis in the bottle incubations (experiment A) ranged from ca. 3 1 to 61 hmol g DW-’ h-l for the 14C and 57-73 for the O2 methods (Table 1). The molar ratio of net apparent 0, evolution to

1326 Kemp et al.

Table 1. Comparison of 14C incorporation and O2 production (both in pmol g DW-’ h-l) by individual shoots of Potamogeton perfoliatus incubated in bottles (experiment A, R + s, n = 5). (ND-Not detectable.)

‘T flux

Day* Part. incorporation DOC production Total Apparent 0, productiont Molar ratio*

63 57.7+ 12.9 0.17~0.10 57.9k13.1 67.1+ 14.2 1.16 77 54.1 k8.5 O.lOkO.35 54.2k9.2 65.4k8.7 1.21 91 42.3+ 17.9 ND 42.3+ 17.9 69.2+ 17.2 1.64

120 30.8k9.3 ND 30.8k9.3 73.Ozk29.6 2.37 134 31.0+11.1 ND 31.0+ 11.1 56.9k20.2 1.83 163 61.2+ 12.3 ND 61.2k12.3 66.4k20.2 1.08

* This refers to the time (d) since the initial establishment of the experimental microcosm community. f Net apparent 0, production is the net O2 evolution in excess of daytime respiratory consumption. :[ The ratio (0, : ‘“C) of net apparent O2 production to total ‘“C incorporation.

14C incorporation (0 2 : 14C) increased from 1.16 to 1.64 over the first four dates, with a decrease from 1.83 to 1.08 over the last two. The ratio for the fourth date (2.37) was exceptionally high, perhaps reflecting the relatively large variance for O2 measure- ments on that occasion (Table 1). The mean ratio (0, : 14C) in this study was 1.55 for all dates and 1.38 excluding the fourth exper- iment; these ratios are slightly higher than typical values suggested for long term mean photosynthetic quotients (e.g. Westlake 1965; McRoy and McMillan 1977). In part, the higher values of (0, : 14C) may indicate that these plants were deriving their N from N03- rather than NH,+ (Westlake 1965), since relatively high concentrations of N03- (2-l 0 PM) were occasionally available in these microcosm waters (Cunningham et al. 1984).

These differences in rates are generally comparable with those reported for bottle incubations with other submersed plants. McCracken et al. (1975) provided both O2 (net) and 14C data for Myriophyllum spi- catum production in bottles. Although these measurements were not contemporaneous, plants were taken from the same popula- tion, and incubations were conducted on days with similar insolation. Assuming that the observations are comparable, we cal- culated ratios (0, : 14C) of 1.4-l .7, similar to those in Table 1. Moeslund et al. (198 1) reported direct bottle measurements of both 0, and DIC exchange for seven species of submersed plants, with ratios of gross ap- parent O2 production to DIC consumption ranging from 0.33 to 1.73. Unusually low values of (0,: 14C) (0.30-0.35) from bottle

incubations with leaves of the seagrasses Cymodocea sp. and Posidonia sp. (Drew 1978) may be attributed to anomalously high photorespiration at elevated O2 concentra- tions (Downton et al. 1976). Titus et al. (1979) reported 14C rates averaging 7% higher than direct measurements of DIC de- creases for in vitro incubations of M. spi- catum shoot sections.

The photosynthetic inhibitor used here (experiment B) has been shown elsewhere to reduce production significantly in P. per- foliatus at concentrations 2 100 pg liter-l (Cunningham et al. 1984; Kemp et al. 1985). The mode of action for this phytotoxic agent involves blockage of the Hill reaction on the reducing side of Photosystem II (Souza- Machado et al. 1978). Thus, both carbox- ylation of CO, and splitting of H-O molec- ular bonds in water would be inhibited, and both O2 production and 14C incorporation would be affected equally. We found a very strong correlation between net apparent O2 evolution and 14C incorporation for exper- imental plants (Fig. l), with a mean molar ratio >0.9 (r = 0.96, P < 0.01).

Methods comparisons: Intact popula- tions-The 14C and O2 techniques are fun- damentally different in approach, one in- volving incorporation of labeled isotope and the other, net exchange of metabolic gas. Measurements of net exchange for O2 and DIC offer a more direct basis for compari- son. The data shown in Fig. 2 are for net apparent production and night respiration of the community rather than macrophytes alone; however, the P. perfoliatus contri- bution to total metabolism in these systems was between ca. 50 and 90% (Lewis 1980;

Macrophyte production 1327

I / /

I / 0 Control

/ 0.4 /

D lOfig liter-’ n?

I 0 IOOpg liter-’ / / I

0.3

- t 5

& / 0 (I: .h a

0. I .& Y = 0.93X+ 0.006

0” 0’ / ( I = 0.9 6) /

/ I I I I 1

0 0. I 0.2 0.3 0.4 0.5

14C UPTAKE

Fig. 1. Net apparent 0, production and 14C incor- poration (both in mmol g DW-’ h-l) by Potamogeton perfoliatus plants incubated in BOD bottles and treated with two levels of a photosynthetic inhibitor (2-chloro- 4-ethylamino-6-isopropylamino-s-triazine) plus con- trols (experiment B).

Cunningham et al. 1984). Although there was a considerable range in the ratio of O2 evolution to DIC consumption over our study, the mean value approaches unity (Fig. 2). Measurements of nighttime O2 con- sumption and DIC production were signif- icantly correlated (r = 0.79, P < 0.01) with a mean molar respiration ratio (CO2 pro- duced per O2 consumed) of 1.48.

Ratios of 0, and CO, metabolism have been reported for other intact macrophyte communities. For example, mean values for the molar ratio of gross O2 production to CO2 consumption were 0.85-0.90 for a Danish river system dominated by sub- mersed macrophytes (Kelly et al. 1983). Mean molar respiration ratios of 0.7 and 0.5 have been reported from experimental communities dominated by the macro- phytes Vallisneria sp. (Beyers 1963) and Egeria densa (Strange 1976). Community respiration ratios greater than or less than unity may partially reflect anaerobic respi- ration or chemoautotrophic production by sediment bacteria (Rich 1979).

While observations on exchange of met- abolic gases and incorporation of 14C pro- vide estimates for instantaneous rates of primary production, net accumulation of plant biomass represents a more temporally

A

.46 ’ l /

./ l ’ l h

/ ’ 3b

2.

jL 02: DIC=O.S6f 0.24

L 8 I I

/ B

/ /

/ / 30

.3b . 29

t

4’ 50 0

67 .5 ‘. / DIC:O, q 1.48f0.22

/

0 50 100 150 200

CARBON METABOLISM, mmol mm2d-’

Fig. 2. A. Net apparent 0, production and dis- solved inorganic carbon (DIC) consumption. B. Night respiration of 0, and DIC in water columns of exper- imental communities containing the submersed macrophyte, Potamogeton perfoliatus (experiment D). Mean molar ratios of O2 and DIC exchange are given (*SD); numbers beside points indicate sequence of biweekly observations.

integrated measure of plant growth. In our large (700 liter) aquaria, plant biomass in- creased over 6 weeks, with the root : shoot biomass ratio increasing from 0.4 to 0.6 and biomass-specific growth rate (difference in biomass between successive dates divided by mean biomass for the two dates) decreas- ing continually (experiment C, Fig. 3A). The overall rate of net biomass production in- creased to a maximum of 1.23 g DW me2 d-l between weeks 3 and 4 and decreased in the last 2 weeks to about half the maxi- mum rate (Fig. 3B). Over the same period, O2 production for P. perfoliatus (incubated in bottles) followed a similar pattern. How- ever, O2 production continued to increase

1328 Kemp et al.

Biomass - Specific

60

B 0 0,Evolution in Bottler

n Biomarr Accumulation I .s I

I 2 3 4 5 6

WEEK FROM START

Fig. 3. Weekly measurements for experimental populations of Potamogeton perfoliatus (experiment C). A. Biomass (g DW-’ m-2) (mean f range) of shoots (stems and leaves) and roots (plus rhizomes) and bio- mass-specific growth (mg g DW-’ d-l) (difference in biomass between the successive weeks f mean biomass for those 2 weeks). B. Net productivity (g m-2 d-l) (mean +- SE) as biomass accumulation (g DW), and 0, evolution by plants incubated in bottles (diel net pro- duction equal to net apparent 0, production integrated over light hours minus dark respiration integrated over night hours).

for a week beyond week 4, with rates de- clining only in the last week. It appears that the photosynthetic potential for these plants remained relatively high for a week after the net accumulation of biomass had begun to decrease. Presumably, natural autumnal mortality of senescing plants (week 6 was in early October) had become significant.

Values for O2 evolution in Fig. 3 represent “diel net production,” which we define as net apparent production integrated over the light period minus dark respiration inte- grated over the night period. Although we measured O2 production only once a week, the conditions of constant temperature, ir- radiance, and photoperiod in these experi- mental systems reduce the potential error of extrapolation to mean weekly rates. The ratio of carbon to dry mass was 0.32-0.3 7

Macrophyte production 1329

for P. perfoliatus (Lewis 1980; Twilley et al. 1985), slightly lower than values reported for related macrophytes (cf. Westlake 1965). Therefore, a mass ratio of O2 evolution to dry matter accumulation of 1 .O is stoichio- metrically equivalent to a molar ratio (0, production to CO,-C consumption) of l.O- 1.2 for this species (since mol C02-C per mol 0, equals 12/32 or 0.375). It is inter- esting that 0, production was consistently 70-80% of biomass accumulation during the first 3 weeks, perhaps resulting from under- estimates due to bottle-effects (e.g. Wetzel 1974). Conversely, in the final 2 weeks fol- lowing peak biomass, rates of O2 production were 30-50% higher than biomass accu- mulation, possibly reflecting elevated rates of leaf mortality and excretion of dissolved organic matter (Westlake 1965), although there were no obvious plant tissue losses. Total biomass accumulation over the 6-week experimental period was virtually identical to total integrated 0, evolution. Although several previous studies have reported both community 0, production and macrophyte biomass net accumulation (e.g. Fisher and Carpenter 1976; Thyssen 1982), no other comparisons of O2 evolution and biomass accumulation are available for macrophytes alone.

We also measured production of intact P. perfoliatus populations and associated au- totrophic communities using O2 and 14C techniques in our smaller aquaria (experi- ment E). In three replicate systems, both plankton and benthos showed net O2 con- sumption in the light, which represented a minor fraction (O-7%) of gross apparent O2 production by the macrophyte populations (Table 2). In this experiment epiphyte pro- ductivities (from 14C incorporation) were only 2-3% of the combined macrophyte- epiphyte values. Production by periphytic algae on aquarium walls was also negligible. Since it would be difficult to separate mac- rophytic and epiphytic O2 production for this experiment, and since epiphytic con- tributions were small, we calculated values of O2 production : 14C incorporation for the macrophyte-epiphyte complex. While the magnitudes of productivity varied among replicates over a factor of two, the ratio (0, :

14C) was consistent, averaging about 0.9 based on net and 1.2 on gross apparent O2 production (Table 2).

Wetzel (1964) made a similar comparison of 14C and 0, production measurements for intact submersed plant populations in a North American lake, and molar rates of 0, production were 2-4 times 14C uptake; he attributed the relatively high O2 rates to pro- duction by other autotrophs which were not measured directly. Lindeboom and DeBree ( 1982) compared net 0, evolution for intact Zostera marina plus epiphytes (similar to our method in Table 2) with 14C incorpo- ration by excised leaves (excluding macro- epiphytes) incubated in bottles (as in our Table 1). In general, they found relatively higher rates using the 0, method, with O2 : 14C ranging from 1.5 to 2.5. However, the basic difference in their two methods makes rate comparisons difficult.

Metabolic gases and macrophyte lacu- nae- The lacuna1 cavities characterizing many submersed angiosperms confer the physical advantage of providing the buoy- ancy for them to remain upright and the physiological advantage of entrapping met- abolic gases that can be reused efficiently (Sculthorpe 1967). Such lacunae may also provide a means by which O2 and CO2 can be translocated between roots and leaves (Brown 1913), and it appears that lacunae are more fully developed in macrophytes inhabiting quiescent rather than flowing waters, where O2 and CO, deficiencies are less likely to occur (Westlake 1967). Al- though the storage and transport of meta- bolic gases may be selectively advantageous to submersed macrophytes, it has been sug- gested (e.g. Zieman and Wetzel 1980) that these processes also tend to confound the measurement of photosynthesis in such plants. There are three obvious mechanisms by which macrophyte lacunae can influence the interpretation of measurements of pho- tosynthesis by gas exchange or 14C tech- niques: these involve storage, recycling, and translocation of the metabolic gases, 0, and cop

Hartman and Brown (1967) concluded from their experiments that O2 storage in lacunae precluded the use of 0, techniques

1330 Kemp et al.

for estimating photosynthesis of submersed macrophytes. These results have been often cited as a general argument against the use of 0, techniques for macrophytes (e.g. Bit- taker and Tverson 1976; Penhale 1977); however, in a contemporaneous paper Westlake (1967) had suggested that steady state 0, production was achieved for sub- mersed plants within 30 min, as long as the surrounding water was well circulated. In later detailed experiments with stirred sys- tems (Westlake 1978; Kelly et al. 198 1), lag periods of about 5-25 min were observed between initial illumination and initial evo- lution of O2 to surrounding water and be- tween incipient darkness and cessation of O2 release for several submersed angio- sperms. These phase shifts, which were greater for plants with larger lacunae (West- lake 1978), were attributed to temporary in- ternal O2 storage. Moeslund et al. (198 1) also demonstrated short lags of < 10 min for CO2 exchange between submersed plants and external water. Although such lags be- tween O2 production and evolution to the water might occur due to changes in lacuna1 storage, this effect would be taken into ac- count as transients in longer (1 h) sequential measurements of 0, (Nixon and Oviatt 1972).

None of these previous studies has ex- amined the potential internal storage of O2 in P. perfoliatus. Since this species has la- cunae and commonly inhabits waters with moderate to poor circulation (Sculthorpe 1967) 0, storage might make problems in the use of gas exchange methods for pro- ductivity measurements. A first-order esti- mate of the potential for such problems can be established with a simple calculation of the time (t,) required to saturate the lacuna1 volume with photosynthetically produced 02, as indicated in the expression t,y = KCsgWW441 f KCJ(P)I. Here, Gg t-ewe- sents the saturated concentration of a gas at standard temperature and pressure (44.6 pmol cmM3), p0, is the partial pressure of O2 in the lacunae at saturation, 4 is the frac- tion of plant volume taken up by gas-space, P,, is biomass-specific net apparent O2 pro- duction (in excess of respiration) for P. per- foliatus (250 pmol g DW-1 h-l: Twilley et al. 1985), and p is plant density (0.11 g DW

cm-3: Westlake 1965). Typical values ofp0, and 4 for aquatic macrophyte lacunae are ca. 0.2 and 0.3 (e.g. Hartman and Brown 1967; Armstrong 1978; Dacey 1981; Pen- hale and Wetzel 1983), which would give a value oft, = 6 min. A very conservative set of assumptions, with $ and p0, both equal to 0.5, still yields t, < 25 min.

This model is similar to one proposed by Kelly et al. (198 l), in which it is assumed that O2 produced photosynthetically fills the lacuna1 volume before any release of O2 to the external water. Unless the lacunae are devoid of 0, at the beginning of a produc- tion experiment, the time lag between pho- tosynthetic production and incipient evo- lution of 0, to the external water would be far less than t,. Exposure of P. perfoliatus to light before starting an O2 production ex- periment would tend to minimize this lag by partially filling the lacunae with photo- synthetic 0,.

More than 70 years ago, Brown (19 13) postulated that O2 liberated in macrophyte photosynthesis was stored in lacunae of sub- mersed plants for subsequent use in respi- ration. Zieman and Wetzel (1980) and oth- ers have suggested that this internal recycling of O2 would cause an underestimation of productivity by conventional 0, tech- niques. Errors associated with such 0, re- cycling were not evident in our data, and rates of productivity based on 14C and O2 bottle methods were similar (Table 1, Fig. 1). Recycling of O2 through respiration dur- ing the day is traditionally taken into ac- count in estimates of gross apparent pho- tosynthesis by using direct measurement of dark respiration, and this correction can be applied, regardless of whether the imme- diate source of 0, for respiration is internal or external to the plant. The assumption is that light and dark respiration are equiva- lent. Photorespiration has been shown to occur in many submersed angiosperms, al- though rates are rarely >, 10% of net pro- duction (Hough 1974; Hough and Wetzel 19 7 8; Sondergaard 197 9; Sondergaard and Wetzel 1980). At supersaturated concentra- tions of 02, photorespiration may become more significant (e.g. Sondergaard and Wetzel 1980). By adjusting container size and incubation period to the weight of plant

Macrophyte production 1331

material used, such excessively high O2 levels can be avoided in experimental sys- tems, thus reducing the error due to induced photorespiration (cf. Ssndergaard and Sand- Jensen 1979a).

The adaptive importance of O2 transport and release from roots has been well estab- lished for terrestrial plants growing in flood- ed soils (e.g. Joshi and Hollis 1977; Arm- strong 1978), as well as seagrasses (Penhale and Wetzel 1983). The ability of various submersed plants to elevate the redox po- tential of their associated sediment pore- waters has been described (Wium-Andersen and Andersen 1972; Carpenter et al. 1983). Some recent studies have reported quanti- tative rates of 0, transport to and rcleasc from roots for selected submersed plants (Sand-Jensen et al. 1982; Carpenter et al. 1983; Thursby 1984), where O2 release rates capable of supporting significant microbial oxidation of reduced ions were observed (Iizumi et al. 1980). However, in none of these studies has the rate of O2 release been compared with rates of photosynthetic O2 production of P. perfoliatus to estimate the potential magnitude of this error with O2 productivity methods.

In Fig. 4 we present results of direct mea- surements of O2 consumption and release by P. perfoliatus roots in comparison with apparent 0, production by plant shoots (ex- periment F). Less than 1% of the photosyn- thetically produced O2 was released from roots. The total rate of O2 translocation from shoots to roots, including root respiration, was < 10% of net apparent production. Pre- sumably, however, root respiration would be taken into account as O2 consumption in dark incubations. In similar experiments under a range of light conditions for P. per- foliatus shoots of various ages, root release ranged from about 0 to 15% of apparent production, with typical values for natural populations being ca. 3-7% (W. M. Kemp and L. Murray unpubl. data),

Sand-Jensen et al. (1982) reported results of comparable experiments for eight species of submersed angiosperms in which root re- lease of O2 ranged from 1 to 100% of pro- duction. In five experiments with Potamo- geton spp., root release rates were 2-4% of production. We conclude, as did Sand-Jen-

0 1.0 ‘. \ J 2 1 “*\.,

0 0.5 ‘\ ., .

(0.9mQ liter-‘) t- , , , , ,

2 4

TIME ELAPSED,h

Fig. 4. Cumulative net exchanges of O2 (amount of 0, accumulated in respective chambers, mg) between (A) Potamogeton perfoliatus shoots (P,) and external aqueous medium and (B) between roots (~5,) and ex- ternal water during daylight incubation of plants in experimental chambers with shoots and roots separat- ed (cxpcriment F). Root respiration (C) for intact plants incubated in dark (R,.), with roots and shoots separated and initial and final 0, concentrations (in parentheses) of 4.1 and 0.9 mg liter-l. All rates are specific to total biomass of shoots plus roots (g DW).

sen et al. (1982), that while O2 translocation from shoots to roots may be significant for the survival of submersed plants and may influence sediment chemistry markedly, it is of little importance in the measurement of productivity with O2 methods for mac- rophytes such as P. perfoliatus.

Transport of inorganic carbon in the op- posite direction (from the rhizosphere to sites of carboxylation in plant leaves) rep- resents another potential source of error in macrophyte productivity measurements with in situ 14C techniques. Significant sed- iment sources of unlabeled CO2 would ef- fectivcly decrease the specific activity of in- organic carbon. By enriching roots with C02, Bristow (1969) and Wium-Andersen (197 1)

1332 Kemp et al.

enhanced the growth of two species of fresh- water macrophytes, suggesting the impor- tance of root uptake of CO,. Kinetic exper- iments with the isoetid, Littoreila unijlora, demonstrated that this lacustrine species obtained most of its inorganic carbon re- quirement by root uptake from sediment sources (Sondergaard and Sand- Jensen 19793). However, other experiments with seagrasses and freshwater macrophytes (Wetzel and Penhale 1979; Penhale and Thayer 1980; Loczy et al. 198 3) reported relatively insignificant uptake of inorganic carbon by roots. In our experiments with H14C03- labeling of water column DIC for intact P. perfoliatus populations, rates of 14C incorporation and O2 production were sim- ilar (Table 2).

Implications for measurement of macro- phyte production -In laboratory aquaria, where plant mortality rates were relatively low, our nondestructive method for esti- mating temporal changes in biomass pro- vided time-series measurements of P. per- foliatus production which were 1 week out of phase with instantaneous measurements of 0, evolution in bottle incubations (Fig. 3). Other methods, more appropriate for use in natural environments, are also available for estimating net accumulation of P. per- foliatus biomass (e.g. Mathews and West- lake 1969; Carpenter 1980). Measurements of 14-labeled DIC incorporation by P. per- foliatus provided a convenient technique for estimating productivity both in vitro and in situ, allowing measurement of both macro- phyte DOC excretion and epiphytic pro- duction for intact systems (Tables 1 and 2).

Measurements of changes in O2 concen- tration in external water provided estimates of both net apparent 0, production and dark respiration for P. perfoliatus intact and in bottles. Mean molar ratios of O2 to carbon metabolism for our experiments ranged from 0.9 to 1.6, within the range of physi- ologically possible metabolic quotients. Di- rect measurements and simple calculations suggest that lacuna1 O2 storage and recy- cling, as well as root release of 0, to the rhizosphere, all represent minor sources of error in estimating P. perfoliatus production by conventional O2 techniques. Overall, we conclude that all of the available methods

for measuring productivity of this species and related submersed vascular plants are potentially useful for specific studies. Each method has its particular strengths and weaknesses, and the use of more than one method is highly recommended.

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Submitted: 6 November 1984 Accepted: 16 April 1986