Growth, biomass allocation and nutrient use efficiency in Cladium jamaicense and Typha domingensis as affected by phosphorus and oxygen availability

Aquatic Botany 70 (2001) 117–133

Growth, biomass allocation and nutrient use efficiency in Cladium jamaicense and Typha domingensis as affected by phosphorus and oxygen availability Bent Lorenzen a,∗ , Hans Brix a , Irving A. Mendelssohn b , Karen L. McKee c , Shi Li Miao d a

Department of Plant Ecology, University of Aarhus, Nordlandsvej 68, 8240 Risskov, Denmark Wetland Biogeochemistry Institute, Louisiana, State University, Baton Rouge, LA 70803, USA c US Geological Survey — National Wetlands Research Center, 700 Cajundome Blvd., Lafayette, LA 70506, USA d South Florida Water Management District, P.O. Box 24680, 3301 Gun Club Road, West Palm Beach, FL 33416-4680, USA b

Received 12 May 2000; received in revised form 3 November 2000; accepted 2 January 2001

Abstract The effects of phosphorus (P) and oxygen availability on growth, biomass allocation and nutrient use efficiency in Cladium jamaicense Crantz and Typha domingensis Pers. were studied in a growth facility equipped with steady-state hydroponic rhizotrons. The treatments included four P concentrations (10, 40, 80 and 500 ␮g l−1 ) and two oxygen concentration (8.0 and <0.5 mg O2 l−1 ) in the culture solutions. In Cladium, no clear relationship was found between P availability and growth rate (19–37 mg g−1 d−1 ), the above to below ground biomass ratio (A/B) (mean = 4.6), or nitrogen use efficiency (NUE) (mean = 72 g dry weight g−1 N). However, the ratio between root supported tissue (leaves, rhizomes and ramets) and root biomass (S/R) (5.6–8) increased with P availability. In contrast, the growth rate (48–89 mg g−1 d−1 ) and the biomass ratios A/B (2.4–6.1) and S/R (5.4–10.3) of Typha increased with P availability, while NUE (71–30 g dry weight g−1 N) decreased. The proportion of root laterals was similar in the two species, but Typha had thinner root laterals (diameter = 186 ␮m) than Cladium (diameter = 438 ␮m) indicating a larger root surface area in Typha. The two species had a similar P use efficiency (PUE) at 10 ␮g P l−1 (mean = 1134 g dry weight g−1 P) and at 40 and 80 ␮g P l−1 (mean = 482 dry weight g−1 P) but the N/P ratio indicated imbalances in nutrient uptake at a higher P concentration (40 ␮g P l−1 ) in Typha than in Cladium (10 ␮g P l−1 ). The two species had similar root specific P accumulation rate at the two lowest P levels, whereas Typha had 3–13-fold higher P uptake rates at the two highest P levels,

∗ Corresponding author. Tel.: +45-8942-4717/+45-8942-3188; fax: +45-8942-4747. E-mail address: [email protected] (B. Lorenzen).

0304-3770/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 0 1 ) 0 0 1 5 5 - 3

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indicating a higher nutrient uptake capacity in Typha. The experimental oxygen concentration in the rhizosphere had only limited effect on the growth of the two species and had little effect on biomass partitioning and nutrient use efficiency. The aerenchyma in these species was probably sufficient to maintain adequate root oxygenation under partially oxygen depleted conditions. Cladium had characteristics typical for plants from nutrient poor habitats, which included slow growth rate, low capacity for P uptake and relatively inflexible biomass partitioning in response to increased P availability. In contrast, Typha demonstrated a high degree of flexibility in growth, biomass partitioning, and nutrient accumulation to P availability, similar to species from nutrient rich habitats. Although the N/P ratio indicated that Typha was more nutrient stressed at the low P levels, Typha had a higher capacity for P uptake and was more competitive than Cladium at the applied P concentrations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sawgrass; Cattail; Nutrient use efficiency; Phosphate; Macrophyte; Hydroponics; Relative growth rate; Steady-state rhizotron; Controlled environments

1. Introduction The two wetland macrophytes Cladium jamaicense Crantz and Typha domingensis Pers. have similar growth form and occupy similar habitats. Both species develop extensive monospecific stands (Dykyjova and Kvet, 1978; Alexander, 1971) and reproduce by seeds and vegetative propagation from persistent rhizomes (Miao and Sklar, 1998). Cladium is adapted to a low-nutrient environment (Steward and Ornes, 1983), while most Typha species are typical of disturbed and nutrient-rich habitats (Dykyjova and Kvet, 1978). The two species are native to the Everglades, Florida, the largest subtropical freshwater ecosystem in the United States. Historically, the Everglades was a phosphorus (P) limited habitat with sheet flow and distinctive annual wet and dry seasons (Davis and Ogden, 1994). The vegetation, dominated by Cladium marshes intermixed with wet prairie, submerged aquatic communities and tree islands (Loveless, 1959), evolved in response to low nutrient input in conjunction with the hydrology (Davis, 1994). Historically, the abundance of Typha was limited to small and scattered patches (Alexander, 1971), but during recent decades, Typha has replaced much of the original vegetation in the northern Water Conservation Area 2A (WCA-2A), an impounded area south-east of Lake Okeechobee (Jensen et al., 1995). Typha abundance is associated with human impact in the Everglades. In this century, much of the northern Everglades was turned into agriculture and urban uses. The construction of dikes and channels has modified the hydrology and water quality in the remaining Everglades (Davis, 1994). Nutrient analyses of the top 30 cm of sediment cores from WCA-2A during the last decade have shown a gradient in total P from approximately 1300 mg P kg−1 peat dry weight in the most impacted areas in the north to approximately 450 mg P kg−1 peat dry weight in the central and southern undisturbed parts of the WCA-2A. A similar but more variable gradient in plant available P (SRP) is also apparent (DeBusk et al., 1994). Increased abundance of Typha is particularly evident in the most impacted areas along the channels and inflow structures (Jensen et al., 1995; Davis, 1994). A number of studies have led to the conclusion that the expansion of Typha relative to Cladium and submerged aquatic plant communities was related to increased P availability, perhaps in combination with alterations in hydrology (Newman et al., 1998;

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Miao and Sklar, 1998; Davis, 1994). Experimental work supports the field observations. For example, mesocosm experiments indicated that Typha responded to P availability with higher tissue P content and higher growth rate, whereas Cladium did not respond (Newman et al., 1996). The observations indicated that Typha has a high capacity for P uptake and is more competitive than Cladium at intermediate and high P availability, whereas Cladium is more competitive than Typha at low phosphorus availability. Plants generally respond to low nutrient availability by increasing resource allocation to roots (Loneragan and Asher, 1967), modifying root system morphology (Keerthisinghe et al., 1998; Drew and Saker, 1978), producing thinner and longer roots (Powell, 1974) and mycorrhizal infections (Clarkson, 1985). Physiological responses to low nutrient availability include slow growth rate, low nutrient requirements and high nutrient use efficiency (Raghothama, 1999; Chapin III, 1991). Changes in ion-specific carriers in the plasma membrane of root cells may promote a higher capacity for nutrient uptake and possibly higher affinity (Lee and Rudge, 1986). Nutrient limitations may also influence the accumulation of carbohydrates and plant water relations (Reinbott and Blevins, 1999; Chapin III, 1991), and reduced P availability may affect the uptake of other essential nutrients (Reinbott and Blevins, 1997). Plant adjustments to low nutrient availability may influence the ability of the plants to supply roots with sufficient oxygen. Thinner and longer roots restrict oxygen transport more than shorter and thicker roots. Aerenchyma is present in rhizomes and roots of both species, but convective gas flow has only been observed in shoot systems of Typha and not in Cladium (Sorrell et al., 2000; Brix et al., 1992). The capacity to supply the root system with sufficient oxygen may therefore differ between the two species. While the ability of a species to compete in low P environments may depend on inherent adaptations to low nutrient levels and a high P utilization efficiency, the ability to compete in intermediate or high P environment may depend more on a high P acquisition efficiency and the ability to adjust plant performance to increased availability. Cladium and Typha may differ in their responses to P availability; i.e. Cladium inherently shows adaptive characteristics to low P supply rate while Typha adjusts to low P availability with modification in biomass allocation and morphology. We hypothesize that Typha is more competitive at high and intermediate P levels and that Cladium, due to inherent adaptive characteristics, competes better at low P levels. In order to test the effect of low oxygen on growth, biomass allocation and nutrient use efficiencies in the two species the experimental design included a low and a high root oxygen level.

2. Methods Experiments with the two species were carried out using a factorial design with Typha and Cladium grown at four P levels; P10, P40, P80 and P500 (10, 40, 80 and 500 ␮g P l−1 ), and two oxygen levels in hydroponic culture solutions. A low O2 level, where the O2 concentration was maintained below 0.5 mg O2 l−1 by flushing the nutrient solutions with nitrogen, and a high oxygen level (8.0 mg l−1 ), where the solutions were aerated with atmospheric air, were used. The low O2 level was selected to provide an oxygen concentration in the culture solutions below the critical oxygen concentration for root growth and that reduce

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aerobic root respiration (Armstrong and Gaynard, 1976). Each treatment combination was replicated three or four times. 2.1. Plant material Seeds of Typha and Cladium were collected from different populations of the two species in the undisturbed interior of the WCA-2A area during August 1996, and germinated on vermiculite in a 14:10 h, 25:10◦ C photo- and thermoperiod, a climatic regime shown to be optimal for germination of the two species (Lorenzen et al., 2000). The seedlings were watered with a basic nutrient solution (Table 1) and allowed to establish in this medium before they were transferred to a hydroponic nursery system with four independent growth units. The nursery occupied a controlled growth cabinet operated with the experimental conditions listed in Table 2. Each growth unit of the nursery consisted of one or two 30 l aerated growth tanks with up to 22 plants. The tanks of each growth unit were connected to a 360 l nutrient solution reservoir. The nutrient solution was recirculated between the reservoir and the growth tanks through pumps delivering 6 l min−1 of solution to each growth unit. The growth condition and the chemical composition of the experimental nutrient solution are provided in Table 1. Daily from Monday through Friday of each week, temperature and conductivity were recorded, pH was adjusted to pH 6.5 and 240 ␮g FeSO4 l−1 was added to each unit. The concentrations of NH4 + and PO4 2− were measured using automated Table 1 Experimental conditions and composition of the basic nutrient solution and the P addition solution applied in the controlled environment and nurserya Basic nutrient solution

P addition solution

Major adjustment

Condition PH Conductivity Temperature Oxygen

6.5 1 mS cm−1 27◦ C <0.5, or 8.0 mg l−1

6.5

NaOH, H2 SO4 Intermediate renewal

Element Phosphorus Nitrogen Potassium Calcium Sulfur Magnesium Sodium Chloride Silicium Borum Manganese Zinc Copper Molybdenum Iron

10, 40, 80, 500 ␮g l−1 2.4 mg l−1 3.4 mg l−1 130 mg l−1 98 mg l−1 41 mg l−1 50 mg l−1 216 mg l−1 351 ␮g l−1 27 ␮g l−1 11 ␮g l−1 13 ␮g l−1 13 ␮g l−1 4.8 ␮g l−1 112 ␮g l−1

50 mg l−1

KH2 PO4 (NH4 )2 SO4 K2 SO4 , KH2 PO4 CaSO4 , CaCl2 (NH4 )2 SO4 , CaSO4 , MgSO4 MgSO4 NaCl NaCl, CaCl2 Na2 SiO3 H3 BO3 MnSO4 ZnSO4 CuSO4 Na2 MoO4 FeSO4

a

802 mg l−1 291 mg l−1 690 mg l−1 114 mg l−1 12 mg l−1 18 mg l−1 5.4 mg l−1 2.2 mg l−1 2.6 mg l−1 2.6 mg l−1 1.0 mg l−1

The chemical reagents used for adjustment are indicated.

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Table 2 Diurnal changes in the shoot environment of the controlled growth facility (PNT) and in the nursery during experimental periodsa Period (transition)

Duration (h) Light (%) Temperature (◦ C) Humidity (%)

Night → day

Day

Day → night

Night

1 0 → 100 25 → 30 90 → 85

13 100 30 85

1 100 → 0 30 → 25 85 → 90

9 0 25 90

a Maximum light (100%) above the canopy was 1200 and 350 ␮mol m−2 s−1 PAR in the PNT and nursery, respectively.

colorimetric methods (Lachat Instr. Milwaukee, USA). Orthophosphate detection was based on the ascorbic acid method for orthophosphate detection (Method EPA-600/4-79-020, 1983, US Environmental Protection Agency) and NH4 + was detected using the salicylate method (Ammonia in waters 1981, London, Her Majesty’s Stationary Office). The P level was adjusted daily to the desired set point (10, 40, 80 or 500 ␮g P l−1 ) using a P addition solution (Table 1). The NH4 + level was adjusted daily by addition of a 1 M (NH4 )2 SO4 solution. Changes in conductivity during operation of the nursery were minimized by intermediate renewal of the culture solutions when conductivity reached 2 mS cm−1 . 2.2. Controlled environment The experiments were carried out in the PhytoNutriTron (PNT); a computer controlled hydroponic growth facility at the Department of Plant Ecology, University of Aarhus (Lorenzen et al., 1998). The hydroponic rhizotron system consisted of four independent growth units each containing eight root vessels built into a controlled growth chamber in a block design. The growth chamber regulated air temperature, humidity and light intensity according to Table 2. Each of the four growth units was connected to a separate temperature, pH and oxygen controlled reservoir (180 l) through which the nutrient solution was recirculated. The reservoirs were equipped with UV-sterilization units, and the concentrations of NH4 + and PO4 3− were monitored continuously through an auto-analyzer using standard colorimetric methods (Lachat Instr. Millwaukee, USA). Nutrient concentrations were maintained at constant levels through computer-mediated feedback regulation of peristaltic pumps that delivered a stock solutions of P addition solution (Table 2) and (NH4 )2 SO4 to the reservoirs. Nutrients were supplied continuously at rates equivalent to their depletion. In order to regulate oxygen concentration, the nutrient supply units and root vessels were sealed from the atmosphere and flushed with either gaseous nitrogen or atmospheric air. Each root vessel (700 mm, ∅ 80 mm) had a lid with two openings for plants and the nutrient solution was circulated through each vessel at a rate of 4 l min−1 . 2.3. Nutrient solutions The basic nutrient solution (Table 1) applied in the PNT, the nursery and for seedling establishment, were developed to resemble the pore water concentrations of the major

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nutrient ions in the interior oligotrophic area of WCA-2A (Miao and Sklar, 1998; DeBusk et al., 1994). The P addition solution compensated for the uptake of nutrients by plants during growth and was added relative to the depletion of phosphorus. The use of the P addition solution and the intermediate renewal of the culture solutions ensured that concentrations of the major nutrients were maintained within ±10% of the desired set point during experimental periods. 2.4. Experiments The plants were allowed to acclimate to the experimental conditions in the nursery for at least 3 months before they were used for experimental purposes. The plants were considered ready for experimental use when the shoot systems were approximately 70 cm tall and the plants had entered a growth stage where rhizomes and ramets were produced by the primary shoot system. Each replicate (n = 3 or 4) of a treatment was carried out with 12 pre-acclimated and numbered plants from the nursery culture with the desired phosphate level. The plants were selected at random from the stock solution of plants and mounted in one of the four growth units of the PNT. Rhizomes two nodes from the shoot base were removed together with senescent plant material before the plants were mounted in the PNT. After a minimum of 10 days pre-incubation in the PNT, the plants were weighed by a standardized weighing procedure, the length of the root and the shoot system was measured and the number of leaves was recorded. On day t0 , t12 and t18 of the experiment 2–4 plants from each replicate were harvested at random and divided into the main shoot system (leaves and shoot base), new ramets, rhizomes and roots. The fractions were weighed, washed in distilled water and then oven-dried in a forced ventilated oven at 80◦ C until constant weight for water content and dry weight determination. On t18 , approximately 10% of the root system was selected at random from one of the plants in each replicate and further fractionated into main roots (primary) and root laterals (secondary). Microscopic inspection of roots was used to check for the presence of root hairs and to determine root diameter in the 10 and 80 ␮g P l−1 treatments. The root diameter was expressed as the mean of the diameter measured at the root base, at the middle, and 5–10 mm behind the root tip. The initial fresh weight and average fresh to dry weight ratios were used to calculate the dry weight of each individual plant at t0 . The dried plant material was ground and analyzed for nitrogen content using an N-protein analyzer (Na2000, Carlo Erba, Italy) and P content using a ICP-AES (Plasma II, Perkin-Elmer, Connecticut, USA) after HNO3 and H2 O2 digestion of the samples. Based on the biometric and plant biomass parameters, the water content (%), plant relative growth rate (RGR = (ln final weight − ln initial weight)/days), root to shoot length ratio (RL/SL) and the biomass fraction of primary roots relative to the total root biomass were calculated. Two ratios of biomass partitioning were calculated, the ratio of above to below ground biomass (A/B) and the ratio of root supported tissue (rhizomes, main shoot system and new ramets) to the biomass of roots (S/R). The P use efficiency (PUE) was calculated as the inverse plant P concentration (gram dry weight g−1 P) and the N use efficiency (NUE) as the inverse plant nitrogen concentration (gram dry weight g−1 N) (Loneragan and Asher, 1967). Root specific N and P accumulation rates were calculated as the average daily accumulation of the nutrients

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during the experimental period per average root biomass (milligram plant nutrient per gram root dry weight d−1 ). 2.5. Statistics Mean and standard error (S.E.) of the parameters were calculated and the differences between species (sp.), P and oxygen treatments were examined by analysis of variance (ANOVA). Statistical calculations were performed using Statgraphics ver. 3, Manugistics, Inc., MD, USA. A factorial model was used for ANOVA in which the effects of P, species, and oxygen were tested using residuals. The residuals from ANOVA models were tested for normality and transformed when necessary. Multiple levels of main effects were compared by multiple range tests using the least square difference procedure.

3. Results At the beginning of the experimental periods (t0 ), Typha and Cladium plants had similar dry weight (5.4 ± 0.6 and 4.0 ± 0.3 g dry weight), shoot length (83 ± 7 and 77 ± 3 cm) and similar number of leaves (7.0 ± 0.3 and 7.4 ± 0.3), respectively. Neither deficiency symptoms nor senescence of plant tissue was observed during the experimental periods. 3.1. Plant growth and biomass allocation Plant characteristics and biomass allocation were significantly affected by P and O2 treatments and the two species differed in their response to P and O2 availability. The shoot length of Typha increased more than shoot length of Cladium (P < 0.001) and at the end of the experimental period, plants of Typha and Cladium had an average of 11.6 and 11.3 leaves (P = 0.374), and a shoot length of 97 and 82 cm, respectively. In both species, P and O2 level had a significant influence on leaf number. Plants in low oxygen treatments had 1.1 leaves more than plants in aerated solutions and plants from the P10 treatment had 1.8 leaves fewer than plants from the P500 treatment. The shoot lengths were similar at the P10, P40 and P80 levels, averaging 93 and 79 cm for Typha and Cladium, respectively, but in the P500 treatment, shoots were 16 and 13 cm taller, respectively. Cladium had a significantly (P < 0.001) lower relative growth rate than Typha (Fig. 1, Table 3), and the species responded differently to P availability as indicated by a significant P × sp. interaction (P = 0.001). In Typha, the relative growth rate (mg g−1 d−1 ) increased (P < 0.001) from 48 (P10) to 89 (P500), but RGR was not affected by oxygen in the culture solutions. The relative growth rate of Cladium varied between 19 and 37 mg g−1 d−1 , approximately 40% of the growth rates observed in Typha, and showed no clear response to P level. Cladium had similar RGR at P40 and P500 (32 mg g−1 d−1 ) and similar RGR at P10 and P80 (22 mg g−1 d−1 ). Low oxygen had a positive influence on the growth of Cladium (Fig. 1) but the effect was only significant at the highest P level (P500). Typha had significant (P < 0.001) higher water content than Cladium (Fig. 1, Table 3). In Typha, the water content increased (P < 0.001) from 87.8% at P10 to 92.9% at P500. In contrast, Cladium plants from the different P treatments had similar water content (75.6–74.6%)

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Fig. 1. Mean relative growth rate (RGR) (A) and mean water content (B) of Typha domingensis and Cladium jamaicense grown in aerated (8.0 mg O2 l−1 ) and low oxygen (<0.5 mg O2 l−1 ) culture solutions with four steady state P levels. Results of Typha domingensis are illustrated with solid (aerated) and / hatched (low oxygen concentration) columns, and Cladium jamaicense with open (aerated) and – hatched (low oxygen concentration) columns. Error bars indicate standard errors.

(P = 0.520). Oxygen level in the culture solutions did not have any influence on the water content of the two species (Table 3, P = 0.173). Most of the morphological changes observed in response to P and O2 availability occurred in the below ground structures. In both species, the root system was shorter than the shoot. The average root system measured 52 cm in Typha and 39 cm in Cladium, but the RL/SL ratio was similar in the two species and they had a similar response to P and oxygen availability (Fig. 2A). The plants in P10 solutions had a 1.5-fold greater RL/SL ratio than plants at high P level (P500), and plants in aerated solutions had a 1.6-fold higher RL/SL ratio than plants Table 3 Analyses of variance (ANOVA) of plant characteristics and nutrient relations in Cladium jamaicense and Typha domingensis against P level (10, 40, 80 and 500 ␮g P l−1 ), species (sp.) and oxygen level (O2 ) (8.0 and <0.5 mg O2 l−1 ) in the culture solutionsa Variable

RGR Water content RL/SL ratio Primary root A/B ratio S/R ratio NUE N accumulation rate PUE P accumulation rate N/P ratio a

Source of variation P

sp.

O2

P × sp.

P × O2

sp. × O2

P × sp. × O2

0.000 0.002 0.000 0.006 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.852 0.019 0.000 0.001 0.000 0.000 0.028 0.001 0.001

0.306 0.173 0.000 0.087 0.922 0.024 0.008 0.634 0.252 0.264 0.761

0.001 0.000 0.297 0.561 0.011 0.000 0.000 0.000 0.000 0.000 0.012

0.910 0.022 0.031 0.725 0.322 0.723 0.546 0.936 0.410 0.715 0.438

0.203 0.705 0.972 0.088 0.164 0.787 0.287 0.314 0.514 0.475 0.192

0.420 0.069 0.178 0.977 0.790 0.070 0.325 0.360 0.861 0.834 0.597

Values in the Table are the probability of a greater F value (P level) and figures in bold indicate P-values <0.05.

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Fig. 2. Root and biomass allocation of Typha domingensis and Cladium jamaicense grown in aerated (8.0 mg O2 l−1 ) and low oxygen (<0.5 mg O2 l−1 ) culture solutions with four steady state P levels: (A) root to shoot length ratio (RL / SL), (B) percent of primary roots of total root biomass (dry weight), (C) above to below ground biomass ratio (dry weight), (A/B) and (D) the ratio of supported biomass (shoot, ramets, rhizomes) to root biomass (dry weight) (S/R). Results of Typha domingensis are illustrated with solid (aerated) and – hatched (low oxygen concentration) columns, and Cladium jamaicense with open (aerated) and – hatched (low oxygen concentration) columns. Error bars indicate standard errors.

in low oxygen solutions (Fig. 2A, Table 3). The plants in low oxygen solutions adjusted root length less in response to low P than plants in aerated solutions as indicated by the significant P × O2 interaction (P = 0.031, Table 3). Although not significant, Typha tended to adjust relative root length more to low P level than Cladium. Plants, regardless of species, had significantly (P = 0.006) lower primary root fractions in the P10 and P40 treatments than plants in higher P levels (P80 and P500), indicating that low P level stimulated allocation of biomass into root laterals (Fig. 2B). The greater allocation of biomass to laterals was a result of both longer and a greater number of laterals. Typha and Cladium grown in low oxygen solutions had similar primary root fractions, but Typha had a tendency for higher primary root fraction in aerated solutions and therefore allocated less biomass to root laterals than Cladium (P = 0.019, Table 3). The increase in laterals was tantamount to a higher proportion of thinner roots. The primary roots of Typha and Cladium had similar (P = 0.782) diameter, 2.9 and 2.4 mm, respectively, but the diameter of secondary roots differed (P < 0.001) between the species: 186 and 438 ␮m, respectively. Cladium produced short second order root laterals (<5 mm) with an average

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root diameter of 168 ␮m but the biomass of these laterals was low and their diameter only slightly lower than the diameter of laterals of Typha. There was no significant effect of P or oxygen on root diameter. However, these measurements were only performed at the P10 and P80 treatments, and the response to oxygen and P may have been different at high P levels. Root hairs were observed in both species and in all treatments. Biomass allocation of the two species differed in response to P and O2 availability (Fig. 2C, Table 3). Typha had 40% lower A/B ratio than Cladium in the P10 treatment whereas A/B was similar for the two species in the high P500 treatment. The two species had similar S/R ratio at the two lowest P levels (P10 and P40) indicating similar biomass allocation to root at low P availability whereas the species differed in root allocation at higher P levels (Fig. 2D). Cladium had an average A/B of 4.6 and there was no significant effect of P level (P = 0.457), or oxygen level (P = 0.654). However, the production of rhizomes increased with increasing P availability, therefore, a root unit of Cladium supported more biomass (8.0 g dry weight) at P80 and P500, than at the P10 and P40 levels (5.6 g dry weight) (Fig. 2D, P = 0.029). In contrast, the A/B ratio in Typha increased significantly (P < 0.001) from 2.4 to 6.1 with increasing P level. In the P80 treatment, more of the below ground biomass was allocated into rhizomes than at the two lower P concentrations and a root unit supported similar amount of biomass at P80 and P500. A root unit of Typha supported 10.4 g dry weight of biomass at the two highest P levels, but only 5.4 g dry weight at the two lowest P levels (P < 0.001). A root unit of Typha and Cladium plants, therefore, supported similar amount of other plant structures at P10 and P40, but at the two highest P levels Typha roots supported approximately 37% more biomass than the roots of Cladium.

3.2. Nutrient utilisation The plants were acclimated to the oxygen and P treatments and dilution of tissue N and P concentrations did not occur during experimental periods (paired t-test). Phosphorus treatment had a significant effect on the N and P accumulation in the two species (Fig. 3), and the species responded differently to P availability (significant P × sp. interactions, Table 3). In Typha, N use efficiency (gram dry weight g−1 N) was 71 at low but only 30 at high P availability (Fig. 3A), indicating that plant nitrogen concentration decreased with decreasing P availability in spite of the high steady-state nitrogen concentration in the culture solutions. Although the roots of Typha had a high capacity for N uptake (54 mg N g−1 root dry weight d−1 ) in the high P treatments (Fig. 3C), the N accumulation rate was much reduced at P10 (2.9 mg N g−1 root dry weight d−1 ) and at the P levels of P40 and P80 (10.1 mg N g−1 root dry weight d−1 ) (P < 0.001). Oxygen did not have an effect on NUE or nutrient accumulation rate in Typha, except in plants grown in aerated solutions at P40 where plants had a lower NUE than plants in low oxygen culture solutions (Table 3, P = 0.008). In contrast, Cladium plants had similar NUE (72 g dry weight g−1 N) and similar nitrogen accumulation rate (2.7 mg N g−1 dry weight d−1 ) at all P levels, and a NUE and an accumulation rate similar to Typha plants at the low P10 level. Oxygen in the culture solutions did not affect N use efficiency or N accumulation rate of Cladium. The PUE decreased more than three-fold from low to high P availability and the difference was most pronounced between P10 (average of both species: 1134 g dry weight g−1 P) and

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Fig. 3. Nutrient use efficiency and root specific accumulation rates of Typha domingensis and Cladium jamaicense, grown in aerated (8.0 mg O2 l−1 ) and low oxygen (<0.5 mg O2 l−1 ) culture solutions with four steady state P levels: (A) N use efficiency (NUE), (B) P use efficiency (PUE), (C) N accumulation rate, and (D) P accumulation rate. Results of Typha domingensis are illustrated with solid (aerated) and – hatched (low oxygen concentration) columns, and Cladium jamaicense with open (aerated) and – hatched (low oxygen concentration) columns. Error bars indicate standard errors.

P40 (average of both species: 529 g dry weight g−1 P) (Fig. 3B). Typha and Cladium had similar PUE in the P10, P40 and P80 treatments. At the high P level (P500) the P use efficiency of Typha was significantly lower (P = 0.028) than that of Cladium. In Typha, there was a linear relationship between relative growth rate and P use efficiency (Fig. 4), indicating a maximum P use efficiency close to 1825 g dry weight g−1 P. In Cladium, PUE was similar at P40, P80 and P500 (453 g dry weight g−1 P), and PUE was not related to relative growth rate of the plants (Fig. 4). The two species had similar root specific P accumulation rates at P10 and P40, but at P80 and P500, the accumulation rate was 3–13-fold greater in Typha than in Cladium (Fig. 3D). In Cladium, there was a significant difference between the root P accumulation rate (0.2 mg P g−1 root dry weight d−1 ) at low P10 and intermediate P40 levels (0.7 mg P g−1 root dry weight d−1 ), while the P accumulation rate at the two other P levels was between these two extremes. Since there was no significant difference between P accumulation rate at P40, P80 and P500, the result indicated a maximum P accumulation rate (Vmax ) for Cladium of approximately 0.5 mg P g−1 root dry weight d−1 (the average accumulation rate at P40, P80 and P500) and a half saturation constant (K1/2 ) for P accumulation between 10 and

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Fig. 4. The relationship between relative growth rate and P use efficiency of Typha domingensis (solid) and Cladium jamaicense (open) grown in aerated (8.0 mg O2 l−1 ) and low oxygen (<0.5 mg O2 l−1 ) culture solutions with four steady state P levels.

40 ␮g P l−1 . In contrast, the phosphorus accumulation rate of Typha increased significantly with P level from 0.2 to 8 mg P g−1 root dry weight d−1 at P10 and P500, respectively. Thus, maximum P accumulation rate (Vmax ) is above or similar to 8.0 mg P g−1 root dry weight d−1 and the K1/2 is higher than 80 ␮g P l−1 . Oxygen availability in the culture solutions did not affect P use efficiency and root specific P accumulation rate of the two species. P availability had a significant (P < 0.001) effect on the atomic N/P ratio of the plants while oxygen availability did not (Fig. 5, Table 3). For both species, the N/P ratio decreased from 32–37 at P10 to 14–15 at P80 and P500, and the species had similar N/P ratios except at the intermediate P level (P40). At P40, Typha plants had similar N/P ratio (37) as plants grown at P10 while Cladium plants had similar N/P ratio (14) as plants grown at higher P levels.

Fig. 5. The atomic N/P ratio in the total biomass of Typha domingensis and Cladium jamaicense grown in aerated and low oxygen culture solutions with four steady state P levels and aerated (8.0 mg O2 l−1 ) and low oxygen (<0.5 mg O2 l−1 ) culture solutions. Results of Typha domingensis are illustrated with solid (aerated) and – hatched (low oxygen concentration) columns, and Cladium jamaicense with open (aerated) and – hatched (low oxygen concentration) columns. Error bars indicate standard errors.

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4. Discussion This experiment examined whole-plant physiological performances of both Cladium and Typha related to leaf and root growth, biomass allocation, and particularly related to nutrient uptake, utilisation, and use efficiency under steady-state P concentrations in hydroponic systems. The results indicated low uptake capacity and half saturation constant for P accumulation in Cladium relative to Typha. While Typha could adjust biomass and nutrient allocation and nutrient use efficiency in response to P availability, Cladium could not. These findings are consistent with field observations (Newman et al., 1998; Miao and Sklar, 1998) and other field experiments (Craft and Richardson, 1997), suggesting that both species develop different life history strategies adapted to contrasting nutrient environments. Cladium is a species adapted to low-P environments, while Typha is a species adapted to high-P habitats. The above to below ground ratio and the ratio between root supported tissue and root biomass only varied slightly in response to P availability in Cladium but increased approximately two-fold in Typha. However, compared to the 3–19-fold variation in root to shoot ratio as a response to P availability in species considered fast growing and the 1–3-fold variation in slow growing species (Christie and Moorby, 1975; Nassery, 1970), both species responded little in above to below ground ratio and in the ratio between root supported tissue and root biomass. It may be difficult to compare shoot to root relations from culture solutions with ratios measured in natural habitats. However, based on the data from Miao and Sklar (1998), the A/B ratio and the S/R ratio of Cladium and Typha was 1.3–2-fold lower in plants along a nutrient gradient in the Everglades than in the culture solutions, indicating higher biomass allocation to roots under natural conditions. The lower biomass allocation to roots in culture solutions might be due to a higher proportion of roots involved in nutrient uptake in culture solutions than in sediments. The high flow rate of the culture solutions through the root vessels and the steady-state nature of the PNT, with a continuous supply of nutrients from stock solutions, minimise nutrient depletion in the boundary layers of roots (Lorenzen et al., 1998). In the oligotrophic sediments of the Everglades, P solubility and diffusion rate is very low and P depleted boundary layers of active roots may develop (Nye, 1977). Roots located in nutrient depleted sediment zones do not play an active role in nutrient uptake, whereas all roots in culture solution are in contact with nutrient ions. In addition, a generally higher nutrient availability in the culture solutions than in the natural environment may also be responsible for the lower biomass allocation to roots in culture solutions. The species had similar root to shoot length ratio and similar proportion of primary roots. However, due to differences in the diameter of root laterals between the species, the root surface area of Typha increased relatively more in response to low P availability than did that of Cladium. Root length and root diameter is important factors affecting the extraction of nutrients from soils. At low soil nutrient availability, the diffusion of nutrients to the root surface often limits uptake. Therefore, at low nutrient availability, a larger nutrient absorbing area will be more advantageous for plants than an increase in root uptake capacity or affinity (Clarkson, 1985). The fact that Typha had a larger nutrient-absorbing surface than Cladium due to thinner root laterals, may explain the higher growth rates of Typha than observed in Cladium.

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Waterlogging and reduced oxygen availability have been shown to reduce root and shoot growth in Typha and Cladium (Kludze and DeLaune, 1996). However, oxygen availability had very little influence on biomass allocation in this experiment. The root systems were shorter under low oxygen concentration than in aerated solutions, as indicated in the smaller RL/SL ratios. Reduced root length at low oxygen availability may reduce the amount of sediment volume exploited by plants in natural habitats and may have a negative effect on nutrient acquisition. Rubio et al. (1997) found that waterlogged plants had higher P uptake and responded with production of thinner roots. Low oxygen availability did encourage the production of laterals slightly in Typha but not in Cladium, and nutrient uptake was not increased by low oxygen. It seems that the constitutive aerenchyma in these species was sufficient to maintain adequate root oxygenation under partially oxygen-depleted conditions. However, the natural sediments in the Everglades may have a high oxygen demand, and reduce internal oxygen availability in the plants by accelerating loss from roots. Cladium has a less efficient root aeration system than Typha, and the growth of Cladium may be more affected by sediment oxygen demand in the natural environment than Typha. Insufficient nutrient supply reduces hydraulic conductivity of roots and leaf stomatal conductance (Reinbott and Blevins, 1999) and may affect plant water content. Furthermore, nutrient deficiency reduces the demand for carbon and causes accumulation of non-structural carbohydrates (Chapin III, 1991). Both responses may lead to a decrease in plant water content. The water content of Typha but not of Cladium was affected by P availability indicating physiological responses in Typha. The N/P ratio indicated that imbalances in nutrient utilization occurred at a higher P level (P40) in Typha than in Cladium (P10). Plants minimize imbalances among environmental resources but compensations are seldom complete (Chapin III, 1991). In Typha, the NUE increased concurrently with increased PUE. However, the increase in NUE in Typha was less than for P, and NUE of Cladium did not change in a similar manner as PUE. A relative increase in one nutrient may function as storage at high environmental concentrations but may also be a disadvantage and indicate plant stress at low supply (Bloom and Chapin III, 1985). The changes in N/P ratio and the change in dry weight ratio observed in Typha might be an early indication of P deficiency, and indicate that Typha experienced P deficiency at a higher P level than Cladium. The two species had similar root specific N and P accumulation rates at the two lowest P levels, but for different reasons. The N and P uptake capacity of Cladium was probably close to its maximum, whereas the nutrient accumulation rates in Typha were limited by plant adjustments to a reduced demand for N and by P availability. The results of this study indicated a Vmax of 0.53 mg P g−1 root dry weight d−1 and a K1/2 between 10 and 40 ␮g P l−1 for P accumulation kinetic in Cladium, whereas it was only possible to suggest a minimum Vmax (8 mg P g−1 root dry weight d−1 ) and a minimum K1/2 (80 ␮g P l−1 ) value for P uptake in Typha. The Vmax and K1/2 for P uptake in Typha was considerable higher than for Cladium, which agrees with the general hypothesis that plants adapted to low nutrient availability have a low maximum uptake capacity (Chapin III, 1980). The low maximum uptake capacity in species from low resource habitats also implies a low K1/2 for nutrient absorption. Nutrient use efficiency is one of the critical characteristics of annual plants growing in nutrient-enriched and nutrient poor environments (Veerkamp et al., 1980). However, this may not be true for perennials like Typha and Cladium, because perennial plants can

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recycle and withdraw nutrients from senescing plant parts such that nutrients can be reused (Vitousek, 1982). Typha and Cladium had similar PUE at low and intermediate P supply, whereas Typha had lower use efficiency than Cladium at high nutrient availability. The result was consistent with field observations of P content in the two species (Miao and Sklar, 1998; Davis, 1991). Along a phosphorus gradient in WCA-2A, the tissue P concentration of Typha responded more to increased P availability than did that of Cladium (Miao and Sklar, 1998), and a 2-year mesocosm experiment showed greater leaf nutrient concentration in Typha than in Cladium (Newman et al., 1996). Based on Miao and Sklar (1998), the leaf P use efficiency along a phosphorus gradient in the WCA 2A varied between approximately 700–1700 and 1100–4300 g plant dry weight g−1 P for Typha and Cladium, respectively, with the lowest PUE in the nutrient rich areas. Cladium and Typha in the low P treatment had PUE similar to field grown plants, whereas plants at intermediate and high P levels had lower use efficiencies, indicating that the range of P availability considered in this study generally was higher than for unenriched areas of WCA 2A. This study suggests that the PUE of Typha in the unenriched areas was close to a maximum PUE of 1825 g dry weight g−1 P, indicating that the phosphorus availability in the unenriched areas of WCA 2A is close to the lower limit for the survival of Typha. Although the species had similar biomass allocation and P utilisation, the results showed that Typha demonstrated a high degree of plasticity to nutrient availability while Cladium responded less and had characteristics similar to plants from nutrient poor environments. The results indicated low uptake capacity and half saturation constant (K1/2 ) for P accumulation in Cladium, whereas the P accumulation kinetic parameters were considerably higher for Typha. However, the differences in biomass allocation, nutrient acquisition and utilisation between the two species was less than expected based on the differences in growth performance of species from nutrient rich and nutrient poor environments observed in similar studies. It can, therefore, be concluded that Cladium demonstrates inherent adaptive characters to low phosphorus availability while Typha adjusts to low P levels by biomass allocation to roots, allocation of biomass into root laterals and increased PUE. However, it cannot be concluded that Cladium is a better competitor for phosphorus within the range of P levels applied in this study. The suggested K1/2 values indicated that Cladium may be a better competitor for phosphorus at lower P levels but it depends on the ability of Typha to adjust to lower P levels by a further increase in biomass allocation to roots. The growth condition applied in the present study with steady-state nutrient concentrations in the rhizosphere is very different from field situations. However, comparing physiological performances of plants grown at steady-state P supply rates and those grown in the field can generate valuable information of how the Everglades system supports plant growth and on the growth restrictions inherent in the field.

Acknowledgements We thank Robert Johnson, South Florida Water Management District, West Palm Beach, Florida, USA for logistic support in the Water Conservation Area 2A. The project was supported by a grant from the South Florida Water Management District, West Palm Beach, Florida (Macrophyte Nutrient Kinetics, Contract C-6642).

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