Aquatic Botany 87 (2007) 7–14 www.elsevier.com/locate/aquabot Chara hispida beds as a sink of nitrogen: Evidence from growth, nitrogen uptake and decomposition Marı́a A. Rodrigo a,*, Carmen Rojo a, Miguel Álvarez-Cobelas b, Santos Cirujano c a Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Apartado Oficial 2085, E-46071 Valencia, Spain b Institute of Natural Resources, CSIC, Serrano 115 dpdo, E-28006 Madrid, Spain c Royal Botanical Garden, CSIC, Pza. Murillo, 2, E-28014 Madrid, Spain Received 1 March 2006; received in revised form 8 January 2007; accepted 19 January 2007 Abstract Chara hispida forms dense beds (0.78–0.95 kg DW m2) in Colgada Lake. The ability of Chara meadows to act as a nitrogen source or sink was evaluated by the following methods: (1) investigating Chara growth, (2) nitrogen incorporation and decomposition laboratory experiments and (3) relating experimental results to field conditions. Sediment oospores were germinated in large aquaria and observed growth rates were 0.001 m day1 (shoot length) and 0.0002 g day1 (dry weight). Nitrogen uptake rates were determined by addition of K15NO3 during two different periods of Chara growth and the rates were 1.21 and 3.86 mM g DW1 h1 when charophytes were 166 days old (not sexually mature) and 323 days old (sexually mature), respectively. After the uptake experiments, the same charophytes were allowed to decompose within two types of litter bags (3 mm-pore litter bags and entire, non-porous plastic litter bags). Decomposition rates of Ch. hispida were 0.016 and 0.009 day1 in perforated and non-perforated bags, respectively, and fit a negative exponential model. The nitrogen release rate, calculated as the disappearance of N content from Chara tissues, was 0.012 day1 and there were no statistically significant differences between the values from the two different bag types. The dissolved organic nitrogen concentrations in aquarium and non-perforated litter bags waters increased linearly with time due to the leaching of soluble compounds from Chara. The rate of N loss from Chara tissues, total nitrogen and dissolved organic nitrogen release rates and the decrease in initial dry weight rate were all lower than the daily rate of Chara N uptake. By extrapolating laboratory data to field situations, we determined that approximately 38% of the N taken up by charophytes in Colgada Lake during the growth period is retained. Given the high charophyte biomass in the lake, its ability to incorporate nitrogen, its low decomposition rate and its ability to over-winter, we conclude that Chara beds could be acting as nitrogen sinks in this ecosystem. # 2007 Elsevier B.V. All rights reserved. Keywords: Submerged macrophytes; Charophytes; 15 N; Uptake; Decomposition rates; Ruidera lakes 1. Introduction Charophytes are a group of submerged macrophytes that dominate hard-water lakes (Hutchinson, 1975). These macroscopic algae are thought to play a significant role in the nutrient cycle of lakes (Kufel and Kufel, 2002), especially in areas where they develop dense meadows. Charophyte properties such as uptake of nutrients through the shoot are likely more important in charophytes than in vascular plants (Vermeer et al., 2003). Their relatively slow decomposition rates and possible prolonged storage over winter in plant tissues make char- * Corresponding author. Tel.: +34 963543596; fax: +34 963543670. E-mail address: maria.a.rodrigo@uv.es (M.A. Rodrigo). 0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.01.007 ophytes more efficient nutrient sinks in comparison to vascular macrophytes growing in lakes (Kufel and Kufel, 2002). Nitrate pollution of natural waters is an extended problem throughout Europe and particularly in Spain. It is especially serious in agricultural areas due to the use of nitrogen fertilizers (Vitousek et al., 1997). In recent years, a trend of increasing concentrations of phosphorus, and especially nitrogen, has been detected in a chain lake system located in Central Spain (Ruidera lakes; Álvarez Cobelas et al., 2006a,b). This lake system is composed of 18 lakes, most of which are deep and are characterized by the development of dense meadows of submerged macrophytes, mainly charophytes belonging to the species Chara hispida L. In lakes which show high nitrate concentrations in comparison to dissolved inorganic phosphorus, nitrophilous macroalgae can control nitrate availability, 8 M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 as demonstrated in brackish and coastal marine ecosystems (Nielsen et al., 2004). Macroalgae such as Chara species take up nitrogen from both water and sediment (Box, 1986, 1987; Vermeer et al., 2003) through the shoots and the rhizoids, respectively, and, as stated above, decomposition features and other characteristics are thought to make charophytes efficient nutrient sinks in water bodies. However, studies examining nitrogen uptake of charophytes using stable isotope methodologies are scarce. Studies focusing on the decomposition of freshwater charophytes, which would be necessary to evaluate their role as a source or sink of nutrients, are also limited (Kufel et al., 2004), especially in comparison with those studying the decomposition of emergent macrophytes (Polunin, 1984; Vargo et al., 1998). However, in brackish and estuarine ecosystems, one can find several studies dealing with nitrogen cycle and macroalgae (Rice and Tenore, 1981; Valiela et al., 1985; Buchsbaum et al., 1991; Nielsen et al., 2004). Specifically, decomposition rates for Chara species have been reported in several studies (Hunter, 1976; Bastardo, 1979; Pereyra-Ramos, 1981), though only two references on charophyte decomposition are reported in the 2002 revision paper by Kufel and Kufel. Also, scientific studies on charophytes in deep lakes are few due to the difficulties of collection (Sosnovsky et al., 2005). As far as we know, no scientific papers describe both the incorporation of nutrients (i.e. nitrogen) and the release of compounds by decomposition using the same algal material. This study aimed to estimate the capacity of Chara beds in Colgada Lake (a deep Spanish lake, in the Ruidera lake system, affected by nitrate pollution) to take up and retain nitrogen. For that, two kinds of laboratory experiments were designed in order to assess: (i) nitrogen uptake, with nitrate being the main form of nitrogen, using 15N isotopic methods in large aquaria and (ii) nitrogen release during the decomposition phase of senescent macroalgal biomass. A field extrapolation of laboratory data was also attempted in order to assess whether the macroalgae bed acts as a nitrogen source or sink in the lake. 2.2. Field measurements and Chara biomass sampling Lake temperature was recorded monthly between June 2003 and December 2004 at 1 m-intervals with an appropriate sensor. Biomass of Ch. hispida beds was estimated in September 2003 and September 2004 from samples collected by a professional diver in 18 stations (9 transects) of the lake, with two replicates taken at each station. Charophyte biomass was also determined in some points of the lake during November 2003, January, May and June 2004. Algal biomass in a 0.25 m2 square was removed, washed to eliminate residues of sediment and epiphytic algae, dried at 65 8C, and weighed. Carbonate content of charophytes was eliminated using a weak acid before analysis. See Sosnovsky et al. (2005) and Álvarez Cobelas et al. (2006a) for further information on charophytes. 2.3. Experimental set up Sediment samples containing Ch. hispida oospores were taken from Colgada Lake. A sediment layer approximately 7cm thick was placed in 178 L plastic aquaria (two replicates) which were filled with dechlorinated tap water. The aquaria were subject to a 12-h photoperiod, a photon irradiance of 40 mmol photons m2 s1 and a temperature of 19 8C. Nitrate was added to the aquarium water to reproduce the mean concentrations usually found in Colgada Lake (0.57–0.71 mM, Table 1). After germination, the growth of macrophytes was followed by extracting 10 characeae individuals from each aquarium every 15–30 days and recording the length and both fresh and dry weights. 2.4. Nitrogen incorporation experiments Nitrogen uptake was determined twice: (1) at 165 days after germination and (2) at 323 days after germination, when sexual organs had been developed. K15NO3 (98 at.% 15N; Isotec Inc.) was added to the aquaria, representing 2% of total nitrate. After 2. Material and methods 2.1. Study site Colgada Lake is part of a system of 18 natural basins and a reservoir (Ruidera lakes, Central Spain). They are connected groundwater- and riverine-fed lakes, and are small (0.1–103 ha), shallow or moderately deep (0.5–10.4 m of mean depth), with SE–NW flow (Álvarez Cobelas et al., 2006a,b). Colgada Lake (408550 N, 58400 W) is the largest lake in the system, it has a maximum depth of 18 m, a mean depth of 8 m and a volume of 9 hm3. It is a mesotrophic and monomictic lake, and its thermal stratification period generally lasts from May to October (PiñaOchoa et al., 2006). The water transparency ranges from 4 to 9 m. Its waters are rich in calcium bicarbonate and the conductivity oscillates between 600 and 700 mS cm1. Colgada Lake has the highest charophyte cover of all lakes in the system (42 and 37 ha in 2003 and 2004, respectively, Sosnovsky et al., 2005; Álvarez Cobelas et al., 2006a,b). Table 1 Values of water and sediment variables in Colgada Lake during the growth of Chara hispida and during 15N uptake experiments in the microcosms Water temperature (8C) Water pH Water dissolved oxygen (mg L1) Water nitrate concentration (mM) Water soluble reactive phosphate (SRP) concentration (mM) Total phosphorus (mM) Water chlorophyll a (mg L1) Sediment total nitrogen (mmol g1 DW) Sediment total phosphorus (mmol g1 DW) Sediment carbon content (mmol g1 DW) Colgada Lake Aquaria 9.7–22 7.6–8.0 8.6–13.5 0.51–0.80 0–0.58 18.9–19.2 7.2–7.6 9.5–10.3 0.57–0.71 0.10–0.21 0.20–2.33 0.8–7 0.23–0.25 4.83–6.45 – 0.50–1.9 2–5.5 0.24 3.55 10 The data for Colgada Lake correspond to minimum and maximum values relative to the averaged values for the 14 m-deep water column (the maximum depth at which charophytes were found) from June 2003 to December 2004. DW: dry weight. M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 40, 70 and 120 h of incubation, two to three charophyte individuals were removed from the microcosms, rinsed with distilled water to eliminate water containing 15N from the charophyte surface, dried, weighed and ground by mortar to be analyzed for the 15N-isotopic signal by a Flash 1112 elemental analyzer and subsequent mass spectrometry of Delta C isotopic relation through a CONFLO III (ThermoFinnigan) interface. During the analyses, each sample was measured three times to look for a 15N-isotopic signal and the three signals were averaged (the relative standard deviation was <5%). Previous to the addition of 15N, some charophyte specimens were removed to determine their natural 15N content. The nitrogen, carbon and phosphorus content per unit of dry weight of algal tissue were also determined. N uptake was determined from the appearance of 15N in the algal tissue (Glibert and Capone, 1993) and was calculated according to equations in Frenette et al. (1996): specific N uptake rate ðV N ; h1 Þ ¼ %atom 15 Nsample  %atom 15 Nblank þ DINÞÞ 100Þ %atom 15 Nblank Þt (1) ððð15 Nadded =ð15 Nadded N incorporation rate ðmM N h1 Þ ¼ PON  V N (2) where %atom 15Nsample is the 15N atomic percentage in the charophyte tissues, %atom 15Nblank represents the natural abundance of 15N in charophytes, DIN the concentration of dissolved inorganic nitrogen in water, t the incubation period (in h) and PON is the particulate organic nitrogen contained in the charophytes (in mM g DW1). Once the N incorporation experiments were finished, all remaining charophytes in the microcosms were used to determine decomposition rates. 2.5. Decomposition experiments Two experiments of decomposition were carried out simultaneously. One was conducted using 3 mm-perforated litter bags and the other experiment used entire and unperforated plastic bags. Charophytes were extracted from the microcosms, dry blotted, weighed and approximately the same quantity of algal material was placed in each bag. Separate samples of Ch. hispida were taken to determine initial dry content of elements (nitrogen, carbon and phosphorus) at the beginning of the experiments. Non-perforated bags containing charophyte samples were filled with 500 mL of water from the location at which charophytes had grown. Several plastic bags, filled only with 500 mL water from the microcosm, were used as a control of possible changes in water chemistry. Other plastic bags were filled with 500 mL of distilled water for possible input of microcosm water into the bags. All unperforated litter bags were closed perfectly and immersed in the microcosm water from where the charophytes came. All perforated litter bags were closed and immersed in a smaller aquarium containing only microcosm water and no sediment. The charophytes were exposed to decomposition 9 conditions for periods of 10, 30, 60 and 90 days. Fluorescent lights were removed from the upper part of the microcosms and the decomposition experiments were performed at room light (4 mmol photons PAR m2 s1 at water surface level). The water temperature during the decomposition experiment oscillated between 18.4 and 18.9 8C. On respective days, three replicates from each experiment were removed each time. In experiments using perforated bags, plant material was carefully recovered, placed on pre-weighed filter paper, dried at 80 8C during 2 days, weighed and finally ground for elemental analyses. For closed bags, the water volume was measured to account for possible water losses and water was filtered through 200 mm mesh size nytal filters (also previously weighed). The water was kept for chemical determination and the plant material retained in the filter was dried, weighed and ground as described above. A water sample from the small aquarium was also taken on respective days for N and P chemical determinations. The decomposition rate was estimated by means of the weight loss of macroalgal material and the results are presented as a percentage of the initial dry weight. A negative exponential model was used to calculate the decomposition rate (Xie et al., 2004). The decomposition rates calculated from the laboratory experiments were transformed to decomposition rates in the field. A monthly decomposition rate was calculated considering the lake water temperature at the depths where the charophytes live (1–14 m) and averaging this value for the whole decomposition period. The Q10 model (Vant-Hoff’s formula) was used to estimate the decomposition rates in the lake, taking a value of 2.4 for thermal coefficient (Asaeda et al., 2000). 2.6. Determinations of physical and chemical variables in water and charophytes Measurements of nitrate, phosphate, dissolved oxygen concentration, temperature and pH were made throughout the growth and N incorporation experiments. In vivo chlorophyll a concentrations in water were also determined as a proxy for phytoplanktonic biomass. For the determination of nitrate and phosphate, we used standard analytical procedures (A.P.H.A., 1992). Chlorophyll a was measured with an Aquafluor (Turner Designs) fluorometer and was calibrated against chlorophyll standards. Temperature, pH and oxygen measurements were made with a multi-parametric WTW-Multiline F/Set3 device with the appropriate sensors. Photon irradiance was measured with a LI-COR (LI-250) sensor. During the decomposition experiments, ammonia, nitrite, nitrate, total nitrogen, total dissolved nitrogen, orthophosphate and total phosphorus were measured in the water contained in the entire bags and in the aquarium water following A.P.H.A. (1992) recommendations. Dissolved inorganic nitrogen was calculated as the sum of nitrate, nitrite and ammonia concentrations, while dissolved organic nitrogen was evaluated as the difference between total dissolved nitrogen and inorganic dissolved nitrogen. Particulate organic nitrogen was calculated as the difference between total nitrogen and dissolved total nitrogen. 10 M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 The nitrogen and carbon content in charophyte tissues was determined by means of a Perkin-Elmer 2400 Series 2 CNHS/O elemental analyzer. 2.7. Statistical methods A Kolmogorov–Smirnov test was used to test the normality of the data, while a Levene test was used to test the homogeneity of variance for the variables. Two-way analyses of variance (ANOVA), with type of litter bag and harvest time as the main factors, were used to detect differences between both types of decomposition experiments. All statistical analysis (linear and exponential fits, Pearson correlations, etc.) were carried out with the computational software SPSS1 (version 12.0.1). Fig. 1. Average growth of individual shoots and rhizoids of Chara hispida in the aquaria. Vertical bars indicate standard errors (n = 10). 3.2. Nitrate uptake of Ch. hispida 3. Results 3.1. Growth of Ch. hispida in the Lake and the microcosms The distribution of Chara beds was quite homogeneous in Colgada Lake with the exception of the eastern littoral zone of the lake where several urban buildings are located and the human activities negatively affect the biomass of charophytes (Sosnovsky et al., 2005). Charophytes were found from 1-m deep waters up to a depth of 14 m, and the underwater light climate of the population varied with the depth at which the charophyte individuals were located; many charophytes were below 1% of incident light. The height of the charophytes decreased with depth and the maximal height observed was between 50 and 60 cm. The total biomass was 446  103 and 380  103 kg DW for 2003 and 2004, respectively (0.95  0.55 and 0.78  0.60 kg DW m2 for 2003 and 2004, respectively). The differences in the two consecutive years resulted from a wastewater point source in July 2004 at the SE end of the lake where the urban area is located. The annual growth period of Chara beds in Colgada Lake began in March and extended to September–November depending on the year. The decay of charophyte biomass was observed the rest of the year. The range of water temperature at the macrophyte depths within the decomposition period was 7–18 8C. Table 1 summarizes the environmental conditions for Chara germination and growth in the laboratory and the range of these variables in Colgada waters for the studied period. The values of the variables in the laboratory are quite close to those found in the lake. The growth of Ch. hispida in microcosms determined from the increase in shoot length, fresh weight and dry weight (Fig. 1) showed linear fits (r = 0.957, p = 0.003; r = 0.988, p < 0.0001; r = 0.902, p = 0.005, respectively). The growth rates were 0.001 m day1 for shoot length, 0.001 g day1 for fresh weight and 0.0002 g day1 for dry weight. The rhizoids did not significantly change in length during the first period of cultivation. At day 166, the charophyte individuals were, on average, 16 cm long and 0.02 g in mass (dry weight). At day 323, the averaged length and dry weight were 31 cm and 0.09 g, respectively. The background atomic percentage of 15N of total N in dry weight was determined to be 0.368%  0.0005 (n = 2) for untreated Chara, which is in close agreement with previously observed values (0.366%, Vermeer et al., 2003). The atomic percentage of 15N in the algal material increased in comparison to the background value when incubated with 15N (Fig. 2), indicating that 15N was indeed taken up by the charophytes. This increment was linear (r = 0.998, p < 0.000; r = 0.997, p = 0.003 for the 166- and 323-day old charophytes, respectively) and the 15N enrichment in the macroalgae did not decay during the 5 days of our experiments (Fig. 2). The specific N uptake rates were 0.00055–0.0018 h1. Nitrogen incorporation rates were 1.21 mM g DW1 h1 in the first experiment when macrophytes were 166 days old and 3.86 mM g DW1 h1 in the second experiment, when charophytes were 323 days old and had reached sexual maturity. 3.3. Decomposition of Ch. hispida and N release The decomposition of Ch. hispida, as estimated by the reduction of dry weight (Fig. 3A), fit a negative exponential Fig. 2. Increase of the 15N at.% in the charophyte tissues together with incubation time in the 166 and 323 day-grown charophytes. Each point of the graph corresponds to an averaged value of three 15N-isotopic signals on the same sample (relative standard deviation <5%). M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 11 significant differences in the loss of C and N in Chara tissues between the different types of litter bag used during decomposition, but there were significant differences in the content of C and N with time ( p = 0.001). In both cases, the interaction type of litter bag  time was not statistically different. The nitrogen release rate (kN) calculated from the decrease in N content from Chara tissues (Fig. 4B) was estimated to be 0.011–0.012 day1. The phosphorus content in Chara decomposing tissues increased slightly until day 30 and then decreased (Fig. 4C). There were no large differences between the two kinds of bags. The analysis of nitrogen compounds in the aquarium water containing the perforated litter bags with charophytes (the ‘‘aquarium’’ hereafter) and non-perforated litter bags (Fig. 5) reveals that ammonia (Fig. 5A) and nitrite concentrations initially increased with decomposition time in both aquarium and bag waters. This is likely the result of leaching that takes place the first few days of Chara decomposition and ammonification processes, because the ammonia and nitrite concentration remained stable in the water from non-perforated bags without charophytes (used as a control). For the first 30 Fig. 3. (A) Disappearance of Chara hispida during the decomposition experiments in perforated and non-perforated litter bags. Vertical bars indicate standard error of three replicates. Statistics are two-way ANOVAs with harvest time and type of litter bag as main factors. *p < 0.05, **p < 0.005. (B) Daily decaying rates in the different incubation periods in perforated and nonperforated bags. Vertical bars indicate standard errors of three replicates. model in both perforated (r = 0.964, p = 0.008) and nonperforated litter bags (r = 0.978, p = 0.004). The decomposition rates (k) were 0.016 and 0.009 day1, respectively. A two-way ANOVA, with type of litter bag and harvest time as the main factors, showed that the decomposition of Ch. hispida was significantly affected by the type of litter bag used ( p < 0.05) and by time ( p < 0.005). The interaction between type of litter bag and time was not statistically significant, since both types of litter bags showed the same pattern over time. After 90 days of exposition, 25% of the initial dry weight remained in the case of perforated litter bags and 43% in the case of non-perforated litter bags. When the decaying rates between harvest periods were compared (Fig. 3B), maximal rates were observed in both perforated and non-perforated bags in the first 10 days of incubation (mainly because of the decomposition of rapid decomposable material), but rates were higher in the former. The rates gradually slowed down afterwards. The carbon content in charophyte tissues decreased exponentially with incubation time in both perforated and non-perforated litter bags (Fig. 4A). The disappearance of nitrogen from the Chara tissues during the decomposition experiments (Fig. 4B) also fits an exponential model. Two-way ANOVA analyses revealed that there were no statistically Fig. 4. Changes of carbon, nitrogen and phosphorus content in decomposing Chara hispida tissues. Percentage of dry weight, mean values and standard errors for three replicates are shown. The rates of decrease obtained from the adjustment to negative exponential models for carbon and nitrogen are also indicated. 12 M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 Fig. 5. Time course of the main forms of nitrogen in water of non-perforated litter bags containing Chara hispida (mean and standard errors of three replicates) and in water of the aquarium where the perforated litter bags were set up. Total phosphorus concentration is also shown. Vertical bars indicate standard errors for three replicates. days of incubation, the increase rate in ammonia concentration was 0.0023 day1 in aquarium water and 0.0028 day1 in water from non-perforated litter bags. At day 60, the ammonium concentrations had decreased substantially due to nitrification. In contrast, nitrate decreased with exposure time until day 30 and increased thereafter (Fig. 5B) and water from litter bags without charophytes showed fairly constant nitrate concentrations once again. The final balance in DIN concentration showed the same pattern in both aquarium and litter bag water (r = 0.994, p = 0.006): a decrease during the first 10 days, an increase later on. Total nitrogen (TN) concentrations (Fig. 5C) showed a significant (r = 0.918, p = 0.028) exponential fit in aquarium water, which yielded a N release rate of 0.010 day1. Total dissolved nitrogen (Fig. 5D) showed the same pattern as TN. The dissolved organic nitrogen (DON) concentrations (Fig. 5E) increased linearly over time at a rate of 0.0014 day1 for aquarium water (r = 0.991, p = 0.008) and 0.0019 day1 in water from non-perforated litter bags (r = 0.976, p = 0.047) due to the release of soluble algal organic compounds from Chara. Total phosphorus concentrations (Fig. 5F) also increased linearly in both aquarium and non-perforated litter bags water, changing from 1 to 2 mM at time 0 to 37–61 mM after 90 days of incubation. TP concentrations in the control bags remained at approximately 1 mM. 3.4. Combining field and laboratory data to calculate nitrogen retention in the lake The data on N incorporation by Ch. hispida obtained throughout the laboratory experiments can be extrapolated to estimate the nitrogen incorporation by Chara beds in Colgada Lake. The immobilization of nitrogen within charophytes at the end of its growth period was estimated to be 11.4 g N m2, while the charophyte biomass was 453  159 g DW m2 (not including the calcium carbonate incrustations in the form of calcite located on the charophytes, which represent approximately 60% of total DW) and the nitrogen content of charophytes was 2.52% (DW percentage). Although, we do not have quantitative data regarding the over-wintering resistance of Ch. hispida meadows in Colgada Lake, we have M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 evidence that quite a substantial part of its biomass does not decompose. Assuming that an average of 70% of the charophyte biomass decomposes, as reported in other lakes with dense beds of Chara (Pereyra-Ramos, 1981; Królikowska, 1997), we have calculated the nitrogen retention by charophytes after decomposition. We obtained an average monthly decomposition rate (percentage of DW disappearance) of 0.0049 day1 using decaying rates obtained in the laboratory and calculated rates for the lake in each month of the decomposition period. Assuming a negative exponential function for decomposition (as demonstrated in the laboratory experiments), this indicates that the Chara biomass in March (at the end of the decomposition period) would be approximately 152 g DW m2. As the charophyte tissues contained only 0.57% of N at final decomposition stages, the amount of nitrogen in the Chara beds at the end of the decomposition period would have been approximately 4.3 g N m2, implying that approximately 38% of the nitrogen taken up during the growth period is retained by charophytes in Colgada Lake. 4. Discussion The data on nitrogen uptake rates by Chara species determined by 15N incorporation techniques are scarce in the scientific literature. Kufel and Kufel (2002) cite only a daily uptake of N of 0.04–0.52 mM g DW1 by Chara vulgaris based on the conversion of maximum photosynthesis data reported by Hough and Putt (1988). Box (1987) determined nitrogen daily uptake rates in the laboratory for Ch. hispida and obtained values of 0.02 and 0.18 mM g DW1 for the decrease of nitrate in culture medium at nitrate concentrations of 0.04 and 0.18 mM, respectively, at 22 8C. Our daily N uptake rates by Ch. hispida (0.03–0.09 mM g DW1) fall in the same range as these reported values. As in situ uptake experiments with charophytes cannot be made easily (Box, 1987), especially in deep lakes such as Colgada Lake, indoor experiments can provide good estimations of N uptake to determine the contribution of charophytes to the nutrient cycling in lakes. With our indoor N uptake experiments we attempted to mimic the main environmental conditions in Colgada Lake (Table 1) to get values of nitrogen incorporation by Chara. However, nitrogen uptake in charophytes can depend on the concentration of N compounds in water, on the nutritional status of the cells with respect to N and on the growing status of the plants, among other factors (Vermeer et al., 2003). In this study, we have confirmed how the N uptake by Ch. hispida differ with the age of the charophytes, with N uptake in sexually mature charophytes being more than three times higher than that of younger plants. Decomposition of charophytes is an important process in the sense that nutrients incorporated in the macroalgal biomass are released to the water during this process. A slow decomposition rate means that nutrients trapped in the macrophyte tissues will be retained for a longer period and will not be accessible for other organisms or processes in the water. We have demonstrated here that the decomposition rates for Ch. hispida are low, as previously reported by Pereyra-Ramos (1981), and 13 our observed rates were close to those reported by Bastardo (1979). There have been few studies on the decomposition of Chara, so we shall compare our results with those about other submerged aquatic plants. Our values are similar to those obtained by Xie et al. (2004) for Potamogeton maackianus (decomposition rate = 0.009–0.012 day1) in indoor experiments. Hootsmans (1994) reported values for decomposition rates of P. pectinatus of 0.010–0.082 day1 and Godshalk and Wetzel (1978) described a decomposition rate of 0.037 day1 for Myriophyllum heterophyllum. Kufel et al. (2004) found in situ decomposition rates of 0.0003–0.009 day1 for Ceratophyllum demersum, however, Battle and Mihuc (2000) obtained higher rates for the same species (0.008–0.049 day1). Several factors, including methodological differences, the specific character of the experimental sites, and the species, greatly influence decomposition rates. The percentage of Chara initial dry weight remaining in the perforated litter bags in our experiment after 90 days of exposure (25%) was within the range reported by Pereyra-Ramos (1981) for three Masurian lakes in northern Poland (14–44%). Conversely, our study found that Ch. hispida became poorer in nitrogen than living Chara after decomposing for 90 days our. The loss of nitrogen from Chara tissues occurred slightly faster than that for carbon (Fig. 4A and B). Buchsbaum et al. (1991) also found few differences between rates of nitrogen and carbon loss in marine macroalgae. In our study, phosphorus content in decomposing Chara tissues did not decrease in the first stages of decomposition, rather it increased (Fig. 4C). This was also observed by Belova (1993) in a decomposition study of freshwater macrophytes in the littoral zone of lakes. This author concluded that P is gradually lost from plant tissues as they decay, and is likely utilised by microbial decomposers, which sometimes leads to enrichment of plant litter with P. Nitrogen compounds in the water during decomposition showed typical temporal patterns for DON, ammonification and nitrification (Nielsen et al., 2004). On the other hand, the decomposition was higher in the case of perforated litter bags, though we did not find significant differences in the loss of N and C (Fig. 4A and B) from Chara tissues between different litter bag types. Additionally, we did not find large differences between perforated and non-perforated bags when comparing the N and P compounds released into the water (Fig. 5). This means that the chemical and microbiological processes had the same magnitude in both kinds of litter bags. The higher loss of macroalgal biomass in perforated litterbags might be due to the loss of smaller decomposing charophyte material (fine particles that were less than the mesh size), which could have left the perforated litter bag, diminishing, the weight of charophytes without affecting concentrations of dissolved compounds in the water. The ability of charophytes to act as nutrient sinks in lakes was recently reviewed by Kufel and Kufel (2002). In the paper, the authors suggest that Chara beds can be considered as efficient nutrient traps in lakes with high charophyte cover, as in Colgada Lake. In our study, all values related to the role of charophytes as a source of nutrients for the environment (rate of loss of N from Chara tissues, organic nitrogen release rate, total 14 M.A. Rodrigo et al. / Aquatic Botany 87 (2007) 7–14 nitrogen release rate, decrease in initial dried weight rate) were lower than the daily rates of Chara N uptake. Chara species are capable of over-wintering (Kufel and Kufel, 2002) and PereyraRamos (1981) found young Chara rudis throughout the year, even in winter time under 0.5 m of ice. Also, green shoots of Chara aspera were observed by Królikowska (1997) under the ice in Lake Luknajno. We also have evidence of such an overwintering ability in the Chara meadows in Colgada Lake. Thus, the nutrient storage in plant biomass may extend beyond the growing season. 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