W M Quan

J D Han

A L Shen

X Y Ping

P L Qian

C J Li

L Y Shi

Y Q Chen
email: yq_chen@citiz.net

Uptake and distribution of N, P and heavy metals in three dominant salt marsh macrophytes from Yangtze River estuary, China

Keywords: Yangtze River estuary, Nutrients, Metals, Salt marshes, Phragmites, Spartina

We examined the variation in aboveground biomass accumulation and tissue concentrations of nitrogen (N), phosphorus (P), copper (Cu), zinc (Zn) and lead (Pb) in Phragmites australis (common reed), Spartina alterniflora (salt cordgrass), and Scripus mariqueter throughout the growing season (April-October 2005), in order to determine the differences in net element accumulation and distribution between the three salt marsh macrophytes in the Yangtze River estuary, China. The aboveground biomass was significantly greater in the plots of S. alterniflora than in the plots of P. australis and S. mariqueter throughout the growing season (P<0.05). In August, the peak aboveground biomass was 1246±89 gDW/m 2 , 2759±250 gDW/m 2 and 548±54 gDW/m 2 for P. australis, S. alterniflora and S. mariqueter, respectively. The concentrations of nutrients and heavy metals in plant tissues showed similar seasonal patterns. There was a steady decline in element concentrations of the aboveground tissues from April to October. Relative element concentrations in aboveground tissues were at a peak during the spring sampling intervals with minimum levels during the fall. But the concentrations of total nitrogen and total phosphorus in the belowground tissues were relatively constant throughout growing season. Generally, trace metal concentrations in the aboveground tissues of S. mariqueter was the highest throughout the growing season, and the metal concentrations of S. alterniflora tissues (aboveground and belowground) were greater than those of P. australis. Furthermore, the aboveground pools of nutrients and metals were consistently greater for S. alterniflora than for P. australis and S. mariqueter, which suggested that the rapid replacement of native P. australis and S. mariqueter with invasive S. alterniflora would significantly improve the magnitude of nutrient cycling and bioavailability of trace metals in the salt marsh and maybe transport more toxic metals into the water column and the detrital food web in the estuary.

Introduction

Macrophytes play important roles in the biogeochemistry of salt marsh ecosystems through their active and passive circulation of elements [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Weis | Release into the environment of metals by two vascular salt marsh plants[END_REF].

Many estuarine salt marshes near urbanized areas receive substantial quantities of anthropogenic materials, including nutrients and metals. When marsh sediment accumulates high concentrations of nutrients and metals, macrophyte roots can absorb these materials and translocate them to aboveground biomass, which may lead to the increase of nutrient and metal bioavailability in the salt marsh [START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF][START_REF] Windham | Effects of an invasive reedgrass (Phragmites australis) on nitrogen cycling in brackish tidal marsh of New York and New Jersey[END_REF]. Macrophytes in salt marshes are widely believed to decrease the rate of tidal flow, cause suspended particulate matter to sediment, enhance the retention rates of nutrients and metals from overlying water, and reduce the import of nutrients and metals to estuarine waters [START_REF] Cacador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritime and Halimione portulacoides from Tagus estuary salt marshes[END_REF][START_REF] Daehler | Status, prediction and prevention of introduced cordgrass Spartina spp. invasions in Pacific estuaries, USA[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF][START_REF] Windham | Effects of an invasive reedgrass (Phragmites australis) on nitrogen cycling in brackish tidal marsh of New York and New Jersey[END_REF]. Therefore, salt marshes are often considered efficient sinks for nutrients and metals; they can ameliorate the environmental quality in coastal ecosystem [START_REF] Romero | Restored wetlands as filters to remove nitrogen[END_REF][START_REF] Coelho | Phosphorus speciation and availability in intertidal sediments of a temperate estuary: relation to eutrophication and annual P-fluxes[END_REF][START_REF] Zhang | China's Yangtze Estuary: I. Geomorphic influence on heavy metal accumulation in intertidal sediments[END_REF]. Salt marsh macrophytes have high primary productivity and assimilative capacity for nitrogen (N) and Phosphorus (P) [START_REF] Romero | Restored wetlands as filters to remove nitrogen[END_REF][START_REF] Teal | Salt marsh values: retrospection from the end of the century[END_REF]. With denitrification as an exception, plant uptake is thought to be the important mechanism for salt marsh ecosystems to remove inorganic nutrients (N and P) from overlying water [START_REF] White | Long-term 15 N-nitrogen retention in the vegetated sediments of a New England salt marsh[END_REF][START_REF] Teal | Salt marsh values: retrospection from the end of the century[END_REF][START_REF] Rozema | Nitrogen and vegetation dynamics in european salt marshes[END_REF]. With increasing N supply, salt marshes can show greater primary productivity and withstand large additions of nutrients without severe damage [START_REF] Adcock | Growth characteristics of three macrophyte species growing in a natural and constructed wetland system[END_REF][START_REF] Brix | The use of aquatic macrophytes in water-pollution control[END_REF][START_REF] White | Long-term 15 N-nitrogen retention in the vegetated sediments of a New England salt marsh[END_REF]. Previous studies focusing on P dynamics show that mud flats colonised by Spartina marshes have higher adsorption capacity, and-through primary production activity-these macrophytes were able to control phosphate efflux from the sediment [START_REF] Teal | Salt marsh values: retrospection from the end of the century[END_REF][START_REF] Coelho | Phosphorus speciation and availability in intertidal sediments of a temperate estuary: relation to eutrophication and annual P-fluxes[END_REF]. Therefore, salt marsh plant activities significantly influence the biogeochemistry of nutrients and increase the retention rate of N and P in salt marshes.

Metal dynamics in salt marshes may be influenced by the composition of plant communities [START_REF] Williams | Phytoremediation in wetland ecosystems: progress, problems, and potential[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. Recent investigations demonstrate that concentrations of metals in plant tissues are highly variable between species [START_REF] Fitzgerald | Copper and lead concentrations in salt marsh plants on the Suir Estuary, Ireland[END_REF][START_REF] Ye | Zinc, lead and cadmium tolerance, uptake and accumulation by the common reed, Phragmites australis (Cav.) Trin. ex Steudel[END_REF]. Differences in uptake rates and allocation patterns between species can generate different rates of metal retention in salt marsh vegetation and perhaps further influence the biogeochemistry of salt marshes [START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF][START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF][START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Ornes | Comparison of selected element concentrations in tall and short forms of Spartina alterniflora[END_REF].

In many salt marshes on the Atlantic coast of North America, Phragmites australis has been invading the low marsh zone dominated by Spartina alterniflora. Several studies demonstrate that P.

australis expansion could alter edaphic conditions [START_REF] Windham | Effects of Phragmites australis (common reed) on aboveground biomass and soil properties in brackish tidal marsh of the Mullica River, New Jersey[END_REF][START_REF] Bart | Environmental determinants of Phragmites australis expansion in a New Jersey salt marsh: an experimental approach[END_REF], accelerate nutrient cycling [START_REF] Windham | Effects of an invasive reedgrass (Phragmites australis) on nitrogen cycling in brackish tidal marsh of New York and New Jersey[END_REF][START_REF] Meyerson | A comparison of Phragmites australis in freshwater and brackish marsh environments in North America[END_REF], and increase metal bioavailability [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF]Weis et al., 2002;[START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. The genus Spartina is native to the Atlantic and Gulf Coasts of North America, but it has been introduced to many coastal and estuarine regions of the world [START_REF] Daehler | Status, prediction and prevention of introduced cordgrass Spartina spp. invasions in Pacific estuaries, USA[END_REF][START_REF] Chen | Local competitive effects of introduced Spartina alterniflora on Scirpus mariqueter at Dongtan of Chongming island, the Yangtze River estuary and their potential ecological consequences[END_REF]). On the Pacific Coast of North America, four introduced cordgrass species have seriously invaded several estuaries. These invading Spartina have outcompeted the native salt marsh plant, reduced open mud feeding habitat for shorebirds and fish, caused suspended sediment to precipitate, and altered the trophic function of salt marsh ecosystems [START_REF] Daehler | Status, prediction and prevention of introduced cordgrass Spartina spp. invasions in Pacific estuaries, USA[END_REF][START_REF] Levin | Invasive cordgrass modifies wetland trophic function[END_REF]. In China, S. alterniflora was intentionally introduced from North America in 1979 for erosion control, soil melioration, and dike protection. Today, it is spreading along the east coast of China from Tianjin to Baihai in Guangxi.. Small patches of S. alterniflora were first found at Dongtan wetland on Chongming Island of the Yangtze River estuary in 1995, which are believed to have invaded the mudflats through seed dispersal from Qidong by water flow. More importantly, S.

alterniflora was transplanted to Dongtan and Jiuduansha wetlands for rapid sediment accretion in the late 1990s when many salt marshes were regularly reclaimed in the Yangtze River estuary. Since then, S. alterniflora has rapidly expanded in salt marshes and gradually replaced the native plants (P.

australis and Scirpus mariqueter). S. alterniflora is now a dominant salt marsh macrophyte in the Yangtze River estuary [START_REF] Chen | Comprehensive Surveys on Shanghai Jiuduansha Wetland Nature Reserve, the Yangtze River estuary[END_REF].

The shift in plant community toward S. alterniflora monoculture may lead to different patterns of nutrient and metal cycling in salt marsh ecosystems. Few studies have examined the impact of the S.

alterniflora invasion on the element cycling of the salt marsh ecosystem in the Yangtze River estuary.

Our objective is to explore the influence of S. alterniflora invasion on nutrient and metal cycling in the salt marsh ecosystem in the Yangtze River estuary. We describe herein the uptake and distribution of N, P and heavy metals (Cu, Zn and Pb) in the plant tissues of P. australis, S. alterniflora and S.

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mariqueter over a growing season (April-October, 2005).

Materials and methods

Study site

The Dongtan wetland at Chongming Island, Shanghai City, is in the Yangtze River estuary of China (Figure 1). In this wetland, P. australis, S. alterniflora and S. mariqueter were the three salt marsh macrophytes. Each plant can form a dense monoculture in the proper marsh zone. Monocultures of P. australis are often found around the high marsh zone, while S. mariqueter, a pioneer plant, dominates low marsh zones. In the late 1990s, S. alterniflora was transplanted to the middle marsh zone and is now quickly expanding toward the higher and lower marsh zones. At this study site, concentrations of heavy metals in the sediment are moderate, compared with other salt marshes in Asia and Europe [START_REF] Zhang | China's Yangtze Estuary: I. Geomorphic influence on heavy metal accumulation in intertidal sediments[END_REF]. Average metal concentrations in the sediment during the growing season of 2005 were Cu-42±11mg/kg; Zn-122±23 mg/kg; Pb-37±10 mg/kg. (Quan, unpublished data).

Field sampling and analysis methodology

We visited the study site every other month throughout the growing season (April, June, August, and October of 2005). During each field sampling, replicate 1m 2 plots (n=6) were staked in each vegetation type (P. australis, S. alterniflora and S. mariqueter) for collection of standing live aboveground biomass. Six soil cores (diameter 15cm and depth 30cm) were sampled in each vegetation type. All cores were washed over a 1mm mesh screen. The retained live roots and rhizomes (white) were collected for analysis. All the samples were sent immediately to a laboratory for processing.

All plant samples were washed thoroughly with tap water and then cleaned with deionised water.

Samples were dried at 60℃ to constant weight for approximately 48h. The dry plant samples were then ground to powder with a mortar and pestle. Sub samples for each sampled plot and tissue type were analyzed for elemental concentrations (TN, TP, Cu, Zn and Pb). N concentration of the plant tissues was determined by the Kjeldahl method. 0.2g sub samples of plant tissues were digested with HNO 3 -HCl (3:1 v/v) at 200℃, and then P in the solution was determined by the spectrophotometer method. Ten millitres of HNO 3 /HClO 4 (5:1 v/v) were added to 0.2g of dry plant sample. The mixture was heated slowly to approximately 100℃, until the formation of nitrous fumes stopped. Then the mixture was boiled to approximately 200℃, until most of the liquid evaporated. The remaining extract was diluted with deionised water to 25ml. The metal concentrations (Cu, Zn and Pb) in the solution were determined by the flame atomic absorption spectrophotometer (3500G, HP inc). All the chemical reagents were GR grade. Inter-laboratory standard reference material and method blanks were digested and analyzed for Cu, Zn and Pb with the same method. The concentration on average deviated 3.24% (Cu), 6.71% (Zn) and 5.10% (Pb) from certified average values. The aboveground biomasses of the three salt marsh macrophytes were calculated on a dry weight basis (g/m

2 ). The elemental concentration and biomass of aboveground tissues were multiplied to yield aboveground element pools.

Data analysis

All data was log-transformed to meet the parametric assumptions prior to statistical analysis.

One-way analysis of variances (ANOVA) was carried out to compare the aboveground biomass and elemental concentrations among the three plant species using STATISTICA 6.0 (StatSoft Inc, 2001).

Duncan's test method was used to test the significant difference between the three salt marsh macrophytes. A set of linear regressions were performed between the elemental concentrations in plant tissue and time for the three species. Each pair of regressions was compared with covariance analyses (ANCOVA). Differences were considered significant when the probabilities for non-significance were smaller than 0.05.

Results

Aboveground biomass

S. mariqueter was not emergent aboveground in the first field sampling (April of 2005). The aboveground biomasses of the three species showed the same seasonal variation pattern. From April of 2005, the aboveground biomasses of the three species increased gradually. By August of 2005, the macrophytes reached maximum aboveground biomasses (Figure 2). One-way ANOVA found that the aboveground biomass was significantly greater in the plots of S. alterniflora than in the plots of the two native plants (P. australis and S. mariqueter) (Figure 2, P<0.05). The aboveground biomasses for S. alterniflora were 120-186% greater than P. australis and 300-600% greater than S. mariqueter, The aboveground biomass in the plots of P.australis had no significant difference from the the plots of S.

mariqueter, except in August. The results showed that S. alterniflora had greater aboveground biomass than native plant across the whole growing season.

Elemental Concentration

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Seasonal patterns of total N (TN) and total P (TP) concentration in plant tissue were relatively consistent among the three species (Figure 3). From April to October 2005, concentrations of TN and TP in the aboveground tissues showed a gradually decreasing trend, whereas the concentrations of TN and TP in the belowground tissues were relatively constant over the growing season (Figure 3). A linear regression between nutrient concentrations in plant tissues and time was established for the three species (Table 1). A significantly negative regression relationship was found between aboveground nutrient concentrations and time for P. australis and S. alterniflora(Table 1, P<0.01). The regression between belowground nutrient concentrations and time was not significant for the three species.

Analysis of covariance revealed significant differences in the regression between belowground TN concentration and time for the three species (Table 1, ANCOVA, P<0.01). The differences in the regression between aboveground TN vs. time, aboveground TP vs. time, and belowground TP vs. time

were not significant for the three species (Table 1, ANCOVA, P>0.05).

For the three plant species, the concentrations of TN and TP were obviously greater in the aboveground plant tissues than the belowground tissues (Figure 3). TN concentrations in the aboveground tissues of P. australis and S. alterniflora were similar throughout the growing season and lower than S.mariqueter. TN concentrations of aboveground tissues were significantly greater in S.

mariqueter than in both P. australis and S. alterniflora, especially in October (Figure 3, P<0.05). For the belowground tissues, from April to August, TN concentrations were similar among the three species. In October, however, TN concentrations were significantly greater in P. australis than in both S. alterniflora and S. mariqueter (Figure 3, P<0.05).

In the aboveground tissues, TP concentration in P. australis was the lowest of the three species over the growing season (Figure 3). In June, TP concentration of the aboveground tissue was significantly lower in P. australis than in S. alterniflora and S. mariqueter (Figure 3, P<0.05). By October, similar to TN results, TP concentration of the aboveground tissue was significantly greater in S. mariqueter than in P. australis and S. alterniflora (Figure 3, P<0.05). But in the belowground tissues, TP concentration in P. australis and S. alterniflora were similar and consistently lower than S.

mariqueter. In August and October, TP concentrations in S. mariqueter were significantly greater than P. australis and S. alterniflora (Figure 3, P<0.05).

Metal concentrations in plant tissues over the growing season were shown in Figure 4. The aboveground and belowground tissues in the three species contained different levels of Cu, Zn, and Pb.

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Metal concentrations were greater in belowground tissues than aboveground tissues. But seasonal variation patterns of metal concentrations in plant tissues were similar between the three species. Early in the growing season (April), relatively higher metal concentrations accumulated in the tissues of the three macrophytes (Figure 4). From April to October, metal concentrations in the plant tissues showed a gradual decreasing trend. At the same time, a negative regression relationship between metal concentrations in plant tissues and time was established for the three species (Table 2). Analysis of covariance found no significant differences in the regression between aboveground Cu vs. time and belowground Cu and Zn vs. time. Analysis of covariance, however, revealed significant differences in the regression between aboveground Zn and Pb vs. time (

Aboveground pools

The aboveground pools of given elements were calculated by combining elemental concentration with aboveground biomass. Aboveground elemental pools showed clear seasonal variation patterns throughout the growing season. Seasonal patterns of both nutrients and metals had the minima in April and the maxima in August at the peak of the growing season (Figure 5, Figure 6).

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Throughout the growing season, the aboveground pools of both N and P were significantly greater in S. alterniflora than P. australis and S. mariqueter (Figure 5, P<0.05), owing to greater aboveground biomass in the plots of S. alterniflora (Figure 5). The aboveground N pools were 23-192% greater in S. alterniflora and 190-265% greater in S. mariqueter than P. australis. The aboveground TP pools of S. alterniflora were approximately 3.1 and 3.4 times higher than P. australis and S. mariqueter, respectively. Except for TN in August, the aboveground TN and TP pools showed no significant difference between the two native species.

Similar to the aboveground pools of N and P, the aboveground pools of metals (Cu, Zn and Pb)

showed the same seasonal variation pattern (Figure 6). In August, all the plants reached maximum pools of metals. The aboveground pools of metals were significantly greater in S. alterniflora than in both P. australis and S. mariqueter (Figure 6, P<0.05). The aboveground metal pools showed no significant difference between the two native plants throughout the growing season, especially for Cu in August. The results showed that exotic plant S. alterniflora could translocate significantly more nutrients and metals to the aboveground tissue than the two native plants.

Discussion

Our measurements of peak aboveground biomass were not equal to the annual net production of the aboveground parts due to significant biomass losses from shoot mortality and leaf shedding. Shoot growth started in March, at the expense of rhizome resources, and the aboveground biomass peaked in August. Then the plants started to senesce , transferring a large portion of the aboveground biomass to the belowground organs. Similar seasonal variation patterns are documented in previous studies [START_REF] Asaeda | Seasonal fluctuations in live and dead biomass of Phragmites australis as described by a growth and decomposition model: implications of duration of aerobic conditions for litter mineralization and sedimentation[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. Compared with Windham's results, we found an equivalent maximal aboveground biomass of P. australis (1246 g DW /m 2 in Dongtan vs. 1117 g DW/m 2 in Hackensack), though under a Mediterranean climate, the peak of aboveground biomass in P.

australis could reach 2296 g DW/m 2 [START_REF] Asaeda | Seasonal fluctuations in live and dead biomass of Phragmites australis as described by a growth and decomposition model: implications of duration of aerobic conditions for litter mineralization and sedimentation[END_REF]. The aboveground biomass of S. alterniflora was obviously greater in Dongtan wetland than in the Hackensack Meadowlands (2795 g DW/m 2 in Dongtan vs. 679 g DW/m 2 in Hackensack). Therefore, aboveground primary production varies widely in salt marsh plants from different sites [START_REF] Turner | Geographic variation in salt marsh macrophytes production: a review[END_REF][START_REF] Ibañez | Structure and productivity of microtidal Mediterranean coastal marshes[END_REF].

Our study found that the aboveground biomass of S. alterniflora was significantly greater than both P. australis and S. mariqueter. [START_REF] Chen | Comprehensive Surveys on Shanghai Jiuduansha Wetland Nature Reserve, the Yangtze River estuary[END_REF] have also found that S. alterniflora had a much greater primary productivity than S. mariqueter and P. australis in the Yangtze River estuary. In less

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than 10 years, exotic S. alterniflora has invaded the salt marsh of the Yangtze River estuary and is rapidly replacing the native plants, P. australis and S. mariqueter. [START_REF] Chen | Comprehensive Surveys on Shanghai Jiuduansha Wetland Nature Reserve, the Yangtze River estuary[END_REF] have shown that S. alterniflora occupied 13.2% of the Jiuduansha Island's area in autumn 2002, while its monocultures contributed 25.3% to the total plant biomass production of the islands. Thus, the S.

alterniflora invasion would significantly increase the primary productivity of the salt marsh ecosystem in Yangtze River estuary and potentially have further influence on the rest of the ecosystem.

Currently, we don't know the mechanism that allows S. alterniflora to invade the salt marsh and replace the native plants in the Yangtze River estuary. Recent studies demonstrate that available N can affect the patterns of plant community diversity and succession in a salt marsh [START_REF] Levine | Nitrogen effects on an interaction chain in a salt marsh community[END_REF][START_REF] Rozema | Nitrogen and vegetation dynamics in european salt marshes[END_REF]. As a result of the physiological differences in growth rate, plant growth form and plant morphology between plant species, plants that assimilate N well will dominate salt marshes [START_REF] Rozema | Nitrogen and vegetation dynamics in european salt marshes[END_REF]. Therefore, we hypothesize that there may be two possible mechanisms that allow S. alterniflora to invade the salt marsh and replace native plants in the Yangtze River estuary: Firstly, S. alterniflora can more rapidly and efficiently assimilate inorganic N than the two native species; secondly, the exotic can improve the mineralization rate and bioavailability of N in the sediment of salt marsh. This hypothesis needs to be tested further.

Bioavailabilities of nutrients and metals are affected by a wide range of environmental characteristics of the sediments, most notably the pH and redox potential [START_REF] Sundareshwar | Phosphorus limitation of coastal ecosystem processes[END_REF][START_REF] Cacador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritime and Halimione portulacoides from Tagus estuary salt marshes[END_REF][START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF]. In coastal salt marshes, the sediments tend to be reduced. The anoxic condition may restrain N mineralization and facilitate denitrification. Thus, N is conventionally thought to limit primary production in coastal salt marshes. [START_REF] Sundareshwar | Phosphorus limitation of coastal ecosystem processes[END_REF][START_REF] Teal | Salt marsh values: retrospection from the end of the century[END_REF]. Phosphate can be present in the form of iron, aluminium and calcium oxides or adsorbed on the surface of minerals and organic materials. Among these forms, calcium-bound P is the most important fraction in the salt marsh of the Yangtze River estuary (Gao, Xu, & Zhang, 2001). [START_REF] Sundareshwar | Phosphorus limitation of coastal ecosystem processes[END_REF] found that P possibly limited the N-transforming bacteria communities and would affect carbon fixation, storage and primary production in some circumstances. In a reduced condition, most of the metal often binds to sulfides in the sediment. This form of metals is unavailable to plant uptake [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Williams | Phytoremediation in wetland ecosystems: progress, problems, and potential[END_REF].

Salt marsh macrophytes can oxidize the sediment in the root zone through the movement of oxygen downwards through aerenchyma tissue, however, which changes the sediment Eh and pH condition.

At the same time, this oxidation can increase the magnitude of nutrient cycling and improve the availability of nutrients and metals in wetland sediment [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF]Moorhead & Reddy,1988). Additionally, salt marsh macrophytes may act as a conduit for the movement of nutrients and toxic metals from the sediment to the surface and near coastal seas [START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF].

The roots of salt marsh plants can accumulate high concentrations of metals, but only a low proportion of the metal is allocated to aerial tissues, which indicates a restriction of transport upwards [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. Similar to previous studies, our results demonstrate that metal concentrations in the aboveground tissues are lower than in belowground tissues. It is accepted that salt marsh plants possibly employ three strategies to translocate metals to aboveground tissues: the excluder strategy, indicator strategy and accumulator strategy [START_REF] Baker | Accumulators and excluders-strategies in the response of plants to heavy metals[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF].

Many studies show that P. australis and S. alterniflora function as excluders. The shoot concentrations of metal are kept low until a critical soil concentration is reached, toxicity ensues, and unrestricted metal transport results [START_REF] Alberts | Metal concentrations in tissues of Spartina alterniflora (Loisel) and sediments of Georgia salt marshes[END_REF][START_REF] Peverly | Growth and trace metal absorption by Phragmites australis in wetlands constructed for landfill leach treatment[END_REF].

Our study shows a relatively consistent seasonal variation pattern of N and P concentrations in the aboveground tissues of three salt marsh plants. The concentrations of TN and TP in the aboveground tissues decreased gradually across the growing season. [START_REF] Roman | Aboveground and belowground primary production dynamics of two Delaware Bay tidal marshes[END_REF] presented similar results: the relative N concentration of standing live S. alterniflora tissue was at a peak during the spring, and there was a steady decline until a minimum was reached at the end of the growing season. The variation patterns of N and P in the aboveground tissues were correlated with the growth dynamic of the salt marsh plants. In the early stage of the growing season, a high nutrient proportion was found to accumulate in the newly emerged photosynthetic tissue (stem and leaves) of the macrophytes. The concentrations of N and P in the aboveground tissues decreased gradually with the growth of salt marsh plants. The nutrients in the aboveground tissue were slowly translocated to the belowground tissue as the plants started to senesce (Roman & Daiber, 194;[START_REF] White | Long-term 15 N-nitrogen retention in the vegetated sediments of a New England salt marsh[END_REF].

In regards to the seasonal pattern of metal concentrations, our results are consistent with patterns observed elsewhere [START_REF] Gleason | Seasonal and environmental variation in Mn, Fe, Cu and Zn content of Spartina alterniflora[END_REF][START_REF] Heller | Seasonal study of speciation of mercury (II) and monomethyl mercury in Spartina alterniflora from Great Bay Estuary, NH[END_REF][START_REF] Cacador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritime and Halimione portulacoides from Tagus estuary salt marshes[END_REF]. Two studies [START_REF] Gleason | Seasonal and environmental variation in Mn, Fe, Cu and Zn content of Spartina alterniflora[END_REF][START_REF] Heller | Seasonal study of speciation of mercury (II) and monomethyl mercury in Spartina alterniflora from Great Bay Estuary, NH[END_REF] discuss decreasing concentrations of metals in the leaf tissue of S. alterniflora across the growing season. The decline is attributed to a growth dilution effect. Additionally, [START_REF] Cacador | Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritime and Halimione portulacoides from Tagus estuary salt marshes[END_REF] suggest that the seasonal variations in root metal concentrations of Spartina maritima and Halimone portulacoides are not linearly correlated to aboveground tissues [START_REF] Ennabili | Biomass production and NPK retention in macrophytes from wetlands of the Tingitan Peninsula[END_REF].

The aboveground size of nutrient pools were mainly attributed to the biomass of the salt marsh plants. Maximum nutrient accumulations in the aboveground shoots corresponded to peak biomass, which occurred in mid to late summer. Similar trends were generally observed in two Delaware Bay tidal marshes [START_REF] Roman | Aboveground and belowground primary production dynamics of two Delaware Bay tidal marshes[END_REF]. As a result of the significant difference in the size of nutrient pools between the exotic and native plants, the exotic plant invasion may significantly influence the biogeochemistry process of the salt marsh ecosystem. [START_REF] Windham | Effects of an invasive reedgrass (Phragmites australis) on nitrogen cycling in brackish tidal marsh of New York and New Jersey[END_REF] examined the extent to which a change in plant species altered N cycling in a salt marsh. His results suggest that P. australis invasions appear to increase the magnitude of N cycles within salt marshes by sequestrating more N through plant and litter uptake, stimulating the soil mineralization rates, increasing the rates of coupled nitrification-denitrification in the sediment, and generating a shift of the dominance of N export pathways-from the export of available N (ammonium) to the export of unavailable N (N 2 ) to the atmosphere. Our results show that the aboveground nutrient pools of S. alterniflora were significantly greater than P. australis and S. mariqueter throughout the growing season as result of greater aboveground biomass, which shows that the S. alterniflora invasion significantly improved the rate of N and P assimilation and possibly increased the magnitude of nutrient cycles and altered the nutrient flux between the salt marsh and estuary.

Although most of the metals taken into the salt marsh plants remain belowground, amounts sufficient for concern are transported to the aboveground tissues [START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF][START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF]. By comparing with the aboveground metal pools of three salt marsh plants, we found that the aboveground metal pools of S. alterniflora were significantly greater than the two native species. This result is mainly the result of greater net primary production in plots of S. alterniflora. Many studies compare the aboveground pool sizes of trace metals in P. australis and S. alterniflora under field conditions [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF] and have found that P. australis appears to have a tighter restriction on the upward movement of metals, while S. alterniflora transports greater amounts to the aboveground tissues, especially the leaves. [START_REF] Windham | Lead uptake, distribution, and effects in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF] demonstrate that the replacement of S. alterniflora with P. australis may reduce Pb bioavailability by sequestering a greater proportion of its metal burden in the belowground tissues, which are likely to be ACCEPTED MANUSCRIPT permanently buried. Later, they found that the document replacement of S. alterniflora with invading populations of P. australis would lead to a decrease in mercury and chromium export from the aboveground biomass into the water column and the detrital pathway in low salt marshes and a possible increase in Cu and Zn export [START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. Our study demonstrates that a S. alterniflora invasion will possibly increase the import of Cu, Zn and Pb from the sediment into the estuarine water column and detrital food web in the Yangtze River estuary.

S. alterniflora can release large amount of metals through the excretion of metal-containing salts produced by leaf salt glands [START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF]Weis et al., 2002). [START_REF] Kraus | Accumulation and excretion of five heavy metals by the salt marsh cordgrass Spartina alterniflora[END_REF] estimated that S.

alterniflora had the theoretical potential to export 145 g cadmium, 260 g Pb, 104 g chromium, 260 g

Cu and 988 g nickel per ha/year through salt excretion. Greater metal release at higher salinities was observed as a result of more salt excretion. Therefore, it is certain that S. alterniflora can release larger quantities of metals into the marsh environment than native plants. When salt marshes flood, metals excreted from the leaves are resolubilized and can dissolve in the water column, which may influence the metal flux between a salt marsh and estuary [START_REF] Marinucci | Trophic importance of Spartina alterniflora production and decomposition to the marsh-estuarine ecosystem[END_REF]Weis et al., 2002).

In addition, [START_REF] Drifmeyer | Sorption and desorption of trace elements by Spartina alterniflora detritus[END_REF] report that metals from S. alterniflora detritus are extracted quickly and may be in a form readily available to consumers. Furthermore, two studies [START_REF] Heyes | Mercury and methylmercury in decomposing vegetation of a pristine and impounded wetland[END_REF]Helller & Webber, 1998), show a more toxic methylated form of mercury was present in S. alterniflora, and the percent of methylmercury in the total mercury increased from May to July (up to 48%). They speculate that either S. alterniflora was capable of methylating mercury, or there was a selective uptake from sediments. Relatively clean Spartina detritus may act primarily as a cation exchanger, adsorbing metals from sediments [START_REF] Giblin | Uptake and losses of heavy metals in sewage sludge by a New England salt marsh[END_REF].

Consequently, an invasion of S. alterniflora may promote metal bioavailability by transporting greater amounts of metals into the aboveground tissue-readily decomposable tissues-and releasing more metals to salt into the environment through the leaves [START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF]Weis et al., 2002).

When the aboveground tissues die and turn into detrius, metal stored in the tissues are likely to become available to deposit feeders and enter the estuarine food web [START_REF] Weis | Growth, survival, and metal content of marsh invertebrates fed diets of detritus from Spartina alterniflora Loisel and Phragmites australis Cav. Trin. ex Steud. Ffrom metal-contaminated and clean sites[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. Therefore, wetland managers and ecologists should be concerned with the effects of S. alterniflora invasions on the biogeochemistry processes of salt marsh ecosystems and particularly take measures to control the spreading of S. alterniflora in the Yangtze

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River estuary.

This study suggests that invasive S. alterniflora holds greater aboveground element pools than the two native plants and infers that its invasion will significantly increase the magnitude of nutrient cycling and bioavailability of metals in the salt marsh and transport more metals into the water column and the detrital food web in the estuary, which is harmful for the health of the ecosystem. To prove this position, three areas require further study: (1) the dynamic change of nutrients and metal concentrations in the litter decomposition process of three salt marsh plants, (2) whether consumption of contaminated detritus has effects on macro invertebrates and nekton, and (3) the influence of S.

alterniflora invasion on the bioavailabilities of nutrients and metals in the sediment and element flux between the salt marsh and estuary.

Conclusion

During the growing season of 2005, we studied the seasonal patterns of the aboveground biomass accumulation, tissue element concentration, and aboveground element pools of three dominant salt marsh macrophytes in the Yangtze River estuary. The aboveground biomass was significantly greater in plots of S. alterniflora than in plots of P. australis and S. mariqueter throughout the season, which shows that the S. alterniflora invasion significantly increased the aboveground primary productivity of the salt marsh ecosystem. The variation patterns of the nutrients and metals in the aboveground tissues were similar and showed a decreasing trend from April to October. Furthermore, the aboveground pools of nutrients and metals were consistently greater in the plots of S. alterniflora than P. australis and S. mariqueter. We suggest that the rapid replacement of the native plants (P. australis and S. mariqueter) by the exotic plant (S. alterniflora) will significantly increase the magnitude of nutrient cycling and bioavailability of metals in the salt marsh, transport more trace metals into the water column and the detrital food web, and further influence the biogeochemistry process of the Yangtze River estuary. Further study of the dynamic change of nutrient and trace metal concentrations in the process of salt marsh plant litter decomposition and the influence of exotic plant invasions on the biogeochemistry cycling process of salt marsh ecosystems is necessary.

Table 1 Linear regressions of TN and TP against time calculated for three salt marsh macrophytes.

Covariance analysis tests the difference among the slopes of three species, and r 2 estimates the proportion of the total variation explained by the regression model.

Regression analysis

Fig. 2

 2 Figure 2

Fig. 3 Fig. 4

 34 Figure 3

  Figure 5

Fig. 6

 6 Figure 6

Table 2 ,

 2 ANCOVA, P<0.05) and belowground Pb vs. time (Table2, ANCOVA, P<0.01) for the three species.

Among the metals analyzed, Zn was the most abundant in all parts of the plants. Cu concentrations in the aboveground tissues of S. mariqueter were the highest among the three species, except in April. In October, Cu and Pb concentrations in the aboveground tissues were significantly greater in S. mariqueter than P. australis and S. alterniflora (Figure

4

, P<0.05). From June to October, relatively low metal concentrations accumulated in the aboveground tissues of P. australis. In June, Cu concentrations in the aboveground tissues were significantly lower in P. australis than S. alterniflora and S. mariqueter (Figure

4

, P<0.05). The same resulted for Zn in August.

Belowground concentrations of the three metals were more variable between the species throughout the growing season. From April to August, Cu and Zn concentrations in S. alterniflora were the highest of the three species. Cu and Zn concentrations in the belowground tissue were significantly greater for S. alterniflora in April and August than for both native plants (Figure

4

, P<0.05). Similar to the aboveground tissues, the metal concentrations in belowground tissues of P. australis were the lowest among the three plants in October. For Zn and Pb, the belowground concentrations were significantly lower in P. australis and S. alterniflora than S. mariqueter in October (Figure

4

, P<0.05).

Table 2

 2 Linear regressions of Cu, Zn and Pb concentrations against time calculated for three salt marsh macrophytes. Covariance analysis tested the difference among the slopes of three plant species, and r 2 estimates the proportion of the total variation explained by the regression model.

	Factors	Plants	Regression analysis Intercept Slope	r 2	Covariance analysis F ratio P
	Aboveground Cu Phragmites australis	0.0231	-0.0015	0.58**	2.75	NS
		Spartina alterniflora	35.9800	-3.6317	0.79**
		Scirpus mariqueter	12.9297	-0.0763	0.21
	Aboveground Zn Phragmites australis	157.1804	-16.12	0.05	5.031	*
		Spartina alterniflora	110.7700	-8.9935	0.86**
		Scirpus mariqueter	83.8454	-5.7074	0.73**
	Aboveground Pb Phragmites australis	18.3300	-1.7289	0.15	3.575	*
		Spartina alterniflora	15.3895	-1.0884	0.24
		Scirpus mariqueter	2.9675	0.3382	0.07
	Belowground Cu Phragmites australis	84.6622	-8.1062	0.21*	2.719	NS
		Spartina alterniflora	125.2146	-11.6098	0.67**
		Scirpus mariqueter	38.5035	-2.5135	0.15
	Belowground Zn Phragmites australis	173.3475	-15.58	0.58**	1.901	NS
		Spartina alterniflora	291.9246	-24.3172	0.79**
		Scirpus mariqueter	174.9108	-13.2795	0.21
	Belowground Pb Phragmites australis	39.4440	-3.7542	0.05	6.755	**
		Spartina alterniflora	29.8884	-2.5782	0.86**
		Scirpus mariqueter	-1.8796	1.2606	0.14
	**=P<0.01; *=P<0.05; NS= P>0.05

and P at 2.76 g/m 2 in the

Acknowledgements

We thank Zheng L and Zhang KJ for providing help during each field visit, professor Zhang QP, Dr Chen Jiquan and Lise Waring for reviewing the manuscript and Dr Chao M for

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the biomass increment and that the greatest increase in metals occurs at the beginning of the growth period (March). This may be the result of modifying the chemistry of the rhizosphere during the plants' growth periods and consequently changing the metal availability at the interface of plant root-sediment. The same seasonal variation patterns in metal levels were also observed in other salt marsh plants, such as Aster tripolum, Puccinellia maritime and Spartina anglica [START_REF] Leendertse | Fate and effects of nutrients and heavy metals in experimental salt marsh ecosystems[END_REF]. But several other studies show different seasonal variation in metal concentrations in salt marsh plants. For example, [START_REF] Drifmeyer | Geographic variability in trace element levels in Spartina alterniflora[END_REF] note that standing dead S. alterniflora leaves had higher metal concentrations of Mn, Fe, Cu, Zn and Ni than the leaves of live plants, whereas P. australis showed a different seasonal pattern with metals [START_REF] Larsen | Macrophyte cycling of zinc, copper, lead, and cadmium in the littoral zone of a polluted and a non-polluted lake: II. Seasonal changes in heavy metal content of above-ground biomass and decomposing leaves of Phragmites australis (Cav.)[END_REF]. At this point, it is difficult to generalize about seasonal changes in metal levels [START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF].

P. australis concentrates a greater proportion of its body-burden of metals in the root and rhizome tissue than S. alterniflora [START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF][START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Windham | Lead uptake, distribution, and effects in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF]. [START_REF] Kraus | Accumulation and excretion of five heavy metals by the salt marsh cordgrass Spartina alterniflora[END_REF] reported that P. australis had higher root concentrations of Ni, Cadmium and Pb than S. alterniflora, and leaf levels of the metals were comparable in the two plants. In general, our study demonstrates that the metal concentrations of plant tissues (aboveground and belowground) were greater in S. alterniflora than in P. australis, which may be related to the difference in root system and metal accumulation pattern of the two plant species [START_REF] Breteler | Trace element enrichments in decomposing litter of Spartina alterniflora[END_REF][START_REF] Weis | Metal uptake, transport and release by wetland plants: implication for phytoremediation and restoration[END_REF][START_REF] Ye | Zinc, lead and cadmium tolerance, uptake and accumulation by the common reed, Phragmites australis (Cav.) Trin. ex Steudel[END_REF]. The larger root system and increased number of fine roots of S. alterniflora compared with P. australis can oxidize the rhizosphere to a greater extent and increase the availability of metals. [START_REF] Breteler | Trace element enrichments in decomposing litter of Spartina alterniflora[END_REF] found that the mercury concentration in S. alterniflora increased when growing in mercury-contaminated sediment, while different populations of P. australis from a contaminated site and the reference marsh had a similar metal uptake of Zn, Pb and cadmium [START_REF] Ye | Zinc, lead and cadmium tolerance, uptake and accumulation by the common reed, Phragmites australis (Cav.) Trin. ex Steudel[END_REF]. [START_REF] Windham | Lead uptake, distribution, and effects in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF] also show a fundamental difference in Pb allocation and accumulation pattern of S. alterniflora and P. australis. S. alterniflora allocated significantly more of the pool to aboveground biomass than P. australis.

In this study, the aboveground N pools in August were 19.9 g N /m 2 for P. australis, 24.4 g N /m 2 for S. alterniflora, and 8.4 g N /m 2 for S. mariqueter, which were equivalent to a N assimilation rate of 199 kg N/(ha•yr), 244 kg N/(ha•yr), and 84 kg N/(ha•yr). Our results are similar to those observed in other studies. In the salt marsh of the Wadden sea area, the rate of N assimilation is 95~230kg N/(ha•yr), average 160 kg N/(ha•yr) [START_REF] Rozema | Nitrogen and vegetation dynamics in european salt marshes[END_REF]. In an Australian treatment pond, the peak

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