Accepted Manuscript

Title: Effects of experimental warming and nitrogenenrichment on leaf and litter chemistry of a wetland grass,Phragmites australis

Author: Sabine Flury Mark O. Gessner

PII: S1439-1791(14)00041-3DOI: http://dx.doi.org/doi:10.1016/j.baae.2014.04.002Reference: BAAE 50779

To appear in:

Received date: 10-11-2013Revised date: 11-3-2014Accepted date: 12-4-2014

Please cite this article as: Flury, S., & Gessner, M. O.,Effects of experimentalwarming and nitrogen enrichment on leaf and litter chemistry of awetland grass, Phragmites australis, Basic and Applied Ecology (2014),http://dx.doi.org/10.1016/j.baae.2014.04.002

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Effects of experimental warming and nitrogen enrichment on leaf

and litter chemistry of a wetland grass, Phragmites australis

Sabine Flurya,b,c,*

and Mark O. Gessnera,b,d,e

5

a) Department of Aquatic Ecology, Eawag: Swiss Federal Institute of Aquatic Science and

Technology, Seestrasse 79, Kastanienbaum, Switzerland

b) Institute of Integrative Biology (IBZ), ETH Zürich, Universitätstrasse 16, Zürich,

Switzerland

c) Department of Chemical analytics and Biogeochemistry, Leibniz Institute of Freshwater 10

Ecology and Inland Fisheries (IGB), Müggelseedamm 310, Berlin, Germany

d) Department of Experimental Limnology, Leibniz Institute of Freshwater Ecology and

Inland Fisheries (IGB), Alte Fischerhütte 2, Stechlin, Germany, email: gessner@igb-

berlin.de)

e) Department of Ecology, Berlin Institute of Technology (TU Berlin), Ernst-Reuter-Platz 1, 15

Berlin, Germany

Present addresses:

3) Department of Chemical analytics and Biogeochemistry, Leibniz Institute of Freshwater

Ecology and Inland Fisheries (IGB), Müggelseedamm 310, Berlin, Germany 20

4) Department of Experimental Limnology, Leibniz Institute of Freshwater Ecology and

Inland Fisheries (IGB), Alte Fischerhütte 2, Stechlin, Germany

5) Department of Ecology, Berlin Institute of Technology (TU Berlin), Ernst-Reuter-Platz 1,

Berlin, Germany

25

*Manuscript

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Running title: Global-change effects on leaf chemistry

Number of words: 4248 (incl. Abstract, excluding Reference List)

Address for correspondence:

* Department of Chemical Analytics and Biogeochemistry 30

Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB)

Müggelseedamm 310, 12587 Berlin, Germany

Phone: +49 (0)30 6418 1960, Fax: +49 (0)30 6418 1700, Email: sabine.flury@yahoo.com

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Abstract 35

As global climate is warming and the nitrogen cycle accelerates, plants are likely to

respond not only by shifting community composition, but also by adjusting traits such as

tissue chemistry. We subjected a widespread wetland plant, Phragmites australis, to increased

nitrate supply and elevated temperature in enclosures that were established in a littoral

permanently submerged freshwater marsh. The nitrogen (N) and phosphorus (P) 40

concentrations in green leaves ranged from 11.4 to 13.8 mg N and from 1.5 to 2.0 mg P g-1

dry mass. While N content remained roughly the same, P decreased to 0.53 - 0.65 mg P g-1

dry mass in brown litter. Neither experimental warming of the water and sediment surface,

nor nitrate enrichment during the growing season affected nitrogen or phosphorus

concentrations in green leaves. Concentrations of the two major structural carbon compounds 45

in plant litter, cellulose and lignin, were also unaffected, ranging from 32.1 to 34.2 % of dry

mass for cellulose and from 16.3 to 17.7 % of dry mass for lignin. Warming, however,

significantly increased the nitrogen concentration of fully brown leaf litter. Thus, temperature

appears to be more important than the supply of dissolved N in the water, especially in

affecting leaf litter N concentrations in P. australis, even when only water but not air 50

temperature is increased. This result may have implications for decomposition processes and

decomposer food webs, which both depend on the quality of plant litter.

Keywords:

Climate change, experimental warming, nitrogen loading, Phragmites australis, plant tissue 55

quality, nutrient stoichiometry, lignin, mesocosm experiment

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Introduction

Increased emissions of greenhouse gases by human activities have led to warming of

the Earth’s atmosphere by approximately 0.13-0.33 °C per decade over the past century 60

(Trenberth, Jones, Ambenje, Bojariu, Easterling et al., 2007). Scenarios for this century

anticipate further warming by about 1.8-3.4 °C (Meehl, Stocker, Collins, Friedlingstein, Gaye

et al., 2007), a projected trend that is paralleled by increasing water temperatures of lakes,

rivers and wetlands (North, Livingstone, Hari, Koester, Niederhauser et al., 2013). Local

thermal pollution by cooling-water discharge from power plants and manufactories also 65

increases temperatures of the receiving water bodies (Kirillin, Shatwell & Kasprzak, 2013;

Prats, Val, Armengol & Dolz, 2010). Simultaneously, humans have greatly altered the global

nitrogen cycle by producing industrial fertilizer, intensifying agriculture, and releasing

geologically fixed N by combusting fossil fuels (Galloway, Townsend, Erisman, Bekunda,

Cai et al., 2008). As a result, global NO3- and NH4

+ deposition (wet and dry) has 70

approximately doubled from pre-industrial times (7.4·109 kg NO3

- yr

-1, 11.6·10

9 kg NH4

+ yr

-1)

to the present (14.8·109 kg NO3

- yr

-1, 24.6·10

9 kg NH4

+ yr

-1) and is predicted to increase

further, reaching about 19-25·109 kg NO3

- yr

-1 and 15-18·10

9 kg NH4

+ yr

-1 by 2100 (Luo,

Zender, Bian & Metzger, 2007).

Alterations in temperature, nitrogen supply and other factors of environmental change 75

can affect ecosystems through effects on plant community composition and traits such as

tissue chemistry (Chapin, Shaver, Giblin, Nadelhoffer & Laundre, 1995; Macek &

Rejmánková, 2007; Macek, Rejmánková & Lepś, 2010; Lü, Reed, Yu, He, Wang, et al.,

2013). For example, litter nutrient contents of wetland species is increased by exposure to

elevated N and P concentrations during shoot growth (Macek et al., 2007; Macek et al., 2010). 80

Such variation in plant tissue chemistry can have knock-on effects on trophic relationships in

ecosystems (Chapin et al., 1995; Hines, Megonigal & Denno, 2006). Both herbivore (Treydte,

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Heitkonig & Ludwig, 2009) and detritivore food webs (Hines et al., 2006) can be affected,

because differences in the tissue chemistry of life plants tend to persist after plant death (e.g.

Garten, Brice, Castro, Graham, Mayes et al., 2011) in spite of important chemical changes 85

during plant senescence. Ecosystem processes such as litter decomposition can also be

influenced because decomposition rates depend on litter chemistry, particularly

concentrations of lignin (Rahman, Tsukamoto, Yoneyama & Mostafa, 2013), nutrients (Aerts

& deCaluwe, 1997; Enriquez, Duarte & Sand-Jensen, 1993; Rejmánková & Houdková, 2006)

and secondary metabolites (Li, Zeng, Yu, Fan, Yang et al., 2011). 90

The aim of this study was to assess the effect of simulated increases in water

temperature and N deposition on the tissue chemistry of a widespread wetland plant

(Phragmites australis (Cav.) Trin. ex Steud.) that often assumes the role of a foundation

species in freshwater and brackish marshes. We hypothesized that N content would increase

in green leaves and leaf litter of plants subject to elevated NO3- supply (Macek et al., 2007; 95

Ruiz & Velasco, 2010). Furthermore, we expected a decrease in the lignin content of plants

exposed to increased N supply and elevated temperature due to a reduced investment in

sclerenchyma by rapidly growing shoots exposed to high nutrient supplies (Engloner, 2009)

and warming (Henry, Cleland, Field & Vitousek, 2005). We tested these hypotheses by

collecting leaves of P. australis naturally established in a permanently flooded littoral reed 100

stand and subjected, or not, to experimental warming and/or nitrogen enrichment in situ.

Green leaves collected in the middle of the growing season and fully brown leaf litter

collected from standing-dead shoots after plant senescence and partial decomposition

(Gessner, 2001) was subsequently analyzed for nutrients (N and P in both green and brown

leaves) and major structural carbon compounds (lignin and cellulose in brown leaves). 105

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Methods

Study site

The experimental site was a littoral freshwater marsh on the eastern shore of Lake Hallwil

(46°54’ N; 6°54’ E) in Central Switzerland. The lake is located at an altitude of 449 m above 110

sea level and is surrounded by gently rolling hills. It has a surface area of 10.2 km2, a volume

of 0.29 km3 and a mean and maximum depth of 28.6 and 48 m, respectively (Buesing &

Gessner, 2006). Vegetation in the littoral zone near the shoreline is composed of common

reed, Phragmites australis (Cav.) Trin. ex Steud., whose annual above-ground biomass

production has been estimated at 600 g C m-2

yr-1

(Buesing et al., 2006). P. australis is a tall 115

wetland grass with a world-wide distribution and that is thus likely to persist under future

warmer climate (Guo, Lambertini, Li, Meyerson, & Brix, 2013). As a foundation species in

brackish and freshwater marshes and along rivers and lake margins, it often develops

monospecific stands under various nutrient conditions. It is also often successful at colonizing

disturbed wetland areas. Due to its aggressive growth habit, it often invades wetlands and 120

replaces the local wetland species in North America (Marks, Lapin, & Randall, 1994), while it

usually does not displace other plant species in well-established European wetlands (Van Der

Werff, 1990). Development from seeds is rare so that spread occurs mostly vegetatively. Once

established, P. australis can be highly productive (e.g., Gessner, Schieferstein, Müller,

Barkmann & Lenfers, 1996), and since herbivore pressure during the growing season is low, 125

this results in a large production of plant litter in the fall.

Experimental set-up

Enclosures were installed at the site in early spring 2004. They were arranged in 4

blocks, each consisting of 4 circular enclosures (1.42 m diameter) and one unenclosed control 130

plot of equal size in the open marsh (Hines, Hammrich, Steiner & Gessner, 2013). Two

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enclosures in each block were heated to raise the ambient water temperature by 4 °C, the

other two were kept at ambient lake temperature. One heated and one unheated enclosures

also received extra nitrate to assess the effect of increased nitrogen loading in combination

with warming. This resulted in a 2x2 factorial design and an extra control in the open marsh 135

to assess potential enclosure effects.

Heating was achieved with 3-4 outdoor aquarium heaters controlled by

microprocessor-based temperature regulators (Type FCR-13A; Roth + Co AG, Switzerland)

(Hines et al., 2013). Aquarium pumps (Universal hobby centrifugal pump EHEIM; 300 l/h)

were connected to the heaters to achieve an even temperature distribution and to maintain a 140

weak but constant circulation in the enclosures. This pumping system differs from the one

described in Hines et al. (2013).

Every fourth week, ⅓ to ½ of the water in the enclosures was exchanged to limit

temporal changes in water chemistry compared to the open marsh (Hines et al., 2013).

Nitrogen was added each time after water had been partly exchanged. It was added as 145

Ca(NO3)2·4H2O dissolved in deionized H2O to minimize changes in ionic lake water

composition (Hines et al., 2013). The amount of nitrate added was calculated based on nitrate

concentrations measured in the lake in previous years at about the time of N addition.

Consequently, concentrations of the added solution varied over the months. The target

concentration in the water body was 5 the estimated ambient concentration of NO3-. The 150

total annual N load during the growing season before the dead leaves were collected was 10.7

g m-2

. Nitrate concentrations were measured from water samples collected each time after

exchanging water in the enclosures, and after NO3- addition. The water samples were brought

back to the laboratory and immediately filtered through cellulose acetate filters (0.45 μm pore

size, Sartorius AG, Göttingen, Germany). Concentrations were determined 155

spectrophotometrically based on the formation of 3- and 5-nitro-salicylate when nitrate is

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mixed with salicylate in acidic solution (DEV, 2012). Hines et al. (2013) provide a detailed

description of the experimental facility and the seasonal dynamics of water chemistry.

Litter chemistry 160

Fully developed green leaf blades of P. australis were collected in summer 2005 when

reed biomass peaked and leaf litter was collected from the enclosures in the same year in the

fall (November) when leaves had turned fully brown but were still attached to standing-dead

shoots. The leaves were collected at a defined height above the water level (1.3 ± 0.2 m). Both

green leaves and leaf litter were frozen and later freeze-dried and ground to pass a 0.2-mm 165

mesh screen. The resulting powder was used for all chemical analyses.

Total N and P concentrations in green leaves and leaf litter were determined

spectrophotometrically following simultaneous wet digestion of the leaf powder according to

the method by Ebina, Tsutsui & Shirai (1983). Briefly, 3 mg of ground leaf material was

placed in a 100-ml Duran glass bottle containing 50 ml of potassium peroxodisulfate solution 170

(20 g K2S2O8 plus 7 ml of 32% NaOH per liter of distilled water; pH = 12.7). All nitrogen

compounds are oxidized to nitrate at this pH. Samples were autoclaved for 1 h at 121 °C and

1100 hPa. The heat treatment transforms K2S2O8 to KHSO4. As a result, the pH drops to 2 and

organic phosphorus compounds are hydrolyzed to ortho-phosphate. After filtering the

resulting solution, nitrate and o-phosphate were quantified by measuring absorbance at 540 175

and 865 nm, respectively. Blank samples were routinely carried through the whole procedure.

Lignin and cellulose concentrations of the litter were determined gravimetrically

following a downscaled acid-detergent fiber method originally developed by Goering and Van

Soest (Gessner, 2005). About 280 mg of ground leaf material was weighed to the nearest 0.1

mg and placed in a Sovirell tube capped with Teflon-lined screw caps. Twenty ml of acid-180

detergent solution (0.5 M sulphuric acid plus 20 g l-1

CTAB = hexadecyltrimethylammonium

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bromide) was added and samples were incubated in a boiling water bath (96 ºC) for 60 min

with occasional swirling. The tube contents were filtered on tared Gooch crucibles placed on a

filter manifold. The mat formed by the filtered debris was broken up with a spatula and

washed 3× with hot distilled water (90 - 100 °C). This washing step was repeated with 185

acetone at ambient temperature until no more colour was removed. Care was taken that the

solvents were in contact with all particles. After drying the samples overnight at 105 °C, they

were cooled to room temperature in a desiccator and weighed to the nearest 0.1 mg. Cellulose

in the remaining fraction was hydrolyzed for 3 h by repeatedly overlaying the mat with 72%

H2SO4 and breaking it up and mixing it thoroughly with a spatula. Care was taken that all 190

material was constantly covered by acid. Subsequently, the samples were abundantly washed

with hot water until they were free from acid and then dried overnight at 105 °C. After

cooling and weighing, the crucibles were placed for 3 h in a muffle furnace at 550 °C to

combust all remaining organic material. The remaining ash was weighed after equilibrating

crucibles to room temperature in a desiccator. ADF (mass remaining after extraction with acid 195

CTAB), cellulose (mass loss upon hydrolysis with 72% sulphuric acid) and lignin (mass loss

upon final ignition) were subsequently calculated. Fiber analyses were only made with

powder of leaf litter, not green leaves.

Data analysis 200

A two-factorial ANOVA with temperature and N enrichment as factors and location in

the reed stand as blocking factor was used to test for differences in green leaf and litter

chemistry variables. These analyses were performed with SYSTAT 13 (Systat Software, Inc,

Chicago, IL, USA). Green leaves and leaf litter from one enclosure were excluded from the

analysis since temperature regulation was not properly working during parts of the 205

experiment. Lake control plots were also excluded from the analyses to take advantage of the

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2x2 factorial design to test for interactive effects of warming and N enrichment. To test for

differences between the unenclosed control plots and the unheated control enclosures,

location in the reed stand was included as blocking factor in a general linear model.

210

Results

Temperature and nitrogen enrichment

Temperature in heated enclosures exceeded that in the unheated enclosures and control

plots by an average of 3.2 ºC between November 2004 and November 2005 (Fig. 1A). This

indicates that experimental warming was successful overall, although temperature differences 215

between heated and unheated enclosures varied over time and the target temperature

difference of 4 °C was not perfectly achieved. Technical failure also prevented proper

warming in one of the heated enclosures; therefore, data from this enclosure were excluded

from the statistical analysis.

Nitrogen enrichment of the enclosures was also successful (Fig. 1B), resulting in 220

concentrations up to 14 times higher than the background shortly after N additions. More

detailed information on water chemistry responses to the NO3- addition are given in Hines et

al. (2013) for a different year.

Leaf and litter chemistry 225

The average N concentration in green leaves and brown leaf litter varied between 11.4

and 13.8 and between 11.5 and 14.1 mg N g-1

dry mass, respectively (Fig. 2A, D). Neither

nitrogen enrichment nor warming of enclosures during plant growth had an effect on the N

concentration in green leaves. However, the N concentration was significantly increased in

leaf litter from plants grown in heated enclosures (Fig. 2, p = 0.038), whereas nitrate addition 230

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had no effect on leaf litter N concentration. Furthermore, N concentrations did not differ

between control enclosures and unenclosed control plots.

The average P concentration of green leaves varied between 1.5 and 2.0 mg P g-1

dry

mass and decreased by about two thirds during senescence, resulting in P concentrations in

litter between 0.53 and 0.65 mg P g-1

dry mass (Fig. 2B, E). Neither warming nor nitrate 235

addition had an effect on the P concentration in fresh green leaves or brown leaf litter.

Concentrations tended to be higher especially in litter when temperature was elevated

(average of 18%), although the difference to the plants grown in control enclosures was not

significant (p = 0.091). There was also no significant difference in P concentrations of either

green leaves or leaf litter between control enclosures and unenclosed control plots. The molar 240

N:P ratios increased threefold during senescence, from 15.8 - 17.0 in green leaves to 45.3 -

52.3 in leaf litter, with no effect of either warming or N addition (Fig. 2C, F). These values

are within the range generally found for fresh biomass and litter in terrestrial plants

(Güsewell, 2004). Similarly, differences in green leaf and litter nutrient chemistry between

control plots and unheated enclosures for N, P or N:P ratios were small and never statistically 245

significant (p > 0.201).

Concentrations (across all treatments and controls) of lignin varied from 16.3 to 17.7%

of leaf litter dry mass, cellulose concentrations ranged between 32.4 and 34.2% and acid-

detergent fibre (ADF) from 50.7 to 53.4% of leaf dry mass (Fig. 3A-C). The lignin:N ratios

ranged between 12.5 and 15.5 (Fig. 3D). Although leaf litter from plants grown in heated 250

enclosures had slightly higher lignin:N ratios, the apparent difference was not statistically

significant (p = 0.090). Similarly, neither elevated temperature nor nitrogen enrichment

affected the concentrations of ADF, cellulose or lignin in the leaf litter. Differences in any of

these variables between unenclosed control plots and control enclosures were also small and

never statistically significant (p > 0.175). 255

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Discussion

Our experiment showed that environmental changes may significantly affect the tissue

chemistry of leaf litter of Phragmites australis growing in natural freshwater marshes, even

when effects on green leaf tissue are not apparent. The most important results of our study 260

were that concentrations of leaf litter nutrients, notably N, responded to experimental

warming, whereas periodic nitrogen enrichment of water had no effect on any of the tissue

chemistry variables assessed in either green leaves or leaf litter of P. australis.

The unexpected lack of N enrichment effects we observed contrasts with results of

other nutrient addition experiments conducted with emergent wetland plants (Macek et al., 265

2007; Rejmánková et al., 2006; Tylová, Steinbachová, Soukup, Gloser, & Votrubová, 2013).

Given that the saturation concentration of nitrate uptake by P. australis is much higher (100

µM; Araki, Mori & Hasegawa, 2005) than the background concentrations in the marsh water

at our study site (Fig. 1B), we had expected to see an increase especially in leaf N content of

the plants exposed to experimentally elevated N supplies. That P. australis did not, however, 270

show a clear response to nutrient addition could have multiple causes. In particular, it is

uncertain whether a notable fraction of the added N was effectively available to the plants.

The nitrate pulses we applied periodically to simulate increased N supply following distinct

rain events (Hines et al., 2013) may have been largely scavenged by algae and heterotrophic

microbes rather than being assimilated by P. australis. The added nitrate was indeed subject 275

to intense transformation processes, as is shown by nitrate concentrations in the water

invariably dropping to background levels within four weeks, or less, after nitrate enrichment

of the enclosures, even after years of experimentally enhanced N supply (Hines et al., 2013).

Shoots of P. australis at our study site developed very little adventitious roots extending into

the water column from submerged portions of the stems, and most of the root biomass was 280

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located at the litter-sediment interface (M.O. Gessner, pers. obs.). This suggests that the plants

primarily tapped nutrients released from freshly mineralized organic matter at the top

sediment layer, rather than using the pool of dissolved N in the overlying bulk water phase.

15N tracer studies could elucidate this hypothesis, but the isotope data required to clarify the

issue are not currently available. An additional fact to consider is that molar N:P ratios of 285

dissolved nutrients in the water (16-2600 in lake control plots, 18 to 26000 in enclosures)

point to P as the limiting nutrient, potentially weakening P. australis’ responsiveness to

increased experimental N addition. Furthermore, as has been discussed in Güsewell (2004),

plants down-regulate nitrate uptake by feedback inhibition when nitrate accumulates in roots.

One alternative hypothesis to account for the lack of an N addition effect is that leaf 290

tissue of P. australis is homeostatic in the sense that elemental stoichiometric relationships

vary little in response to changes in nutrient supply (Yu, Elser, He, Wu, Chen et al., 2011).

Tight homeostasis (Yu et al., 2011), however, can practically be ruled out as a mechanism

accounting for our results, because there was at least some scope for variation in leaf nutrient

concentrations as revealed by significantly higher N concentrations (and a possible tendency 295

to higher P concentrations) in response to our experimental warming treatment. This finding

agrees with data presented by Lessmann, Brix, Bauer, Clevering & Comin (2001) showing

that P. australis grown in warmer climates had increased leaf N and P concentrations.

Similarly, elevated N concentrations in leaf litter of various trees grown in warmer conditions

had been noted (Liu, Berg, Kutsch, Westman, Ilvesniemi et al., 2006; Huttunen, Aphalo, 300

Lehto, Niemela, Kuokkanen et al., 2009). In addition, leaf nutrient chemistry of P. australis

can also change in response to variation in other types of environmental conditions (e.g.

Schaller, Brackhage, Gessner, Bäuker & Dudel, 2012). Therefore, taken together, this

evidence provides little support for the hypothesis of homeostasis as a major mechanism

accounting for our result of lacking N enrichment effects. 305

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The increased concentrations of N in leaf litter that we observed in response to

experimental warming could be due to several non-mutually exclusive mechanisms. These

include influences of temperature on 1) nutrient uptake at the sediment surface, 2) nutrient

allocation within the plants, 3) the timing of leaf senescence, and 4) nutrient resorption

efficiency. Enhanced nutrient uptake is a straightforward mechanism, since elevated 310

temperature stimulates sediment microbial activity, thus accelerating nutrient recycling from

organic matter (e.g. Frank & Malkomes, 1993) and increasing the nutrient supply rate for P.

australis (Liikanen, Murtoniemi, Tanskanen, Vaisanen & Martikainen, 2002; Søndergaard,

Jeppesen, Kristensen & Sortkjaer, 1990). This is supported by a study by Tylová et al. (2013)

who found an elevated N assimilation in P. australis when the plants were exposed to 315

elevated pore water NO3- and NH4

+ concentrations. Furthermore, N resorption proficiency has

been reported to decrease with high N availability (e.g. van Heerwaarden, Toet, & Aerts,

2003). Additionally, nutrient acquisition could be directly promoted through a temperature-

induced stimulation of enzyme activities. Changes in N allocation patterns in response to

warming have been documented in Douglas fir (Tingey, McKane, Olszyk, Johnson, 320

Rygiewicz et al., 2003). However, the proposed mechanisms (i.e. increased protein

incorporation into lignin and up-regulation of photosynthesis leading to increased N demand

in foliar tissue) by Tingey et al. (2003) are unlikely to apply in our experiment, because no

warming effects on litter lignin contents occurred. Furthermore experimental warming of

water and sediment surface in the enclosures had little effect on plant canopy temperatures. 325

For this reason, and because we collected leaf litter at the fully-brown stage when nutrient

concentrations of P. australis leaves converge (Gessner, 2001), it is unlikely that delayed

senescence of plants exposed to warming (Yuan & Chen, 2009) are responsible for the

elevated nutrient content. This leaves enhanced nutrient uptake and availability in surface

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sediment layers due to warming as the most parsimonious mechanism behind the observed 330

warming effect on leaf N concentration.

Concentrations of the two major structural leaf constituents, cellulose and lignin, were

unaffected by warming and enhanced N supply, reflecting notable variation in responses

among plant species depending on environmental conditions and biotic interactions (Moura,

Bonine, Viana, Dornelas & Mazzafera, 2010). For example, Ford, Morrison & Wilson (1979) 335

found a positive correlation between temperature and lignin content in temperate but not in

tropical grass species, whereas Henry et al. (2005) found a decreased lignin content in grasses

but not in forbs in response to warming. The slight, though non-significant, decrease in the

lignin:N ratio in heated enclosures resulted mainly from an increased N concentration in the

litter, not from an effect on litter lignin. Similarly, acid detergent fiber (ADF), essentially the 340

sum of cellulose and lignin, failed to respond to nutrient supply in our experiment, as also

found in other temperate and tropical grasses (Johnson, Reiling, Mislevy & Hall, 2001; Tran,

Salgado & Lecomte, 2009). Clearly, the increases in water temperature and nitrogen supply in

our experiment to simulate two important aspects of global environmental change were

insufficient to alter concentrations of major structural carbon compounds in P. australis 345

leaves.

In conclusion, experimental warming of marsh water and surface sediments during

plant growth increased nitrogen concentrations of P. australis leaf litter, whereas increased

external nitrate supply had no effect on tissue N or P concentrations of green leaf or leaf litter.

Furthermore, none of the treatments had an effect on concentrations of lignin and cellulose, 350

the two primary structural carbon compounds in plant tissue. Temperature thus appears to be

more important than N supply in the overlaying water column in affecting leaf litter nitrogen

concentration in P. australis through decreased N resorption proficiency.

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Although the overall effects were not large in our experiment, warming-induced

changes in litter chemistry may have multiple other direct and indirect consequences in 355

aquatic ecosystems beyond immediate effects on plants. For example, i) plant litter

decomposition could be fostered by increased microbial or detritivore activity, which (ii) in

turn could stimulate nutrient release and hence (iii) plant nutrient uptake, potentially leading

(iv) to increased litter quality that could further stimulate decomposition (Enriquez et al.,

1993; Hines & Gessner, submitted). Furthermore, growth stimulation of plants through 360

increased nutrient availability may positively affect herbivorous consumers and even

predators due to greater amounts or quality of food resources (Hines et al., 2006). Although

these relationships are likely to be more complex in real ecosystems than described above,

they provide an initial framework for assessing effects of global warming beyond plants at the

food web and ecosystem level, at which initial effects could be either dampened or amplified 365

(Jeppesen, Moss, Bennion, Carvalho, DeMeester et al., 2010).

Acknowledgments

We thank A. Hammrich, D. Steiner, D. Hohmann, S. Käppeli, T. Neuenschwander and

M. Schindler for their assistance during field and laboratory work, and Richard Illi and the 370

AUA lab at Eawag for water and plant tissue analyses. This study was funded by the Swiss

National Science Foundation (SNF; grant no. 3100A0-108441) and the Swiss State Secretariat

for Education and Research (SER) through the Euro-limpacs project supported under the 7th

Framework Programme of the EU Commission.

375

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Figure legends

Figure 1. (A) Frequency distribution of average water temperature differences between

control and warmed enclosures from November 2004 to 2005. Data from one enclosure pair

were excluded because of poor temperature regulation during parts of the experiment. (B) 545

NO3- concentrations (mean ± 1 SE) of enclosure water between November 2004 and 2005

shortly after monthly fertilizer additions. L = unenclosed control, 0 = ambient temperature, H

= heated (ambient+4 ºC), 0N = ambient temperature + NO3- addition, HN = heated + NO3

-

addition. Only one error bar is shown where error bars are overlapping; where error bars are

invisible, they are smaller than the symbols. 550

Figure 2. Nitrogen concentration, phosphorus concentration and molar N:P ratios in green

leaves (A, B, C) and in fully brown leaf litter (D, E, F), respectively, of Phragmites australis

grown under two temperature and NO3- supply regimes. L = unenclosed control, 0 = ambient

temperature, H = heated (ambient + 4 ºC), 0N = ambient temperature + NO3- addition, HN = 555

heated + NO3-. Bars represent means ± 1 SE with n=3 to 4.

Figure 3. Concentrations of lignin (A), cellulose (B) and acid detergent fiber (C), and the

lignin:N ratio (D) of Phragmites australis leaf litter from shoots grown under two temperature

and NO3- supply regimes. L = unenclosed control, 0 = ambient temperature, H = heated 560

(ambient + 4 ºC), 0N = ambient temperature + NO3- addition, HN = heated + NO3

-. Bars

represent means ± 1 SE with n=3 to 4.

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0 2 4 6 80

5

10

15

20Fr

eq

ue

ncy

(%

)

Temperature di!erence (°C)

(A)

N J M M J S N0

2

4

6

NO

- 3 -

N (

mg

l-1

)

L 0 0N H HN

(B)

D F A J A O

Figure 1

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Leaf litter

0

5

10

15

20

0.0

0.5

1.0

1.5

2.0

2.5

L 0 H 0N HN0

20

40

60

(D)

(E)

(F)

0

5

10

15

20

N (

mg

g-1

DM

)Green leaves

0.0

0.5

1.0

1.5

2.0

2.5

L 0 H 0N HN0

20

40

60

Mo

lar

N:P

(A)

(B)

(C)

P (

mg

g-1

DM

)Figure 2

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L 0 H 0N HN0

10

20

30

40

Lig

nin

:N

Ce

llu

lose

(%

DM

)

AD

F (

% D

M)

Lig

nin

(%

DM

)

0

5

10

15

20

0

20

40

60

L 0 H 0N HN0

5

10

15

20

(A)

(B)

(C)

(D)

Figure 3

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Zusammenfassung

Im Zuge der globalen Klimaerwärmung und der Beschleunigung des

Stickstoffkreislaufs ist es wahrscheinlich, dass die Vegetation nicht nur durch

Veränderungen der derzeit etablierten Artengemeinschaften, sondern auch durch

Verschiebung von Pflanzenmerkmalen wie der chemischen Gewebezusammensetzung

reagieren. In einem permanent überfluteten natürlichen Uferröhricht errichteten wir

Mesokosmen, um die Reaktion einer weit verbreiteten Feuchtgebietspflanze

(Phragmites australis) auf erhöhte Nitratverfügbarkeit und erhöhte

Wassertemperaturen zu testen. Die Stickstoff- und Phosphorkonzentrationen der

grünen Blätter variierten zwischen 11.4 und 13.8 mg N bzw. zwischen 1.5 und 2.0 mg

P g-1

Trockenmasse. Während sich der P Gehalt in den braunen Blättern auf 0.53 -

0.65 mg P g-1

Trockenmasse verringert hat, blieb der N Gehalt während der

Seneszenz unverändert. Weder die experimentelle Erwärmung des Wassers noch die

Erhöhung des Nitratangebots während der Wachstumsperiode hatte einen Einfluss auf

die Stickstoff- oder Phosphorkonzentrationen grüner Blätter. Ebenfalls unbeeinflusst

war der Gehalt der wichtigsten Kohlenstoffverbindungen - Cellulose und Lignin - in

der Blattstreu , wobei der Cellulose- und Ligningehalt zwischen 32.1 und 34.2 %

bzw. 16.3 und 17.7 % der Trockenmasse schwankte. Erwärmung des Wassers führte

dagegen zu einem signifikanten Anstieg des Stickstoffgehalts der Blattstreu. Diese

Ergebnisse weisen darauf hin, dass die Temperatur eine wichtigere Rolle für die

Stickstoffkonzentration in Blattstreu spielt als die Verfügbarkeit von gelöstem

Stickstoff im Wasser, selbst wenn nur die Wasser-, nicht aber die Lufttemperatur

direkt erhöht ist. Daraus ergeben sich mögliche Konsequenzen für Abbauprozesse und

Nahrungsnetze von Destruenten, die beide maßgeblich von der Qualität der

Pflanzenstreu abhängen.

Abstract in German