Influences of elevated ozone and carbon dioxide in growth responses of lowland hay meadow mesocosms

Environmental Pollution 144 (2006) 101e111 www.elsevier.com/locate/envpol

Influences of elevated ozone and carbon dioxide in growth responses of lowland hay meadow mesocosms Kaisa Ra¨mo¨ a,*, Teri Kanerva a, Suvi Nikula a, Katinka Ojanpera¨ b, Sirkku Manninen a a

Department of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 1, P.O. Box 65, 00014 Helsinki, Finland b MTT, Agrifood Research Finland, Environmental Research, 31600 Jokioinen, Finland Received 5 September 2005; received in revised form 4 January 2006; accepted 4 January 2006

Species- and parameter-specific differences found in O3 responses and CO2 amelioration in wild plants under competitive environment. Abstract We studied the effects of relatively low levels of O3 (40e50 ppb) and CO2-enrichment (þ 100 ppm) on a northern European lowland hay meadow during the summers 2002e2004 using open-top chambers (OTCs) and ground-planted mesocosms. Ozone reduced the aboveground biomass of the community (up to 40%), and four out of seven species (Campanula rotundifolia, Fragaria vesca, Trifolium medium, Vicia cracca) showed either significant growth reduction and/or visible injuries under elevated O3. However, the reductions in aboveground biomass were not reflected as changes in the dominance of different functional groups or in the total community root biomass. Elevated CO2 did not amend the detrimental effects of O3 on aboveground biomass. Elevated CO2 alone had only minor effects. An O3-induced reduction in the aboveground biomass and N pool of the community are likely to have important consequences in the nutrient cycling of the ecosystem. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Semi-natural vegetation; Visible O3 injury; Biomass; Nitrogen concentration; Open-top chamber

1. Introduction The concentrations of tropospheric ozone (O3) and carbon dioxide (CO2) are rising due to human activity (IPCC, 2001; Vingarzan, 2004). Even though O3 is regarded as a significant threat to biodiversity in Europe (Catizzone et al., 1998), scientists have only recently commenced to study the potential changes taking place in the semi-natural plant communities. Experimental evidence on the interactive effects of CO2 and O3 on wild plants and plant competition is limited, and the outcome is difficult to predict since elevated CO2 and O3 have contrasting ways of affecting plant growth. The direct effects of CO2 are mainly beneficial, including stimulation in photosynthesis, growth, and resource allocation to roots

* Corresponding author. Tel.: þ358 9 191 57769; fax: þ358 9 191 57763. E-mail address: [email protected] (K. Ra¨mo¨). 0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2006.01.009

(Bazzaz, 1990; Jablonski et al., 2002), whereas O3 is a toxic compound that may cause visible injuries and substantial reductions in photosynthesis, growth, and seed production in a variety of wild species (Black et al., 2000; Davison and Barnes, 1998; Franzaring et al., 2000). However, not all species react similarly to O3 and CO2, and there is considerable variation both between and within wild species (e.g. Bassin et al., 2004; Fuhrer et al., 2003; Poorter and Navas, 2003). Most studies have been conducted as single plant or monoculture studies (Davison and Barnes, 1998). That study design is, however, in contrast with the fierce interspecific competition plants experience in natural communities. Differences in sensitivity or competitive ability may define the outcome of competition in altered atmospheric environments (e.g. Andersen et al., 2001; Ashmore et al., 1996; Barbo et al., 1998, 2002; Bazzaz, 1990). According to some simple competition experiments, the relative abundance of grasses increases on account of herbs when subjected to elevated O3 (e.g. Nussbaum et al.,

102

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

1995; Rebbeck et al., 1988). Results of more complex communities have indicated alterations in structure and species diversity and suggest increases in grass biomass at the expense of legumes with increasing O3 concentrations (e.g. Ashmore and Ainsworth, 1995; Ashmore et al., 1995). However, a recent multi-year study by Tonneijck et al. (2004) suggested that interspecific competition may not always enhance plants’ responses to O3. Elevated CO2 mainly acts indirectly by intensifying competition for light, water, and nutrients in a competitive environment (Bazzaz and McCounnaughay, 1992). Contrary to O3 responses, legumes and herbs may increase their proportion when grown in competition with grasses in a CO2-enriched environment (e.g. Owensby et al., 1999; Poorter and Navas, 2003). The responses of wild plants to a combination of elevated O3 and CO2 have received little attention, and the existing literature is controversial. As elevated CO2 generally decreases stomatal conductance and increases C fixation and stress tolerance, it may thereby protect plants from the negative effects of O3 (Allen, 1990). Several studies suggest that elevated CO2 ameliorates O3 injury in some crop plants (Booker and Fiscus, 2005; Booker et al., 2005; Cardoso-Vilhena et al., 2004; Fiscus et al., 2005; Heagle et al., 1999, 2000; McKee et al., 1997, 2000; Morgan et al., 2003; Mulholland et al., 1997a; Volin et al., 1998), but not in others (Balaguer et al., 1995; Barnes et al., 1995; Heagle et al., 2002, 2003). To date, there are few studies conducted on interspecific competition under elevated O3 and CO2 (Johnson et al., 1996; Mortensen, 1997). Although the current O3 levels have not been reported to cause visible injuries or growth reductions in wild plants in Finland (Timonen et al., 2004), the forecast increases in O3 concentrations within the next decades may have unpredictable effects. The climatic conditions, especially the long summer days and high relative humidity, are potentially favorable to high O3 uptake (Benton et al., 2000; DeTemmerman et al., 2002; Pleijel et al., 1999). Furthermore, low mean temperature and high soil water content may increase the susceptibility of plants to O3 (e.g. Bungener et al., 1999a,b). The relatively cold and varying winter characteristic of Finland is an interesting additional feature, because successful over-wintering may be a critical point in determining the community structure, as the two gases may alter the root-to-shoot ratios, stolon densities, and energy reserves of various species (Bazzaz, 1990; Rebbeck et al., 1988; Wilbourn et al., 1995). The purpose of the present study was to find out how a long-term exposure to O3 and/or CO2 alters species performance and community structure on a lowland hay meadow. We hypothesized that the proportion of grasses would increase on account of especially N2-fixing herbs (i.e. legumes) under elevated O3, whereas under elevated CO2, the proportion of grasses would decrease. Under the combination of elevated O3 and CO2, we expected that the detrimental effects of O3 would be diminished and the proportion of the species of intermediate O3 tolerance would increase. Our study design contrasts with previous studies in that the competition experienced among plants in natural communities is a component of the experiment. Differences in sensitivity to the treatment gases combined with competitive ability should help delineate

species responses in a lowland hay meadow under altered atmospheric environments. 2. Materials and methods 2.1. Cultivation and fumigations To study the effects of O3 and CO2 on a meadow community, we used large mesocosms with species typical to lowland hay meadows (EU Council directive 92). The mesocosms were designed to hold the major functional types of plants that occur on northern European lowland hay meadows: grasses (Agrostis capillaris L., Anthoxanthum odoratum L.), herbs (Fragaria vesca L., Campanula rotundifolia L., Ranunculus acris L.,) and legumes (Trifolium medium L., Vicia cracca L.). The above-mentioned perennial species grow in open, dry habitats and tolerate disturbance (Ha¨met-Ahti et al., 1998). The grasses are archaeophytes, while the herbs and legumes are native and common to Finland. According to previous studies conducted on individual plants, in general, the species belonging to the functional type ‘‘grasses’’ have been classified as O3-tolerant and species belonging to the functional type ‘‘legumes’’ as O3-sentitive (Ashmore et al., 1996; Mortensen, 1992, 1994; Power and Ashmore, 2002). The seeds were obtained from a commercial supplier (Kukkiva Niitty) who had collected the seeds from natural populations in south-western Finland. A commercial supplier and pooled seeds were used to obtain an adequate number of seedlings in a short period of time. The open-top chamber (OTC) experiment was carried out in 2002e2004 in Jokioinen (60
490 N, 23
280 E) in south-western Finland, at 100 m above sea level. At the end of March 2002, the seeds were germinated in peat-sand mixture in the boreal greenhouses of the University of Helsinki. At the end of May 2002, the seedlings were transplanted to small pots containing peat-sand mixture and transferred to the experimental field where they were allowed to grow under ambient air. During June 18e20, 2002, 25 seedlings of F. vesca, C. rotundifolia, R. acris, A. odoratum, and A. capillaris, and five T. medium and eight V. cracca seedlings were randomly transplanted to mesocosms of approximately 2.25 m2 (diameter 1.5 m; for illustrations see Kanerva et al., 2005). The spacing between individuals was approximately 20 cm. A semitransparent net (green plastic, mesh size 1.5  2 mm) was dug into the soil (up to 20 cm above soil surface) around the mesocosm to restrict the expansion of the experimental plants. If alien plants were found inside the mesocosms, they were removed by hand. Open-ground planting was chosen instead of pots as it is less limiting to root growth and has a minor edge effect (Wilbourn et al., 1995), thus allowing for more natural root competition. Before the onset of the experiment, T. medium and V. cracca were inoculated with proper Rhizobiums. Each individual received 5 ml of Rhizobium water mixture resulting in 50  106 living cells/seedling. The soil of the mesocosm was sand: peat mixture (sand 86.5%/silt 11.9%/clay 1.6%) and pH was 6.7e7.0. The mesocosms were fertilized with a soluble NPK fertilizer (N-P2-O5-K2O 19-10-24) (Kekkila¨ Taimi-Superex) after transplanting the seedlings in early July 2002. Each mesocosm received an addition of N 2.38 mg L1. The fertilizing process was repeated once later in July. The soil physical and chemical properties have been summarized by Kanerva et al. (2005). Any occurring insects were manually removed and the plants were then sprayed with slightly alkaline water-soap solution (Havin Ma¨ntysuopa, Henkel Finland Oy). The OTC treatments (three replicates in each) were as follows: NF (nonfiltered ambient air), NF þ O3 (1.5  ambient O3), NF þ CO2 (1.3  ambient CO2) and NF þ O3 þ CO2 (1.5  ambient O3 and 1.3  ambient CO2). Three open-field ambient air plots (AA) were established as controls for the chamber effect. The target O3 and CO2 concentrations were chosen to simulate the predicted ambient concentrations in the year 2050 with a yearly increase of 0.5e 2% in O3 (Vingarzan, 2004) and a moderate 0.5% increase in CO2 (IPCC, 2001). The CO2 fumigation does not, however, replicate the projected real world situation completely accurately because instead of 24-h exposure we fumigated the mesocosms only for 9 h per day (from 10:00 to 19:00 h), seven days a week. The soil was not fumigated. The exposure lasted from July 1 to August 28 in 2002, from June 3 to August 31 in 2003, and from May 18 to August 22 in 2004. The OTCs (3 m in diameter, 2.8 m in height) with added frustum were used. All OTCs were equipped with blowers to exchange 3 air volumes per

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111 minute. The OTCs were placed on the experimental field in a completely randomized design, and the PCV covers were removed for the winter. Ozone was generated by electric discharge using pure oxygen with a Fischer 502 O3 generator, and the gaseous CO2 was distributed into the air circulation system of each CO2 chamber using Kyto¨la¨ EK-8937 rotameters for CO2. The gases were monitored at approximately 1 m above the soil surface. In July 2004, ambient O3 concentrations were also measured from a 3-m high mast to enable comparisons with local EMEP station data (Fig. 1). Relative air humidity and temperature were measured from two NF OTCs and one AA plot. In the summer of 2002, the mesocosms were regularly watered with equal amounts of water, and the soil water content (5 cm depth) was determined with a portable capacitance sensor (TDR) (Theta Meter, type HH1, Delta D Devices, Cambridge, UK). The plots received ambient rainfall and were additionally watered with tap water when necessary (TDR readings lower than

1 July

O3 concentration (ppb)

70 60 50 40 30 20 10

0

0

:0

23

0

:0 21

0

:0

19

0

:0

17

0

15

:0

0

:0

:0

13

00

11

00

9:

00

7:

00

5:

3:

1:

00

0

Time of day 15 July

O3 concentration (ppb)

70 60 50 40 30 20 10

0 5: 00 7: 00 9: 00 11 :0 0 13 :0 0 15 :0 0 17 :0 0 19 :0 0 21 :0 0 23 :0 0

30 July

70 60 50 40 30 20 10

Visible O3 injuries were recorded according to the ICP-Vegetation protocol (UN-ECE, 2003) throughout the three-year study. Each mesocosm was monitored regularly at one-week intervals during the growing seasons of 2002e 2003. During the growing season of 2004, visible O3 injuries were initially scored once a fortnight, but after the appearance of injuries, recordings were made weekly. The following scores were used to assess the proportion of both injured individuals and injured leaves per mesocosm: 0, no injury; 1, very slight injury, occurrence of the first symptoms; 2, slight injury, 1e5%; 3, moderate injury, 5e25%; 4, heavy injury, 25e50%; 5, very heavy injury, 50e90%; and 6, total injury, 90e100% of the individuals or leaves in each mesocosm are injured. The aboveground biomass of each species in each mesocosm was harvested separately at the height of 3 cm on 2e5 September 2003 and 31 Auguste3 September 2004. Plants were dried to constant weight at 60
C for the minimum of 72 h and weighed subsequently. The dried plant biomass was then returned to the mesocosms to maintain the nutrient balance. After the first growing season, 2002, no harvest was made to assure mesocosm establishment and successful over-wintering. We also scored growth onset and flowering, but the results will be presented in another paper. Root biomass was determined at the end of the growing season of 2004 by taking five soil core samples (Cope´k soil core; 25 cm  5 cm diameter) from each mesocosm. The cores were taken from between the plants and at least 25 cm from the mesocosm edge and other cores. The cores were divided into vertical samples of 0e5 cm and 7.5e12.5 cm, so that each sub-sample contained 98 cm3 of soil. Sub-samples were combined to a bulk sample for each mesocosm (2  490 cm3 soil) and stored in a freezer (18
C). The bulks were later thawed, and the roots carefully separated from the soil by sinking the soil core into a bowl of water. This was repeated until the roots were carefully washed. Floating fine roots were collected using tweezers. Subsequently, roots were dried to constant weight at 60
C and weighed. Nitrogen concentrations were analyzed from dried and milled (1 mm sieve) aboveground plant samples with a Leco-CNS 1000 analyzer (Leco Corp., USA). In the summers of 2003 and 2004, samples from each mesocosm for all species but C. rotundifolia (only in 2004) were collected a week after the fumigations ceased. The total community N pool was calculated by multiplying the N concentration by the biomass of each species, and summing the values of each species to a total community N pool for each mesocosm.

Within-year effects of all treatments (including AA plots) on each species and functional groups were analyzed by one-way analysis of variance (ANOVA) (SPSS 12.0.1 for Windows), with LSD post hoc tests. The effects of OTC treatments (excluding AA plots) and years (or time) on different plant parameters were analyzed using a factor ANOVA. For statistical analysis, a continuous variable for the class of visible injuries was formed of the categorical variable using the following median values for the classes: I, 0.5%; II, 2%; III, 15%; IV, 37.5%; V, 70%; and VI, 95%. Repeated-measures-ANOVA was used to evaluate the differences between the treatments in the timerepeated measurements of visible injuries. Spearman correlations were performed to observe possible correlations between total aboveground biomass and N concentration of the species. If the assumptions of normality and homogeneity of variances were not met, the data was log10 transformed. The results were considered significant at p < 0.05 and marginally significant at p < 0.10.

3:

00 5: 00 7: 00 9: 00 11 :0 0 13 :0 0 15 :0 0 17 :0 0 19 :0 0 21 :0 0 23 :0 0

0

00

O3 concentration (ppb)

2.2. Plant measurements

2.3. Statistics Time of day

1:

0.20 m3 m3). From the summer of 2003 onwards, the mesocosms were equipped with tensiometers (Soil Moisture Equipment Corp., Santa Barbara, CA) to maintain the desired (5e30 kPa) moisture regimes. The water content in the soil was determined with gypsum blocks. For details, see Kanerva et al. (2005).

3: 0

1: 0

0

0

103

Time of day AA

NF+O3

3. Results mast

Fig. 1. Variation in daily O3 concentrations in the NF þ O3 and AA (1 m height) treatments and ambient air mast (3 m height) at three different occasions in July 2004. Arrows indicate the onset and offset of the fumigations. Values are 1-h means of three NF þ O3 OTCs, two AA plots and a single mast.

3.1. Climate data and air quality There was considerable variation in the climate over the three years (Table 1), the summer (MayeAugust) 2002 being

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

104

Table 1 Mean temperature (
C) and monthly precipitation (mm) at the Jokioinen observatory in MayeAugust 2002e2004, and the long-term average for the site (Finnish Meteorological Institute, 2004) 2002

2003

2004 9.6 12.2 15.5 15.7

Temperature (
C)

May June July August

11.3 15.6 18.2 17.9

9.7 12.9 19.7 15.0

Precipitation (mm)

May June July August

32 95 66 14

82 72 68 80

60 122 129 86

30-year average 9.5 14.1 16.1 14.5 35 47 80 83

the warmest and driest, and the summer 2004 the coolest and wettest. Mean air temperatures in the OTCs did not differ from those measured in the AA plots in any of the summers ( p > 0.1), but relative humidity tended to be slightly higher ( p ¼ 0.096) in the AA plots than in the OTCs in the summer 2002. Ambient 1-h mean O3 concentrations during the exposures (10:00 to 19:00 h) were generally below 30 ppb in the treatments receiving ambient O3, and between 40 and 50 ppb in the treatments receiving supplemental O3 (NF þ O3 and NF þ O3 þ CO2). Accumulated exposures above a threshold value of 40 ppb (AOT40) ranged from 85 to 674 in the ambient O3 treatments and from 3132 to 10,331 ppb in the treatments receiving supplemental O3, resulting in a more than twofold AOT40 value in the last summer of 2004 (Table 2). The 3-m high mast yielded approximately 20% higher O3 concentrations compared to those on the AA plot (both 24-h and 9-h Table 2 9-h mean O3 (ppb) and CO2 (ppm) concentrations and AOT40 values (ppb h) for each treatment, mean temperature (
C), and relative humidity (%) for two OTCs and one AA plot in 2002e2004 Year

AA

NF

NF þ O3 NF þ CO2 NF þ O3 þ CO2

2002a O3 33 31 CO2 353 351 AOT40 674 375 Temperature 17.5 18.1 Relative humidity 83.1 79.5

47 355 5260

31 455 409

41 448 3132

31 27 2003b O3 CO2 352 354 AOT40 456 85 Temperature 17.3 18.1 Relative humidity 76.6 75.6

39 357 4782

24 455 93

40 449 5014

31 28 52 2004c O3 CO2 410 407 410 AOT40 309 138 10331 Temperature 15.7 16.6 Relative humidity 81.6 76.0

28 530 139

53 530 10886

The gas concentrations from the OTCs are measured 1 m above ground during the fumigation periods. a 1 July to 28 August. b 3 June to 31 August. c 19 May to 23 August.

means), which measured O3 at the height of 1 m (Fig. 1). Also, the CO2 concentrations in both ambient and elevated treatments were somewhat higher in the final year 2004. 3.2. Visible O3 injuries In the last summer 2004, the two leguminous species, T. medium and V. cracca, showed significant visible foliar injury in the NF þ O3 and NF þ O3 þ CO2 treatments as compared to the NF treatments when measured as the percentage of injured individuals (p < 0.001; data not shown) and percentage of leaves showing O3 injury ( p < 0.001; Fig. 2). Visible injuries were manifested on both species as white stippling on the upper leaf surface. There was no difference in whether O3 was given alone or in combination with CO2. The extent of the visible injuries was higher in T. medium, of which 59% of the leaves showed visible injuries compared to that of 14% in V. cracca, in late August 2004. Ozone induced foliar injury was not observed in the legumes during the first two summers. Neither did we observe O3-induced visible injuries in the other species in any of the years. 3.3. Biomass The total aboveground biomass was reduced by 30% in 2003 and by 40% in 2004 in the NF þ O3 treatment compared to the NF treatment (p < 0.05; Fig. 3). CO2 did not induce any growth enhancement. The total aboveground biomass tended to be smaller (p ¼ 0.070) in the NF þ O3 þ CO2 treatment compared to the NF treatment in year 2004. The total aboveground biomass production of the mesocosms increased from 2003 to 2004 ( p < 0.001, Fig. 3 and Table 3), and at the end of the experiment in 2004, it was on the average 408 g dry weight m2. With regard to the individual species, the biomass of C. rotundifolia and F. vesca decreased in the summer 2003 when exposed to elevated O3 alone and in combination with CO2 (p < 0.05; Fig. 4). However, the biomass of F. vesca did not differ in either of the supplemental O3 treatments when compared to the NF treatment in the summer 2004. The mean dry weight of V. cracca was 47% and 61% lower in the NF þ O3 treatment as compared to the NF treatment in 2003 and 2004, respectively, but the differences were only marginally significant (p ¼ 0.097 and p ¼ 0.078) due to the marked variation between the three replicate OTCs. In the summer 2003, the V. cracca biomass in the NF þ O3 þ CO2 treatment was also lower than in the NF treatment, but this difference did not persist to the year 2004. No growth enhancement by CO2 was observed in any of the species. A. capillaris was the most dominant species in all treatments but AA, in which T. medium dominated. The least abundant species, irrespective of treatment, was C. rotundifolia. The treatments caused comparatively few and temporary changes in the relative biomass of different species and functional groups (Table 4). For instance, in summer 2003, the proportion of C. rotundifolia of the total aboveground biomass was reduced in the NF þ O3 and NF þ O3 þ CO2 treatments

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

T. medium

V. cracca

100

100

AA

NF

NF+O3

NF+CO2

18.8.

13.8.

8.8.

0

3.8.

0

29.7.

10

24.7.

20

10

19.7.

20

14.7.

30

9.7.

40

30

4.7.

40

29.6.

50

24.6.

50

19.6.

60

14.6.

70

60

9.6.

70

3.6.

80

29.5.

80

24.5.

90

19.5.

90

Daily max 1-h O3 concentration (ppb)

18.8.

0

8.8.

0

13.8.

10

3.8.

10

29.7.

20

24.7.

20

19.7.

30

9.7.

30

14.7.

40

4.7.

40

29.6.

50

24.6.

50

19.6.

60

14.6.

60

9.6.

70

3.6.

70

29.5.

80

24.5.

90

80

Daily max 1-h O3 concentration (ppb)

100

90

19.5.

% of leaves showing visible O3 injury

100

% of leaves showing visible O3 injury

105

NF+O3+CO2

Plant biomass (g dw m-2)

600

p = 0.070

Fig. 2. Development of visible O3 injuries (% of leaves showing injury) in T. medium and V. cracca in the different treatments (symbols and lines) and daily 1-h maximum O3 concentrations (gray bars) in the NF þ O3 and NF þ O3 þ CO2 treatments in summer 2004.

a ab

500

ab b

400

b

A

AB BC

300 200

AB

C

3.4. Plant N concentrations and community N pool

100 0

AA

( p ¼ 0.025). This change did not, however, persist in the following summer (2004). The highest root biomass was recorded from the NF and NF þ CO2 treatments and the lowest from the NF þ O3 treatment, but there were no statistically significant differences between the treatments in the root biomass at either depth (p > 0.1; data not shown). Irrespective of treatment, the root biomass was always higher in the top soil.

NF

NF+O3

NF+CO2

NF+O3+CO2

Treatments Fig. 3. Total aboveground biomass (mean, S.E.) in the different treatments in late August to early September 2003 (open box) and 2004 (filled box). The letters indicate statistically significant differences ( p < 0.05) between the treatments within each year. Statistical tendency (compared to the NF treatment of the same year) is marked with the exact p value.

The N concentrations varied significantly among the different species and years ( p < 0.001), but few treatment effects were recorded (Table 3). The highest N concentrations were measured in V. cracca (2.4%) and the lowest (0.8%) in R. acris (Fig. 5). The NF þ O3 þ CO2 and NF þ CO2 treatments decreased the overall N concentration of V. cracca during the last summer 2004 ( p < 0.05). In summers 2003 and 2004, the N concentration of F. vesca was lower in the OTCs than in the plants grown in the AA plots ( p ¼ 0.001; Fig. 5). There

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

106

Table 3 Statistics for year, OTC treatments and their interaction on the absolute biomass and N concentration for different species Species

Total

A. capillaris

A. odoratum

C. rotundifolia

F. vesca

R. acris

T. medium

V. cracca

Absolute biomass

Year Treatment Year  Treatment

0.002 n.s. n.s.

0.007 n.s. n.s.

0.007 0.007 n.s.

n.s. 0.006 n.s.

0.017 n.s. n.s.

0.013 n.s. n.s.

n.s. 0.017 n.s.

<0.001 0.010 n.s.

N concentration

Year Treatment Year  Treatment

<0.001 n.s. n.s.

<0.001 n.s. n.s.

e e e

<0.001 n.s. n.s.

<0.001 n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s., not significant.

different species A. capillaris benefited most from growing in the NF treatment compared to the AA plots. In summers 2003 and 2004, the proportion of A. capillaris was higher in the OTCs than in the AA plots ( p < 0.05; Fig. 4), and in the summer 2004, the relative biomass of grasses increased in the OTCs irrespective of gas exposure ( p ¼ 0.042; Table 4). In summer 2003, also the biomass of C. rotundifolia and F. vesca were higher in the NF OTCs than in the AA plots (Fig. 4). In both years, 2003 and 2004, the biomass of V. cracca tended to be lower in AA plots than in the NF treatment

was no correlation between the total aboveground biomass and N concentration of the species. During the last summer of 2004, the total community N pool tended to be ( p ¼ 0.087) lower in the NF þ O3 and NF þ O3 þ CO2 treatments when compared to the NF treatment (Fig. 6). 3.5. Chamber effect The NF OTC increased the total biomass production markedly compared to the AA plots in both years (Fig. 3). Of

2003

700

Plant biomass (g dw)

600 500 p = 0.097

a

400

a a

300

ab a a a a

200 b a a a a bc

0

a

A. capillaris

A. odoratum

a c b c

b a b

C. rotundifolia

a

b

F. Vesca

b

a a a a a

R. acris

T. medium

V. cracca

2004

700

a

600

a

500

ab

a

a

a

400

a

a

p = 0.078

a

300

a

b 200

a

100 0

ab b

b

100

Plant biomass (g dw)

a

A. capillaris

a

a

a a

A. odoratum

c

a ab a c b c

a a a a a

C. rotundifolia

F. vesca

ab bc ab

a a a a R. acris

T. medium

V. cracca

Species AA

NF

NF+O3

NF+CO2

NF+O3+CO2

Fig. 4. Aboveground biomass of the species (mean, S.E.) in the different treatments in late August to early September 2003 and 2004. Letters indicate statistically significant differences ( p < 0.05 unless stated otherwise) between treatments within the species.

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

107

Table 4 Mean relative biomass (% of total biomass per mesocosm) of the species and functional types in the late August to early September 2003 and 2004 in the different treatments Species

Functional types

A. capillaris

A. odoratum

C. rotundifolia

F. vesca

R. acris

T. medium

V. cracca

Grasses

Herbs

Legumes

1.00 1.50 0.59 1.01 0.49

ab a b ab b

1.98 3.43 3.04 4.69 2.38

a a a a a

4.73 4.97 5.47 6.10 5.63

a a a a a

37.65 19.32 27.52 21.64 29.51

a a a a a

22.40 24.68 16.86 24.99 15.92

a a a a a

33.2 47.6 47.1 42.6 46.6

7.7 a 9.9 a 9.1 a 11.8 a 8.5 a

60.1 44.0 44.4 46.6 45.4

a a a a a

2.43 2.48 4.33 2.12 1.99

a a a a a

5.17 1.93 3.94 2.47 2.23

a a a a a

46.83 26.52 26.76 27.94 29.19

a a a a a

10.10 19.43 13.49 19.47 15.99

a a a a a

33.02 47.79 49.93 47.01 50.12

10.06 a 6.26 a 9.82 a 5.58 a 4.70 a

56.92 45.95 40.25 47.41 45.18

0.022 n.s n.s.

n.s n.s. n.s.

2003

AA NF NF þ O3 NF þ CO2 NF þ O3 þ CO2

24.79 42.22 40.57 36.21 42.08

b a a a a

7.46 3.89 5.95 5.36 4.01

a a a a a

2004

AA NF NF þ O3 NF þ CO2 NF þ O3 þ CO2

21.73 42.95 43.73 41.85 44.83

a a a a a

11.29 a 4.84 a 6.20 a 5.16 a 5.30 a

2.46 1.85 1.55 1.00 0.48

p Values

Year Treatment Year  Treatment

n.s n.s. n.s.

n.s. n.s. n.s.

n.s. 0.012 n.s.

n.s. n.s. n.s.

0.004 n.s. n.s.

n.s n.s. n.s.

n.s. n.s. n.s.

a a a a a b a a a a

n.s n.s. n.s.

a a a a a a a a a a

Letters indicate statistically significant differences ( p < 0.05) between the treatments within each species and functional type. p Values of the statistical analysis for the OTC treatments are also given. n.s., not significant.

(Fig. 4). In the last summer of 2004, total community N pool tended to be (p ¼ 0.087) lower in the AA treatment when compared to the NF treatment (Fig. 6).

Ashmore, 2002; Tonneijck et al., 2004). Yet, four out of seven species (Campanula rotundifolia, Fragaria vesca, Trifolium medium, Vicia cracca) showed either growth reduction, visible ozone injury, or both as a response to elevated O3. This observation supports the findings of Power and Ashmore (2002) who found that species typical to fens and -meadows may be particularly sensitive to O3. The mean aboveground biomass per area unit (408 g dry weight m2) of all of the mesocosms irrespective of treatment at the end of the experiment, in 2004, corresponds to that of natural meadows (Marissink et al., 2002; Titlyanova et al., 1988). The greenhouse gas emissions of the mesocosms were also at the same level as those of three natural meadows (Kanerva et al., 2005), suggesting that the results obtained from the present study may well be applicable to intact meadows.

4. Discussion 4.1. O3 effects The cumulative O3 exposure AOT40 values in all three summers in the NF þ O3 and NF þ O3 þ CO2 treatments exceeded the critical level (3000 ppb h over a 3-month period) for protecting semi-natural vegetation as proposed by the UN-ECE convention (Karlsson et al., 2003). However, the values were generally below those measured in the O3 enhancement studies in Central Europe (e.g. Power and 3,5

a

N concentration (% dw)

3

a a

2,5

a a

ab

a a

b b

2 a

1,5 a

a a

a

a

b a

a

a

a

1

ab a a

a

a

a

a a

a

a

a

a a a

0,5 0

A. capillaris

A. odoraturm

C. rotundifolia

F. vesca

R. acris

T. medium

V. cracca

Species AA

NF

NF+O3

NF+CO2

NF+O3+CO2

Fig. 5. The N concentrations (mean, S.E.) of the species in the different treatments in late August to early September 2004. Letters indicate statistically significant differences ( p < 0.05) between the treatments within each species.

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

108 3000

Total community N pool

A

2500 2000

B

AB B

B

1500 1000 500 0

AA

NF

NF+O3

NF+CO2

NF+O3+CO2

Treatments Fig. 6. Total community N pool (mean, S.E.) of the mesocosms in the different treatments in late August to early September 2004. Letters indicate statistically significant tendencies ( p < 0.1).

The reduction in the total aboveground biomass by the NF þ O3 treatment is in agreement with studies on monocultures that report severe growth reductions in numerous wild species under O3 exposure (see review by Davison and Barnes, 1998). We did not detect significant and persistent changes in the relative biomass of functional groups as previously reported by Ashmore and Ainsworth (1995), Ashmore et al. (1995) and Wilbourn et al. (1995). The growth reductions of grasses A. capillaris and A. odoratum in NF þ O3 treatment were rather large (38% and 36% in 2004, respectively), but these decreases were not significant. The high O3 tolerance of grasses has been reported by several previous studies (e.g. Ashmore et al., 1996; Mortensen, 1992, 1994). The most sensitive functional group in terms of growth reduction was the group of non-leguminous herbs, especially C. rotundifolia, which showed reduced biomass production in both 2003 and 2004. Previous studies on C. rotundifolia monocultures have reported on both no responses (Mortensen and Nilsen, 1992) and reductions of aboveground biomass (19.2%; Ashmore et al., 1996) when exposed to O3. Ashmore et al. (1996) also studied the performance of C. rotundifolia in a community, but found no changes in the absolute or relative biomass. Aboveground biomass reduction of F. vesca has previously been reported on by Mortensen (1993), who observed O3-induced growth reductions at the concentration of 90 ppb, but not at 45 ppb. The initial reductions and following compensation in the aboveground biomass of F. vesca observed in the present study in 2003 and 2004 may be explained as physiological and morphological adaptation (Bungener et al., 1999b). An alternative, and more likely, explanation may be attributed to the weaker competition in the O3 treatments. Wild strawberries thrive in sunny spots, and their fast vegetative growth can benefit from reductions in total community coverage and thus reduced competition. R. acris has been reported to show both visible injuries and growth reductions when exposed to high levels of O3 (90 ppb; Mortensen, 1993), but no such effects were observed in this experiment with lower O3 concentrations (40e50 ppb). In the present study, O3 effects on legumes were mainly manifested as visible injuries, which appeared only in the summer 2004. There are several explanations for this response.

The exposure level during the summer of 2004 was significantly higher than that of the two previous summers, and the occurrence of high peak values early in the growing season may have enhanced the emergence of visible injuries (Pihl Karlsson et al., 1995; Timonen et al., 2004). Additionally, the exceptionally wet summer of 2004 may have increased O3 uptake, and hence the development of visible injuries (Benton et al., 2000). Ozone exposure had, however, only slight effects on the growth of the legumes. Unlike previous community studies on other clover species (Ashmore and Ainsworth, 1995; Ashmore et al., 1996; Gimeno et al., 2004), zigzag clover (T. medium) did not prove to be a particularly O3-sensitive species with regard to growth reductions. In addition to interspecific differences, individuals of Trifolium species have shown to exhibit intraspecific differences in O3 sensitivity (Nebel and Fuhrer, 1994). As the seeds used in the study were pooled from several populations, we cannot exclude the possibility that the effects of O3 were hidden by intraspecific differences. Power and Ashmore (2002) studied V. cracca in monocultures and found no growth reductions either above- or below-ground in a season-long experiment, even though the AOT40 value was 9200 ppb h at the end of the experiment. In the present study, the AOT40 values were only 5000 ppb in the growing season of 2003, and yet reduced growth was observed in V. cracca. All in all, we observed growth reductions at rather low concentrations of O3 as compared to some previous studies (e.g. Power and Ashmore, 2002; Tonneijck et al., 2004). As we did not directly assess the competitive interactions, but rather reported on the growth of plants under competition, we cannot point out a single cause for the potentially increased sensitivity. Firstly, the sensitivity may be altered by other species and the characteristics and number of the species involved (Davison and Barnes, 1998; Nussbaum et al., 2000). Secondly, the prevailing conditions in different studies are always dissimilar. Long days and high relative humidity of the northern latitudes allow abundant O3 uptake for many hours, and because of the short nights, plants have only a limited time over which to recuperate from the oxidative stress (DeTemmerman et al., 2002; Embersson et al., 2000). Moreover, the O3 sensitivity of plants may also be modified by nutrient supply (Davison and Barnes, 1998), and nutrient stress can augment the growth reductions caused by O3 (Pa¨a¨kko¨nen et al., 1995; Whitfield et al., 1998). As the soil in the present study was relatively low-N (Kanerva et al., 2005), it is possible that some of the effects of O3 were enhanced by nutrient stress. Supporting this view, the N concentrations of most O3-responsive non-leguminous herbs (1.0% dry weight on average) were lower than those generally measured from other grassland forbs (between 1.2 and 1.95% dry weight; Marissink et al., 2002; Reich et al., 2001). On the other hand, N concentrations of grasses and legumes were comparable or higher to those reported in the literature (Marissink et al., 2002; Reich et al., 2001). The root biomass was not affected by the treatments, an observation that fits amongst the controversial data on the known effects of O3 on wild plants. Previous studies on wild plants (e.g. Power and Ashmore, 2002; Reiling and Davison, 1992)

K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

have reported on stimulation, reductions, and no changes in the biomass allocation to roots. We did not, however, assess rootto-shoot ratios of individual plants or the whole community root biomass, but took bulk samples from the soil. This may in part have caused the absence of significant growth responses. Although O3 reduced neither the frequency of the legumes nor the N% of individual species, it tended to reduce the total community N pool. This may have important consequences in the nutrient cycling of the ecosystem. 4.2. CO2 alone and in combination with O3 By itself, effects of CO2 were minimal. A number of studies (e.g. Ainsworth and Long, 2005; Bazzaz, 1990; Jablonski et al., 2002; Jongen and Jones, 1998; Lu¨scher and No¨sberger, 1997) have reported CO2 to enhance growth, increase allocation to roots, and decrease the grasselegume ratio, but no such stimulation or change was observed in the present study. However, Reich et al. (2001) reported on only modest and negligible increases in biomass production of wild C3 plants under CO2 enrichment (560 ppm). The lack of response is most likely due to the low levels of CO2 and the restricted exposure period. In the present study, CO2 concentration was increased only by approximately 100 ppm, whereas several studies have doubled the CO2 concentrations (e.g. Jongen and Jones, 1998; Marissink et al., 2002; Mulholland et al., 1997a,b). In addition, CO2 was added only for 9 h per day. Furthermore, although the total biomass production kept increasing year by year, the communities grew on relatively nitrogen-poor soil (Kanerva et al., 2005) and the plants may not have been able to benefit from the augmentation of CO2 (Poorter and Pe´rez-Soba, 2001). This is, however, not supported by the fact that the N-fixing legumes did not increase growth or percent composition of mesocosm. Finally, Poorter and Navas (2003) reported on negative correlation between CO2 response and plant density. The weak responses may indeed be a consequence of increased competition. Increase in the ambient CO2 concentration in the final year 2004 is most likely a consequence of the unordinarily wet summer, which may have enhanced decomposition and decreased CO2 uptake by plants. Also the low measurement height (1 m) might have increased the sensitivity of measurements to environmental fluctuations. Elevated CO2 has been reported to decrease plant N concentrations (e.g. Marissink et al., 2002; Reich et al., 2001), which is in line with our results on V. cracca. Reich et al. (2001) studied the tissue N concentrations of 12 different C3 species and reported that moderately elevated CO2 concentrations (560 ppm) tended to reduce tissue N concentrations of all functional types including legumes, grasses, and herbs, but that different species exhibited large variability. Supplemental CO2 alone did not affect the total community N pool either in the present study or in the investigation by Reich et al. (2001). Previous studies have shown that CO2 may ameliorate the negative O3 responses (e.g. Booker and Fiscus, 2005; Booker et al., 2005; Cardoso-Vilhena et al., 2004; Fiscus et al., 2005; Heagle et al., 1999, 2000; McKee et al., 1997, 2000; Morgan et al., 2003; Mulholland et al., 1997a,b; Volin et al., 1998),

109

while others have shown that CO2 did not protect against O3 (Balaguer et al., 1995; Heagle et al., 2002, 2003; Kull et al., 1996). In the present study, CO2 generally ameliorated only slightly the O3-induced reductions in aboveground biomass, and elevated CO2 did not modify the visible O3 injuries, either. The lack of compensation in individual species may be connected to the low CO2 concentrations. Heagle et al. (1993) studied Trifolium repens and noticed that only the highest CO2 concentration (710 ppm) used protected clover from the effects of tropospheric O3. Some studies have reported that elevated CO2 may indeed enhance the detrimental O3 effects on photosynthesis and growth (Johnson et al., 1996; Kull et al., 1996; Wustman et al., 2001). CO2 was not able to ameliorate the effects of O3 on the total community N pool. Based on the present study, most of the deleterious effects of tropospheric O3 are not diminished by a moderate increase in CO2. 4.3. Chamber effect Apart from the O3 and CO2 effects, there were also rather strong chamber effects. OTC increased, although non-significantly, the temperature, decreased relative humidity, and filtered the ambient O3. The fact that OTCs filtered some of the ambient O3 was mainly reflected as differences in cumulative AOT40 values as the measured O3 concentrations were generally below or just barely over 40 ppb, and amounts exceeding the threshold concentration were small. The concentrations in the AA plots, however, were rather low and it is unlikely that any differences in growth responses between the NF OTCs and AA plots would be due to differences in the O3 concentrations. OTCs had a positive effect on the total aboveground biomass production, an observation which is in good agreement with the earlier studies (e.g. Danielsson et al., 1999; Owensby et al., 1993, 1999), and increased the proportion of grasses, especially that of A. capillaris. Moreover, the NF OTC enhanced the growth of C. rotundifolia and F. vesca in 2003 as compared to the AA plots, but these enhancements did not persist. This is most likely due to the fact that mesocosms were not fully developed until the third summer. 5. Conclusions The observed growth reductions of the studied species under competition verify the O3 sensitivity of meadow species and indicate that nitrogen-poor meadows are potentially a very sensitive ecosystem. Growth reductions were recorded after only two years of moderate (40 ppb) exposure. Ozone reduced the total community biomass production and the growth of three species out of seven, but these reductions were not reflected as persistent changes in the dominance of different functional groups. The biggest reductions were, however, seen in the least abundant species (C. rotundifolia and F. vesca), which may indicate O3-induced suppression of the weak competitors. CO2 only slightly amended the detrimental effects of O3 on the biomass production, and no amelioration was observed on the visible injuries of the individual species. The minor effects of CO2 were attributed to the low levels of

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K. Ra¨mo¨ et al. / Environmental Pollution 144 (2006) 101e111

CO2 enhancement. Long-term predictions are difficult to make, but the study indicates that the growth of several herb species decreases with increasing atmospheric CO2 and O3 concentrations, and that these changes may possess a threat to the biodiversity of meadows. An increase in the temperature may, however, compensate for some of these changes. Ozoneinduced reductions in the total community biomass production and N pool are likely to have important consequences in the nutrient cycling of the ecosystem. Acknowledgements This study was financially supported by funds from the Helsinki University Environmental Research Center and the University of Helsinki. We are grateful to Mr. Peter Huhtala and Mr. Oiva Hakala for their contribution to the development and maintenance of fumigations throughout the study. Our thanks go to the staff of MTT/MPY for all the assistance. We would also like to thank Dr. Kustaa Niini for providing valuable information on the sowing of the seeds and Dr. Tuomas Laurila from the Finnish Meteorological Institute for lending us the O3 analyzer. References Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist 165, 351e372. Allen Jr., L.H., 1990. Plant responses to rising carbon dioxide and potential interactions with air pollutants. Journal of Environmental Quality 19, 15e34. Andersen, C.P., Hogsett, W.E., Plocher, M., Rodecap, K., Lee, H., 2001. Blue wild-rye competition increases the effects of ozone on ponderosa pine seedlings. Tree Physiology 21, 319e327. Ashmore, M.R., Ainsworth, N., 1995. The effects of ozone and cutting on the species composition of artificial grassland communities. Functional Ecology 9, 708e712. Ashmore, M.R., Thwaites, R.H., Ainsworth, N., Cousins, D.A., Power, S.A., Morton, A.J., 1995. Effects of ozone on calcareous grassland communities. Water Air and Soil Pollution 85, 1527e1532. Ashmore, M.R., Power, S.A., Cousins, D.A., Ainsworth, N., 1996. Effects of ozone on native grass and forb species: a comparison of responses of individual plants and artificial communities. Towards a critical level of ozone for natural vegetation. In: Ka¨renlampi, L., Ska¨rby, L. (Eds.), Critical Levels for Ozone in Europe: Testing and Finalizing the Concepts. UNECE Workshop Report. University of Kuopio, Department of Ecology and Environmental Science, pp. 193e197. Balaguer, L., Barnes, J.D., Panicucci, A., Borland, A.M., 1995. Production and utilization of assimilates in wheat (Triticum aestivum L.) leaves exposed to elevated O3 and/or CO2. New Phytologist 129, 557e568. Barbo, D.N., Chappelka, A.H., Somers, G.-L., Miller-Goodman, M.S., Stolte, K., 1998. Diversity of an early successional plant community as influenced by ozone. New Phytologist 138, 653e662. Barbo, D.N., Chappelka, A.H., Somers, G.L., Miller-Goodman, M.S., Stolte, K., 2002. Ozone impacts on loblolly pine (Pinus taeda L.) grown in a competitive environment. Environmental Pollution 116, 27e36. Barnes, J.D., Ollerenshaw, J.H., Whitfield, C.P., 1995. Effects of elevated CO2 and/or O3 on growth, development and physiology of wheat (Triticum aestivum L.). Global Change Biology 1, 129e142. Bassin, S., Ko¨lliker, R., Creton, C., Bertossa, M., Widmer, F., Bungener, P., Fuhrer, J., 2004. Intra-specific variability of ozone sensitivity in Centaurea jacea L., a potential bioindicator for elevated ozone concentrations. Environmental Pollution 131, 1e12.

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