Response to heat stress of populations of two Sphagnum species from alpine bogs at different altitudes

Environmental and Experimental Botany 74 (2011) 22–30

Contents lists available at ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Response to heat stress of populations of two Sphagnum species from alpine bogs at different altitudes Renato Gerdol ∗ , Renzo Vicentini Department of Biology and Evolution, University of Ferrara, Corso Ercole I d’Este 32, I-44121 Ferrara, Italy

a r t i c l e

i n f o

Article history: Received 25 December 2010 Received in revised form 12 April 2011 Accepted 18 April 2011 Keywords: Adaptation Bog Climate change Heat wave Nitrogen Temperature

a b s t r a c t Sphagnum mosses are a fundamental component of bog vegetation in northern regions, where these plants play a major role in controlling important ecosystem processes. As heat waves are expected to become increasingly intense and frequent, especially in cold territories, it is important to improve our knowledge of heat resistance in Sphagnum species. We investigated the response to heat stress of S. fuscum and S. magellanicum. Three populations of the two species collected at different altitudes (1090 m, 1870 m and 2100 m) were grown at three daytime temperature levels: 25 ◦ C (AT); 36 ◦ C (MT); 43 ◦ C (HT). The HT treatment decreased concentrations of chlorophyll and nitrogen in the plant tissues, which resulted in lower net CO2 exchange rates and quantum yield of PSII . The plants recovered significantly within six days, probably because temperature in the living tissue did not reach lethal thresholds because of the high water content in the plant tissues. Contrary to our main hypothesis, that S. magellanicum had greater resistance to high temperatures because of its more southern distribution, the two species showed much the same response patterns to heat stress. Supporting our second hypothesis, populations of both species originating from the highest site suffered somewhat stronger, although still reversible, damage when grown at HT. Heat stress brought about by heat waves will unlikely have differential effects on these two Sphagnum species. We also conclude that heat waves are unlikely to exert irreversible damage to the Sphagnum layer in bog ecosystems if high temperatures are not coupled with drought. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Model simulations, supported by observations, point to increasing intensity and frequency of heat waves in the 21st century (Meehl and Tebaldi, 2004; Koffi and Koffi, 2008; Ganguly et al., 2009). Several heat wave events have been documented in temperate and arid regions of the northern hemisphere (Park and Schubert, 1997; Gershunov et al., 2009; Unkasevic and Tosci, 2009; Lobo et al., 2010). Yet, climate warming is expected to be strongest in cold regions, especially at high latitudes (Przybylak, 2002) but also on mid-latitude mountains (Christensen and Christensen, 2007). A number of studies have addressed possible effects of climate warming, and associated environmental changes, on arctic ecosystems (see Hinzman et al., 2005 for review). However, there is poor knowledge of how heat waves could affect ecosystems patterns and processes in cold regions. Marchand et al. (2006) observed strong differences in the response of four vascular plant species to experimental manipulation of soil temperature simulating a prolonged heat wave event in a high arctic ecosystem. As the vegetation of cold habitats usually is very poor in species (Wielgolaski, 1997),

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (R. Gerdol). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.04.010

species-specific responses of dominant plant species to extreme heat events may bring about profound changes in the species composition of cold ecosystems which may in turn exert major effects on ecosystem functioning. Ombrotrophic bogs cover vast areas in northern cold territories, particularly in boreal and (sub)arctic regions and, to much a lesser extent, in mountainous regions. Bog vegetation usually is dominated by a few bryophyte species belonging to the genus Sphagnum (so-called peat mosses), which account for the major fraction of living and dead plant biomass in bogs. Peat mosses play a crucial role in regulating the functioning of these ecosystems since they behave like ecosystem engineers, by creating peculiar environmental conditions such as strong acidity, nutrient poorness and cold soil temperatures, which hinder the establishment of vascular plants, with the exception of a few specialists (Rydin and Jeglum, 2006). Peat mosses are, furthermore, characterized by extremely recalcitrant litter, which accounts for the bulk of carbon (C) sequestration in peat. Recent studies suggested that climate warming in ombrotrophic bogs results in higher evapotranspiration rates implying increased drainage and aeration of peatland surface (Chen et al., 2008) and/or higher mineralization rates improving the nutrient status in the rooting layer (Keller et al., 2004). This will probably enhance growth of vascular plants at the expense of peat mosses, eventually reducing rates of peat C accumulation (Gerdol et al.,

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

2008; Breeuwer et al., 2010). Contrary to the gradual effects of climate warming on the competitive equilibria between peat mosses and vascular plants, growth and even survival of Sphagnum mosses in ombrotrophic bogs may be jeopardized much more abruptly as a consequence of heat waves. Indeed, the unprecedented heat wave that occurred over large areas of central-southern Europe in summer 2003 caused the die-off of peat mosses at several bogs in the southern Alps (Bragazza, 2008). Sphagnum species have been shown to differ from each other as regards both resistance to heat (Balagurova et al., 1996) and resistance to drought (Gerdol et al., 1996; Schipperges and Rydin, 1998). Yet, it is still unknown if Sphagnum species possess differential capacities to withstand heat waves. If so, the occurrence of hot spells may trigger changes in the species composition of the Sphagnum layer of ombrotrophic bogs and may, in turn, affect ecosystem functioning because Sphagnum species also differ to a certain extent from each other as regards production (Gunnarsson, 2005) and decomposition rates (Johnson and Damman, 1993; Limpens and Berendse, 2003; Bragazza et al., 2007). On the other hand, these effects will be attenuated if Sphagnum species are able to adapt to increasing temperatures. Few studies have so far addressed adaptations of Sphagnum species to changing environmental conditions (Sastad et al., 1999; Melosik, 2009; Szovenyi et al., 2009) and none has, to our knowledge, investigated if and to what extent different Sphagnum populations are able to adapt to varying temperature levels. In this study we investigated the response of two Sphagnum species to heat stress. We chose two hummock-forming species [S. fuscum (Schimp) Klinggr. and S. magellanicum Brid.] for our experiment since heat waves are expected to exert maximum impact on hummocks. Although during prolonged arid periods Sphagnum mosses may be damaged by drought in hollow microhabitats (Robroek et al., 2009), on hot sunny days the apical tissues of Sphagnum mosses warm up to a greater extent in hummocks than in the surrounding hollows (Clymo and Hayward, 1982; den Hartog et al., 1994). These conditions can bring about extensive damage to hummock-forming Sphagna if the moss surface dries up because of increased evaporative demand (Bragazza, 2008). We hypothesized that: (1) The species exhibiting more southern distribution, namely S. magellanicum, is more resistant to heat stress compared to the northern species, namely S. fuscum. (2) The populations of both species collected at low-elevation sites are more resistant to heat stress compared to those collected at high-elevation sites.

2. Materials and methods 2.1. Plant material and experimental design In October 2008, two cores (40 cm × 30 cm × 15 cm) of both Sphagnum species (S. fuscum and S. magellanicum) were collected at each of three mires at different altitudes in the south-eastern Alps of Italy, province of Bolzano (Bozen): Rasner Möser (46◦ 48 N, 12◦ 04 E, 1090 m; low altitude, LA); Hirschenlacke (46◦ 35 N, 11◦ 28 E, 1780 m; intermediate altitude, IA) and Schwarze Lacke (46◦ 35 N, 10◦ 28 E, 2100 m; high altitude, HA). The three mires span almost the whole altitudinal range known for these species in mountainous regions of central-southern Europe (Gerdol and Bragazza, 1994; Hájkova and Hájek, 2007). All cores were collected in ombrotrophic sectors of the three mires, those of S. fuscum from high hummocks and those of S. magellanicum from low hummocks. We selected for the collections almost pure stands, with the cover of the target Sphagnum species >95% and the cover of vascular plants <5%. The cores were placed in plastic containers and brought within two days from the collection to the Botanical Garden of Ferrara University. After carefully removing the aboveground tissues of vascular plants, the cores were stored for some

23

weeks in a glasshouse. Water-table depth in all containers was set about 2 cm below surface by adding rain water every two days. The experiment was carried out separately for the two species because of space limitation in the laboratory. Small samples of Sphagnum gametophores (15 samples per species per mire) were randomly picked from the containers and gently placed into paperboard cups. The gametophores were placed at natural density, without gaps, into the cups with about 180 individuals in the S. fuscum samples and 80 individuals in the S. magellanicum samples. The water level, as in Breeuwer et al. (2008), was kept constant at 1 cm below the growing apex (the so-called capitulum) by adding distilled water. Although these species, especially S. fuscum, usually live at greater water-table depths, we chose such high water level because this prevented possible dehydration of the capitula used for the gas-exchange measurements (see below). However, hummock Sphagna have been found to grow well at high water levels they rarely experience in natural conditions (Rydin and McDonald, 1985; Grosvernier et al., 1997; Breeuwer et al., 2008). The samples originating from the three sites were randomly assigned three temperature treatments: ambient temperature (AT), medium temperature (MT) and hot temperature (HT). For each species, the experiment lasted four days when the temperature treatments consisted in manipulating air temperature during the daytime period (12 h) by varying the height of the greenhouse lights which provided illumination. Photosynthetic photon flux density at the capitulum surface was calibrated at 280 ± 20 ␮mol m−2 s−1 independent of lamp height, while daytime air temperature rapidly achieved 25 ◦ C in the AT temperature, 36 ◦ C in the MT temperature and 43 ◦ C in the HT treatment, respectively. Ambient temperature was similar to daytime temperature in the greenhouse during the acclimation period. In addition, AT simulated temperature levels that the plants usually experience during warm sunny days at the LA mire. Our increased temperature levels (MT and HT) exceeded by far (>5 ◦ C) AT, both being higher than those ever recorded in the sampling region. Temperature at the capitulum surface was measured manually several times during the daytime period. Capitulum temperature was: 22–28 ◦ C in the AT treatment 30–41 ◦ C in the MT treatment and 37–49 ◦ C in the HT treatment. During the night time period, air temperature gradually decreased to a minimum of 22 ◦ C for all treatments. An air-conditioning device kept air humidity constant (about 35%) throughout the experimental period. In summary, the experiment was full-factorial with 2 species × 3 levels of altitude × 3 levels of temperature and five replicates for each combination, with 90 samples in total. At the end of the fourth day, the samples were left to recover for six days at the same photoperiod (12 h/12 h) and water level (1 cm below capitula) set up during the experiment, but lower photosynthetic photon flux density (100 ± 10 ␮mol m−2 s−1 ), lower daytime temperature (20 ± 1 ◦ C) and lower night temperature (18 ± 1 ◦ C). Lower temperatures prevented any, albeit unlikely, thermal stress during the recovery period. 2.2. CO2 exchange Prior to the start of the experiment, some capitula in each of the samples were cut off the underlying gametophore tissues and placed into a 2.5 cm × 2.5 cm plastic basket, which was gently re-inserted into the cup at the level of the surrounding capitula. These baskets were used for determining net CO2 exchange rates on two occasions: immediately after the experiment had ended (0 d) and six days after the experiment had ended (6 d). Net CO2 exchange was measured using an open flow system carrying air with ambient CO2 pressure (39 ± 1.5 Pa) from the Botanical Garden into an infrared gas analyser (LCA-4, Hoddesdon, UK) connected to a factory-designed small (625 mm2 ) leaf chamber. The baskets were placed into the leaf chamber equipped with a fan and a water

24

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

cooling system to ensure almost constant temperature at ambient (22 ± 2 ◦ C) level. Illumination was provided by a glasshouse lamp at 520 ± 20 ␮mol m−2 s−1 photosynthetic photon flux density, i.e., above light saturation (Maseyk et al., 1999; Marschall and Proctor, 2004; Gerdol, unpublished; but see Hájek et al., 2009) and below photoinhibition (Harley et al., 1989) for Sphagnum mosses. Initially, we planned to keep each basket in the leaf chamber for 15–20 min in order to achieve (sub)optimum water content in the capitula when determining net CO2 exchange rates (Granath et al., 2009). Net CO2 exchange rates of several samples regrettably became negative a few minutes after the baskets had been placed into the chamber, perhaps due to increased respiration or to pressure change within the chamber. Therefore, we only considered the net CO2 exchange rates determined 1.5 min after placing the basket into the leaf chamber, although these values may not represent maximum photosynthetic rates for Sphagnum mosses (Silvola and Aaltonen, 1984; Schipperges and Rydin, 1998). However, as the capitulum water content was much the same (1900–2200% of dry weight) in all samples, with no difference either between species or among treatments, we feel confident that our data are appropriate for comparative purpose. In order to further facilitate comparison between the two species, the data of net CO2 exchange rates were normalized for each species as follows: Anorm =

(A − Amin ) × 100 (Amax − Amin )

where Anorm is the normalized value (ranging from 0 to 100); A the rate of net CO2 exchange (as ␮mol CO2 m−2 s−1 ) actually measured in the chamber; Amin the minimum rate of net CO2 exchange measured for each species across all treatments on the two measuring dates; Amax the maximum rate of net CO2 exchange measured for each species across all treatments on the two measuring dates. 2.3. Chlorophyll a fluorescence At the end of the experimental period, six capitula in each cup were randomly picked and placed individually in special cuvettes. These cuvettes had a port for the fibre optics measuring probe of a pulse-modulated chlorophyll (chl) fluorometer (OS1FL, Optisciences, USA). The probe was fixed vertically about 1 cm above the capitulum. The capitulum in each cuvette was saturated with distilled water and dark-adapted for 15 min prior to the measurements. The maximum quantum yield of PSII (Fv /Fm ) was determined, as a proxy for stress, on the same dates as for CO2 gas exchange, i.e., on 0 d and 6 d. In the statistical analysis we used, for each cup, the median of the Fv /Fm values of the six capitula. 2.4. Plant tissue chemistry On 6 d, the capitula not used for measuring either CO2 gas exchange or chl a fluorescence were divided into two subsamples that served for determining chl and nitrogen (N) contents in the plant tissues. Chl analysis was carried out as described in Gerdol et al. (1994). Briefly, about 150 mg fresh weight were freeze-dried, powdered using an Omnimixer homogenizer (International Waterbury, CT, USA) and extracted with 4 ml 80% cooled (about 4 ◦ C) acetone, buffered with 20% 0.1 M Tris–HCl at pH 7.2. The extract was then centrifuged at 3500 g for 10 min. On the supernatant, absorptions at 663 nm and 646 nm were read in a spectrophotometer (Ultrospec 2000, Pharmacia Biotech Ltd., Cambridge, UK) to calculate concentrations of chl a and chl b, respectively, based on the Lichtenthaler (1987) equations. Total N concentration was determined on about 50 mg plant material oven-dried at 60 ◦ C for 24 h. This material was ground through a 1-mm mesh titanium mill, digested according to the Kjeldahl procedure and then analyzed

Table 1 P values obtained from repeated-measures three-way ANOVAs of net CO2 exchange and chlorophyll a fluorescence (Fv /Fm ). The statistics were performed on normalized data for net CO2 exchange and non-transformed data for chlorophyll a fluorescence, respectively. Sphagnum species, altitude, temperature treatment and their interaction were the between-subject factors and date the within-subject factor.

Species Altitude Temperature Species × Altitude Species × Temperature Altitude × Temperature Species × Altitude × Temperature Date Date × Species Date × Altitude Date × Temperature Date × Species × Altitude Date × Species × Temperature Date × Altitude × Temperature Date × Species × Altitude × Temperature

Net CO2 exchange

Fv /Fm

0.47 0.73 0.001 0.05 0.08 0.39 0.36 0.17 0.68 0.10 0.001 0.18 0.39 0.78 0.34

0.39 0.13 0.000 0.13 0.51 0.03 0.51 0.000 0.16 0.006 0.000 0.09 0.23 0.001 0.43

Significant (P < 0.05) values are in boldface.

colorimetrically by a flow-injection autoanalyser (FlowSys, Systea, Roma, Italy). 2.5. Statistics The data of net CO2 exchange and Fv /Fm were statistically analyzed by repeated-measures three-way ANOVAs with species, altitude, temperature treatment and their interaction as betweensubject factors and date as within-subject factor. The data of chl a and chl b concentrations, chl a:b ratio and N concentration were analyzed by factorial three-way ANOVAs with species, altitude, temperature treatment and their interaction as fixed factors. Significance of differences in the mean values for pairwise comparisons was assessed by Fisher’s LSD post-hoc tests. All computations were performed using the package STATISTICA (Release 6; StatSoft Inc., Tulsa). 3. Results 3.1. Net CO2 exchange and chl a fluorescence Area-based rates of net CO2 exchange were much higher in S. fuscum (1.74 ± 0.14 ␮mol CO2 m−2 s−1 ) compared to S. magellanicum (0.85 ± 0.11 ␮mol CO2 m−2 s−1 ). These values are very similar to those determined for individuals of S. magellanicum and S rubellum (a species ecologically and taxonomically related to S. fuscum; Laine et al., 2009), grown at optimum temperature and hydration (Gerdol, unpublished; Robroek et al., 2009). Although we could not measure pre-treatment CO2 exchange in all samples, we did determine net CO2 exchange rates in some capitula randomly picked from the containers prior to the beginning of the experiment. The measured rates did not differ significantly, based on one-way ANOVAs, from the maximum rates recorded at the end of the experiment (P = 0.95, n = 6 for S. fuscum; P = 0.94, n = 6 for S. magellanicum). Therefore, we assume that our maximum values are representative of pre-treatment photosynthetic performance for the two species. Normalized net CO2 exchange rates did not vary either with species or with altitude, although a weak Species × Altitude interaction indicates somewhat higher rates at HA for S. fuscum (Table 1, Fig. 1). Net CO2 exchange rates were very significantly affected by the temperature treatment (Table 1), with highest mean values at AT for both species (Fig. 1). Net CO2 exchange rates did not overall differ between the two dates but net CO2 exchange varied differently between the two dates in relation to the temperature

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

S. magellanicum, HA

S. fuscum, HA 100

100

a

90

ab

a

80 70

70

b

b

b

b

50

50

40

40

30

30

20

20

10

10

0

0

AT

MT

b

60

(%)

60

(%)

a

90

80

bc

AT

100

a

a

a

70

60

60

50

HT

a ab

80

70

b

b

b

50

b

b

40

40 30

b

30

20

20

10

10 0

0

AT

MT

AT

HT

ab

80

HT

100

a

90

a

a

80

ab abc

70 60

70 60

(%)

bc

50

c

40

MT

S. magellanicum, LA

S. fuscum, LA

100 90

a

90

(%)

(%)

80

MT

S. magellanicum, IA

S. fuscum, IA 90

bc

c

HT

100

(%)

25

50

b

40

30

30

20

20

10

10

0

b b

b

0

AT

MT

HT

AT

MT

HT

Fig. 1. Mean (+1 SE) values of normalized net CO2 exchange rates measured in the capitula of two Sphagnum species immediately after an experiment of temperature manipulation (black columns) and after six days since the end of the experiment (grey columns). During the experiment, groups of Sphagnum plants collected at three different altitudes (2100 m, HA; 1780 m, IA; 1090 m, LA) were grown at three levels of daytime temperature (25 ◦ C, AT; 36 ◦ C, MT; 43 ◦ C, HT). The means followed by the same letter within each panel do not differ at P = 0.05 based on Fisher’s LSD post-hoc tests.

treatments, as substantiated by the significant Date × Temperature interaction (Table 1). Net CO2 exchange rates on the two dates were much the same in the plants subjected to the AT treatment and to the HT treatment while they were generally higher on 6 d, compared with 0 d, in the plants subjected to the MT treatment (Fig. 1). The mean Fv /Fm in the capitula grown at AT was high (0.72–0.80) and coincided with the values reported for Sphagnum plants at

optimum temperature for photosynthesis (about 20–25 ◦ C; see Maseyk et al., 1999). The temperature treatment strongly affected chl a fluorescence (Table 1). Indeed, the capitulum Fv /Fm values on 0 d were weakly (P = 0.09) lower in the MT treatment, but they were significantly lower on both dates in the HT treatment compared to the AT treatment, (Fig. 2). However, chl a fluorescence in the HT treatment recovered considerably on 6 d (Fig. 2), which resulted in significant effects of date and Date × Temperature

26

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

S. magellanicum, HA

S. fuscum, HA 0.8

a

a

0.8

a

a

a

0.7

a

Fv/Fm

b

0.4

0.3

0.2

0.2

0.1

0.1 0.0

0.0

AT

MT

AT

HT

S. fuscum, IA ab

a

b

a

0.8

a

b

HT

ab

a

a

bc

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0.0

b

c

0.7

Fv/Fm

Fv/Fm

MT

S. magellanicum, IA

0.7

0.0

AT

MT

HT

AT

b

a

b

MT

HT

S. magellanicum, LA

S. fuscum, LA a

0.8

ab

c

0.7

0.7

0.6

0.6

0.5

0.5

Fv/Fm

Fv/Fm

b

c

0.4

0.3

0.8

a

0.5

0.5

0.8

a

0.6

0.6

Fv/Fm

a

0.7

0.4

a

a

a

c

b

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0.0

a

0.0

AT

MT

HT

AT

MT

HT

Fig. 2. Mean (+1 SE) values of Fv /Fm values measured in the capitula of two Sphagnum species immediately after an experiment of temperature manipulation (black columns) and after six days since the end of the experiment (grey columns). During the experiment, groups of Sphagnum plants collected at three different altitudes (2100 m, HA; 1780 m, IA; 1090 m, LA) were grown at three levels of daytime temperature (25 ◦ C, AT; 36 ◦ C, MT; 43 ◦ C, HT). The means followed by the same letter within each panel do not differ at P = 0.05 based on Fisher’s LSD post-hoc tests.

interaction (Table 1). Recovery was slightly faster in S. fuscum, as shown by a weak (P = 0.09) Date × Species × Altitude interaction (Table 1). Altitude did influence to some extent chl a fluorescence, with significant Date × Altitude and Date × Altitude × Temperature interactions (Table 1). This was mainly due to significantly lower Fv /Fm values, especially at the end of the experiment, in HA plants compared to IA and LA plants (Fig. 2).

3.2. Plant tissue chemistry Chl (a + b) concentration and chl a:b ratio in the AT capitula were both within the ranges compiled by Marschall and Proctor (2004) for healthy Sphagnum plants. Chl (a + b) concentration differed significantly between species, with higher values in S. fuscum presumably due to lower amount of hyaline cells

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

S. fuscum

27

S. magellanicum

Chlorophyll (a + b)

Chlorophyll (a + b) 700

900

α

A

800

600

α

700

a

a

500

B

αβ

500

-1

a

β

C

400

nmol g

nmol g

-1

600

A

400

β

β

300

b

b

300

A

A

b

200

200 100

100 0 HA AT HA MT HA HT

IA AT IA MT IA HT

0

LA AT LA MT LA HT

HA AT HA MT HA HT

IA MT

IA HT

4.0

4.0

3.5

3.5

A

a

2.5

b

A

α

A

α

β

b

2.5

β

B

2.0

B

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0

HA AT HA MT HA HT

IA AT IA MT IA HT

LA AT LA MT LA HT

HA AT HA MT HA HT

N concentration α

A

a a

A b

IA AT IA MT IA HT

LA AT LA MT LA HT

N concentration

12

β

12

α

A

β

10

B

8

a

ab

B

α

α

AB

b

-1

8

mg g

mg g-1

β

B

b

10

α

3.0

a

a

LA AT LA MT LA HT

Chlorophyll a : b

Chlorophyll a : b

3.0

IA AT

6

6

4

4

2

2

0

0

HA AT HA MT HA HT

IA AT IA MT IA HT

LA AT LA MT LA HT

HA AT HA MT HA HT

IA AT IA MT IA HT

LA AT LA MT LA HT

Fig. 3. Mean (+1 SE) values of chlorophyll (a + b) concentration, chlorophyll a:b ratio and N concentration in the capitula of two Sphagnum species after six days since the end of the experiment (grey columns). During the experiment, groups of Sphagnum plants collected at three different altitudes (2100 m, HA; 1780 m, IA; 1090 m, LA) were grown at three levels of daytime temperature (25 ◦ C, AT; 36 ◦ C, MT; 43 ◦ C, HT). Within each panel, significance of differences between the means are shown separately for plants collected at HA (small-case letters), IA (upper-case letters) and LA (greek letters). Different letters indicate significantly (P = 0.05) differing mean values based on Fisher’s LSD post-hoc tests.

in the leaves which also implied higher net CO2 exchange rates. In contrast, the chl a:b ratio did not vary between species (Table 2). Both variables responded similarly to the experimental treatment, with significant effects of temperature and Species × Temperature interaction (Table 2). Chl (a + b) concentration and chl a:b ratio were lowest at HT in both species (Fig. 3), but chl (a + b) concentration in S. fuscum and chl a:b ratio in S. magellanicum, respectively, were higher at MT than AT

(Fig. 3). Altitude did not affect photosynthetic pigment composition (Table 2). Nitrogen concentration in the capitula of the plants subjected to the AT treatment was about 10 mg g−1 , i.e., at optimum levels for the functioning of Sphagnum plants (Bragazza et al., 2004), with no differences between the two species (Table 2). Altitude and temperature affected capitulum N concentrations with overall highest values at LA and AT, respectively (Fig. 3). Both factors interacted

28

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

Table 2 P values obtained from three-way factorial ANOVAs of chlorophyll (a + b) concentration, chlorophyll a:b ratio and N concentration in the Sphagnum capitula after six days of recovery.

Species Altitude Temperature Species × Altitude Species × Temperature Altitude × Temperature Species × Altitude × Temperature

Chlorophyll (a + b)

Chlorophyll a:b

Nitrogen

0.000 0.10 0.000 0.55 0.000 0.49 0.34

0.17 0.33 0.000 0.57 0.000 0.34 0.53

0.61 0.000 0.02 0.05 0.008 0.59 0.13

Species, altitude, temperature treatment and their interactions were the fixed factors. Significant (P < 0.05) values are in boldface.

significantly with species (Table 2). Capitulum N concentration was higher in IA than HA for S. magellanicum but not for S. fuscum. On the other hand, capitulum N concentration was higher at MT than HT for S. fuscum but not for S. magellanicum (Fig. 3). 4. Discussion The lower net CO2 exchange rates in the MT plants, compared to the AT plants, were very probably caused by increased respiration rates at higher temperature (Clymo and Hayward, 1982; Kurets et al., 1993; Riis and Sand-Jensen, 1997). Net CO2 exchange rates in the plants experiencing the MT treatment recovered noticeably on 6 d because MT did not impair the photosynthetic machinery. Both species were seriously affected by the HT treatment. Not only did net CO2 exchange rates significantly decline, but Fv /Fm also decreased on 0 d by about 35% in HT compared to AT. A decrease in the maximum quantum efficiency of PSII , as estimated by Fv /Fm values, has been observed in several species of vascular plants grown at temperatures >38–40 ◦ C (Robakowski et al., 2002; Kouˇril et al., 2004). Our data suggest that the decline in Fv /Fm values in the HT plants was probably caused by the same mechanisms described in heat-stressed vascular plants, viz. alteration of thylakoid membranes and degradation of photosynthetic pigments (Pessarakli, 1999). Similarly, Liu et al. (2004) observed chloroplast damage and degreening in gametophores of the moss Plagiomnium acutum immersed in hot water at temperature >40 ◦ C. Indeed, chl concentrations in the capitula of the plants subjected to the HT treatment still were low on 6 d. The chl a:b ratio also was low in the HT plants, perhaps because chl a undergoes more rapid degradation than chl b in Sphagnum mosses (Gerdol et al., 1994). Decreased chl concentrations were accompanied by low capitulum N concentration especially in S. fuscum, i.e., a species exhibiting intrinsically low N concentrations in the capitula (Malmer, 1988). Indeed, N represents a major element in chl as well as in other cell components that can in turn be leached from the cell after membrane alteration (Haque et al., 2006). Interestingly, foliar application of N has been found to partially alleviate the adverse effects of heat stress on the photosynthetic performance of vascular plants (Zhao et al., 2008). As capitulum N concentrations in our plants always were below the threshold indicating N saturation in Sphagnum mosses (Bragazza et al., 2004), this suggests that a moderate increase in atmospheric N influx may alleviate the negative effects of heat stress on the Sphagnum layer in bog ecosystems in the sampling region. Despite severe damage, the plants experiencing the HT treatment did exhibit capacity to recover because the Fv /Fm values increased significantly, reaching almost 90% of optimum levels on 6 d. On the other hand, net CO2 exchange rates in the HT plants did not recover so quickly, presumably because the heat-stressed capitula required high respiration rates to restore the damaged tissues. Active respiration has also been observed in

Sphagnum mosses recoverying from drought stress (Gerdol et al., 1996). Summer warming affects the Sphagnum layer of ombrotrophic bogs primarily by lowering water-table depth (Weltzin et al., 2001) and reducing the moisture holding and transporting capacity of the mosses (Dorrepaal et al., 2003). Lack of precipitation has been observed to negatively affect CO2 uptake in Sphagnum mosses, rather than water-table drawdown itself (Robroek et al., 2009). Heat waves in temperate low-altitude regions in Europe were actually characterized by dry conditions (De Boeck et al., 2010). However, this does not seem to be a rule in mountainous regions. For example, precipitation during the 2003 heat wave was even somewhat higher than normal in part of the south-eastern Alps (Gerdol et al., 2008). Our experiment was, hence, designed to investigate specific effects of high temperature on Sphagnum mosses so that we cultivated the mosses at constant hydration in order to rule out any indirect effects associated with varying capitulum water content. As our plants were grown at very high water content throughout the experiment, temperature in the moss tissues did not rise much above air temperature because of the large thermal capacity of water (see Clymo and Hayward, 1982, and references therein). Should temperature in the living tissues exceed lethal threshold this would imply cell death with no chance of recovery. This apparently was not our case since even the plants grown at about 43 ◦ C, in the HT treatment, did recover fairly well within six days. Our data essentially support the results of previous studies based either on laboratory experiments or on field observations. On one hand, Balagurova et al. (1996) observed lethal temperatures >55 ◦ C for both S. magellanicum and S. fuscum when heating entire moss branches for 5 min while Buchner and Neuner (2010) observed a lethal threshold of 49.9 ◦ C when exposing individual leaves of S. capillifolium, a hummock-forming species taxonomically related to S. fuscum, to high temperatures for 30 min. On the other hand, Bragazza (2008) reported examples of Sphagnum mortality in the south-facing part of hummocks only at bogs in the southern Alps where the ratio of precipitation to temperature was below a critical threshold in the 2003 heat wave. Those irreversibly damaged mosses had quantum yield of PSII equal to zero, neither did they exhibit any sign of recovery after 4 years since the drought (Bragazza, 2008). Similarly, Alm et al. (1999) found irreversible damage in hummock-forming Sphagnum species at a boreal bog in northern Europe during a period when high temperatures were accompanied by prolonged drought in summer 1994. In conclusion, our first hypothesis was not supported by the results of this experiment since the two species presented much similar responses to heat stress as regards both physiological mechanisms and threshold temperature levels. On the other hand, we cannot exclude that varying temperature can affect the competitive abilities of different Sphagnum species growing in mixtures (Gunnarsson et al., 2004; Robroek et al., 2007; Breeuwer et al., 2008). Our second hypothesis was, conversely, supported by the results of this study. Indeed, the plants originating from the highaltitude mire suffered somewhat heavier damage when grown at HT compared to those originating from lower altitude sites. Consistent evidence of this resides in lower Fv /Fm values and lower chl and N concentrations recorded in the HA-HT plants. Increasing attention is being paid to analysing altitudinal adaptation of plant species to changing environment, including higher temperatures, based on the genetic structure of plant populations. Recent studies reported different degrees of altitudinal adaptation in alpine vascular species ranging, for example, from rather high in the native plant Arabis alpina (Manel et al., 2010) to none in the alien invasive plant Erigeron annuus (Trtikova et al., 2010). To our knowledge there are no data available on altitudinal adaptation in Sphagnum populations. Our study suggests that the two Sphagnum species possess moderate altitudinal plasticity to increased temperature

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

although we cannot provide mechanistic explanation to this observation. Altitudinal plasticity of Sphagnum populations with respect to environmental changes certainly deserves more detailed attention in further research. In general, we conclude that heat waves, even stronger than ever recorded, will unlikely bring about die-off of Sphagnum mosses in bog ecosystems unless high temperatures are coupled with drought.

Acknowledgements This research was partly supported by a grant from Ferrara University. We would like to thank Dr. Roberta Marchesini for technical assistance and Mr. Claudio Mutinelli (Province of Bolzano-Bozen) for providing climatic data.

References Alm, J., Schulman, L., Walden, J., Nykanen, H., Martikainen, P.J., Silvola, J., 1999. Carbon balance of a boreal bog during a year with an exceptionally dry summer. Ecology 80, 161–170. Balagurova, N., Drozdov, S., Grabovik, S., 1996. Cold and heat resistance of five species of Sphagnum. Ann. Bot. Fenn. 33, 33–37. Bragazza, L., 2008. A climatic threshold triggers the die-off of peat mosses during an extreme heat wave. Global Change Biol. 14, 2688–2695. Bragazza, L., Tahvanainen, T., Kutnar, L., Rydin, H., Limpens, J., Hájek, M., Grosvernier, P., Hájek, T., Hajkova, P., Hansen, I., Iacumin, P., Gerdol, R., 2004. Nutritional constraints in ombrotrophic Sphagnum plants under increasing atmospheric nitrogen depositions in Europe. New Phytol. 163, 609–616. Bragazza, L., Siffi, C., Iacumin, P., Gerdol, R., 2007. Mass loss and nutrient release during litter decay in peatland: the role of microbial adaptability to litter chemistry. Soil Biol. Biochem. 39, 257–267. Breeuwer, A., Heijmans, M.M.P.D., Robroek, B.J.M., Berendse, F., 2008. The effect of temperature on growth and competition between Sphagnum species. Oecologia 156, 155–167. Breeuwer, A., Heijmans, M.M.P.D., Robroek, B.J.M., Berendse, F., 2010. Field simulation of global change: transplanting northern bog mesocosms southward. Ecosystems 13, 712–726. Buchner, O., Neuner, G., 2010. Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium. Protoplasma 243, 63–71. Chen, J.Q., Bridgham, S., Keller, J., Pastor, J., Noormets, A., Weltzin, J.F., 2008. Temperature responses to infrared-loading and water table manipulations in peatland mesocosms. J. Integr. Plant Biol. 50, 1484–1496. Christensen, J.H., Christensen, O.B., 2007. A summary of the PRUDENCE model projections of changes in European climate by the end of this century. Clim. Change 81, 7–30. Clymo, R.S., Hayward, P.M., 1982. The Ecology of Sphagnum. In: Smith, A.J.E. (Ed.), Bryophyte Ecology. Chapman and Hall, London. De Boeck, H.J., Dreesen, F.E., Janssens, I.A., Nijs, I., 2010. Climatic characteristics of heat waves and their simulation in plant experiments. Global Change Biol. 16, 1992–2000. den Hartog, G., Neumann, H.H., King, K.M., Chipanshi, A.C., 1994. Energy budget measurements using eddy-correlation and Bowen-ratio techniques at the Kinosheo lake tower site during the northern wetlands study. J. Geophys. Res.-Atmos. 99, 1539–1549. Dorrepaal, E., Aerts, R., Cornelissen, J.H.C., Callaghan, T.V., Van Logtestijn, R.S.P., 2003. Summer warming and increased winter snow cover affect Sphagnum fuscum growth, structure and production in a sub-arctic bog. Global Change Biol. 10, 93–104. Ganguly, A.R., Steinhaeuser, K., Erickson, D.J., Branstetter, M., Parish, E.S., Singh, N., Drake, J.B., Buja, L., 2009. Higher trends but larger uncertainty and geographic variability in 21st century temperature and heat waves. PNAS 106, 15555–15559. Gerdol, R., Bragazza, L., 1994. The distribution of Sphagnum species along an elevational gradient, in the southern Alps (Italy). Bot. Helv. 104, 93–101. Gerdol, R., Bonora, A., Poli, F., 1994. The vertical pattern of pigment concentrations in chloroplasts of Sphagnum capillifolium. Bryologist 97, 158–161. Gerdol, R., Bonora, A., Gualandri, R., Pancaldi, S., 1996. CO2 exchange, photosynthetic pigment composition, and cell ultrastructure of Sphagnum mosses during dehydration and subsequent rehydration. Can. J. Bot. 74, 726–734. Gerdol, R., Bragazza, L., Brancaleoni, L., 2008. Heatwave 2003: high summer temperature, rather than experimental fertilization, affects vegetation and CO2 exchange in an alpine bog. New Phytol. 179, 142–154. Gershunov, A., Cayan, D.R., Iacobellis, S.F., 2009. The great 2006 heat wave over California and Nevada: signal of an increasing trend. J. Clim. 22, 6181–6203. Granath, G., Wiedermann, M.M., Strengbom, J., 2009. Physiological responses to nitrogen and sulphur addition and raised temperature in Sphagnum balticum. Oecologia 161, 481–490.

29

Grosvernier, P., Matthey, Y., Buttler, A., 1997. Growth potential of three Sphagnum species in relation to water table level and peat properties with implications for their restoration in cut-over bogs. J. Appl. Ecol. 34, 471–483. Gunnarsson, U., 2005. Global patterns of Sphagnum productivity. J. Bryol. 27, 269–279. Gunnarsson, U., Granberg, G., Nilsson, M., 2004. Growth, production and interspecific competition in Sphagnum: effects of temperature, nitrogen and sulphur treatments on a boreal mire. New Phytol. 163, 349–359. Hájek, T., Eeva-Stiina Tuittila, E.-S., Ilomets, M., Laiho, R., 2009. Light responses of mire mosses – a key to survival after water-level drawdown? Oikos 118, 240–250. Hájkova, P., Hájek, M., 2007. Sphagnum distribution patterns along environmental gradients in Bulgaria. J. Bryol. 29, 18–26. Haque, M.M., Hamid, A., Khanam, M., Biswas, D.K., Karim, M.A., Khaliq, Q.A., Hossain, M.A., Uprety, D.C., 2006. The effect of elevated CO2 concentration on leaf chlorophyll and nitrogen contents in rice during post-flowering phases. Biol. Plant. 50, 69–73. Harley, P.C., Tenhunen, J.D., Murray, K.J., Beyers, J., 1989. Irradiance and temperature effects on photosynthesis of tussock tundra Sphagnum mosses from the foothills of the Philip Smith Mountains, Alaska. Oecologia 79, 251–259. Hinzman, L.D., Bettez, N.D., Bolton, W.R., Chapin, F.S., Dyurgerov, M.B., Fastie, C.L., Griffith, B., Hollister, R.D., Hope, A., Huntington, H.P., Jensen, A.M., Jia, G.J, Jorgenson, T., Kane, D.L., Klein, D.R., Kofinas, G., Lynch, A.H., Lloyd, A.H., McGuire, A.D., Nelson, F.E., Oechel, W.C., Osterkamp, T.E., Racine, C.H., Romanovsky, V.E., Stone, R.S., Stow, D.A., Sturm, M., Tweedie, C.E., Vourlitis, G.L., Walker, M.D., Walker, D.A., Webber, P.J., Welker, J.M., Winker, K.S., Yoshikawa, K., 2005. Evidence and implications of recent climate change in northern Alaska and other arctic regions. Clim. Change 72, 251–298. Johnson, L.C., Damman, W.H., 1993. Decay and its regulation in Sphagnum peatlands. Adv. Bryol. 5, 249–296. Keller, J.K., White, J.R., Bridgham, S.D., Pastor, J., 2004. Climate change effects on carbon and nitrogen mineralization in peatlands through changes in soil quality. Global Change Biol. 10, 1053–1064. Koffi, B., Koffi, E., 2008. Heat waves across Europe by the end of the 21st century: multiregional climate simulations. Clim. Res. 36, 153–168. ˇ Kouˇril, R., Lazár, D., Ilík, P., Skotnica, J., Krchnák, P., Nauˇs, J., 2004. High-temperature induced chlorophyll fluorescence rise in plants at 40–50 ◦ C: experimental and theoretical approach. Photosynth. Res. 81, 49–66. Kurets, V.K., Talanov, A.V., Popov, E.G., Drozdov, S.N., 1993. Light-temperature dependencies of apparent photosynthesis and dark respiration in Sphagnum. Russ. J. Plant Physiol. 40, 608–611. Laine, J., Harju, P., Timonen, T., Laine, A., Tuittila, E.S., Minkkinen, K., Vasander, H., 2009. The intricate beauty of Sphagnum mosses – a Finnish guide to identification, Publications 39, University of Helsinki, Department of Forest Ecology. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic membranes. Method Enzymol. 148, 350–382. Limpens, J., Berendse, F., 2003. How litter quality affects mass loss and N loss from decomposing Sphagnum. Oikos 103, 537–547. Liu, Y.D., Li, Z.Y., Cao, T., Glime, J.M., 2004. The influence of high temperature on cell damage and shoot survival rates of Plagiomnium acutum. J. Bryol. 26, 265–271. Lobo, A., Maisongrande, P., Coret, L., 2010. The impact of the heat wave of summer 2003 in SW Europe as observed from satellite imagery. Phys. Chem. Earth 35, 19–24. Malmer, N., 1988. Patterns in the growth and the accumulation of inorganic constituents in the Sphagnum cover on ombrotrophic bogs in Scandinavia. Oikos 53, 105–120. Manel, S., Poncet, B.N., Legendre, P., Gugerli, F., Holderegger, R., 2010. Common factors drive adaptive genetic variation at different spatial scales in Arabis alpina. Mol. Ecol. 19, 3824–3835. Marchand, F.L., Verlinden, M., Kockelbergh, F., Graae, B.J., Beyens, J., Nijs, I., 2006. Disentangling effects of an experimentally imposed extreme temperature event and naturally associated desiccation on Arctic tundra. Funct. Ecol. 20, 917–928. Marschall, M., Proctor, M.C.F., 2004. Are bryophytes shade plants? Photosynthetic light responses and proportions of chlorophyll a, chlorophyll b and total carotenoids. Ann. Bot. 94, 593–603. Maseyk, K.S., Green, T.G.A., Klinac, D., 1999. Photosynthetic responses of New Zealand Sphagnum species. N. Z. J. Bot. 37, 155–165. Meehl, G., Tebaldi, C., 2004. More intense, more frequent, and longer lasting heat waves in the 21st century. Science 305, 994–997. Melosik, I., 2009. The effect of the ploidy level and genetic background of Sphagnum denticulatum on its morphology and ecological requirements. Oceanol. Hydrobiol. St. 38, 153–164. Park, C.K., Schubert, S.D., 1997. On the nature of the 1994 East Asian summer drought. J. Clim. 10, 1056–1070. Pessarakli, M., 1999. The Effect of High Temperature Stress on the Photosynthetic Apparatus. Marcel Dekker Inc, New York. Przybylak, R., 2002. Changes in seasonal and annual high-frequency air temperature variability in the arctic from 1951 to 1990. Int. J. Climatol. 22, 1017–1032. Riis, T., Sand-Jensen, K., 1997. Growth reconstruction and photosynthesis of aquatic mosses: Influence of light, temperature and carbon dioxide at depth. J. Ecol. 85, 359–372. Robakowski, P., Montpied, P., Dreyer, E., 2002. Temperature response of photosynthesis of silver fir (Abies alba Mill.) seedlings. Ann. Forest Sci. 59, 163–170. Robroek, B.J.M., Limpens, J., Breeuwer, A., Schouten, M.G.C., 2007. Effects of water level and temperature on performance of four Sphagnum mosses. Plant Ecol. 190, 97–107.

30

R. Gerdol, R. Vicentini / Environmental and Experimental Botany 74 (2011) 22–30

Robroek, B.J.M., Schouten, M.G.C., Limpens, J., Berendse, F., Poorter, H., 2009. Interactive effects of water table and precipitation on the CO2 assimilation of three co-occurring Sphagnum mosses differing in distribution above the water table. Global Change Biol. 15, 680–691. Rydin, H., Jeglum, J., 2006. The Biology of Wetlands. Oxford University Press, Oxford. Rydin, H., McDonald, A.J.S., 1985. Tolerance of Sphagnum to water level. J. Bryol. 13, 571–578. Sastad, S.M., Pedersen, B., Digre, K., 1999. Habitat-specific genetic effects on growth rate and morphology across pH and water-level gradients within a population of the moss Sphagnum angustifolium (Sphagnaceae). Am. J. Bot. 86, 1687–1698. Schipperges, B., Rydin, H., 1998. Response of photosynthesis of Sphagnum species from contrasting microhabitats to tissue water content and repeated desiccation. New Phytol. 140, 677–684. Silvola, J., Aaltonen, H., 1984. Water content and photosynthesis in the peat mosses Sphagnum fuscum and S. angustifolium. Ann. Bot. Fenn. 21, 1–6.

Szovenyi, P., Hock, Z., Korpelainen, H., Shaw, A.J., 2009. Spatial pattern of nucleotide polymorphism indicates molecular adaptation in the bryophyte Sphagnum fimbriatum. Mol. Phylogenet. Evol. 53, 277–286. Trtikova, M., Edwards, P.J., Güsewell, S., 2010. No adaptation to altitude in the invasive plant Erigeron annuus in the Swiss Alps. Ecography 33, 556–564. Unkasevic, M., Tosci, I., 2009. An analysis of heat waves in Serbia. Global Planet. Change 65, 17–26. Weltzin, J.F., Harth, C., Bridgham, S.D., Pastor, J., Vonderharr, M., 2001. Production and microtopography of bog bryophytes: response to warming and water-table manipulations. Oecologia 128, 557–565. Wielgolaski, F.E., 1997. Polar and Alpine Tundra, Ecosystems of the World. Elsevier, Amsterdam. Zhao, W.Y., Xu, S., Li, J.L., Cui, L.J., Chen, Y.N., Wang, J.Z., 2008. Effects of foliar application of nitrogen on the photosynthetic performance and growth of two fescue cultivars under heat stress. Biol. Plant. 52, 113–116.