Responses of the green algal foliose lichen Platismatia glauca to increased nitrogen supply

Authors

  • Kristin Palmqvist,

    1. Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå, Sweden;
    Search for more papers by this author
  • Lena Dahlman

    1. Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå, Sweden;
    2. Bioresources Research Centre (BRC), University College Dublin, Earlsfort Terrace, Dublin 2, Ireland
    Search for more papers by this author

Author for correspondence: K. Palmqvist Tel: +46 90 786 91 84 Fax: +46 90 786 67 05 Email: Kristin.Palmqvist@emg.umu.se

Summary

  • • Nitrogen (N) availability and light exposure were manipulated under field conditions to study responses to altered resource supply in the green algal lichen Platismatia glauca.
  • • The lichen was fertilized with different concentrations and frequencies of ammonium, nitrate or glutamine under different light regimes for 2–3 months. Responses were followed from the intact thallus to the cellular level.
  • • Despite significant differences in overall light exposure, light conditions were not significantly different among treatments when the lichens were wet and active. Ammonium was the preferred N source, followed by glutamine and then nitrate. Thallus N concentration as well as the chlorophyll a (Chl a) concentration increased 3–4-fold at the highest ammonium concentration, while the mycobiont ergosterol concentration remained unaltered. Growth was significantly enhanced by the enhanced N supply, with the increase in dry weight varying from 3 to 30%. Variation in Chl a concentration explained 31% of this variation, suggesting a causal link to the increased growth rate.
  • • Platismatia glauca responded to increased N availability by increasing its growth rate and carbon assimilation capacity through increased investments in the photobiont cells. This suggests a tight regulation of resource investments and metabolic pathways between the symbionts of this lichen.

Introduction

Plant growth is simultaneously subject to genetic, ontogenetic and environmental control (Grime & Hunt, 1975). For instance, to secure future development, carbon (C)-acquiring (shoot) and mineral-acquiring (root) tissues must continuously be balanced both in relation to each other and in relation to resource supplies (Chapin, 1991). Lichens that are symbiotic associations between a nutritionally specialized fungus (mycobiont) and an algal or cyanobacterial photobiont (Honegger, 1991) face a similar, albeit possibly more complex, problem in that growth of two nonrelated organisms must be co-ordinated in relation both to each other and to environmental resource supply (Honegger, 1991; Palmqvist, 2000). One reason for the requirement for regulated investments between the photobiont cells and the fungal hyphae of lichens is related to the necessity to maintain a positive balance between C gain and expenditures in the thallus under varying environmental conditions (Palmqvist, 2000). This notion is supported by a recent study on Cladina rangiferina, which found a significant increase in the producer-to-consumer ratio, i.e. the photobiont-to-mycobiont ratio, with increasing temperatures to match the increased respiratory requirements of warmer habitats (Friedmann & Sun, 2005; Sun & Friedmann, 2005). A few studies carried out both between (Sonesson et al., 1992; Smith & Griffiths, 1998; Sancho et al., 2000) and within lichen populations (MacKenzie et al., 2001) have shown that lichens also regulate their thallus composition in relation to an altered light supply.

In addition to temperature and light, nitrogen (N) is another environmental resource that may affect the C balance of lichens and require an adjustment of the photobiont-to-mycobiont ratio. This is because increased investments of nitrogenous compounds in the thallus without a concomitant increase in photosynthesis may lead to a negative C balance. This is related to the generally increased respiration of N-rich cells (Turpin, 1991; Grace, 1997; Lambers et al., 1998), and has also been demonstrated for lichens (Palmqvist et al., 2002). Increased N deposition is, moreover, an environmental stress factor that has received increased attention in the lichen literature in recent years (Søchting, 1995; Van Dobben & De Bakker, 1996). Support for the view that increased N supply might result in an altered balance between the bionts in lichen thalli has been provided by some studies. Area gain and hyphal extension were, for instance, inhibited in N-fertilized Nephroma arcticum thalli, although weight gain and photobiont growth continued (Sundberg et al., 2001; Dahlman et al., 2002). The N-sensitive lichen Evernia prunastri responded similarly, showing reduced soluble C pool concentrations in the mycobiont and a decreased mycobiont (ergosterol) concentration with increased N assimilation (Gaio-Oliveira et al., 2004).

However, increased N availability might also be beneficial to the C balance because photosynthetic capacity is generally increased with increasing N content of photosynthetic tissue, which can be explained by the N investment costs of the photosynthetic proteins (Evans, 1989). This appears to be the case for lichens, in which photosynthetic capacity, Rubisco content and photosynthetic pigment concentration increase with increasing thallus N concentration both inter- and intraspecifically (Tretiach & Carpanelli, 1992; Tretiach & Pecchiari, 1995; Palmqvist et al., 1998, 2002; Palmqvist & Sundberg, 2000; Sundberg et al., 2001; Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2006). Environmental N supply may hence be limiting for lichens (Crittenden, 1991; Crittenden et al., 1994), and their capacity to assimilate both inorganic and organic N forms is generally high (Smith, 1960a,b; Crittenden, 1996, 1998; Kielland, 1997; Dahlman et al., 2004). However, different N forms are assimilated with different efficiency (Dahlman et al., 2004), and the N source may have indirect effects on the C balance of lichens. Ammonium (NH4+) uptake is, for instance, more passive than amino acid and nitrate (NO3) assimilation (Brown et al., 1994), and the reduction of NO3 is associated with additional C costs for its reduction. In contrast, the lichen may acquire both C and N when assimilating organic N sources (Smith et al., 1994).

The aim of this study was to investigate the responses of a lichen to altered N supply using the foliose, epiphytic, green algal (Trebouxia sp.) lichen Platismatia glauca as a model species. In addition to alteration of the N supply, the lichen was exposed to different light regimes, with the aim of manipulating the C availability. P. glauca can thrive in both light-exposed habitats and deep shade (Smith & Griffiths, 1998), and can assimilate both inorganic and organic N forms (Dahlman et al., 2004). It was also able to survive 15 years of intensive N fertilization (Dahlman et al., 2003), indicating that this is an N-tolerant lichen. More specifically, the following questions were addressed. (i) How rapidly can lichens assimilate and adapt to an enhanced N supply? (ii) What are the major responses? (iii) Are the responses dependent on the supplied N form or light availability? The thallus N concentration was measured before and after the experiment, and the fertilizer was spiked with the stable isotope 15N to determine the N uptake. The photosynthetic pigment chlorophyll a (Chl a) was used as an indirect marker for the photobiont, and ergosterol as a marker for the mycobiont. We used Chl a because the concentration of this pigment covaries with the Rubisco concentration (Palmqvist et al., 1998), the photosynthetic capacity (Sundberg et al., 1997; Palmqvist et al., 1998, 2002), and growth (Palmqvist & Sundberg, 2000; Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2006) in unstressed lichens. Growth was measured to assess the vitality of the whole thallus.

Materials and methods

The foliose, epiphytic, green algal (Trebouxia sp.) lichen Platismatia glauca (L.) W. Culb. was fertilized with different N forms at various concentrations and frequencies under natural field conditions, in a closed forest stand at Omagaliden, Vindeln during the summer of 2003 and at a meadow site at Täfteå, Umeå during the summer of 2004. Both sites are situated in the boreal forest zone of north-east Sweden.

Low-intensity N fertilization and N preference experiment (closed forest stand)

Omagaliden (64°09′ N, 19°50′ E) is a dense 70–80-year-old Norway spruce (Picea abies (L.) Karst) forest intermixed with Pinus sylvestris L., Betula pubescens Ehrh., Betula verrucosa Ehrh. and Salix. caprea L., located on a north-facing slope above Vindelälven. The stand has a lush epiphytic lichen flora with Alectoria spp., Bryoria spp., Nephroma spp., Lobaria spp., Platismatia glauca and Usnea spp., with P. glauca being dominant on the lower spruce branches. The site was used in a previous transplantation experiment carried out between June 2000 and September 2001; for a detailed description of the climatic conditions from an epiphytic lichen perspective, see Gaio-Oliveira et al. (2003). Four spruce trees in close proximity to each other were selected for this experiment. P. glauca-rich branches situated 1.5 to 2 m above ground, together covering a 0.25 m2 projected area were chosen on each tree, and assigned to one of the following treatments: irrigation with artificial rainwater (control), or fertilization with ammonium (NH4+), nitrate (NO3) or glutamine (Gln). The N was added weekly (15 times) between 10 June and 29 September 2003 at 16:00 h, as 0.55 mm (NH4)2SO4, 1.1 mm KNO3 or 0.55 mm Gln (C5H10N2O3), dissolved in artificial rainwater (Tamm, 1953) of the following composition (values in milligrams per litre): 8.8 mg l−1 K2CO3, 4.6 mg l−1 Na2CO3, 5 mg l−1 CaCO3, 4.4 mg l−1 NaH2PO4, 0.25 mg l−1 Fe2SO47H2O, and 0.6 mg l−1 MnSO4H2O. The fertilizer was dissolved in artificial rainwater to avoid the osmotic stress of using distilled water in the unfertilized control treatment. The same rainwater recipe has been used in recent studies (Dahlman et al., 2002; Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2004), allowing comparisons between experiments. The lichens in each treatment were gently sprayed with 2.5 l of the respective solution for 10–15 min. The total N dosage corresponded to 25 kg N ha−1 administered over the experimental period, and the artificial rainwater dosage corresponded to 150 mm precipitation on top of 250 mm of natural precipitation (Table 1); climatic conditions were monitored as described in the section ‘Climate monitoring’ below. Background deposition of N from precipitation amounted to 0.5–2 kg N ha−1 year−1 in the area (Lövblad et al., 1992; Forsum et al., 2006).

Table 1.  Climatic conditions during the low-intensity (closed forest, 2003) and the high-intensity (meadow, 2004) fertilization experiments
 Closed forestShaded meadowExposed meadow
  1. Values are mean ± standard error (SE) unless otherwise indicated.

  2. The parameters were derived as described in the Materials and methods, averaging the light and water content data for the four treatments in the closed forest, and the five treatments in each respective light regime at the meadow site. Mean values ± 1 SE followed by a different letter are significantly different at P < 0.05 [one-way analysis of variance, Tukey (HSD (honestly significant difference) comparison of means)].

  3. Itot, the total irradiance received by the lichens during the whole period; Iwet, the accumulated irradiance intercepted by metabolically active thalli; PFDtot, photon flux density for the whole transplantation period; PFDwet, photon flux density for periods when the lichens had a water content above 5% of maximum; Tair, air temperature.

Total time (h)266317621762
Natural precipitation (mm) 250 131 131
Irrigation (mm) 150  50  50
Wet time (h) 927 ± 54a 675 ± 27b 651 ± 16b
Wet in darkness (h) 163 ± 14 139 ± 7 132 ± 12
Wet occasions  27 ± 2  58  58
Itot (mol m−2) 106 ± 15a 244 ± 34b 340 ± 29c
Iwet (mol m−2)  52 ± 15  46 ± 3  40 ± 3
Mean daytime PFDtot (µmol m−2 s−1)  16 ± 2a  48 ± 7b  67 ± 5c
Mean daytime PFDwet (µmol m−2 s−1)  19 ± 4  25 ± 1  24 ± 2
Maximum PFDtot (µmol m−2 s−1) 198 ± 50a 760 ± 86b1064 ± 85c
Maximum PFDwet (µmol m−2 s−1) 174 ± 34a 508 ± 19b 548 ± 135b
Mean Tair for total time (°C)11.814.214.2
Mean Tair for wet time (°C)10.2 ± 0.1a12.3 ± 0.2b12.0 ± 0.2b

At harvest, each tree was irrigated with 10 l of artificial rainwater to remove forest debris and traces of the different N sources that might have been passively attached to the lichens. A large number of thalli were collected from the treated branches and brought to the laboratory, cleaned of debris and epiphytic algae, dried at 15°C in low light (10–15 mmol m−2 s−1), and thereafter stored at −18°C for a few months before analysis of their N uptake capacity in an N preference experiment, as in Dahlman et al. (2004). For this, the lichens were removed from the freezer, temperature-equilibrated for 10–15 min, and thereafter sprayed with water to reactivate and stabilize their metabolism in a controlled climate chamber at 15°C, 95% relative humidity (RH), 4 h dark: 12–17 h light, with a photon flux density (PFD) of 50 µmol m−2 s−1 over the waveband 400–700 nm. Briefly, 18 thalli from each treatment were placed in 20 ml of an incubation solution containing NH4NO3 and Gln, with equal concentrations of N in the three forms and a total N concentration of 1.0 mm. The incubation solution contained additional nutrients and the pH was adjusted to 5.0 (Persson & Näsholm, 2002). Three different N solutions were prepared, in each of which one of the three N forms was labelled with 15N: 15NH4NO3Gln, NH415NO3Gln or NH4NO315 Gln (Cambridge Isotope Laboratories, Andover, MA, USA). The 18 thalli from each field treatment were randomly assigned to one of the three solutions (six to each). The thalli were of similar size, varying in dry weight (DW) from 0.09 to 0.17 g, with an average of 0.12–0.14 g, not varying significantly among the four field treatments. A small piece of each thallus was removed before the incubation to determine its initial 15N content. Three additional thalli from each field treatment were incubated in each of the three N solutions in the presence of 100 µm carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically inhibiting ATP-dependent transport across cellular plasma membranes (cf. Martin et al., 1991; Persson & Näsholm, 2002). The lichens were completely submerged for 30 min in darkness at 15°C in the incubation solution and heavily bubbled with air to prevent anaerobic conditions. The lichens were thereafter placed in an unlabelled incubation solution (10 mm total N), and washed twice in a 1 mm CaCl2 solution to wash off labelled substrate passively attached to the cell walls. Following the washing procedure, the lichen thalli were immediately frozen in liquid N2, freeze-dried, weighed and thereafter stored in a freezer (−80°C). Subsequently their isotopic N composition and their total N, chlorophyll, and ergosterol concentrations were analysed as described in the section ‘Chemical analysis’.

High-intensity fertilization, growth and N uptake analysis (meadow site)

At a meadow site closer to Umeå University (Täfteå; 63°50′ N, 20°28′ E), more intensive (daily) fertilization was possible. For this, P. glauca thalli were collected from a nearby relatively open forest of spruce mixed with deciduous trees (Ulterviken; 63°48′ N, 20°27′ E), a site situated within two kilometers from the coast with a lush epiphytic lichen flora, used in previous transplantation studies in which the climatic conditions are described in detail: from September 1995 to September 1996 (Palmqvist & Sundberg, 2000), from June to October 1997 (Sundberg et al., 2001), and from June to September 1999 (Dahlman & Palmqvist, 2003). A total of 150 lichen thalli were assigned to four fertilization (50 and 100 kg NH4+, 50 kg NO3 and 50 kg Gln) and one control treatment, duplicated in one shaded and one exposed light regime, i.e. 15 thalli per treatment. All thalli were cut into two halves, one used to obtain data on the characteristics of the lichens at the start of the experiment, and the other for the treatments. In this way, all changes between the start of the experiment and harvest could be measured for each individual thallus. The ‘start’-halves were freeze-dried (Lyovac GT 2; Steris GmbH, Hürth, Germany) and stored at −80°C until the end of the experiment. The N sources were identical to those in the low-intensity fertilization experiment, this time using 0.64 mm or 1.28 mm (NH4)2SO4, 1.28 mm KNO3, or 0.64 mm Gln (C5H10N2O3) dissolved in artificial rainwater, as specified above. The control thalli were treated with artificial rainwater. The N solutions were spiked with the stable isotope 15N to measure N uptake over the whole experimental period. The transplantation rods (see next paragraph) in each treatment were gently sprayed for 10 min with 75 ml of the respective solution at 18:00 h each day between 19 June and 29 August 2004 (65 times), each thallus receiving c. 5 ml. No treatments were carried out on the few days on which there was heavy rain. The lichens were wet and active 58 times (Table 1), as some wet periods exceeded 24 h. The daily N dosage per thallus was c. 90 µg N in the lower NH4+, the NO3 and the Gln treatments and c. 180 µg N in the higher NH4+ treatment. Based on the area of the transplantation frame (see next paragraph) this corresponded to a 50 kg ha−1 dosage for the lower NH4+, the NO3 and the Gln treatments, and 100 kg ha−1 for the higher NH4+ treatment. Natural precipitation during the experiment was 131 mm, and the irrigation corresponded to 50 mm (Table 1). All thalli were sprayed with a surplus of artificial rainwater immediately before harvest as in the other experiment.

Growth of the treated thallus halves was determined by measuring their DW under controlled conditions in the laboratory before and after the experiment (Dahlman & Palmqvist, 2003). The thalli were mounted on 1-m-long rods made from Norway spruce, using a thin, transparent nylon thread. A photograph of each thallus was taken both before and after it was mounted on the rod. The rods were positioned 70 cm above the ground, with the lichens facing the sky at a 90° angle. The rods were fastened onto plastic-covered metal frames, fixed firmly in the soil, placed among lower shrubs (2–4 m high) close to a meadow. The shaded frame was exposed to the north-east and more enclosed by shrubs, while the exposed frame was placed outside the shrubs and was exposed to the south. The rods with the transplants were brought back to the laboratory after the experiments for DW determinations, and new photographs were taken of each thallus before and after dismounting. Thalli that had lost visible parts during the experiment, as judged from the photographs, were excluded from the analysis: in shaded light, one in the control, two in the 50-kg NH4+, two in the 100-kg NH4+, three in the NO3 and two in the Gln treatment, and in exposed light, one in the control and two in the 100-kg NH4+ treatment. Start values for these thalli were also excluded, and as a result, 137 of the 150 treated thalli are included in the data presented.

Sampling of thalli for the experiment had been biased towards relatively large and homogenously healthy-looking individuals to enable enough material for all analyses on both the start and the transplantation halves of each individual, the DW of the intact thalli ranging between 0.20 and 1.48 g, and the transplanted halves between 0.08 and 0.98 g at the start of the experiment (average = 0.27 g), and between 0.09 and 1.02 g at harvest (average = 0.31 g) (excluding the damaged thalli).

Chemical analysis

The thalli from the N preference experiment and the freeze-dried start and harvest halves from the high-intensity fertilization experiment were ground to a homogeneous powder using a ball mill and vials that had been pre-cooled in liquid N2. The powder from each sample was aliquoted for different assays and stored dry at −80°C, and thereafter extracted and analysed within 1 wk after homogenization.

Chlorophyll pigments were quantified after extraction in MgCO3-saturated dimethyl sulphoxide (DMSO), at 60°C for 40 min (Palmqvist & Sundberg, 2001), using 10 mg of material and 1.5 ml of DMSO.

Ergosterol was quantified by high-performance liquid chromatography (HPLC) after extraction in 99.5% ethanol (Dahlman et al., 2001) using 10 mg of material and 1 ml of ethanol.

Arabitol, glucose, mannitol and ribitol were extracted and quantified from 10 mg of material in 1 ml of sterilized water, incubated on a shaker for 30 min at 20°C, and centrifuged at 20 000 g for 15 min. The resulting supernatant was transferred to a new tube and stored in a freezer (−20°C) before analysis using ion chromatography (Metrohm AG, Herisau, Switzerland). The soluble carbon compounds were separated on a Metrosep Carb-250 column (250 cm × 4 mm) using 0.1 m NaOH as isocratic eluent and a flow rate of 1 ml min−1 (20 min per sample), and quantified using d-(+)-arabitol (Fluka, Buchs, SG, Switzerland), d-(+)-glucose (Sigma, Buchs, SG, Switzerland), d-mannitol (Sigma), and adonitol (i.e. ribitol) (Fluka). Quantifications were obtained using standard curves. A mixture of known concentrations of the standards was run every 20th sample to check for accuracy of the assays.

Total thallus N concentration, and the total thallus C concentration of the start halves from the high-intensity fertilization, excluding the control thalli, were determined with a CHN elemental analyser (Perkin-Elmer Model 2400 CHN, Perkin-Elmer, Boston, MA, USA). The total thallus C and N concentrations of the thalli from the laboratory N preference experiment, the start halves of the control thalli, and all harvested thalli from the high-intensity fertilization experiment were analysed with a CN analyzer (ANCA-NT System, Solid/Liquids Preparation Module; Europa Scientific, Crewe, UK) coupled to a Europa 20-20 Isotopic Ratio Mass Spectrometer (IRMS; Europa Scientific), to determine the ratios of 14N to 15N, and 12C to 13C, respectively, according to Olsson & Wallmark (1999). These C and N analyses were purchased from certified laboratories at the Faculty of Forest Sciences at The Swedish University of Agricultural Sciences, Umeå, Sweden, whose instruments are calibrated according to international standard procedures. The total thallus N and C concentrations derived from the two analysers can therefore be compared on an absolute scale.

N uptake in both the N preference experiment and the high-intensity fertilization experiment was calculated as:

image( Eqn 1)

[At%s, the atomic percentage of 15N in the sample; At%c, the atomic percentage of 15N in unlabelled control thalli; total Ns, the total N concentration of the sample (g g−1 DW); fraction 15N corrects for the amount of label in the fertilizer or incubation solution, being 100%15N in NH4+ and NO3, and 50%15N in Gln in the N preference experiment, and 10%15N in NH4+ and NO3, and 5% in Gln in the high-intensity fertilization experiment.]

Climate monitoring

A climate station recorded macro- and microclimatic conditions at the two experimental sites, the closed forest stand (summer 2003) and the meadow site (summer 2004), using the same sensors, techniques, and data processing as described in detail previously (Sundberg et al., 1997, 2001; Palmqvist & Sundberg, 2000; Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2003). The sensors were read every minute and averaged (rain was summed) over 30-min intervals in the closed forest, and over 15-min intervals at the meadow site. The closed forest was visited weekly for fertilization, collection of climatic data, and checking of the sensors. The lichens at the meadow site were fertilized daily, and the sensors were also inspected daily.

Microclimatic conditions in each treatment were extracted from the logger data as described previously (Palmqvist & Sundberg, 2000) (Table 1). Briefly, impedance values of fully hydrated thalli were set to 100% water content (WCmax), and the lichens were assumed to be wet and metabolically active when their water content was above 5% of WCmax. The accumulated irradiance, i.e. the accumulated light dose, intercepted by metabolically active thalli, denoted Iwet (mol photons m−2), was obtained by summing all photons received by the lichens when their WC was above this limit. The light intensity (PFD) was calculated for periods when the PFD was above 0 µmol m−2 s−1, denoted daytime PFD, where PFDtot reflects the whole transplantation period and PFDwet the periods when the lichens were above 5% of WCmax.

Statistical analysis

Regressions and analyses of variance (ANOVAs) were performed using the statistical package statistix 7 (Analytical Software, Tallahassee, FL, USA). All post hoc tests were made with Tukey's honestly significant difference (HSD) test.

Results

Climatic conditions

The microclimatic conditions and the differences between the two field sites and the two light regimes at the meadow site are presented in Table 1, as determined by one-way ANOVA and Tukey (HSD) comparison of means, with P < 0.05. The microclimatic conditions did not differ within the four treatments in the closed forest, or within the five treatments in each light regime at the meadow site (not shown). The treatment period was longer in the closed forest (2663 h) than at the meadow site (1762 h). Despite this, the total irradiance received by the lichens during the whole period (Itot) was significantly lower in the forest than in the meadow, which can be explained by the significantly lower light intensity (PFDtot) in the forest, with both the mean daytime PFDtot and the maximum PFDtot being significantly lower in the forest (Table 1). The light intensity (maximum and mean daytime PFDtot) and subsequently the total light dose (Itot) were also significantly lower in the shaded than in the exposed site at the meadow (Table 1), so the attempt to expose the lichens to two different light regimes had been successful. However, despite the significant difference in overall light exposure between the sites, this difference was diminished when the lichens were wet and active. The mean daytime PFDwet was similarly low (19–25 µmol m−2 s−1) and not significantly different at the three sites (Table 1). In agreement with this, the ratio between Iwet and Itot, which compensates for the difference in experimental period, was lowest in the exposed meadow and highest in the closed forest.

Nitrogen uptake

When the lichens were given a mixture of the three N forms in a 30-min laboratory experiment, NH4+ uptake was highest, Gln uptake somewhat lower, and NO3 uptake lowest (Fig. 1). This pattern was similar when the uptake was related to DW (Fig. 1, upper panel) and thallus N concentration (Fig. 1, lower panel). CCCP inhibited 50 ± 5 (mean ± 1SE), 68 ± 5 and 78 ± 4% of NH4+, Gln and NO3 uptake, respectively, with a significantly higher inhibition of NO3 compared with NH4+ uptake [one-way ANOVA, Tukey (HSD) comparison of means, P < 0.05].

Figure 1.

Nitrogen (N) uptake in Platismatia glauca in relation to thallus dry weight (top panels) or thallus N concentration (bottom panels) during a 30-min laboratory experiment. The lichens were pretreated with artificial rainwater (control), NH4+, NO3 or glutamine (Gln) for 3.5 months in a low-intensity fertilization regime in the field, and were exposed to a 1.0 mm mixture of all the three N forms in the laboratory experiment, as further described in the Materials and methods. Bars (mean + 1 standard error; n = 6) with a different letter are significantly different for the interaction term field treatment × labelled N form [two-way analysis of variance, Tukey (HSD (honestly significant difference)) comparison of means, P < 0.05].

As the lichens had been treated with only one of the three N forms in the preceding low-intensity fertilization experiment, the laboratory experiment also tested if this had altered the preferences of the lichens for using a particular N form. There were some indications of such alterations, with NH4+ uptake being highest in the NO3-treated thalli and lowest in the NH4+-treated thalli, when expressed on thallus N concentration basis (Fig. 1, lower panel). Moreover, both the NO3- and the Gln-treated thalli showed lower Gln uptake compared with the control and the NH4+ treatment when expressed either on thallus DW or thallus N concentration basis (Fig. 1).

As in the laboratory experiment, uptake of NH4+ was the highest of the three N forms in field conditions (Fig. 2). Moreover, N uptake was higher in the higher (100 kg ha−1) than in the lower (50 kg ha−1) NH4+ treatment, being c. 11 and 7 mg g−1 DW, respectively. In contrast to the laboratory experiment, the uptakes of Gln and NH4+ were similar in the field, ranging from 4.4 to 4 mg g−1 DW (Fig. 2). There was no difference in these uptake patterns between the shaded and the exposed light regimes (Fig. 2), reflecting the fact that light conditions were similar when the lichens were wet and active (Table 1).

Figure 2.

Accumulated nitrogen (N) uptake from the fertilizer during 2.5 months of high-intensity N fertilization with NH4+, NO3 or glutamine (Gln) in the shaded (SL) and the exposed (EL) light regimes, as described in the Materials and methods. Bars (mean + 1 standard error) with a different letter are significantly different for the interaction term N source × light regime [two-way analysis of variance, Tukey (HSD (honestly significant difference)) comparison of means, P < 0.05]. The number of samples ranged from 12 to 15, as specified in Materials and methods.

Effects of N uptake

Thallus N concentration  Fertilization with each of the three N forms resulted in a significant increase in the lichen thallus N concentration. This was manifested as a higher N concentration in the NH4+-treated thalli compared with control under low-intensity (25 kg ha−1) fertilization, with N concentration being 6.2 and 4.5 mg g−1 DW, respectively (Table 2). Under high-intensity fertilization this was manifested as an increase in N concentration from c. 6 mg g−1 DW at the start of the experiment to 16–22 mg g−1 DW at harvest in the two NH4+ treatments (50 and 100 kg ha−1) (Fig. 3). The Gln-treated thalli also showed an increase in N concentration, relative to control under low-intensity fertilization (Table 2) and relative to control and start values under high-intensity fertilization (Fig. 3). The NO3 treatment resulted in a significant increase in N concentration only in the high-intensity fertilization experiment (Table 2, Fig. 3).

Table 2.  Concentrations of total thallus nitrogen (N), chlorophyll (Chl) a, Chl b, and ergosterol at harvest for the 18 Platismatia glauca thalli from each tree under low-intensity fertilization used in the N-preference experiment (Fig. 1)
TreatmentNtot (mg g−1 DW)Chl a (mg g−1 DW)Chl b (mg g−1 DW)Ergosterol (mg g−1 DW)
  1. Values are mean ± 1 standard error.

  2. Values followed by a different letter are significantly different for P < 0.05, testing each compound separately [one-way analysis of variance, Tukey (HSD (honestly significant difference)) comparison of means].

  3. DW, dry weight; Gln, glutamine; Ntot, total nitrogen concentration.

Control4.49 ± 0.08b0.55 ± 0.02c0.16 ± 0.005c0.60 ± 0.02ab
25 kg NH4+6.23 ± 0.42a0.88 ± 0.05a0.25 ± 0.01a0.68 ± 0.04a
25 kg NO35.23 ± 0.11b0.51 ± 0.02c0.17 ± 0.004c0.57 ± 0.02b
25 kg Gln6.22 ± 0.12a0.75 ± 0.02b0.22 ± 0.006b0.62 ± 0.03ab
Figure 3.

Platismatia glauca thallus nitrogen (N), chlorophyll (Chl) a and b, and ergosterol concentrations at the start of the experiment and at harvest in the high-intensity N fertilization experiment; see the Materials and methods for details. Bars (mean + 1 standard error) with a different letter, for each compound, are significantly different for the interaction term occasion × treatment [two-way analysis of variance, Tukey (HSD) comparison of means, P < 0.05]. Data from the two light regimes were pooled so the number of samples ranged from 26 to 28, as specified in Materials and methods.

The increase in thallus N concentration under high-intensity fertilization (Fig. 3) was well correlated to the uptake of the respective N forms (Fig. 2). However, in all the fertilization treatments, the increase in thallus N concentration was higher than the uptake from the 15N-labelled fertilizer, and there was also an increase in N in the control (Fig. 3). This suggests an additional background source of N at the meadow site.

Chlorophyll and ergosterol  In the low-intensity fertilization experiment, there was an increase in Chl concentration concomitant with the increase in thallus N in the NH4+ and Gln treatments (Table 2). There was also a significant increase in Chl concentration in the high-intensity fertilization experiment associated with the increase in N (Fig. 3), with many individuals displaying more than a doubling in Chl a concentration in the NH4+ treatments (Fig. 4). These effects (occasion × N treatment × light treatment) were tested with ANOVA [general linear model, Tukey (HSD) comparison of means, P < 0.05].

Figure 4.

Platismatia glauca chlorophyll (Chl) a concentration as a function of the thallus nitrogen (N) concentration at the start of the experiment (open symbols) and at harvest for the control thalli (circles) and thalli treated with 50 kg NH4+ (squares), 100 kg NH4+ (squares with cross), 50 kg NO3 (triangles) or 50 kg glutamine (Gln) (diamonds) in the shaded (grey symbols) and the exposed (closed symbols) light regimes, in the high-intensity fertilization experiment. Data were obtained as described in the Materials and methods. Data for the two light regimes and the two NH4+ concentrations were pooled so the number of samples ranged from 27 to 54, as specified in Materials and methods. The slope of the regression line was significant for P < 0.001 in all plots. The slope (b) of the regression did not differ (P > 0.05) among the treatments at the start of the experiment, ranging from 0.19 to 0.23 for the equation Chl a = a + (b × N) (a, y-axis intercept; b, slope of the line; N, thallus N concentration), and was significantly lower in all treatments except Gln at harvest, ranging from 0.06 to 0.14. The r2 values of each regression are displayed in italics.

The Chl a concentration was positively, and linearly, correlated to thallus N status both at the start of the experiment and at harvest. However, the slope of the Chl a to N regression line was shallower at harvest compared to the start of the experiment in all treatments except for Gln, as described in the legend of Fig. 4. This emphasizes that the thallus N concentration increased more than the Chl a concentration in the control, NH4+ and NO3 treatments during the 3-month experiment.

The fungal marker ergosterol was not significantly different between the start of the experiment and harvest or among treatments in the high-intensity fertilization experiment (Fig. 3). In the low-intensity fertilization experiment, ergosterol was higher in the NH4+ treatment than in the NO3 treatment (Table 2).

Total and soluble carbon  The mean total C concentration was 450 ± 6 (mean ± 1SE) mg g−1 DW at the start of the experiment and remained unaltered in the control and NO3 treatments, but increased in the Gln treatment to 460 ± 10 mg g−1 DW. The total C concentrations in the two NH4+ treatments were significantly higher than those in the three other treatments, being increased to 467 ± 10 in the higher and 473 ± 10 mg g−1 DW in the lower NH4+ treatments. The total C concentration of the thalli was therefore slightly increased with their increased thallus N concentration. There was no significant effect of the light regime on the total C status of the lichens at harvest, again reflecting the fact that light conditions were similar when the lichens were wet and active (Table 1). Effects (occasion × N treatment × light treatment) were tested with ANOVA [general linear model, Tukey (HSD) comparison of means, P < 0.05].

The Trebouxia photobiont of P. glauca exports polyol ribitol which is rapidly metabolized by the mycobiont into arabitol, and further to arabinose, ribinose, fructose and finally to the temporary storage pool mannitol (Fahselt, 1994). Ribitol, arabitol and mannitol were quantified in this study, together with glucose, which may be present in both bionts. Arabitol was the largest soluble C pool, the mannitol and ribitol pools were smaller with similar concentrations, and the glucose pool was the smallest (Fig. 5). The significantly smaller size of the glucose pool can be explained by its more direct participation in metabolism compared with the other three compounds (Fahselt, 1994). The summed DW concentration of these four pools ranged from 21 to 26 mg g−1 DW at the start of the experiment and from 23 to 28 mg g−1 DW at harvest, increasing in all except the highest NH4+ treatment (Fig. 5, upper panel). The N-based concentrations of all four compounds decreased with the thallus N concentration of the lichens (Figs 3, 5), showing that the increase in N concentration was greater than that of the soluble C concentration.

Figure 5.

Soluble carbon concentration (ribitol, arabitol, mannitol and glucose) in Platismatia glauca in relation to thallus dry weight (top panels) or nitrogen (N) concentration (bottom panels) at the start of the experiment and at harvest in the high-intensity N fertilization experiment. Data from the two light regimes were pooled so the number of samples ranged from 26 to 28, as specified in the Materials and methods.

At the start of the experiment, there was a significant and positive linear relation between the concentration of each of the four soluble C compounds and thallus Chl a concentration (not shown). Of these, the mannitol to Chl a relation was the strongest, with an r2 of 0.37 (Fig. 6, start). There was no significant correlation between arabitol and Chl a at harvest, while the correlation persisted between Chl a and each of the other three compounds (P < 0.05). The mannitol to Chl a relation was still the strongest, with an r2 of 0.60 when all the treated thalli were pooled (Fig. 6, harvest). The slope of the mannitol to Chl a regression line was steeper at the start of the experiment compared with harvest, as described in the legend to Fig. 6.

Figure 6.

Mannitol concentration as a function of the Platismatia glauca thallus cholorophyll (Chl) a concentration at the start of the experiment and at harvest for the control thalli (circles) and thalli treated with 50 kg NH4+ (squares), 100 kg NH4+ (squares with crosses), 50 kg NO3 (triangles) or 50 kg glutamine (Gln) (diamonds) in the shaded (grey symbols) and the exposed (closed symbols) light regimes, in the high-intensity fertilization experiment. Data were obtained as described in the Materials and methods. The slope (b) of the regression was significant for P < 0.001 in both plots, being significantly different between the start of the experiment (b = 2.8) and harvest (b = 2.0), for the equation mannitol = a + b × Chl a (a, y-axis intercept). Note that scales are different in the two panels.

Growth responses  The biomass gain of the lichens in the high-intensity fertilization experiment ranged from 0.003 to 0.3 g g−1 DW, i.e. from 3 to 30% (Fig. 7), relating the weight change to the initial thallus weight. The relative growth rate (RGR = [(ln DWharvest − ln DWstart)/Δt]; Hunt, 1982) ranged from 0.4 to 3.8 mg g−1 DW d−1 based on the 73-d experimental period (not shown). There was no significant effect of the light regime, although there was a tendency for a lower weight gain in the shaded than in the exposed light regime in the NO3 treatment (Fig. 7). The variation in Chl a concentration could alone explain a large part (18–41%) of the variation in weight gain among the thalli in each treatment (Fig. 7). When all treatments were pooled, 31% of the variation in weight gain could be explained by the variation in Chl a concentration.

Figure 7.

Platismatia glauca weight gain as a function of the thallus chlorophyll (Chl) a concentration at harvest, for the control thalli and thalli treated with 50 kg NH4+ (squares), 100 kg NH4+ (squares with cross), 50 kg NO3 or 50 kg glutamine (Gln) in the shaded (grey symbols) and the exposed (closed symbols) light regimes, in the high-intensity fertilization experiment. Data were obtained as described in the Materials and methods. The slope of the regression line was significant for P < 0.01 in all plots. The slope (b) was not significantly different (P > 0.05) among the four treatments, ranging from 0.09 to 0.13 for the following equation: weight gain (g g−1) = a + b × Chl a (a, y-axis intercept) (mg g−1 DW). Note that the x-axis scale is different in the control and NO3 treatments compared with NH4+ and Gln.

Area growth is difficult to accurately quantify for P. glauca, but the photographs taken of all thalli before and after the high-intensity fertilization experiment indicated that thallus expansion proceeded unhampered, even in the fastest-growing individuals in the highest NH4+ treatment (not shown).

Discussion

Effects of light regime

With the aim of determining whether differences in net C gain have an effect on lichen responses to increased N supply, the lichens in the high-intensity fertilization experiment were exposed to two contrasting light regimes (Table 1), which theoretically should affect their photosynthetic activity and potential for growth (Sundberg et al., 1997; Palmqvist, 2000; Dahlman & Palmqvist, 2003). However, when the lichens were wet and metabolically active, the light conditions were identical, which provides an explanation for the similar responses in the two contrasting light regimes. Possible interactions between photosynthesis and C metabolism, on the one hand, and N assimilation and its metabolism, on the other, thus remain to be explored in future studies of lichens. Because of the apparent absence of light effects, the discussion below is focused primarily on the N treatments. The observation that differences in light intensity between sites may be diminished when lichens are wet and metabolically active (Table 1) indicates that care must be taken when evaluating light conditions in lichen habitats.

Effects of N source, supply intensity and concentration

The lichens were fertilized with NH4+, NO3 or the amino acid glutamine, with the aim of separating the effects of these forms of N and of determining whether the lichens could acclimate to a particular N form. In accordance with previous studies, the lichen assimilated NH4+ more efficiently than NO3 and glutamine (Figs 1, 2, Table 2), which can be explained by the higher energy costs of assimilating NO3 compared with NH4+ (Chapin et al., 1987). Moreover, NH4+ uptake may be more passive than amino acid and NO3 assimilation (Brown et al., 1994), as reflected here by the lower CCCP inhibition of NH4+ uptake (see Results). Overall, however, there were only minor differences in the responses to increased N that could be related to N form: a slight shift in relative N preferences after low-intensity fertilization (Fig. 1), and an unaltered Chl a to N regression line after high-intensity glutamine fertilization (Fig. 4). Instead, the general response to the increase in thallus N concentration with an increased Chl a and mannitol concentration (Figs 3, 5), a maintained ergosterol concentration (Fig. 3), and increased growth (Fig. 7) seemed to be insensitive to the particular N form. Insensitivity to N form has previously also been suggested for the nitrophytic lichen Xanthoria parietina (Gaio-Oliveira et al., 2005). However, the apparent lack of an ‘N-form effect’ in the present study must be interpreted with caution, in the light of the unknown background N in the high-intensity fertilization experiment (see Results). More controlled studies are therefore needed to explore the possibility that different N forms might act differently on lichens.

The increase in thallus N concentration in the control treatment and an apparently higher thallus N concentration in the N treatments than could be accounted for by the 15N-spiked fertilizer (Figs 2, 3) suggested that the background N contributed to the lichen N concentration at harvest. This implies that the fertilization treatments were below saturation, which may seem surprising as the fertilizer was administered daily at a relatively high concentration. However, the thalli were sprayed with c. 90 or 180 µg of N on each occasion, and the average thallus weight was 270 mg (see Materials and methods). This amount of N would theoretically be assimilated within 40 min in the lower NH4+ treatment, within 1.5 h in the higher NH4+ treatment, within 1 h in the glutamine treatment, and within 8 h in the NO3 treatment, using the uptake rates presented in Fig. 1. As the lichens were wet and metabolically active for considerably longer than 10 h after most of the fertilization events (Table 1), the added N was probably depleted long before desiccation of the lichens, even if uptake rates are likely to decrease with decreasing N concentration. In addition, several fertilization occasions were followed by heavy rain (not shown) which would have diluted the fertilizer, and the foliage above the lichens was likely to leak even more assimilates.

With these points in mind, the following discussion is focused on how the lichens handled the significant N changes in their thalli, regardless of N form or whether this N originated from the fertilizer or background deposition.

Effects on the symbionts

The chlorophyll concentration increased significantly with the increase in thallus N concentration (Table 2, Figs 3, 4), in agreement with previous studies showing that the photosynthetic pigment concentration remains relatively constant in relation to thallus N concentration both inter- and intraspecifically in lichens (Palmqvist et al., 1998, 2002; Sundberg et al., 2001; Gaio-Oliveira et al., 2005). The photosynthetic capacity of lichens is furthermore well correlated to their Chl a concentration, with the capacity increasing significantly with increasing Chl a (Tretiach & Carpanelli, 1992; Tretiach & Pecchiari, 1995; Palmqvist et al., 1998, 2002; Palmqvist, 2000). The concentration of the primary carboxylating enzyme Rubisco, which contains the major part of the N found in chloroplasts, is also well correlated to the Chl a concentration of lichens (Palmqvist et al., 1998). Thus, it is possible that the N-fertilized thalli in this study used the assimilated N to increase investment in overall photosynthetic capacity, and not only in additional Chl pigments and light-absorbing capacity. Such a response may further be competitively advantageous as photosynthetic capacity appears to increase more than respiration with increasing Chl a status, increasing net C gain across species (Palmqvist et al., 2002) as well as the light use efficiency intraspecifically (Sundberg et al., 2001; Dahlman & Palmqvist, 2003). We may then conclude that the significantly increased Chl a concentration following the increase in thallus N concentration was an entirely appropriate response (Table 2, Figs 3,4), serving both to support the (possibly) increased maintenance respiration of the N-richer thallus (Chapin et al., 1987; Grace, 1997; Lambers et al., 1998; Palmqvist, 2000; Palmqvist et al., 2002) and potentially to increase the net C gain capacity.

Ergosterol is a component of fungal plasma membranes and can be used to indirectly assess the activity of the mycobiont in lichens (Sundberg et al., 1999; Gaio-Oliveira et al., 2004), and in mycorrhiza (Ekblad et al., 1998). Similar to Chl a, the ergosterol concentration increases with increasing N concentration across lichen species (Palmqvist et al., 2002), while the intraspecific ergosterol to N relation is less clear (Sundberg et al., 2001). Both negative and positive effects of increased N supply have been observed, with the N-sensitive lichen E. prunastri showing significant decreases in ergosterol concentration (Gaio-Oliveira et al., 2004), while P. glauca in a 15-year fertilization experiment displayed an increased ergosterol concentration (Dahlman et al., 2003). The ergosterol concentration remained constant across treatments and thallus N concentrations in the present study (Fig. 3), indicating that the mycobiont was not negatively affected by the enhanced N concentration. The mycobiont mannitol pool was as well correlated to thallus Chl a and N concentration after the treatments as before the treatments (Fig. 6). The C flow from photobiont to mycobiont thus appeared to proceed unconstrained with increasing thallus N concentration, indicating that the mycobiont of P. glauca was equally vital in the high-N thalli.

Growth and metabolism

Growth was measured to assess the overall activity of the lichen thalli in relation to the increased N supply. It appears that growth was significantly stimulated by this treatment, as the weight gain of the thalli was significantly increased when the Chl a concentration was increased (Fig. 7). This is a strong indication that both partners benefited from the increased N supply, and supports the above suggestion that net C gain capacity may be increased with increasing Chl a concentration. Moreover, there were no signs of impaired thallus expansion, which has been observed in lichens that are stressed by high N (Sundberg et al., 2001). A previous study in the same area of Sweden (Vindeln, Västerbotten) showed an average weight gain of 4–7% in P. glauca thalli when Iwet was 30–70 mol m−2 (Sundberg et al., 1997). Assuming a similar impact of Iwet on growth in the present study [see Palmqvist & Sundberg (2000) for a more detailed discussion], the 40–50 mol m−2 light dose (Table 1) should have yielded a weight gain in the range of 3–5%, thus being in the lower range compared with the observed growth (Fig. 7). However, the Chl a concentration of the P. glauca thalli in Sundberg et al. (1997) was on average significantly lower (0.4 mg Chl a g−1 DW) than that in the thalli of the present study (Fig. 7), providing further support for the theory that the lichens had used the increased N availability to increase not only their Chl pigment concentration but also their overall net C gain capacity.

Despite the significant correlation between thallus Chl a concentration and growth observed here and elsewhere for lichens (Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2006), as well as for other phototrophic organisms (Enriquez et al., 1996), it must be emphasized that the concentration of this pigment is not always causally linked to photosynthetic activity (Evans, 1989). The realization of the growth potential of a lichen is also regulated by environmental factors such as water availability, light intensity, and relative length of active periods in darkness vs light (Sundberg et al., 1997; Palmqvist & Sundberg, 2000; Dahlman & Palmqvist, 2003; Gaio-Oliveira et al., 2003, 2005). Neighbouring thalli with a similar Chl a concentration may further differ in their water holding capacities (Dahlman & Palmqvist, 2003), or in the concentration of other growth-promoting nutrients such as phosphorous (Crittenden et al., 1994), and already existing pools of both N and C compounds required for growth. Such differences between thalli might then explain the relatively low r2 values of the Chl a to growth relation of the control and NO3-treated thalli in Fig. 7.

Finally, although the metabolic links between photosynthesis in the photobiont and carbon metabolism and storage in the mycobiont have not been fully elucidated, the relatively constant ratio between Chl a and mannitol concentrations across the various N treatments (Fig. 6) and growth rates (Fig. 7) is intriguing. Furthermore, the ratio between these two compounds obtained here is similar to the ratio obtained previously for this lichen, when it was exposed to completely different environmental conditions, and contained even more thallus N (Dahlman et al., 2003). It can be speculated that this may suggest that the C flow through the mycobiont mannitol pool is quite strongly linked to the magnitude of the C input from photobiont photosynthesis. This further suggests that the C flow from photobiont to mycobiont proceeded unhampered in the lichen studied here, in contrast to the situation in the more N-sensitive lichen Evernia prunastri, where the soluble C pools in the mycobiont were depleted with increased N uptake (Gaio-Oliveira et al., 2004).

Conclusions

Platismatia glauca displayed no signs of reduced vitality in this study, indicating that this lichen can apparently acclimate to large variations in environmental N supply, with respect to frequency, N concentration, and N source. The lichen was able to assimilate N efficiently, thereby increasing the thallus N concentration (Table 2, Figs 2, 3), as well as the photobiont concentration (Figs 3, 4), without any negative effects on the mycobiont (Table 2, Fig. 3). The increased photobiont concentration was further correlated with an increased growth of the whole thallus (Fig. 7), and an increased mannitol pool in the mycobiont (Figs 5, 6). These responses were similar, irrespective of supply intensity or N concentration, and the magnitude of the response was related to the magnitude of the N uptake. The lichens in the high-intensity fertilization experiment were metabolically active for less than 700 h, divided among 58 occasions (Table 1). During this period, some thalli displayed a 4–5-fold increase in thallus N and Chl a concentrations (Figs 3, 4), and a 20–30% increase in DW (Fig. 7), which could largely be explained by the increased Chl a concentration (r2 = 0.31). Thus, the relatively rapid response of P. glauca to varying N availability suggests that the symbionts of this lichen are well integrated, with tight regulation of major investments and metabolism between the two partners.

Acknowledgements

The authors are grateful to Karl Jansson and Olle Palmqvist for help with the fertilization, Reiner Giesler for providing analytical equipment, and Margareta Zetherström for skilful technical help and advice. Torgny Näsholm, Annika Nordin, Åsa Forsum and Gisela Gaio-Oliveira provided useful input throughout. This study was supported by grant 993194 from the Centre for Environmental Research (CMF, Umeå, Sweden) to LD and by grant 24.0795/97 from FORMAS, Sweden to KP. We would also like to thank the three unknown referees who made valuable suggestions on the manuscript.

Ancillary