Planta

, Volume 220, Issue 5, pp 794–803

Nitrogen uptake in relation to excess supply and its effects on the lichens Evernia prunastri (L.) Ach and Xanthoria parietina (L.) Th. Fr.

Authors

    • CEBV—Centre for Ecology and Plant Biology, Departamento de Biologia VegetalFaculdade de Ciências da Universidade de Lisboa
    • Department of Ecology and Environmental ScienceUmeå University
  • Lena Dahlman
    • Department of Ecology and Environmental ScienceUmeå University
  • Kristin Palmqvist
    • Department of Ecology and Environmental ScienceUmeå University
  • Maria Amélia Martins-Loução
    • CEBV—Centre for Ecology and Plant Biology, Departamento de Biologia VegetalFaculdade de Ciências da Universidade de Lisboa
  • Cristina Máguas
    • CEBV—Centre for Ecology and Plant Biology, Departamento de Biologia VegetalFaculdade de Ciências da Universidade de Lisboa
Original Article

DOI: 10.1007/s00425-004-1396-1

Cite this article as:
Gaio-Oliveira, G., Dahlman, L., Palmqvist, K. et al. Planta (2005) 220: 794. doi:10.1007/s00425-004-1396-1
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Abstract

The aim of this study was to compare the physiological responses to increased nitrogen (N) supply between the nitrophytic lichen Xanthoria parietina (L.) Th. Fr. and the acidophytic lichen Evernia prunastri (L.) Ach. The two lichens were exposed to a weekly dosage of 0.05, 0.1, 0.2, 0.6 or 2.4 g N m−2 for 2 months, administered as NH4NO3 dissolved in artificial rainwater (1 l m−2). After the treatments, in vivo chlorophyll a fluorescence was determined to assess vitality; concentrations of total N, ammonium, nitrate and dominant amino acids, including glutamate, glutamine and arginine, were quantified in order to follow changes in N status; and the polyols ribitol, arabitol and mannitol were quantified to follow changes in the lichens’ carbon (C) status. The uptake of N was quantified by labelling the fertiliser with 15N in the ammonium position; chlorophyll a was used as an indirect marker for algal activity, and ergosterol as an indirect marker of fungal activity. Nitrogen uptake was higher in E. prunastri than in X. parietina, although the latter species may have used the mannitol reserves to obtain C skeletons and energy for N assimilation. Chlorophyll a and ergosterol concentrations remained unaltered in X. parietina irrespective of N dosage while ergosterol decreased with increasing N uptake in E. prunastri. The latter species had accumulated a large pool of ammonium at the highest N dosage, whilst in X. parietina a significant nitrate pool was instead observed. Taken together, these short-term responses to high N supply observed in the two lichens, and the differences between them, can partly explain the higher tolerance of X. parietina towards increased atmospheric N levels.

Keywords

AmmoniumEverniaNitrateNitrogen assimilationNitrogen uptakeXanthoria

Abbreviations

Arg

Arginine

DW

Dry weight

Gln

Glutamine

Glu

Glutamate

Introduction

Lichens are symbiotic associations in which nutritionally specialized fungi (mycobionts) derive carbon (C) and in some cases nitrogen (N) from algal or cyanobacterial photobionts (Honegger 1991). Lichen growth is primarily dependent on reduced C compounds, required for biosynthesis and energy. Growth is also dependent on mineral nutrients, such as N and phosphorous (P), needed for the synthesis of new membranes, DNA and proteins (Palmqvist 2000). In contrast to lichens with a cyanobacterial photobiont, those with green algae are not able to fix N2, and are therefore solely dependent on deposition of combined N, such as ammonium or nitrate, on the thallus (Rai 1988). However, if deposited in high concentrations, N compounds can be quite toxic for lichens. Because N assimilation requires C skeletons and energy (Turpin 1991), a reduced or even lack of assimilative capacity can lead to a build up of N within the thallus and consequently to toxicity. Nevertheless, lichens can present different degrees of sensitivity towards increased N deposition, due to diverse mechanisms of avoiding excessive uptake (Hyvärinen and Crittenden 1998; Gaio-Oliveira et al. 2001), or an ability to assimilate N into non-toxic forms, such as arginine (Dahlman et al. 2003).

Lichens can be classified as nitrophytic or acidophytic in relation to their resistance to air-pollution (De Bakker 1989; Van Dobben 1996; Van Dobben and Ter Braak 1998). Nitrophytic lichens are found in regions with high atmospheric N levels whereas acidophytic lichens are found in areas with low N (De Bakker 1989; Ruoss 1999; Van Herk 2001). In regions where N deposition has increased during recent decades there has subsequently been a replacement of acidophytic lichens by nitrophytic ones (De Bakker 1989; Van Dobben and De Bakker 1996; Van Herk 2001). Several studies have related these changes to concomitant increases in bark pH (De Bakker 1989; Van Dobben 1996; Van Dobben and Ter Braak 1998). However, changes in substratum pH cannot alone explain the dominance of nitrophytic lichens in areas with increased N deposition (Van Herk 2001). The acidophytic lichens might, for instance, be more vulnerable to parasitic attacks when N is supplied in excess, as for some low-N-adapted plants (Strengbom et al. 2002), or they might lack mechanisms for avoiding excessive N uptake or lack regulatory mechanisms to metabolise excessively assimilated N (Dahlman et al. 2003). Thus, to understand the disappearance of acidophytic lichens from N-polluted areas, one needs to study the effects of high N levels as such on lichen performance.

The green algal lichens Xanthoria parietina and Evernia prunastri are examples of nitrophytic and acidophytic associations, respectively (De Bakker 1989; Van Dobben 1996; Ruoss 1999; Van Herk 2001). Xanthoria parietina is reported to increase its abundance in areas where atmospheric N levels are increased (De Bakker 1989; Ruoss 1999; Gaio-Oliveira et al. 2001; Van Herk 2001), whilst E. prunastri is described as one of the first lichens to decrease in density or even disappear when N deposition is increased (De Bakker 1989; Van Herk 1999, 2001). Thallus total N concentrations can vary from 10 to 34 mg g−1 DW in X. parietina (Rai 1988; Gaio-Oliveira et al. 2001, 2004), reflecting an ability of this lichen to handle both low and high tissue N concentrations (Gaio-Oliveira et al. 2004). Total N concentrations vary much less in E. prunastri, reported to be close to 10 mg g−1 DW in several studies and contrasting populations (Rai 1988; Gaio-Oliveira et al. 2001; Palmqvist et al. 2002), suggesting that this lichen tolerates low N dosages in nature. The two lichens also differ significantly with respect to cation affinity, with a 5-fold higher cation-exchange capacity in E. prunastri compared to X. parietina (Gaio-Oliveira et al. 2001). Other lichen associations, considered as neutrophytic (i.e. lichens indifferent towards increased N levels; De Bakker 1989; Van Dobben 1996; Van Herk 1999), have cation-exchange capacities in between those of the acidophytic E. prunastri and the nitrophytic X. parietina (Gaio-Oliveira et al. 2001). The lower cation-exchange capacity of X. parietina might to some extent explain the higher tolerance of this species towards high N exposure, in the form of ammonium, in this way avoiding excessive uptake. In the present study these two lichens were exposed to a gradient of increased N supply during a short-term (2 months) field experiment, aiming to (i) examine how they responded physiologically to increased N, by comparing their responses, and (ii) assess whether any such putative differences might explain their different tolerances towards N-pollution.

Materials and methods

Lichen material and N fertilisation

Both Xanthoria parietina (L.) Th. Fr. and Evernia prunastri (L.) Ach. have green alga in the genus Trebouxia as photobiont (Tschermak-Woess 1988). The mycobiont of E. prunastri belongs to the ascomycete family Parmeliaceae, and the mycobiont of X. parietina to the Teloschistaceae (Eriksson and Winka 1998). These lichens have different growth forms, X. parietina being a foliose lichen, whilst E. prunastri is fruticose. For linguistic reasons, each lichen association will be referred to as “a species” in the following.

A large number of thalli of both species were exposed to a gradient of increased N supply (see below) while remaining on their natural substratum and in their native habitat, X. parietina on clay roof tiles and E. prunastri on Olea europaea twigs. Twenty-six clay roof tiles with X. parietina thalli, devoid of other lichens, were treated while remaining on a small roof on a house in Santo António (near Serra de Aire e Candeeiros Natural Park, central Portugal; 43°74′ N, 5°23′ W). Only the lowermost row of tiles was used in order to avoid cross-contamination among the different treatments. The tiles were assigned to six treatment groups: treatments 1–5, consisting of four tiles each (0.44 m2) and treatment 6 (control) with six tiles (0.66 m2). The control was divided into three sets of two tiles each, and placed across the roof among the five treatments. There were no significant differences between the three control sets (not shown), so the data from these tiles were pooled in the presented results. Several thalli were collected randomly from each treatment to obtain start values on 25 January 2002.

More than 120 Olea europaea twigs, with E. prunastri thalli, were collected on 25 January 2002 at Alvados forest (Serra de Aire e Candeeiros Natural Park, central Portugal; 43°77′ N, 5°20′ W) and then taken to the laboratory where the twigs were randomly allocated to six treatment groups as for X. parietina. The twigs assigned for each treatment were then divided into two sets: one set was used to obtain start values and the other set was immediately attached with flax wire to a 0.25-m2 plastic net for each treatment. Other lichen species on the twigs were not removed. The transplantation nets were brought back to the Alvados forest a few days later and attached with thin wires to tree trunks in such a way that the small twigs were exposed to similar environmental conditions as before.

The two lichen species were thereafter treated identically. The control treatment was sprayed once a week with 1 l m−2 of artificial rainwater without P (Tamm 1953; Dahlman et al. 2002). The fertilised treatments received 1.8, 3.4, 6.8, 21 or 86 mM of NH4NO3 (0.05, 0.1, 0.2, 0.6 or 2.4 g N m−2), with 1% of (15NH4)NO3, dissolved in the same amount of artificial rainwater as the control, and were also sprayed once a week. The pH of all solutions was adjusted to ca. 7 immediately before use. The fertilisations were started by wetting the thalli with a small amount of the solution and continued for ca. 5 min per treatment and species. The experiment lasted for 2 months (1 February to 30 March 2002), and in total the lichens were sprayed nine times. The NH4NO3 additions thus corresponded to an accumulated exposure to 0.45, 0.9, 1.8, 5.4 or 21.6 g N m−2 in the five treatments, respectively. Although one could argue that the concentrations used in the performed treatments were high, this was essential to understand the different responses these two species have towards increased N levels. The chosen concentrations also represented a compromise between the reported high sensitivity of E. prunastri and the high tolerance of X. parietina towards N (De Bakker 1989; Van Herk 1999, 2001; Gaio-Oliveira et al. 2001, 2004).

Sampling procedures

Sampling for in vivo chlorophyll (Chl) a fluorescence, N uptake from ammonium, total N, ammonium, nitrate, Chl a, ergosterol, soluble carbohydrates and amino acid analyses (see below) was done at the start, as described above, and at harvest. In X. parietina, sampling was done by randomly collecting pieces of lichen thallus from the tiles in each treatment. In the case of E. prunastri the nets were taken to the laboratory at harvest followed by random sampling from the twigs in each treatment. The vitality of the lichens was determined in the laboratory using the Chl a fluorescence parameter Fv/Fm determined at room temperature for five thalli of each treatment and species, using a portable Mini-PAM (Waltz, Effeltrich, Germany). The fluorescence samples were hydrated with deionised water and activated for 12 h in darkness followed by 12 h in light (130 μmol photons m−2 s−1) at 15°C in a reactivation chamber with a high relative humidity. Prior to the fluorescence measurements the lichens were dark-adapted for 15 min. Mean Fv/Fm values were 0.67 at the start and 0.74 at harvest, for both species, with no significant differences among the treatments (not shown).

The sampled thalli, including the fluorescence samples, were thereafter pooled to six larger samples for each species and treatment. Three of these were used for total N and 15N analyses and the other three for biont marker and metabolite analyses.

Analysis of 15N and total N

The samples used for analysis of 15N and total N were dried at 60°C for 24 h and then ground to a homogeneous powder. The samples were analysed by Stable Isotope Mass Ratio Spectrometry by Continuous Flow (IRMS–CF) for 15N/14N ratio (only the samples from harvest) and total N concentration (all samples) in an Isoprime (GV Instruments, Manchester, UK) coupled to an Elemental Analyser (EuroVector, Milan, Italy), following standard methods at the Stable Isotopes Laboratory (LIE, ICAT, Faculty of Sciences, Lisbon).

N uptake from ammonium was calculated as in Eq 1:
$${\text{N}}\;{\text{uptake}} = {\left[ {{\left( {{\text{At}}\% _{{\text{s}}} - {\text{At}}\% _{{\text{c}}} } \right)} \times {\text{Total}}\;{\text{N}}_{{\text{s}}} } \right]}/0.01$$
(1)
where At%s is the atomic percentage of the 15N of the sample, At%c the atomic percentage of the 15N of the control thalli, Total Ns the total N concentration of the sample (g g−1 DW) and the value 0.01 corrects for the fraction of 15N in the NH4NO3 additions (1%).

Biont marker and metabolite analyses

The samples used for the following analyses were freeze-dried and ground to a homogeneous powder, and aliquots were taken for analysis of ammonium, nitrate, Chl a, ergosterol, polyols, and amino acids. Ammonium was determined by the Berthelot reaction (Rhine et al. 1998) and nitrate was determined by the method of electrophilic replacement of sodium salicylate (Yang et al. 1998). Chl a was used as a marker for the photobiont, because it correlates well with photosynthetic capacity in lichens (Palmqvist et al. 1998, 2002), being quantified after extraction in MgCO3-saturated dimethyl sulfoxide (DMSO), at 60°C for 40 min (Palmqvist and Sundberg 2001). Ergosterol, the main sterol of fungal plasma membranes, was used as an indirect marker for active mycobiont tissue in the lichens (Palmqvist et al. 2002; Dahlman et al. 2003), being quantified by HPLC after extraction in 99.5% ethanol (Dahlman et al. 2001). Soluble carbohydrates were analysed according to Dahlman et al. (2003) with a Varian 3800 GC connected to a Varian Saturn 2000 ion-trap MS (Varian, Walnut Creek, CA, USA). The polyols ribitol, arabitol and mannitol could be quantified in both lichens, whereas the glucose, sucrose and fructose peaks were below the detection limit. Amino acids were extracted in 10 mM HCl for 1 h and then analysed according to Dahlman et al. (2003). Sixteen amino acids were detected and the following major ones were quantified: asparagine, aspartate, threonine, serine, glycine, alanine, glutamine, glutamate and arginine.

Statistical analysis

Regressions and ANOVAs were done using the statistical package Statistix 7 (Analytical Software, Tallahassee, FL, USA).

Results

N uptake and total N

After the 2 months exposure to NH4NO3 fertilisation, E. prunastri had taken up between 0.5 and 6 mg N g−1 DW from ammonium while X. parietina had taken up between 0.4 and 4.5 mg N g−1 DW (Fig. 1). The magnitude of N uptake increased with NH4NO3 exposure in both lichens, displaying a saturating, curvilinear, response to the N dosage. E. prunastri had a significantly higher uptake than X. parietina except at the lowest N exposure (Fig. 1).
Fig. 1

Average (±1SE) N uptake from ammonium (mg g−1 DW) as a function of NH4NO3 exposure (mM) in Xanthoria parietina (black symbols) and Evernia prunastri (white symbols). Different letters refer to significant differences among both species and treatment (2-way ANOVA and the interaction term)

The initial total N concentration was 33±3 mg g−1 DW in X. parietina and 7±1 mg g−1 DW in E. prunastri (Table 1). Final total N concentrations were, for both species, positively correlated with N uptake, ranging from 30 to 39 mg g−1 DW in X. parietina (final total N = 0.85 × N uptake + 31.23; r2=0.41; P=0.0044) and from 9 to 16 mg g−1 DW in E. prunastri (final total N = 0.92 × N uptake + 10.48; r2=0.87; P<0.0001) (Fig. 2). The N concentrations were then more than doubled in E. prunastri, but only 30% higher in X. parietina at the highest N exposure. The slope of the relation between thallus N concentration and N uptake was similar for the two species (P=0.76), with a slope value of 0.85±0.26 (r2=0.41) in X. parietina and 0.92±0.09 (r2=0.87) in E. prunastri (Fig. 2).
Table 1

Average (±1SD) concentrations of the biont markers and the metabolites measured at the start of the experiment in Evernia prunastri and Xanthoria parietina. Average values with a different letter refer to significant differences (n=3; P<0.05) between the two species; n.d. not detectable. Values have been rounded

E. prunastri

X. parietina

Biont markers

  Chlorophyll a (mg g−1 DW)

0.13±0.03b

0.30±0.06a

  Ergosterol (mg g−1 DW)

0.30±0.08b

1.25±0.22a

Soluble polyols

  Total (mg g−1 DW)

19.6±8.3

15.5±5.4

  Ribitol (mg g−1 DW)

3.9±1.5a

1.4±0.6b

  Arabitol (mg g−1 DW)

15.0±6.0a

5.0±2.4b

  Mannitol (mg g−1 DW)

1.4±0.03b

8.4±3.1a

Amino acids

  Total (mg g−1 DW)

2.6±0.5b

6.7±1.6a

  Total minus Gln, Glu, Arg (mg g−1 DW)

1.5±0.3b

2.0±0.5a

  Arginine (Arg) (mg g−1 DW)

0.5±0.1

0.5±0.1

  Glutamine (Gln) (mg g−1 DW)

0.2±0.1b

1.9±0.6a

  Glutamate (Glu) (mg g−1 DW)

0.4±0.1b

2.3±0.9a

  Gln:Glu ratio

0.5±0.2b

1.0±0.4a

Total N (mg g−1 DW)

6.6±1.2b

33.0±3.3a

Ammonium (μmol g−1 DW)

32.2±0.6

33.3±1.1

Nitrate (μmol g−1 DW)

n.d.

n.d.

Fig. 2

Final thallus N concentration (mg g−1 DW) as a function of N uptake from ammonium (mg g−1 DW) in X. parietina (black symbols) and E. prunastri (white symbols) exposed to different NH4NO3 concentrations. Total dosage during the treatment period was: 0 (circles), 0.45 (squares), 0.90 (triangles), 1.8 (inverted triangles), 5.4 (diamonds) and 21.6 g N m−2 (hexagons) (see Materials and methods for details). A positive linear relation was obtained for X. parietina: final total N = 0.85 × N uptake + 31.23; r2=0.41; P=0.0044. A positive linear relation was also obtained for E. prunastri: final total N = 0.92 × N uptake + 10.48; r2=0.87; P<0.0001 (confidence interval of 95%)

Ammonium and nitrate concentrations

The initial ammonium concentration was similar in the two species, being ca. 33 μmol g−1 DW (Table 1). The final ammonium concentration ranged from 33 to 100 μmol g−1 DW in X. parietina and from 28 to 244 μmol g−1 DW in E. prunastri, depending on the treatment (Fig. 3a). At the highest NH4NO3 exposure (86 mM; 21.6 g N m−2), E. prunastri had an ammonium concentration of 244 μmol g−1 DW, being more than twice that of X. parietina at this exposure (Fig. 3a).
Fig. 3a, b

Final ammonium (a) and nitrate (b) concentrations (μmol g−1 DW) as a function of NH4NO3 exposure (mM) in X. parietina (black symbols) and E. prunastri (white symbols)

No nitrate was detected in any of the two species at the start of the experiment (Table 1), while the final nitrate concentration ranged from close to zero to 145 μmol g−1 DW in X. parietina and from zero to 16 μmol g−1 DW in E. prunastri, depending on the treatment (Fig. 3b). The nitrate pool was significantly increased between the two highest NH4NO3 dosages in X. parietina, where the 4-fold increase in exposure (from 5.4 to 21.6 g N m−2; 21 to 86 mM) resulted in an 8-fold increase in the nitrate pool (Fig. 3b).

Biont markers

The initial Chl a concentration was 0.30 mg g−1 DW in X. parietina and 0.13 mg g−1 DW in E. prunastri (Table 1). The Chl a concentration was significantly higher in both lichens at harvest, with the average final Chl a concentration ranging from 0.9 to 1.1 mg g−1 DW in X. parietina and from 0.4 to 0.6 mg g−1 DW in E. prunastri, without any significant treatment effect (Fig. 4a). The significantly higher Chl a concentration of the control at harvest compared to start (Table 1, Fig. 4a) followed the increase in the Fv/Fm values from 0.67 to 0.74. This probably reflects an annual variation in thallus Chl a concentration and concomitantly in quantum yield efficiency (Mackenzie et al. 2001).
Fig. 4a, b

Average (±1SE) final chlorophyll a (a) and ergosterol (b) concentrations (mg g−1 DW) as a function of average (±1SE) N uptake from ammonium (mg g−1 DW) in X. parietina (black symbols) and E. prunastri (white symbols). A negative linear relation was obtained for E. prunastri between average final ergosterol concentration and average N uptake from ammonium (b): average final ergosterol concentration = −0.05 × average N uptake + 0.62; r2=0.66; P=0.0492 (confidence interval of 95%)

The initial ergosterol concentration was 1.25 mg g−1 DW in X. parietina and 0.30 mg g−1 DW in E. prunastri (Table 1). The average final ergosterol concentration ranged from 1.0 to 1.1 mg g−1 DW in X. parietina and from 0.3 to 0.8 mg g−1 DW in E. prunastri, with a clear negative treatment effect in the latter species (Fig. 4b). The initial ratio of Chl a to ergosterol was 0.2 in X. parietina and 0.4 in E. prunastri being increased to 0.9 and 0.7, respectively, in the control at harvest (derived from Table 1 and Fig. 4b). Since there was a negative correlation between final ergosterol and N uptake in E. prunastri (average final ergosterol = −0.05 × average N uptake + 0.62; r2=0.66; P=0.0492) (Fig. 4b), this species also displayed an increased ratio of Chl a to ergosterol with increased N uptake (Chl a:ergosterol = 0.13 × N uptake + 0.77; r2=0.57; P=0.0003).

Soluble C pools

The only soluble C compounds that could be detected were the polyols ribitol, arabitol and mannitol (see Materials and methods). The summed concentration of the three polyols was ca. 20 mg g−1 DW in both species at the start (Table 1). The ribitol concentration varied around an average of 1–4 mg g−1 DW in both species, irrespectively of N exposure and N uptake (Table 1, Fig. 5a). The arabitol pool was also similar in all treatments, but consistently higher in E. prunastri than in X. parietina (Table 1, Fig. 5b). Mannitol was the largest polyol pool in X. parietina, whilst the arabitol pool was the largest in E. prunastri (Table 1). The mannitol concentration was not affected by the treatments in E. prunastri, while being negatively correlated to N uptake in X. parietina (average final mannitol concentration = −1.73 × average N uptake + 15.28; r2=0.92; P=0.0024) (Fig. 5c).
Fig. 5a–c

Average (±1SE) final ribitol (a), arabitol (b) and mannitol (c) concentrations (mg g−1 DW) as a function of average (±1SE) N uptake from ammonium (mg g−1 DW) in X. parietina (black symbols) and E. prunastri (white symbols). A negative linear relation was obtained for X. parietina between average final mannitol concentration and average N uptake from ammonium (c): average final mannitol concentration = −1.73 × average N uptake + 15.28; r2=0.92; P=0.0024 (confidence interval of 95%)

Amino acids

The initial total amino acid concentration was ca. 3 mg g−1 DW in E. prunastri and ca. 7 mg g−1 DW in X. parietina (Table 1). Glutamine (Gln), glutamate (Glu) and arginine (Arg) comprised half of the total amino acid pool in E. prunastri, and even more than that in X. parietina (Table 1). Xanthoria parietina initially had overall higher Gln and Glu concentrations than E. prunastri (Table 1). In E. prunastri, the ratio Gln:Glu increased from 0.5 at the start to 1–2 at harvest, without any clear treatment effect (Table 1, Fig. 6a). In X. parietina, the Gln:Glu ratio decreased from 1 at the start to 0.7 in the control at harvest (Table 1, Fig. 6a), consistently being lower than the ratio in E. prunastri in all treatments (Fig. 6a).
Fig. 6a, b

Average (±1SE) final glutamine:glutamate ratio (a) and average (±1SE) final arginine concentration (mg g−1 DW) (b) as a function of average (±1SE) N uptake from ammonium (mg g−1 DW) in X. parietina (black symbols) and E. prunastri (white symbols)

Initial Arg concentration was similar in the two species, being 0.5 mg g−1 DW (Table 1). The average final Arg concentration ranged from 0.3 to 0.4 mg g−1 DW in X. parietina and from 0.3 to 1.5 mg g−1 DW in E. prunastri (Fig. 6b), the wide range in the latter species resulting from a large variability among the thalli exposed to the two highest N dosages (Fig. 6b). The Arg concentrations were not affected by the N supply in either of the two lichens (Fig. 6b). Moreover, when comparing Arg concentrations in E. prunastri thalli from the harvest control with the concentrations in thalli from the highest treatments (Fig. 6b), no significant differences were found, with P<0.05 (statistic not shown).

Discussion

The two lichen species responded differently to the short-term N fertilisation treatment, E. prunastri taking up more of the added ammonium than X. parietina (Fig. 1). The higher N uptake from ammonium observed in E. prunastri (Fig. 1) may in part be explained by its higher cation-exchange capacity (Miller and Brown 1999; Gaio-Oliveira et al. 2001). Moreover, being a fruticose lichen, E. prunastri has a higher ratio of surface area to volume than the foliose X. parietina, which might contribute to the higher uptake of ammonium of the former lichen (Fig. 1).

The different habitat preferences of the two lichens may also contribute to their different N uptake capacities (Fig. 1), E. prunastri being confined to more oligotrophic environments than X. parietina (De Bakker 1989; Van Herk 1999, 2001). This implies that the higher uptake capacity of E. prunastri is an adaptation to deal with low N concentrations, a trait which might be less beneficial when the N supply is increased. Xanthoria parietina, on the other hand, is able to deal with both low and high N concentrations in nature (Gaio-Oliveira et al. 2001; Van Herk 2001). The habitat N availability was probably lower for E. prunastri than for X. parietina also in the present study. This can be inferred from the different thallus N concentrations of the two species already at the start of the experiment, being 33 and 7 mg g−1 DW in X. parietina and E. prunastri, respectively (Table 1), reflecting the fact that lichen N concentrations generally mirror N availability in the recent past (Boonpragob et al. 1989; Søchting 1995; Poikolainen et al. 1998). The duration of the metabolic activity periods may also have been different for the two lichens, because E. prunastri was growing in a sheltered, shaded habitat (Q. faginea canopy), whilst X. parietina was growing in an open, sun-exposed habitat (roof tiles). Although not measured, one could then assume that the former lichen might have had the longest activity periods, associated with longer hydration periods after each irrigation event. Thus, this is an additional explanation for the higher N uptake in E. prunastri compared to X. parietina (Fig. 1).

Lichens that are sensitive towards increased N levels in the thallus might not only suffer from N toxic concentrations per se (Turpin 1991) but also from a possible change of the balanced exploitation of N between the symbionts, if one symbiont is favored over the other due to altered nutrient supplies (Gries 1996). This may have been the case in E. prunastri, where the significant decrease in ergosterol with increasing N uptake suggests that the mycobiont was more affected than the photobiont by the increased N supply (Fig. 4b). However, this was only observed at the highest N dosages (Fig. 4b), meaning that the fungal partner was not negatively affected when exposed to NH4NO3 concentrations below 6.8 mM (0.2 g N m−2). Xanthoria parietina, on the other hand, was able to maintain a balanced ratio of Chl a to ergosterol with increasing N uptake, irrespective of N dosage (Fig. 4), being in agreement with the higher tolerance of this species (Brown et al. 1994; Ruoss 1999; Gaio-Oliveira et al. 2001; Van Herk 2001). Previous lichen studies have also shown that the mycobiont is more affected than the photobiont when exposed to excessive N supply (Dahlman et al. 2002, 2003; Gaio-Oliveira et al. 2004). The reason for this has not been fully elucidated, but the fungus dominates in terms of biomass in lichen thalli (Honegger 1991), so the higher sensitivity of the mycobiont can simply be related to its relatively higher surface area being exposed to the added N. Also, the photobiont has, through its photosynthesis, a direct access to the C skeletons and energy required for assimilating N into cellular compounds (Turpin 1991). In a scenario where the photobiont is also exposed to an increased N supply, this partner might subsequently use more of its assimilated C for its own requirements, resulting in a shortage of C flow for N assimilation in the mycobiont (Turpin 1991; Palmqvist 2000; Dahlman et al. 2003). Nevertheless, the lack of a treatment effect on Chl a concentrations in both species (Fig. 4a) suggests that a general up-regulation in photosynthesis did not occur in the present study, making unlikely the hypothesis of photosynthesis as a potential source of C skeletons for N assimilation.

The lichens investigated here have Trebouxia photobionts, which export C to the mycobiont in the form of ribitol (Richardson 1985). After ribitol transfer, the fungus rapidly metabolizes it into arabitol, and further to arabinose, ribose, fructose, and finally mannitol via the pentose phosphate pathway (Lines et al. 1989; Feige and Jensen 1992), thereby making it unavailable to the photobiont (Galun 1988). Pool sizes and flow rates of these compounds in lichens during stress have been poorly studied so far (cf. Dahlman et al. 2003), and there is no clear consensus concerning the role of arabitol vs. mannitol in different lichens and various processes (cf. Fahselt 1994). However, mannitol is considered the most important form of low-molecular-weight reserve sugar (Sturgeon 1985). The size of the mannitol pool was un-altered by the N fertilisation in E. prunastri, whereas a decrease was observed in X. parietina at the two highest N treatments (Fig. 5c). The mannitol pools were moreover significantly lower in the former species across all treatments (Fig. 5c). From these observations, one could hypothesize that X. parietina may have been able to use its larger mannitol pool to obtain the C skeletons and energy needed for N assimilation (Turpin 1991), when exposed to the two highest N dosages (Fig. 5c). This decrease in mannitol coincided with an ability of X. parietina to maintain a low concentration of ammonium in the thallus at the highest N dosage (Fig. 3a), as a 2-fold increase in N uptake (from 2.2 to 4.5 mg g−1 DW) led to an increase in thallus ammonium concentration of only ca. 25% (Fig. 3a). On the other hand, the stable concentrations of mannitol in E. prunastri, even with the highest N treatments (Fig. 5c), coincided with a dramatic increase in the ammonium levels with the same treatments (Fig. 3a). Taken together, these observations seem to support the assumption that X. parietina may have been able to use the mannitol pool for N assimilation, in contrast to E. prunastri. However, the data from the present study do not allow a definite conclusion to be drawn. The importance of mannitol pools for N assimilation requires further research, namely through the use of labeling techniques.

Glutamine and Glu are key amino acids in N metabolism, as N assimilation into amino acids occurs primarily via the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway (Turpin 1991). The overall smaller Gln and Glu pools of E. prunastri compared to X. parietina (Table 1) suggest that overall metabolic turnover is relatively lower in the former lichen, as also evidenced by its overall lower N, Chl a and ergosterol status (Table 1; cf. Palmqvist et al. 2002). The Gln:Glu ratio was shifted towards relatively higher Gln levels in E. prunastri with increasing N uptake whereas the opposite was observed in X. parietina (Fig. 6a). The continuous synthesis of Glu from the GS/GOGAT cycle requires the input of additional C skeletons, while a shortage of C might lead to a build-up of Gln (Turpin 1991; Martins-Loução and Cruz 1999). The higher Gln:Glu ratio of E. prunastri therefore provides additional evidence that N assimilation in this species may have suffered from C limitation.

The presence of a nitrate pool in X. parietina at the two highest N dosages is intriguing (Fig. 3b). One can argue that this might be the result of both nitrate and ammonium being assimilated from the fertiliser. Although nitrate can be assimilated by lichens (Crittenden 1996), it is known that green algal lichens have a much higher affinity for ammonium compared to nitrate (Crittenden 1996; Dahlman et al. 2002, 2004). This higher affinity for ammonium is supported by the results presented here, as there was nearly a 1:1 relation between final thallus N and N uptake from ammonium (Fig. 2) for both lichens, implying that nitrate uptake was minor. In X. parietina, an increase in the uptake from 2 to 4 mg g−1 DW led to an increase in thallus N from 32.9 to 34.6 mg g−1 DW (Fig. 2), that is, an increase of 1.7 mg g−1 DW. This means that ca. 85% of the increase in thallus N was attributable to ammonium uptake. Likewise, in E. prunastri an increase in uptake from 4.5 to 6 mg g−1 DW led to an increase in thallus N from 14.62 to 16 mg g−1 DW (Fig. 2), meaning that ca. 92% of the increase in thallus N was attributable to ammonium uptake. In this way, only 15% and 8% of the increase in thallus N in X. parietina and E. prunastri, respectively, could be accounted from other sources. Nevertheless, an increase in the nitrate pools with increasing N uptake was only observed in X. parietina, these pools displaying a 7-fold increase, from 21 to 140 μmol g−1 DW, at the highest treatment (Fig. 3b). Being so high, this increase in nitrate cannot be explained only by a possible nitrate uptake by X. parietina from external sources. It then appears that X. parietina might have been able to oxidize surplus ammonium into nitrate, which is a non-toxic form of N storage. A conversion of ammonium into nitrate has previously been observed in some vascular plant species tolerant to surplus ammonium supply, although the mechanism behind this reaction remains to be resolved (Martins-Loução and Cruz 1999; Millar et al. 2002). There is also evidence for the presence in fungi of nitric oxide synthase, the main enzyme responsible for nitrate production (Ninnemann and Maier 1996). Obviously, the hypothesis of such a mechanism in lichens requires further investigation.

One could argue that the presence of apothecia in X. parietina, which does not occur in E. prunastri, could constitute an advantage for the former species, as apothecia could function as N and C sinks. When exposed to high N concentrations X. parietina might be able to shuttle some of the N that had been taken up for the development of these reproductive structures. In a previous study, however, no relation was found between different N concentrations in X. parietina thalli and the number of apothecia in the same thalli (unpublished data). Nevertheless, the role of apothecia as N and C sinks in N-tolerant lichen species is another question that requires further investigation.

Conclusion

The acidophytic lichen E. prunastri showed a higher N uptake from ammonium than the nitrophytic lichen X. parietina, this coinciding with both different morphological features and habitat conditions of the two species. Chlorophyll a and ergosterol concentrations remained un-altered in X. parietina irrespective of the N uptake, and a decrease in the mannitol pool was observed at the highest N dosages, as well as an increase in the nitrate pool at the same dosages. Ergosterol concentrations decreased with increasing N uptake in E. prunastri and an increase in the ammonium pool was observed at the highest N dosages for this lichen. Taken together, the results of the present study can partly explain the reported higher tolerance of X. parietina towards increased atmospheric N levels. Nevertheless, the mechanisms behind N assimilation in the two lichen species require further investigation.

Acknowledgements

The authors are thankful to Cristina Cruz for helpful discussions, Pedro Pinho for help with the experimental work, Carla Rodrigues for the analysis of the total N, Rodrigo Maia for the analysis of the 15N, Alice Tavares for the analysis of ammonium and nitrate, Torgny Näsholm for the use of his HPLC laboratory and Margareta Zetherström for skilful technical advices. The authors also thank the reviewers for fruitful and opportune comments. This study was supported by grants from FCT to Gisela Gaio-Oliveira (PRAXIS XXI/BD/19622/99), from Centrum for Environmental Research (CMF, Umeå, Sweden) (993194) to Lena Dahlman and from FORMAS, Sweden (24.0795/97) to Kristin Palmqvist.

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© Springer-Verlag 2004