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Keywords:

  • depsidones;
  • growth rate;
  • herbivore defence;
  • lichens;
  • melanins;
  • photosynthetic acclimation;
  • photoprotection;
  • secondary compounds.

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    This study analysed relationships between secondary chemistry, lichen growth rates and external habitat factors for two groups of UV-B-absorbing secondary compounds in the lichen Lobaria pulmonaria in order to test some hypotheses on their formation and function.
  • • 
    Medullary depsidones and cortical melanins were quantified in thalli transplanted to three successional forest stands (shaded young forest, open old forest, sun-exposed clear-cut area) and subjected to different watering regimes (spraying with water, water + nitrogen, no spraying). Growth rates were already known.
  • • 
    The total concentration of all seven depsidones was constant across the entire range of growth rates and sun exposures, showing that these depsidones serve functions other than photoprotection. Thalli from the well-lit transplantation sites had the highest synthesis of melanins. Within each forest type there was a trade-off between growth and melanin synthesis. Melanins and photosynthetic acclimation enhanced survival on a subsequent exposure to high light intensity, despite excessive temperatures resulting from higher absorption of solar energy in melanic thalli relative to pale thalli.
  • • 
    In conclusion, the highly responsive melanic pigments play a photoprotective role in light acclimation, whereas the constant amount of depsidones across a wide spectrum of growth ranges and irradiances is consistent with herbivore defence functions.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A lichen is a symbiotic association between a species-specific fungal partner, the mycobiont and its autotroph photobiont (green algae and/or cyanobacteria). The mycobiont depends on the delivery of photosynthates from its photobiont that often grows inside a dense mass of screening fungal tissues. The fungal partner uses the photosynthates as an energy source and for new thallus growth (as reviewed by Palmqvist, 2000), allowing both partners to colonize new ground. At the same time, the mycobiont invests a substantial part of the fixed carbon in a diverse secondary chemistry with > 800 secondary compounds identified so far (Huneck & Yoshimura, 1996; Huneck, 2001). Individual species can contain as much as 30% of dry weight (DW) (Huneck, 1973), but the relationship between lichen growth and investments in secondary compounds is not well known.

A few secondary compounds, such as the orange parietin, the yellow usnic acid, the brown melanins and the colourless atranorin, are located in the upper cortex directly above the photobiont layer. During the last decade, there has been a focus on cortical lichen compounds. There is now solid evidence that some coloured cortical compounds are induced by UV-B (Solhaug & Gauslaa, 1996; Solhaug et al., 2003; McEvoy et al., 2006), boosted by photosynthates (Solhaug & Gauslaa, 2004; McEvoy et al., 2006), correlated with solar radiation at spatial (Gauslaa & Solhaug, 2001; Gauslaa & Ustvedt, 2003) and temporal scales (Bjerke et al., 2005; Gauslaa & McEvoy, 2005), and that they screen (Gauslaa & Solhaug, 2001) and protect underlying photobionts against excessive solar radiation (Gauslaa & Solhaug, 2004; Vráblikováet al., 2006). These functions serve to optimize lichen photosynthesis in a changing light environment and hence expand the organic carbon pool in a lichen thallus. Among the mentioned cortical compounds, melanins are the only compounds that cannot be extracted. They are complex and poorly known polymers of phenolic compounds (Plonka & Grabacka, 2006), and can only be quantified by indirect methods.

The vast majority of secondary lichen compounds are located in the medulla. Most are colourless, but with strong UV-B absorbance bands. Medullary compounds often occur in largest quantities in the photobiont layer, which may still be consistent with a photoprotective role (as hypothesized by, for example, Fahselt & Alstrup, 1997). However, the ecological role of medullary compounds is not clear. Additional hypotheses for functional roles exist, such as antiherbivore, antimicrobial and antifungal agents and mineralization agents (as reviewed by Fahselt, 1994). One hypothesis supported by experimental evidence, infers herbivore deterrent roles of medullary compounds for lichen-feeding snails (Lawrey, 1980; Gauslaa, 2005) and insects (Giez et al., 1994; Pöykkö & Hyvärinen, 2003; Pöykköet al., 2005). The compounds are carbon-based, and most ought to be present in large concentrations in order to deter herbivores efficiently (Gauslaa, 2005). However, the natural variation in medullary compound concentration is still unknown for most species.

This study deals with the tripartite foliose lichen, Lobaria pulmonaria (L.) Hoffm., a spectacular and large old forest species. It contains both green algae (main photobiont) and cyanobacteria in small, internal cephalodia. The upper cortex forms visible cortical melanins that screen UV and PAR radiation from the photobiont (Gauslaa & Solhaug, 2001). In addition to melanins, UV-absorbing depsidones are deposited as numerous tiny crystals outside medullary hyphae. It is not known whether these two groups of secondary metabolites are regulated by common factors. If the depsidones add to the protection against excessive UV-B, the content of both compound types should be highest in sun-exposed habitats. If depsidones serve other functions, one would expect the melanin content to follow a light gradient, but the depsidones may not necessarily concur.

The concentration of secondary compounds is in some way related to the carbon metabolism. They may be produced as a sink for excess carbon (Mosbach, 1973) under some environmental stresses during which major parts of the fixed carbon cannot be invested in new thallus growth. If this is the case, a trade-off between lichen growth and synthesis of secondary compounds would be expected. A trade-off may also occur between the synthesis of melanins and depsidones if they are formed from a common resource pool. On the other hand, if the depsidones serve as a vital protection against herbivores, as recently shown by Gauslaa (2005) in grazing experiments on L. pulmonaria, one would not expect depsidone concentration to vary with lichen growth rate and/or solar radiation in its habitat. To test such hypotheses, lichen growth and secondary compounds need to be quantified. So far, we are not aware of lichen studies relating the concentration of secondary compounds to growth. Recently, growth was assessed in 600 L. pulmonaria thalli transplanted in three successional forest stands with contrasting light climates, in which three irrigation treatments were applied in each stand (Gauslaa et al., 2006b). Growth varied substantially among the replicates because of a dependency on a combination of external and internal factors (Gauslaa et al., 2006b). The main objective of our study is to quantify depsidones and melanins in these transplants, and use these new results to analyse relationships between the two categories of compounds, lichen growth and habitat factors.

Another objective is to study the sun-screening function of melanic compounds in a wider perspective, by including experiments quantifying side-effects of melanins, such as their ability to cause excess heating during strong solar radiation (Gauslaa, 1984). Lobaria pulmonaria is susceptible not only to high light intensity, but also to excessive temperatures (Lange, 1953; Gauslaa & Solhaug, 1999). High light screening by melanins may come at a price, as the darkened thalli absorb more solar radiation, causing the temperature to rise beyond tolerable values. In order to evaluate the effect of various acclimation traits on the total tolerance of excessive light, we also aim to estimate the ability of the photosynthetic apparatus to acclimate by dissipating excess light by increased nonphotochemical quenching (NPQ) achieved after a sudden exposure to clear-cut conditions. Previously, NPQ has only been studied in L. pulmonaria during gradual seasonal variations in light in intact forests (MacKenzie et al., 2001, 2002). Finally, we aim to assess the high light susceptibility of the transplants, acclimated under defined forest stand-specific conditions and with known acclimation traits, by exposing them to natural full, direct sunshine.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lichen material and transplantation experiment

A collection of approx. 600 full-size lobes of Lobaria pulmonaria (L.) Hoffm., used in a previous growth study (Gauslaa et al., 2006b), formed the basis material for the new experiments and analyses in this paper. The lobes are referred to as thalli, and their area and dry matter growth, as well as chlorophyll (Chl) content, were assessed by Gauslaa et al. (2006b). These thalli were transplanted to three boreal spruce forest stands, young forest, old forest and a clear-cut area in south-east Norway (59°22′N 9°45′E, 500 m asl) for 100 d (July–October 2004; with a total natural rainfall of 487 mm during 51 rainy days). Thalli were randomly located on 153 frames (15.9 × 15.9 cm2), tied to a nylon mesh. Three frames representing three irrigation regimes (discussed later) with four thalli on each were placed in one frame-holder. Seventeen frame-holders were randomly placed within each of the three types of forest stands. In this way, the transplant group was subdivided into three treatment groups (68 thalli in each) in each forest stand: those sprayed once a day with deionized water (0.63 mm each time) in late afternoon during 31 of the dry days; those sprayed at the same days with nutrients (13 mg N m−2) dissolved in the same amount of water; and the nonsprayed controls. Representative hemispherical photographs from the three stand types are shown in Fig. 1. Indirect site factors (Anderson, 1964) were computed by Gauslaa et al. (2006b) from such photographs taken at all the frame-holders to quantify canopy openness. Before transplantation, size and Chl fluorescence parameters did not differ between the samples of thalli selected for the different forest treatments (Gauslaa et al., 2006b), meaning that the dataset should be well suited for detecting differences resulting from treatment. After the completion of the transplantation experiment, thalli were stored air dry at –20°C, as recommended by Honegger (2003), between further measurements and analyses presented in this paper.

image

Figure 1. Hemispherical photos taken at one representative transplantation location in each of the three successional Picea abies stands. Mean indirect site factor (± SE, n = 17), as computed by Gauslaa et al. (2006b): young forest (c. 30 yr old), 0.141 ± 0.003; old forest (c. 120 yr old), 0.246 ± 0.011; clear-cut area, 0.645 ± 0.012.

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Indirect assessment of the nonextractable melanic compounds

Melanic pigments are not extractable from lichens. Thus, the content was indirectly measured recording reflectance spectra (Gauslaa & Solhaug, 2001) of all thalli from the transplantation experiment (n = 594). Thalli were gently pressed to give a more even surface for measurement. The colour difference between treatments was most conspicuous among desiccated thalli, and therefore reflectance was measured in the dry state. Reflectance spectra (450–900 nm) were recorded using an integrating sphere (ISP-50-REFL OceanOptics, Eerbeek, the Netherlands) placed directly on the upper surface of each thallus. A halogen lamp (DH2000, OceanOptics) illuminated the thallus via a 600 µm optical fibre, connected to the sphere. Reflectance was measured at one random position per thallus with a spectrometer (model SD2000, OceanOptics) connected to the output port of the sphere with a 400-µm-thick fibre. The percentage reflection was calculated on the basis of a dark spectrum and a reference spectrum from a white reference tile (WS-2, OceanOptics). The browning reflectance index (BRI), calculated as BRI = (1/R550 – 1/R700)/R750 (Chivkunova et al., 2001), was used as a quantitative estimate of melanic compounds.

Nonphotochemical quenching (NPQ) and quantum yield of PSII

Xanthophyll pools are strongly influenced by the irradiance during the last hydrated period. It is therefore important to specify irradiance values during the last moist days before harvest on 19 October in order to analyse NPQ data correctly. In October, PAR cannot exceed 300 µmol m−2 s−1 in the open clear-cut area under a cloud-free sky (Gauslaa et al., 2006b). The last 6 d in the field were cloudy and rainy, resulting in a substantially lower PAR during the last 150 h before harvesting. All thalli were moist during harvesting, and desiccated in darkness overnight. Thereafter, FV/FM was measured as reported in Gauslaa et al. (2006b). Thalli were then dried at 20°C before being put in the freezer, implying a hydration of 30 h in the laboratory at low light intensity.

From the total pool of harvested transplants, one random thallus for the NPQ analyses was selected from each of the 151 transplantation frames. Selected thalli were rehydrated and placed on wet filter papers in plastic boxes allowing air circulation, and preconditioned for 3 h at 20°C and low light intensity (2–3 µmol m−2 s−1) to allow relaxation of short-term down-regulation of photosystem (PS) II resulting from desiccation. Chl a fluorescence was measured with a portable, modulated fluorometer (PAM-2000, Walz, Effeltrich, Germany). Quenching analysis was carried out at a central position on each thallus and nonphotochemical quenching, inline image (Bilger & Schreiber, 1986), and the light-adapted quantum yield of PSII, inline image (Genty et al., 1989), were computed (FM, maximum Chl fluorescence of a dark-adapted thallus; inline image, maximum Chl fluorescence of a light-adapted thallus; FS, steady-state Chl fluorescence of a light-adapted thallus). FM was determined after 10 min darkness using a saturating pulse (15 000 µmol photons m−2 s−1) lasting 0.6 s. Determination of Chl fluorescence parameters in a light-adapted state followed: after the determination of FM, the actinic light (100 µmol photons m−2 s−1) was turned on. This actinic light intensity was used since it is common in open clear-cut areas under cloudy and moist conditions and/or during early mornings when lichens are photosynthetically active, and because it is high enough to induce a high amount of NPQ in L. pulmonaria (MacKenzie et al., 2002). Steady-state Chl fluorescence (FS) was reached after 5 min when another saturating light pulse (15 000 µmol photons m−2 s−1, duration 0.6 s) was applied and inline image was recorded.

Quantification of extractable secondary compounds

After completing the NPQ measurements, the concentration of acetone-soluble secondary compounds in these 151 thalli was quantified by HPLC. Each thallus was pulverized, and 45–55 mg of the powder was extracted with 1 ml acetone for 20 min. The extraction was repeated four times. The combined extracts were evaporated to dryness under N2 in a warm bath and the sample was then stored in the freezer. Extracts were re-dissolved in 500 µl acetone (every second sample contained 1.5 mm benzoic acid as an internal standard) and analysed on an ODS Hypersil column, 60 × 4.6 mm, using a HP 1100 series HPLC (Aligent Technologies, Waldbronn, Germany). The injection volume was 5 µl and the flow rate was 2 ml min−1. The mobile phases of A (millipore water, 0.25% orthophosphoric acid and 1.5% tetrahydrofuran) and B (100% methanol) were run following an adapted gradient of Feige et al. (1993). Compounds were detected at 245 nm. Identification was based on retention times, UV spectra and comparison with a norstictic acid standard (Gaia Chemical Corporation, Gaylordsville, CT, USA). Results are presented as w/w.

Setup for measuring the thallus temperature-dependence on light

A new random selection of 10 transplants from each of the three forest stands was analysed to test how thallus pigmentation formed during the transplantation period affected thallus temperature under varying light intensity. Micro-quantum and temperature sensors (Model 2060-M, Walz), connected to a modulated fluorometer (PAM-2000, Walz), were pinned in position on a small piece of white polystyrene, placed 30 cm in front of a projector (Leica Pradovit P 2002, bulb 24 V, 250 W). One desiccated thallus at a time was placed over the thermocouple held in position with pins. The quantum sensor was placed 3 mm from the thermocouple. The distance to the projector was adjusted to focus the light beam on both the thallus and quantum sensor simultaneously. After 1 min adjustment time, the temperature and light intensity were recorded. The polystyrene sheet was then moved slightly closer to the projector and new readings were taken. This was repeated four more times until the sheet was approx. 15 cm from the light source, equivalent to 2000 µmol m−2 s−1. This procedure was repeated for each of the 30 thalli. The experiment was performed indoors at 27°C.

Measurements of high-light tolerance achieved during acclimation

A final random selection of 144 thalli, 48 from each forest stand, was made from the remaining pool of transplants. These thalli were used in an exposure experiment to test the response of the acclimated thalli to ambient summer PAR and UV intensities. The experiment was carried out in a fully exposed site at Ås, south-east Norway (59°40′N, 10°45′E), during the period June 1–13 2005 (12 d). Thalli were secured with pins to four 30 × 30 × 3 cm white polystyrene boards. Each board had 12 thalli from each forest stand. The thalli were positioned in a fixed pattern. Starting at the top left corner of the board; a random thallus from forest stand 1 was pinned in position, then one from stand 2, one from stand 3, etc., until all 36 thalli were placed in six rows. Each polystyrene board was centrally placed underneath either: (i) one of two 70 × 70 cm UV-transmitting Plexiglass screens (Perspex, GS2458, 4 mm, Finn Løken, Ås, Norway); or (ii) one of two similar sized mirrors (4 mm, Ås Glass Mester, Ås, Norway). Screens and mirrors were placed 10 cm above ground level, leaving sufficient space for air circulation around the thalli. PAR readings were taken with a PAR sensor (model SKP 215, Skye Instruments Ltd, Llandrindod Wells, Powys, UK) below one UV-transmitting screen and one mirror. Mean values were recorded with a DataHog data logger (Skye Instruments) every 20 min. The UV-B intensities below the screen were monitored with a UV-B sensor (model SKU 430, Skye Instruments). Mean values were logged every 20 min with a SpectroSense logging meter (Skye Instruments). The average daily PAR dose under the screen was 36.4 mol m−2, with daily maxima of approx. 2000 µmol m−2 s−1 in 8 out of 12 d. The average UV-B per day was 15.8 kJ m−2, with daily maxima of approx. 1 W m−2. The irradiance recorded underneath the mirror was negligible. The mean daily air temperature and relative humidity, recorded 2 m above ground level at the experimental site, were 11.4°C and 69%, respectively, with maximum values up to 21.6°C and 99%. The moisture in the air hydrated the thalli during several nights.

The Chl a fluorescence of each thallus was recorded immediately before and after the exposure experiment. Before measurement, all thalli were sprayed with deionized water and left in the hydrated state for 24 h at 15°C under low incandescent light (2–3 µmol photons m−2 s−1) to allow for relaxation of short-term down-regulation of PSII. They were then preadapted in the dark for 15 min. Chl a fluorescence induction curves were recorded with a portable fluorometer (Plant Efficiency Analyser, Hansatech, King's Lynn, Norfolk, UK.) The fluorometer calculated FV/FM values from the curves recorded at an irradiance of 1500 µmol m−2 s−1, for 5 s, from light-emitting diodes. Afterwards, these thalli were again hydrated for 1 min, divided in two from lobe base to lobe tip and one half was selected for Chl assessment. The area of the selected half was measured with a leaf area meter (LI3100, Li-Cor, Lincoln, NE, USA) before being submerged in separate aluminium foil-covered glass vials containing 10 ml of N,N-dimethylformamide (DMF). Pigment extraction was complete after 90 h in the dark at 4°C, and Chl was quantified according to Porra et al. (1989).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Acetone soluble secondary compounds

The stictic–norstictic acid chemosyndrome comprising a total of seven depsidone compounds occurred in the L. pulmonaria thalli (Table 1). The compounds were identified as stictic, constictic, norstictic, peristictic and methylnorstictic acids, and two other spectrally similar derivatives were labelled numbers 1–2. The most abundant compound was stictic acid (24.0 ± 0.5 mg g−1 DW; mean ± SE, n = 150), followed by constictic acid (10.2 ± 0.2 mg g−1) and norstictic acid (6.5 ± 0.3 mg g−1). The remaining compounds were present in small amounts only. The mean total concentration of all depsidones amounted to 4.3 ± 0.1% of DW (total range 2.2–9.3%). The total concentration of depsidones, as well as the content per unit area, was not significantly affected by forest stand during the transplantation. A detailed analysis, however, revealed that the minor compound methylnorstictic acid (< 0.1% of DW) had slightly higher concentrations in the clear-cut area than in the two other forest stands (P = 0.003, Table 1), but did not differ with respect to irrigation regime (two-way anova; data not shown). The concentration of most depsidones correlated positively with each other (Table 2). Norstictic, peristictic and methylnorstictic acids and derivative 1 were strongly coupled, as were stictic and constictic acids and derivative 2. These two compound groups were weakly associated with each other, although the majority of coefficients were highly significant (Table 2).

Table 1.  Concentration of depsidones in Lobaria pulmonaria transplanted to three boreal forest stands
Lichen depsidones (mg g−1) Young forestOld forestClear-cut areaanova P
  1. Values are means ± SE, n = 50.

  2. The results from an anova are included. A two-way anova with irrigation regime in addition to forest stand did not yield any significant differences (data not shown). ns, not significant.

Stictic acid24.3 ± 1.023.1 ± 0.924.5 ± 0.9ns
Constictic acid10.3 ± 0.4 9.9 ± 0.410.3 ± 0.5ns
Norstictic acid 6.7 ± 0.4 5.8 ± 0.4 6.9 ± 0.6ns
Peristictic acid 1.3 ± 0.1 1.2 ± 0.1 1.4 ± 0.1ns
Methylnorstictic acid0.58 ± 0.03 (a)0.64 ± 0.04 (a)0.73 ± 0.04 (b)0.0027
Derivative 20.48 ± 0.020.47 ± 0.020.50 ± 0.02ns
Derivative 10.22 ± 0.020.20 ± 0.010.23 ± 0.03ns
Total43.8 ± 1.441.4 ± 1.444.5 ± 1.7ns
Table 2.  Pearson correlation coefficients (upper part) and corresponding level of significance (lower part; n = 150) between the concentrations of the various measured secondary compounds, including melanic compounds (expressed as browning reflectance index, BRI) in Lobaria pulmonaria
 Norstictic acidDerivative 1Peristictic acidMethylnorstictic acidConstictic acidDerivative 2Stictic acidBRI
  1. ns, not significant.

Norstictic acid   –   0.6360.6660.6340.1950.3970.3680.114
Derivative 1< 0.0001   –0.8790.6040.1650.3180.1470.085
Peristictic acid< 0.0001< 0.0001    –0.6190.1270.3130.3540.149
Methylnorstictic acid< 0.0001< 0.0001< 0.0001    –0.3570.5310.3120.140
Constictic acid  ns  ns   ns< 0.0001    –0.5130.6680.048
Derivative 2< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001    –0.6750.137
Stictic acid< 0.0001  ns< 0.00010.0001< 0.0001< 0.00010.121
BRI  ns  ns   ns   ns   ns   nsns

No significant correlations (P < 0.05) were found between total concentration of depsidones and DW growth (r = −0.030), area growth (r = 0.045) or canopy openness assessed by means of indirect site factor (r = 0.088; calculated from photos similar to those in Fig. 1), despite the substantial variation in lichen growth and site factors as given by Gauslaa et al. (2006b). Among the individual compounds, only the minor compound methylnorstictic acid (0.1% of DW) showed a weakly negative correlation with DW growth (r = −0.200; P = 0.02).

Formation of cortical melanic pigments

Melanin content, expressed in terms of BRI, did not show any significant correlations with detected depsidones (Table 2). Unlike the depsidones, however, melanins were highly significantly influenced by both forest stand and irrigation treatment (Fig. 2), also evidenced by a strong correlation (r = 0.756; P < 0.0001) with the indirect site factor as measured by Gauslaa et al. (2006b). The irrigation regime had no significant effect in the shaded young forest, in which brown pigments were hardly visible. The effect of irrigation on the BRI increased with increasing light exposure and thereby increased melanin formation. Irrigation with water only increased the synthesis of melanic compounds in the two most light-exposed forest stands, whereas the addition of N significantly lowered the melanin formation in the clear-cut area (Fig. 2).

image

Figure 2. The effect of forest stand and irrigation treatment regime on the colour of Lobaria pulmonaria thalli, expressed as a browning reflectance index (BRI). Values are mean ± 1 SE (n = 66). The inserted table shows the two-way anova of BRI data that was square-root-transformed. Significant differences in BRI (P < 0.05, Student–Newman–Keul method) are indicated with small letters above the columns. The inset graph shows mean visible (450–700 nm) and near-infrared (700–900 nm) percentage reflectance spectra ± 95% confidence limits (dotted lines, n = 196; the confidence interval is narrow and barely seen) from the upper surface of desiccated L. pulmonaria thalli after the 100 d transplantation experiment in clear-cut areas, young and old forests. A two-way anova (data not shown) showed a highly significant effect of forest stand on reflectance in the PAR (P < 0.0001) and infrared (IR) range (P < 0.0001), as well as of irrigation treatment (PAR, P = 0.012; IR, P = 0.0009). The forest stand × irrigation interaction was significant for PAR reflectance (P = 0.0309) but not for IR.

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The BRI was based on spectral reflectance from the upper surface (Fig. 2, insert). Therefore, the reflectance curves and mean visible reflectance showed the same pattern as the BRI with the lowest visible reflectance from the thalli from the clear-cut area. The clear-cut area thalli, rich in melanic compounds, reflected on average 34.7% less light than thalli from the old forest, 39.6% less than the pale young forest thalli in the PAR range, and 20.1 and 27.8%, respectively, in the infrared range. Like BRI, the reflectance curves based on the thalli from two forest stands with canopies were relatively similar compared with the clear-cut area. However, the BRI discriminated much better between forest stands and especially irrigation regimes compared with reflectance spectra. Therefore, the BRI appeared also for lichens to be a powerful way to reduce a complex reflectance spectrum to one simple measure of browning.

The average value of BRI in each of the three stands increased with average area growth, as a result of a common positive response to light for both variables (Fig. 3 inset). However, within the two forested stands, weak, but highly significant, negative correlations between BRI and growth occurred (Fig. 3). Fast-growing thalli from each of the two forested stands showed a smaller scatter with respect to BRI than those with slow growth rates. In the clear-cut area, browning tended to be less in thalli with both the lowest and the highest growth, with the largest scatter in BRI occurring in between. The irrigation regimes did not form clusters in the plot, and were not shown.

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Figure 3. The relationship between thallus area growth of Lobaria pulmonaria (Gauslaa et al., 2006b) and colour, expressed as browning reflectance index (BRI), for the three forest types, young (triangles, n = 201, r = 0.324, P < 0.0001), old (open circles, n = 197, r = 0.267, P = 0.0002) and clear-cut area (closed circles, n = 196, r = 0.143, P = 0.053). Lines show the linear regression for each forest type. Inset, the relationship between the mean area growth and mean BRI in each forest type (vertical and horizontal bars represent 1 SE).

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NPQ and quantum yield of PSII

Nonphotochemical quenching increased from young forest thalli to clear-cut area thalli, with the old forest thalli in between (Fig. 4a). The thalli showed stand-specific amounts of NPQ (P < 0.0001; anova), but with no significant irrigation regime effects. Unlike the more exponential response of BRI from shady young forest via open old forests to clear-cut areas (Fig. 3 inset), the NPQ showed a more even increase from the darkest to the sunniest stand (Fig. 4a). These results showed that the thalli had acclimated to stand-specific light regimes during the transplantation period. Finally, there was a persistent and constant amount of the quantum yield of PSII at 100 µmol photons m−2 s−1 in all forest stands after the acclimation period, including even the sun-exposed clear-cut areas (Fig. 4b), indicating that the photosynthetic capacity remained unchanged.

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Figure 4. Nonphotochemical quenching (NPQ) (a) and quantum yield (b) of PSII of Lobaria pulmonaria after the 100 d transplantation period in three successional forest stands (means ± 1 SE, n = 50). Inset (a): results of a two-way anova of NPQ, data were square-root-transformed before analysis. Significant differences in NPQ (P < 0.05, Student–Newman–Keul method) are indicated with letters above the columns. Inset (b): results of a two-way anova of quantum yield of PSII; data were squared before analysis.

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Melanic compounds increase the thallus temperature

Thallus temperatures were measured in desiccated thalli, because lichens dry rapidly under a direct sun. The temperature at a given irradiance was significantly higher in thalli acclimated to the clear-cut area by melanin formation than in the pale, melanin-deficient thalli from the shaded young forest (Fig. 5). Furthermore, the excess temperature in melanic thalli relative to that of pale thalli increased with increasing irradiance. An irradiance equivalent to maximal values during clear weather in a boreal summer (2000 µmol m−2 s−1) caused 3°C higher temperatures on the lower side of melanic (> 20°C in excess of ambient temperatures) compared with nonmelanic thalli (Fig. 5).

image

Figure 5. The relationship between irradiance and thallus-underside temperature for pale, young forest (open circles, r = 0.979) and melanic, clear-cut area thalli of Lobaria pulmonaria (closed circles, r = 0.988) measured in the laboratory. Air temperature and relative humidity were 27°C and 50%, respectively. Separate regression lines for each site (P < 0.0001) and 95% confidence limits (dotted lines) are given.

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High irradiance tolerance

The clear-cut area (Fig. 1) was shaded in early morning and late evening hours by adjacent forest edges, whereas the site for the exposure experiment in June 2005 had no shading elements. All thalli suffered from such a full exposure to the sun compared with those placed under mirrors excluding most PAR and UV, despite acclimation traits such as high melanization and enhanced NPQ in clear-cut area thalli. Nevertheless, the habitat source influenced the amount of damage (Table 3a). The maximal PSII efficiency (FV/FM) was less depressed in thalli acclimated to the clear-cut area than in forest thalli (Fig. 6a), with a highly significant difference between clear-cut area and young forest thalli (P < 0.0001) and a weak but significant difference between young and old forest thalli (P = 0.018). Relative to the pre-exposure value, thalli under the shading mirrors experienced an increase in FV/FM (Fig. 6a). Chlorophyll bleaching of the forest thalli, especially those from the dark young forest, was evident at the end of the 12 d period under the screens transmitting UV and PAR. Therefore, photobiont tolerance was also assessed in terms of effects of forest stand and screen type on total Chl content (Table 3b). Young forest thalli had significantly less Chl (% of start) than clear-cut area thalli (P < 0.05, Fig. 6b), although values of absolute Chl content at the end of the exposure were not significantly different within screen type (Fig. 6c). Irrigation treatments applied during the transplantation period did not affect the photobiont's response to the subsequent high light exposure.

Table 3.  Analyses of variance of the factors influencing FV/FMa and total chlorophyllb during the outdoor exposure experiment with extreme sunshine
Sourced.f.F-ratioP
  • a

    A three-way anova of FV/FM (% of start), with screen type, forest stand and irrigation treatment as factors showed that the interaction term screen × forest was highly significant (P < 0.001, results not shown). Therefore a two-way anova of the effect of forest and irrigation within both screen types (solar radiation transmitting and excluding) was carried out and the results presented here. Data was log-transformed before analysis to improve the distribution requirements for the anova analysis.

  • b

    Effect of forest stand and screen type on thallus Chl content during the outdoor exposure experiment. Two-way anovas of Chl content for all three forest stands for both mirrors and light screens.

FV/FM
Comparison within mirror
 Forest stand  2 16.09< 0.001
 Irrigation treatment  2  1.450.243
 Forest stand × irrigation  4  1.390.249
Comparison within UV screen
 Forest stand  2 11.05< 0.001
 Irrigation treatment  2  0.350.707
 Forest stand × irrigation  4  0.740.567
Total Chl (% of start)
 Screen  1107.64< 0.0001
 Forest stand  2  3.460.034
 Screen × forest stand  2  1.020.362
 Total143  
Total Chl (µmol m−2)
 Screen  1 43.73< 0.0001
 Forest stand  2  0.300.743
 Screen × forest stand  2  2.180.117
 Total143  
image

Figure 6. Photochemical quantum yield and chlorophyll content for Lobaria pulmonaria thalli preacclimatized for 100 d in a clear-cut area, young and old forest, and thereafter placed under UV- and PAR-transmitting screens (black columns) or behind mirrors excluding PAR as well as UV (grey columns) during a 12 d field exposure with high solar irradiation. Columns show mean ± 1 SE (n = 24) after the final field exposure. (a) Maximal PSII efficiency (FV/FM) measured after 24 h at low light to allow relaxation of short-term down-regulation of PSII; (b) content of chlorophyll a+b per thallus area expressed as a percentage of pre-transplantation period values; (c) actual total chlorophyll content after the exposure.

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Multiple linear regression analyses (data not shown) were run using the total Chl, thallus mass per unit area and BRI as explanatory variables for the FV/FM measured after the high light exposure, for thalli under the UV- and PAR-transmitting screens. Total Chl was the most critical factor (P < 0.0001) in determining FV/FM, explaining 43% of the variation. Adding BRI (P < 0.0001) increased the explained part of the variation to 54%.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Forest stand strongly influenced the L. pulmonaria thalli studied, especially with respect to the solar radiation-induced synthesis of cortical melanins (Figs 3, 4) and increased NPQ (Fig. 6). These acclimation traits presumably played a role in the high growth rates during the 100 d transplantation period, with an average DW gain of 23.1% for the clear-cut area thalli (total range 0–45%; Gauslaa et al., 2006b). Despite the strong light-induced variation in growth responses during the transplantation period (Gauslaa et al., 2006b), the total concentration of medullary depsidones was conserved (Table 1), apparently regardless of external factors such as the indirect site factor reflecting the openness of the canopy. This is strong evidence against the hypothesis inferring a solar radiation-protective role for UV-B-absorbing medullary depsidones, violating the inducibility criterion (Cockell & Knowland, 1999).

The lack of any relationship between thallus growth rate and production of medullary depsidones also suggests that these compounds do not function as waste products, or as a sink for excess carbon during oxidative stress, as was proposed by Mosbach (1973). In plants, various hypotheses exist regarding the effects of environmental factors on secondary metabolite concentrations. The carbon-nutrient balance (CNB) hypothesis (Bryant et al., 1983) states that the concentration of carbon-based secondary compounds (CBSC) will increase whenever excess amounts of carbohydrates accumulate in relation to growth requirements, or, put another way, factors that inhibit photosynthesis (shading) or stimulate growth more than photosynthesis (fertilization) will decrease the carbohydrate pool available for CBSC. A second hypothesis, the growth-differentiation balance (GDB) hypothesis (Loomis, 1932; Lorio, 1986; Herms & Mattsson, 1992; Stamp, 2004), states that growth is limited by water and nutrients, whereas differentiation depends on the available carbohydrates. Therefore, differentiation, and, hence, the production of CBSC, dominate when conditions (excluding carbohydrate supply) are suboptimal for growth. The growth of the L. pulmonaria thalli used in our study has previously been shown to be determined by a combination of external (forest stand, site factors) and internal factors (Chl content and thallus area; Gauslaa et al., 2006b). Application of the plant CNB and GDB hypotheses to our transplants would suggest that the concentration of depsidones varies in accordance with growth rate, higher growth rates implying lower concentrations of secondary compounds. This was not the case for any of the depsidones. A fixed percentage of the weight gain was deposited in medullary depsidones regardless of growth rate, with the exception of the minor methylnorstictic acid that occurred at the highest concentrations in the fastest-growing clear-cut area thalli.

Our results are, however, consistent with a herbivore defence role of depsidones. Grazing marks from snails are frequent on L. pulmonaria in calcareous broadleaved deciduous forests (Gauslaa et al., 2006a). Nondestructive acetone rinsing, in reducing the concentration of secondary compounds, consistently increases the grazing by snails of this particular lichen (Gauslaa, 2005). In a herbivore-defensive perspective it makes sense for the long-lived L. pulmonaria to maintain a certain concentration of depsidones regardless of growth and light availability.

The cortical melanins in L. pulmonaria, quantified by means of BRI, were highly influenced by habitat-specific indirect site factors during the transplantation period. In the present paper, we also showed that the BRI, compared with plain reflectance values, is a simple and more responsive quantitative estimator of the brown pigments located in the upper parts of the upper cortex of L. pulmonaria. Melanins screen solar radiation (Gauslaa & Solhaug, 2001) and are induced by UV-B (Solhaug et al., 2003). Therefore, the low reflectance of the clear-cut area thalli was expected (Fig. 2). Irrigation increases melanin formation in sites where ambient UV-B, according to Solhaug et al. (2003), is sufficient for a strong induction of melanin synthesis. This is consistent with the view that melanin formation is an active physiological process requiring hydration. Furthermore, the addition of nutrients reduced the formation of melanic pigments in the clear-cut stand. These thalli also had a slightly higher area gain than those sprayed with water alone (Gauslaa et al., 2006b). In contrast to the depsidones, melanic pigment production appears to respond to nutrient addition and/or thallus area growth. A trade-off between melanin synthesis and lichen growth is evident in Fig. 5, showing negative significant correlations between BRI and area gain within each forest stand. All thalli were pale at the start of the experiment, and earlier induction experiments have shown that melanin formation is rapid, provided there are sufficient intensities of inducing UV-B. Therefore, the decreasing BRI with increasing growth is hardly caused by dilution of the browning resulting from hyphal expansion. The trade-off suggests that melanin formation and growth compete to some extent for a common resource pool. Whereas eumelanins and pheomelanins are nitrogen-based, most allomelanins are carbon-based, as reviewed by Plonka & Grabacka (2006). However, as long as the identity of the L. pulmonaria melanins is unknown, the trade-off can be caused by either nitrogen or carbon pools. Low BRI values at both ends of the growth spectrum (clear-cut area thalli) were probably the result of Chl bleaching in some thalli. Browning requires an active metabolism, and some clear-cut area thalli had severe Chl degradation (Gauslaa et al., 2006b), which probably occurred before the protective melanins were synthesized.

Acclimation of NPQ also occurred during the transplantation period as a strong response to stand-specific light intensities. According to Demmig-Adams & Adams (2006), both sustained and flexible forms of thermal energy dissipation may occur. The long low light hydration period before NPQ measurements (see Materials and Methods section) suggests that the clear-cut area thalli have achieved a long-lasting increased NPQ value resembling the state in evergreen plants during winter (Demmig-Adams & Adams, 2006). This sustained high NPQ may explain the lower FV/FM values in the clear-cut area thalli recorded by Gauslaa et al. (2006b). In addition to the NPQ reported in Fig. 4, flexible energy dissipation dependent on rapid function of the xanthophyll cycle presumably occurs during daily fluctuations in solar radiation. Such a flexible xanthophyll-based NPQ could hardly have been detected after the prolonged low light hydration period before harvesting. The thalli may have reached higher NPQ values if measured directly after natural high light exposure. Acclimation of L. pulmonaria thalli to seasonal fluctuations in ambient light and temperature has been shown to be temperature-dependent in deciduous forests, but the ability to quench is dependent on seasonal light intensities (MacKenzie et al., 2001, 2002). A higher Chl a : b ratio in clear-cut area thalli at the end of the transplantation period represents another trait of high-light acclimation (Gauslaa et al., 2006b). Quantum yield of PSII, on the other hand, was not forest stand-specific (Fig. 4). However, it is noticeable that the quantum yield of PSII did not become depressed in clear-cut area thalli, because laboratory (Gauslaa & Solhaug, 1996, 1999) and field studies (Gauslaa & Solhaug, 2000) show that L. pulmonaria is susceptible to sudden exposures of high light. One reason for the successful acclimation in the clear-cut area was probably the small clear-cut area size, meaning that lichens were shaded from direct sun by neighbouring canopies 2 h every morning after sunrise (Gauslaa et al., 2006b), allowing repair of photoinhibitory damage and acclimation at low light when hydrated by morning dew. The forest stand-specific acclimation acquired during the 100 d field transplantation greatly influenced their success in the subsequent field exposure to extreme sun (Fig. 6). Higher cortical melanic pigment content, affording better solar protection for the photobiont (Gauslaa et al., 2001; Solhaug et al., 2003), is the likely reason for the increased tolerance exhibited by the air-dry clear-cut area thalli.

Cockell & Knowland (1999) proposed the following set of criteria for determining whether a compound has a screening role: (i) the compound should absorb the relevant radiation; (ii) biosynthesis of the pigment must be inducible by the radiation; (iii) screening activity should be demonstrated in vivo; and (iv) enhanced survival under elevated radiation should be shown to be the result of the compound when other physiological processes are not functioning. Criteria (i)–(iii) have been fulfilled for the melanic compounds in L. pulmonaria (Gauslaa & Solhaug, 2001; Solhaug et al., 2003). Melanic pigmentation reduced photoinhibition in L. pulmonaria under high light exposure in the desiccated state, according to our regression analysis, although a high content of Chl before exposure was highly beneficial. DNA repair mechanisms do not function when thalli are desiccated (Buffoni-Hall et al., 2003), as is probably the case for other physiological repair processes. Thus, the enhanced survival of the darker brown thalli provides the evidence to fulfil the fourth requirement of Cockell & Knowland (1999), assuming that NPQ does not function in desiccated thalli.

Lobaria pulmonaria is a heat-sensitive lichen when desiccated (Lange, 1953; Gauslaa & Solhaug, 1999). Melanin synthesis should ideally reach a concentration that sufficiently shields the photobiont from high irradiance without jeopardizing photochemical processes with excess heat. The excess temperature in melanic thalli relative to pale thalli increased with increasing irradiance (Fig. 5). Air-dry L. pulmonaria thalli experience a linear decrease in maximal PSII efficiency, from below normal to almost zero with increasing temperatures from 40 to 54°C during a 24 h period (Gauslaa & Solhaug, 1999). Together, these results suggest that melanic thalli are more susceptible to heat damage than their paler counterparts at temperatures > 35°C. Thallus temperatures during the outdoor exposure experiment were not recorded, but with irradiance values approaching or exceeding 2000 µmol m−2 s−1 for 8 out of 12 d, it can be assumed that temperatures > 40°C were frequently achieved under the UV/PAR-transmitting screens. Thus, the duration of the exposure to elevated thallus temperatures can be a critical factor for melanic thalli, with adverse effects narrowly tuned against the beneficial photoprotection provided by the melanins. In a long-term perspective, the beneficial light-shielding effect of melanins is more crucial than the detrimental heat-absorbing one, at least in cool climate zones and/or in natural forest habitats.

In conclusion, our study has shown that melanic compounds protect transplanted thalli against high irradiance stress, whereas the depsidones maintain a fixed concentration, regardless of sun exposure and lichen growth rate. The nonresponsiveness of depsidones to climatic factors during transplantation is consistent with a herbivore defence function rather than with the irradiance screening hypothesis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to Prof. J. Elix for his advice regarding the nature of the depsidones, and to Annie Aasen and Dr Line Nybakken for their assistance with the extractions. The transplantation study was funded by the Research Council of Norway (project 154442/720 given to Prof. Mikael Ohlson).

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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