OPTIMAL QUANTUM YIELD OF PHOTOSYSTEM II AND CHLOROPHYLL DEGRADATION OFLOBARIA PULMONARIAIN RELATION TO pH

Lichenologist 28(3): 267–278 (1996)

OPTIMAL QUANTUM YIELD OF PHOTOSYSTEM II AND CHLOROPHYLL DEGRADATION OF LOBARIA PULMONARIA IN RELATION TO pH* Yngvar GAUSLAA‡, Cor KOPPERUD‡ and Knut Asbjørn SOLHAUG‡

Abstract: The FV/FM ratio of Lobaria pulmonaria decreased with decreasing pH in samples from a boreal forest influenced by acid rain in SE Norway, suggesting that low pH has an effect on the optimal quantum yield of PSII. The pH was measured of distilled deionized water in which lobes from different habitats were rinsed, probably indicating the ionic environment of the lichen. The best prediction of FV/FM was found in a multiple regression model including the explanatory variables: pH, a/b-ratio and chlorophyll degradation measured as the 666/666a ratio, all correlated positively with FV/FM, but not with each other. The commonly used chlorophyll degradation 435/415 ratio cannot be used for L. pulmonaria because of an unidentified, habitat-influenced brownish pigment with high absorbance in the 415–435 nm wavelength range. ? 1996 The British Lichen Society

Introduction Many lichens are sensitive to elevated SO2 concentrations, since high levels cause chlorophyll degradation (Pearson & Skye 1965; Rao & LeBlanc 1966; LeBlanc & Rao 1973), resulting in a breakdown of symbiosis between the fungal partner and its photobiont. The toxicity of SO2 for lichens is higher when the pH is low (Hill 1971; Puckett et al. 1973; Manrique et al. 1989), but low pH in the absence of SO2 also seems to affect some lichens. There is an increasing awareness that some species, especially in the Lobarion, are threatened by acid rain even in areas with low SO2 concentrations (Gilbert 1986; Looney & James 1988; Farmer et al. 1991a,b, 1992; Gauslaa 1995). Exposure of Lobaria pulmonaria to simulated acid rain (pH=2·6) reduces photosynthesis (Sigal & Johnston 1986). Since most Lobarion species contain cyanobacterial photobionts, it has been hypothesized that low pH affects nitrogen fixation, based upon studies of L. pulmonaria (Dennison et al. 1977; Sigal & Johnston 1986). However, until now, adverse effects of low pH under experimental conditions have been reported for pH well below the lower range of the natural substratum pH for L. pulmonaria (pH 4·5–5·0, Gauslaa 1985; Rose 1988). A study of the Lobarion in a nature reserve in south-eastern Norway with low levels of SO2 (Gauslaa 1995), showed that pH of the substratum was often at *Abbreviations: FO =minimal fluorescence in the dark-adapted stage; FM =maximal yield of fluorescence; FV =variable yield of fluorescence (i.e. FM "FO); FV/FM =optimal quantum yield of PSII; PSII=photosystem II; 666/666a=absorbance ratio measured at 666 nm for fresh lobes and fully acidified lobes (a); 435/415=absorbance ratio measured at 435 nm and 415 nm. ‡Department of Biology and Nature Conservation, Agricultural University of Norway, P.O. Box 5014, N–1432 Ås, Norway. 0024–2829/96/030267+12 $18.00/0

? 1996 The British Lichen Society

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the lower end of the pH range that allows a healthy Lobarion to develop. Dead portions were observed in 20% of the lobes, and in some cases as much as 25% of the lobe area was dead, probably because of airborne acidic deposition (Gauslaa 1995) as the mean rainfall pH is 4·3. The area annually receives around 950 mg wet+dry deposited S m "2 and 1100 mg wet+dry deposited N m "2 (Statens Forurensningstilsyn 1992). The major deposition of S was not of marine origin, as the area is more than 100 km from more exposed seashores, and to leeward of dominating strong maritime winds. The present study deals with L. pulmonaria samples collected along a natural pH gradient in this oligotrophic area, which is further described in Gauslaa (1995). Degradation of chlorophylls to phaeophytin has frequently been used as a measure of damage caused by high SO2 concentrations in lichens (Ronen & Galun 1984; Manrique et al. 1989) or to assess the effects of heavy metal pollution in transplanted epiphytic lichens (Ronen et al. 1983; Garty et al. 1985) and aquatic bryophytes (López & Carballeira 1989). One objective was to study whether chlorophyll degradation takes place in L. pulmonaria along this natural pH gradient. Another objective was to evaluate the reliability of the commonly used phaeophytinization ratio measured at 415 and 435 nm (Ronen & Galun 1984; Manrique et al. 1989; Balaguer & Manrique 1991; Garty et al. 1992) for L. pulmonaria. Since the optimal quantum yield of photosystem II (FV/FM) has been found to decrease as a response to various environmental stresses (e.g. Krause & Weis 1984; Jensen & Feige 1991), a final objective was to study whether actual pH values affect FV/FM. Materials and Methods Plant material Two sets of reference material of L. pulmonaria for standard curves of chlorophyll degradation were collected: (1) a rich Lobarion locality with healthy and fertile specimens of L. pulmonaria, L. amplissima and L. virens on Quercus in S. Norway, Vestfold, Larvik, Fjærvardåsen, 59)11*20+N, 10)03*E, 190 m.a.s.l.; (2) in S. Norway, Sør Trøndelag, Malvik, Hommelvik, Storfossen in Homla, 63)22*48+N, 10)47*E, 150 m.a.s.l., on Sorbus aucuparia. Three to seven separate lobes of L. pulmonaria of around 160 mg dry weight each were taken from each of 20 investigated localities (Populus tremula, Salix caprea, Acer platanoides and Betula with a bark pH range of 4·6–7·2) in a 12·5-km2 conifer-dominated forest reserve area in Østmarka, south-eastern Norway, located at 59)50*N, 11)03*E, altitude 200–345 m, described by Gauslaa (1995). Samples were collected from May to September 1991.

Pretreatment Lobes were air-dried at room temperature for 24 h before they were frozen ("20)C) until 1992. Freezing in an air-dry condition is a commonly used way of storing lichens for later physiological studies (Larson 1977; Feige & Jensen 1987; Demmig-Adams et al. 1990; see below). Air-dried lobes were removed from the freezer, weighed after 30 min adjustment to room temperature, dried further under vacuum for 18 h to standardize the degree of drying as much as possible, and reweighed. Each lobe was rinsed separately in a vial containing 5 ml distilled deionized water for 1 h. Demmig-Adams et al. (1990) found that a 2-h submersion of L. pulmonaria in water did not negatively influence photosynthesis. The lobes were thereafter placed on a wet filter paper in a moist atmosphere for 18 h in darkness at 18)C. Excess water was then gently blotted with filter paper, and fresh weight, approximating the water-saturated weight, was immediately recorded.

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pH pH was measured in the vial containing the 5 ml distilled deionized water in which each lobe had been moistened and rinsed for 1 h. Repeated measurements within the same vial showed negligible variation. pH was also measured at five separate positions on the upper surface of each moist lobe after rinsing, immediately before fresh weight determination, with a surface ROSS electrode, model 81-35. The mean value for each lobe was treated as one observation. One bark sample consisting of several pieces of maximum thickness of 3 mm was collected beneath thalli of L. pulmonaria for measuring substratum pH. Bark samples were air dried at room temperature and finely ground in a ball-mill with grinding vessels and balls made of sintered alumina. pH was measured in two replicates each of 0·5 g air-dried bark mixed with 5 ml distilled deionized water, closed to prevent CO2 contamination and left overnight before measuring pH. Only one measurement of bark pH was made for each locality (n=20) because each bark sample consisted of subsamples.

Pigment studies Since lichen substances can degrade extracted chlorophyll (Brown & Hooker 1977), a preliminary experiment with the reference material (1) was made involving ten dry lobes that were rinsed with six serial washings of 100% acetone to remove lichen substances and ten control lobes. Lobes were then moistened with water and chlorophyll was extracted by dimethyl sulphoxide (DMSO) as described below. No significant effects of acetone rinsing were observed and chlorophyll extraction without a previous rinsing was subsequently used as the standard extraction routine. One sample consisting of four circular discs with a total area of 1·59 cm2 was taken from each lobe with a cork borer; thallus edges were avoided. Immediately afterwards, while still in a moist condition, the sample was put in 5 ml DMSO; chlorophylls were completely extracted after 4 h at 65)C in darkness. Forty minutes, as recommended by Ronen & Galun (1984), was not enough for complete extraction. Absorbance was measured with a Shimazdu UV-2101PC spectrophotometer at 415, 435, 649, and 666 nm (band width 1 nm). Absorbances at 649 and 666 nm were used for chlorophyll calculations according to Shoaf & Lium (1976) and Hiscox & Israelstam (1979). Chlorophyll degradation was estimated according to the method used by Moss (1967) for algae, adapted for lichens by Ronen & Galun (1984). Standard curves were prepared by mixing different proportions of intact pigments with 100% acidified pigments and, for each, absorbance was measured at 415, 435, and 666 nm. Standard curves were based on three replicates. Both reference materials (1 and 2) showed an almost linear response of both absorbance ratios, 666 (fresh)/666a (acidified) and 435/415 (Fig. 1). Acidification of test extracts was made by adding 15 ìl of 1  HCI to each 2 ml of extract, left for 30 min, and remeasured at 666 nm. This amount of acid was found to break down the chlorophyll completely. In order to study the influence of carotenoids, chlorophyll a/b ratio, and other pigments on the chlorophyll degradation ratio 435/415, photosynthetic pigments were separated by thin-layer chromatography. Lobes were homogenized in a cooled mortar with acetone, quartz sand and CaCO3. The homogenate was filtered, and applied to a thin-layer plate (12 g Kieselguhr, 3 g silica gel, 3 g CaCO3, 0·02 g Ca(OH)2, 50 ml ascorbic acid solution (0·008 ; layer thickness 0·2 mm), newly prepared according to Hager & Bertenrath (1966). The plate was developed for about 30 min in a solvent system composed of 100 ml petrol ether (b.p. 100–140)C), 12 ml isopropanol and 0·25 ml distilled water. Immediately afterwards each pigment band was scraped off the plate while still moist into a tube with 5 ml DMSO. Tubes were shaken and centrifuged before absorbance spectra were recorded for each pigment separately, including the non-mobile residual.

Optimal quantum yield of PSII Hydrated thalli were left at 18)C for 18 h in darkness in order to recover before FV/FM was measured with a portable fluorometer (Plant Efficiency Analyzer, Hansatech Ltd., Kings Lynn, UK) using excitation light of about 1500 ìmol photons m "2 s "1. A 15-min predarkening period was used before measurements of chlorophyll fluorescence parameters at three separate positions on each lobe; the mean value was used as one observation.

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1.6

1.4 Absorbance ratio

666/666a 1.2

1.0 435/415 0.8

0.6 0

20 40 60 80 Percentage acidified pigments

100

F. 1. Chlorophyll degradation ratios (1, 5: 435 nm/415 nm and -, ,: 666 nm/666 nm acidified) of a series of mixtures of intact total pigments and acidified total pigments (n=3) according to the method given by Ronen & Galun (1984). The ratios were made from one DMSO extract from the healthy and fertile reference material of Lobaria pulmonaria of, respectively, Fjærvardåsen in Vestfold (filled symbols) and Storfossen in Sør Trøndelag (open symbols). Standard errors are smaller than symbol size.

Some additional longer cultivation experiments at 18)C with the reference material (1) were done. Thalli were sprayed once a day with distilled water, and replicates experienced a 12-h photoperiod with 65 ìmol photons m "2 s "1.

Other measurements The area of each lobe in a moist condition was measured with a Licor Leaf Area Meter LI 3100. The specific thallus weight is mg dry weight (vacuum-dried) cm "2. The water-holding capacity in mg H2O cm "2 (Delf 1912) represents the difference between the water-saturated weight cm "2, immediately after blotting, and specific thallus weight.

Results Chlorophyll degradation The two phaeophytinization ratios (435/415 and 666/666a (Table 1) were closely correlated (r=0·999, P<0·001, n=11) when extraction was done with the reference material (1) (Fig. 1) with a rinsing water pH of 6·3. Repeating measurements of such standard curves on a set of lobes from Sør Trøndelag with apparently less pollution (reference material 2), but with a slightly lower rinsing water pH of 6·0, showed that especially the position of the 435/415 curve was significantly lowered (Fig. 1), indicating that there was a habitat factor that complicated the use of the 435/415 ratio as a measure of

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T 1. Summary statistics for measured characteristics of Lobaria pulmonaria* Parameter

Total range (min–max)

Mean&

4·8–6·7 4·0–5·8 4·7–7·2 551–1521 1290–3723 292–2681 235–689 0·201–0·737 0·910–1·422 1·486–1·677

5·60&0·05 4·95&0·04 5·61&0·17 1137&22 2778&57 1642&50 454&10 0·577&0·010 1·214&0·010 1·617&0·004

0·387–2·629 0·104–0·735 0·492–3·347 0·379–3·122 2·84–4·61 10·5–34·4 8·1–22·6

1·450&0·048 0·416&0·014 1·866&0·062 1·629&0·069 3·515&0·037 16·54&0·48 12·07&0·28

pH (rinsing water) pH (lichen surface) pH (bark) FO FM FV TM FV/FM Chlorophyll degradation ratio 435/415 Chlorophyll degradation ratio 666/666a Chlorophyll a (mg dm "2) b (mg dm "2) a+b (mg dm "2) a+b (mg g "1) a/b ratio Water-holding capacity (mg H2O cm "2) Specific thallus weight (mg dry matter cm "2) *n=90 for all means apart from bark pH where n=20.

chlorophyll degradation in L. pulmonaria. Both sets of test lobes grew on bark with a pH of 6·2. The two phaeophytinization ratios were, therefore, not surprisingly, poorly correlated within the study area (r=0·442, P<0·001, n=90). Many lobes from the study area had a higher 435/415 ratio than the reference material, which appeared more healthy than any of the lobes from the study area. The percentage degradation of chlorophyll could therefore not be computed. A multiple regression analysis was made to see whether other factors could improve the correlation between the two ratios. A better prediction of the 435/415 ratio was obtained in a multiple model adding specific thallus weight (mg dry weight cm "2), and water-holding capacity (mg H2O cm "2 in the water-saturated state) to the 666/666a ratio as explanatory variables (r=0·538, P<0·001, n=90), the least significant variable, water-holding capacity, was then significant at p=0·003. A measurement of the 435/415 ratio for the separate pigments from the reference material (1) showed that chlorophyll b and most carotenoids slightly increased the total 435–415 ratio compared to chlorophyll a only, whereas a brownish non-mobile residual reduced the ratio significantly (Table 2). None of the isolated pigments apart from chlorophylls had any absorbance at 666 nm. Optimal quantum yield of PSII FV/FM increased rapidly after wetting to 0·704&0·011 (mean&standard error of means, n=10), along with a decrease in standard errors of means (Fig. 2) for the fertile and healthy reference material (1) that was kept in the

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T 2. Absorbance at 415 and 435 nm, and 435/415 ratio for each component in an acetone extract of a moist sample of Lobaria pulmonaria separated by thin-layer chromatography Pigments

415 nm

435 nm

435/415 ratio

Chlorophyll a Chlorophyll b Lutein (possibly including zeaxanthin) Violaxanthin â-carotene Neoxanthin Non-mobile residual

1·191 0·096 0·244 0·124 0·077 0·089 0·225

1·625 0·226 0·396 0·174 0·121 0·102 0·164

1·36 2·35 1·62 1·40 1·57 1·15 0·72

Chlorophyll a+b Carotenoids Chlorophylls+carotenoids All components

1·287 0·534 1·821 2·046

1·851 0·793 2·644 2·808

1·44 1·48 1·45 1·37

0.8

FV/FM

0.6

0.4

0.2

0.0 0

5 10 15 20 Time since thallus saturation (h)

25

F. 2. Chlorophyll fluorescence measured as FV/FM in lobes of Lobaria pulmonaria as a function of time after wetting the lobes with distilled water. The first measurement at time=0 h was done on air-dried lobes. Vertical bars indicate 95% confidence limits when larger than symbol.

freezer only. Extending the pretreatment in darkness to 48 h did not increase FV/FM further. Accordingly, 18 h pretreatment after wetting was considered sufficient. Cultivation of the lobes for 35 days with a 12 h photoperiod of 65 ìmol photons m "2 s "1, increased FV/FM to 0·744&0·007, which is a typical value of healthy and freshly collected L. pulmonaria , indicating that a 1 year freezing of air dry samples followed by submersion in water caused no major damage. Additional drying under vacuum reduced FV/FM slightly, but highly significantly (P<0·001), to 0·620&0·011 (n=10). The vacuum-dried weight was 91·4&0·1% (n=10) of the air-dried weight. The level of FM was not significantly influenced by vacuum drying, reaching the same level as air-dried

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thalli, approaching 3100 with both treatments, while FO increased from 940&26 to 1110&32 (n=10). The test lobes had a significantly lower FV/FM (0·577&0·010, n=90) compared to the reference material (0·620&0·011, n=10), with slightly higher FO values, but a significantly lower FV (1642&50, n=90) compared to the reference material (1881&58, n=10). pH and its effect on chlorophyll degradation and FV/FM Many lobes decreased the pH of the rinsing water, whereas others increased the pH. The pH of the rinsing water varied from 4·8 to 6·7 with a mean value of 5·6 (Table 1). The pH of the rinsing water, thallus surface and the substratum were intercorrelated; there was a weak, but highly significant positive correlation between pH of the thallus surface and pH of the rinsing water (r=0·331, P=0·001, n=90, linear regression), as well as a positive correlation between bark pH and thallus surface pH (r=0·513, P=0·021, n=20). The pH of the bark had a similar range to the pH of the water in which lobes were washed, whereas the surface pH of the thalli was much lower (4·0–5·8, mean value 4·95, Table 1). A separate study of surface pH on 12 different loosely attached thalli (reference material 1) showed that the upper surface had a pH of 5·04&0·03, the lower surface 5·27&0·09 with the highest value nearest the centre (pH=5·86) in closest contact with the bark (pH=6·2). None of the two degradation ratios were significantly correlated with any of the pH measurements. However, there was a weak, but positive correlation between total chlorophyll content per unit surface area and bark pH (r=0·513, P=0·021, n=20), as well as with pH measured on the thallus surface (r=0·263, P=0·012, n=90), and an almost significant correlation with pH in the water. FV/FM decreased with decreasing pH measured in the rinsing water (r=0·317, P=0·002, n=90). Lobes that gave a high pH were healthy with respect to the photochemical efficiency of PSII, whereas lobes that gave a lower pH showed a larger variation in FV/FM (Fig. 3). A high FV/FM was associated with a low degree of chlorophyll degradation, measured as the 666/666a ratio (r=0·428, P<0·001, Fig. 4), showing that degradation of chlorophyll was often associated with a lower optimal quantum yield of PSII. Many lobes were apparently healthy, but many also had low vitality with dead portions. A considerably better regression model for predicting FV/FM included the following explanatory variables: the 666/666a-ratio, the chlorophyll a/b ratio and the pH of the rinsing water (r=0·614, P<0·001, n=90). All correlated positively with FV/FM, but not with each other. Discussion The reliability of chlorophyll degradation ratios The 435/415 ratio has previously been used as a measure of damage for transplanted lichens subjected to SO2 (Ronen & Galun 1984; Manrique et al. 1989; Balaguer & Manrique 1991). The ratio was also shown to be lowered

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0.8

0.7

0.6

FV/FM

0.5

0.4

0.3

0.2

0.1 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 pH of rinsing water F. 3. The relationship between chlorophyll fluorescence measured as FV/FM and pH in distilled and deionized water in which lobes of Lobaria pulmonaria were rinsed for 1 h (r=0·317, P=0·002, n=90).

when thalli of Ramalina duriaei were submerged in extremely acidic water (pH=2 or below) for 30 min (Garty et al. 1992). The ratio is, however, measured at wavelengths where chlorophylls and carotenoids absorb much light, and also where a variation in chlorophyll a/b ratio (Barnes et al. 1992) can influence the 435/415 ratio. Chlorophyll a is more susceptible to phaeophytinization than chlorophyll b (Brown & Hooker 1977) but none of the two degradation ratios showed any correlation with the chlorophyll a/b ratio in the study area. Chlorophyll a/b ratio (Dale & Causton 1992) and carotenoids (Demmig-Adams & Adams 1992) often change with light intensity and could, therefore, explain at least some of the measured variation in the 435–415 ratio. The chlorophyll a/b ratio is not especially high, indicating that the chlorophyll pool is mainly of algal origin; the internal cephalodia occupy a minor part of the photobiont layer. Lobaria pulmonaria contains, in addition, a habitat-related factor, most probably the brownish pigment that is included in the non-mobile residual in Table 2. This residual influences the 435/415 ratio so much that the ratio cannot be used as a measure of chlorophyll degradation. A high concentration of brownish cortical pigments in L. pulmonaria is frequently observed in relatively open habitats with ample light. High light intensity also causes high specific thallus weight and water-holding capacity in lichens (Riehmer 1932; James & Henssen 1976; Snelgar & Green 1981), two parameters that significantly improved the prediction of the 435/415 ratio, as demonstrated by a multiple regression analysis. Habitat variation in long-term light intensity is

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0.7

FV/FM

0.6

0.5

0.4

0.3

0.2 1.5

1.6 666/666a

1.7

F. 4. The relationship between chlorophyll fluorescence measured as FV/FM and chlorophyll degradation ratio 666/666a in lobes of Lobaria pulmonaria (r=0·428, P<0·001, n=90).

the most likely underlying factor. The more laborious ratio 666/666a (Ronen & Galun 1984) is, therefore, the only adequate measure of chlorophyll degradation for species with high amounts of pigments absorbing at 415 and 435 nm. FV/FM as a tool in ecological studies FV/FM of hydrated thalli is generally reversibly decreased during temporarily elevated light intensities (e.g. Demmig-Adams et al. 1990) and reduced values are normally considered to be an effect of photoinhibition (e.g. Krause 1988; Krause & Weis 1991). It is therefore important to let samples collected in the field recover in low light or in darkness before measurements when FV/FM is used to assess effects of other environmental stresses rather than high light intensities. Unpublished data on healthy L. pulmonaria showed that typical values of FV/FM were around 0·74, which corresponds well to levels given by Demmig-Adams et al. (1990) as well as to levels obtained on the reference material after 1 month cultivation. Considerably lower levels of FV/FM around 0·6 have been reported for healthy specimens of a variety of lichen species in the family Parmeliaceae (Manrique et al. 1993). For future studies it is probably more important to eliminate damage by vacuum drying than maintaining a strict control of water content. Vacuum drying affected FO, which might indicate damage to the photochemical apparatus, whereas reversible decreases in FM could indicate increased harmless energy dissipation (Krause 1988). However, since all samples were treated the same way, the significantly lower values of FV/FM in test samples

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compared to the reference material is probably an indicator of lower vitality within the study area, as observed in a field study (Gauslaa 1995). FV/FM could probably be a more sensitive measure of early stress, due to unfavourable environmental conditions, than chlorophyll degradation. Chlorophyll degradation (Galun & Ronen 1988) and a change in pigment status (Richardson & Nieboer 1983) apparently represent one of the later stages in photosynthetic damage. However, the two variables were correlated in the study area, and it is possible that some degradation of chlorophyll can take place without a concomitant reduction in FV/FM, as the pigment status and optimal quantum yield of PSII represent different aspects of the photosynthetic apparatus. Effects of pH on the photosynthetic apparatus The pH of the rinsing water is probably ecologically more representative of the ionic environment in the habitat than the surface pH of lichen thalli, since acidic groups of organic molecules in the thalli are likely to produce a lower surface pH. It is, therefore, reasonable that the best correlation was found between FV/FM and pH of the rinsing water. A good correlation should also be expected between bark pH and FV/FM, but the number of bark pH measurements were too low to give a significant correlation. The elemental composition and pH of the tree bark under L. pulmonaria inhabiting localized nutrient streaks, seemed to reflect mainly the mineral content of soil around the roots of host trees, while bark outside nutrient streaks was apparently influenced by airborne depositions (Gauslaa 1995). Since L. pulmonaria inhabits the interface between the bark surface and the atmosphere, the pH of the rinsing water is likely to be a product of both media, the pubescent lower surface presumably being more closely coupled to the bark chemistry than the upper surface. The present study suggests a low pH effect in L. pulmonaria, measured as a reduction in FV/FM (Fig. 3). Other environmental factors could easily have masked the effects of pH upon FV/FM. One factor is high light intensity causing photoinhibition (Demmig-Adams et al. 1990). There might have been residual effects of photoinhibition in some lobes even after an 18-h recovery period, indirectly indicated by a significantly improved regression model after adding the chlorophyll a/b ratio to pH as an explanatory variable. On a more permanent basis light conditions in the habitat also influence the chlorophyll a/b ratio (Dale & Causton 1992). Another factor that possibly masks the effect of pH on FV/FM is chlorophyll degradation measured as the 666/666a ratio. The 666/666a ratio also added significantly to the two previously mentioned variables to produce an improved prediction of FV/FM in a multiple model. The level of FV/FM in the studied lobes appeared, therefore, to be a product of multiple site factors of which pH is one, so FV/FM cannot easily be used for a routine assessment method of pH damage. The lack of correlation between pH and chlorophyll degradation could possibly be attributed to a 30% reduction in mean concentration of sulphate in air-borne depositions since 1979 (Statens Forurensningstilsyn 1992). However, chlorophyll degradation actually takes place, as shown by patches of decolouration in lobes under field conditions (Gauslaa 1995).

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