Ecotoxicology and Environmental Safety 84 (2012) 341–346

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Ecotoxicology and Environmental Safety

0147-65

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Effect of copper stress on cup lichens Cladonia humilis and C. subconisteagrowing on copper-hyperaccumulating moss Scopelophila cataractaeat copper-polluted sites in Japan

Hiromitsu Nakajima a,n, Kenjiro Fujimoto b, Azusa Yoshitani c, Yoshikazu Yamamoto c,Haruka Sakurai b, Kiminori Itoh a

a Division of Natural Environment and Information Research, Graduate School of Environment and Information Sciences, Yokohama National University, Tokiwadai 79-7,

Hodogayaku, 240-8501 Yokohama, Japanb Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, 278-8510 Noda, Japanc Graduate School of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-nakano, 010-0195 Akita, Japan

a r t i c l e i n f o

Article history:

Received 23 April 2012

Received in revised form

13 July 2012

Accepted 2 August 2012Available online 17 August 2012

Keywords:

Copper pollution

X-ray fluorescence analysis

Visible absorption spectra

Chlorophyll

Allelopathy

Secondary metabolites

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.08.009

esponding author. Fax: þ81 45 339 4354.

ail address: h-nakaji@ynu.ac.jp (H. Nakajima)

a b s t r a c t

We investigated lichen species in the habitats of the copper (Cu)-hyperaccumulating moss Scopelophila

cataractae and found that the cup lichens Cladonia subconistea and C. humilis grow on this moss.

We performed X-ray fluorescence and inductively coupled plasma mass (ICP-MS) analysis of lichen

samples and measured the visible absorption spectra of the pigments extracted from the samples to

assess the effect of Cu stress on the cup lichens. The chlorophyll a/b ratio and degradation of chlorophyll

a to pheophytin a were calculated from the spectral data. X-ray fluorescence analysis indicated that Cu

concentrations in cup lichens growing on S. cataractae were much higher than those in control samples

growing on non-polluted soil. Moreover, Cu microanalysis showed that Cu concentrations in parts of

podetia of C. subconistea growing on S. cataractae increased as the substrate (S. cataractae) was

approached, whereas those of C. humilis growing on S. cataractae decreased as the substrate was

approached. This reflects the difference in the route of Cu ions from the source to the podetia.

Furthermore, ICP-MS analysis confirmed that C. subconistea growing on S. cataractae was heavily

contaminated with Cu, indicating that this lichen is Cu tolerant. We found a significant difference

between the visible absorption spectra of pigments extracted from the Cu-contaminated and control

samples. Hence, the spectra could be used to determine whether a cup lichen is contaminated with Cu.

Chlorophyll analysis showed that cup lichens growing on S. cataractae were affected by Cu stress.

However, it also suggested that the areas of dead moss under cup lichens were a suitable substrate for

the growth of the lichen. Moreover, it suggested that cup lichens had allolepathic effects on

S. cataractae; it is likely that secondary metabolites produced by cup lichens inhibited moss growth.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

Lichens and mosses are ecologically similar (During andVan Tooren, 1990); they frequently occur together and competefor light and substrates (Lawrey, 1977). However, little is knownabout the coexistence and ecology of lichens and mosses in themetal-polluted environments.

Many lichen species occur in metal-polluted environments(Purvis and James, 1985; Nash, 1990; Purvis and Halls, 1996). Forexample, 97 species of lichen were found in copper (Cu)-pollutedsites around an abandoned Cu mine in England (Purvis and James,1985). The Cladonia genus is one of the most common metal-

ll rights reserved.

.

tolerant lichens (Nash, 1990), and 12 Cladonia species are tolerantto Cu and zinc (Tyler, 1989). However, information regarding thelichen flora in Cu-polluted areas of Japan is still lacking, and nolichens of the Cladonia genus have been reported to occur insuch areas.

Scopelophila cataractae is a Cu-hyperaccumulating moss.The dry weight (DW) concentration of Cu in this moss is twopercent (Satake et al., 1988; Kobayashi et al., 2006; Nakajimaet al., 2010, 2011), which is 3�103 times higher than that innormal plants (Taiz and Zeiger, 2006). This moss has a very broad,but discontinuous geographic distribution; it occurs in North,Central, and South America, as well as in Europe and Asia but islargely restricted to metal-rich environments (Shaw, 1990, 1995).In Japan, S. cataractae grows on rock faces or on soil moistenedwith rainwater running off structures, such as Cu roofs or bronzestatues (Noguchi, 1988). Many temples and shrines in Japan have

H. Nakajima et al. / Ecotoxicology and Environmental Safety 84 (2012) 341–346342

Cu roofs, and S. cataractae has been collected mostly from suchsites for its study (Satake et al., 1988; Shaw, 1995; Nakajima et al.,2010, 2011). However, no lichen species have been found tocompete with this moss.

Therefore, investigating lichen species in or around S. cataractae

habitats in Japan should provide valuable information on the ecologyand physiology of lichens in Cu-polluted sites. The purpose of thisstudy was to investigate lichen species in S. cataractae Cu-pollutedsites in Japan, to identify lichen species that compete with this moss,and to assess the effect of Cu on them.

Metal-induced stress in lichens can be assessed by analyzingtheir pigments (Garty, 2001; Boonpragob, 2002; Backor andFahselt, 2004; Backor et al., 2009). Degradation of chlorophyll a

to pheophytin a, which can be evaluated by the ratio of absor-bances at 435 and 415 nm, can be used to assess stress in lichens(Garty, 2001). Cu stress decreases the chlorophyll a/b ratio(Chettri et al., 1998), which can be determined by measuringthe absorption spectra of pigments extracted from lichens (Ronenand Galun, 1984; Wellburn, 1994).

We investigated Cu-polluted sites and found that cup lichensCladonia humilis and C. subconistea grow on S. cataractae. Contraryto our findings, the cup lichen, C. humilis, generally grows on soil(Yoshimura, 1974; Nash et al., 2002; Kowalewska and Kukwa,2003; Dobson, 2005; Yazici et al., 2007; Wang et al., 2011). Itscups, or the funnel-shaped podetia (a podetium is a verticalthallus arising from a primary thallus and is typical of theCladonia genus (Webster and Weber, 2007)), are up to 1 cm tall,and the stalks have a 1 mm diameter at the base and expandabruptly near the rim. It contains fumarprotocetraic acid andatranorin; the thallus turns yellow when KOH solution is applied(Yoshimura, 1974). This lichen has a very broad distribution,occurring in Africa, Asia, Australasia, Europe, and North and SouthAmerica (Nash et al., 2002). Another cup lichen, C. subconistea,also generally grows on soil (Yoshimura, 1974; Nayaka et al.,2009) and is similar to C. humilis in morphology and in colorationin the KOH test, but it contains psoromic acid and atranorin(Yoshimura, 1974). This lichen occurs only in Asia (Yoshimura,1974; Abbas et al., 2001; Nayaka et al., 2009; Baniya et al., 2010;Wang et al., 2011).

Samples of collected cup lichens were analyzed by X-rayfluorescence and inductively coupled plasma mass (ICP-MS)spectroscopy. We found that Cu concentrations in samples ofcup lichens growing on S. cataracae were much higher than thosein control samples growing on non-polluted soil, and confirmedthat C. subconistea growing on S. cataractae was heavily contami-nated with Cu. The absorption spectra of pigments extracted fromsamples were measured, and the chlorophyll concentration anddegradation of chlorophyll a to pheophytin a were calculatedfrom the spectral data. The effects of Cu on chlorophyll in thelichen samples were assessed by visible absorption spectroscopy.

2. Materials and methods

2.1. Sampling and sample identification

Lichen samples growing on S. cataractae were collected in August–September

2010 at Iwakuni (20100818002), Nagoya (20100817001), and Tokyo

(20100929001) in Japan. Control samples growing on non-polluted soil were

collected in August 2010 at Iwakuni (20100818001) and Kamakura

(20100815001). The Iwakuni site, from which the control samples were collected,

was hundreds of meters away from where the specimens were growing on S.

cataractae. Another control sample growing on non-polluted soil was collected in

September 2011 at another Tokyo site (2011092601). This sample was used only

for identification and Cu quantitative analysis. All samples were dried in paper

envelopes for 2 weeks under normal laboratory conditions. The samples were

identified by their morphology and secondary metabolites. The number of podetia

on lichens growing on S. catractae at the Tokyo site was fewer than that growing

on S. catractae at the other sites; hence, the sample growing on S. catractae at the

Tokyo site could not be further analyzed. Moreover, quantitative Cu analysis was

performed only for the C. subconistea sample from Iwakuni and the control C.

humilis sample from Tokyo because of sample shortage.

2.2. Identification of secondary metabolites

Secondary metabolites were extracted overnight with acetone (1 ml) from

dried samples (ca. 10 mg). The acetone solution was analyzed by high-

performance liquid chromatography (HPLC) with a photodiode array detector.

HPLC conditions were as follows: column YMC-Pack ODS-A (150 mm�4.6 mm id,

5 mm, 12 nm) at 40 1C; solvent MeOH:H2O:H3PO4¼80:20:1 at 1 ml/min; detection

wavelength, 254 nm; wavelength range of UV spectrum, 200–400 nm. Peaks were

detected at retention times corresponding to psoromic acid and atranorin for the

sample growing on S. cataractae at Iwakuni and at retention times corresponding

to fumarprotocetraic acid and atranorin for the other samples.

2.3. X-ray fluorescence analysis of Cu

Energy-dispersive X-ray spectra were measured using an analyzer with an

X-ray source (Rh tube) operated at 50 kV, 0.50–1.00 mA, and a silicon semicon-

ductor detector to qualitatively analyze Cu in the dried samples. The intensity of

Cu Ka irradiation at 8 keV from the sample was recorded. The excitation beam was

3 mm in diameter; thus, a microarea of the same size was analyzed. We analyzed

the upper, middle, and lower areas of the podetia by energy-dispersive X-ray

spectroscopy. Similarly, the intensities of Cu irradiation from the primary thallus

(on the substrates) and substrates (1 cm below the primary thallus) of the lichen

samples were also measured.

2.4. ICP-MS analysis of Cu

Cu concentrations in podetia of C. subconistea growing on S. cataractae at

Iwakuni and C. humilis growing on soil at the control site in Tokyo were

determined by ICP-MS spectroscopy. Podetia were dried at 90 1C for 24 h.

Approximately 10 and 40 mg of the dried podetia from the Cu-polluted and

control samples, respectively, were digested in 65 percent HNO3 and 30 percent

H2O2 (2:1, v/v) for 24 h, and the volumes were made up to 20 ml with deionized

water. Cu concentration was measured three times using an ICP-MS spectrometer

(Agilent 7700�).

2.5. Visible absorption spectra of pigments and their analysis

Pigments were extracted from dried samples (5 mg) in dimethyl sulfoxide

(DMSO) (2 ml) for 60 min at 65 1C in the dark. After cooling to room temperature,

the absorption spectra of the pigments in DMSO were measured using a spectro-

photometer. Chlorophyll concentrations were calculated from the spectral data, or

the absorbances at 649 and 665 nm, using Wellburn’s equations (Wellburn, 1994).

The degradation of chlorophyll a to pheophytin a was evaluated by determining

the ratio of absorbances at 435 and 415 nm (A435/A415) (Garty, 2001).

3. Results

3.1. Lichen species in the S. cataractae habitats

We investigated lichen species at the S. cataractae Cu-pollutedsites in Japan near old mines, around Cu-roofed temples andshrines, and under bronze statues. We found cup lichensC. subconistea and C. humilis growing on S. cataractae atCu-polluted sites in Iwakuni (Yamaguchi Prefecture), Nagoya(Aichi Prefecture), and Tokyo. The distances from Iwakuni–Nagoya and Nagoya–Tokyo were 4.6�102 and 2.7�102 km,respectively. Cup lichens in the three habitats formed largecolonies (several centimeters or more in diameter) on theS. cataractae moss cushions. We noted that cup lichens did notoccur directly on soil or rock at any of the three sites, althoughthey invaded the surface of the S. cataractae cushions.

The Iwakuni site is located near the old Kiwada mine in aforested area. This mine was first discovered in the 17th century,and Cu was mined until the early 20th century. Cu-polluted areasare scattered around the old mine. S. cataractae grew on rockwalls along a road at the Iwakuni site, with the moss cushions

Fig. 3. C. humilis growing on S. cataractae around a bronze lantern at a shrine

in Tokyo.

H. Nakajima et al. / Ecotoxicology and Environmental Safety 84 (2012) 341–346 343

covering parts of the walls. C. subconistea grew on the mosscushions, as shown in Fig. 1.

The Nagoya site is located at a temple in an urban area.The Cu-roofed temple was built in 1982, after which the buildingwas polluted with Cu. At the Nagoya site, S. cataractae grew onconcrete and soil around a concrete container into which rain-water flowed through the Cu roof of the adjacent building. Thecushions of this moss covered areas of concrete and soil. C. humilis

grew on these moss cushions, as shown in Fig. 2.The Tokyo site is located at a shrine in an urban area. Bronze

lanterns were donated to the shrine in the 17th century, and Cupollution was found around the bronze lanterns. S. cataractae

grew on soil around the bronze lanterns of the shrine at the Tokyosite, and the cushions of this moss covered the soil. C. humilis

grew on these moss cushions, as shown in Fig. 3.

3.2. Cu analysis

X-ray fluorescence analysis indicated that Cu concentrations ofthe cup lichen samples growing on S. cataractae were muchhigher than those of the control samples growing on soil. Fig. 4aand b shows the intensity of Cu irradiation from the upper,

Fig. 1. C. subconistea growing on S. cataractae near an old mine at Iwakuni,

Yamaguchi Prefecture.

Fig. 2. C. humilis growing on S. cataractae around a Cu roofed temple building in

Nagoya, Aichi Prefecture.

Fig. 4. Intensities of Cu Ka irradiation from the parts (upper, middle, and lower) of

the podetia (a, b) and from the primary thallus and substrates (c, d) of cup lichen

samples: (1) C. subconistea growing on S. cataractae in Iwakuni; (2) C. humilis

growing on S. cataractae in Nagoya; (3) C. humilis growing on soil in Iwakuni; and

(4) C. humilis growing on soil in Kamakura. Error bars represent standard

deviations (n¼3). (a) Cu-Polluted, (b) Control, (c) Cu-Polluted, and (d) Control.

middle, and lower parts of the podetia of these samples. The Cuirradiation intensity from the lower part of C. subconistea growingon S. cataractae at Iwakuni (36.7722.6 cps (average7standarddeviation, n¼3)) was 40 times higher than that of C. humilis

control samples growing on soil at Iwakuni and Kamakura(0.970.6 cps). Moreover, the Cu irradiation intensity from theC. humilis sample on S. cataractae at Nagoya (3.471.4 cps) was3.7 times higher than that of the control samples.

Similar analysis was performed for the primary thallus andsubstrates of cup lichens (Fig. 4c and d), and the results indicatedthat Cu concentrations of the primary thallus and substrates ofcup lichens at the Cu-polluted sites were much higher than those

H. Nakajima et al. / Ecotoxicology and Environmental Safety 84 (2012) 341–346344

of cup lichens at the control sites. Cu irradiation intensities fromthe primary thallus and substrates of C. subconistea growing onS. cataractae at Iwakuni (162.27119.4 and 216.1793.6 cps) were1.6�102 and 2.0�102 times higher than those of the C. humilis

control samples growing on soil at Iwakuni and Kamakura(1.170.3 and 1.170.5 cps). Moreover, the Cu irradiation inten-sities from C. humilis primary thallus and substrates growing onS. cataractae at Nagoya (61.5725.4 and 44.3713.7 cps) were 62and 40 times higher than those of the control samples.

Cu concentrations in podetia of C. subconistea growing on S.

cataractae at Iwakuni and C. humilis growing on soil at the controlsite in Tokyo were 1.0170.01 mg/g DW and 4.170.6 mg/g DW.The former concentration was 2.5�102 times higher than that ofthe latter concentration and confirmed that C. subconistea grow-ing on S. cataractae at Iwakuni was heavily contaminated with Cu.

3.3. Visible absorption spectra and chlorophyll analysis

Fig. 5 shows the visible absorption spectra of the pigmentsextracted from the samples. The concentrations of chlorophyll a

and b, the chlorophyll a/b ratio, and the A435/A415 ratio wereobtained using the spectral data, and the results are summarizedin Table 1. The concentrations of chlorophyll a in C. humilis

growing on moss (S. cataractae) and soil were about two timeshigher than those in C. subconistea growing on moss and C. humilis

growing on soil. Hence, no dependence of chlorophyll concentra-tion on the substrate was observed. However, the chlorophyll a/b

ratios of cup lichens C. subconistea and C. humilis growing on mosswere less than those of C. humilis growing on soil. Moreover, theA435/A415 ratios of cup lichens growing on moss were less thanthose of C. humilis growing on soil.

4000

1

2

3

4

Rel

ativ

e ab

sorb

ance

Wavelength [nm]

C. humilis on moss in NagoyaC. subconistea on moss in IwakuniC. humilis on soil in KamakuraC. humilis on soil in Iwakuni

450 500 550 600 650 700 750

Fig. 5. Representative absorption spectra of pigments extracted from cup lichen

samples. Absorbances at 665 nm were taken to be unity.

Table 1Concentration of chlorophyll (chl.) a in dry weight (DW), chl. a/b ratio, and the

ratio of absorbances at 435 and 415 nm (A435/A415). The moss was S. cataractae.

Values are average7standard deviation (n¼3).

Species Site Substrate Chl. a

(mg/g DW)

Chl. a/b

ratio

A435/A415

C. subconistea Iwakuni Moss 0.9870.18 2.6370.12 0.8670.04

C. humilis Nagoya Moss 1.8470.11 2.4570.07 0.7470.01

C. humilis Iwakuni Soil 1.7970.17 2.8870.02 1.0970.03

C. humilis Kamakura Soil 0.9470.05 2.9570.04 0.9570.02

4. Discussion

The wide distribution of the three habitats of cup lichensC. subconistea and C. humilis suggests that it is common for cuplichens to grow on S. cataractae at Cu-polluted sites in Japan.The findings from the three habitats showed that cup lichens arecompetitors with S. cataractae at the Cu-polluted sites, and this isthe first observation that S. cataractae can be invaded by otherspecies at such sites. Moreover, these findings indicate thatthe surface of the moss cushions is a good substrate for cuplichens at Cu-polluted sites. This is compatible with observations,which state that a moss substrate is most suitable for artificiallydispersed lichens to fasten on to and for the natural settlement oflichens (Roturier et al., 2007).

X-ray fluorescence analysis suggested that cup lichensC. subconistea and C. humilis are Cu tolerant. Moreover, ICP-MSanalysis confirmed that C. subconistea growing on S. cataractae atIwakuni was heavily contaminated with Cu, thereby indicatingthat this lichen is Cu tolerant.

A significant difference was observed in the Cu irradiationintensities from the podetia between C. subconistea growing on S.

cataractae at Iwakuni and C. humilis growing on the moss atNagoya; the former intensity (16.9719.3) was 5.0 times higherthan that of the latter (3.471.4). This corresponded to the resultthat the Cu irradiation intensity from C. subconistea substrates atIwakuni was 4.9 times higher than that of C. humilis at Nagoya.Hence, the difference in Cu irradiation intensities from thepodetia between C. subconistea at Iwakuni and C. humilis atNagoya reflects the difference in the Cu irradiation intensitiesfrom substrates between the two lichen samples.

We found that the distribution of Cu in the podetia reflectedthe route of Cu ions from the source to the podetia. Cu micro-analysis of the podetia of C. subconistea growing on S. cataractae

(Fig. 4a) showed that Cu concentrations in the podetia increasedas the substrate (S. cataractae) is approached. This could bebecause the lower part of the podetia allows the upstream flowof Cu ions in the podetia. At the Iwakuni site, rainwater contain-ing Cu ions flowed down the wall, of which the cushions ofS. cataractae-covered parts, and then the rainwater passedthrough the substrate to the podetia of C. subconistea. In contrast,Cu microanalysis results in podetia of C. humilis growing onS. cataractae (Fig. 4a) indicated that Cu concentrations in thepodetia decreased as the substrate was approached. This can beexplained by considering the C. humilis habitat at the Nagoya siteas follows. The habitat was situated around a building with a Curoof; rainwater containing Cu ions flowed off the roof and fellonto the habitat. Hence, the upper part of the podetia wasupstream of the Cu ion flow in the podetia, unlike that in thepodetia of C. subconistea at the Iwakuni site, which resulted in theopposite distribution of Cu in the podetia.

By comparing the absorption spectra of pigments extractedfrom the lichen samples (Fig. 5), we observed a significantdifference between the spectra of Cu-contaminated samples andthose of control samples, i.e., the slopes of the C. subconistea andC. humilis spectra growing on S. cataractae were negative at theshort wavelength edge, whereas those of C. humilis spectragrowing on soil were positive. Thus, it was possible to use thesign of the slope to determine whether cup lichen was contami-nated with Cu. Furthermore, the slope of the fitting line of thespectra between 400 and 405 nm for C. subconistea growing onS. cataractae (�0.0470.01 (average7standard deviation, n¼3))was four times less than that for C. humilis growing on S.

cataractae (�0.0170.00), suggesting a negative correlationbetween the slope and Cu concentration.

The chlorophyll a/b ratios of C. humilis growing on soil(Table 1) was in accordance with those of the control samples

H. Nakajima et al. / Ecotoxicology and Environmental Safety 84 (2012) 341–346 345

from five other Cladonia species (3.070.2) (Chettri et al., 1998;Backor and Fahselt, 2004; Ochoa-Hueso and Manrique, 2011;Pisani et al., 2011). Therefore, the ratios of C. humilis growing onsoil can be considered as the ratios of the control sample. Thus,we found that the chlorophyll a/b ratios of cup lichens growing onS. cataractae were less than those of the control samples, suggest-ing that the decrease in the chlorophyll a/b ratio of cup lichenswas caused by Cu. This is supported by previous observations thatCu stress decreases the chlorophyll a/b ratios of C. convoluta andC. rangiformis (Chettri et al., 1998). A decrease in chlorophyll a

concentration, an increase in chlorophyll b concentration, or adecrease in the former and an increase in the latter results in adecrease in the chlorophyll a/b ratio. Because concentrations ofchlorophyll aþb were significantly different between samples, itcould not be determined whether a decrease in chlorophyll a

concentration or an increase in chlorophyll b concentrationresulted in a decrease in the chlorophyll a/b ratio. Nevertheless,analysis of the A435/A415 ratio indicated that the chlorophyll a

concentration decreased.The A435/A415 ratios of C. humilis growing on soil (Table 1) was

in accordance with those previously reported for C. pleurota

control samples (1.0670.11) (Backor and Fahselt, 2004). Thus,we found that the A435/A415 ratios of cup lichens growing onS. cataractae were lower than those of the control samples,indicating that the degradation of chlorophyll a to pheophytin a

occurring in cup lichens growing on S. cataractae was more thanthat in observed in the control samples. Accordingly the concen-trations of chlorophyll a decreased, resulting in a decrease in thechlorophyll a/b ratios of the samples growing on S. cataractae.Moreover, our results suggest that the decrease in the A435/A415

ratios in cup lichens was induced by Cu stress. This is compatiblewith previous findings that the A435/A415 ratio of C. pleurota at ametal- (including Cu) polluted site was less than that at a controlsite (Backor and Fahselt, 2004).

The chlorophyll a/b and A435/A415 ratios of C. humilis growingon S. cataractae at Nagoya were smaller than those of C. sub-

conistea growing on moss at Iwakuni (Table 1), suggesting thatthe two C. humilis ratios at Nagoya were affected by Cu stressmore than those of C. subconistea at Iwakuni, although Cuconcentration in podetia of C. humilis at Nagoya was much lowerthan that of C. subconistea at Iwakuni (Fig. 4a). This can beexplained by the morphological difference between the twospecies as follows. C. humilis has soredia, which are non-corticate combinations of photobiont cells and fungal hyphae,whereas C. subconistea does not have such structures (Yoshimura,1974). Photobiont cells of the soredia lie outside the cortex ofpodetia are directly affected by Cu stress; therefore, chlorophyllsin soredia photobiont cells on the podetia are much moresensitive to Cu stress than those in photobiont cells inside thecortex of podetia. This is why the two C. humilis ratios weresmaller than those of C. subconistea, although Cu concentration inthe podetia of C. humilis was much lower than that of C.

subconistea.Our findings regarding the chlorophyll a/b and A435/A415 ratios

demonstrate that when cup lichens C. humilis and C. subconistea

grow on S. cataractae they are affected by stress from thesubstrate itself, Cu, or both. However, we found that cup lichenscan grow on Cu-hyperaccumulating moss despite this stress.This raises another question: what are the advantages for cuplichens growing on S. cataractae under such stressful conditions?If the lichen covers areas on the surface of the moss cushions,these covered areas will die. However, the dead areas of S.

cataractae will not rot because this moss contains high concen-trations of Cu. Hence, the dead areas of moss are a suitablesubstrate for lichen growth. This can be a survival advantage forcup lichens growing on Cu-hyperaccumulating moss.

If the growth rate of cup lichens is lower than that ofS. cataractae, cup lichens cannot cover the moss cushion areas ofS. cataractae. The existence of cup lichen colonies on mosscushions indicates that the growth rate of cup lichens was higherthan that of S. cataractae or that cup lichens have allolepathiceffects on S. cataractae. Secondary metabolites from lichens canfunction as allelopathic agents (Lawrey, 1986; Molnar and Farkas,2010); it is likely that such metabolites produced by cup lichenscan inhibit the growth of S. cataractae. This is supported byprevious observations that psoromic and fumarprotocetraricacids, which are metabolites produced by C. subconistea and C.

humilis, inhibit the germination of spores from the moss Funaria

hygrometrica (Gardner and Mueller, 1981; Lawrey, 1986).

5. Conclusions

We investigated lichen species at Cu-hyperaccumulating mossS. cataractae Cu-polluted sites in Japan and found cup lichensC. subconistea and C. humilis growing on this moss. X-ray fluores-cence analysis showed that Cu concentrations in cup lichensgrowing on S. cataractae were much higher than those growingon soil in control samples, and that the Cu distribution in thelichen samples reflected the route of Cu ions from the sourceto the sample. Moreover, ICP-MS analysis confirmed thatC. subconistea growing on S. cataractae at Iwakuni was heavilycontaminated with Cu, thereby indicating that this lichen isCu tolerant. We found a significant difference between thevisible absorption spectra of pigments extracted from theCu-contaminated and control samples. Hence, the spectra couldbe used to determine whether a cup lichen is contaminated withCu. Chlorophyll analysis showed that cup lichens growing onS. cataractae were affected by Cu stress. However, it suggestedthat areas of dead moss under cup lichens were suitable sub-strates for lichen growth. Furthermore, it suggests that they haveallolepathic effects on S. cataractae; it is likely that the secondarymetabolites produced by cup lichens inhibit moss growth.

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