Hypogymnia Physodes and Its Genetic Diversity

Ann. Bot. Fennici 48: 473–482 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 30 December 2011 © Finnish Zoological and Botanical Publishing Board 2011

Depsides and depsidones in populations of the lichen Hypogymnia physodes and its genetic diversity

Katalin Molnár* & Edit Farkas

Institute of Ecology and Botany, Hungarian Academy of Sciences, H-2163 Vácrátót, Hungary (*corresponding author’s e-mail: [email protected])

Received 18 Oct. 2010, revised version received 20 Dec. 2010, accepted 22 Dec. 2010

Molnár, K. & Farkas, E. 2011: Depsides and depsidones in populations of the lichen Hypogymnia physodes and its genetic diversity. — Ann. Bot. Fennici 48: 473–482.

The aim of this study was to determine the extent and geographical pattern of intraspe- cific chemical and genetic variability of the lichen Hypogymnia physodes by compar- ing populations from different habitats. We analyzed the secondary lichen substances and their relative concentrations using HPTLC and HPLC in samples collected from sites with different environmental conditions. We identified seven lichen substances: the cortical atranorin and chloroatranorin, and the medullary physodalic, physodic, protocetraric, 3-hydroxyphysodic, and 2´-O-methylphysodic acids. The samples were uniform qualitatively, which means that H. physodes has only one chemotype. We detected quantitative chemical differences between the samples without any geographical pattern. We investigated 21 samples in order to study the connection between genotypic diversity of populations and geographical distribution. We determined the sequences of five loci (ITS, nucSSU, nucLSU, mitSSU, EF1α). We found no significant genetic differentiation among populations collected from different areas.

Introduction 1989, Pfeiffer & Barclay-Estrup 1992, Bruteig 1993, Werner 1993, Bennett et al. 1996, Poličnik Hypogymnia physodes is a common foliose et al. 2004, Rusu et al. 2006, Hauck 2008). lichen in boreal and temperate forests through- Hypogymnia physodes produces several out the northern hemisphere (Smith et al. 2009). secondary lichen substances, which are located This species occurs primarily as an epiphyte and either in the cortex or in the medulla, and are preferentially grows on acidic bark and wood, biologically active in varying ecological roles. but occasionally occurs on mosses or on soil as Cortical depsides play an important role in solar well. In Hungary, it is most frequently found on radiation screening, while medullary depsidones bark of various coniferous and deciduous trees, function as antiherbivores (Solhaug et al. 2009). sometimes also on siliceous rock (Verseghy The depsidone physodic and protocetraric acids 1994). Due to its abundance and its moderate have relatively strong antimicrobial effects sensitivity to sulphur dioxide and heavy metals, against numerous microorganisms, includ- H. physodes is frequently used as an indicator of ing human, animal and plant pathogens, food- air quality (Farkas et al. 1985, Farkas & Pátkai spoilage organisms and producers of mycotoxins 474 Molnár & Farkas • Ann. BOT. Fennici Vol. 48

(Ranković & Mišić 2008, Ranković et al. 2008). cal sites, there may also be genetic variability, Acetone extracts of H. physodes exhibit a strong as is reported in the literature for some species antifungal effect on some plant pathogenic fungi (DePriest 1993, Beard & DePriest 1996, Zoller (Halama & Van Haluwyn 2004). The secondary et al. 1999, Lindblom & Ekman 2006, Zhou metabolites of H. physodes selectively inhibit the et al. 2006). Therefore, we studied the genetic intracellular uptake of Cu2+ and Mn2+, whereas diversity among populations of H. physodes as the uptake of Fe2+ and Zn2+ is not affected by the well. The nuclear ribosomal gene complex [com- lichen substances (Hauck 2008). These impacts prising nucSSU (18S) rDNS, ITS rDNS (5.8S, are consistent with the ecology of H. physodes, ITS1 and ITS2), nucLSU (28S) rDNS, IGS rDNS i.e., Cu2+ and Mn2+ might be toxic in ambient (5S, IGS1 and IGS2)], as well as the mitSSU concentrations on acidic bark, but Fe2+ and Zn2+ rDNS and EF1α loci are suitable for population do not limit the survival of this species. Physo- studies in lichens (e.g., Beard & DePriest 1996, dalic acid is the most effective at controlling Zoller et al. 1999, Ekman & Wedin 2000). metal homeostasis (Hauck & Huneck 2007). Physodic, physodalic, and protocetraric acids increase the adsorption of Fe3+ (Hauck et al. Material and methods 2007). Atranorin, physodalic, and physodic acids are responsible for allergic reactions to lichens HPTLC analyses (Thune & Solberg 1980, Cabanillas et al. 2006). Secondary metabolites of H. physodes at natu- We screened 297 H. physodes specimens from ral concentrations act as antiherbivore agents Hungary and 44 specimens from other countries (Pöykkö et al. 2005). Physodalic acid exhibits (mostly European) by HPTLC according to Arup mutagenicity against Salmonella typhimurium et al. (1993) in three solvent systems, most fre- (Shibamoto & Wei 1984). quently in “B”. We mostly analyzed dried herbar- Though several details are known about the ium specimens; the oldest one was from 1852. We secondary substances in H. physodes, we found also collected fresh material between 1997 and no data on lichen substances of this species in the 2006. To study within-population chemical diver- literature on Hungarian specimens, and only few sity, we analyzed specimens from the same popu- quantitative results were found (e.g., Czeczuga et lations as well. Additionally, we included infra- al. 2000, Białońska & Dayan 2005). Therefore, specific taxa, varieties and forms of H. physodes. we studied specimens found in the three major We soaked approximately 5 mm ¥ 5 mm thallus Hungarian lichen herbaria: Hungarian Natural fragments in 0.1–0.2 ml acetone for 30 minutes History Museum Herbarium (BP), Eszterházy in order to extract the lichen substances. We used Károly College Herbarium (EGR), Institute of pretreated (50 °C for 5 min, CAMAG TLC Plate Ecology and Botany of the Hungarian Academy Heater III), 10 cm ¥ 10 cm thin layer chroma- of Sciences Herbarium (VBI). Many lichen spe- tographic plates (Merck, Kieselgel 60 F254). We cies have different chemotypes, sometimes with applied 6–8 µl acetone extracts to each position (5 specific geographical distributions (e.g., Hale mm from each other) of the plate using a microap- 1962, Moore 1968, Sheard 1974, Filson 1981, plicator (CAMAG Micro Applicator I). We used Halvorsen & Bendiksen 1982, Culberson et al. Platismatia glauca (atranorin), Cladonia symphy- 1984, Randlane & Saag 1989, Calatayud & Rico carpia (atranorin, norstictic acid), Pleurosticta 1999). In the current study, we examined the acetabulum (atranorin, norstictic acid), and Heter- chemical diversity of populations from differ- odermia leucomelos (atranorin, zeorin) as control ent habitats (particularly from Hungary) of H. specimens. After chromatographic development, physodes, a toxitolerant lichen species, focusing we examined the plates under UV light (254 and on the possible qualitative and quantitative dif- 366 nm), then sprayed (CAMAG TLC Sprayer) ferences in the production of depsides and depsi- with a 10% sulphuric acid solution and heated dones among populations. at 110 °C for 5–10 minutes. Finally, we cooled In addition to chemical diversity in popula- the plates to room temperature and studied them tions of a lichen species from various geographi- under UV light (366 nm). Ann. BOT. Fennici Vol. 48 • Chemical and genetic variability of Hypogymnia physodes 475

HPLC analyses 110 150 375 350 360 350 225 605 500 170 130 850 (m) 1347 Altitude We analyzed 13 samples (Table 1) by HPLC with reversed-phase column and gradient elution to compare their secondary compounds. We E N NE NE NE NE SW NW NW NW followed the standardized method described by NW from ESE NNE

Feige et al. (1993) with some modifications. We Direction settlement* extracted approximately 10 mg air-dried lichen thalli in 1 ml acetone. We added benzoic acid and bis-(2-ethylhexyl)-phthalate to the extrac- tion liquid (acetone) as internal standards (20 mg 2 km 5 km 8 km 8 km 2 km 1 km 4 km 11 km 11 10 km 1.5 km 5.5 km 2.5 km of each per 1000 ml acetone). Huovinen et 0.5 km Distance from center al. (1985) reported that these internal standards of settlement are suitable. We performed the analyses with a Shimadzu High Performance Liquid Chromato- graph using LC-10ADvp and LC-10AS pumps, 2500 8000 8000 2300 2000 2200 1400 43000 62000 and SPD-10Avp UV/VIS Detector. We used Phe- 20000

® Number 2000000 2000000 nomenex Prodigy 5 µ ODS-3V column, onto 2000000 which we injected 20 µl samples. We used two of inhabitants solvent systems: 1% ortho-phosphoric acid in bidistilled water (solvent system A) and 100% methanol (solvent system B). We monitored the elution of the lichen substances at 245 nm. We calculated two kinds of retention indices for pollution source Salgótarján Zalaegerszeg Budapest Budapest Nearest Budapest settlement or Komárom Visonta Felsőtárkány Felsőtárkány Egerszalók C sévharaszt Gârda de Sus the lichen compounds (data not shown): I value Štrba Tatranská (Feige et al. 1993) and RI (Huovinen et al. 1985). Concentrations of substances are given in relative terms (percentage of area: area%).

DNA analyses samples analyzed by HPL C . We collected 21 lichen samples (Table 2) from different geographical sites for DNA analysis.

Salgótarján Zalaegerszeg Budapest, Remete-hegy Budapest, Hosszúerdő-hegy Locality Budapest, Látó-hegy

Almásfüzitő-felső Tarjánka Mátra, Bükk, Kolozs-lápa Egerszalók Avenul Zgurasti Avenul We extracted genomic DNA from dried lichen Štrbské Pleso thalli using a protocol modified from Zolan and Pukkila (1986). We used 2% sodium dodecil sulphate (SDS) as a lysis buffer. We precipitated

the DNA by isopropanol, then washed the pellet Hypogymnia physodes

Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary c sákpilis Hungary Hungary Romania c sévharaszt with ca. 70%–87% ethanol and dried at room Slovakia temperature for 1 hour. We resuspended the iso- lated genomic DNA in 50 µl sterile bidistilled water, and we prepared a 1:10 dilution for PCR amplification. We amplified and sequenced the following loci: internal transcribed spacer (ITS

EGR1439 EGR4383 no. 24/ C [BP] no. 192/a [BP] Herbarium c ountry no. 208/a [BP] specimen BP89961 EGR4215 EGR4613 EGR4598 EGR4239 EGR1444 N/A rDNA), nuclear small subunit ribosomal DNA EGR1445 (nucSSU rDNA), nuclear large subunit ribosomal DNA (nucLSU rDNA), mitochondrial small subunit ribosomal DNA (mitSSU rDNA), elonga-

0 5 0 4 0 3 0 2 Table 1. List and characterization of Table Sample 0 1 The direction of prevailing winds at the sites is NW. *

0 6 0 7 0 8 0 9 10 11 12 tion factor 1 alpha (EF1α), RNA polymerase II 13 476 Molnár & Farkas • Ann. BOT. Fennici Vol. 48 α * * * * * * * * * * * * * * * EF1

* RPB1 (f–g)

* RPB1 (a–f)

* * RPB2 (7–11)

* * * * * * * * * * * RPB2 (5–7) * * * * * * * * * * * * * * * * * * * * * mSSU

* * * nucSSU

* * * * * * * * * * * * * * * * * * ollecting sites set in boldface are characterized in Table 1. Table samples investigated. C ollecting sites set in boldface are characterized nucLSU

* * * * * * * * * * * * * * * * ITS Hypogymnia physodes Bükk, Kolozs-tető Bükk, Balázs-kő Alsó-Kecskor Bükk, Bükk, Kolozs-lápa Bükk, Kövesdi-kilátó Bükk, Kövesdi-tető Bükk, Csákpilis Bükk, Ökrös-fertő Bükk, Borzlyuk-tető Szilvásvárad Csévharaszt Strbské Pleso Mátra, Tarjánka-völgy Tarnalelesz Egerszalók Bükk, Felsőtárkány Bükk, Oldal-völgy Mátra, Ilona-völgy Bükk, Vöröskő-völgy Aggtelek Bükk, Jómarci-fertő

0507/LL 0520/LA 0504/LA 0514/LB 0515/LK 0503/LB 0510/L C 0513/L C 0506/OA 0508/LW 0524/LW Herb. no. EGR1442 EGR1444 EGR1445 EGR4215 EGR4238 EGR4239 EGR4240 EGR4241 EGR4322 23-08-2005/A c oll. or c ollecting site

no.* 1966 AFTOL 15 16 17 18 19 20 21 Type of DNA sequences and locality data of of DNA Type 2. Table Sample 0 1 of Life) project. Tree (Assembling the Fungal AFTOL * Sequences deposited in the database of 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 10 11 14 12 13 Ann. BOT. Fennici Vol. 48 • Chemical and genetic variability of Hypogymnia physodes 477 largest subunit (RPB1), and RNA polymerase II nucleic acid gel stain, then purified by a Mon- second largest subunit (RPB2). We sequenced tage PCR Centrifugal Filter Device (Millipore). RPB1 and RPB2 only for the AFTOL project, We performed sequencing reaction in a 10 µl and not for population studies. The primers used final reaction volume using 3 µl purified PCR in this study are listed in Table 3. We prepared product, 0.75 µl Big Dye (ABI PRISM ver. 3.1), PCR mix for a 25 µl final volume containing 3.25 µl Big Dye buffer, 1 µl primer (10 µM), and 1 µl 1:10 dilution of genomic DNA, 2.5 µl PCR 2 µl bidistilled water. buffer (with 15 mM MgCl2, Denville), 2.5 µl We assembled and edited the sequences using dNTPs (2mM each, Denville), 2.5 µl BSA (10 the software package Sequencher 4.5 (Gene mg ml–1), 1.25 µl primers (10 µM), 0.15 µl Taq Codes Corporation), and aligned them manu- polymerase (5 U µl–1, Choice Taq, Denville), and ally in MacClade 4.06 (Maddison & Maddison 13.85 µl sterile bidistilled water. We prepared a 2003). In order to determine the similarities of slightly different PCR mix for the protein-coding the populations, we ran Maximum Parsimony RPB1, RPB2, and EF1α containing 3 µl 1:10 (MP) quick tests for ITS and EF1α conducted in dilution of genomic DNA, 2.5 µl PCR buffer PAUP* 4.0b10.

(Invitrogen), 0.75 µl MgCl2 (50 mM), 2.5 µl dNTPs (2 mM each, Denville), 2.5 µl BSA (10 mg ml–1), 2 µl primers (10 µM), 0.2 µl Taq Results polymerase (5 U µl–1, Platinum Taq, Invitrogen), and 9.55 µl sterile bidistilled water. PCR ran on HPTLC a PTC-200 Peltier thermal cycler (MJ Research) under the following conditions for nuclear and We screened 297 Hypogymnia physodes speci- mitochondrial ribosomal genes: one initial cycle mens from Hungary and 44 specimens from var- of 1 min at 95 °C (denaturation), followed by 35 ious, mostly European localities by HPTLC. We cycles of 45 s at 95 °C, 40 s at 52 °C (annealing), identified five lichen substances: protocetraric 2–4.5 min at 72 °C (elongation; time depends on acid, 3-hydroxyphysodic acid (conphysodic or fragment size), and one cycle of 10 min at 72 °C oxyphysodic acid), physodalic acid, physodic as a final extension. For protein-coding genes, acid, and atranorin. We detected no significant there was one initial cycle of 3 min at 95 °C, differences among specimens from different followed by 35 cycles of 45 s at 95 °C, 90 s at habitats, nor among specimens collected at dif- 52 °C, 2 min at 72 °C, and one cycle of 10 min ferent times. There were no significant chemical at 72 °C. We applied cloning as needed using a differences among the varieties and forms of this TOPO TA Cloning Kit (Invitrogen). We exam- species. In some cases, the spots of protocetraric ined PCR products by gel electrophoresis using acid and atranorin were absent or very pale due 1% agarose gel and SYBR Green I (Invitrogen) to their very low concentrations. While one of

Table 3. List of primers for the loci sequenced in this study. Oligonucleotide sequences are available at http://www. lutzonilab.net/primers/index.shtml.

Locus Primers

ITS ITS1, ITS5, ITS1F, ITS4 nucSSU nssu131, NS24, NS22; Sequencing primers: nssu897R, nssu1088R, SR7R, nssu1088, nssu634, SR7 nucLSU LROR, LR5, LR6, LR7; Sequencing primers: LR3R, LR3 mitSSU mrSSU1, mrSSU2R, mrSSU3R

RPB1 regions A–F RPB1-Af, VH6R

RPB1 regions F–G VH6Fa, RPB1-G2r RPB2 regions 5–7 fRB2-5F, fRB2-7cR, RPB2-1554R RPB2 regions 7–11 fRB2-7cF, fRB2-11aR, RPB2-3053R, RPB2-3053bR EF1α EF1α-1F, EF1α-1R, EF1α-2218R 478 Molnár & Farkas • Ann. BOT. Fennici Vol. 48

Fig. 1. HPLC chromatogram of acetone extract of Hypogymnia physodes (Sample 2, cf. Table 1) at 245 nm. Peaks: a: acetone, b: benzoic acid (internal standard), c: protocetraric acid, d: 3-hydroxyphysodic acid, e: physodalic acid, f: 2´-O-methylphysodic acid, g: physodic acid, h: atranorin, i: chloroatranorin, j: bis-(2-ethylhexyl)-phthalate (internal standard).

nol depsidones physodic, 3-hydroxyphysodic, and 2´-O-methylphysodic acids (Figs. 1 and 2). According to peak areas on the HPLC chroma- tograms, medullary depsidones represented the major secondary compound pool, while cortical depsides occurred in substantially lower concentrations. Relative concentration (area%) of atranorin was 1.71–15.18 area% and chloroatranorin was 0.79–1.93 area%. The values for physodalic acid were 7.95–23.39 area%; Fig. 2. Bootstrap tree resulting from Maximum Par- protocetraric acid, 0.26–1.87 area%; physodic simony analysis of EF1α sequences of Hypogymnia acid, 0.01–5.14 area%; 3-hydroxyphysodic acid, physodes samples collected from various environmen- 22.22–41.72 area%; and 2´-O-methylphysodic tal conditions. Samples are indicated by numbers as in acid, 4.44–15.00 area% (Table 4). There was Table 2. a large range in the concentrations of various substances, especially for atranorin and phys- these substances was absent in one specimen odic acid. Protocetraric acid, chloroatranorin, of a certain variety or form, we detected it in and physodic acid occurred in low concentration other specimens of the same variety or form. We in all samples (minor lichen substances), while identified the cortical location of atranorin and 3-hydroxyphysodic acid occurred at the high- chloroatranorin by analyzing different parts of est concentration in all specimens, but in lower the thallus (intact thallus, medullary layer, sor- concentrations in some polluted areas (i.e., Zala- alia). The other lichen substances are located in egerszeg, Salgótarján). the medulla.

DNA analyses HPLC We sequenced DNA of 21 samples collected We analyzed thirteen H. physodes thalli grow- from different populations to study the genetic ing naturally under various environmental diversity. DNA amplification and sequencing conditions by HPLC. Seven lichen substances yielded 16 ITS (6 kb), 18 nucLSU (1.4 kb), 3 occurred in all thalli: the cortical β-orcinol nucSSU (1.6 kb), 21 mitSSU (8 kb), 1 RPB1 para-depsides atranorin and chloroatranorin, the (A–D region 1.2 kb; D–G region 1.8 kb), 10 medullary β-orcinol depsidones physodalic and RPB2 (5–7 region 1.2 kb; 7–11 region 9 kb), protocetraric acids, as well as the medullary orci- and 15 EF1α (1.4 kb) sequences. ITS, nucLSU, Ann. BOT. Fennici Vol. 48 • Chemical and genetic variability of Hypogymnia physodes 479 nucSSU, mitSUU and EF1α can be informative at the population level in lichens. We found no differentiation at all among the mitSSU 1 1.7 1.42 1 0.9 1.67 1.93 1.22 0.887 1.333 1.32 0.79 0.99 1.243 0.358 sequences. The lengths of the nucLSU sequences were variable, 14 specimens contained an intron (400 bps). The nucSSU sequences varied in the number of introns (with 200 bps length in the 2.97 3.86 6 1.708 6 9.92 4.91 4.581 7.908 4.52 1.87 7.45 5.913 3.650

342nd and 1367th positions). We detected only 15.178 Atranorin c hloroatranorin small differentiation among the ITS and EF1α sequences of populations, confirmed by the MP analysis (Fig. 2). 0.5 5.143 0.08 1.594 0.4 0.229 3.68 0.29 0.34 0.007 0.1 0.265 0.25 0.37 0.896 Physodic acid Discussion

Applying HPTLC in order to analyze the secondary compounds in the entire Hungarian population of a lichen species is unique and novel. Due 4.44 7.38 2.873 6.583 6.39 9.45 9.37 7.84 6.3 9.015 11.2 11 11.43 to the high number of samples analyzed, various 15 10.816 further comparative studies are possible. Accord- ing to TLC and HPTLC, H. physodes specimens from sites of different environmental conditions 2´- O -methyl-physodic acid do not show any qualitative differences in terms of secondary lichen compounds. Chemical con- habitats. samples collected from different 4.408 tents of old and freshly collected specimens were 8.429 7.95 11.78 11.28 11.254 23.39 16.03 10.88 similar, which means that herbarium specimens 10.984 17.37 17.38 10.121 10.296 12.857 physodes are suitable for taxonomic and monitoring stud- Physodalic acid ies after even a long period of storage. We identified all seven lichen substances by HPLC in all specimens, including varieties and forms, thus confirmed the HPTLC results, i.e., Hypogymnia the samples were uniform qualitatively, which 8.996 40.6 39.5 23.68 22.224 25.18 38.65 50.75 41.72 27.4 37.1 24.151 25.74 30.75 32.880 means that this lichen species has only one chemotype. We detected quantitative chemical differences between samples without any geo- 3-hydroxy-physodic acid graphical pattern (Table 4). From this we con- clude that microclimatic conditions or age of the thalli may be responsible for the differences. 1.22 1.87 0.26 Medullary depsidones were the major sec- 0.869 1.242 1.27 1.32 1.15 1.03 1.001 1.095 1.08 1.12 1.117 0.351 ondary compound pool (with physodic and protocetraric acids as minor lichen substances) in all Protocetraric acid samples. Cortical depsides showed substantially lower concentrations. Atranorin, 3-hydroxyphysodic, physodalic, and 2´-O-methylphysodic acids are major compounds of this species, whereas protocetraric and physodic acids, as well as chlo- Bp., Hosszúerdő-h. Locality Bp., Látó-hegy Bp., Remete-h. roatranorin are minor substances. Zeybek et al. Zalaegerszeg Salgótarján Almásfüzitő-felső Tarjánka Mátra, Bükk, Kolozs-lápa Bükk, C sákpilis Egerszalók Zgurăşti Avenului Štrbské Pleso (1993) showed slightly different results for H. physodes samples collected mainly from Turkey 0 2 Table 4 . Relative concentrations (area%) of lichen compounds in Table Sample 0 1 0 3 (Table 5). 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 11 0 12 sévharaszt c 0 13 Mean SD 480 Molnár & Farkas • Ann. BOT. Fennici Vol. 48

The high relative concentrations of physo- spread clonal reproduction. We detected only dalic acid in our samples growing naturally very low variability in the ITS, nucLSU, nucSSU, in polluted sites (Budapest: Hosszúerdő-hegy, and EF1α sequences of populations from differ- Látó-hegy, Remete-hegy, Almásfüzitő-felső, Sal- ent habitats, without any geographical patterns. gótarján) are not surprising, since this substance Haplotype network would also be a possibil- might be effective against pollution stress as ity for presenting our results as was suggested Białońska and Dayan (2005) suggested based recently (B. McCune pers. comm.). We are going on increased levels in transplanted H. physodes to check the results by further investigation of samples. The samples with the highest levels additional specimens and other genetic markers, of physodalic acid in the present investigation such as microsatellites and minisatellites. in Budapest were collected two decades ago when pollution by heavy metals and acidic inorganic sulphur compounds was high (Farkas et al. Acknowledgments 1985). However, the results for other substances cannot be compared with that study. Białońska Our work was supported by the Hungarian Scientific and Dayan (2005) found remarkable changes in Research Fund (OTKA T047160, T048736, K81232) and the levels of secondary compounds in H. phy- the AFTOL project of NSF (grant no. DEB-0228725). The authors are grateful to Molly McMullen (Duke University, sodes thalli transplanted to areas polluted with Durham) for revision of the text. Our sincere thanks to Prof. heavy metals and acidic inorganic sulphur com- François Lutzoni (Duke University, Durham) for his support pounds, while the current study showed various during the DNA analysis. We would like to thank Dr. Beáta concentrations without regard to pollution level. Bóka (Eszterházy Károly College, Eger) for her help in Many details about the secondary metabolism HPLC analyses. We are grateful for technical help given by Ms. Emily Fraker in laboratory work and Dr. Jolanta Miad- of this widespread toxitolerant lichen species likowska (Duke University, Durham) in phylogenetic analy- are still not entirely understood, and further sis. We are also thankful to Dr. László Lőkös (Hungarian studies are needed to determine its usefulness Natural History Museum, Budapest) for his help at various in bioindication research. Moreover, the study stages of this study and during preparation of the manuscript. of other more sensitive and rarer lichen species We thank anonymous reviewers for helpful criticism of the manuscript. are necessary as well, in order to determine whether natural environmental factors influence the biosynthesis of lichen compounds, and to identify those substances. Statistical sampling References and experimental conditions might also be used Arup, U., Ekman, S., Lindblom, L. & Mattsson, J. 1993: for more detailed studies. High performance thin layer chromatography (HPTLC), We found no significant genetic differentia- an improved technique for screening lichen substances. tion among populations collected from different — Lichenologist 25: 61–71. areas, indicating high gene flow and/or wide- Beard, K. H. & DePriest, P. T. 1996: Genetic variation within and among mats of the reindeer lichen, Cladina subte- nuis. — Lichenologist 28: 171–182. Bennett, J. P., Dibben, M. J. & Lyman, K. J. 1996: Element Table 5. Quantity of lichen substances established in concentrations in the lichen Hypogymnia physodes (L.) the current study and in Zeybek et al. (1993). Nyl. after 3 years of transplanting along Lake Michigan. — Environ. Exp. Bot. 36: 255–270. Lichen substances Samples Białońska, D. & Dayan, F. E. 2005: Chemistry of the lichen

Hypogymnia physodes transplanted to an industrial This study Turkish region. — J. Chem. Ecol. 31: 2975–2991. Hypogymnia physo- Protocetraric acid minor–trace minor Bruteig, I. E. 1993: The epiphytic lichen des 3-hydroxyphysodic acid major major as a biomonitor of atmospheric nitrogen and sulphur Physodalic acid major major deposition in Norway. — Environ. Monit. Assess. 26: 2´-O-methylphysodic acid major–minor traces 27–47. Physodic acid minor–trace major Cabanillas, M., Fernández-Redondo, V. & Toribio, J. 2006: Atranorin major–minor major Allergic contact dermatitis to plants in a Spanish derma- Chloroatranorin minor–trace minor tology department: a 7-year review. — Contact Derm. 55: 84–91. Ann. BOT. Fennici Vol. 48 • Chemical and genetic variability of Hypogymnia physodes 481

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