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 geo- graphical 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). nomenex Phe used We Detector. UV/VIS SPD-10Avp and graph using LC-10ADvp and LC-10AS pumps, Shimadzu High Performance Liquid Chromato are suitable. We performed the analyses with a al of each per 1000 (20 standards internal as (acetone) liquid tion ml acetone). Huovinen and bis-(2-ethylhexyl)-phthalate to the extrac thalli in 1 extracted approximately 10 mg air-dried lichen Feige followed the standardized method described by tion to compare their secondary compounds. We with reversed-phase column and gradient We elu analyzed 13 samples (Table 1) by HPLC HPLC analyses A tion factor 1 alpha ( unit ribosomal DNA ( ( DNA ( rDNA), nuclear small subunit ribosomal DNA following loci: internal transcribed spacer ( amplification.We amplified and sequenced the water, and we prepared a 1:10 dilution for PCR lated genomic DNA in 50 µl sterile bidistilled temperature for 1 resuspended We hour. the iso with ca. 70%–87% ethanol and dried the by at DNA isopropanol, then washed the room pellet sulphate (SDS) precipitated as a We lysis buffer. Pukkila (1986). We used 2% thalli using a sodium protocol modified fromdodecil Zolan and We extracted genomic DNA from dried lichen different geographical sites for DNA analysis. We collected 21 lichen samples (Table analyses DNA relative terms(percentageofarea:area%). 1985). Concentrations of substances are given in (Feige the lichen compounds (data not shown): calculated two kinds of elution retention of the lichen indices substances at 245 for nm. We methanol (solvent system B). monitored We the bidistilled water (solvent system A) and 100% solvent systems: 1% ortho-phosphoric acid which we in injected 20 µl samples. We used two nucSSU NN . (1985) reported that these internal standards . BOT. et al nucLSU et al rDNA), nuclear large subunit ribosomal subunit large nuclear rDNA), ® F Prodigy 5 . (1993) with We some modifications. ENNI ml acetone. We added benzoic acid . 1993) and RI (Huovinen rDNA), mitochondrial small sub c I Vol. EF1α mitSSU 48 µ ODS-3V column, onto ), RNA polymerase II • rDNA), elonga Chemical andgeneticvariabilityofHypogymniaphysodes