Photosynthetic characteristics of foliose lichens in response to light, temperature and water status of thallus

Barták, M., Gloser, J., Hájek, J., Dubová, J., Vráblíková, H., Váczi, P., Komárek, O. & Hájková, M.
Department of Plant Physiology and Anatomy, Masaryk University, Kotlárská 2, 61137 Brno, Czech Republic

Introduction. Majority of lichens are capable to cope with extremes and rapid fluctuations in environmental factors, availability of water, light, and air temperature in particular. Photosynthesis in lichens is controlled by these factors and actual rate of photosynthetic processes reflects their interactive effect.
Material and Methods. To study lichen photosynthesis both in situ (mainly maritime Antarctica) and in a laboratory, fluorometric and gas exchange methods were used. In addition, spectral reflectance, photosynthetic pigments and antioxidants analyses, oxymetric evaluation of photosynthesis in photobionts, anatomic studies were applied in Lasallia pustulata, Umbilicaria hirsuta, U. antarctica, and U.decussata.
Results and discussion. Temperature response curves revealed that primary processes of photosynthesis (Fv/Fm – capacity of photochemical reactions in photosystem II, FII – quantum yield of PS II) had optimum at 18-22 oC, and hold high level (about 80 % of maximum) within the range of –5 to 18 oC. Temperature minimum was found at –20 to –15 oC. Hydration response curves showed that maximum values of Fv/Fm, FII retained to water saturation deficit (WSD) of 50-60 % and then decreased rapidly with desiccation. In most species, critical WSD was 70-80 % which corresponds to water potential (WP) of – 35 to –25 MPa.
In situ measurements of net photosynthesis (Pn) showed Pn of 1-2 mmol (CO2) m-2 s-1 for well-hydrated U.antarctica. When exposed to high light in wet state in a laboratory, thalli showed decrease in Fv/Fm, FII and consequent recovery in dark. Chlorophyll fluorescence imaging showed intrathalline differences in the sensitivity of U. antarctica to photoinhibition. During photoinhibion, non-photochemical quenching (NPQ) and zeaxanthin formation increased simultaneously. Photosynthetic processes (FII) were linearly related to oxygen evolution rate in non-stressed symbiotic algae. Under osmotic stress, however, FII to OER relation was curvilinear.


Seasonal variation of chlorophyll fluorescence in parmelioid lichens along an altitudinal gradient

Baruffo, L. & Tretiach, M.
Dipartimento di Biologia, Università degli Studi di Trieste, Via L. Giorgieri 10, I-34127 Trieste, Italy

Chlorophyll fluorescence was measured in the five most frequent parmelioid lichens (Flavoparmelia caperata, Parmelia saxatilis, P. sulcata, Parmotrema chinense and Punctelia subrudecta) found in four stations along an altitudinal gradient from the Tyrrhenian coast to Mt Amiata (Southern Tuscany, Italy). The stations are representative of the four main forest types found in the survey area: evergreen horn oak (Quercus ilex) forest, and the deciduous downy oak (Q. pubescens), chestnut (Castanea sativa) and beech (Fagus sylvatica) forests.
In situ measurements were carried out with a photosynthesis yield analyser Mini-PAM (Walz, Effeltrich, Germany) every three months from December 2003 to June 2004 on the same portions of ten dark-adapted thalli of each species in each stand.
The results show that the lichens had remarkable, asynchronous changes in Fv/Fm, correlated with the climatic conditions of each period and the light regime of the understorey. During the year, surprisingly higher values than those reported in the literature were recorded in the station where the frequency of each species was highest.
The importance of in situ measurements along ecological gradients for a
better understanding of species ecology is thoroughly discussed.


Threadlike crystals on thallus surfaces of selected sorediate lichen species

Bayerová, S. (1) & Haas, K. (2)
(1) Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pruhonice, Czech Republic; (2) Institute of Botany, University of Hohenheim, Garbenstr. 30, D-70593 Stuttgart, Germany

The thallus surfaces of about 50 species of sorediate lichens, e.g. Cladonia sp. div., Lecanora sp. div., Lepraria sp. div., were investigated using scanning electron microscopy (SEM). Threadlike crystals on the surface of soredia were observed in terpenoid-containing specimens, e.g. Chrysothrix chlorina, Cladonia deformis, Lecanora subaurea, Lepraria incana. In contrast, lichen species not containing triterpenoids proved to lack such crystals. Therefore the authors conclude that triterpenoids are responsible for the formation of the threadlike forms. This enriches our current knowledge and corroborates previous observations of other authors, e.g. T. Tønsberg. Some representative SEM micrographs are presented.


Nitrogenase activity in Thyrea spp. – preliminary results

Crittenden, P. D. (1), Llimona, X. (2) & Sancho, L. G. (3)
(1) University of Nottingham, Nottingham, UK; (2) Universitat de Barcelona, Barcelona, Spain; (3) Universidad Complutense, Madrid, Spain

Nitrogenase activity (NA) in heterocystous cyanobacteria is confined to heterocysts where the enzyme is spatially separated from oxygenic photosynthesis in vegetative cells. Heterocystous cyanobacteria achieve maximal rates of NA in the light, partially fuelled by photophosphorylation carried out by photosystem I in the heterocysts. Unicellular cyanobacteria cannot achieve spatial separation between NA and photosynthesis. However, there is now abundant evidence that unicells fix nitrogen during dark periods and thus utilize temporal separation between NA and photosynthesis. The large majority of studies on nitrogen fixation in lichenized cyanobacteria have been undertaken on lichen species containing heterocystous photobionts (e.g. Collema, Nephroma, Peltigera, Stereocaulon). The pattern of diurnal variation in NA in these genera is similar to that in free-living cyanobacteria. However, several lichen genera (e.g. Anema, Peccania, Thyrea) contain unicellular cyanobacteria (e.g. Gloeothece, Gloeocapsa). It is not known whether NA in these lichens is confined to dark periods as has been shown previously for free-living unicells. Here we report a preliminary study of diurnal variation in acetylene reduction rates in Thyrea pulvinata and T. girardii in the field and under laboratory conditions. We briefly discuss the ecophysiological implications of daytime vs night time nitrogen fixation in lichens.


Association of the mycobiont cell aggregates of Tremolecia atrata with heavy metals

Fujii, H. (1), Hara, K. (1), Komine, M. (1), Yamamoto, Y. (1), Ozaki, T. (2) & Ohnuki, T. (2)
Kaidoubata-nishi, Shimoshinjyo-nakano, Akita 010-0195, Japan (1); Advanced Science Research Center, Japan Atomic Energy Research Institute, 2, Shirakata, Tokai, Ibaraki 319-1195, Japan

Lichens accumulate heavy metals in the thalli. We studied the association mechanisms of lichen mycobionts with heavy metals, for the purpose of the application of lichens to the remediation of contaminated soils with heavy metals. Screening tests for 25 lichen mycobiont species in terms of accumulation and tolerance of Cu and Cd gave Tremolecia atrata being the highly tolerant among them. Using the mycobiont, we investigated the effects of Cu and Cd on the growth. The amounts of the accumulated Cu or Cd in the mycobiont cultured in liquid medium containing either were determined by ICP-AES. Spatial distributions of Cu and Cd in the mycobiont cell aggregates were analyzed by Micro Particle Induced X-ray Emission (Micro-PIXE). The mycobiont grew to be aggregates in spherical shape of several mm in diameter with up to 100 mg/l Cu or up to 16.8 mg/l Cd. The largest growth of the mycobiont was observed at approximately 25 mg/l Cu. The growth of mycobiont was not accelerated in the presence of Cd. The mycobiont accumulated approximately 3.8 mg/g of Cd or 9 mg/g of Cu on a dry weight basis from the medium containing 11.2 mg/l Cd or 75 mg/l Cu, respectively, during 2 weeks. Micro-PIXE analyses of the aggregates grown with 75 mg/l Cu showed that most of the Cu absorbed was distributed in the inner region of the aggregates. On the contrary, Cd was found to be distributed homogeneously in the aggregates. Phosphorous, which is one of essential elements and is concentrated in the active cells, was distributed in the outer region. This fact suggests that the cell viability is lower in the inner region than the outer region. It is considered that Cu was accumulated in the inner region because the inner region is less sensitive to the metal toxicity and can be a sink for toxic metals like the vacuole. It suggests that localization of Cu in the aggregates of T. atrata is one of the mechanisms for hyper-metal-accumulation and -tolerance.


New histochemical techniques for the localisation of heavy metal ions in the lichen thallus

Giordani, P. (1), Bombardi, V. (1), Crisafulli, P. (2), Modenesi, P. (1), Monaci, F. (3), Rinino, S. (1) & Tretiach, M. (2)
(1) DIP.TE.RIS., University of Genova, Corso Dogali 1/c, I-16136 Genova, Italy; (2) Dipartimento di Biologia, University of Trieste, Via Giorgieri 10, I-34127 Trieste, Italy; (3) Dipartimento di Scienze Ambientali "G. Sarfatti" University of Siena, Italy

A study was carried out to assess the pattern of heavy metals ions localisation in Pseudevernia furfuracea (L.) Zopf. var. furfuracea, a species used in a parallel study for biomonitoring persistent atmospheric pollutants with lichen bags.
Thalli of known elemental composition collected in a pristine area of the Carnic Alps (Italy) were immersed for three days in a 100µg/ml solution of different salts at pH 5.5 to introduce target cations (Al3+, Cd2+, Cu2+, Fe3+, Fe2+, Hg2+, Mn2+, Ni2+, Pb2+, Zn2+). Histochemical techniques derived from classical methods routinely used in human histopathology were used. They had been opportunely modified in order to preserve dye-metal complexes and to reduce artefacts caused by diffusion and solubilization. Generally, methods originally described for fresh material were preferred; they were applied with good results on thin glycol methacrylate resin-embedded sections. The following precautions were taken: 1) different fixation methods were tested, and alcoholic fixatives refused; 2) results of different histochemical reagents for the same metal were compared; 3) methods involving chelate formation were preferred; 4) histochemical differentiation was used to study reaction specificity. Metals localization was compared with histochemical tests for proteins, neutral and acidic polysaccharides, polyanions, and melanin pigments.
The results show that the selected metal ions differ characteristically in the distribution within the lichen compartments. Al3+ and Hg2+ occur only intracellularly in all thalline districts (medulla, algae, upper and lower cortex and isidia). Pb2+ and Zn2+ occur extracellularly in the conglutinated scleroplectenchyma of the bi-layered upper and lower cortex. The other metals occur both extra- and intracellularly. The importance of melanin pigments as ligands for Cu2+, Fe3+, Fe 2+ is pointed out. This pattern may certainly explain why some heavy metals only are toxic for the myco- and/or photobiont cells.


Visualization of high light effects on intrathalline variability of photosynthetic processes in hydrated Lasallia pustulata

Hájek, J. & Barták, M.
Department of Plant Physiology and Anatomy, Masaryk University, Kotlárská 2, 61137 Brno, Czech Republic

Introduction. It is expected that hydrated lichen thalli are more susceptible to negative effects of high light (HL) under low rather then optimum temperature of thallus. In our experiments, we investigated response of foliose lichen species (Lasallia pustulata) to HL stress at low temperature under laboratory conditions.
Material and Methods. Hydrated thalli were exposed to HL of 500 mmol (CO2) m-2 s-1 for 30 min under 5 oC. Response of photosynthetic parameters (Fv/Fm – potential yield of PS II photochemistry, FII - quantum yield of PS II photochemistry, qN – non-photochemical quenching, qT+I – photoinhibition-related quenching) was monitored during HL treatment and subsequent 6 h recovery in dark using chlorophyll (Chl) fluorescence imaging technique False color images of lichen thallus showing heterogeneity of the above-specified Chl fluorescence parameters over the thalli were taken using a kinetic fluorometric CCD Camera (FluorCam FL-400, PSI, Czech Republic). Subsequent image analysis allowed us to specify zones and anatomic structures exhibiting different values of Chl fluorescence parameters.
Results and discussion. When considering whole lichen thallus, HL treatment led to 23 and 20 % decrease in Fv/Fm and FII, respectively. Dark recovery led to a gradual increase in both parameters. Fast phase of recovery, attributed to regulatory and protective mechanisms, was finished after 90 min recovery in dark. Slow phase of recovery, attributed to reparatory mechanisms, lasted several hours. When considering different thallus zones, there were big differences in Chl fluorescence parameters. Before HL treatment, Fv/Fm and FII, reached 0.585 and 0.446 in umbilicus zone. Close-to-umbilicus zones showed higher values of 0.649 and 0.523, resp. Marginal zones showed Fv/Fm and FII ranges of 0.599-0.699 and 0.312-0.615. HL treatment led to differential decrease of Fv/Fm and FII in the above-specified zones showing their different sensitivity to HL effects on photosynthesis.


Characterization of acidophilic mycobiont from lichen Cetraria aculeata

Hara, K., Kuroki, M., Fujii, H., Komine, M. & Yamamoto Y.
Akita Prefectural University, Akita, Japan

Through the symbiotic associations, lichens can survive in extreme environments characterized by high or low temperatures, pressures, irradiation, salinities, and acidities. In symbiotic associations of lichens, mycobionts play two important roles: to protect photobionts, and to absorb mineral nutrients. These abilities are expected to be useful for bioremediation. On the other hand, lichens are well-known as living indicators of environmental problems because of their sensitivity to air pollution. Thus many lichens are damaged by acid rain. Then lichens are supposed to respond to acid stress uniquely. However, the mechanism is poorly understood.
We had previously screened mycobiont cultures for the acidophilic ones. As a result, the only mycobiont derived from lichen Cetraria aculeata found to grow well in the acid liquid medium (pH 2.5). In this study, we investigated the acid stress response of the acidophilic mycobiont at the molecular level. The optimum pH level for growth was pH 3.0, and differences in the medium or in the mycobiont cells between two pH conditions, pH 3.0 and pH 6.0, were analyzed respectively. By TLC analyses, an unknown spot was only detected in the extract derived from acid-treated medium as a difference, indicating activated secondary metabolism by acid stress. Proteomic analyses resulted in differential protein expression patterns between two conditions. These will be discussed in detail.


Conserved cAMP signaling in fungi: Are lichenized fungi different?

Joneson, S. & Lutzoni, F.
Duke University, Durham, NC, USA

The cyclic AMP (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways are conserved from mammals to fungi, and are involved in regulating cell function. In fungi in particular, the cAMP pathway plays an integral role in morphogenesis, sexual development, and pathogenicity (when present). For example, in Aspergillus fumigatus cAMP signaling is involved in PKS activity and conidiation, in Magnaporthe grisea it is associated with appressorium formation, and in Ustilago maydis it is linked with virulence and filamentous growth. Could cAMP signaling be essential for the establishment of lichenized fungal symbiosis? We have begun to characterize one gene of this signaling pathway in Endocarpon pusillum and Cladonia grayi. In Saccharomyces cerevisiae, GPA2 encodes the G-protein alpha-subunit of a G-protein coupled receptor. Upon ligand binding to this receptor, the -subunit is released from its heterotrimeric conformation and activates adenylyl cyclase and cAMP production. A 500bp region of the GPA2 homologue in E. pusillum. and in C. grayi have been sequenced, and we are further characterizing the loci and their expression in these fungi grown alone and in resynthesis.


Regeneration of lichen thalli of Parmotrema tinctorum, P. austrosinense and Rimelia clavulifera in the field

Kon, Y. (1) & Kashiwadani, H. (2)
(1) Kohoku Senior High School, Tokyo, Japan; (2) National Science Museum, Tokyo, Tsukuba, Japan

Parmotrema tinctorum (Nyl.) Hale is a foliose lichen widely distributed in temperate and subtropical zones including Japan. Fragments were cut off in circle (6 mm in diameter) by a circular cutter from marginal parts of thalli of P. tinctorum. These fragments were transplanted on trunks of Cryptomeria japonica D. Don near the collecting sites. They were covered with pieces of nylon mesh, which were stapled to the bark at the corners. The growth of them was measured by weight and size in every three, six and nine months after transplantation. An external and internal structure was examined with a scanning electron microscope and a light microscope. Weights of excised thalli increased by 1.3, 2.1 and 2.2 times after three, six and nine months respectively after transplantation. They have increased to 1.3, 1.9, and 2.0 times in size after three, six and nine months later respectively after transplantation.
The initiation of cell division in the fungal and algal partner of P. tinctorum was found to start in the algal layer after three months of the transplantation, and globose primordial stages of new lobes (cylindrical protuberances) appeared along the exposed algal layer along cut edges. Palmate new lobules were formed by fusion of cylindrical protuberances after six months. Following the formation flight lobules, the lower cortex changed brown in color and formation of rhizines was observed. After nine months cylindrical isidia were also formed on the upper surface of dorsiventral lobules. Although the cortex and the algal layer of the new lobes were stratified and connected with mother thallus, they are extremely thin. In addition, algal cells in the lobules are extremely smaller, ca 1/3 in size compare with the original ones.
The similar results were observed in sorediate species, Parmotrema austrosinense and Rimelia clavulifera. However, the lobules formed along the adage were relatively small in number in both P. austrosinense and R. clavulifera.


Induction of usnic acid synthesis by UV irradiance in Xanthoparmelia somloensis

McEvoy, M., Solhaug, K. A., Nybakken, L. & Gauslaa, Y.
Department of Ecology and Natural Resource Management, Agricultural University of Norway, P.O. Box 5003, 1432 Ås, Norway

Acetone rinsed (usnic acid-free) Xanthoparmelia somloensis thalli were placed beneath UV-B-transmitting or UV-B-absorbing screens in the field for a 20-day period during summer 2003. Thalli were either sprayed with water or with a 5 g ribitol L-1 solution at dusk each day. Post-harvest acetone extraction clearly showed that thalli exposed to UV-B resynthesised usnic acid, while those receiving PAR radiation only, failed to do so. Carbon supplements (in the form of ribitol) had no significant positive effect on usnic acid resynthesis, with or without UV radiation. Reflectance measurements of thalli after exposure showed lower values in the visible region of the spectrum for those thalli exposed to UV radiation. This suggested the synthesis of an additional lichen pigment, which was confirmed by a visible blue-tint in UV-exposed thalli. Such a colour can sometimes be seen in various Parmeliaceae lichens in nature. Acetone rinsing failed to extract this unidentified pigment.


A steppe fire as an enemy of lichens.

Merkulova, O. S.
Institut of Steppe of the Ural Branch of Russian AS, Russia

The Orenburg region is mainly situated in the steppe zone. Thereby problems related to a pioneering (such as plowing, cattle-breeding, abandoning of a land, etc.) are here very topical. The problem of steppe fires is in Orenburg region especially acute. Since 2000 we carry out a work of lichen diversity research in the steppe zone of Southern Urals. Large-scale fires have regularly taken place in the researched area, that vary complicated our work. There is an opinion, that fires are good and even necessary for a steppe. It is very difficult to judge of this about lichens, because there is virtually no information about species diversity of lichens in bygone days. Nevertheless, being considering our recent dates, we affirm, that steppe fires are disastrous for on-soil lichens. Most of epigeous lichens dies, and a reconstruction of an on-soil cover proceeds very slowly.
Let us give an example. 40 species of lichens grew within the territory of Burtinskaya steppe of the state natural reserve Orenburgskiy after the fire of 1998 and before the fire of autumn 2003. In the spring of 2004 we have found only 33 species of lichens, which are mainly epilithous ones. We have not found 5 species of on-soil and 2 species of epiphytic lichens, which grew in this territory formerly. Such on-soil growing genus as Cladonia, Neofuscelia, Xanthoparmelia, are also especially damaged, and dimensions of their populations are shrunk. Researches, which are carried out in other territories, also confirm that steppe fires are enemies of lichens.


Anatomical structure and photobiont properties of lichens growing in an urban environment

Paukov, A. G.
Urals State University, Ekaterinburg, Russia

Changes of the morphology and anatomical layers thickness in lichens growing in polluted environments are reported by several authors (Goyal, Seaward, 1982; Holopainen, 1983). These changes are considered as degenerative or attributed to the fertilizing effect. The aim of this work is to estimate the response of anatomical structure and photobiont properties of lichen species to an urban environment.
Two foliose species – the tolerant Phaeophyscia orbicularis (Necker) Moberg and moderately tolerant Parmelia sulcata Tayl. had been chosen. Specimens were collected in Ekaterinburg (Middle Urals, Russia) and 40 km SE from the town. The major pollutants are formaldehyde, heavy metals, nitrogen dioxide and sulphur dioxide.
Layers thickness in Phaeophyscia orbicularis didn't show any significant changes in the specimens growing in the town compared with the rural ones. Algal layer in Parmelia sulcata is almost twice as thick in specimens from the centre of the town and upper cortical, medullar and lower cortical thickness is respectively 5 %; 12 % and 2 % lower in the same thalli. Algal cells volume counted per volume of the thallus in the town center significantly rises by 10 % but it is 50 % lower if counted per volume of the algal layer.
The increase of algal layers thickness and the volume of algal cells per thallus volume can be regarded as an adaptive feature that enables the thalli of Parmelia sulcata to thrive in the centre of the town. The medullar and two other heterotrophous layers thickness decreases at the same time which might also compensate the possible breakdown of chlorophyll in photobiont in the polluted environment. Meanwhile the thinner medulla fails to act as a water-storage reservour in the thalli growing in the town. We can suppose that such an enormous increase in algal layer thickness besides the ability to contain more algal cells can also be used as an additional water tank that is very important in a drier atmosphere of a town.


Lichen morphology, thallus water content and photosynthetic performance. Looking for a single trait

Sancho, L. G., Pintado, A., Blanquer, J. M., Raggio, J. & Vilches, R.
Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain

Both in lichens and in other photosynthetic organisms, the aim of plant ecological theory is to understand the adaptive strategies that have driven their evolution. However, plant ecologists recognize that this objective is unlikely to be achieved without some form of simplification. The most widely used axis is probably one related to the capacity to exploit resources of the environment. In the search for a single trait that expresses the ecophysiological information of this axis in vascular plants,it is clear that specific leaf area (SLA, leaf area per unit dry mass) is the most powerful parameter, showing a direct correlation with Amax, and photosynthetic efficiency. However, a practical difficulty is how to measure the leaf area in certain plants, such as cacti or leafless stems, and in most lichens and mosses. Recently, another leaf trait has been proposed: the leaf water content (LWC), sometimes referred to as tissue density or as leaf dry matter content. The trait relates dry weight and fresh weight of the leaves at maximum turgor. Different authors have shown clearly that it is much less variable and is a better predictor than SLA. The same could be applied to lichens if we consider maximum water content (MWC) in place of leaf water content at maximum turgor. To check the relationship between MWC, Amax, and chlorophyll content six fruticulose and seven foliose lichen species from Mediterranean mountains were selected. We found a good correlation within each morphotype, however, no or very weak correlation appeared when all studied species were considered as a single cluster. These results stresses the importance of lichen morphology for photosynthetic performance and shows that for lichens MWC may become a good an indicator of adaptive strategies as LWC is for vascular plants.


Studies on tropical mycobionts

Sangvichien, E. (1) & Whalley, A. J. S. (2)
(1) Faculty of Science, Ramkhamhaeng University, Hua Mark, Bangkok 10240, Thaliand, eks@ram1.ru.ac.th; (2) School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

Tropical lichens from variety of locations in Thailand were collected over a period of five years. Investigations on ascospore discharge, ascospore germination and development of axenic mycobionts cultures were undertaken and the resulted are presented. In a number of species high discharge rates and ascospore germination were obtained e.g. Trypethelium eluteriae, Laurera benguelensis but in others e.g. Rinodina sp. and Placythiella sp. the discharge was low with usually low percentage germination. A number of species discharged ascospores over a relatively long period but in all cases discharge was poor or did not occur during the dry season (March to May). The ability of germlings to develop as cultures was also followed under both solid and liquid growth conditions. The use of physical carrier e.g. sponge resulted in enhanced growth in the liquid media.


Cloning and sequencing of the complete beta-tubulin gene of Xanthoria parietina

Scherrer, S., Zippler, U. & Honegger, R.
Institute of Plant Biology, University of Zürich, Switzerland

The beta-tubulin gene is currently receiving increasing attention for multilocus phylogenetic studies of non-lichenized and lichen-forming fungi (Geiser et al. 1998, Mycologia 90: 831-845; Myllys et al. 2001, Mycologia 93: 335-343; etc.). We present the first complete sequence of the beta-tubulin gene (BTUB1) from a lichen-forming ascomycete. Degenerate primers designed after sequences from non-lichenized ascomycetes served to amplify a fragment of the beta-tubulin gene in Xanthoria parietina. The product was used to screen the genomic lambda phage library of X. parietina that was previously constructed in our laboratory for the entire gene. A region of 2.8 kb was cloned and sequenced. The beta-tubulin gene spans 1.77 kb and contains 8 introns which were confirmed by sequencing of PCR products derived from cDNA. Five of the introns are located at the 5’-end of the gene. The value of this region for phylogenetic studies is currently being tested and compared to other regions of the gene (see contribution of C. Eichenberger et al.). It is crucial to know the number of copies of the studied gene in the genome for phylogenetic studies. For many species several beta-tubulin copies are known. However, genomic DNA blot analyses after restriction digests with different enzymes suggest that BTUB1 of X. parietina is a single copy gene. Additionally, in the course of our investigations, we discovered a polymorphism with two allelic forms in the promotor region of BTUB1. This polymorphism might provide a useful tool as a genomic marker in population studies.

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