Extracellular reactive oxygen production and metabolism by lichens

Beckett, R. P. (1) & Minibayeva, F. V. (2)
(1) School of Botany and Zoology, University of KwaZulu-Natal, Pbag X01, Scottsville 3209, South Africa; (2) Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O.Box 30, Kazan 420111, Russia

In preliminary work we discovered that some lichens actively producesuperoxide radicals extracellularly. We therefore conducted a survey,testing for production in 34 species from different taxonomic groupingsand contrasting habitats before and after desiccation stress. All 20species tested from Suborder Peltigerineae produced superoxideextracellularly at high rates, even when they were not stressed. Inaddition, some of these species showed a burst of superoxide productionduring rehydration following desiccation. Extracellular production ofsuperoxide was almost absent from 14 species from other lichen groups.Superoxide production was best developed in metabolically active speciesthat contain low concentrations of secondary metabolites. In suchlichens, production of superoxide may form an alternative to secondarymetabolites in protecting thalli from pathogen attack following stress.Our preliminary results on the nature of the enzymes that producesuperoxide suggest that they do not possess the classicalcharacteristics of those suggested to produce reactive oxygen species inhigher plants.The superoxide radical has a very short half life, and the radicalsproduced by members of the Peltigerinae will be rapidly converted tohydrogen peroxide, either spontaneously or by the action of the enzymesuperoxide dismutase. However, our studies showed that unstressedlichens produce almost no hydrogen peroxide, and that desiccationinduces only a small short-lived peak of hydrogen peroxide formation. Wehypothesized that lichens can rapidly metabolize hydrogen peroxide, andmeasured metabolism rates in 20 species. While considerable diversity inthese rates existed, rates were on average double in members of thePeltigerinae. Experiments involving inhibitors indicated thatperoxidases are responsible for the metabolism of hydrogen peroxide.Presumably, peroxidase activity protects lichens from the harmfuleffects of their own reactive oxygen species.


Influence of isidia development on CO2 gas exchanges in the epiphytic lichen Pseudevernia furfuracea (L.) Zopf var. furfuracea

Crisafulli, P. (1), Modenesi, P. (2), Pittao, E. (1), Roccotiello, E. (2) & Tretiach, M.(1)
(1) Dipartimento di Biologia, Università di Trieste,Via L. Giorgieri 10, I-34 127 Trieste, Italy; (2) DIP.TE.RIS., Università di Genova , Corso Dogali 1/c, I-16136 Genova, Italy

The events related to the development of isidia in thalli of Pseudevernia furfuracea, and the effects of these structures on CO2 gas exchanges were studied in samples collected in an open Larix decidua wood, at 1500 m in the Carnic Alps (North-eastern Italy).The developmental events leading to the formation of isidia were studied by comparison of sections stained with different histochemical tests and SEM observations. A high cell turnover rate in both symbiotic partners is the first signal of isidium development, followed by an increased aplanosporogenesis of algae and growth of neighbouring medullary hyphae which become upward oriented. Large nuclei, visible with DAPI staining, and an intense cytoplasm activity, e.g. in dephosphorilative processes involved in active intercellular movements of glucids, characterise mycobionts cells. The surface of very young isidia shows an irregular structure of spherical to ovoid protruding tips of perpendicular cortical hyphae, that are later organised in a pseudomeristematic area that is similar to that observed in the apex of growing lobes.CO2 gas exchanges carried out in the laboratory confirmed the high metabolic activity of isidia. At optimal water content and favourable light conditions, isolated isidia had higher gross photosynthesis and dark respiration than non isidiate laciniae (c. 6.3 and -1.4 mg CO2 g-1 h-1, respectively, corresponding to c. 130% and 150% of the values measured in non isidiate laciniae). Surprisingly, however, intact isidiate laciniae had very low CO2 gas exchanges. Isolated isidia had also a very low CO2 saturation point, probably because of their favourable surface/volume ratio, and seemed to affect only negligibly the water relations of the thallus.The different roles played by isidia in the biology of Pseudevernia furfuracea are thoroughly discussed, and the presence of thalloconidia is briefly mentioned.


Mating systems and genetic diversity in the genus Xanthoria

Honegger, R., Zippler, U. & Scherrer, S.
Institute of Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland

Genetic variability among sterile cultured single ascospore isolates of Xanthoria parietina, X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei was investigated with RAPD-PCR. If available 5 out of 8 ascospores per ascus were analysed with up to 10 primers. In some samples multispore and mycelial isolates from ascomata were included in the analysis. Ascospore germination rates and phenotypic features such as growth rate, pigmentation and secondary metabolites were uniform in X. parietina sporelings of the same ascus, but varied among the progeny of meiosis in all other species. Phenotypic features correlated with genetic variability. X. parietina revealed polymorphisms among specimens from different worldwide locations, but 9 out of 10 sets of sibling spores were genetically uniform, with only 2% polymorphism in the remaining set, indicating that X. parietina might be homothallic. X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei revealed 9-66% polymorphic loci and therefore are considered heterothallic. Xanthoria mating-type genes are currently investigated (see contribution of S. Scherrer et al.). Can we conclude on mating systems on the basis of numbers of ascomata per thallus? Species with no, few to many ascomata per thallus (X. calcicola, X. ectaneoides, X. capensis and X. resendei) were heterothallic, but species with lots of ascomata of all age classes were either homothallic (X. parietina) or heterothallic (X. polycarpa). Having seen that X. parietina is presumably homothallic we investigated the genetic structure of 4 systematically sampled european populations from coastal, rural and urban sites. RAPD-PCR data from multispore isolates were analysed. None of these populations revealed a clonal structure. All were genetically diverse, the reasons for this diversity being a matter of debate. Simultaneously the genetic diversity of the green algal photobiont was investigated (see contribution of S. Nyati et al.).


Imaging of photosynthetic acitivity of lichens using chlorophyll fluorescence: the water suprasaturation effect

Jensen, M. & Rosenkranz, P.
Universtität Duisburg-Essen, Campus Essen, Universitätsstr. 5, D-45117 Essen, Germany

Chlorophyll fluorescence (CF) analysis is an efficient and convenient tool for estimation of photosynthetic activity of lichens. Therefore, it can be used to replace more difficult gas exchange measurements – as long as there is a linear relationship between the two types of measurement. However, the fluorescence signal (photon use efficiency, CF parameter FPSII) and the CO2 fixation rate (measured by infrared gas analysis) do not always conincide, e.g. at high water content of lichen thalli. During transition from optimal water content to water suprasaturation, CO2 fixation was diminished but photon use efficiency was almost unaffected. As we wanted to know whether this effect can be easily explained by increased photorespiration or not, we used the novel CF imaging method parallel to gas exchange measurements. The 2-dimensionally resoluted CF was analysed for 5 different green algal lichen species. Since the CF parameter NPQ (indicating light stress) did not change the same way for all of the 5 species, their should be different ways how photosynthesis is influenced by high water content. In addition, we observed special effects at the margin of some thalli, and some variation of the CF signals within the thallus area was normal. The
consequences of these findings for the interpretation of CF signals are discussed.


Population structure and habitat-correlated genetic differentiation in Xanthoria parietina

Lindblom, L. & Ekman, S.
Dept. of Biology, University of Bergen, Bergen, Norway

Little is known about the structuring of genetic variation in lichenised fungi. We have investigated the genetic structure on a local scale and on different substrates in a common and wide-spread lichenised ascomycete, the foliose species Xanthoria parietina. Seven populations on the island Storfosna located off the west coast of Trøndelag, Norway, were investigated. Four populations were collected from sea-shore rock and three populations were collected from bark. Sequences (total ITS and partial IGS) were obtained from c. 30 specimens sampled from each population. Intraspecific variation is high compared to other lichenised fungi: until now, we have found c. 15 haplotypes for each of the gene fragments. An exact test of population differentiation and population pairwise fixation indices FST was performed. The major contributing factor predicting FST was substrate. In addition, there was a possible isolation-by-distance effect in the bark populations, but not in the rock populations at the present scale. The populations on rock generally contain a lower number of haplotypes than the populations on bark. Genetic differentiation between the populations growing on different substrates is evident, indicating that Xanthoria
parietina should not be considered as one panmictic population at the study site but instead forms distinct, sea-shore rock and bark populations, comparable to ecotypes.


High diversity of cryptic fungi inhabiting healthy lichen thalli in a temperate and tropical forest

Miadlikowska, J., Arnold, A. E., Hofstetter V. & Lutzoni, F.
Duke University, Department of Biology, Durham, NC 27708-0338, USA

Studies of lichenicolous fungi (secondary fungi associated with lichen thalli) have been restricted, almost exclusively, to fungal species with visible reproductive structures on lichen surfaces. These visible lichenicolous fungi are abundant in nature and more than 1100 species have been described. However, the potential for fungi to occur asymptomatically within thalli (i.e., as endolichenic fungi, analogous to endophytes of plants) remains mostly unexplored. We used a gradient of surface-sterilization to examine fungal communities associated with leafy lichens of the genus Peltigera (Peltigeraceae, Peltigerales, Ascomycota) from forests in North Carolina and Costa Rica, with the goals of assessing diversity, species composition, and geographic structure of endolichenic fungi.
From two thalli per site, two 2 cm2 pieces were subjected to different washing treatments: water only (30 seconds), or washing in water followed by immersion in 0.5% sodium hypochlorite and 70% ethanol for 10s, 30s, or 120s each. Following treatment, thallus pieces were pressed lightly against 2% malt extract agar (MEA) to harvest viable fungi from external surfaces, and then were cut into small pieces and transferred to 2% MEA plates to harvest all cultivable fungi in and on the thallus. A total of 325 fungal isolates were obtained, representing 96 unique genotypes as defined by 90% sequence similarity at the nuclear ribosomal internal transcribed spacer (ITS rDNA). Endolichenic fungi represent lineages that are phylogenetically distinct from better-known (visible) lichenicolous fungi, and are taxonomically congruent with lineages of Ascomycota frequently isolated as endophytes of plants. Whereas fungi occurring on lichen surfaces differed markedly between North Carolina and Costa Rica, there was a surprising congruence of genotypes among endolichenic fungi from the two sites. The implications for the ecology and evolution of the lichen symbiosis are discussed.


Phylogeography of homing endonucleases found in Pleopsidium (Acarosporaceae) and its implication for group I intron mobility

Reeb, V. (1), Haugen, P. (2), Lutzoni, F. (1) & Bhattacharaya, D. (2)
(1) Duke University, Dept. of Biology, Durham NC 27708-0338 USA; (2) University of Iowa, Biological Sciences Dept., Iowa City 52242 USA.

The sporadic distribution of group I introns in different kingdoms, genomes and genes is more simply explained by their lateral transfer from one organism to another, then by their inheritance and subsequent losses. The two models invoked in lateral transfer of group I introns are reverse-splicing at the RNA level and homing events mediated by a homing endonuclease (HE) at the DNA level. HEs are encoded by an open reading frame found within the introns themselves. To date, only 22 HEs have been found in group I introns of eukaryotic nuclear ribosomal genes, out of more than 1500 group I introns which have been reported within these genes. We recently found one of these HEs at position S943 of the small subunit ribosomal nuclear gene of the lichen-forming fungus Pleopsidium (Acarosporaceae). The nucleotide sequence of this HE is extremely conserved between members of Pleopsidium, suggesting that the enzyme may still be active. The ability of homing endonuclease to spread quickly from HE plus alleles to HE minus alleles of heterozygous cells lead to their rapid fixation within a population (90% of the meiotic products inherit HE genes). However, once fixed, the HE genes are expected to degenerate, as there are no remaining HE minus alleles to be cut. The only way for HEs to persist over evolutionary time would be by occasionally moving to new locations, genomes or hosts. We show here, that the presence/absence of HEs within group I intron S943 of populations of Pleopsidium follow a geographical pattern independently of the phylogenetic history of the genus. We will reconstruct the ancestral states for absence/presence of the HE on a phylogeny of the Acarosporaceae to determine if the HE was vertically inherited and subsequently lost, or was acquired by horizontal transfer between the different Pleopsidium species within a broad geographical context.


Investigating lichen life cycles on transparent substrates placed in situ

Sanders, W. B.
Centro de Ciencias Medioambientales, CSIC, Calle Serrano 115 bis, 28006 Madrid, Spain; University Herbarium, University of California, Berkeley, USA

There are major gaps in our understanding of the life histories of the fungi and algae that participate in lichen symbioses. While the visible symbiotic thallus is relatively easy to study, a much more difficult challenge is to detect and observe propagule behavior, aposymbiotic phases and microscopic early stages of lichen ontogeny under natural conditions. One recent approach that has proven useful involves the placement of transparent substrates in the field, where they may be colonized by propagules whose development can be then observed with light microscopy. This approach has been successful in foliicolous lichen communities, where the smooth surface of transparent plastic appears to be sufficiently similar to that of the natural leaf substrate. A study carried out in the Atlantic forest of northeastern Brazil (in collaboration with Robert Lücking) documented a variety of reproductive strategies, including various types of symbiont co-dispersal and aposymbiotic dispersal followed by relichenization. Early stages of thallus ontogeny and different patterns of symbiont coordination were also observed. In a revisit to that field site, fertile thalli of Opegrapha filicina, Mazosia rotula, cf. Gyalectidium filicinum, and others were observed on the plastic cover slips at 20 months after placement. Currently, similar studies are being attempted in temperate corticolous lichen communities in several habitats in Spain. Plastic cover slips placed among these communities show intial stages of germination of fungal spores and lichen propagules. However, further stages of ontogeny and establishment of lichen thalli have not (yet) been seen. A notable result is that non-lichenized populations of Trebouxia and other algae can be readily observed using this technique.


Characterisation of the mating-type locus in the genus Xanthoria

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

In filamentous ascomycetes mating is controlled by one mating-type (MAT) locus which is biallelic in heterothallic species. Conversely, in homothallic species the offspring of one meiotic event is genetically uniform, also at the MAT locus. This study is a first approach to clone and sequence the MAT locus of representatives of the genus Xanthoria. With PCR-based methods we were able to amplify the conserved HMG domain of the MAT1-2 gene in several species, including X. flammea, X. calcicola, X. polycarpa and X. parietina. A PCR-amplified HMG fragment was used as a probe to screen the genomic lambda phage library of Xanthoria parietina which had previously been constructed in our laboratory. After subcloning a region was sequenced which spans 6.7 kb and includes both flanking regions. In the MAT locus of X. parietina one putative ORF was identified which shows homology to the MAT1-2 gene of filamentous non-lichenized ascomycetes and an intron at conserved position. Irritatingly, the ORF is disrupted by a stop codon; this was confirmed by sequencing samples from different geographic locations. Upstream of MAT1-2, a gene with high homology to SLA2 (encoding a cytoskeleton assembly control protein) of Saccharomyces cerevisiae was found. Downstream of the MAT locus a DNA lyase gene was identified which was also found in close proximity of the mating-type genes in other ascomycetes. To test the presence of the MAT1-2 gene in the offspring of one meiotic event, a PCR assay of the HMG domain was performed in at least five single spore isolates derived from one ascus. In the presumably homothallic X. parietina (see contribution of R. Honegger et al.) a product was obtained in all isolates in contrary to heterothallic species where it could not be detected in all samples. To conclude, MAT1-2 is the first mating-type gene of a lichen-forming fungus described.


Diversity and evolution of secondary metabolite encoding genes in the Pertusariaceae

Schmitt, I. & Lumbsch, H. T.
The Field Museum, Department of Botany, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA

Elucidating the nature of secondary metabolites in lichens has been an issue of interest for several years. While many lichen substances have been chemically characterized, and their value for taxonomy has been proven, their biological functions are still largely unknown. One way to explore the nature and evolutionary history of secondary metabolites is by comparing sequences of genes that encode biosynthetic enzymes. In the current study we investigate phylogenetic relationships of fungal genes encoding putative polyketide synthases (PKSs) that are predicted to synthesize type I polyketides. Our model group is the Pertusariaceae, a family which is phylogenetically and chemically well characterized, and which contains secondary metabolites from various substance classes, such as depsides, depsidones, depsones, and chlorinated xanthones. The presence of certain secondary metabolites is correlated with monophyletic groups in a phylogeny based on rRNA genes. Aims of this project include assessing the diversity of PKS genes in the Pertusariaceae, reconstructing the evolutionary history of PKS genes in this group, and identifying putative subgroups of type I PKS genes. We are fishing for PKS genes using a PCR based approach. Segments of the KS domain are targeted with degenerate primers and cloned into E. coli. Up to six different PKSs could be extracted with a single primer pair. The sequences were subjected to a phylogenetic analysis to reveal clusterings of certain types of PKSs, or sequence similarities to PKSs of known function retrieved from GenBank.


Breeding systems in the lichen-forming fungal genus Cladonia

Seymour, F. A., Crittenden, P.D., Dickinson, M. J., Paoletti, M., Montiel, D. & Dyer, P. S.
School of Biology, University Park, University of Nottingham, Nottingham,
NG7 2RD, UK

Sex in lichenised fungi has been assumed to equate with outcrossing, but recent evidence using DNA fingerprinting methods has demonstrated homothallism (selfing) in the crustose lichens Graphis scripta and Ochrolechia parella. In further studies, we are examining the breeding systems in species of the lichen-forming fungal genus Cladonia that exhibit contrasting ecological lifestyles. Specimens of C. floerkeana, C. portentosa and C. galindezii were collected from single locations (from Derbyshire, England; Caithness, Scotland; and Adelaide Island, Antarctica, respectively), with replicate thalli collected at spots more than 10 metres apart. Three fruiting bodies from each thallus were excised and each used to generate eight single-spore sibling progeny. These were grown in axenic culture and DNA extracted for molecular investigations. Breeding systems were assessed using RAPD-PCR and AFLP analyses. The DNA fingerprinting techniques revealed that spores from the same apothecium were not genetically uniform, suggesting a heterothallic breeding system (obligate outbreeding) in these species. In addition to fingerprinting, we have also used a novel approach based on the analysis of mating-type genes, to determine breeding systems in Cladonia. Degenerate PCR primers were designed to amplify the high mobility group (HMG) gene characteristic of MAT-2 ascomycete mating-type loci. These primers were successfully used to amplify a region of the MAT-2 gene from C. galindezii and C. floerkeana. Significantly, the primers only produced amplicons from approximately 50% of the progeny tested. This result concurred with results obtained using DNA fingerprinting methods, indicating a heterothallic breeding system. Failure of PCR was thought to be due to presence of a MAT-1 rather than a MAT-2 gene at the mating-type idiomorph locus.


Testing of ecological roles of secondary lichen compounds

Solhaug, K. A. & Gauslaa, Y.
Department of Ecology and Natural Resource Management, Agricultural University of Norway, P.O.Box 5003, NO-1432 Ås, Norway

Secondary compounds can be extracted from air-dry thalli of many living lichen species with acetone without affecting metabolic activities of the organisms. This method has made it possible to expose lichen thalli with and without secondary compounds to various stresses and treatments to test hypotheses on assumed roles of these compounds in lichens. One hypothesis is a screening role of the secondary compounds against high levels of irradiation; another hypothesis is that they deter grazing.
We have used the acetone-rinsing method to show that the blue light absorbing cortical pigment parietin from Xanthoria parietina protects the lichen against high levels of photosynthetic active radiation (PAR). Although parietin also absorb UV-B strongly, exposure to natural UV-B levels in summer had no adverse effects on parietin-free thalli. In other species we have not found evidence of a UV-B protecting role of the UV absorbing cortical pigments atranorin and usnic acid. Cortical UV-B
transmittance is very low also after acetone rinsing.
Removal of colourless lichen compounds significantly increases grazing by snails. Therefore, colourless lichen compounds rather deter grazing than function as sunscreens. However, the coloured parietin protects against high PAR, not against snail grazing.
Resynthesis of parietin in parietin-free acetone-rinsed thalli was clearly dependent on UV-B exposure. Thalli exposed to visible light only showed hardly any resynthesis of parietin. Resynthesis of parietin by the mycobiont is dependent on photosynthates from the photobiont, since photosynthetically active irradiation stimulates parietin synthesis in UV-B exposed thalli. Exogenously added carbohydrates like ribitol, which is the carbohydrate delivered by the photobiont, increase the resynthesis substantially.


Does disturbance matter? A landscape genetic approach for Lobaria pulmonaria

Werth, S., Kalwij, J., Csencsics, D., Cornejo, C. & Scheidegger, C.
WSL Swiss Federal Research Institute, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland

Recent studies hypothesised that dispersal limitation leads to the susceptibility of lichens to forest management related disturbances. Dispersal and genetic diversity of the endangered epiphytic lichen L. pulmonaria were studied by molecular-genetic analyses of 39 demes (subpopulations) within a geographically restricted area in western Switzerland. In total, 811 thalli were analysed. Based on six polymorphic mycobiont-specific microsatellite loci, both polymorphic and monomorphic demes were found. There was significant genetic differentiation among thalli from the same tree, among thalli from different trees within demes and among demes. Only a weak positive trend was found between deme size and genetic diversity, and this effect was most pronounced at small deme sizes. Furthermore, genetic diversity was lower in demes that had formerly been disturbed by a stand-replacing fire as compared demes not affected by this disturbance. Genetic diversity was similar in those undisturbed demes and demes growing in habitats disturbed by former charcoal production. In the latter, overlapping tree generations were always present. We suggest that monomorphic demes at disturbed sites might either be single remnant genotypes or the result of recent colonisation by a single genotype. Significant positive spatial autocorrelation of genotypes at distances up to 100 m indicated dispersal of vegetative propagules at this spatial scale. The results suggest that in L. pulmonaria dispersal limitation prevents the colonisation of habitats at distances exceeding a few hundred meters.


How much do we know about morphogenesis and secondary growth of a lichen thallus?

Zavarzin, A.
St.Petersburg State University, St.Petersburg, Russia

Growth and developmental morphology of lichens has being randomly investigated since 19th century (Schwendener, 1860; Babikoff,1878; Bitter,1904; Rosendahl, 1907) with the significant progress beingmade recently (Honegger,1987, Poelt, 1995; series o studies by Jahns, Ott and others). Morphologically similar process of thallus
initial development was discovered (Ott, 1987; Jahns,1987, 1988) coupled with common developmental mechanisms employed at a later stages (Honegger, 1991, 1998). The secondary growth of the lichen thallus (in a form of either regeneration or development of vegetative propagules) is found to be also based on a few principles that seem to be equally realized by unrelated groups even when the resulting structures are morphologically different. Hence the lichens are representing ecological evolutionary line parallel to higher plants in terrestrial ecosystems, they possess a number of morphogenetic analogies (configuration of the three dimensional body, patterns of anatomical differentiation, organ-like structures of different origin). These analogies have been derived from the similar basic requirements for optimizing photosynthesis and metabolism, though based on a principally different "structural material" – fungal dimorphism in close contact with algal cells. Lichens obviously employ the full range of morphological abilities existing among fungi, however, the precise mechanisms of pattern formation remain poorly understood. The present knowledge suggests the existence of only limited number of pattern formation processes both during primary and secondary growth in lichens. However, these few principles are resulting in a significant diversity of morphological structures. True understanding of the morphogenetic mechanisms in lichens require comparative studies of development of morphologically similar but phylogeneticaly distant lichens and involvement into analysis available data on growth patterns in phanerogams.

top