Fungal Diversity in Rock Beneath a Crustose Lichen as Revealed by Molecular Markers

Lichen-forming fungi have been assumed to be more or less restricted to the surface of the substrate on which they grow, Conclusive identification of hyphae or an assessment of the fungal diversity inside lichen-covered rock has not been possible using methods based on direct observation. We circumvented this problem by using a DNA sequencing approach. Cores were drilled from a Devonian arcosic sandstone rock harboring the crustose lichen Ophioparma ventosa (L.) Norman on the surface. The cores were cut vertically, and DNA was extracted from the pulverized rock slices. A series of polymerase chain reactions using fungal-specific primers as well as Ophioparma ventosa specific primers were employed to amplify the internal transcribed spacer region of the nuclear ribosomal DNA. The results show that hyphae of O. ventosa penetrate approximately 10–12 mm into the rock. Consequently, the hyphal layer formed by the lichen fungus inside the rock could be 7–12 times as thick as the symbiotic thallus at the surface of the rock. In addition, eight non-lichenized fungal taxa and five that could not be identified to species were encountered. One fungal species in the order Helotiales occurs in six of the eight cores. The significance of these results to the colonization and weathering of rock by lichenized fungi is discussed.     

Lichenized species of Omphalina (Tricholomataceae) in the Iberian Peninsula

Abstract:A taxonomic revision of the lichenized species of the genus Omphalina in the Iberian Peninsula is presented, based upon fungal and lichen herbarium material, as well as on fresh collections. Relevant morphological and ecological features are discussed and a diagnostic key is provided. Four lichenized species, O. ericetorum, O. hudsoniana, O. meridionalis and O. velutina, are recognized. A squamulose vegetative thallus (Coriscium -type) is present only in O. hudsoniana, while the other three species form a thallus of hyphal globules (Botrydina -type). Anatomical features of the vegetative thallus cannot be used to distinguish between these last three species. Whilst O. hudsoniana is known only from the montane belt of the Eurosiberian Region and O. meridionalis seems to be restricted to the Mediterranean Region, living in more or less humid sites of the Supramediterranean belt, the other two species, O. ericetorum and O. velutina, are widespread.     

Generation of primary amide glucosides from cyanogenic glucosides

The cyanogenic glucoside-related compound prunasinamide, (2R)-β-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H₂O₂ has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.     

El género Ochrolechia A. Massai. (Pertusariaceae,liquenes) en Espana y Portugal

The genus Ochrolechia is widespread in Spain and Portugal and now includes 14 species, 1 form and 4 chemical races (11 species and 3 chemical races are epiphytes, 2 species, 1 form and 1 chemical race are saxicolous and one species is muscicolous). We propose a new combination: O. dalmatica (Erichsen) Boqueras. Eight species are excluded from the Spanish and Portuguese floras: O. aggregata (Bagl.) Verseghy, O. anomala Verseghy, O. glaucescens (Hue) Zahlbr., O.frigida (Sw.) Lynge, O. pseudotartarea (Vain.) Verseghy, O. rosella Müll. Arg., O. subpallescens Verseghy, O. tenuissima Verseghy, O. crozalsiana Clauzade et Vêzda is included in O. tartarea. The criteria for the identification of these species are: 1 ) the chemical substances (the presence/absence of gyrophoric acid in the thallus and/or in the apothecia determines the groups inside of the genus, the presence of alectoronic acid determines the chemical races); 2) thallus with or without soredia or isidia; 3) the presence of pruina on the disc; and 4) the substratum. A key for the identification to all mentioned species is included.     

Antioxidant and antimicrobial properties of the lichens <Emphasis Type="Italic">Anaptychya ciliaris,Nephroma parile,Ochrolechia tartarea</Emphasis> and <Emphasis Type="Italic">Parmelia centrifuga</Emphasis>

The aim of this study was to characterize the antioxidant and antimicrobial activity of the methanol extracts of: Anaptychya ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga. The methanol extract of the P. centrifuga showed a strong antioxidant activity, in comparison to the extracts from A. ciliaris, O. tartarea and N. parile which were relatively weaker. Furthermore, the methanol extract of the lichen P. centrifuga was shown to contain the highest total phenol content (54.19 mg/g of the dry extract). Interestingly, a statistically significant positive relationship between the antioxidant activity and the total phenol content was observed. The minimum inhibitory concentration (MIC) against six bacterial and eleven fungi was established for the methanol extracts from each of species of lichens. The methanol extracts of the lichens P. centrifuga and O. tartarea showed the strongest both antibacterial and antifungal activity. Taken together, the results from this study suggest that the lichens P. centrifuga and O. tartarea may be used as a natural source of antioxidants in addition to providing certain antimicrobial features.     

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research I. Morphological and Anatomical Characteristics

Lichens are symbioses of two organisms, a fungal mycobiont and a photoautotrophic photobiont. In nature, many lichens tolerate extreme environmental conditions and thus became valuable models in astrobiological research to fathom biological resistance towards non-terrestrial conditions; including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. All studies demonstrated the high resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides other adaptations, this study focuses on the morphological and anatomical traits by comparing five lichen species—Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, Buellia frigida, Pleopsidium chlorophanum—used in present-day astrobiological research. Detailed investigation of thallus organization by microscopy methods allows to study the effect of morphology on lichen resistance and forms a basis for interpreting data of recent and future experiments. All investigated lichens reveal a common heteromerous thallus structure but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum, and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa, as well as pigmented cortices and basal thallus protrusions in P. chlorophanum. Thus, generalizations on morphologically conferred resistance have to be avoided. Such differences might reflect the diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting extreme terrestrial conditions and may explain the high resistance of lichens found in astrobiological research.     

Sequence Variation of the tRNALeu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA^Leu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA^Leu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA^Leu (UAA) intron can be of great value when examining cyanobacterial diversity.     

Copper toxicity to five Parmelia lichens in vitro

Treatment of Parmelia caperata, P. perlata, P. subrudecta, P. sulcata and P. tiliacea with CuSO₄ resulted in a time- and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus, indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached intracellular sites in the thallus, and K⁺/Cu²⁺ exchange was not electroneutral. After 5 h of exposure to copper, the extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with copper absorption levels, but only at 0.05<P<0.10, suggesting that membrane damage was proportional to the amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall. Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic, and usnic acids. Titration of the extracts with copper and assay of the free Cu²⁺ concentration revealed the presence of copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu²⁺ to each of the lichen substances detected in the extracts. However, no significant correlation (P>0.10) was found between the Cu²⁺-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a simple function of the Cu²⁺-binding properties of the lichen substances present in the thallus. Several hypotheses were formulated to interpret the results.     

Krogia coralloides,a new lichen genus and species from Mauritius

Abstract:The new corticolous lichen genus Krogia and species K. coralloides are described from humid forest in Mauritius. The genus shows affinities with Phyllopsora, but differs in characters of the ascus, ascospores, and thallus anatomy. The ascal characters make its inclusion in the Bacidiaceae orPhyllopsoraceae problematic. The lichen contains boninic acid and an apparently related compound.     

Starting from scratch: Evolution of the lichen thallus in the basidiolichen Dictyonema (Agaricales: Hygrophoraceae)

Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought to represent only a single genus with few species, includes several well-supported genus-level clades, all of which form associations with a unique lineage of obligately lichenized cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nuclear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny obtained via maximum likelihood and Bayesian approaches indicates the presence of five genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species, a paraphyletic grade representing Dictyonema s.str., and three clades representing the genera Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the lichenized thallus in the group, ancestral character state reconstruction was done using six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal sheath and cyphelloid–stereoid basidiocarps partially incorporated into the lichen thallus in Dictyonema, to squamulose–foliose thalli with corticioid basidiocarps entirely supported by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that, at least in this case, lichenized thalli may have evolved from reproductive structures in their nonlichenized ancestors.     

New species or interesting records of foliicolous lichens. VIII. Two new taxa from tropical Africa,with a key to sorediate Fellhanera species

Abstract:Two new species belonging to the lichen families Pilocarpaceae andEctolechiaceae are described from tropical Africa: Calopadia lucida sp. nov. (Ectolechiaceae) from Tanzania, being similar to C. puiggarii but differing in the UV fluorescent thallus and the pruinose apothecia, andFellhanera ivoriensis sp. nov. (Pilocarpaceae) from the Ivory Coast, differing from the related F. rhapidophylli by the sorediate thallus and from other sorediate taxa by the combination of pale soralia, dark brown apothecia, and the ellipsoid-bacillar conidia. A key to the nine sorediate species of Fellhanera is provided.     

Colonization by lichens and the development of lichen-dominated communities in the maritime Antarctic

Three long-term studies of lichen growth and colonization have been undertaken at Signy Island, South Orkney Islands, in the maritime Antarctic. Small individual thalli of several crustose species and uncolonized plots on 12 fresh rock surfaces were photographically monitored at intervals of 3–4 years over a period of up to 20 years. The development of Ochrolechia frigida colonies on a regenerating moss bank, recently uncovered by a receding glacier, was similarly monitored. The results indicate that many lichens growing in sites enriched by nitrogenous compounds derived from populations of sea birds, have relatively rapid colonization and growth rates. Mean percentage increase in thallus area can be as high as 15–32% per annum in some nitrophilous saxicolous species (e.g. Acarospora macrocyclos, Xanthoria elegans and species of Buellia and Caloplaca), but as low as 0·4–4% in nitrophobous species (Lecanora physciella, Lecidea sp., Rhizocarpon geographicum). Umbilicaria antarctica and Usnea antarctica also yielded data indicating high growth rates, with colonist plants reaching several centimetres after 20 years. Colonization by mixed assemblages of lichens of new rock surfaces can attain 40→90% cover after 20 years in nutrient-enriched sites, and even 20–25% in non-biotically influenced sites. Colonization by or increase in extant O. frigida on the regenerating moribund moss bank was also quite rapid. It is suggested that the ~exceptionally large thalli of several lichen species and the locally extensive dense lichen fellfield communities in the maritime Antarctic may be much younger than previously supposed.     

Secondary chemistry of cultured mycobionts: formation of a complete chemosyndrome by the lichen fungus of Lobaria spathulata

Abstract:The study aimed to optimize culture conditions and nutrient requirements for the production of secondary metabolites by the cultured mycobiont Lobaria spathulata. This species proved to be an excellent model system for such studies, as the complete chemosyndrome found in the natural lichen thallus was repeatedly formed in the cultured mycobiont with differentiated, aerial mycelia. Nutrient media containing the disaccharide, sucrose, were found to favour both rapid growth and the production of typical lichen substances. Higher proportions of the secondary compounds were detected in the developing mycobiont than in mature lichen thalli.     

Rinodina turfaceoides,a new corticolous,blastidiate species from the Iberian Peninsula

Abstract:A new corticolous lichen, Rinodina turfaceoides, is described from the Serra de Estrela (Portugal). It is characterized mainly by the blastidiate thallus, large apothecia covered by a thick, yellow-orange pruina, large Physcia -type ascospores and chemistry. The new species is compared with the closely related but mainly muscicolous, R. turfacea, as well as with other corticolous Rinodina species with vegetative propagules, including R. colobinoides and R. disjuncta. Habit photographs of the new species and illustrations of its ascospores are also provided.     

Kroswia,a new genus in the Pannariaceae (lichenized ascomycetes)

Abstract:A characteristic, sterile lichen genus with homoiomerous thallus containing terpenoids is described, with one subtropical species, Kroswia crystalliferum sp. nov., occurring scattered in Africa from Kenya south to the Cape, southern India (with Sri Lanka) and Taiwan. Another temperate species, Kroswia gemmascens (Nyl.) comb. nov., is restricted to Eastern Asia. In spite of its gelatinous, homoiomerous thallus, the genus appears to belong to thePannariaceae .     

Effect of low water potential on photosynthesis in intact lichens and their liberated algal components

Earlier experiments (T.D. Brock 1975, Planta124, 13–23) addressed the question whether the fungus of the lichen thallus might enable the algal component to function when moisture stress is such that the algal component would be unable to function under free-living conditions. It was concluded that the liberated phycobiont in ground lichen thalli could not photosynthesize at water potentials as low as those at which the same alga could when it was present within the thallus. However, our experience with lichen photosynthesis has not substantiated this finding. Using instrumentation developed since the mid-1970's to measure photosynthesis and control humidity, we repeated Brock's experiments. When applying “matric” water stress (equilibrium with air of constant relative humidity) we were unable to confirm the earlier results for three lichen species including one of the species,Letharia vulpina, had also been used by Brock. We found no difference between the effects of low water potential on intact lichens and their liberated algal components (ground thallus material and isolated algae) and no indication that the fungal component of the lichen symbiosis protects the phycobiont from the adverse effects of desiccation once equilibrium conditions are reached. The photosynthetic apparatus of the phycobiont alone proved to be highly adapted to water stress as it possesses not only the capability of functioning under extremely low degrees of hydration but also of becoming reactivated solely by water vapor uptake.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

Lichens from gambia,with a new black-fruiting isidiate caloplaca on savannah trees

Twenty-five lichen species are reported from Gambia. The corticolous isidiate Caloplaca gambiensis is described as new to science. It is characterized by a blackish, isidiate thallus and brown to black apothecia. It occurs on exposed trees in savannah area, where the lichen flora is dominated by Physciaceae. All 27 species mentioned are new records for Gambia; Trypethelium nigroporum is also new to Africa.     

Do secondary substances in the thallus of a lichen promote CO2 diffusion and prevent depression of net photosynthesis at high water content?

Many lichens show seriously depressed net photosynthesis (NP) at high thallus water contents due to increased carbon dioxide diffusion resistance through blockage of diffusion pathways by water. The soil lichen Diploschistes muscorum, however, shows no depression and NP is close to maximal even at the highest thallus water content. We investigated whether lichen substances (lecanoric and diploschistesic acids) in the cortex and medulla contributed to this ability to maintain high NP. Dry thalli were extracted with water-free acetone and, after this treatment, were found to be fully viable to the extent of continued growth after replanting in the field. No differences were found in the response of NP to thallus water content between the normal and extracted thalli, in fact the response curves were often nearly identical. Thus, in this species it seems that lichen substances did not maintain the water-free diffusion pathways and some other explanation, possibly structural, needs to be sought. Received: 5 April 1997 / Accepted: 26 April 1997