Differenze Ultrastrutturali Di Alcune Specie Di Trebouxia Poste in Condizioni Di Illuminazione Differenti

Abstract

Ultrastructural changes in some species of « Trebouxia » under different light conditions. — Some species of the phycobiont alga Trebouxia (Tr. decolorans and Tr. albulescens), both isolated and grown on synthetic medium and still in the lichen, were examined in order to study the effect of light on the plastid ultrastructures. The species isolated from Buellia punctata and Xanthoria parietina were very sensitive to light condition and lost their chlorophyll content quickly. Striking ultrastructural changes were found in the algae grown under small light intensities and those which become achloric owing to strong light. In the latter, modifications of the Iamellar System were observed. The disappearance of Chlorophyll pigments was followed by a reduced electron density of the whole Iamellar system, as if were lacking the Iipidic compounds which are usually present and absorb fixators and dyers, thus allowing a good view. On the contrary, normal light conditions did not affect cultures of Trebouxia humicola, a free living alga. In the chloroplasts of the phycobiont species, unlike in the free living alga, grana were very close and sometimes formed very thick masses towards the edge of the chloroplast. It could not be ascertained whether such changes corresponded to a different composition of the lipoproteic compounds of the lamellar system.

Xanthoria parietina could grow in very lighted environments with no damage of the algae present in its thallus. The lichen thalluses, under different light conditions, showed very different colourings: the overlighted ones were rusty-red and the shadowed ones deep green. The chlorophyll content of the lichen thalluses with various shades (table 1) were very similar. The ultrastructural changes induced by strong light intensities in the phycobiont algae, kept in the lichen, were very small in respect of those observed in the same algae isolated and grown on synthetic medium and concerned the Iamellar system and the pyrenoid, above all. The rusty-red lichen showed a great number of stromatic lamellae, often with a parallel trend, so as to simulate a Iamellar system not organized in grana and often presented groups of lamellae concentrically arranged. In the pyrenoid of the algae from rusty-red thalluses, compared with the green ones, a much greater number of electron dense masses was observed, which are very thick and occupy the whole stromatic portion of the pyrenoid. But the Chlorophyll content did not decrease. Unlike the results of PEVELING, we noted that the electron dense masses (cited by the Author as « osmiophilic plastoglobules) were visible even after fixation with permanganate; the different numbers of these globules might depend on environmental factors. The phycobiont alga, when in the lichen thallus, could perhaps support strong light intensities, because pigments or compounds formed with the mycobiont or by it alone prevented the photooxidation of chlorophyll. Hypothetically a relationship might exist between the sensitivity of the phycobiont algae to light intensities and the content in antraquinonic pigments in the lichen thallus. But also using filters with absorption maxima similar to those of these pigments, the « in vitro » cultures of the phycobiont algae became achloric in the same time as the control ones.

Some Authors had found in Trebouxia humicola a different relationship between Chlorophyll pigments and carotinoids from that observed in the phycobiont species and had ascribed to it the greater resistence to strong light of the free living alga. Pigments or other substances present in the mycobiont can have a protective action on the Chlorophyll content and on the ultrastructures. In the phycobiont algae the resistence to strong light might be explained by an exchange of compounds between mycobiont and phycobiont, ending with the structural changes of the pyrenoid.     

Spatial organization of the three‐component lichen Peltigera aphthosa in functional terms

The cephalolichen Peltigera aphthosa (L.) Willd. is characterized by lateral heterogeneity, which manifests itself in the presence of three thallus zones, referred to as the apical, basal and medial zone. These zones differ in terms of interaction between lichen bionts and their physiological activity. The apical thallus zone is more efficient in establishing a contact with cyanobacteria, because of a higher lectin content and a larger overall thallus surface area due to the presence of numerous mycobiont hyphae. Cephalodia are formed in this zone. The interaction between the mycobiont and cyanobiont is more intense in the medial zone. However, the establishment of the contact with cyanobacteria in this zone less probable. The spatial distribution of lectins in the thallus was determined. To reveal the differences in photosynthetic activity in three thallus zones, transient analysis of chlorophyll a fluorescence and the assessment of non‐photochemical quenching of excited chlorophyll states were performed. Assimilation of absorbed light energy was more effective in the medial zone. The basal zone was characterized by decreased photosynthetic activity, lichen dissociation and thallus death.     

Proteins from the liehenXanthoria parietina which bind to phycobiont cell walls Localization in the intact liehen and cultured mycobiont

Summary A protein fraction, previously isolated from the lichenXanthoria parietina and known to bind to the appropriate culturedTrebouxia phycobiont, was visualized in the intact lichen thallus and cultured mycobiont by an indirect immunoperoxidase assay. The protein was localized in both the upper and lower cortices of the lichen thallus; it was also present in the cell walls of the mycobiont culturedin vitro. The possible role of this protein in the recognition, or initial interaction, between separated lichen symbionts is discussed.     

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.     

Developmental stages and secondary compound biosynthesis of mycobiont and whole thallus cultures of <Emphasis Type="Italic">Buellia subsororioides</Emphasis>

Lichen species have unique culture media preferences, and established cultures are known for the synthesis of secondary metabolites. This paper reports observations on the developmental stages and secondary compound biosynthesis by the mycobiont and whole thallus cultures of Buellia subsororioides. It also investigates the suitable media compositions for the culture growth, the role (nutrient or stressor) of sucrose concentrations on the growth stages, biomass, secondary compound profiles, and the quantity of biosynthesized known compounds/g of culture biomass for each treatment using mycobiont cultures. The ascospore-derived mycobiont cultures and thallus macerate-derived whole thallus cultures of B. subsororioides were established and grown using malt yeast extract (MY) medium. Mycobiont cultures were subcultured in MY medium supplemented with sucrose and its concentrations ranging from 0 to 30 % (with 2 % increment between treatments) for 120 days. The molecular identity of cultures was confirmed using nuclear ribosomal Internal transcribed spacer (ITS) DNA sequences obtained from the cultured mycobiont and from the natural thallus. The ITS DNA sequences of the mycobiont showed 99 % similarity with the sequences of the natural thallus. The mycobiont cultures under varying sucrose concentrations initiated as white cottony stages and transformed to brown compact mycelia, with optimum biomass and biosynthesis of nine secondary compounds in MY 10 %. The number of compounds (1–9) varies according to treatments. The whole thallus cultures (MY 0 %) showed a profile of secondary compounds similar to that of the natural thalli along with a trace of one unknown compound. The obtained results are encouraging for the synthesis of the desired quantities of lichen secondary compounds through cultures for relevant applications.     

Does aposymbiotically cultivated fungus Ramalina produce isolichenan?

The main α-glucan synthesized by lichens of the genera Ramalina in the symbiotic state is isolichenan. This polysaccharide was not found in the aposymbiotically cultivated symbionts. It is still unknown if this glucan is produced by the mycobiont only in the presence of a photobiont, in a lichen thallus, or if the isolichenan suppression is influenced by the composition of culture medium used in its aposymbiotic cultive. Consequently, the latter hypothesis is tested in this study. Cultures of the mycobiont Ramalina complanata were obtained from germinated ascospores and cultivated on 4% glucose Lilly and Barnett medium. Freeze-dried colonies were defatted and their carbohydrates extracted successively with hot water and aqueous 10% KOH, each at 100 °C. The polysaccharides nigeran, laminaran and galactomannan were liberated, along with a lentinan-type β-glucan and a heteropolysaccharide (Man : Gal : Glc, 21 : 28 : 51). Nevertheless, the α-glucan isolichenan was not found in the extracts. It follows that it was probably a symbiotic product, synthesized by the mycobiont only in this particular microenvironment, in the presence of the photobiont in the lichen thallus. A discussion about polysaccharides found in the symbiotic thallus as well as in other aposymbiotic cultivated Ramalina mycobionts is also included.     

Algal and Fungal Diversity in Antarctic Lichens

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7–100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen‐associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen‐associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen‐associated fungi was not evident.     

Cyanolichens can have both cyanobacteria and green algae in a common layer as major contributors to photosynthesis

Background and Aims

Cyanolichens are usually stated to be bipartite (mycobiont plus cyanobacterial photobiont). Analyses revealed green algal carbohydrates in supposedly cyanobacterial lichens (in the genera Pseudocyphellaria, Sticta and Peltigera). Investigations were carried out to determine if both cyanobacteria and green algae were present in these lichens and, if so, what were their roles.

Methods

The types of photobiont present were determined by light and fluorescence microscopy. Small carbohydrates were analysed to detect the presence of green algal metabolites. Thalli were treated with selected strengths of Zn²⁺ solutions that stop cyanobacterial but not green algal photosynthesis. CO₂ exchange was measured before and after treatment to determine the contribution of each photobiont to total thallus photosynthesis. Heterocyst frequencies were determined to clarify whether the cyanobacteria were modified for increased nitrogen fixation (high heterocyst frequencies) or were normal, vegetative cells.

Key Results

Several cyanobacterial lichens had green algae present in the photosynthetic layer of the thallus. The presence of the green algal transfer carbohydrate (ribitol) and the incomplete inhibition of thallus photosynthesis upon treatment with Zn²⁺ solutions showed that both photobionts contributed to the photosynthesis of the lichen thallus. Low heterocyst frequencies showed that, despite the presence of adjacent green algae, the cyanobacteria were not altered to increase nitrogen fixation.

Conclusions

These cyanobacterial lichens are a tripartite lichen symbiont combination in which the mycobiont has two primarily photosynthetic photobionts, ‘co-primary photobionts’, a cyanobacterium (dominant) and a green alga. This demonstrates high flexibility in photobiont choice by the mycobiont in the Peltigerales. Overall thallus appearance does not change whether one or two photobionts are present in the cyanobacterial thallus. This suggests that, if there is a photobiont effect on thallus structure, it is not specific to one or the other photobiont.     

Differential responses of lichen symbionts to enhanced nitrogen and phosphorus availability: an experiment with Cladina stellaris

BACKGROUND AND AIMS: Lichens can be both nitrogen- (N) and phosphorous- (P) limited and thus may be susceptible to nutrient enrichment. Nutrient enrichment with N and P may have differing impacts on the lichen structure because of different physiological responses of fungal and algal partners to these nutrients. The hypothesis was tested that the differential responses of lichen symbionts to enhanced availability of N and P is reflected in the lichen thallus structure and the wall-to-wall interface between the algal and fungal cells. METHODS: Lichen cushions of Cladonia stellaris were treated with one P and two N concentrations alone and in combination that yielded total depositions of approx. 300 (moderate) and 1000 (high) mg N m(-2) and 100 (high) mg P m(-2) over an experiment lasting 14 weeks. The effects of N and P inputs on the relative volumes of fungal and algal cell in the medullary tissue and on the thallus structure were studied using light microscopy. The interface between algal and fungal cell walls was examined using transmission electron microscopy. KEY RESULTS: The influence of excess P on the lichen thallus structure was stronger than that of additional N. Addition of P reduced the N : P ratio in podetia, the proportion of the medullary layer volume occupied by the algal cells, the thallus volume occupied by the internal lumen, and the algal cell-wall area covered by fungal hyphae. CONCLUSIONS: Ecologically realistic changes in the availability of key macronutrients can alter the growth of symbionts. Reduction in the proportion of photobiont cells indicates that the application of P either stimulates fungal hyphal growth in the medullary tissue or impairs the cell division of the algal cells. The results suggest that both the N and P availability and thallus N : P ratio affect the growth rates of lichen symbionts.     

Fine structure of the vegetative thallus of the lichen,Peltigera polydactyla

Summary An investigation was made of the vegetative thallus of the lichen Peltigera polydactyla. Using a modified embedding technique it was shown that the ultrastructure of the mycobiont was not radically different from that of nonlichenized Discomycetes, and that the ultrastructure of the phycobiont was like that of the blue-green alga Nostoc. In addition to what were considered healthy blue-green algal cells other cells were present which obviously were breaking down. Well defined heterocysts were also present. No haustoria were found in the thallus.     

Endolichenic Fungal Community Analysis by Pure Culture Isolation and Metabarcoding: A Case Study of Parmotrema tinctorum

Lichen is a symbiotic mutualism of mycobiont and photobiont that harbors diverse organisms including endolichenic fungi (ELF). Despite the taxonomic and ecological significance of ELF, no comparative investigation of an ELF community involving isolation of a pure culture and high-throughput sequencing has been conducted. Thus, we analyzed the ELF community in Parmotrema tinctorum by culture and metabarcoding. Alpha diversity of the ELF community was notably greater in metabarcoding than in culture-based analysis. Taxonomic proportions of the ELF community estimated by metabarcoding and by culture analyses showed remarkable differences: Sordariomycetes was the most dominant fungal class in culture-based analysis, while Dothideomycetes was the most abundant in metabarcoding analysis. Thirty-seven operational taxonomic units (OTUs) were commonly observed by culture- and metabarcoding-based analyses but relative abundances differed: most of common OTUs were underrepresented in metabarcoding. The ELF community differed in lichen segments and thalli in metabarcoding analysis. Dissimilarity of ELF community intra lichen thallus increased with thallus segment distance; inter-thallus ELF community dissimilarity was significantly greater than intra-thallus ELF community dissimilarity. Finally, we tested how many fungal sequence reads would be needed to ELF diversity with relationship assays between numbers of lichen segments and saturation patterns of OTU richness and sample coverage. At least 6000 sequence reads per lichen thallus were sufficient for prediction of overall ELF community diversity and 50,000 reads per thallus were enough to observe rare taxa of ELF.     

Asymmetric Co-evolution in the Lichen Symbiosis Caused by a Limited Capacity for Adaptation in the Photobiont

It is proposed that lichen photobionts, compared to mycobionts, have very limited capacity to evolve adaptations to lichenization, so that the symbionts in lichens do not co-evolve. This is because lichens have (a) no sequential selection of photobiont cells from one lichen into another needed for Darwinian natural selection and (b) no photobiont sexual reproduction in the thallus. Molecular studies of lichen photobionts indicate no predictable patterns of photobiont lineages that occur in lichens so supporting this proposal. Any adaptation by photobionts accumulating beneficial mutations for lichenization is probably insignificant compared to the rate of mycobiont adaptation. This proposal poses questions for research relating the photobiont sexual cycle (genetic and cellular), the fate of photobiont lineages after lichenization, whether lineages of photobionts in thalli change with time, thallus formation by from spores as well as carbohydrate movement from photobionts to mycobionts and regulation of co-development of the symbionts in the thallus.     

The most ancient terrestrial lichen <Emphasis Type="Italic">Winfrenatia reticulata</Emphasis>: A new find and new interpretation

Silicified fossils from Rhynie cherts in Scotland are studied. A lichen belonging to the genus Winfrenatia is detected and studied. This oldest terrestrial lichen is dated to the Pragian (=Siegenian) of the Early Devonian. New characters of the lichen are described, and their new interpretation is given. The main component of the lichen thallus is a filamentous cyanobacterium (Nostocales). Structures which were interpreted as fungal hyphae are probably hollow sheaths of this cyanobacterium. Mycobiont hyphae develop at the base of the thallus and symbiose with a coccoid cyanobacterium. Thus, Winfrenatia reticulata is a three-parted organism, constituted of a mycobiont and filamentous and coccoid cyanobacteria.     

Can Antarctic lichens acclimatize to changes in temperature?

The Antarctic Peninsula, a tundra biome dominated by lichens and bryophytes, is an ecozone undergoing rapid temperature shifts. Such changes may demand a high physiological plasticity of the local lichen species to maintain their role as key drivers in this pristine habitat. This study examines the response of net photosynthesis and respiration to increasing temperatures for three Antarctic lichen species with different ecological response amplitudes. We hypothesize that negative effects caused by increased temperatures can be mitigated by thermal acclimation of respiration and/or photosynthesis. The fully controlled growth chamber experiment simulated intermediate and extreme temperature increases over the time course of 6 weeks. Results showed that, in contrast to our hypothesis, none of the species was able to down‐regulate temperature‐driven respiratory losses through thermal acclimation of respiration. Instead, severe effects on photobiont vitality demonstrated that temperatures around 15°C mark the upper limit for the two species restricted to the Antarctic, and when mycobiont demands exceeded the photobiont capacity they could not survive within the lichen thallus. In contrast, the widespread lichen species was able to recover its homoeostasis by rapidly increasing net photosynthesis. We conclude that to understand the complete lichen response, acclimation processes of both symbionts, the photo‐ and the mycobiont, have to be evaluated separately. As a result, we postulate that any acclimation processes in lichen are species‐specific. This, together with the high degree of response variability and sensitivity to temperature in different species that co‐occur spatially close, complicates any predictions regarding future community composition in the Antarctic. Nevertheless, our results suggest that species with a broad ecological amplitude may be favoured with on‐going changes in temperature.     

Symbiotic performance,nitrogen flux and growth of lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) varieties inoculated with different indigenous strains of rhizobia

Nitrate uptake and nitrogen inclusion into amino acids were studied in the intact thallus and isolated bionts of the lichen Parmelia sulcata with the aid of mass spectroscopic tracing of heavy isotope ¹⁵N. The isolated photobiont, the green algae Trebouxia sp. did not take up nitrate, whereas the mycobiont and intact thalli were enriched in ¹⁵N when incubated with Na¹⁵NO₃. Pulse feeding experiments with intact thalli followed by separation of photobiont showed that the labelled nitrate was originally assimilated by the mycobiont and only after that was detected in the photobiont. The isolated mycobiont after pulse labeling excreted labeled compounds into the incubation medium. Amino acids were detected in the exudate. The quantities of two amino acids considerably exceeded those of the others. One was identified as alanine, the other could not yet be identified with certainty. Both of these high-quantity compounds were also much more enriched in ¹⁵N than the others. These two compounds are proposed to be the transport forms of nitrogen within the Parmelia sulcata thallus.     

Culture studies on the mycobiont isolated from Parmotrema reticulatum (Taylor) Choisy: metabolite production under different conditions

A strain of the lichen mycobiont isolated from a thallus of Parmotrema reticulatum was cultured axenically on different media. The morphology, anatomy, growth of the colonies, and metabolite production were studied. The isolated fungal colonies developed well and showed a remarkable morphogenetic capacity on most of the assayed solid media, e.g., malt extract 2%-yeast extract 0.2% (MEYE), malt extract 1%-yeast extract 0.4%-sucrose 10% (MY10), and the original Lilly & Barnett medium (LB). The identity of the isolated fungus was confirmed by its ITS rDNA-sequence. Atranorin, the major cortical lichen depside, was produced when the colonies were grown over 5 and 10 months on solid LB medium, combined with a dessication treatment. Atranorin was identified by matching of UV spectra obtained from HPLC running and a reference substance in a spectrum library. Colonies grown on MEYE and MY10 with a dessication treatment did not produce any lichen secondary metabolite. Mycobionts grown for 5 months on solid MEYE without a dessication treatment produced triacylglycerides as the major metabolites, and the fatty acids were characterized as their methyl esters. Analysis by TLC and HPLC-DAD of extracts of colonies grown on LB and MY10 without dessication revealed that the typical secondary compounds of the natural lichen were not produced. The major metabolites of the natural lichen thallus were identified by chromatographic and spectroscopic methods.     

INVESTIGATIONS ON LICHEN SYNTHESIS

Ahmadjian , V. (Clark U., Worcester, Mass.) Investigations on lichen synthesis. Amer. Jour. Bot. 49(3): 277–283. Illus. 1962.—Separated fungal and algal components of the lichen, Acarospora fuscata (Nyl.) Am., were recombined under controlled laboratory conditions to form structures comparable to those of the naturally occurring lichen thallus. The primary condition for this artificial synthesis was the absence of organic and inorganic supplements to the agar substrate. Thus, in effect, the mycobiont was forced into union with the phycobiont. It was demonstrated that both alga and fungus derived benefit from the lichenized association.     

A new species of Phyllopsora (Lecanorales, lichen-forming Ascomycota) from Dominican amber, with remarks on the fossil history of lichens

Phyllopsora dominicanus sp. nov. (Bacidiaceae, Lecanorales,lichen-forming Ascomycota) is described and illustrated fromDominican amber. The diagnostic features of the lichen includea minute subfolious thallus of lacinulate, ascending squamules,a well-developed upper cortex, and a net-like pseudocortex onthe lower surface. The algal symbionts are unicellular greenalgae, forming a distinct layer immediately below the uppercortex. The fossil demonstrates that distinguishing featuresof Phyllopsora have remained unchanged for tens of millionsof years. The fossil also provides the first detailed viewsof mycobiont–photobiont contacts in Tertiary green algallichens. The mycobiont hyphae formed apical and intercalaryappressoria by pressing closely against the photobiont cells.This indicates that a conserved maintenance of structure isalso seen in the fine details of the fungal–algal interface. Key words: Amber, fossil, fungi, lichen, Phyllopsora, symbiosis, Tertiary Received 30 May 2007; Revised 30 November 2007 Accepted 21 December 2007     

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.