Tripartite Lichen Research Articles

The Tripartite Lichen Ricasolia virens: Involvement of Cyanobacteria and Bacteria in Its Morphogenesis.

Ricasolia virens is an epiphytic lichen-forming fungus mainly distributed in Western Europe and Macaronesia in well-structured forests with ecological continuity that lack eutrophication. It is considered to be threatened or extinct in many territories in Europe (IUCN). Despite its biological and ecological relevance, studies on this taxon are scarce. The thalli are tripartite, and the mycobiont has a simultaneous symbiotic relationship with cyanobacteria and green microalgae, which represent interesting models to analyse the strategies and adaptations resulting from the interactions of lichen symbionts. The present study was designed to contribute to a better understanding of this taxon, which has shown a clear decline over the last century. The symbionts were identified by molecular analysis. The phycobiont is Symbiochloris reticulata, and the cyanobionts (Nostoc) are embedded in internal cephalodia. Light, transmission electron and low-temperature scanning microscopy techniques were used to investigate the thallus anatomy, ultrastructure of microalgae and ontogeny of pycnidia and cephalodia. The thalli are very similar to its closest relative, Ricasolia quercizans. The cellular ultrastructure of S. reticulata by TEM is provided. Non-photosynthetic bacteria located outside the upper cortex are introduced through migratory channels into the subcortical zone by the splitting of fungal hyphae. Cephalodia were very abundant, but never as external photosymbiodemes.

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones.

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen-fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well-supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.

Sectorial land snail damage to the lichen Argopsis friesiana could be explained by metabolite profiles

Notodiscus hookeri is the only native land snail of the sub-Antarctic present in Crozet Archipelago. Two shell ecophenotypes co-exist on Possession Island and feed exclusively on lichens [1] that are symbiotic organisms widely distributed in sub-Antarctic islands. To improve our understanding of lichen-snail trophic interactions, the endemic tripartite lichen Argopsis friesiana was selected. The snail belonging to the organic ecophenotype occurs sympatrically with A. friesiana. Profiling and quantification of the major secondary metabolites in the whole lichen were carried out by LC-DAD-MS. Two main compounds were identified, the depside atranorin (8.2 mg g-1 DM) and the depsidone argopsin. Besides, a screening of primary metabolites was carried out to determine which compounds could be useful for the snail energetic demands. Glutamate, glutamine, gamma-aminobutyric acid and alpha-alanine were the main amino acids found in the lichen in low concentrations (0.4 mg·g-1 DM). Carbohydrates, including arabitol (10.2 mg g-1), mannitol (4.0.10-4 mg g-1) and sucrose (3.0.10-4 mg g-1), were also identified. In cultivation, N. hookeri snails were fed with fructified thalli of A. friesiana. Snails (n = 20) ate the podetia cortex and phyllocladia but they clearly avoided apothecia, the reproductive organs. This sectorial damage of the lichen by the snail suggested a differential distribution of the metabolites. Therefore, a profiling of secondary compounds was performed by Direct Analysis in Real Time – Mass Spectroscopy (DART-MS) [2] on each morphological part of the lichen (phyllocladia, apothecia, podetia and cephalodia). The identification and distribution of secondary metabolites, fatty acids, amino acids and carbohydrates should help us to determine whether metabolites affect snail feeding choice and which of them could be involved.

The diversity and community dynamics of hazelwood lichens and bryophytes along a major gradient of human impact

Background: Oceanic hazelwoods in western Scotland are hypothesised to be unmanaged post-glacial relicts, representing an unusual type of old-growth forest habitat in Europe. They are characterised by an exceptionally high epiphytic diversity, including their status as ‘hotspots’ for indicators of woodland ecological continuity. Aims: The aim of this study was to evaluate the effects of climate, pollution and management on the occurrence of epiphytic lichens and bryophytes on Corylus avellana. Methods: Thirteen hazelwood study sites were systematically sampled along a climate, pollution and management gradient in Britain. Epiphyte composition and richness were examined in a strict hierarchical framework, and compared against site-, stool- and stem-scale environmental predictors. Results: The study showed that along the gradient from ‘clean-air’ relict sites to ‘polluted’ coppiced sites: (i) epiphytic local stem-scale diversity declined, (ii) there was a loss of late-successional species including foliose cyano- and tripartite lichens and bryophytes and (iii) stem sizes were reduced, providing a further limit to the accumulation of species richness within a site. Conclusions: Relict hazelwoods in western Scotland are confirmed as an example of the most intact epiphyte communities. In particular, we show that the transition to coppicing can be clearly linked to ecological processes causing species loss.

Gas chromatographic analysis to compare the fatty acid composition of fifteen lichen species, with a focus onStereocaulon

AbstractThe composition of fatty acids (FAs) is known to discriminate families within genera of bacteria, fungi, octocorals and algae. Here, we applied the more sensitive gas chromatographic (GC) analysis to investigate the taxonomic value of using FA composition to discriminate 15 species of lichen, including three chlorolichens (twoCladoniaspecies andStereocaulon scutelligerum), nine tripartite lichens (Stereocaulonspecies) and three cyanolichens (Lichina pygmaea,Collema cristatum,Peltigera membranacea). One macroscopic cyanobacterium (Nostocsp.), corresponding to the photobiont partner ofPeltigeraandCollemacyanolichens, was included for comparison. Five lipid extraction methods were evaluated, usingStereocaulon scutelligerum. Shaking at 80 °C with chloroform/methanol (2:1, v/v) was the most efficient and reproducible method. The total FA composition of the 16 species was compared, using GC coupled with flame ionization detection or mass spectrometry. A statistical hierarchical cluster analysis was used to determine the similarity between the FA profiles, based on total, saturated, unsaturated and branched-chain FAs (BCFAs). The BCFA profile allowed a distinction between theStereocaulonspecies and the cyanolichens, in contrast to all the other types of FAs. A detailed characterization of eight FA subclasses, provided by the GC analysis, suggested a preferential pathway for the biogenesis of unsaturated FAs through desaturases, which was especially favoured in the cyanolichens.

Characterization of the diversity of mycosporine-like amino acids in lichens from high altitude region of Himalaya.

Lichens are tolerant to a number of environmental variables including high-intensity solar radiations, which is mainly due to the presence of chemical substances in the thallus. Especially, cyanobacterial lichens synthesize a unique class of chemical substances known as mycosporine-like amino acids (MAAs) the primary characteristic of which is strong ultraviolet (UV) absorption between 300 and 360 nm. In view of its UV-protecting potential, the applicability of mass spectral fragmentation using electrospray ionization tandem mass spectrometric analysis for the characterization of MAAs in lichen samples was explored. MAA compounds were characterized in four cyanobacteria-containing lichen species belonging to genus Peltigera, Stereocaulon and Lobaria. Among them, Peltigera and Lobaria are true cyanobacteria containing lichens (cyanolichens) while Stereocaulon is a tripartite lichen, as it contains both green algae (in the thallus) and cyanobacteria (in the cephalodia), collected from higher altitudes of Himalaya (Tungnath-Chopta in Garhwal Himalaya, 3432 m) from an exposed locality experiencing high light intensity. Mass spectral data of distinctive fragmentation pattern revealed that all the four species have good diversity of MAA compounds, especially Lobaria retigera was found to be enriched with highest diversity of oxo and imino MAAs. Overall, different numbers of oxo and imino MAA compounds were detected in the remaining lichen species. Good diversity of imino MAAs has ecological significance which is required to be investigated further. Moreover, the impressive diversity characterized in each lichen species suggests that lichens should be thoroughly studied for their MAAs contents.

Fluorescence quenching in the lichen Peltigera aphthosa due to desiccation

Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.

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

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. 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 Zn(2+) solutions that stop cyanobacterial but not green algal photosynthesis. CO(2) 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. 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 Zn(2+) 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. 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.

Genetic structure in a fragmented Northern Hemisphere rainforest: large effective sizes and high connectivity among populations of the epiphytic lichenLobaria pulmonaria

An extraordinary diversity of epiphytic lichens is found in the boreal rainforest of central Norway, the highest-latitude rainforest in the world. These rainforest relicts are located in ravine systems, and clear cutting has increased the distance between remaining patches. We hypothesized that the relatively small lichen populations in the remaining forest stands have suffered a depletion of genetic diversity through bottlenecks and founder events. To test this hypothesis, we assessed genetic diversity and structure in the populations of the tripartite lichen Lobaria pulmonaria using eight SSR loci. We sampled thalli growing on Picea abies branches and propagules deposited in snow at three localities. Contrary to expectations, we found high genetic diversity in lichen and snow samples, and high effective sizes of the studied populations. Also, limited genetic differentiation between populations, high historical migration rates, and a high proportion of first generation immigrants were estimated, implying high connectivity across distances <30km. Almost all genetic variation was attributed to variation within sites; spatial genetic structures within populations were absent or appeared on small scales (5-10m). The high genetic diversity in the remaining old boreal rainforests shows that even relict forest patches might be suitable for conservation of genetic diversity.

Photoprotection of reaction centers: thermal dissipation of absorbed light energy vs charge separation in lichens‡

During desiccation, fluorescence emission and stable light-dependent charge separation in the reaction centers (RCs) of photosystem II (PSII) declined strongly in three different lichens: in Parmelia sulcata with an alga as the photobiont, in Peltigera neckeri with a cyanobacterium and in the tripartite lichen Lobaria pulmonaria. Most of the decline of fluorescence was caused by a decrease in the quantum efficiency of fluorescence emission. It indicated the activation of photoprotective thermal energy dissipation. Photochemical activity of the RCs was retained even after complete desiccation. It led to light-dependent absorption changes and found expression in reversible increases in fluorescence or in fluorescence quenching. Lowering the temperature changed the direction of fluorescence responses in P. sulcata. The observations are interpreted to show that reversible light-induced increases in fluorescence emission in desiccated lichens indicate the functionality of the RCs of PSII. Photoprotection is achieved by the drainage of light energy to dissipating centers outside the RCs before stable charge separation can take place. Reversible quenching of fluorescence by strong illumination is suggested to indicate the conversion of the RCs from energy conserving to energy dissipating units. This permits them to avoid photoinactivation. On hydration, re-conversion occurs to energy-conserving RCs.

Recent literature on lichens—217

Abbott, B. F. M. 2009. Checklist of the Lichens and Lichenicolous Fungi of Greece. Bibliotheca Lichenologica No. 103. J. Cramer in der Gebruder Borntraeger Verlagsbuchhandlung. Berlin and Stuttgart. 368 pages. [Exhaustive catalog of Greek lichens based on both literature records and unpublished specimens. Includes extensive listings of synonyms and Greek place names.] Abonyi, D. O., M. U. Adikwu, C. O. Esimone & E. C. Ibezim. 2009. Plants as sources of antiviral agents. African Journal of Biotechnology 8(17): 3989–3994. [Lichens are mentioned as one of the many plant sources.] Ahti, T. 2009. A tribute to John Walter Thomson (1913–2009). Lichenologist 41(6): 561–563. [Short article remembering his life and work, especially on arctic lichens.] Allen, A. & B. Hilton. 2010. [Review] C. W. Smith, A. Aptroot, B. J. Coppins, A. Fletcher, O. L. Gilbert, P. W. James & P. A. Wolseley (eds.). The Lichens of Great Britain and Ireland. The British Lichen Society, Botany Department, Natural History Museum, London. x + 1046 pages. 2009. Lichenologist 42(1): 123–125. Alstrup, V., J. Kocourkova, M. Kukwa, J. Motiejūnaitė, W. Von Brackel & A. Suija. 2009. The lichens and lichenicolous fungi of South Greenland. Folia Cryptogamica Estonica 46: 1–24. [Notes on 744 lichens, 156 lichenicolous and 6 allied fungi. Many species new to Greenland. New: Naetrocymbe kentrospora (Branth) Alstrup comb. nov.] ——— & U. Sochting. 2009. Galloea cladoniicola, a new hyphomycetous lichenicolous fungus from Denmark. Graphis Scripta 21(2): 33–35. [New: Galloea cladoniicola gen. et sp. nov.] Aptroot, A. 2009. A revision of the lichen genus Stirtonia. Lichenologist 41(6): 615–625. [Treatment of 13 species; key. Stirtonia neotropica (Netherlands Antilles), S. schummii (Seychelles) and S. curvata Aptroot spp. nov. (Indonesia).] ———. 2009. Diversity and endemism in the pyrenocarpous lichen families Pyrenulaceae and Trypetheliaceae in the Malesian flora region. Blumea 54(1–3): 145–147. [‘‘Species numbers in both families seem highest in Australia and Papua New Guinea, but some potentially rich regions are much undercollected. The numbers of local endemics are also highest in these areas, at least in the Pyrenulaceae, and remarkably high for lichens.’’] Armaleo, D. & S. May. 2009. Sizing the fungal and algal genomes of the lichen Cladonia grayi through quantitative PCR. Symbiosis 49(1): 43–51. [Authors report first genome size for a lichen, 28.6 Mb (mycobiont) and 106.7 Mb (Asterochloris photobiont). DNA was obtained from culture of soredia.] Armstrong, R. A. & S. N. Smith. 2009. Carbohydrates in the hypothallus and aerolae of the crustose lichen Rhizocarpon geographicum (L.) DC. Symbiosis 42(2): 95–100. [Lichen material from Wales. Authors report 8 carbohydrates present. The results suggest that the ‘‘ ... hypothallus has a lower demand for carbohydrates than the areolae or there is limited transport ...’’ and the ‘‘ ... increased trehalose in the non-lichenised hypothallus may be an adaptation to withstand stress and desiccation ....’’] Asplund, J. & Y. Gauslaa. 2010. The gastropod Arion fuscus prefers cyanobacterial to green algal parts of the tripartite lichen Nephroma arcticum due to low chemical defense. Lichenologist 42(1): 113–117. [Gastropods in experiments preferred cephalodia which lack secondary lichen metabolites. This feeding behavior reduces N concentration 1 Author e-mail: regan@unomaha.edu The cumulative database for this series is available in searchable form on the World Wide Web at http:// www.nhm.uio.no/botanisk/bot-mus/lav/sok_rll.htm. I owe special thanks to Bill Buck for providing copies of papers by other authors, which were otherwise unavailable to me, and to the cooperating authors who send reprints or electronic versions of their works to me for inclusion in this series. DOI: 10.1639/0007-2745-113.2.390

PHYLOGENETIC PATTERNS AMONG NOSTOC CYANOBIONTS WITHIN BI- AND TRIPARTITE LICHENS OF THE GENUS PANNARIA(1).

Phylogenetic relationships between Nostoc cyanobionts in the lichen genus Pannaria were studied to evaluate their correlation to geography, habitat ecology, and other patterns previously reported. The 16S rRNA gene sequences of a total of 37 samples of 21 Pannaria species from seven countries from the Northern and Southern hemispheres were analyzed and compared with 69 free-living and symbiotic cyanobacterial strains. The sequences from Pannaria were distributed throughout a branch of Nostoc sequences previously called "the Nephroma guild," and within two subgroups from another branch, referred to as the "Peltigera guild," although there was a gradual transition between the two major groups. There is a more diverse pattern of relationships between Nostoc sequences from bipartite versus tripartite lichen species in Pannaria, compared with other well-studied genera, such as Nephroma and Peltigera. Cyanobionts from several tripartite Pannaria species from the Southern Hemisphere and corticolous bipartite species from both hemispheres were grouped together. Four sequences of Pannaria and Pseudocyphellaria cyanobionts from rocks in the Chilean Juan Fernández Islands were nested within corticolous cyanobionts, whereas the terricolous "Pannaria sphinctrina clade" was placed with other terricolous strains. The cluster patterns derived from phylogenetic analysis were partly reflecting lichen taxonomy, in two groups of lichen species, possibly indicating coevolution. The phylogram partly also reflected lichen ecology. Three Pannaria species have very different cyanobiont strains when they grow in different habitats.

Effects of manganese on chlorophyll fluorescence in epiphytic cyano- and chlorolichens

Incubation with 10 mM MnCl 2 for 1 h decreased the effective quantum yield of photochemical energy conversion in photosystem 2 in the bipartite chlorolichen Hypogymnia physodes as well as in the bipartite cyanolichens Leptogium saturninum and Nephroma helveticum, but not in the tripartite lichen Lobaria pulmonaria. Among the bipartite species, Mn sensitivity increased in the order H. physodes < N. helveticum < L. saturninum. This equals the sequence heteromerous chlorolichen < heteromerous cyanolichen < homoiomerous (gelatinous) cyanolichen. MnCl 2 reduced non-photochemical quenching of chlorophyll fluorescence in the bipartite cyanolichens and in H. physodes; in the latter, however, this decrease was limited to light intensities above the adapted growth light intensity. Photochemical quenching was increased in H. physodes, but reduced in the bipartite cyanolichens. The results indicate that the bipartite cyanolichens L. saturninum and N. helveticum are even more sensitive to high Mn concentrations than the chlorolichen H. physodes, the low Mn tolerance of which has been already demonstrated. This agrees with results of field studies from western North America, where conifer bark under cyanolichens (including L. saturninum and N. helveticum) was found to contain less Mn than bark which only supported chlorolichens. The high sensitivity of the bipartite cyanolichens probably results from high sensitivity of the Nostoc photobiont. The high Mn tolerance of L. pulmonaria is probably not due to its being a tripartite lichen, but might be caused by high tolerance of the green-algal primary photobiont Dictyochloropsis, which is, however, not experimentally proven. The high Mn tolerance of the highly SO 2-sensitive L. pulmonaria shows that different mechanisms are responsible for Mn and SO 2 toxicity in lichens.

Lichen fungi have low cyanobiont selectivity in maritime Antarctica.

• The cyanobionts of lichens and free-living Nostoc strains from Livingston Island (maritime Antarctica) were examined to determine both the cyanobiont specificity of lichens and the spatial distribution of Nostoc strains under extreme environmental conditions. • We collected five different lichen species with cyanobacteria as primary or secondary photobiont (Massalongia carnosa, Leptogium puberulum, Psoroma cinnamomeum, Placopsis parellina and Placopsis contortuplicata) and free-living cyanobacteria from different sample sites and analysed them using the tRNALeu (UAA) intron as a genetic marker to identify the cyanobacterial strains. • Our results showed that the same Nostoc strain was shared by all five lichen species and that an additional strain was present in two of the lichens. Both Nostoc strains associated with lichen fungi also occurred free-living in their surrounding. Bi- and tri-partite lichens were not different in their cyanobiont selectivity. • Contrary to studies on different lichen species in temperate regions, the Antarctic lichen species here did not use species-specific cyanobionts; this could be because of a selection pressure in this extreme environment. Limiting factors under these ecological conditions favor more versatile mycobionts. This results in selection against photobiont specificity, a selection pressure that may be more important for lichen distribution than the effect of cold temperatures on metabolism.

CO2 exchange and thallus nitrogen across 75 contrasting lichen associations from different climate zones.

Aiming to investigate whether a carbon-to-nitrogen equilibrium model describes resource allocation in lichens, net photosynthesis (NP), respiration (R), concentrations of nitrogen (N), chlorophyll (Chl), chitin and ergosterol were investigated in 75 different lichen associations collected in Antarctica, Arctic Canada, boreal Sweden, and temperate/subtropical forests of Tenerife, South Africa and Japan. The lichens had various morphologies and represented seven photobiont and 41 mycobiont genera. Chl a, chitin and ergosterol were used as indirect markers of photobiont activity, fungal biomass and fungal respiration, respectively. The lichens were divided into three groups according to photobiont: (1) species with green algae, (2) species with cyanobacteria, and (3) tripartite species with green algal photobionts and cyanobacteria in cephalodia. Across species, thallus N concentration ranged from 1 to 50mg g-1 dry wt., NP varied 50-fold, and R 10-fold. In average, green algal lichens had the lowest, cyanobacterial Nostoc lichens the highest and tripartite lichens intermediate N concentrations. All three markers increased with thallus N concentration, and lichens with the highest Chl a and N concentrations had the highest rates of both P and R. Chl a alone accounted for ca. 30% of variation in NP and R across species. On average, the photosynthetic efficiency quotient [K F=(NPmax+R)/R)] ranged from 2.4 to 8.6, being higher in fruticose green algal lichens than in foliose Nostoc lichens. The former group invested more N in Chl a and this trait increased NPmax while decreasing R. In general terms, the investigated lichens invested N resources such that their maximal C input capacity matched their respiratory C demand around a similar (positive) equilibrium across species. However, it is not clear how this apparent optimisation of resource use is regulated in these symbiotic organisms.

Cyanobacterial lichen symbiosis: the fungal partner as an optimal harvester

Lichen symbiosis has been traditionally treated as a model case of mutualism in which both partners, the fungus and the photobiont, gain benefits reciprocally. Some recent evidence, however, supports an alternative view that lichen symbiosis may represent an association largely controlled by the commensal or even parasitic fungal partner. The latter gains photosynthates from the photobiont (algae and/or cyanobacteria) which may not always substantially benefit from the symbiosis. We analyze from this perspective how a lichen fungus may maximize photosynthetic gains in bipartite and tripartite associations. We treat the frequency of nitrogen‐fixing cells called heterocysts in cyanobacteria and the relative proportion of green algal cells vs. that of cyanobacteria per unit fungus as the variables to be manipulated for maximal carbon gain. The model predicts that even with a negligible cost of cephalodia (compartments containing cyanobacteria) it is in the interest of the tripartite lichen, first, to increase the heterocyst frequency, and second, keep the relative number of cyanobacteria considerably lower than that of green algae. Hence, the lichen fungus achieves higher fitness by making the cyanobacterial partner to specialize on N fixation. The available empirical data support these predictions as the reported heterocyst frequencies in bipartite lichens range from 2 to 8%, and in tripartite lichens between 10 and 55%. It is concluded that interaction asymmetry (i.e. commensalism or parasitism rather than mutualism) provides a sound basis to understand the high phenotypic plasticity expressed by fungi‐forming bipartite and tripartite associations with cyanobacteria and green algae.

Growth, nitrogen uptake, and resource allocation in the two tripartite lichens Nephroma arcticum and Peltigera aphthosa during nitrogen stress

Summary Lichen responses towards nitrogen stress, both increased exposure and deprivation of N, were investigated by measuring N uptake, growth, ergosterol, chitin and Chla in two tripartite nitrogen‐fixing species, Nephroma arctium and Peltigera aphthosa. The lichens were irrigated with different N forms, enriched in 15N to assess N uptake, during 3 months in the field, with a total N dosage of 500 mg m−2. Nitrogen deprivation was induced by removing the nitrogen‐fixing cephalodia. The lichens took up 11–134 mg N m−2 of the added N, corresponding to 1–4% of their total thallus N. Uptake was 4 times higher for NH4+ than for NO3−, and the highest 15N concentrations were found in newly synthesized tissue. Both forms of N stress affected thallus expansion rates in both species. It is concluded that the two lichens were able to maintain a balanced tissue N concentration despite large variations in N supply, and that assimilated N might be transported to growing apices. Alternatively, N assimilation from external sources might be greater in the margins than in the mature thallus. Thallus expansion was sensitive to N stress, apparently being tightly coupled to N assimilation.

Cyanolichens

CYANOLLICHENS Amar N. Rai and Birgitta Bergman Amar N. Rai (corresponding author; e-mail: rai_amar Ohotmail.com), Biochemistry Department, North Eastern Hill University, Shillong-793022, India; Birgitta Bergman, Department of Botany, Stockholm University, SE-10691 Stockholm, Sweden. INTRODUCTION This short review provides an overview of some physiological and biochemical aspects of cyano lichens. For more details of lichens in general and the aspects covered here in particular: see Ahmadjian (1993), Galun (1988), Nash (1996), Rai (1990) and Rai et al. (2000). Of all known fungi, 20% form lichens (13,500 species). Most are ascomycetes (98%), 1.6% deuteromycetes and 0.4% basidiomycetes. About 10% of lichen species are bipartite and 3-4% tripartite cyanolichens. The latter contain a green alga (phycobiont) as the primary photobiont. Sometimes both bipartite and tnrpartitemorpho types occur in the same thallus (chimeroid thallus). Lichens have a worldwide distribution, occurrng in the highest, hottest, coldest, wettest and driest habitats, yet they are extremely sensitive to pollution. Lichens have a stable and organised thallus morphology quite distinct from any of the sym bionts. They appear as crustose (small lobes or scales; e.g. Collema), foliose (flat and dorsiventral lobes; e.g. Peltigera) or fruticose (round or flat thalli, upright or hanging down from the substra tum; e.g. Stereocaulon). In the thallus, the partners remain extracellular to each other and can be isolated and grown in culture (facultative symbio sis), but the symbiosis remains stable under a var iety of environmental conditions in nature. The partners involved undergo balanced and synchron ised growth and development. This prevents any one partner outgrowing the other(s). Close physi cal contacts occur between the cyanobiont and the mycobiont. The cell walls are thinner (less sheath material), and specialised mycobiont hyphae and haustoria, showing transfer cell ultrastructure, have been noted. As the bulk of the thallus consists of mycobiont, the thallus interior ismicroaerobic. A lichen thallus may develop from propagules (phyllidia, isidia, soredia, and hormocystangia) of a pre-existing thallus or from fresh synthesis. The latter involves fresh acquisition of the cyanobiont from the environment. Similar modes of cyanobiont acquisition also apply to cephalodia development. THE CYANOBIONT A wide range of cyanobionts is found in lichen symbioses. Most common are the heterocystous fonns Nostoc, Scytonema, Calothrix and Fischerella (including Hyphomorpha, Stigonema and Mastigocladus). Several unicellular forms are also reported. These include Gloeocapsa (also Chroococ cus), Gloeothece and Synechocystis (alsoAphanocapsa). In bipartite lichens, cyanobionts are either dispersed throughout the thallus (e.g. Collema) or occur as a distinct layer below the upper cortex (e.g. Peltigera canina). In tripartite lichens, the cyanobionts occur in cephalodia. The latter occur at the upper' surface of the thallus (external cephalodia; e.g. in P. aphthosa) or inside the medulla (internal cephalodia; e.g. Nephroma arcticum). Intemal cephalodia could be close to the lower surface of the thallus (e.g. in P. venosa). Entry of the cyanobiont for development of inter nal cephalodia is from the lower surface of the thallus, but occasionally, when the cyanobiont enters from above, the phycobiont layer is pressed deep into the medulla. In tripartite lichens, there is never a direct contact between the cyanobiont and the phycobiont. Lectins of mycobiont origin have been impli cated in the recognition of the cyanobiont by a mycobiont. However, direct observations, lectin binding experiments and tRNALeU intron analysis all indicate a broad or low cyanobiont-mycobiont specificity. Different lichen species can have the same cyanobiont, and different cyanobionts have been reported among cephalodia of a single lichen thallus. Different Nostoc strains have been found in different lichen species from the same site, whereas different lichen species from distant places had the same Nostoc strain. In chimeroid thalli, both bipart ite and tnrpartitemorphotypes are reported to have the same cyanobiont strain. Overall, there is a great deal of cyanobiont diversity among lichens, and much of itmay be due to themode of cyanobiont acquisition during the development of the lichen thallus and/or cephalodia. STRUCTURAL-FUNCTIONAL CHANGES In the lichen thallus, cyanobionts show structural functional changes that permit close interaction and the development of nutrient exchange be tween the partners. The filamentous habit tends to become aseriate, and sometimes a filamentous cyanobiont appears unicellular. Other changes...

The effect of nitrogen on growth and key thallus components in the two tripartite lichens, Nephroma arcticum and Peltigera aphthosa

ABSTRACTRelationships between growth, nitrogen and concentration of unique biont components were investigated for the tripartite lichens Nephroma arcticum (L.) Torss. and Peltigera aphthosa (L.) Willd. Nitrogen availability was manipulated during 4 summer months by removing cephalodia and their associated N2 fixation activity, or by weekly irrigation with NH4NO3. Chlorophyll and ribulose 1·5‐biphosphate carboxylase/oxygenase (Rubisco), and chitin and ergosterol were used as photobiont and mycobiont markers, respectively. Nitrogen concentrations were similar in older and newer parts of the same thallus, varying between 2 and 5 g m−2, with P. aphthosa having higher concentrations than N. arcticum. Both chlorophyll (Chl a) and chitin were linearly correlated with thallus N, but N. arcticum invested more in fungal biomass and had lower Chl a concentrations in comparison with P. aphthosa at equal thallus N. During the 4 months, control and N‐fertilized thalli of N. arcticum increased in area by 0·2 m2 m−2 and P. aphthosa by 0·4 m2 m−2. Thallus expansion was significantly inhibited in samples without cephalodia, but there was no effect on lichen weight gain. Mean relative growth rate (RGR; mg g−1 d−1) was 3·8 for N. arcticum and 8·4 for P. aphthosa, when time (d) reflected the lichen wet periods. RGR was 2–3 times lower when based on the whole time, i.e. when including dry periods. The efficiency (e) of converting incident irradiance into lichen biomass was positively and linearly correlated with thallus Chl a concentration to the same extent in both species. The slower growth rates of N. arcticum, in comparison with P. aphthosa, could then be explained by their lower nitrogen and Chl a concentrations and a subsequently lower light energy conversion efficiency. Functional and dynamic aspects of resource allocation patterns of the two lichens are discussed in relation to the above findings.

Cyanobiont specificity in some Nostoc‐containing lichens and in a Peltigera aphthosa photosymbiodeme

The cyanobacterial symbionts in some Nostoc‐containing lichens were investigated using the nucleotide sequence of the highly variable cyanobacterial tRNALeu (UAA) intron. When comparing different Nostoc‐containing lichens, identical intron sequences were found in different samples of the same lichen species collected from two remote areas. This was true for all species where this comparison was made (Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and Nephroma arcticum (L.) Torss.). With one exception, a specific intron sequence was never found in more than one lichen species. However, for two of the species, Peltigera aphthosa and Nephroma arcticum, two different cyanobionts were found in different samples. By examining a P. aphthosa photosymbiodeme it could be shown that the same Nostoc is present in both bipartite and tripartite lobes of this lichen. It is thus possible for one cyanobiont/Nostoc to form the physiologically different symbioses that are found in bipartite and tripartite lichens. The connection between photobiont identity and secondary chemistry is discussed, as a correlation between differences in secondary chemistry and different cyanobionts/Nostocs in the species Peltigera neopolydactyla (Gyeln.) Gyeln. was observed. It is concluded that more knowledge concerning the photobiont will give us valuable information on many aspects of lichen biology.