The green algal photobionts of 12 Xanthoria, seven Xanthomendoza, two Teloschistes species and Josefpoeltia parva (all Teloschistaceae) were analyzed. Xanthoria parietina was sampled on four continents. More than 300 photobiont isolates were brought into sterile culture. The nuclear ribosomal internal transcribed spacer region (nrITS; 101 sequences) and the large subunit of the RuBiSco gene (rbcL; 54 sequences) of either whole lichen DNA or photobiont isolates were phylogenetically analyzed. ITS and rbcL phylogenies were congruent, although some subclades had low bootstrap support. Trebouxia arboricola,T. decolorans and closely related, unnamed Trebouxia species, all belonging to clade A, were found as photobionts of Xanthoria species. Xanthomendoza species associated with either T. decolorans (clade A), T. impressa, T. gelatinosa (clade I) or with an unnamed Trebouxia species. Trebouxia gelatinosa genotypes (clade I) were the photobionts of Teloschistes chrysophthalmus,T. hosseusianus and Josefpoeltia parva. Only weak correlations between distribution patterns of algal genotypes and environmental conditions or geographical location were observed.

Photobiont diversity within populations of Xanthoria parietina was studied at the species level by means of ITS analyses and at the subspecific level with fingerprinting techniques (RAPD-PCR) applied to sterile cultured algal isolates. Populations from coastal, rural and urban sites from NW, SW and central France and from NE Switzerland were investigated. Between 8 and 63 samples per population, altogether 150 isolates, were subjected to phenetic and ordination analyses. Epiphytic samples of X. parietina associated with different genotypes of Trebouxia decolorans but saxicolous samples contained T. arboricola. For comparison the T. gelatinosa photobiont of a small population of Teloschistes chrysophthalmus (4 samples) was investigated. ITS sequences of T. decolorans isolates from different geographic locations were largely similar. In all populations a surprisingly high diversity of genotypes was observed in Trebouxia isolated from lichen thalli growing side by side. As Trebouxia spp. are assumed to be asexually reproducing haplonts, the genetic background of this diversity is discussed. Fingerprinting techniques are a powerful tool for obtaining valuable insights into the genetic diversity within the algal partner of lichen-forming fungi at the population level, provided that sterile cultured isolates are available.

The genetic diversity within and among populations of Xanthoria parietina was studied at the subspecific level with a fingerprinting technique (RAPD-PCR) applied to sterile cultured multispore isolates, each being derived from a single apothecium. Populations from coastal, rural and urban sites from NW, SW and central France and from NE Switzerland were investigated. Between 1 and 8 microsites of a few decimetres square, each comprising 13 to 27 thalli of X. parietina, were analysed per population. A total of 132 isolates from epiphytic and 3 isolates from epilithic specimens were investigated. Phenotypic variation was recorded among some of the thalli in the field and among sterile cultured isolates in the laboratory. A high diversity of genotypes was observed, even among thalli growing side by side in phenotypically homogenous populations. An average of 73·5 % polymorphism was found in all samples. As shown with Principal Coordinates Analysis (PCO), most of the genetic variation (90%) resided within, not among, populations. As X. parietina had previously been shown with molecular and fingerprinting techniques to be homothallic, the potential genetic background of this diversity is discussed. Intense genotype rather than gene (allele) flow seems to be an important element in X. parietina populations.

Three new Xanthoria species are described from South Africa. Xanthoria hirsuta sp. nov. has hairs on the surface of the thallus and apothecia, best visible in young, growing parts. Dust particles and sand granules stick to this hairy surface, giving the thallus a somewhat dirty appearance. Xanthoria inflata sp. nov. has inflated lobes similar to a Menegazzia. It carries numerous crystals on its medullary hyphae, which are ivory-coloured in young, but intensely orange coloured in old lobes. Xanthoria doidgeae sp. nov. has relatively small lobes with pruinose margins. All three species are fertile, none of them forms symbiotic propagules.

Specimens of Xanthoria parietina were collected from worldwide locations and ascospore discharge used to establish axenic cultures of the mycobiont. DNA was extracted and RAPD-PCR fingerprinting of 59 isolates was successfully achieved, resulting in 58 unique fingerprints. 110 multilocus RAPD markers were generated and used to construct a dendrogram. Two main groups were distinguished (75% bootstrap support): the first comprising samples from the Iberian Peninsula, the Balearic and Canary Islands; the second comprising all other worldwide samples including isolates from throughout Europe and North America. Samples from Australia and New Zealand clustered with the second group except one additional, phenotypically distinct specimen, not belonging to X. parietina, which formed an outgroup. However, comparative DNA sequence analyses are required to verify this interpretation.

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 five out of eight ascospores per ascus were analysed. 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. In contrast nine out of ten 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.

The symbiotic phenotype of a lichen arises through the interaction and cooperation of two or more genetically unrelated partners. Ultrastructural and molecular methods were used to investigate the changes that take place during early stages of the lichenization process. The resynthesis of prethallus stages of Baeomyces rufus was studied by co-culturing under sterile conditions the isolated, axenically grown mycobiont and its green algal photobiont Elliptochloris bilobata. The lichenization process was monitored by SEM. One day after co-culture, symbionts were bound together by a newly secreted mucilage. By day 12, photobiont induced, morphological changes in the mycobiont were visible. Aerial hyphae grew around photobiont cells, showed a high frequency of branching and formed appressoria on the algal wall surface. By day 28, many photobiont cells were completely engulfed by hyphae and soredia-like clusters were observed. These morphological developments resemble lichenized structures formed in the natural lichen. cDNA-AFLP was used to investigate gene expression profiles on day 12 of co-culture. Differential gene expression patterns revealed that few genes were induced, and many fungal and algal genes seemed to be suppressed in the early stages of lichenization.

Lichen mycobionts are typical representatives of their fungal classes but differ from non-lichenized taxa by their manifold adaptations to symbiosis with a population of minute photobiont cells. Most interesting are the morphologically complex macrolichens, the fungal partner of which competes for space above ground and contains photobiont cells optimally positioned for gas exchange and illumination. Such thalli are the product of an amazing hyphal polymorphism, with multiple switches between polar and apolar growth and hydrophilic or hydrophobic cell wall surfaces. Hydrophobic sealing of the apoplastic continuum between the partners by means of mycobiont-derived hydrophobic compounds canalizes the fluxes of solutes during the often quite dramatic de- and rehydration processes and keeps the algal layer gas-filled at any level of hydration. The impressive tolerance of drought, heat and cold stress of most lichen-forming fungi and their photobionts is due to a very interesting combination of protective and repair mechanisms at the cellular level, the molecular bases of which remain to be explored. Contemporary experimental lichenology is analysed and strategies are proposed aimed at better integration into mainstream biology.

Under favourable climatic conditions the mycobiont of Coniocybe furfuracea bears masses of conidia in chains on macronematous conidiophores. The same type of conidia were also formed by axenically grown mycobionts which had been isolated from single ascospores. Germinating conidia were found on the thalli. Coniocybe furfuracea is one of the very few lichen mycobionts so far known with a teleomorph and hyphomycetous anamorph.

Cytological aspects of the mycobiont-phycobiont contact were investigated in the lichen species Peltigera aphthosa, Cladonia macrophylla, Cladonia caespiticia and Parmelia tiliacea by means of freeze-etch and thin sectioning techniques, and by replication of isolated fragments of myco- and phycobiont cell walls.

In the symbiotic state of the mycobionts investigated a thin outermost wall layer with a distinct pattern was observed mainly in the hyphae contacting phycobiont cells and in the upper medullary layer. No comparable structures were noted on the hyphal surface of the cultured mycobionts of the Cladonia and Parmelia species investigated. A distinct rodlet layer was found on the hyphal surface of the mycobiont of Peltigera aphthosa, while mycobionts of Cladonia macrophylla, C. caespiticia and Parmelia tiliacea had a mosaic of small, irregular ridges, each corresponding in its size to a bundle of rodlets on the outermost wall layer. Comparable surface layers have been described in aerial hyphae of a great number of non-lichenized fungi.

The rodlet layer of the mycobiont wall surface of Peltigera aphthosa adheres tightly to the outermost layer of the sporopollenin-containing cell wall of the Coccomyxa phycobiont. Mature trebouxioid phycobiont cells of the Cladonia and Parmelia species investigated in the symbiotic state had an outermost wall layer which was structurally indistinguishable from the tessellated surface layer of the mycobiont cells. A rodlet pattern was detected in the outermost wall layer of Trebouxia autospores still surrounded by the cellulosic mother cell wall. In mature Trebouxia cells the bundles of rodlets became increasingly covered by a homogeneous material, and thus attained the same tessellated pattern which was observed on the mycobiont wall surface. No comparable structures were found on the wall surface after culturing the Trebouxia phycobionts axenically in liquid media. Confluence of the tessellated surface layers of fungal and algal origin was noted at the contact sites of growing hyphal tips and young Trebouxia cells.

The possible correlations between these cytological features and published immunological data on the cell surface of cultured and symbiotic lichen myco- and phycobionts are discussed.

The contact sites of pycnidia and the terminal cells of trichogynes in Cladonia furcata were investigated using either freshly fixed material, or ascomatal primordia and pycnidia from which the gelatinous material either on the primordial surface, or in the pycnidial cavity, had been removed. The sickle-shaped conidia fused, tip first, with the cell wall of trichogynes. Circular holes of about the diameter of the conidia found in the cell walls of trichogynes arise from enzymatic degradation of the wall material by fusing conidia. As the conidia appear to stick on any gelatinous surface material of the thallus the adhesion process is presumed to be unspecific.

This comparative investigation on ascus fine structure and function substantiates the findings of Chadefaud (1960) and his coworkers indicating a close taxonomic relationship between the Baeomycetaceae and Leotia, a non-lichenized member of the Helotiales, rather than between the Baeomycetaceae and the Cladoniaceae and other members of the Lecanorales. Besides the distinct differences in ascus structure and function, cytological divergences were noted between the Baeomycetaceae and Leotia on one hand, and Cladoniaceae on the other. The occurrence of glycogen in the Baeomycetaceae and Leotia, but not in the Cladoniaceae and other members of the Lecanorineae, and the differences in phycobiont preference and thus in the mycobiont–phycobiont contact in the Baeomycetaceae and Cladoniaceae were discussed.

On the basis of light microscopic (LM), scanning electron microscopic (SEM) and transmission electron microscopic (TEM) investigations the Pertusaria-type of ascus is described as a particular functional type. The functionally unitunicate Pertusaria-type is characterized by its structure, staining properties, and by its particular mode of dehiscence. Tripartite ascus walls were observed in LM and TEM. The non-amyloid ascus wall is surrounded by a thin, amyloid outer layer. Both become amorphous at maturity and partly disintegrate. An apically thickened, amyloid inner layer reaches the base of the ascus. In its fine structure this amyloid inner layer resembles the material of the amyloid dome of Lecanora-type asci. It plays an important role during dehiscence and spore discharge. An elongation process was observed prior to dehiscence, at the end of which the ascus tip is situated above the hymenial surface. Dehiscence occurs by bursting or splitting of the whole ascus tip. The Pertusaria-type might represent a side-branch of evolution from bitunicate to unitunicate forms within the Lecanorales.

Pertusaria-type asci are restricted to a small number of genera within the Pertusariaceae. A considerable heterogeneity in ascus structure and staining properties was observed within the Pertusariineae sensu Henssen & Jahns (1973) and Henssen (1976).

Ascus structure of eight yellow, two white and two brown Rhizocarpon species has been investigated by light microscopy. Ultrastructure and function in R. atroflavescens subsp. pulverulentum and R. montagnei were studied in TEM.

The Rhizocarpon-type ascus clearly differs from all other ascus types observed in the Lecanorales. It is bitunicate, opening with a slight ‘Jack-in-the-box’-mechanism. Its structure and function are related to patellariacean ascus types, but unlike those the ascus wall cytochemistry shows a certain similarity with Lecanora- and Peltigera-type asci. Rhizocarpon-type asci are embedded in a strongly amyloid hymenial gelatine. The nonamyloid ascus wall is surrounded by the strongly amyloid outer layer. The slightly amyloid expansible inner layer (= endoascus) is apically thickened; it shows the banded and pleated ‘accordion-structure’ characteristic of bitunicate asci. Prior to dehiscence, the ascus wall and its outer layer burst. Thereafter the pleatings of the expansible inner layer are stretched, forming the rather short beak which reaches the hymenial surface. During expansion gliding occurs between the expansible inner layer and an outer part of the endoascus, here described as the ‘inner layer’. In a few sections of aldehyde- fixed material of R. atroflavescens a small laminated plug was observed in the apex of the endoascus.

Rhizocarpon-lype asci are considered to be the most archaic in the Lecanorales. This supports a hypothesis that Rhizocarpon is a phylogenetically basal group, linking the evolved Lecanorineae, and possibly also the Peltigerineae and Teloschistineae with not yet recognized bitunicate ancestral forms similar to those occurring in the Patellariaceae.