The lichen symbiosis has evolved several times within the fungal kingdom, although the total number of lichenization events leading to extant taxa is still unclear. Two lichenized families, the Icmadophilaceae and Baeomycetaceae have been classified in the Helotiales. Because the Helotiales are predominantly nonlichenized, this suggests that these families represent independent evolutionary episodes of lichenization from the Lecanorales. As a first step towards understanding the evolution of the lichen symbiosis within this order, we tested recent hypotheses concerning the segregation of lichen genera between the two lichen families. Specifically, we used phylogenetic analyses of nucleotide sequence data from nuclear small-subunit and large-subunit ribosomal DNA to test the morphology-based hypotheses that Dibaeis is a distinct genus from Baeomyces and that Dibaeis is a member of the Icmadophilaceae rather than the Baeomycetaceae. Phylogenetic analyses of nuclear SSU rDNA and combined SSU and LSU rDNA data support the hypothesis that Dibaeis is more closely related to IcmadophUa than it is to Baeomyces. Therefore, these data support the resurrection of Dibaeis from its previous synonymy with Baeomyces based on the characters of ascocarp colour and ascus morphology. The recognition of two distinct genera is also consistent with character state distribution of unique lichen acids.

Lecanora demissa (Körb.) Zahlbr. is a crustose, lobate lichen that produces soredia and conidiomata but no apothecia. Its placement in Lecanora has long been questioned but nothing better has been proposed. We have studied the nuclear rDNA of the ITS regions and the SSU of L. demissa. In an alignment of the ITS regions of several representatives of Lecanora s. lat. it could clearly be shown by a PAUP analysis, using Aspicilia caesiocinerea as outgroup, that L. demissa does not belong to Lecanora. In a PAUP analysis of sequences of the SSU from representatives of the order Lecanorales, using members of Saccharomycetales as outgroup, L. demissa clustered on a well-supported branch with Caloplaca chlorina. In a further analysis of the ITS sequences of L. demissa together with representatives of Caloplaca and Xanthoria using Protoparmelia as outgroup two most parsimonious trees were found. In these trees the L. demissa branch was well within a strongly supported clade with C. cerina, the type species of the genus Caloplaca. The sister taxon to L. demissa in this analysis was C. variabilis. Chemical data and characters of the conidiomata support the affinity with Caloplaca and the new combination C. demissa (Körb.) Arup & Grube is therefore proposed. A lectotype for Imbricaria demissa has been designated. The phylogenetic analysis of several representatives of the genera Caloplaca and Xanthoria suggests that these genera are not monophyletic as presently circumscribed. Two large, monophyletic groups of species could be recognized, one with Xanthoria species mixed with lobate and crustose members of Caloplaca, and one with mainly crustose Caloplaca, including both species with orange or black apothecia.

The phylogenetic relationship between the Alectoriaceae and the Parmeliaceae (lichenized ascomycetes) were studied using sequences of the small subunit of the nuclear ribosomal DNA (SSU rDNA). The Alectoriaceae was represented by specimens of Alectoria ochroleuca and A. sarmentosa, and the Parmeliaceae by Bryoria capillaris, Cetraria islandica, Cornicularia normoerica, Evernia prunastri, Hypogymnia physodes, Parmelia saxatilis, Platismatia glauca, Pleurosticta acetabulum, Usnea florida, Vulpicida juniperina, V. pinastri and Xanthoparmelia conspersa. The results suggest that the Alectoriaceae is derived from within the Parmeliaceae, and it is suggested the families should be treated as synonyms. Morphological and anatomical characters are discussed, and it is concluded that earlier investigations might have overemphasized minor variations in ascus structure.

Phylogenetic trees based on group I intron sequences and on internal transcribed spacer (ITS) sequences of mycobiont ribosomal genes were calculated and compared. Eight cetrarioid and four non-cetrarioid species of the Parmeliaceae were compared. The phylogeny based on group I intron sequences is partly congruent with the ITS sequence phylogeny. Group I intron sequences are presumably less informative for infragenic studies. The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. All introns analysed are located between the positions 1516 and 1517 of the fungal 18S ribosomal RNA gene. Cetrarioid lichens form a non-homogeneous group within the Parmeliaceae according to both group I intron and ITS sequences.

Morphological and chemical data from 64 genera of Parmeliaceae, including the most common genera in Europe, were analysed by means of neighbour–joining and parsimony analysis. Two presence/absence matrices were constructed, one for the 64 genera and a smaller one for the most common European genera. Probabilities were too low to allow grouping of the genera and only a few pairs of genera had a probability of >50% for both matrices. A molecular matrix (nuclear rDNA-ITS) of the most common genera in Europe was constructed and analysed by the method of maximum likelihood. These results were much more informative than the non-molecular matrix for establishing phylogenetic relationships among the genera.

The ability of the internal transcribed spacers (ITS regions) of ribosomal DNA to resolve phylogenetic relationships within the euascomycetous order Arthoniales, focusing on the family Roccellaceae was investigated. The effect of alignment on phylogenetic hypotheses was evaluated. A data matrix from the ITS regions was constructed from 33 specimens representing 14 genera, including the outgroup Arthothelium spectabile. Six different alignments were analysed cladistically using parsimony jackknifing. Most groups in the six trees were congruent and well supported under the different alignment settings. In a conservative analysis, where only unambiguously alignable regions were included, the resolution was low. These results indicate that the ITS regions contain phylogenetic structure, and all information, including the variable regions, should be utilised. A data matrix from the SSU rDNA sequences was constructed for the same taxa. The SSU rDNA tree was less resolved than the ITS trees. There were only minor conflicts between the two sources of data and an incongruence test confirmed that the ITS and SSU rDNA data matrices were not significantly incongruent. The six differently aligned data matrices generated from the ITS regions were each combined with the SSUrDNA data. Simultaneous analysis of the combined data sets is the best approach as it uses all available evidence. As with the ITS trees, most groups in the combined trees were congruent and well supported. The SSU rDNA provided resolution within one clade, otherwise the ITS sequences provided most of the signal in the combined analysis, both at the basal nodes and at the tips of the tree. Molecular data clearly indicates that the fruticose/crustose habits have evolved multiple times even in comparatively small groups as in the family Roccellaceae and that the characters such as fruticose-crustose may be overemphasized in morphological analyses.

The lichen family Umbilicariaceae is accepted by most lichenologists as consisting of two genera, Lasallia and Umbilicaria. The monophyly of these two genera was examined by phylogenetic analyses of nucleotide sequences of ITS 1 and ITS2 rDNA. Sequences of these regions from three Lasallia and 17 Umbilicaria species were aligned to those of seven representatives of the outgroup taxa including Eurotiales, Onygenales and Caliciales (Mycocaliciaceae) and subjected to maximum parsimony, maximum likelihood and neighbour-joining analyses. The resulting phylogenetic hypotheses supported the monophyly of the representative species of Lasallia. However, the species of Umbilicaria did not form a monophyletic sistergroup to Lasallia due to the basal placement of other Umbilicaria species in some analyses. Based on these analyses, if Lasallia is recognized as a separate genus then Umbilicaria appears to be paraphyletic. Although further taxon sampling is required to resolve the monophyly of Umbilicaria, for the present we recommend retaining the current treatment of Lasallia as separate from Umbilicaria.

Biatora helvola is a corticolous crustose lichen occurring in boreal and montane spruce and spruce-fir-beech forests. After the last glaciation, spruce reinvaded Europe from three refugia situated in the Carpathians, southeastern parts of the Alps and the Ural Mts., resulting in a slightly disjunct distribution. Our aim was to find out whether the glacial fragmentation of the distributional area of spruce is reflected by genetic differences in a typical spruce-forest lichen. Collections of Biatora helvola from Scandinavia and various parts of Central Europe were investigated using RAPD analysis. Algal free periclinal sections of the apothecia were obtained using a freezing microtome and transferred directly into PCR tubes. Six different RAPD primers were used. The data were analysed using PAUP*. It was shown that genetic differences between samples of B. helvola reflect the glacial disjunction of spruce in Europe.

The photobiont inventory of a stand of the Acarosporetum sinopicae, lichen community comprising saxicolous, chalcophilous lichens, has been analysed. Investigated lichen species were Acarospora rugulosa, A. sinopica, Bellemerea diamartha, Lecanora polytropa, L. subaurea, Lecidea silacea, L. lapicida, Rhizocarpon geographicum, and Umbilicaria cylindrical. For all these lichen species this is the record of the photobionts, except for L. lapicida. The photobionts were cultured axenically and investigated using light microscopical and molecular methods (ITS-sequence analyses). Every lichen species contained only one photobiont species. All photobionts belong to Trebouxia jamesii, but two different subspecies were found with the morphological differences corresponding to molecular differences. The new subspecies T. jamesii subsp. angustilobata is described, differing from the typical T. jamesii by a crenulate chloroplast but identical to the latter taxon in respect to the pyrenoid structure in the light microscope. These results are discussed with respect to the photobiont inventory of the Physcietum adscendentis, analysed in an earlier study.

Four primers for the amplification of mitochondrial DNA of lichenforming ascomycetes are presented. The primers match the conserved regions U2, U4, and U6, respectively, of mitochondrial small subunit (SSU) ribosomal DNA (rDNA). Polymerase chain reaction using different combinations of the primers produced single amplification products from DNA of eight lichen-forming fungal species but did not amplify DNA of two axenic cultured algal species. The amplification product obtained from Lobaria pulmonaria was sequenced and the 894-bp sequence was compared with the mitochondrial SSU rDNA sequence of Podospora anserine. The two sequences revealed more than 76% identity in the conserved regions U3 to U5 demonstrating that we amplified mitochondrial DNA. The primers matching U2 and U6 yielded amplification products of 800–1000 bp depending on the species examined. The variation observed suggests that mitochondrial SSU rDNA may be useful for phylogenetic analyses of lichen-forming ascomycetes.

Factors affecting the outcome of a PCR is discussed. Nested PCR is described and a protocol that has been shown to work well for the amplification of ribosomal genes in ascomycetes is described. It is claimed that DNA extracts of lichenized fungi, contrary to many other organism groups, are often contaminated by other fungi. This poses a problem due to the preferential amplification of short intron-lacking sequences over longer, intron-containing ones, particularly when extension time is limited. A simple protocol for optimizing and troubleshooting PCRs is presented.

RNase activity in Lasallia hispanica, Parmelia omphalodes and Cornicularia normoerica collected at different times and in different habitats, have shown variability that cannot be solely attributable to variation in habitat or season. Electromorph pattern of RNase activity in L. hispanica and C. normoerica, showed variations among different collection times; some bands are constant but other showed high variability, which could be related to physiological changes. It is also noteworthy that in both species one band is exclusively present in thalli from one of the sampling localities. This could be the result of an adaptation to this habitat and/or due to genetic differences.