New insights into the evolutionary history of Fungi from a 407 Ma Blastocladiomycota fossil showing a complex hyphal thallus.

Zoosporic fungi are key saprotrophs and parasites of plants, animals and other fungi, playing important roles in ecosystems. They comprise at least three phyla, of which two, Chytridiomycota and Blastocladiomycota, developed a range of thallus morphologies including branching hyphae. Here we describe Retesporangicus lyonii gen. et sp. nov., an exceptionally well preserved fossil, which is the earliest known to produce multiple sporangia on an expanded hyphal network. To better characterize the fungus we develop a new method to render surfaces from image stacks generated by confocal laser scanning microscopy. Here, the method helps to reveal thallus structure. Comparisons with cultures of living species and character state reconstructions analysed against recent molecular phylogenies of 24 modern zoosporic fungi indicate an affinity with Blastocladiomycota. We argue that in zoosporic fungi, kinds of filaments such as hyphae, rhizoids and rhizomycelium are developmentally similar structures adapted for varied functions including nutrient absorption and anchorage. The fossil is the earliest known type to develop hyphae which likely served as a saprotrophic adaptation to patchy resource availability. Evidence from the Rhynie chert provides our earliest insights into the biology of fungi and their roles in the environment. It demonstrates that zoosporic fungi were already diverse in 407 million-year-old terrestrial ecosystems.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.

Fertile Prototaxites taiti: a basal ascomycete with inoperculate, polysporous asci lacking croziers.

The affinities of Prototaxites have been debated ever since its fossils, some attaining tree-trunk proportions, were discovered in Canadian Lower Devonian rocks in 1859. Putative assignations include conifers, red and brown algae, liverworts and fungi (some lichenised). Detailed anatomical investigation led to the reconstruction of the type species, P. logani, as a giant sporophore (basidioma) of an agaricomycete (= holobasidiomycete), but evidence for its reproduction remained elusive. Tissues associated with P. taiti in the Rhynie chert plus charcoalified fragments from southern Britain are investigated here to describe the reproductive characters and hence affinities of Prototaxites Thin sections and peels (Pragian Rhynie chert, Aberdeenshire) were examined using light and confocal microscopy; Prídolí and Lochkovian charcoalified samples (Welsh Borderland) were liberated from the rock and examined with scanning electron microscopy. Prototaxites taiti possessed a superficial hymenium comprising an epihymenial layer, delicate septate paraphyses, inoperculate polysporic asci lacking croziers and a subhymenial layer composed predominantly of thin-walled hyphae and occasional larger hyphae. Prototaxites taiti combines features of extant Taphrinomycotina (Neolectomycetes lacking croziers) and Pezizomycotina (epihymenial layer secreted by paraphyses) but is not an ancestor of the latter. Brief consideration is given to its nutrition and potential position in the phylogeny of the Ascomycota.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.

Bacterial epibionts and endolichenic actinobacteria and fungi in the Lower Devonian lichen Chlorolichenomycites salopensis.

The charcoalified fragment of the dorsiventrally organized, internally stratified presumed green algal lichen Chlorolichenomycites salopensis from the Lower Devonian Lochkovian strata in the Welsh Borderland carries bacterial colonies on the upper surface, i.e. the cortex, and actinobacterial filaments in the medulla underneath the photobiont layer. Moreover relatively thin hyphae of presumed endolichenic fungi were found. As in extant lichens, which are best regarded as consortia with an unknown number of participants, this internally stratified, fossil thallus fragment of a presumed green algal lichen harbours a diverse microbial community.

The earliest records of internally stratified cyanobacterial and algal lichens from the Lower Devonian of the Welsh Borderland.

Lichenization is assumed to be a very ancient mode of fungal nutrition, but fossil records are rare. Here we describe two fragments of exceptionally preserved, probably charred, lichen thalli with internal stratification. Cyanolichenomycites devonicus has a cyanobacterial and Chlorolichenomycites salopensis a unicellular, presumably green algal photobiont. Fruiting bodies are missing. Cyanolichenomycites devonicus forms asexual spores in a pycnidium. All specimens were examined with scanning electron microscopy techniques. The fossils were extracted by maceration. Extant lichens and free-living cyanobacteria were either experimentally charcoalified for comparison or conventionally prepared. Based on their septate hyphal structure, both specimens are tentatively interpreted as representatives of the Pezizomycotina (Ascomycota). Their presence in 415 million yr (Myr) old rocks from the Welsh Borderland predates existing Late Cretaceous records of pycnidial conidiomata by some 325 Myr and Triassic records of lichens with broadly similar organization by some 195 Myr. These fossils represent the oldest known record of lichens with symbionts and anatomy as typically found in morphologically advanced taxa today. The latter does not apply to Winfrenatia reticulata, the enigmatic crustose lichen fossil from the Lower Devonian, nor to presumed lichen-like organisms such as the Cambrian Farghera robusta or to the Lower Devonian Spongiophyton minutissimum.

Phylogenetic analysis of LSU and SSU rDNA group I introns of lichen photobionts associated with the genera Xanthoria and Xanthomendoza (Teloschistaceae, lichenized Ascomycetes).

We studied group I introns in sterile cultures of selected groups of lichen photobionts, focusing on Trebouxia species associated with Xanthoria s. lat. (including Xanthomendoza spp.; lichen-forming ascomycetes). Group I introns were found inserted after position 798 (Escherichia coli numbering) in the large subunit (LSU) rRNA in representatives of the green algal genera Trebouxia and Asterochloris. The 798 intron was found in about 25% of Xanthoria photobionts including several reference strains obtained from algal culture collections. An alignment of LSU-encoded rDNA intron sequences revealed high similarity of these sequences allowing their phylogenetic analysis. The 798 group I intron phylogeny was largely congruent with a phylogeny of the Internal Transcribed Spacer Region (ITS), indicating that the insertion of the intron most likely occurred in the common ancestor of the genera Trebouxia and Asterochloris. The intron was vertically inherited in some taxa, but lost in others. The high sequence similarity of this intron to one found in Chlorella angustoellipsoidea suggests that the 798 intron was either present in the common ancestor of Trebouxiophyceae, or that its present distribution results from more recent horizontal transfers, followed by vertical inheritance and loss. Analysis of another group I intron shared by these photobionts at small subunit (SSU) position 1512 supports the hypothesis of repeated lateral transfers of this intron among some taxa, but loss among others. Our data confirm that the history of group I introns is characterized by repeated horizontal transfers, and suggests that some of these introns have ancient origins within Chlorophyta.

Consumption of fa cai Nostoc soup: a potential for BMAA exposure from Nostoc cyanobacteria in China?

Grown in arid regions of western China the cyanobacterium Nostoc flagelliforme--called fa cai in Mandarin and fat choy in Cantonese--is wild-harvested and used to make soup consumed during New Year's celebrations. High prices, up to $125 USD/kg, led to overharvesting in Inner Mongolia, Ningxia, Gansu, Qinghai, and Xinjiang. Degradation of arid ecosystems, desertification, and conflicts between Nostoc harvesters and Mongol herdsmen concerned the Chinese environmental authorities, leading to a government ban of Nostoc commerce. This ban stimulated increased marketing of a substitute made from starch. We analysed samples purchased throughout China as well as in Chinese markets in the United States and the United Kingdom. Some were counterfeits consisting of dyed starch noodles. A few samples from California contained Nostoc flagelliforme but were adulterated with starch noodles. Other samples, including those from the United Kingdom, consisted of pure Nostoc flagelliforme. A recent survey of markets in Cheng Du showed no real Nostoc flagelliforme to be marketed. Real and artificial fa cai differ in the presence of beta-N-methylamino-L-alanine (BMAA). Given its status as a high-priced luxury food, the government ban on collection and marketing, and the replacement of real fa cai with starch substitutes consumed only on special occasions, it is anticipated that dietary exposure to BMAA from fa cai will be reduced in the future in China.

Interactions of sterile-cultured lichen-forming ascomycetes with asbestos fibres.

Sterile cultured isolates of lichen-forming ascomycetes have not yet been used to investigate mycobiont-mineral substrate interactions under controlled conditions. In this study Candelariella vitellina, Xanthoparmelia tinctina and Lecanora rupicola mycobionts were isolated and inoculated with chrysotile fibres in the laboratory, in order to verify whether physical and chemical weathering processes, which were already described in the field, may be reproduced in vitro. Tight adhesion of hyphae to chrysotile fibres was observed in all species. The adhering hyphae affected the chemical composition of asbestos fibres, with the selective depletion of magnesium being a prominent feature, as is the case in field conditions. Oxalic acid and pulvinic acid, mycobiont-derived metabolites of X. tinctina and C. vitellina, were involved in the weathering action. Time and environmental factors and the absence of biological synergisms strongly limited the chemical weathering in vitro compared with what was observed in the field. Nevertheless, the results show that in vitro incubation of sterile-cultured lichen-forming fungi with minerals is a practicable experimental system to investigate the weathering effects of different mycobionts and fungal compounds under controlled conditions.

Mating systems in representatives of Parmeliaceae, Ramalinaceae and Physciaceae (Lecanoromycetes, lichen-forming ascomycetes).

The progeny of meiosis of eight Parmeliaceae, two Ramalinaceae and seven Physciaceae were subjected to fingerprint analysis using RAPD-PCR applied to single spore isolates. The sample set included common and widespread rarely fertile species (Parmelia sulcata, Pseudevernia furfuracea, Physcia tenella), local to common, infrequently fertile species (Melanelixia glabra, Parmelina tiliacea, Xanthoparmelia conspersa, X. stenophylla, Anaptychia runcinata, Diploicia canescen, Physconia distorta), local to rare, infrequently or regularly fertile species with declining distributions (Parmelina carporrhizans, P. quercina, Ramalina fastigiata, R. fraxinea, Anaptychia ciliaris), and local to common, regularly fertile species (Physcia aipolia, P. stellaris). All species turned out to be heterothallic, polymorphisms among RAPD markers ranging from 10-87%. The significance of these findings for population genetics and conservation biology, and potential reasons for infrequent ascoma formation in some of the species are discussed.

Characterisation of the mating-type locus in the genus Xanthoria (lichen-forming ascomycetes, Lecanoromycetes).

Conserved regions of mating-type genes were amplified in four representatives of the genus Xanthoria (X. parietina, X. polycarpa, X. flammea, and X. elegans) using PCR-based methods. The complete MAT locus, containing one ORF (MAT1-2-1) coding for a truncated HMG-box protein, and two partial flanking genes, were cloned by screening a genomic lambda phage library of the homothallic X. parietina. The flanking genes, a homologue of SLA2 of Saccharomyces cerevisiae and a DNA lyase gene, served to amplify the two idiomorphs of the X. polycarpa MAT locus. Each idiomorph contains a single gene: MAT1-2-1 codes for a HMG-box protein, MAT1-1-1 encodes an alpha domain protein. The occurrence of mating-type genes in eight single spore isolates derived from one ascus was studied with a PCR assay. In the homothallic X. parietina a HMG fragment, but no alpha box fragment was found in all isolates, whereas in X. elegans, another homothallic species, all tested isolates contained a fragment of both idiomorphs. Conversely, isolates of the heterothallic X. polycarpa contained either a HMG or an alpha box fragment, but never both.

Mating systems in the genus Xanthoria (lichen-forming ascomycetes).

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.

Hydrophobins DGH1, DGH2, and DGH3 in the lichen-forming basidiomycete Dictyonema glabratum.

Dictyonema glabratum is a lichen-forming basidiomycete whose symbiotic phenotype shares similarities to both lichens and its non-lichen-forming relatives. In the photobiont layer of D. glabratum intercellular gas-filled spaces are present even when the lichen is water-saturated. The walls of hyphae lining air cavities are covered by a hydrophobic, rodlet-patterned layer, assumed to be formed by hydrophobins. Hot SDS-insoluble, but trifluoroacetic acid-soluble lichen cell wall extracts contained seven proteins. The N-terminal sequence of the most abundant 14-kDa protein was used to carry out cDNA cloning by RT-PCR. The deduced amino acid sequence of the amplified fragment encoded a class I hydrophobin, called DGH1. The cDNA sequence encoding the signal peptide was cloned by RACE-PCR, which also coamplified cDNA fragments encoding two additional class I hydrophobins, DGH2 and DGH3. The three proteins share 54 to 66% amino acid identity. The D. glabratum hydrophobin extract containing either all proteins or primarily DGH1 self-assembled and formed a rodlet mosaic similar to the one observed in situ. Concentration of the protein extract was shown to influence the length of the self-assembled rodlets.

Insect-mediated reproduction of systemic infections by Puccinia arrhenatheri on Berberis vulgaris.

• Witches' brooms on Berberis vulgaris are induced by a systemically infecting rust fungus, Puccinia arrhenatheri. These witches' brooms bear yellow discolored leaves on which the fungus exposes its gametes in a sugary nectar. During the spermatial stage of the fungus the infected leaves emit a strong, flowery scent. • An exclusion-experiment was used to evaluate whether fungal reproductive success, defined by the ability of the fungus to produce aeciospores, depended on gamete transfer by insects. To determine whether insects were attracted to the infected leaves, and if so, why, visitation to infected and uninfected leaves was quantified and volatiles produced by leaves, infected leaves and flowers were analyzed. • The production of aeciospores was significantly higher on witches' brooms with insect visitation. Visitation rates were higher and visits were longer on witches' brooms than on uninfected branches. A wide diversity of visitors, mainly Diptera and Hymenoptera, was observed. The volatiles emitted by infected leaves were composed of sweet floral fragrances and insect pheromones. • Our results suggest that sexual reproduction of the pathogen requires out-crossing by insects and that infected leaves attract insects by floral mimicry (bright yellow color and the production of sugary nectar and volatiles).

Developmental biology of lichens.

Lichen-forming fungi are a large, taxonomically diverse group of nutritional specialists which acquire fixed carbon from a population of minute green algal or cyanobacterial cells. Mycobionts of foliose or fruticose lichens differ from the rest of the fungi by expressing morphologically and anatomically complex symbiotic phenotypes. The extracellularly located photobiont cell population of these macrolichens is housed and controlled by the quantitatively predominant fungal partner which competes for space above ground, secures adequate illumination and facilitates gas exchange. This review summarizes data on the ontogeny, functional morphology, growth patterns and internal thalline differentiation of macrolichens. Contents Summary 659 I. Introduction 659 II. Establishment of the lichen symbiosis 662 III. Growth and cell turnover 669 IV. Outlook 674 Acknowledgements 674 References 674.