Bioactive phenolic compounds from a medicinal lichen, Usnea longissima

Natural products, longissiminone A (1) and longissiminone B (2), were isolated along with a known compound, glutinol (3), from a medicinal lichen, Usnea longissima. The structures of compounds 1 and 2 were determined with the help of spectroscopic studies. Compound 1 was found to possess potent anti-inflammatory activity in a cell-based contemporary assay. Cytotoxicity activity measured by cell viability assay showed 100% viability in the presence of 200 microg/mL conc. of these compounds.     

Unconstrained gene flow between populations of a widespread epiphytic lichen Usnea subfloridana (Parmeliaceae,Ascomycota) in Estonia

Few studies have investigated the genetic diversity of populations of common and widespread lichenized fungi using microsatellite markers, especially the relationships between different measures of genetic diversity and environmental heterogeneity. The main aim of our study was to investigate the population genetics of a widespread and mainly clonally reproducing Usnea subfloridana at the landscape scale, focusing on the comparison of lichen populations within hemiboreal forest stands. Particular attention has been paid to the genetic differentiation of lichen populations in two geographically distinct regions in Estonia and the relationships between forest characteristics and measures of genetic diversity. We genotyped 578 Usnea thalli from eleven lichen populations using seven specific fungal microsatellite markers. Measures of genetic diversity (allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of multilocus genotypes, the number of private alleles, and the minimum number of colonization events) were calculated and compared between Usnea populations. Shared haplotypes, gene flow and AMOVA analyses suggest that unconstrained gene flow and exchange of multilocus genotypes exist between the two geographically remote regions in Estonia. Stand age, mean circumference of the host tree, size of forest site and tree species composition did not show any significant influence on allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of private alleles, and the minimum number of colonization events of U. subfloridana populations. Therefore it was concluded that other factors of habitat heterogeneity could probably have a more significant effect on population genetics of U. subfloridana populations.     

Molecular phylogeny of parmotremoid lichens (Ascomycota, Parmeliaceae)

Parmotrema is one of the larger genera segregated from Parmelia s. lat. Additional genera recently have been segregated from this large genus based mainly on morphological and chemical features. We have employed molecular data from three genes to continue a revision of the generic concept within the parmelioid lichens. A Bayesian analysis of nuclear ITS, LSU rDNA and mitochondrial SSU rDNA sequences was performed. The genera Canomaculina, Concamerella, Parmelaria and Rimelia appear nested within Parmotrema. Alternative hypotheses to maintain the independence of Canomaculina, Concamerella and Rimelia are shown to be highly unlikely and are rejected. As a consequence these three genera are reduced to synonymy with Parmotrema. An alternative topology segregating Parmelaria from Parmotrema s. lat. cannot be rejected with the dataset at hand. However we have established that this genus is closely related to Parmotrema rather than to cetrarioid species as was considered previously. The revised genus Parmotrema includes species that have an upper cortex consisting of a palisade plectenchyma or rarely paraplectenchyma with vaults, have a pored or fenestrated epicortex, lack pseudocyphellae, have or lack cilia, have laminal, perforate or eperforate apothecia, usually have simple rhizines and filiform, cylindrical, bacilliform or sublageniform conidia. It is closely related to Flavoparmelia but the status of these genera requires further investigation. Nineteen new combinations are made.     

Punctelia, a new lichen genus in the Parmeliaceae

The genus Punctelia Krog gen. nov., comprising subgen. Punctelia (the Parmelia borreri group) and subgen. Flavopunctelia Krog subgen. nov. (the Parmelia flaventior group), is here segregated from Parmelia s. str. The main differences between the two genera lie in the development of the pseudocyphellae, the chemical properties of the medulla, and the centres of distribution. Parmelia s. str. has its highest number of species in East Asia and the Australian region, Punctelia in the Americas and Africa.     

Remototrachyna, a newly recognized tropical lineage of lichens in the Hypotrachyna clade (Parmeliaceae, Ascomycota), originated in the Indian subcontinent

Biogeographical studies of lichens used to be complicated because of the large distribution ranges of many species. Molecular systematics has revitalized lichen biogeography by improving species delimitation and providing better information about species range limitations. This study focuses on the major clade of tropical parmelioid lichens, which share a chemical feature, the presence of isolichenan in the cell wall, and a morphological feature, microscopic pores in the uppermost layer. Our previous phylogenetic studies revealed that the largest genus in this clade, Hypotrachyna, is polyphyletic with a clade mainly distributed in South and East Asia clustering distant from the core of the genus. To divide the Hypotrachyna clade into monophyletic groups and to reevaluate morphological and chemical characters in a phylogenetic context, we sampled ITS, nuclear large subunit (nuLSU) and mitochondrial small subunit (mtSSU) rDNA sequences from 77 species. We are erecting the new genus Remototrachyna for a core group of 15 former Hypotrachyna species. The segregation of Remototrachyna from Hypotrachyna receives support from morphological and chemical data, as well from maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses of the DNA. We used a likelihood approach to study the geographic range evolution of Remototrachyna and Bulbothrix, which are sister groups. This analysis suggests that the ancestral range of Remototrachyna was restricted to India and that subsequent long-distance dispersal is responsible for the pantropical occurrence of two species of Remototrachyna.     

Understanding Phenotypical Character Evolution in Parmelioid Lichenized Fungi (Parmeliaceae,Ascomycota)

Parmelioid lichens form a species-rich group of predominantly foliose and fruticose lichenized fungi encompassing a broad range of morphological and chemical diversity. Using a multilocus approach, we reconstructed a phylogeny including 323 OTUs of parmelioid lichens and employed ancestral character reconstruction methods to understand the phenotypical evolution within this speciose group of lichen-forming fungi. Specifically, we were interested in the evolution of growth form, epicortex structure, and cortical chemistry. Since previous studies have shown that results may differ depending on the reconstruction method used, here we employed both maximum-parsimony and maximum-likelihood approaches to reconstruct ancestral character states. We have also implemented binary and multistate coding of characters and performed parallel analyses with both coding types to assess for potential coding-based biases. We reconstructed the ancestral states for nine well-supported major clades in the parmelioid group, two higher-level sister groups and the ancestral character state for all parmelioid lichens. We found that different methods for coding phenotypical characters and different ancestral character state reconstruction methods mostly resulted in identical reconstructions but yield conflicting inferences of ancestral states, in some cases. However, we found support for the ancestor of parmelioid lichens having been a foliose lichen with a non-pored epicortex and pseudocyphellae. Our data suggest that some traits exhibit patterns of evolution consistent with adaptive radiation.     

Biogeography and Genetic Structure in Populations of a Widespread Lichen (Parmelina tiliacea,Parmeliaceae, Ascomycota)

The genetic diversity and population structure of the foliose lichenized fungus Parmelina tiliacea has been analyzed through its geographical range, including samples from Macaronesia (Canary Islands), the Mediterranean, and Eurosiberia. DNA sequences from the nuclear ribosomal internal transcribed spacer, the mitochondrial large subunit ribosomal RNA gene, and the translation elongation factor 1-α were used as molecular markers. The haplotypes of the three markers and the molecular variance analyses of multilocus haplotypes showed the highest diversity in the Canary Islands, while restricted haplotypes occurred at high frequencies in Mediterranean coastal samples. The multilocus haplotypes formed three unevenly distributed clusters (clusters 1-3). In the Canary Islands all the haplotypes were present in a similar proportion, while the coastal Mediterranean sites had almost exclusively haplotypes of cluster 3; cluster 2 predominated in inland Mediterranean sites; and cluster 1 was more abundant in central and northern Europe (Eurosiberian area). The distribution of clusters is partially explained by climatic factors, and its interaction with local spatial structure, but much of the variation remains unexplained. The high frequency of individuals in the Canary Islands with haplotypes shared with other areas suggests that could be a refugium of genetic diversity, and the high frequency of individuals of the Mediterranean coastal sites with restricted haplotypes indicates that gene flow to contiguous areas may be restricted. This is significant for the selection of areas for conservation purposes, as those with most genetic variation may reflect historical factors and biological properties of the species.     

Unveiling annual growth chronologies from inter-nodal branch elongations in a fruticose lichen in southern Europe

Techniques for retrospective analysis of size dynamics at annual resolution remain poorly developed in lichens in general, and fruticose lichens in particular. Only a few attempts in very high latitudes suggested that growth might be studied as a chronosequence of inter-nodal branch elongations. Here we evaluated, for the first time, this hypothesis in a dry Mediterranean environment using the lichen Cladonia rangiformis as a case study. Mixed models supported a strong positive relationship between humidity measured as precipitation/PET and inter-nodal branch elongations. Importantly, model selection suggested that (i) the number of intermodal elongations were a major determinant of stem elongation, and (ii) a second-order temporal autocorrelation denoted legacies of environmental influences at least over the next 2 y. The strong growth–humidity relationship, along with the potential legacies observed, support the idea that inter-nodal branch elongations could be used to reconstruct growth chronologies at annual resolution in drylands. This finding highlights the high vulnerability of these organisms to rising aridity, and opens a new venue for climate reconstruction and other potential applications in Ecology and Earth Science disciplines.     

Phylogenetic significance of morphological characters in the tropical Hypotrachyna clade of parmelioid lichens (Parmeliaceae, Ascomycota)

Lichen-forming ascomycetes exhibit often complex morphologies of the vegetative thallus that are usually not found in non-lichenized fungi. This includes the thallus organization and appendical structures associated with the main thallus, such as cilia and rhizines. Such morphological characters are widely employed in the taxonomy of parmelioid lichens, especially at generic level. Within parmelioid lichens, several monophyletic groups can be distinguished, the Hypotrachyna clade being one of them, which includes mostly tropical taxa. In this first molecular study focused specifically on the Hypotrachyna clade, we used maximum parsimony and Bayesian analyses of a combined data set of nuclear ITS and mitochondrial SSU rDNA sequences to (1) test the monophyly of genera presently accepted within the clade and (2) evaluate the phylogenetic value of the morphological characters used to circumscribe genera in parmelioid lichens. Out of the 89 mtSSU and 88 nuITS sequences included in the present study, 121 sequences were newly obtained. Our results show that the taxa within the clade fall into two major groups and that the genus Hypotrachyna is polyphyletic. Everniastrum and Parmelinopsis are nested within Hypotrachyna sensu stricto, the latter being also polyphyletic. Bulbothrix is paraphyletic with Parmelinella nested within and is basal to the second major Hypotrachyna clade. Monophylies of Bulbothrix and Hypotrachyna are significantly rejected. The phylogenetic analysis demonstrates that morphological characters currently used to circumscribe genera in parmelioid lichens, such as cortical anatomy, lobe configuration, cilia, and rhizines have been overestimated and have only minor value in identifying monophyletic groups.     

The Caloscyphaceae (Pezizomycetes, Ascomycota), with a new genus

The family Caloscyphaceae with a single genus, Caloscypha, has been considered to include a single species, C. fulgens. Study of an overlooked second species, Caloscypha incarnata from North Africa and Italy, using SSU, LSU rDNA, and morphology allows placement of this species in a new genus, Kallistoskypha, in the Caloscyphaceae. This fungus is found in association with Eucalyptus species. The species was recently redescribed from Spain under the name Marcelleina parvispora. Caloscypha fulgens, the type species of the genus Caloscypha, shows sequence variation from across its range.     

New species and new records of Xanthoparmelia (lichenized Ascomycota,Parmeliaceae) from Western Australia

The following new species of Xanthoparmelia (Parmeliaceae) are described from Western Australia: X. cranfieldii Elix, X. elevata Elix, X. kimberleyensis Elix, X. monadnockensis Elix,X. nanoides Elix, X. nortegeta Elix, X. sleei Elix and X. toolbrunupensis Elix. In addition, X. luminosa (Elix) Hale and X. victoriana Elix & J. Johnst. are reported from Western Australia for the first time.     

Phenotypic plasticity of primary thallus in selected Cladonia species (lichenized Ascomycota: Cladoniaceae)

The paper presents the results of comprehensive analyses concerning the phenotypic variability of the primary thallus in five chosen Cladonia species in relation to habitat conditions. Morphology and anatomy, as well as the cortex ultrasculpture, of primary squamules were examined in individual specimens from sunny and shady populations of each species. The Mann-Whitney U test and Principal Component Analysis (PCA) revealed several clear correlations between the type of habitat and the organisation of squamules. Significant differences refer mainly to anatomical features, especially to epinecral layer frequency and cortex thickness. On the contrary, examination of the surface ultrasculpture of squamules under SEM did not show any important differences between populations. Regardless of the habitat factors, a full range of cortical surface rugosity in samples from both habitats was noticed, and a high variability often refers to a singular squamule. The results indicate that some features are largely modified by environmental factors and/or depend on the development stage of individual specimens, and examination of these relationships should be the first step towards the evaluation of the taxonomic usefulness of the considered features.     

Characterization of mycobiont adaptations in the foliose lichen Xanthoparmelia chlorochroa (Parmeliaceae)

A cross section of the vagrant soil lichen Xanthoparmelia chlorochroa was analyzed using proton microprobe PIXE. Data were used to generate quantitative, two-dimensional element distribution maps for Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, and Sr. Element maps show differential element partitioning between the stratified layers of the thallus. These data document transfer of inorganic nutrients across the thallus to the algal layer. Inorganic particle entrapment was also evident in the element maps. Dense accumulations of calcium oxalate at the junction of the medulla and the algal layer on the order of 10% by dry mass were discovered. Scanning electron microscopy and thermogravimetric analyses were used to characterize the calcium oxalate region. These data provide evidence for possible functional roles of the calcium oxalate layer, including regulation of water and light. Data also provide support for a mutualistic interpretation of the lichen association.     

Genetic variation of Usnea filipendula (Parmeliaceae) populations in western Germany investigated by RAPDs suggests reinvasion from various sources

Random amplified polymorphic DNA (RAPD) markers were characterized for 25 specimens of Usnea filipendula to evaluate the genetic diversity of populations reinvading formerly uninhabited regions in Northrhine-Westphalia due to decreasing sulfur dioxide levels. With six 10-mer randomly amplified polymorphic DNA (RAPD) primers, a 66 character by 25 specimens matrix was generated. Phenetic analysis (UPGMA) showed no obvious groupings. The reinvading populations are distributed over the phenogram and are not genetically closely related. The results suggest that the reinvading populations of this usually sterile species are derived from different sources and do not consist of a particular clone capable of re-entering the area.     

Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity

Parmelioid lichens comprise about 1500 species and have a worldwide distribution. Numerous species are widely distributed and well known, including important bioindicators for atmospheric pollution. The phylogeny and classification of parmelioid lichens has been a matter of debate for several decades. Previous studies using molecular data have helped to establish hypotheses of the phylogeny of certain clades within this group. In this study, we infer the phylogeny of major clades of parmelioid lichens using DNA sequence data from two nuclear loci and one mitochondrial locus from 145 specimens (117 species) that represent the morphological and chemical diversity in these taxa. Parmelioid lichens are not monophyletic; however, a core group is strongly supported as monophyletic, excluding Arctoparmelia and Melanelia s. str., and including Parmeliopsis and Parmelaria. Within this group, seven well-supported clades are found, but the relationships among them remain unresolved. Stochastic mapping on a MC/MCMC tree sampling was employed to infer the evolution of two morphological and two chemical traits believed to be important for the evolutionary success of these lichens, and have also been used as major characters for classification. The results suggest that these characters have been gained and lost multiple times during the diversification of parmelioid lichens.     

Identification of crystalline material found in the thallus of the lichen, Myelochroa leucotyliza

Lichens are symbiotic associations of fungi and algae (or cyanobacteria). Fungal cells produce large amounts of lipid, assisted by algae, and secrete them out of the cells. Some of the secreted lipids crystallize in the thallus of the lichen. The crystalline materials sometimes occupy 30% of total dry weight of the thallus. This unusual amount of lipid crystal led to our interest in investigating the mechanism of lipid secretion. To begin the cell biological study of lipid secretion and to know the significance of the existence of such crystals in the thallus, it is essential to identify the crystal. The lipid crystal extracted from the thallus of a lichen, Myelochroa leucotyliza, was studied by EM observations, TLC analysis, and EM and X-ray diffraction methods. Atranorin is the predominant component of the crystalline materials in the lipids extracted.     

Miocene and Pliocene dominated diversification of the lichen-forming fungal genus Melanohalea (Parmeliaceae, Ascomycota) and Pleistocene population expansions

ABSTRACT: BACKGROUND: Factors promoting diversification in lichen symbioses remain largely unexplored. While Pleistocene events have been important for driving diversification and affecting distributions in many groups, recent estimates suggest that major radiations within some genera in the largest clade of macrolichens (Parmeliaceae, Ascomycota) vastly predate the Pleistocene. To better understand the temporal placement and sequence of diversification events in lichens, we estimated divergence times in a common lichen-forming fungal genus, Melanohalea, in the Northern Hemisphere. Divergence times were estimated using both concatenated gene tree and coalescence-based multilocus species tree approaches to assess the temporal context of major radiation events within Melanohalea. In order to complement our understanding of processes impacting genetic differentiation, we also evaluated the effects of Pleistocene glacial cycles on population demographics of distinct Melanohalea lineages, differing in reproductive strategies. RESULTS: We found that divergence estimates, from both concatenated gene tree and coalescence-based multilocus species tree approaches, suggest that diversification within Melanohalea occurred predominantly during the Miocene and Pliocene, although estimated of divergence times differed by up to 8.3 million years between the two methods. These results indicate that, in some cases, taxonomically diagnostic characters may be maintained among divergent lineages for millions of years. In other cases, similar phenotypic characters among non-sister taxa, including reproductive strategies, suggest the potential for convergent evolution due to similar selective pressures among distinct lineages. Our analyses provide evidence of population expansions predating the last glacial maximum in the sampled lineages. These results suggest that Pleistocene glaciations were not inherently unfavorable or restrictive for some Melanohalea species, albeit with apparently different demographic histories between sexually and vegetatively reproducing lineages. CONCLUSIONS: Our results contribute to the understanding of how major changes during the Miocene and Pliocene have been important in promoting diversification within common lichen-forming fungi in the northern Hemisphere. Additionally, we provide evidence that glacial oscillations have influenced current population structure of broadly distributed lichenized fungal species throughout the Holarctic.     

Origin and diversification of major clades in parmelioid lichens (Parmeliaceae, Ascomycota) during the Paleogene inferred by Bayesian analysis

There is a long-standing debate on the extent of vicariance and long-distance dispersal events to explain the current distribution of organisms, especially in those with small diaspores potentially prone to long-distance dispersal. Age estimates of clades play a crucial role in evaluating the impact of these processes. The aim of this study is to understand the evolutionary history of the largest clade of macrolichens, the parmelioid lichens (Parmeliaceae, Lecanoromycetes, Ascomycota) by dating the origin of the group and its major lineages. They have a worldwide distribution with centers of distribution in the Neo- and Paleotropics, and semi-arid subtropical regions of the Southern Hemisphere. Phylogenetic analyses were performed using DNA sequences of nuLSU and mtSSU rDNA, and the protein-coding RPB1 gene. The three DNA regions had different evolutionary rates: RPB1 gave a rate two to four times higher than nuLSU and mtSSU. Divergence times of the major clades were estimated with partitioned BEAST analyses allowing different rates for each DNA region and using a relaxed clock model. Three calibrations points were used to date the tree: an inferred age at the stem of Lecanoromycetes, and two dated fossils: Parmelia in the parmelioid group, and Alectoria. Palaeoclimatic conditions and the palaeogeological area cladogram were compared to the dated phylogeny of parmelioid. The parmelioid group diversified around the K/T boundary, and the major clades diverged during the Eocene and Oligocene. The radiation of the genera occurred through globally changing climatic condition of the early Oligocene, Miocene and early Pliocene. The estimated divergence times are consistent with long-distance dispersal events being the major factor to explain the biogeographical distribution patterns of Southern Hemisphere parmelioids, especially for Africa-Australia disjunctions, because the sequential break-up of Gondwana started much earlier than the origin of these clades. However, our data cannot reject vicariance to explain South America-Australia disjunctions.