Larry L. St. Clair

Lichens: a promising source of antibiotic and anticancer drugs

Lichens are symbiotic associations between fungi and a photosynthetic alga and/or cyanobacteria. Lichenized fungi have been found to produce a wide array of secondary metabolites, most of which are unique to the lichenized condition. These secondary metabolites have shown an impressive range of biological activities including antibiotics, antifungal, anti-HIV, anticancer, anti-protozoan, etc. This review focuses primarily on the antibiotic and anticancer properties of lichen secondary chemicals. We have reviewed various publications related to antibiotic and anticancer drug therapies emphasizing results about specific lichens and/or lichen compounds, which microbes or cancer cells were involved and the main findings of each study. We found that crude lichen extracts and various isolated lichen compounds often demonstrate significant inhibitory activity against various pathogenic bacteria and cancer cell lines at very low concentrations. There were no studies examining the specific mechanism of action against pathogenic bacteria; however, we did find a limited number of studies where the mechanism of action against cancer cell lines had been explored. The molecular mechanism of cell death by lichen compounds includes cell cycle arrest, apoptosis, necrosis, and inhibition of angiogenesis. Although lichens are a reservoir for various biologically active compounds, only a limited number have been tested for their biological significance. There is clearly an urgent need for expanding research in this area of study, including in depth studies of those compounds which have shown promising results as well as a strong focus on identifying specific mechanisms of action and extensive clinical trials using the most promising lichen based drug therapies followed by large scale production of the best of those compounds.

Species delimitation and evolution in morphologically and chemically diverse communities of the lichen-forming genus Xanthoparmelia (Parmeliaceae, Ascomycota) in western North America

PREMISE OF THE STUDY Accurate species delimitation is important for understanding the diversification of biota and has critical implications for ecological and conservation studies. However, a growing body of evidence indicates that morphology-based species circumspection in lichenized fungi misrepresents fungal diversity. The foliose lichen genus Xanthoparmelia includes over 800 species displaying a complex array of morphological and secondary metabolite diversity. METHODS We used a multifaceted approach, applying phylogenetic, population genetic, and genealogical analyses to delimit species in a single well-supported monophyletic clade containing 10 morphologically and chemically diverse Xanthoparmelia species in western North America. Sequence data from four ribosomal and two low-copy, protein-coding markers, along with chemical and morphological data were used to assess species diversity. KEY RESULTS We found that traditionally circumscribed species are not supported by molecular data. Rather, all sampled taxa were better represented by three polymorphic population clusters. Our results suggest that secondary metabolite variation may have limited utility in diagnosing lineages within this group, while identified populations clusters did not reflect major phylogeographic or ecological patterns. CONCLUSIONS In contrast to studies revealing previously undiscovered fungal lineages masked within lichen species circumscribed by traditional morphological and chemical concepts, the present study suggests that species diversity has been overestimated in the species-rich genus Xanthoparmelia.

Species delimitation in taxonomically difficult lichen-forming fungi: an example from morphologically and chemically diverse Xanthoparmelia (Parmeliaceae) in North America.

Mounting evidence suggests many morphology-based species circumscriptions in lichenized ascomycetes misrepresent fungal diversity. The lichenized ascomycete genus Xanthoparmelia includes over 800 described species displaying a considerable range of morphological and chemical variation. Species circumscriptions in this genus have traditionally been based on thallus morphology, medullary chemistry, and the presence or absence of sexual or asexual reproductive structures. Notwithstanding concerted effort on the part of taxonomists to arrive at a natural classification, modern taxonomic concepts for the most part remain unclear. Here we assess the evolution of characters traditionally regarded as taxonomically important by reconstructing a phylogenetic hypothesis based on sequence data from four nuclear ribosomal markers as well as fragments from two protein-coding nuclear loci. A total of 414 individuals were tested, representing 19 currently accepted species. Most sampled species, as currently circumscribed, were recovered as polyphyletic, suggesting that major diagnostic characters have evolved in a homoplasious manner. The vagrant growth form, distinct medullary chemistries, and production of vegetative diaspores appear to have evolved independently multiple times. Application of a population assignment test resulted in the recognition of 21 species-level genetic clusters, each of which was supported by a comparison of genetic distances as well as a Bayesian species delimitation method calculating probabilities associated with speciation events. Inferred clusters are largely incongruent with traditionally circumscribed species due to the prevalence of cryptic diversity and, in some cases, high levels of intraspecific morphological and chemical variation. These results call for a major taxonomic revision of Xanthoparmelia species in western North America.

Element Accumulation Patterns in Foliose and Fruticose Lichens from Rock and Bark Substrates in Arizona

Abstract Growth form and substrate influences on elemental accumulation patterns were investigated in four lichen species. Two fruticose species (Usnea amblyoclada on rock and Usnea hirta on bark) and two foliose species (Flavoparmelia caperata on rock and Flavopunctelia flaventior on bark) were collected below Massai Point in Chiricahua National Monument in southeastern Arizona, U.S.A. Samples were analyzed for 14 elements. A two-way ANOVA model was used to examine the relationships between substrate and growth form (independent variables) on element accumulation (dependent variable) patterns in lichen samples. In the ANOVA model the growth form variable was significant for K, Ca, Ti, Ba, Fe, Ni Cu, Zn, Pb, Rb, and Sr while the substrate variable was significant for K, Ti, Mn, Fe, Ni, Rb, and Sr. A significant interaction between the two class variables was observed for P, K, Ti, Mn, Fe Ni, Rb, and Sr. Accumulation of sulfur appeared to be independent of both growth form and substrate influences. In this study growth form was a key factor affecting element accumulation patterns in lichens. It is proposed that thallus continuity and orientation, which partially define growth form characteristics, influenced the accumulation of elements from airborne and substrate sources.

Pelletized cyanobacterial soil amendments: Laboratory testing for survival, escapability, and Nitrogen fixation

The escape and growth of Microcoleus vaginatus from alginate pellets onto soil crusts under controlled laboratory conditions were examined. Soil crust samples were collected and placed in petri dishes. Selected samples were sterilized, and three treatmerits were established: 75 g m−2 Microcoleus pellets added to sterilized soil, 75 g m−2 blank pellets added to sterilized soil, and unsterilized soil with no additions. Chlorophyll a readings were significantly higher in unsterilized soils. Microcoleus biovolumes were highest in sterilized inoculated soils and significantly higher than sterilized untreated soils. Microcoleus filament density was significantly higher in Microcoleus‐inoculated soils than in the other two treatments. Nitrogen fixation activity was recorded in unsterilized soils after only 1 month, and rates increased as the experiment progressed. Microcoleus‐inoculated soils had significant nitrogen‐fixing activity during the last sampling period.

Influence of growth form on the accumulation of airborne copper by lichens

Lichens are known to accumulate airborne elements. This characteristic makes them useful as biomonitors of air quality. However, direct correlations of element concentrations in the air with element concentrations in lichen thalli are generally unavailable. The purpose of this study was to quantitatively examine the relationship between concentrations of copper in ambient air samples and thalli of foliose and fruticose lichens. Lichen samples from four sites along an air copper gradient were collected and analyzed. Foliose specimens consistently accumulated more than twice as much copper as fruticose specimens at all four sites. The relationship between copper concentrations in foliose and fruticose lichens and ambient air samples along an airborne copper gradient was examined using a stepwise regression model. An R2 value of (0.84) and an F-statistic of (182.5, p<0.001) for the model indicate that variability in lichen copper concentrations between sites is explained by airborne copper concentrations. Based on the regression analysis both foliose and fruticose growth forms appear to accurately predict airborne copper concentrations. A significant interaction between the airborne copper and growth form variables and large differences in the calculated slopes suggests that foliose lichens more efficiently accumulate airborne copper than fruticose lichens.

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.

Pleistocene Speciation in North American Lichenized Fungi and the Impact of Alternative Species Circumscriptions and Rates of Molecular Evolution on Divergence Estimates

Pleistocene climatic fluctuations influenced patterns of genetic variation and promoted speciation across a wide range of species groups. Lichens are commonly found in habitats that were directly impacted by glacial cycles; however, the role of Pleistocene climate in driving speciation in most lichen symbionts remains unclear. This uncertainty is due in part to limitations in our ability to accurately recognize independently evolving lichen-forming fungal lineages and a lack of relevant fossil calibrations. Using a coalescent-based species tree approach, we estimated divergence times for two sister clades in the genus Xanthoparmelia (Parmeliaceae) restricted to western North America. We assessed the influence of two different species circumscription scenarios and various locus-specific rates of molecular evolution on divergence estimates. Species circumscriptions were validated using the program BP&P. although speciation was generally supported in both scenarios, divergence times differed between traditional species circumscriptions and those based on genetic data, with more recent estimates resulting from the former. Similarly, rates of evolution for different loci resulted in variable divergence time estimates. However, our results unambiguously indicate that diversification in the sampled Xanthoparmelia clades occurred during the Pleistocene. Our study highlights the potential impact of ambiguous species circumscriptions and uncertain rates of molecular evolution on estimating divergence times within a multilocus species tree framework.

Estimating Xanthoparmelia (Parmeliaceae) population density in subalpine communities in southern Utah, U.S.A. using two distance methods, with implications for assessing community composition

Abstract Lichens are often used to monitor air quality and ecosystem health, and to define ecological gradients. Quantitative methods for estimating lichen population density and community composition are essential to effectively assess species and community response to ecological and environmental change. In this study we used two distance methods (point-centered quarter and quartered-neighbor methods), along with a compound estimator (Diggles) to remove bias from non-random spatial patterns, to estimate Xanthoparmelia population density at a generic level on the summit of a high elevation plateau in southern Utah, USA. The sampling was extended to assess species composition and diversity. Population densities and relative species abundance were evaluated between meadow areas, sites, cardinal directions, and general substrate classes. We found that Xanthoparmelia populations varied widely in density and community composition across a relatively homogeneous landscape. Microhabitat, meadow-wide, and plateau-wide factors significantly influenced density and community structure in Xanthoparmelia. Our data reemphasize the inherent variability found in lichen communities and the importance of effective sampling for accurate ecological and biomonitoring studies. The two distance methods implemented here, moderated by Diggles population density estimator, provide an efficient alternative to cover methods for estimating density. This study also indicates that these methods provide an effective approach for estimating population density and community structure in conjunction with ecological and bio-monitoring studies of some types of terricolous and saxicolous lichen communities.

Size is not a reliable measure of sexual fecundity in two species of lichenized fungi

Abstract Since a number of studies have shown a strong positive correlation between apothecia production and lichen thallus size, it has been suggested that size can serve as an easy measure of lichen sexual fecundity. The reliability of the relationship between size and apothecia production among different environments has not been studied. We measured apothecia development and thallus area in populations of the lichenized fungi Xanthoparmelia cumberlandia and Xanthoparmelia coloradoënsis. Our data suggest that while size is a significant predictor of apothecia production, the relationship between size and apothecia production is not consistent among various environments, nor does size adequately explain variation in apothecia production.