Chicita F. Culberson

Improved conditions and new data for identification of lichen products by standardized thin-layer chromatographic method

Abstract A standardized method for the identification of compounds by thin layer chromatography uses three solvent systems and RF classes coded on punched cards. This method, applied to the study of secondary products of lichen-forming fungi, has been imporoved and expanded to include easily prepared hydrolysis and O-methylation products. The data reported allow the confirmation of many substances previously difficult to identify and the proposal of structures for certain types of new compounds extrated from fragments of herbarium specimens. New data are given for 220 compounds and derivatives chromatographed in three standard solvent systems.

A standardized method for the identification of lichen products

Abstract A procedure for the routine identification of the products of lichen-forming fungi by thin-layer chromatography is described. Microextracts of plant fragments are chromatographed in three solvent systems. The spots of unknowns are assigned to R F classes defined by the R F values of marker controls of two lichen substances (atranorin and norstictic acid) chromatographed on every plate. The unknowns are tenatively identified by sorting (by R F classes) punched cards summarizing microchemical data for all compounds previously studied. The preliminary identification is then confirmed by additional microchemical tests. The open-ended system can incorporate new and unknown compounds as well as information from other chromatographic systems. Data obtained by the standardized procedure are given for 104 products.

A Standardized TLC Analysis of f3-Orcinol Depsidones

Many p-orcinol depsidones with low Rf values in the three stan- dardized TLC solvents commonly used in lichenology are better resolved by a solvent system of toluenelethyl acetate/formic acid. Standardized chromatographic data and examples of specific problems resolved by one- and two-dimensional TLC with this solvent system are given. Chemical analyses are reported for 25 lichen species from 17 genera. A vexing problem in the identification of lichen products by thin-layer chromatography (TLC) has been the separation of some 35 low-Rf, p-orcinol compounds many of which are still unknowns. In the three standardized solvent systems (Culberson & Kristinsson 1970; Culberson 1972, 1974; Culberson & Ammann 1979), most of these compounds run below the control substance norstictic acid, which has Rf values between 28 and 40. Even if the spots for these substances were evenly spaced, the Rf values would differ by only about one millimeter. In addition, we now know that these compounds occur in complicated mixtures and consequently accurate identifications will require more sophisticated chro- matographic techniques than those routinely used. Reports in the recent literature of low- Rf compounds (even common ones like salazinic, constictic and connorstictic acids) may be misidentifications based upon insufficient TLC evidence. The purpose of the present paper is to give standardized TLC data for a fourth solvent system (Solvent G) to assist the resolution of difficult mixtures of low-Rf, p-orcinol depsidones by one- and two-dimen- sional TLC. The chromatographic procedure followed the standardized method (Culberson 1972) except that toluene (dried over CaCl2 and redistilled) was used in place of benzene. Solvent G, toluene/ethyl acetate/formic acid (139:83:8, v/v/v; 230 ml), is modified from previously suggested systems (Stahl & Schorn 1961; Hirayama et al. 1976). Formic acid (95-97%) and ethyl acetate were not purified or redistilled; it is not necessary to purify a good grade of toluene for satisfactory results with Solvent G. As with Solvent B, however, a grade of formic acid with low water content is essential. TLC plates were Merck Silica Gel 60 F254 (0.25-mm layer; No. 5765). Chromatographic tanks (Desaga- Heidelberg) were insulated from external temperature fluctuations but were not lined with filter paper. Pre-equilibration of the plates in 60% formic acid (Culberson 1974) did not improve the quality of chromatograms in Solvent G. Unless noted otherwise, the standardized data in Table 1 were from shortened plates (12.5 x 20 cm) developed to 10 cm. Initial spots were 2 cm from the bottom of the plate and 0.5 cm above the solvent. Controls for standardization were included at three positions on every plate. The Rf data in Table 1 are standardized with respect to fumarprotocetraric acid in addition to the usual control substances, atranorin and norstictic acid. Two-dimensional chromatograms were prepared by the standardized method (Culberson & John- son 1976) except that full plates (20 x 20 cm) were used with seven one-dimensional comparisons (including two atranorin-norstictic acid controls) in each direction. In addition, for the second direc- tion, an atranorin-norstictic acid control was added just to the left of the origin on the two-dimensional portion of the plate. This control helps to align spots and to monitor effects of residual first-direction solvent on R, values in the second direction. (In the present report, first- and second-direction solvents

Induction of a complete secondary-product pathway in a cultured lichen fungus

CULBERSON, C. F., AND ARMALEO, D. 1992. Induction of a complete secondary-product pathway in a cultured lichen fungus. Experimental Mycology 16, 52-63. Experimental studies on the secondary metabolism characteristic of lichens have been impeded by the slow growth of the fungi and by the inconsistent results of many attempts to induce the pathways in the fungi isolated from their photosynthetic partners. In the present study, a lichen-specific secondary pathway was consistently induced in a lichen fungus (Cladonia grayi) grown in the absence of the alga. The depside (4-0-demethylsphaerophorin) and two depsidones (grayanic and 4-0-demethylgrayanic acids) found in the natural lichen began to accumulate a few days after the transfer of lightly fragmented mycelia from liquid to solid medium. Induction was enhanced on drier substrates and was correlated with the proliferation of aerial hyphae, where the major product (grayanic acid) accumulated in extracellular patches visible by fluorescence microscopy. The time course was analyzed by quantitative high-performance liquid chromatography of extracts from small cultures grown on nylon filters. Induction was rapid in view of the slow growth of the fungus, and secondary productivity was comparable to that of some nonlichen fungi. These results cotm polyketides; lichen fungi; Cladonia grayi; grayanic acid; depsides; depsidones; aerial hyphae; photobiont; hyphal differentiation.

Xanthones and depsidones of the lichen Lecanora dispersa in nature and of its mycobiont in culture

A chemotype ofthe natural lichen Lecanora dispersa contains 2,7-dichlorolichexanthone as the major secondary compound, but cultured spore isolates, growing without the alga, produced pannarin and related depsidones (HPLC, TLC and MS) instead. HPLC data suggested that traces of the xanthone characteristic of the source lichen might also be in some of the pannarin-producing sporelings, but concentrations were not sufficient for TLC or MS confirmation. Surprisingly, pannarin could not be confirmed in voucher material of the natural source lichen, but the biosynthetic potential for this depsidone was proven for the species by a herbarium survey. When museum samples larger than single apothecia were analyzed, trace to moderate proportions of pannarin were confirmed in some specimens. That the source lichen produced primarily xanthones and the mycobiont produced primarily depsidones is surprising because xanthones are a class of compounds also common in non-lichen fungi whereas depsidones like pannarin are nearly restricted to lichens. Pannarin, which has previously not been reported from an isolated lichen mycobiont, was produced copiously by some cultures.

A standardized two-dimensional thin-layer chromatographic method for lichen products

Abstract Two-dimensional thin-layer chromatography is useful for microchemical studies on mixtures difficult to resolve by the standardized one-dimensional thin-layer chromatographic method now commonly used for lichen products. A modified two-dimensional technique uses the large body of standardized RF data already accumulated for these compounds. In addition, correlations of RF values with chemical structures permit tentative identifications of many trace constituents, including new natural products, resolved from microextracts by the two-dimensional method. The standardized two-dimensional procedure also allows more reliable comparisons of chromatograms and the determination of RF classes of components of complex mixtures. The method is illustrated for the orcinol-type depsides of two closely related species, Parmelia loxodes and P. verruculifera.

Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.

Habitat selection by chemically differentiated races of lichens.

The maritime European lichens of the aggregate species Ramalina siliquosa represent six chemical races. Where the races are sympatric they populate different habitats. Such intensive local ecological sorting of morphologically similar individuals accumulating different, highly specialized metabolic end products appears to be unknown in other plants.

A Phylogenetic View of Chemical Evolution in the Lichens

The distribution of 209 chemical substances among the 2,315 species of lichens that have been reported in a literature of approximately 1,000 papers is summarized. The information is used to assess the extent and the nature of chemical evolution in the lichen-forming fungi and to evaluate the present supraspecific (especially generic and familial) classifications of these plants. The order Lecanorales is the seat of most of the chemical variation, especially in the secondary natural products most useful in systematics, namely the sub- stances formed by coupling of phenolic units such as the orcinol and p-orcinol compounds. Most genera and families that are well defined morphologically and appear to represent natural taxa show highly uniform chemistries of several to many biogenetically related substances or sets of substances. Many genera with chemistries discordant for the families in which they are currently classified also seem to have affinities elsewhere from morphological information. In the lichen-forming fungi, which lack a fossil record, comparative phytochemistry is the most useful independent check that exists to evaluate the naturalness of systems of classification based upon morphology.

Characteristic lichen products in cultures of chemotypes of the Ramalina siliquosa complex

Sixteen characteristic aromatic lichen products were identified in alga-free fungal cultures derived from single-spore isolates of four chemotypes of the Ramalina siliquosa species complex. The chemistries of many cultures are more complex than those of natural thalli. The compounds identified include the major f-orcinol depsidones that characterize the chemotypes as well as biogenetically reasonable precursors not yet proved in natural thalli. Single-spore progeny from natural thalli can be assigned to chemotype without being lichenized with an alga. This result simplifies the use of secondary-product chemistry as a genetic marker to assess the limits of gene flow between chemotypes in natural populations.