**Effect of Environmental Change on Secondary Metabolite Production in Lichen-Forming Fungi**

Christopher Deduke, Brinda Timsina and Michele D. Piercey-Normore *Department of Biological Sciences, University of Manitoba, Winnipeg, MB, Canada*

#### **1. Introduction**

196 International Perspectives on Global Environmental Change

Wainright, S.C.; Haney, J.C.; Kerr, C.; Golovkin, A.N.; Flint, M.V. (1998). Utilization of

Waldrop, M.P.; Zak, D.R. (2006). Response of oxidative enzyme activities to nitrogen

Wilkinson, C.E.; Hocking, M.D.; Reimchen, T.E. (2005). Uptake of salmon-derived nitrogen by mosses and liverworts in coastal British Columbia. *Oikos*, 108, 85-98 Zak, D.R.; Holmes, W.E.; Burton, A.J.; Pregitzer, K.S.; Talhelm, A.F. (2008). Simulated

Pribilof Islands, Bering Sea, Alaska. *Marine Biology*, 131, 63-71

decomposition. *Ecological Applications*, 18, 2016-2027

921-933

nitrogen derived from seabird guano by terrestrial and marine plants at St. Paul,

deposition affects soil concentrations of dissolved organic carbon. *Ecosystems*, 9,

atmospheric NO3- deposition increases soil organic matter by slowing

The production and regulation of secondary metabolites in non-lichenized fungi, mainly ascomycetes, has been reviewed by a number of authors with an emerging understanding of the biosynthesis and the pathways involved in regulation (Keller et al., 2005; Yu & Keller 2005; and others). However, lichenized fungi make up almost half of all known ascomycetes (Kirk et al., 2001) and are known to produce over 800 secondary metabolites, most of which are unique to lichenized fungi. Many of these compounds have bioactive properties (Huneck, 1999) and some studies have shown or suggested that secondary metabolite production is influenced by changes in culture conditions, which might be regarded as environmental changes. Intense investigation of the changes in production of these unique bioactive secondary metabolites from lichen fungi have been hampered by problems associated with isolating and growing cultures of lichen fungi. Lichens have been studied for more than two centuries as morphological entities but experimental lichenology has remained a nearly unexplored scientific field for many decades because of the slow growing nature of lichens. Thomas (1939 in Stocker-Worgotter, 2001) reported the first successful resynthesis of *Cladonia pyxidata*. Since the 1970's, one major goal of experimental lichenology has been the improvement and optimization of culture conditions of lichen fungi. Culture techniques for lichen fungi have improved in recent years allowing for further research on these challenging organisms. Therefore, with greater access to cultures of lichen symbionts and progression of knowledge of non-lichenized fungi, studies are just beginning to accumulate on genes involved in production of secondary metabolites from lichen fungi; and the effects of the environment on the expression of these genes by observations in ecological studies, and through experimentation by manipulating culture conditions.

Fungal secondary metabolism is covered by extensive body of literature (see Bennett & Ciegler, 1983). Secondary metabolism is not required for survival and its products are dispensible whereas primary metabolism is essential for survival with anabolic and catabolic activities to maintain life. Secondary metabolites are chemically diverse but are produced from a few key intermediates of primary metabolism, and are generally categorized by the intermediates from which they are produced. Bennett and Ciegler (1983) summarize six categories of secondary metabolites derived from different primary intermediates. Although fungal secondary metabolites are extensive, they are generally

Effect of Environmental Change on

**fungi** 

examining secondary metabolite diversity.

reproduction, influencing the entire biology of the species.

Secondary Metabolite Production in Lichen-Forming Fungi 199

But there were no changes in thallus dimensions or nitrogen fixation activity. A shift in secondary metabolism to allow survival in a particular habitat may promote changes in species and therefore functional attributes of phenotype. One of the functional changes of lichen-fungi dealt with in this chapter is that of secondary metabolite production. To some extent fungal secondary metabolites reflect taxonomy, but some studies have suggested that secondary metabolites may also be influenced by environmental change. Environmental changes influence many cellular activities and also serve as triggers for a change in mode of

Since most species have diagnostic compounds that are consistently produced because of genetic inheritance and species adaptation to particular niches, chemical diversity can be correlated with taxonomy. The chemical correlation with taxonomy is referred to as chemotaxonomy (reviewed by Hawksworth, 1976; Frisvad et al., 2008). Knowledge of species taxonomic diversity is a first clue to understanding the polyketide diversity in any habitat. *Ramalina americana* was split into two different species (*R. culbersoniorum* and *R. americana*) based on secondary metabolite and nucleotide sequence divergence (LaGreca, 1999). The *Cladonia chlorophaea* complex contains at least five chemospecies, which are named and determined by the secondary metabolite produced (Culberson C. F. et al., 1977a). Other examples exist to show variability among individuals within the same geographic area. Secondary metabolites may also vary even within chemospecies. For example, the diagnostic metabolite for *C. grayi* is grayanic acid, and for *C. merochlorophaea* is merochlorophaeic acid. However, these species may or may not produce fumarprotocetraric acid, a polyketide that is considered to be an accessory compound since it is not consistently produced among individuals within a species. One suggestion for the quantity of accessory compounds to vary is changes in the environment (Culberson C. F. et al., 1977a) affecting

regulatory pathways that depend on fungal developmental and environmental cues.

**2.1 Exploring diversity of secondary metabolites within three genera of lichen-forming** 

Since lichens are named according to their fungal partner (Kirk et al., 2001), 13,500 known species of lichenized fungi are somewhat scattered throughout the ascomycete families and reflect one of several ecological groups of fungi. Other ecological groups of fungi include mycorrhizal fungi, plant and animal pathogenic fungi, and saprobic fungi. These ecological groups may be considered artificial groups that reflect changes in feeding habits because of environmental plasticity that are present in most taxonomic groups. The lichenized fungi are currently classified among three classes of ascomycetes, Sordariomycetes, Lecanoromycetes, and Eurotiomycetes, and approximately 20 species of basidiomycetes. The majority of lichen-forming fungi belong to the Lecanoromycetes (Tehler & Wedin, 2008). Three genera within the Lecanoromycetes include *Cladonia*, a large ground-dwelling genus; *Ramalina*, epiphytes on rocks and trees; and *Xanthoparmelia*, an almost exclusive rockdwelling genus. The substratum on which fungi grow allows for a diversity of nutrients to be available to the fungus (Brodo, 1973). The three genera grow on different substrata, have large thalli, have broad global distributions, and therefore provide a good contrast for

The genus *Cladonia* is a large genus within the family Cladoniaceae comprised of more than 400 species (Ahti, 2000) and contains more than 60 described secondary metabolites with 30 of those being major metabolites in high concentration (Ahti, 2000) and the remaining being

produced by one of just a few major pathways (Moore, 1998). The mevalonic acid pathway produces terpenes, sterols, and carotenoids. The malonate (or acetyl-polymalonyl; Elix & Stocker-Worgotter, 2008) pathway produces polyketides. Other metabolites are produced by the shikimate-chorismate (or shikimic acid; Elix & Stocker-Worgotter, 2008) pathway. This chapter focuses mainly on the polyketides produced by the acetyl-polymalonyl pathway. Polyketides constitute structurally diverse molecules produced by the successive condensation of small carboxylic acids, typically co-enzyme A activated malonate by a mechanism similar to fatty acid biosynthesis (Hopwood & Sherman, 1990). The diversity of polyketide structure produced from this pathway reflects the diversity of their biological activities.

For more thorough reviews of the structure and regulation of secondary metabolites in fungi the reader is referred to (Hopwood, 1997; Drew & Demain, 1977; Katz & Donadio, 1993; Hutchinson & Fujii, 1995; Keller & Hohn, 1997; Bennett & Ciegler, 1983). Reviews and inventories of lichen metabolites are summarized by Culberson C. F. & Elix (1989), Elix & Stocker-Worgotter (2008); Culberson C. F. (1969); Culberson C. F. (1970); Culberson C. F., et al. (1977b); Stocker-Worgotter (2008); and a recent classification of lichen substances (Culberson C. F. & Elix, 1989). The adaptive significance of secondary metabolites produced by lichen fungi has been speculated (Lawrey 1977) and numerous functional studies (reviewed in Huneck, 1999), but few studies have linked adaptation of lichen substances with environmental change.

This chapter provides a synopsis of secondary metabolite production in fungi with a focus on lichenized fungi. The synopsis includes a review of the effect of environmental parameters and fungal development on production and regulation of secondary metabolites by focusing on three genera of lichen-forming fungi but not exclusive to these taxa. The chapter also describes an original ecological study of secondary metabolite production for two species of lichen fungi along a geographic gradient. It concludes by summarizing these findings in light of the significance of secondary metabolic changes in terms of ecological and anthropogenic prospects.
