Molecular and physiological diversity in the bipolar lichen-forming fungus Xanthoria elegans Gareth J. MURTAGH, Paul S. DYER, Peter A. FURNEAUX and Peter D. CRITTENDEN School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK. E-mail : Gareth.Murtagh@nottingham.ac.uk Received 22 February 2002; accepted 8 August 2002. Genetic and physiological diversity was assessed in isolates of the lichen-forming fungus Xanthoria elegans originating from a range of geographic localities and climatic regimes including polar and temperate regions. In vitro cultures were established and isolates examined by production of multi-locus RAPD markers and by sequence comparison of the ITS region of the rDNA tandem repeat unit. Both molecular techniques revealed significant variability. Phylogenetic analysis of RAPD profiles (226 markers) clustered isolates from particular locations together and Mantel’s test showed a correlation between genetic divergence and extent of geographic separation. Phylogenies based on ITS sequence divergence were not well supported and did not correlate with the geographic origin of samples, suggesting a single locus approach to be less informative than RAPD data in a study of this nature. Physiological investigations also revealed significant variability between isolates with different geographic origins. Temperature had a significant effect on relative growth rate (RGR) such that X. elegans originating from sites with lower mean annual temperatures had significantly higher RGRs at all test temperatures between 2 x and 18 xC. Enhanced metabolic activity might be an adaptation for growth in colder climates. The results demonstrate high genetic diversity within this morphologically variable species in response to geographic and environmental factors, and are discussed in relation to data obtained for non-lichenised fungal species and the possibility of cryptic speciation. INTRODUCTION Lichen-forming fungi constitute approximately 21 % of all known fungal species and represent ancient groups of fungi with slow rates of evolution and highly varied geographical distributions (Honegger 1991, Ka¨ rnefelt 1990). Galloway (1996) described 16 principal types of distribution ranging from endemic (e.g. Vulpicida spec- ies ; Mattsson 1993) to bipolar and cosmopolitan (e.g. Cladonia species; Stenroos 1993). Despite their preva- lence, little is known of the population genetics and genetic diversity of lichen-forming fungi, in contrast to advances made with many ‘ free-living ’ species. This is mainly due to the inherent difficulties of working with lichen-forming fungi, such as their very slow growth rates and inability to establish the symbiotic phenotype in vitro (Honegger 1991). Knowledge of the genotypes of lichen-forming fungi will allow insights into several areas of the natural history of lichens. While morpho- species are relatively easily identified, the genetic diver- sity of broadly distributed species is unclear and wheth- er or not local populations are specifically adapted, possibly to the point of speciation, is unknown. The first population markers used with lichens relied on secondary genetic characters, such as presence or absence of specific secondary metabolites, and were used to discriminate distinct populations of the same species on a local scale (Culberson & Culberson 1967, Hageman & Fahselt 1984, Reyes, Lopez-Bilbao & Molina 1996). DNA markers, offering increased resolution, have since been used to quantify genetic divergence both within and between certain species of lichen-forming fungi. DePriest & Been (1992) correlated the presence of different numbers of group I introns within the small subunit (SSU) rDNA with different chemotypes of the species complex Cladonia chlorophaea. Further studies showed a natural population to contain podetia with SSU rDNA of at least four distinct size classes and nine restriction-site patterns (DePriest 1993). Beard & DePriest (1996) used similar molecular markers to dem- onstrate genetic variation in mats of Cladonia subtenuis growing at the same and at different geographic loca- tions, and Crespo et al. (1997, 1999) differentiated sub- populations of Parmelia sulcata on the basis of size differences in the rRNA gene cluster due to the presence of an intron. Zoller, Lutzoni & Scheidegger (1999) de- termined sequence data for six loci and found limited genetic diversity among six Swiss populations of Lobaria pulmonaria. DNA fingerprinting by means of random amplified polymorphic DNA (RAPDs) has Mycol. Res. 106 (11): 1277–1286 (November 2002). f The British Mycological Society 1277 DOI: 10.1017/S0953756202006615 Printed in the United Kingdom.