Molecular Ecology (2005) 14, 743–752 doi: 10.1111/j.1365-294X.2005.02453.x © 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Clonal diversity and population genetic structure of arbuscular mycorrhizal fungi (Glomus spp.) studied by multilocus genotyping of single spores EVA H. STUKENBROCK * and SØREN ROSENDAHL Institute of Biology, Department of Microbiology, University of Copenhagen, Oester Farimagsgade 2D, DK-1353 Copenhagen K, Denmark Abstract A nested multiplex PCR (polymerase chain reaction) approach was used for multilocus genotyping of arbuscular mycorrhizal fungal populations. This method allowed us to amplify multiple loci from Glomus single spores in a single PCR amplification. Variable introns in the two protein coding genes GmFOX2 and GmTOR2 were applied as codomin- ant genetic markers together with the LSU rDNA. Genetic structure of Glomus spp. populations from an organically and a conventionally cultured field were compared by hierarchical sampling of spores from four plots in each field. Multilocus genotypes were characterized by SSCP (single stranded conformation polymorphism) and sequencing. All spore genotypes were unique suggesting that no recombination was taking place in the populations. There were no overall differences in the distribution of genotypes in the two fields and identical genotypes could be sampled from both fields. Analysis of gene diversity indicated that Glomus populations are subdivided between plots within each field. There were however, no subdivision between the fields. Keywords: arbuscular mycorrhiza, gene diversity, Glomus spp., multilocus genotyping, population structure, reproductive mode Received 12 August 2004; revision received 5 December 2004; accepted 5 December 2004 Introduction Arbuscular mycorrhizal (AM) fungi, Glomeromycota (Schüssler et al. 2001) are ancient asexuals which form symbiotic associations with a broad range of host plant species. The fungi are abundant in most terrestrial eco- systems and play an important role for plant biodiversity and ecosystem function (Van der Heijden et al . 1998; Helgason et al . 2002). Although most known species of AM fungi appear to be ubiquitous in their distribution in different ecosystems, it is possible that genetic drift and selection pressure affect the spatial genetic structure of fungal populations. Genetic drift or spatially restricted gene flow may lead to the ran- dom fixation of different alleles in different populations thereby causing divergence of populations (Milgroom & Lipari 1995). Additionally, heterogeneous environments may locally select for different genotypes in localized patches also leading to subdivision of populations. This is perhaps best exemplified by populations of pathogenic fungi where natural selection and genetic drift are expected to play important roles in the spatial genetic structure of pathogen populations resulting from selection for avirulence alleles in gene-for-gene interactions between plants and pathogens (Martens et al . 1970; Keen 1990). Only few studies of AM fungi have attempted to assess spatial structure within populations. Koch et al . (2004) found high levels of genetic variability within in vitro propagated populations of Glomus intraradices isolated from tillage/no tillage systems. Soil treatment had little influence on the distribution of genetic variation whereas a spatial structure between plots was observed. Still, little is known about the extent or distribution of genetic variation in natural populations of AM fungi or how genetic variation is influenced by environmental factors. Correspondence: Eva H. Stukenbrock, *Present address: Institute für Pflanzenwissenschaften, Gruppe Phytopatologie, ETH Zentrum/ LFW, Universitätstrasse 2, CH-8092 Zürich, Switzerland. Fax: 41-1-632-1572; E-mail: eva.stukenbrock@ipw.agrl.ethz.ch