Microbiology (2002), 148, 1253–1262 Printed in Great Britain Fine-scale genetic analyses reveal unexpected spatial-temporal heterogeneity in two natural populations of the commercial mushroom Agaricus bisporus Jianping Xu, 1 Christophe Desmerger 2 and Philippe Callac 3 Author for correspondence : Jianping Xu. Tel : ›1 905 525 9140 ext. 27934. Fax: ›1 905 522 6066. e-mail : jpxu!mcmaster.ca 1 Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1 2 Centre Technique du Champignon, Munet, 49400 Distre ! , France 3 Institute National de la Recherche Agronomique, Unite ! de Recherche sur les Champignons, B.P. 81, 33883 Villenave d’Ornon, France This study examined the fine-scale genetic variation of the commercial mushroom, Agaricus bisporus, over 2 years at two sites in France. One site was a meadow fertilized with horse manure and disturbed regularly by humans ; the other was a Monterey cypress forest free of human disturbance. Altogether, 50 mushrooms were collected and analysed for mitochondrial and nuclear genetic variation marked by RFLPs and multilocus enzyme electrophoretic polymorphisms. Population samples from these two sites were genetically different and both sites contained high levels of genetic diversity. No identical genotypes were found at either site between the 2 years and there was little evidence for extensive vegetative clonality for this species. Contrary to expectations, very limited evidence of pseudohomothallic reproduction was found. Results from tests of Hardy–Weinberg equilibrium and genotypic equilibrium showed that outcrossing and recombination have played significant roles in both populations. The results demonstrated spatial- temporal genetic heterogeneity of A. bisporus in natural populations. Keywords : molecular markers, modes of reproduction, population structure, fungi INTRODUCTION The application of molecular markers has had signifi- cant impact in addressing questions concerning the natural history and population biology of many organ- isms, clarifying modes of reproduction, speciation and the spatial-temporal dynamics of populations. For the commercial button mushroom, Agaricus bisporus Lange (Imbach) (syn. A. brunnescens Peck), large-scale geo- graphic structure has been investigated, primarily due to the potential utility of wild germplasm for commercial breeding purposes (Callac, 1995 ; Kerrigan, 1995 ; Xu et al., 1997, 1998). However, relatively little is known about the mode of reproduction and the fine-scale spatial-temporal dynamics of A. bisporus in natural environments. The purpose of this study was to use highly polymorphic molecular markers to investigate the potential modes of reproduction and patterns of spatial-temporal genetic variation of A. bisporus in two defined locations in France. ................................................................................................................................................. Abbreviations : GE, genotypic equilibrium ; HWE, Hardy–Weinberg equi- librium ; LG, linkage group. The basidiomycete A. bisporus is a major agricultural crop in many countries, with a worldwide annual production worth over US$2 billion (Chang, 1993). This fungus has been cultivated for about three cen- turies, beginning in France during the reign of Louis XIV. The practice subsequently spread to other Euro- pean countries. By the early twentieth century, com- mercial cultivation of A. bisporus was widespread on all populated continents. Recent genetic evidence indicated that escapes of cultivar germplasm to the wild ac- companied the spread of commercial cultivation in many geographic areas (Xu et al., 1997, 1998). The mating system in A. bisporus is controlled by one locus with multiple alleles (Xu et al., 1993, 1996a ; Imbernon et al., 1995). Homokaryons (i.e. mycelium with only one nuclear type) with different alleles at the mating-type locus can mate to form a heterokaryon (i.e. mycelium with two different nuclear types) capable of forming mushroom fruiting bodies. However, the pro- duction of homokaryotic basidiospores is limited in most strains of A. bisporus (Allen et al., 1992 ; Kerrigan et al., 1993; Royse & May, 1982; Summerbell et al., 1989). Most often, the majority of basidia produce only 0002-5291 # 2002 SGM 1253