Of the ten species of European truffle (fungi of the genus Tuber, phylum Ascomycota), some have economic value because of their organoleptic properties (taste and perfume), in particular the black truffle (Tuber melanosporum Vitt.) and the summer and burgundy truffles 1,2 . The black truffle is mainly found in Spain, France and Italy (Fig. 1a), and it shows variation in several traits, including in its famous organoleptic properties, across this geographical range. Here we show that this variation probably results from environmental, rather than genetic, influences.In an attempt to explain the variation in T. melanosporum across its geographical range and to study the distribution of genetic variability within and among populations, we analysed fruiting bodies (ascocarps) from different populations in France and Italy for random amplified polymorphic DNA (RAPD) and microsatellite polymorphism. We found an extremely low level of polymorphism over the whole study area for both types of marker (Fig. 1b). This pattern contrasts dramatically with that of the sympatric summer truffle (T. aestivum Vitt., which forms a species complex with the burgundy truffle, T. uncinatum Chatin), which has distinct internal transcribed spacer alleles 3 and highly variable RAPD patterns 4 .The absence of heterozygotes in T. melanosporum, shown by study of microsatellites, is consistent with a very closed mating system, such as homothallism or even exclusive selfing. As the two putative haploid genomes from an ascocarp are very similar, we considered each variable RAPD band to be a distinct locus exhibiting two alleles (presence or absence of a band). The genetic models used subsequently showed no increase in genetic differentiation with geographic distance (Fig. 1c).The low level of genetic diversity of the black truffle probably cannot be explained by its present large population size. In France, 1-3 ǂ10 4 kilograms of black truffles are now officially sold each year, and more than 10 6 kilograms per year were traded in the last century (the average weight of an ascocarp is about 30-40 grams).A population bottleneck probably occurred during the last, and coldest, glaciation, when the broadleaved forest of Europe was considerably reduced and restricted mainly to the Mediterranean coastal zone 5 . The black truffle ripens in winter (November-February), which probably contributed to its drastic reduction in population size and restriction to its southernmost limits during the glaciation, as ascocarps of contemporary T. melanosporum are susceptible to frost. The present low level of genetic variability in black truffle populations is consistent with such a bottleneck occurring 10,000 years ago, followed by a rapid colonization of southwestern Europe, which would also explain the absence of phylogeographic signals in the few polymorphic markers found. The 'glaciation hypothesis' would be strengthened if more southerly populations (in Spain or Italy) were found to show greater genetic diversity.The seasonal behaviour of the summer and burgu...

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The colonization dynamics of the black true in an arti®cial ®eld were assessed through analyses of microsatellite and RAPD markers. The true ®eld was composed of three tree species and mycelial inoculum of three dierent origins, and was monitored for the ®rst three years of true production. We found very low levels of genetic diversity. Isolation by distance was detected only at the betweentree level. This could be interpreted as local colonization around each tree facilitated by the presence of the tree root system. At the larger spatial scale of the European range, the absence of isolation by distance corroborates the hypothesis of an impact of glaciation on genetic variation, followed by rapid postglaciation demographic expansion. In addition, genetic variation of harvested trues was explained by neither inoculation origin, nor tree species. Our study questions the real impact of manmade inoculation of tree root systems with fungal mycelia.Keywords: biogeography, microsatellite, population genetics, RAPD, true, Tuber melanosporum. IntroductionThe black true (Tuber melanosporum Vitt.) is a hypogeous ectomycorrhizal ascomycete mainly found in France, Italy and Spain (Callot, 1999), and highly appreciated for its remarkable organoleptic properties (taste and odour). Haploid mycelium can be found in the soil, usually in contact with or near to tree root systems. Fruiting bodies (i.e. ascocarps) result from the fusion of two haploid mycelia, and are therefore supposedly diploid (Moore-Landecker, 1996). They are composed of conjunctive tissue (the gleba) that surrounds haploid spores. These spores are produced by meiosis after karyogamy.The black true naturally occurs on calcareous soils, in association with the root systems of trees, most often oaks (Quercus spp.) and hazelnuts (Corylus avellana). Since the 1960s, attempts have been made to create favourable arti®cial habitats to grow and monitor trues, resulting in managed ®elds of planted trees.Traditionally, a true ®eld is created by sowing oak acorns and relying on the presence of true mycelium in the soil to interact with the developing tree root system. Under such conditions the ®rst trues are usually harvested about 6±8 years after sowing. Alternatively, it is now possible to inoculate the roots of young trees with speci®c mycelia before planting these trees directly in the soil (Chevalier & Grente, 1979). Trues are harvested earlier (3±4 years) under these conditions (Chevalier & Dupre , 1988) and can even be grown in areas where the mycelium did not previously occur (e.g. New Zealand and Tasmania; Callot, 1999). However, when T. melanosporum mycelia or spores already exist in the soil (mainly in France, Italy and Spain), the usefulness of planting inoculated trees is unclear. In particular, it has not been established whether the resulting trues originated from the arti®cially generated mycorrhiza or from formerly present local (and potentially competitive) mycelia. In other words, when a black true is harvested around an arti®cially inoculated tree, t...

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