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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 alleles3 and highly variable RAPD patterns4.

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, 1-3×104 kilograms of black truffles are now officially sold each year, and more than 106 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 zone5. 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 burgundy truffles, which ripen in spring and autumn, respectively, would have allowed them to sustain a larger geographical range and population size during the last glaciation, explaining the present high level of genetic variability of this species complex. Moreover, their current geographical range, extending further to the east and north (for example, to Poland and Sweden6), shows that they are more tolerant of colder climates.

Our results show that the morphological and organoleptic differences seen over the geographical range of the black truffles can probably be explained by environmental variation rather than by genetic factors. Research is needed to identify the environmental variables that affect the black truffle's perfume and taste, which are the objects of intense human interest.