Heterothallism in T. melanosporum and T. indicum: warning and lessons from genomics to preserve identity and promote fructification of the Perigord truffle

The last several years have witnessed a leap forward in our understanding of the life cycle and the reproductive biology of truffles . The sequencing of Tuber melanosporum genome has permitted the identification of the MAT locus, which is the genomic region controlling the reproduction mode of fungi . Since T. melanosporum strains harbor either the MAT1-1-1 or MAT 1-2-1 gene at this locus, we concluded that this fungus is heterothallic . The availability of sequence information for T. melanosporum MAT genes gives way to cloning their orthologs in other truffle species. Among these, the Asiatic species T. indicum is the closest relative of T. melanosporum, and produces black truffles of inferior value with respect to the Périgord truffle. The high rate of polymorphism at morphological and molecular traits within black truffles from Asia prompted several authors to claim the presence of several other species in addition to T. indicum . Here, benefiting from the information and genomic tools relative to T. melanosporum MAT genes, we aimed to: a) corroborate the occurrence of a mating type dependent spatial segregation of T. melanosporum strains on host plants and b) clone the orthologs of these genes from T. indicum. With respect to the first point we collected ectomycorrhizas and soil samples from four T. melanosporum grounds located in central Italy and screened them with MAT specific primer pairs. These results lend further support to our contention of a spatial segregation, likely due to a competitive exclusion between genotypes, of strains of opposite mating type on their hosts. Concerning T. indicum, here we show that it is also a heterothallic species since its MAT locus is organized in two idiomorphs harbored by different strains . By virtue of their high evolutionary rates, the MAT genes are good candidates for taxonomic and phylogenetic analyses [10]. On these grounds, the comparative analyses of MAT locus from several Chinese specimen point to the presence of at least two cryptic species within the T. indicum complex and support the hypothesis of vicariance for explaining the allopatric isolation of T. melanosporum from T. indicum populations in Europe and Asia, respectively. Notwithstanding, all T. indicum MAT1-1-1 and MAT1-1-2 genes show a high rate of similarity with their orthologs from T. melanosporum. The evidence that T. indicum is not only the closest relative to T. melanosporum, but it is also heterothallic is pragmatically very relevant. Because T. indicum and T. melanosporum could be sexually compatible, the deliberate or accidental introduction of T. indicum in Europe may represent a serious ecological threat for the indigenous populations of T. melanosporum. Tools and strategies that span from innovative host plant mycorrhization techniques to methods for certifying species identity and mating type of truffle strains will be discussed. These instruments are fundamental to preserve T. melanosporum biodiversity from erosion and counteract gamete limitation that likely occurs in T. melanosporum stands, as a important consequence of the spatial segregation of strains with different mating types.