Multilocus Phylogeography of the Tuber mesentericum Complex Unearths Three Highly Divergent Cryptic Species.

Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing a high degree of genetic variability. In this study, we performed an integrative taxonomic assessment of the T. mesentericum complex, combining a multilocus phylogeographic approach with morphological analyses, and including authentic specimens of Vittadini, and Berkeley and Broome. We performed maximum likelihood phylogenetic analyses, based on single and concatenated gene datasets (ITS rDNA, ß-tubulin, elongation factor 1-α), and including all available sequences from previous studies. Phylogenetic analyses consistently recovered three reciprocally monophyletic and well-supported clades: clade I, with a wide range across Europe; clade II, specimens collected mainly in the Iberian, Italian, and Balkan peninsulas; and clade III, specimens collected almost exclusively in central Italy. Genetic distance between clades ranged from 10.4% to 13.1% at the ITS region. We also designed new primer pairs specific for each phylogenetic lineage. Morphology of spores, asci, and peridium were investigated on specimens representing the three lineages. Macro- and micromorphological analyses of ascomata revealed only a few, but not diagnostic, differences between the three phylogenetic lineages, thus, confirming that they are morphologically cryptic. By studying authentic specimens of Vittadini, and Berkeley and Broome, it was possible to identify the three clades as T. mesentericum, Tuber bituminatum, and Tuber suave sp. nov., and to designate an epitype for T. mesentericum s.s. and a lectotype for T. bituminatum. Future investigations on volatile organic compound (VOC) composition are needed to define the aroma repertoires in this species complex.

Two new species of Tuber previously reported as Tuber malacodermum.

Recent collections of Tuber malacodermum from Spain, Corsica, and Mexico were compared in an integrative morphological and molecular phylogenetic framework, including study of type material. Phylogenetic analyses of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) and nuc 28S rDNA (28S) regions showed that specimens from Spain and Corsica form a monophyletic group closely related to T. melosporum and T. rufum, whereas Mexican specimens form a clade within the T. lyonii species complex. Peridium and ascospore morphology contribute clear morphological distinctions among specimens from Spain and Corsica, Mexico, and the type specimen of T. malacodermum. Based on results of the morphological and molecular phylogenetic analyses, we assigned the specimens from Spain and Corsica to Tuber pustulatum, sp. nov., and the Mexican specimens to Tuber theleascum, sp. nov. We restrict T. malacodermum to the sole type material. Formal descriptions and illustrations of these taxa are provided.