Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis

Extensive morphological convergence or divergence, a common occurrence in fungi, tends to obscure recognition of phylogenetic relationships among Pezizales, widespread filamentous Ascomycetes with either enclosed underground (hypogeous) or exposed (epigeous) fruit bodies, that often establish mutualistic interactions with arboreous plants. Focusing on hypogeous Pezizales commonly known as truffles, we sequenced the 18S rDNA from nine species belonging to three different families (Tuberaceae, Terfeziaceae, and Balsamiaceae). A data set consisting of 1700 secondary structure-aligned sites, including 24 homologous sequences from the GenBank DNA database and using three reconstruction methods, was employed to infer phylogenies in an interval ranging from the subordinal to the subgeneric level. As revealed by the 18S phylogenetic scheme, Balsamiaceae represent a monophyletic clade, comprising the hypogeous taxa Balsamia and Barssia, nested within Helvellaceae. Similarly, the terfeziacean genera Pachyphloeus and Terfezia constitute together with Cazia a distinct hypogeous clade nested within Pezizaceae. The lack of clustering between Terfezia arenaria and Terfezia terfezioides strongly supports the reassignment of the latter taxon to the original monotypic genus Mattirolomyces. Within Tuberaceae, which are sister to the highly evolved Helvellaceae, the genus Tuber cannot be considered monophyletic if Choiromyces is recognized. The paraphyly of Tuber and other relationships that were not supported by high bootstrap values, nor corroborated by morphological evidence, were supported by a parallel analysis of the faster evolving internal transcribed spacer (ITS) rDNA. Distinct episodes of fruit body morphology shifts are discernable in the 18S rDNA phylogenetic tree. In all cases, the shift from an epigeous to a hypogeous form is the most parsimonious interpretation of character transformation, without any instance of character reversal.