Four new Tuber species added to the Rufum group from China based on morphological and molecular evidence

Four new species, Tuber crassitunicatum, T. lishanense, T. piceatum, and T. wanglangense, are described and illustrated based on both morphological and molecular data. Morphologically, T. crassitunicatum is diagnosed by its brown ascomata and ellipsoidal ascospores ornamented by crowded spiny reticulum with more than 10 meshes across the spore width. Tuber lishanense can be recognized by its whitish to pale brown ascomata with a basal cavity, and very broadly ellipsoidal to subglobose ascospores ornamented by densely isolated spines. Tuber piceatum differs in its pale white ascomata and ellipsoidal ascospores with isolated spines, whereas T. wanglangense is characterized by its broad ellipsoidal ascospores with short spines that are connected by lower ridges when examined under scanning electron microscope (SEM). Phylogenetic analyses inferred the four new species in the Rufum group. Each species had less than 96% similarity in the internal transcribed spacer (ITS1-5.8S-ITS2) nuc rDNA ITS sequence to other Tuber species and represented a unique terminal clade in the phylogenetic tree. Our research did not confirm the occurrence in China of the European T. rufum and T. nitidum and the North American T. lyonii, although they are often reported in the literature on Chinese Tuber species.