Phylogeny and diversity of Japanese truffles (Tuber spp.) inferred from sequences of four nuclear loci

The genus Tuber, which includes some highly valued truffles, comprises ascomycetous ectomycorrhizal fungi associated with ecologically important tree species. Although the genus is distributed over northern temperate regions, we know little about the phylogeny and diversity of Tuber species in Japan. We have collected 186 new Tuber ascoma samples in Japan over a 10 y period. The identities and phylogenies of the samples were analyzed with sequences of four nuclear loci (i.e. internal transcribed spacer [ITS] and large subunit [LSU] regions of rDNA, elongation factor 1 alpha [EF1-α], and RNA polymerase II large subunit [rpb2] genes). Based on the species delimitation of 95% sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified 20 Tuber species. The number of observed species did not reach an asymptote with our maximum sampling localities in a species accumulation curve. The Chao2 species richness estimator indicated that at least 40 Tuber species should be present in Japan. Molecular phylogenetic analyses revealed that Japanese Tuber species belong to five major phylogroups, including Macrosporum, which had not been reported previously in Asia. Two Japanese species were morphologically and phylogenetically distinct from other known phylogroups, and here we propose a new Tuber phylogroup, Japonicum. In addition most of the other Japanese species formed separate clades within individual major phylogroups and deserve to be proposed as new species. Detailed molecular phylogeny within individual phylogroups revealed the existence of phylogeographic structures at both continental and within-Asia scales, indicating that migration and allopatric speciation have occurred even between the mainland and islands in Asia. Although our findings substantially advance current understanding of Tuber diversity and phylogeny, comparable richness estimation and multilocus phylogeny in other geographic regions are necessary to unequivocally address global patterns of Tuber diversity and biogeography.