Zn overaccumulating Russula species clade together and use the same mechanism for the detoxification of excess Zn.

It has been firmly established that macrofungi can accumulate large amounts of heavy metals in their sporocarps. However, the mechanisms of the accumulation and storage are being uncovered only recently. We have previously documented that Russula bresadolae can accumulate over 1 g Zn kg-1 dry weight and that sequestration of a substantial proportion of overaccumulated Zn involves binding with peptides, RaZBPs, seen so far only in this species. In this work we examined Zn contents of 360 sporocarp collections from unpolluted environments covering 114 species of the genus Russula. Whilst the concentrations of Zn in most analysed species were in the range of 50-150 mg kg-1 , the species of subgenera Brevipes and Compactae accumulate very low Zn (< 50 mg kg-1 ). We further identified five new Zn-overaccumulating species of subgenus Russula, which form with R. bresadolae a separate phylogenetic subclade in which the sporocarp Zn concentrations ranged from 326 to 845 mg kg-1 . We demonstrate that R. pumila and R. ochroleuca express at least one ZBP gene and when expressed in metal-sensitive S. cerevisiae, all ZBPs protected the yeasts against Zn (and Cd) toxicity equally well. The respective ZBPs were confirmed in the native Zn-complexes of R. pumila and R. ochroleuca, which represented 80% of Zn extracted from their sporocarps. This study is the first extensive genus-wide report of metal accumulation in macrofungi, which further demonstrates that the Zn binding with cytosolic ZBP peptides is not a trait restricted only to R. bresadolae.