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Rhizopogon roseolus Corda (synonym Rhizopogon rubescens Tul.), an economically important edible mushroom associated with the Pinaceae (mostly Pinus sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of R. roseolus varies with the place of origin. R. roseolus strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of R. roseolus and other closely related fungi belonging to Rhizopogon subgenus Roseoli to determine the genetic basis for phenotypic differences among R. roseolus isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within Rhizopogon subgenus Roseoli. Collections from the United States and Europe grouped into four distinct clades. Rhizopogon roseolus isolates found in New Zealand were closely related to those from the United States, likely due to introduction of Pinus radiata from its native California in the United States. In contrast, Japanese R. roseolus isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand R. roseolus isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand R. roseolus isolates to track the establishment of ectomycorrhiza on P. radiata seedlings inoculated with commercially valuable R. roseolus. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on P. radiata in the Southern Hemisphere. [ABSTRACT FROM AUTHOR]