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Among the higher fungi, reproductively isolated cryptic species exist that are morphologically difficult to distinguish owing to a lack of taxonomically useful morphological characters. Mating tests are helpful for detecting reproductive isolation between cryptic species, but are often difficult to perform for higher fungi, especially ectomycorrhizal fungi. In order to identify cryptic species of the ectomycorrhizal genus Strobilomyces more efficiently, lineages were defined based on the nucleotide sequence of two mitochondrial genes. Then the gene flow among lineages was measured using cleaved amplified polymorphic sequences (CAPS) markers designed for single copy nuclear genes. No heterozygosity was observed between different lineages, but within the same lineage heterozygosity was present at the ratio expected given Hardy Weinberg equilibrium. These results show that the mtDNA lineages are separate …