Restriction fragment length polymorphism and sequence analysis of ribosomal DNA for mapping and phylogenetic inference of Lentinula species

Intergenic spacers (IGRs-1 and 2) flanking the 5S ribosomal RNA gene of Lentinula edodes, L. boryana, L. lateritia, and L. novaezelandia were enzymatically amplified via the polymerase chain reaction (PCR). Length heterogeneities of IGR-1 and 2 were observed at both the inter- and intraspecific levels. Amplified IGRs were digested with restriction endonucleases and the restriction sites were mapped. Consistent conservation of mapped restriction sites was observed for the different species, with the exception of L. boryana. A phylogenetic analysis of the mapped restriction sites indicates that L. boryana is the most distantly related of the species of Lentinula, and that L. edodes, L. lateritia and L. novaezelandiae are the most closely related. Reconstruction of the inter- and intraspecies phylogenetic relationships of the genus Lentinula with outgroups of Clitocybula, Collybia and Pleurotus was made possible through restriction fragment length polymorphism (RFLP) analysis of the entire ribosomal DNA (rDNA) repeat of these fungi. The species L. edodes, L. lateritia, and L. novaezelandiae are monophyletic as indicated by their close relationships and comparatively low level of inter-species when compared to L. boryana and the outgroup species in phylogenies based on total rDNA RFLP data. Distinct clades comprised of each of the species of Lentinula were observed. The monophyly of the genus Lentinula remains unresolved, as Collybia maculata grouped with L. boryana, albeit distantly, in the total rDNA analysis. Phylogenies based on the transcribed and non-transcribed rDNA spacers yielded distinct clades for each of the Lentinula species. Use of the non-transcribed spacer data delineated individual isolates of each of the species within their respective clades. These molecular phylogenies support those based on morphological characters for Lentinula and further highlight the phylogenetic differences of the Asia-Australasian species of Lentinula. The DNA from a 115-year-old herbarium specimen of L. guarapiensis, available only in the severly limiting form of a few mold-infested herbarium specimens, was found to be amenable to PCR and direct sequencing. These results indicate that additional herbarium specimens of L. guarapiensis may be valuable resources for future molecular phylogenetic studies of the genus Lentinula.