Extent of Genetic Diversity in a Single Fruiting Body of Parmelia laevior

Abstract

There have been debates in defining species boundary in fungi with emphasis on phenotypic or genotypic characteristics. To test the extent of genetic diversity in lichen-forming fungus, Parmelia laevior, sequence variations of three genes (ITS, RPBII and mtSSU rDNA) were analyzed from 20 spore-germinated cultures from an identical fruiting body. All of the isolates contained identical mtSSU rDNA sequences, whereas two types of ITS and RPBII sequences were observed. Two types of ITS sequences showed 7 nucleotide difference among 639 bases (98.9% similarity). Two types of RPBII sequences showed 16 nucleotide difference among 1192 bases (98.83% similarity). To discriminate mixed cultures from homogeneous single spore cultures, two types of single-copy RPBII gene was specifically detected by oligonucleotide ligation assay (OLA) based upon the single nucleotide polymorphism. As a result, 13 isolates turned out as mixed cultures by showing both types of RPBII sequences. To detect incomplete concerted evolution of ITS sequences, OLA was applied for ITS sequences and it was found that all of the single-spore isolates had only one type of ITS sequences. RPBII and ITS alleles were segregated together in all of the single-spore isolates, implying that they are closely linked on the chromosome. From the identical sequences of mtSSU rDNA and high variation of ITS and RPBII sequences, it is concluded that boundary of species in Parmalia laevior is broader than have been expected from previous researches.