How many DNA markers are needed to reveal cryptic fungal species?

• Inclusion of five DNA loci in multi-gene analyses gave a highly confident separation of cryptic fungal species.
• Several individual genes gave better support than the DNA barcoding marker ITS in resolving phylogenetic species.
• Portions of a few informative genes may give a good indication about the existence of cryptic species in fungi.

In the fungal kingdom there is a high prevalence of morphologically defined species that includes closely related ‘cryptic’ biological species with similar phenotypes. Due to evolutionary processes like incomplete lineage sorting and introgression through hybridization, several independent DNA markers are essential to resolve closely related fungal species. In this study we wanted to analyze how many independent loci are necessary to reveal the cryptic species, using the genus Serpula as a model system. DNA sequences from ten different DNA loci, eight nuclear and two mitochondrial DNA markers, were obtained from various cryptic species within Serpula. The inclusion of five loci gave a highly confident separation of the cryptic species. Several other loci performed better than the standard DNA barcoding marker ITS in separating the cryptic species. The DNA loci tub, hsp, rpb2 and tef gave, on average, best support for the different cryptic species in single gene trees. We conclude that the analyses of a few but informative independent DNA loci, such as tub, hsp, rpb2 and tef in addition to the standard DNA barcode ITS, may give a good indication about the existence of cryptic species in fungi.