A character is a feature or attribute of an organism that can be used as a basis for comparison with other organisms. Fungal phylogenetics has always been based on such characters, and as technology advances, new kinds of characters become available to study. Mycologists use many types of characters to contribute to their evolutionary research, including morphology and anatomy features, and ultrastructural features derived from the electron microscope. Most recently molecular techniques have come to mycology, bringing new characters and analytical tools, which have supported some taxonomic groups, established some new ones and removed a few old ones. A phylogeny is a hypothesis of the genealogy of a group of organisms and their hypothetical ancestors. A variety of genes have been sequenced for fungi to be then involved in comparison studies, including mitochondrial and several protein-coding genes. However, many phylogenetic analyses of molecular sequences have focused on the small ribosomal subunit RNA sequence.

Ribosomal Nucleic Acid Sequences

The most commonly used sequencing tool for the fungal phylogenetic analyses is the nuclear encoded ribosomal RNA genes. Ribosomes contain a small number of RNA molecules (rRNA), which are are designated by their sedimentation coefficients (S) on centrifugation. Some sections of the nucleotide sequence in cytoplasmic rRNA molecules are almost identical throughout the eukaryotes, some however show differences when comparisons are made between higher taxa (such as famalies). RNA sequences that are transcribed from rDNA, but eliminated before RNA is incorporated into the ribosome, vary more, and rDNA sequences that are not transcribed are highly variable. Ribosomal RNA and ribosomal DNA sequences can thus provide information of value at every level of classification. 

There are a number of reasons why such studies have focused on this area of molecular sequencing, mainly all practical considerations:

  • Fungal ribosomal gene clusters are arranged in roughly 200 tandem repeats. Therefore, since each nucleus contains around 200 identical copies of the particular region, there is a much better chance that one intact copy for molecular analysis would be obtained. 

  • The rDNA regions (which transcribe the RNA sequences) permit phylogenetic comparisons and resolutions at a variety of taxonomic levels since the genes of interest are highly conserved and universally present. This is helpful when a phylogenetic framework is needed to compare more than one set of organisms.

  • The relative ease of primer design for the rRNA regions. Nucleotides which define rRNA function are distributed in highly conserved patches - these patches are ideal for designing primers that amplify DNA.

  • They have a unusual base-pair ratio. Therefore, easy to detect.

  • Large molecular genetic databases are now available for rDNA and rRNA sequences since many fungi have now been studied. Sequencing ribosomal genes for just a few ingroup taxa while drawing the remaining ingroups and outgroups from national databases, for example, can answer many questions with minimum effort.

  • RNA can be isolated directly from ribosomes, or rDNA can be amplified with the polymerase chain reaction, a technique that overcomes the problems of dealing with single-copy-number genes that provide only small amounts of DNA template for sequencing. 

The foremost use of such technology is the production of a phylogenetic framework, inferred from comparisons of the small subunit ribosomal RNA sequences, which can describe the evolutionary origin and early branching patterns of the kingdom fungi. The time scale then produced is calibrated using fungal fossil evidence. A sound phylogenetic tree should thus be the product, giving details of major events in the evolution of fungi.