Codon usage frequency

Alternative codon usage frequency was examined in all taxa included in this study. Fungal mt genomes utilize both alternative codes to the Universal Genetic Code (NCBI code 1), i.e., NCBI genetic code 3 (The Yeast mt genetic code) and 4 (The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplama/Spiroplasma Code), and thus, they present a few alternative codons encoding for the amino acids Threonine (T), Methionine (M), and Tryptophan (W). Codon usage frequency was examined using sequence manipulation suite (Stothard 2000), after inserting the gene sequence and the genetic code used by the species’ mt genome. The related information regarding the mt genetic code utilized by each species was retrieved from the NCBI Taxonomy database. In case where codon frequency results were contradictory and unusually different from the rest, the mt genetic code was further examined and verified or even corrected in some species. Seven of the 14 protein coding genes were included in this study, since seven genes of NADH dehydrogenase subunits 1–6 and nad4L are not present in all studied fungal genomes, and thus would provide misleading evidence regarding comparative codon usage frequency of the whole mt genome. After listing the frequency of alternative codons, the RSCU index was calculated as an indication of possible codon usage bias (Sharp et al. 1986). The RSCU value for a codon reflects the observed frequency of that codon, divided by the expected frequency, assuming an equal usage of the synonymous codons for an amino acid (Sharp et al. 1986). The synonymous codons with RSCU values >1.0 have positive codon usage bias and are defined as abundant codons, whereas those with RSCU values <1.0 have negative codon usage bias and are defined as less-abundant codons.

In detail, the frequencies of synonymous codons within each amino acid class were calculated utilizing mt sequences of the protein coding genes cob, atp6, atp8, atp9 and cox1-3. In the above analyses, codon frequencies and RSCU values were measured for each taxon and for each of the above genes. In addition, accumulative mean RSCU values were measured for 1) each codon and each gene separately, by adding the respective values of all taxa (e.g., ATG of cob in all species) and dividing them with the total number of taxa (supplementary table S4, Supplementary Material online), 2) each codon in all genes of each taxon (e.g ATG of cob plus ATG of cox1 of each strain etc.) divided with the number of genes (supplementary table S4, Supplementary Material online), and 3) a final mean RSCU value was calculated for each codon by summing RSCU values occurring in calculation and dividing them with the total number of taxa (supplementary table S4, Supplementary Material online). In all cases, RSCU values were measured separately for 1) species utilizing genetic code 3 and 4 and 2) for each codon within each family or phylogenetic cluster, respectively.