Complete Mitochondrial Genome Sequence of Three Tetrahymena Species Reveals Mutation Hot Spots and Accelerated Nonsynonymous Substitutions in Ymf Genes

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions.