RESUMO
Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.
RESUMO
Mitochondrial translation often exhibits departures from the standard genetic code, but the full spectrum of these changes has certainly not yet been described and the molecular mechanisms behind the changes in codon meaning are rarely studied. Here we report a detailed analysis of the mitochondrial genetic code in the stramenopile group Labyrinthulea (Labyrinthulomycetes) and their relatives. In the genus Aplanochytrium, UAG is not a termination codon but encodes tyrosine, in contrast to the unaffected meaning of the UAA codon. This change is evolutionarily independent of the reassignment of both UAG and UAA as tyrosine codons recently reported from two uncultivated labyrinthuleans (S2 and S4), which we show are not thraustochytrids as proposed before, but represent the clade LAB14 previously recognised in environmental 18S rRNA gene surveys. We provide rigorous evidence that the UUA codon in the mitochondria of all labyrinthuleans serves as a termination codon instead of encoding leucine, and propose that a sense-to-stop reassignment has also affected the AGG and AGA codons in the LAB14 clade. The distribution of the different forms of sense-to-stop and stop-to-sense reassignments correlates with specific modifications of the mitochondrial release factor mtRF2a in different subsets of labyrinthuleans, and with the unprecedented loss of mtRF1a in Aplanochytrium and perhaps also in the LAB14 clade, pointing towards a possible mechanistic basis of the code changes observed. Curiously, we show that labyrinthulean mitochondria also exhibit a sense-to-sense codon reassignment, manifested as AUA encoding methionine instead of isoleucine. Furthermore, we show that this change evolved independently in the uncultivated stramenopile lineage MAST8b, together with the reassignment of the AGR codons from arginine to serine. Altogether, our study has uncovered novel variants of the mitochondrial genetic code and previously unknown modifications of the mitochondrial translation machinery, further enriching our understanding of the rules governing the evolution of one of the central molecular process in the cell.
