Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists

Adaptations of ciliates to hypoxic environments have arisen independently several times.Studies on mitochondrion-related organelle(MRO)metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes.To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis,mass-culture and single-cell transcriptomes of two anaerobic species,Metopus laminarius(class Armophorea)and Plagiopyla cf.narasimhamurtii(class Plagiopylea),were sequenced and their MRO metabolic maps were compared.In addition,we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes(i.e.,Armophorea,Litostomatea,Muranotrichea,Oligohymenophorea,Parablepharismea and Plagiopylea).We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates.The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates,even among closely related species.Notably,our findings indicate the existence of group-specific functional relics of electron transport chains(ETCs).Detailed group-specific ETC functional patterns are as follows:full oxidative phosphorylation in Oligohymenophorea and Muranotrichea;only electron-transfer machinery in Armophorea;either of these functional types in Parablepharismea;and ETC functional absence in Litostomatea and Plagiopylea.These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times.Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates.