Progressive evolutionary trajectories of mitochondrion-related organelles in anaerobic ciliates (Eukaryota, Alveolata) revealed by APM ciliates and a facultatively anaerobic spirotrichean species.

Ciliates are an excellent model for studying convergent transitions from mitochondria to mitochondrion-related organelles (MROs) in protists. Despite our growing knowledge of adaptive evolution in ciliate MROs, the progressive evolutionary trajectories within anaerobic ciliate lineages and the MRO metabolisms of facultatively anaerobic ciliates remain unexplored. In this study, we predicted MRO metabolisms of eight species within the anaerobic monophyletic APM (Armophorea-Muranotrichea-Parablepharismea) clade and a facultative anaerobe from its sister class Spirotrichea. Our main results are as follows: (1) During their adaptation to anaerobic environments, the MRO electron transfer chain (ETC) components and their associated functions have been progressively lost in the APM clade. (2) The MRO of the last common ancestor of Armophorea likely possesses complexes Ⅰ, Ⅱ, and Ⅴ, but lacks functional complexes Ⅲ and Ⅳ. Subsequently, during their adaptation to anaerobic environments, the armophorean lineage has further lost complex Ⅴ in the order Clevelandellida and Metopida. (3) In the MRO of the facultatively anaerobic ciliate Heterodeviata sinica, complexes Ⅲ and Ⅳ are absent, and alternative oxidases (AOX) play a key role in adaptation to fluctuating dissolved oxygen levels. (4) The fused [FeFe]-hydrogenase appears to have been acquired by the last common ancestor of ciliates through horizontal gene transfer (HGT), followed by multiple independent losses. Our results provide insights into the progressive adaptations of anaerobic ciliates to the low-oxygen environments.