Exploring the evolution of anaerobes within ciliate class Prostomatea by transcriptomics

Mitochondrion-related organelles (MROs) enable anaerobic eukaryotes to thrive in anoxic environments, and the independent ciliate lineages of anaerobes serve as excellent candidates for investigating the convergent evolutionary transition from mitochondria to MROs. Previous studies have demonstrated that the adaptations of ciliates to anaerobic conditions may be lineage-specific. However, our understanding of the diverse metabolic peculiarities of MROs is limited to a few ciliate lineages. In this study, we sequenced the transcriptomes of four anaerobic species from two genera (Apolagynus and Holophrya), which are classified within the predominantly aerobic class Prostomatea, and predicted their mitochondrial metabolisms. The ecological niches of prostomatean anaerobes were mapped onto newly constructed phylogenomic trees and small subunit (SSU) rDNA trees. Results showed that paraphyletic class Prostomatea containing six clades (Clade Ⅰ–Ⅵ) has a close relationship with Oligohymenophorea and Plagiopylea. Notably, all prostomatean species within Clade Ⅱ are anaerobic, while anaerobes are only sporadically present in other clades. The MROs of anaerobic prostomatean species display at least two distinct phenotypes. Holophrya ovum in Clade Ⅰ produces ATP by oxidative phosphorylation under aerobic conditions and via substrate-level phosphorylation via acetate: succinate CoA transferase (ASCT) and succinyl CoA synthetase (SCS) as well as adenylate kinase (AK) under anaerobic conditions. In contrast, three species of Apolagynus in Clade Ⅱ possess reduced electron transport chain (ETC), and are capable of ATP generation via substrate-level phosphorylation mediated by ASCT/SCS and propionyl-CoA. Additionally, these three Apolagynus species possess [FeFe] hydrogenase probably producing H₂. A comparison of the ETC pathways among various anaerobic ciliates further showed that the MROs of these organisms have originated from repeated convergent evolution. Our findings shed lights on evolutionary history of anaerobes within the ciliate class Prostomatea.