Comparative genomics provides insights into the evolutionary history of the phylum Ciliophora (Eukaryota, Alveolata) and uncovers the adaptive evolution of anaerobic ciliate classes.

Ciliophora are one of the most diverse phyla of unicellular eukaryotes. Members of this group are widely distributed and have been found in several environments including those that are anaerobic. However, the paucity of sufficient omics data for the major classes of the phylum Ciliophora, especially the anaerobic ciliate lineages, has been a serious impediment to research thus far. In this study, omics data were expanded to include representatives of 16 ciliate classes (excluding Cariacotrichea and Copemetopea) using single‐cell sequencing techniques. Using omics data of 57 ciliates and two apicomplexans, we conducted a comparative genomics study. Phylogenomic analysis based on 156 orthologous genes showed that: there are two superclades within the subphylum Intramacronucleata, i.e., Colpodea + Oligohymenophorea + Nassophorea + Plagiopylea + Prostomatea + Phyllopharyngea (CONThreeP), and Spirotrichea + Armophorea + Litostomatea + Odontostomatea + Muranotrichea + Parablepharismea (SALOMP); with the exception of Armophorea, all ciliate classes are monophyletic; neither Metopus nor Plagiopyla is monophyletic; Paraspathidium has a close affiliation with prostomateans. The results of the divergence time estimations indicated that: the radiation of Ciliophora occurred ca. 1067 million years ago (Mya); the SALOMP and CONThreeP lineages diverged from the intramacronucleatean common ancestor ca. 892 Mya and 851 Mya, respectively; the split between Muranotrichea and Parablepharismea occurred ca. 712 Mya; Odontostomatea are the youngest class, with a crown age estimated at ca. 19 Mya. In addition, we profiled stop codon usage and programmed ribosomal frameshifting (PRF) events. Our analyses revealed that: most of the species investigated use either TAA or TGA as the biased stop codon; stop codon usage in Paraspathidium sp. resembles that of Prostomatea; almost all ciliates included in the analyses have prevalent +1 and −1 PRF events. Finally, through GO enrichment analysis for group‐shared orthogroups of five anaerobic ciliate classes, we successfully identified the biological process and molecular function GO terms that were related to the adaptation of this particular group of ciliates to anaerobic environments. Together, these findings provide new insights into the evolutionary history of the Ciliophora and deepen our understanding of the adaptation of anaerobic ciliate groups to anaerobic environments from an evolutionary perspective. [ABSTRACT FROM AUTHOR]