Comparative genomics reveals the evolutionary history of the unicellular eukaryote class Litostomatea and its adaptive evolution based on biochemical metabolic capacity

Abstract Ciliated protists are unicellular eukaryotic organisms characterized by their morphological diversity, ubiquitous distribution, and the important roles they play in a wide range of biological studies. The class Litostomatea is a morphologically diverse ciliate group that comprises hundreds of free-living and endosymbiotic species. Here, we sequenced 14 predatory litostomateans, i.e. 12 haptorians and two rhynchostomatians. A comparative analysis was performed with other published omics’ data on litostomateans. Our first phylogenomic analysis of litostomateans showed the monophyly of the subclasses Trichostomatia and Rhynchostomatia, the non-monophyly of the subclass Haptoria, and the monophyly of all orders and families that were analysed. Evolutionary history analysis suggested that Litostomatea diverged during the Late Neoproterozoic, the family Chaeneidae was the earliest diverging haptorian lineage, and the Rhynchostomatia probably separated from the order Lacrymariida (subclass Haptoria) during the Early Palaeozoic. Stop codon usage analysis of 28 litostomateans showed that they use TAA as the biased stop codon and reassign the other two stop codons (TAG and TGA) to code for amino acids. In addition, the preferred codons in the 14 newly sequenced litostomateans are strongly biased towards A/U bases in the third position, very probably due to the comparatively low GC content. Genes encoding carbohydrate-active enzymes (CAZymes) are more diversified in the endosymbiotic Trichostomatia than in the free-living predatory Rhynchostomatia and Haptoria, suggesting that trichostomes have the strongest capability of carbohydrate utilization. Notably, we found that three free-living litostomateans (Didinium sp.1, Myriokaryon sp., and Apodileptus visscheri) exhibit substantial differences from other free-living ciliates in terms of their number of CAZymes. Considering the potency and versatility of CAZymes in the degradation and biotransformation of carbohydates, we propose that the multifarious CAZymes in these three ciliates could be a survival strategy for nutrient acquisition and niche adaptation. Finally, the functional annotation of significantly expanded gene families in these three ciliates revealed their vigorous potency in biochemical metabolism. These findings will facilitate wider omic-scale phylogenetic analyses of Litostomatea and deepen our understanding of this group from an evolutionary standpoint.