Contrasting modes of intron evolution in an evolutionarily distinct eukaryotic lineage revealed by comparative genomics of ciliated protists

Introns are widespread in eukaryotic genomes and play important roles in gene regulation, making intron evolution a long-standing focus of genome research. However, the patterns and mechanisms of intron evolution in evolutionarily ancient unicellular eukaryotes remain poorly understood. Here, we investigate intron evolution in ciliates, a morphologically and genetically diverse unicellular lineage, using a newly sequenced high-quality genome and comparative genomic data from derived and deep-branching lineages. Our analyses reveal that: 1) intron evolution in ciliates has been dominated by loss. While derived lineages experienced limited loss, the common ancestor of deep-branching groups, represented by heterotrich ciliates, underwent massive intron loss with almost no detectable gain. 2) Unlike derived ciliates, which display relatively uniform distributions of 3n, 3n+1, and 3n+2 introns or a low proportion of 3n introns, deep-branching ciliates show a striking dominance of tiny 3n introns. These are preferentially retained when generating in-frame premature termination codons (PTCs), likely allowing aberrant transcripts to be eliminated via nonsense-mediated mRNA decay, while introns incapable of forming PTCs tend to avoid insertion within protein domains, suggesting a strategy to reduce the metabolic cost of erroneous splicing. 3) Spliceosome analyses reveal conserved core catalytic components but lineage-specific variation in regulatory factors, suggesting differences in splicing regulation among ciliate lineages. Furthermore, the newly assembled 48.5 Mb Blepharisma genome encodes 27,571 protein-coding genes, and comparative analyses identified two rounds of whole-genome duplication in Blepharisma, one shared with Stentor. These findings provide new insights into intron evolution in unicellular eukaryotes and highlight ciliates as a powerful model for studying unconventional intron evolutionary strategies.