Abstract
Abstract
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.