Structural Remodeling and Enzymatic Replacement Shape the Evolution of Organellar Group II Introns in Ulva

Group II introns are catalytic RNAs that combine self-splicing ribozyme activity with mobility and have played major roles in shaping organellar genome evolution. In green macroalgae of the genus Ulva, organellar genomes are highly compact, yet they harbor unusually diverse and dynamic repertoires of group II introns. To understand how organellar group II introns diversify and persist within compact organellar genomes, we performed a comparative analysis of mitochondrial and chloroplast group II introns across Ulva, integrating secondary structure reconstruction, intron occurrence patterns, and phylogenetic inference based on both conserved intron RNA regions and intron-encoded proteins (IEPs), including reverse transcriptase/maturase (RT/M) and LAGLIDADG homing endonuclease (LHE). A total of 168 mitochondrial and 123 chloroplast introns were identified and classified into 32 families belonging to seven major subgroups (IIA1-RT/M, IIA2-RT/M, IIB1-RT/M, IIB1-LHE, IIB2-RT/M, IIB2-LHE, and IIB-like). Most intron families retain the canonical six-domain architecture (DI-DVI), but four mitochondrial IIA families display a seven-domain configuration generated by the lineage-specific insertion of an additional stem-loop structure (DIIIa). Phylogenetic analyses revealed a high degree of congruence, supporting persistent coevolution between RNA scaffolds and their IEPs. Notably, the LHE-encoding families were scattered across distinct IIB lineages instead of forming a single clade, suggesting that at least two independent invasion events occurred within the IIB1 and IIB2 lineages. Analysis of intron occurrence frequency revealed an evolutionary continuum ranging from structurally intact and broadly distributed families to lineage-specific families exhibiting progressive scaffold degeneration, with the chloroplast infA-62 family representing a stably inherited lineage maintained through vertical transmission. These results suggest that organellar group II introns in Ulva evolve through coordinated scaffold remodeling, enzymatic replacement, and differential distribution patterns across genomic compartments, highlighting Ulva organellar genomes as a valuable comparative model for investigating the long-term evolution of mobile ribozymes within compact genomic environments.