Stepwise evolutionary transitions in Cladophorales plastid genomes reveal origins of hairpin chromosomes

While organellar genomes are typically a single circular molecule, some have undergone extreme evolutionary changes, such as fragmentation. Among plastid genomes, extensive fragmentation is rare but has been observed in the green alga Boodlea composita (Cladophorales), wherein single-stranded chromosomes adopt an unusual hairpin structure, stabilized by inverted repeats. This plastome is also highly reduced in coding content, containing only 21 protein-coding genes that are highly divergent in sequence, yet the plastome comprises a numerous and heterogeneous chromosome population. The lack of plastomes among close relatives has stymied our understanding of the evolutionary origins of Boodlea's unique plastome characteristics. Here, we report plastomes from three additional Cladophorales families. We show that elevated mutation rates and a massive reduction in coding content preceded fragmentation and that fragmentation resulted in a highly heterogeneous population with hundreds of minicircular chromosomes and a concomitant increase in GC content. Long inverted repeats are absent in minicircular chromosomes, suggesting the repetitive sequences that facilitate the formation of hairpin structures are unique to the Boodlea clade. Codon reassignments also occurred concurrently among fragmented and non-fragmented Cladophorales plastomes. Our findings document a clear stepwise progression of plastid topologies and sequence characteristics in Cladophorales. This assists in our understanding of the evolution of fragmented organellar genomes in general, as well as the limits to coding capacity reduction and sequence divergence able to be withstood by a functioning plastid.