Different fates of two mitochondrial gene spacers in early land plant evolution

Plant mitochondrial DNA is generally characterized by slow sequence drift but frequent genomic recombination. Accordingly, gene sequences in plant mitochondria are informative for old cladogenic events, whereas intergenic regions have so far not been considered for phylogenetic reconstruction because high recombinational activity rearranges mitochondrial gene orders too frequently. Here we report that an ancient gene continuity, the nad5-nad4-nad2 gene arrangement, is conserved among the bryophyte lineages: mosses, liverworts, and hornworts. The nad4-nad2 spacer is surprisingly conserved at a size of only 26 bp in all three bryophyte lineages. In striking contrast, the small ancestral nad5-nad4 spacer, of only 57 bp in the alga Chara, is significantly increased in size, to about 600 bp in mosses and 1000-1300 bp in liverworts, and even exceeds 3000 bp in hornworts. A large group II intron fragment and a nad6 pseudogene sequence, respectively, contribute to the expanded spacer sizes in liverworts and in hornworts. The small nad4-nad2 intergenic region is retained in the lycophyte Isoetes lacustris, whereas no linkage between nad5 and nad4 could be detected in this quillwort. Given its size and sequence variability, the intergenic region between nad5 and nad4 holds promise as a locus for phylogenetic analyses within the bryophyte lineages.