*2.5. Fission of ccmF in Land Plants*

To investigate the phylogenetic distribution of the fission of *ccmFn* and conservation of two ORFs *ccmFn1* and *ccmFn2* in *Trifolium*, mitochondrial *ccmF* sequences were assembled using available next-generation sequencing (NGS) reads (Table S2). The expanded taxon sampling confirmed the adjacency of the ORFs *ccmFn1* and *ccmFn2* and that the fission was restricted to *Trifolium*. All examined *Trifolium* species shared the *ccmFc* intron loss. Draft nuclear genome sequences of four species of *Trifolium* (*T. subterraneum*, *T. pratense*, *T. pallescens* and *T. repens*) were examined for intact copies of *ccmFn1* and *ccmFn2*. Fragments of sequences similar to *ccmFn1* and *ccmFn2* were identified in *T. subterraneum* and *T. pratense* but no intact copies were detected. However, intact copies both of *ccmFn1* and *ccmFn2* from *T. pallescens* (chromosome 4) and *T. repens* (chromosomes 4 and 9) were identified and were adjacent as in mitogenomes of *Trifolium*. Eleven *ccmFn* sequences (eight mitochondrial and three nuclear copies) were detected in *Trifolium* (Figure S4a). All nuclear copies were identical to their corresponding mitochondrial copy. Among mitochondrial copies, only three *Trifolium* species (*T. aureum*, *T. grandiflorum* and *T. pallescens*) showed unique sequence and the remaining sequences in the other five species were identical to each other in the coding region (Figure S4b).

Fission of *ccmFc* was analyzed in three species of *Marchantia* and two other genera of the Marchantiales. Sequence alignment revealed that a single nucleotide deletion caused *ccmFc* fission in one species of *Marchantia*, *M. paleacea* (Figure S5).

Examination of *ccmFn* fission in Brassicaceae included 17 taxa (Table S2). The *ccmF* genes were assembled from Cleomaceae (*Cleome violacea*), the sister family of Brassicaceae and two early diverging Brassicaceae genera (*Aethionema* and *Odontarrhena*). The fission of *ccmFn* was shared by all Brassicaceae except *Aethionema* and in all cases *ccmFn1* and *ccmFn2* were found in different loci. *Odontarrhena argentea* was the only member of the Brassicaceae that lost the *ccmFc* intron.

The phylogenetic position of *ccmFn* fission and separation in Fabaceae and Brassicaceae (Table S2), were plotted on cladograms of each of family (Figure 6a,b). The location of the breakpoint of *ccmFn* fission was also compared among the three families Fabaceae, Brassicaceae and Amaryllidaceae (Figure 6c). The fission occurred in different locations in the gene within each family and occurred

in a more basal position in Brassicaceae than Fabaceae. The separation of *ccmFn1* and *ccmFn2* only occurred in Amaryllidaceae and Brassicaceae.

**Figure 6.** Fission events of *ccmFn* in angiosperms. (**a**) Examined genera of Fabaceae are plotted on cladogram from Ellison et al. [42]. Number of examined species is indicated in the parenthesis. The branch where the fission occurred is marked with grey bar. (**b**) Examined tribes of Brassicaceae are plotted on cladogram from Huang et al. [47]. Number of examined genera is indicated in the parenthesis. The branch where the fission and separation occurred is marked with grey bar. (**c**) *CcmFn* gene organization compared among various taxa of angiosperms. Dark brown box indicates conserved domains.
