**2. Results**

## *2.1. Mitogenome Features of Four Trifolium Species*

For each of four *Trifolium*, a single chromosome was assembled that contained all expected mitochondrial coding sequences. The length of the four mitogenomes varied from to 294,911 to 348,724 bp (Table 1). The GC content was conserved among the species at 44.9–45.2 %. Gene content was identical with three rRNAs, 16 tRNAs and 32 protein coding genes while gene order was distinct for each species (Figure 1).

**Table 1.** Assembly information for four *Trifolium* mitogenomes.

**Figure 1.** Linear mitogenome maps of four *Trifolium* species. Fragmented genes caused by duplication or pseudogenization are not depicted. pt indicates tRNAs of plastid origin.

Gene and intron content comparison with other published mitogenomes revealed one gene loss (*rps1*) (Figure S1), which was shared with *Lotus* and two cis-spliced intron losses (ccmFci829 and rps3i174) that were exclusive to *Trifolium* (Figure S2). Sequence alignment of *ccmFn* from *Trifolium* with other IRLC genera revealed a 59 bp deletion that resulted in a frame shift and premature stop codon (Figure 2). a putative downstream start codon for a second open reading frame (ORF) (*ccmFn2*) was also identified.

**Figure 2.** Fission of *ccmFn* in four *Trifolium*. Alignment of six *ccmFn* sequences of mitogenomes from species in inverted repeat lacking clade. The region (grey dashed rectangle, aligned positions 374-591) showing the 59 bp deletion (red dotted box) is enlarged above. Translated amino acid alignments are presented below corresponding nucleotide sequence alignments. Nucleotide coordinates are indicated above consensus of alignment. Sequence identity is shown below consensus (green = 100%, yellow-green = at least 30% and under 100%, red = below 30%).
