*4.2. Annotation and Genome Content Comparison of Mitogenomes*

To compare gene and intron content of *Trifolium* mitogenomes with related taxa, five previously published mitogenomes were acquired—two from IRLC [*Vicia faba* (KC189947) and *Medicago truncatula* (NC\_029641)], one from the robinioid clade [*Lotus japonicus* (NC\_016743)], which is sister to IRLC; and two from millettioid *sensu lato* clade [*Millettia pinnata* (NC\_016742)] and *Glycine max* (NC\_020455)], which is sister to the hologalegina clade (robinioid + IRLC). Annotation of rRNAs, protein coding genes and introns was conducted based on a reference mitogenome of *Liriodendron tulipifera* (NC\_021152) with a set of 41 conserved mitochondrial genes in Geseq [64]. Annotation for protein coding genes was manually corrected in Geneious to fit ORFs. The annotation for tRNAs was cross-checked by tRNAscan-SE v2.0 [65].
