*4.1. Assembly of Trifolium Mitogenomes*

Four species of *Trifolium* from the two subgenera *Chronosemium* (*T. aureum* and *T. grandiflorum*) and *Trifolium* (*T. meduseum* and *T. pratense*) were selected for mitogenome assembly. The 100 bp paired-end raw Illumina (San Diego, CA, US) reads (Table 1) for mitogenome assembly were from Sabir et al. [31]. Assembly and mapping were conducted in Geneious Prime (https://www.geneious.com) using Geneious assembler and mapper, respectively. To assemble mitogenomes, the methods in Choi et al. [8] were followed. First, raw reads from the plastome were excluded by mapping total raw reads to corresponding plastomes [*T. aureum* (NC\_024035.1), *T. grandiflorum* (NC\_024034.1), *T. meduseum* (NC\_024166.1) and *T. pratense* (MT039393)]. De novo assembly was subsequently conducted for each with ~30 million plastome-filtered reads. Among the assembled contigs, mitochondrial contigs were selected by BLAST searches against reference Fabaceae mitogenome sequences at National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/genome/organelle/) using BLASTN 2.8.0+ [63] with default options. Mitochondrial contigs were manually assembled as single chromosomes in Geneious. Finally, draft mitogenomes were refined by mapping total plastome-filtered reads.
