*4.6. Investigation on Status of rps1 in Nuclear and Mitochondrial Genome*

Nuclear and mitochondrial sequences of *rps1* generated for a previous study [39] were acquired from NCBI. Nuclear *rps1* sequences for other species were searched by MegaBLAST using the options described above. Mitochondrial *rps1* of *Vicia faba* was used to query nuclear genomes of *Lotus japonicus*, *Medicago truncatula*, *Trifolium subterraneum*, *T. pratense*, *T. pallescens* and *T. repens* (Table S1). Mitochondrial *rps1* sequences were also extracted from mitogenomes of *Glycine max*, *Millettia pinnata*, *Vicia faba* and *Medicago truncatula*. All *rps1* sequences were aligned with MAFFT v.7.017 [69] using default options. Nucleotide substitution models were evaluated in jModelTest v.2.1.6 [70] by Akaike information criterion. ML analysis (GTR +G with 1000 bootstrap replications) was conducted using *G. max* and *M. pinnata* as outgroups in RAxML v.8 [71] in the CIPRES Science Gateway [72].

The status of mitochondrial*rps1* in *Trifolium* was tested by sequence alignment of the mitochondrial locus containing *rps1* and *nad5* exon1 in*M. truncatula* and the corresponding regions in four mitogenomes of *Trifolium*. Sequences were aligned in MAFFT [69] using default options followed by manual adjustments to minimize gaps and maximize apparent homologous regions.
