**1. Introduction**

Plant cells comprise three genomic compartments (i.e., nucleus, mitochondrion and plastid). Unlike the typically conservative plastid genome (plastome) [1,2], plant mitochondrial genomes (mitogenome) display drastic evolutionary plasticity in size, content and structure, intracellular gene transfer (IGT) and interspecific horizontal gene transfer [3–8]. Substitution rates of mitochondrial protein coding genes, however, are the most conservative among the three genomic compartments [9]. In angiosperms, the relative rate of synonymous substitutions of mitogenome, plastome and nuclear genome is 1:3:16 [10].

Extensive gene loss and IGT of organelle DNA to the nucleus occurred in the early stages of endosymbiosis [11]. Nuclear genome sequences that originate from the mitogenome and plastome are referred to as nuclear mitochondrial DNA sequences (NUMTs) and nuclear plastid DNA sequences (NUPTs), respectively [12,13]. Transfer of mitochondrial DNA to the nuclear genome is an ongoing process in both of plants and animals but functional transfer of mitochondrial genes has almost ceased in animals [14]. Functional transfer of mitochondrial genes in plants has often involved ribosomal protein or succinate dehydrogenase genes [5]. Transfer of mitochondrial genes to the nuclear genome cannot substitute function of the original mitochondrial copy unless the nuclear copy acquires the appropriate expression and targeting signals [15]. Before the acquisition of regulatory signals, NUMTs must survive mutational decay in nuclear genome, which limits the lifespan of the nonfunctional sequences [16]. Mitochondrial IGT events may be successful or unsuccessful in terms of functionality and the phylogenetic distribution of pseudogenization and deletion of mitochondrial genes can be assessed in descendant lineages [17].

Following functional transfer, NUMTs attain higher substitution rates than their mitochondrial counterparts [14] because of substantial differences in the synonymous substitution rate between mitochondrial and nuclear genomes in plants [10]. Hence, functional transfer of mitochondrial genes into the nucleus is often detected by the presence of intact but highly diverged copies in nuclear genome compared to mitochondrial copies [18,19]. On rare occasions, functional transfers of mitochondrial genes exhibit an intriguing situation in which the nuclear and mitochondrial genomes contain different portions of the coding region resulting from mitochondrial gene fission and IGT (e.g., *rpl2* in many of eudicots) [20]. Szafranski [21] named this process "intercompartmental piecewise gene transfer." In plant mitogenome evolution, the protein that most commonly undergoes gene fission is cytochrome c maturation protein *ccmF* [22–25].

In *Escherichia coli*, the eight *ccm* genes (*ccmA*-*H*) are clustered in a single locus [26]. In most plants, three *ccm* genes (*ccmA*, *ccmE* and *ccmH*) have been transferred from the mitogenome to the nuclear genome, two (*ccmD* and *ccmG*) were lost and four (*ccmB*, *ccmC*, *ccmFc* and *ccmFn*) remain in the mitochondrion [27]. Since the fission of *ccmF* into *ccmFc* and *ccmFn* is shared by liverworts and seed plants [3], this event happened early in land plant evolution. In addition, there were independent fissions of *ccmF* in several lineages of land plants, including fission of *ccmFc* into *ccmFc1* and *ccmFc2* in *Marchantia* [22] and fissions of *ccmFn* into *ccmFn1* and *ccmFn2* in Brassicaceae [23,24] and *Allium* (Amaryllidaceae) [25].

Fabaceae are the third largest angiosperm family with approximately 20,000 species in six subfamilies [28]. Most species diversity occurs in subfamily Papilionoideae, which includes many economically important species [29]. The inverted repeat (IR) lacking clade (IRLC) is one of the major groups of Papilionoideae, which is defined by absence of the canonical plastome IR (~25 kb) [30]. Plastome studies of the IRLC elucidated several rare evolutionary phenomena, including high degree of genome rearrangement [31], localized hypermutation [32], genome size expansion with accumulation of dispersed repeats and unique sequences of unknown origin [33–35] and re-acquisition of a large IR [36]. However, mitogenome evolution in IRLC is poorly understood and represented by only two species, *Vicia faba* (tribe Fabeae) [37] and *Medicago truncatula* (tribe Trifolieae) [38]. In Trifolieae, a study of the mitochondrial *rps1* gene documented the existence of functional nuclear copies and putatively pseudogenized mitochondrial copies from three genera (*Medicago*, *Melilotus* and *Trigonella*) [39]. Deletion of mitochondrial *rps1* was also identified from another papilionoid species, *Lotus japonicus* [40]. The status of mitochondrial *rps1* across Trifolieae and related taxa has not been examined until recently. Parallel losses of several mitochondrial genes in Fabaceae were revealed in a previous study, however, whether the losses represent a single ancestral IGT or multiple IGTs was not determined [8].

*Trifolium* is the largest genus (ca. 250 species) of the tribe Trifolieae [41] and is divided into two subgenera (*Chronosemium* and *Trifolium*) [42]. Trifolieae belong to the IRLC and are closely related to Fabeae [30]. Several evolutionary studies of Trifolieae plastid [31,33,34] and nuclear [43–46] genomes have been conducted but mitogenome comparisons of *Trifolium* have been neglected. In this study, gene content, size and repeat structure of mitogenomes of four *Trifolium* species from the two subgenera *Chronosemium* (*T. aureum* and *T. grandiflorum*) and *Trifolium* (*T. meduseum* and *T. pratense*) were examined and compared to related papilionoid species.
