Abstract
Macroptilium (Benth.) Urb. is a forage genus native to regions from North to South America. Phylogenetic analyses place it close to Phaseolus L., but its infrageneric division into two sections is not well supported. Despite its chromosomal number stability (2n = 22), interspecific rDNA loci variation enabled species differentiation, suggesting that a cytogenomic approach might be valuable for inferring species relationships and genome evolution. Here, we (1) characterized nine Macroptilium species through oligonucleotide-based chromosome painting and barcoding (Oligo-FISH); (2) generated genome skimming data for six species and used it to investigate their repeatome dynamics, and (3) performed phylogenomic reconstruction using complete plastomes. Oligo-FISH data unveiled de novotranslocations between chromosomes 2 and 6, and 3 and 11 in species from proposed groups II and III, respectively, in disagreement with the currently proposed phylogenetic hypothesis. Our phylogenomic (plastid) and repeatome (nuclear) analyses supported groups II and III as clades, with shared satDNA families. Group I was paraphyletic and resembled the Ancestral Phaseolinae Karyotype. We demonstrated the efficiency of different cytogenomic approaches to characterize Macroptilium species, providing insights into its genomic evolution and indicating the need for a systematic re-evaluation of the genus. These findings also support the power of these approaches to solve phylogenetic relationships even in groups with chromosome number stability and recent diversification.