Allele phasing is critical to revealing a shared allopolyploid origin of Medicago arborea and M. strasseri (Fabaceae)

Jonna S. Eriksson, Filipe de Sousa, Yann J. K. Bertrand, Alexandre Antonelli, Bengt Oxelman, Bernard E. Pfeil

Whole genome duplication plays a central role in plant evolution. There are two main classes of polyploid formation: autopolyploids which arise within one species by doubling of similar homologous genomes; in contrast, allopolyploidy (hybrid polyploidy) arise via hybridization and subsequent doubling of nonhomologous (homoeologous) genomes. The distinction between polyploid origins can be made using gene phylogenies, if alleles from each genome can be correctly retrieved. We examined whether two closely related tetraploid Mediterranean shrubs (Medicago arborea and M. strasseri) have an allopolyploid origin - a question that has remained unsolved despite substantial previous research. We sequenced and analyzed ten low-copy nuclear genes from these and related species, phasing all alleles. To test the efficacy of allele phasing on the ability to recover the evolutionary origin of polyploids, we compared these results to analyses using unphased sequences. In eight of the gene trees the alleles inferred from the tetraploids formed two clades, in a non-sister relationship. Each of these clades was more closely related to alleles sampled from other species of Medicago, a pattern typical of allopolyploids. However, we also observed that alleles from one of the remaining genes formed two clades that were sister to one another, as is expected for autopolyploids. Trees inferred from unphased sequences were very different, with the tetraploids often placed in poorly supported and different positions compared to results obtained using phased alleles. The complex phylogenetic history of M. arborea and M. strasseri is explained predominantly by shared allotetraploidy. We also observed that an increase in woodiness is correlated with polyploidy in this group of species and present a new possibility that woodiness could be a transgressive phenotype. Correctly phased homoeologues are likely to be critical for inferring the hybrid origin of allopolyploid species, when most genes retain more than one homoeologue. Ignoring homoeologous variation by merging the homoeologues can obscure the signal of hybrid polyploid origins and produce inaccurate results.