Understanding early stages of speciation: Allopatric divergence, introgression and chromosomal dynamics in the...

Accurate species delimitation is essential for understanding biodiversity and evolutionary processes, yet it remains challenging in taxonomically complex groups shaped by recent divergence and reticulate evolution. Nevertheless, such groups offer unique insights into the earliest stages of speciation and its driving forces. The genus Erysimum (Brassicaceae), notable for its karyological diversity and high endemism, represents an excellent model for such studies. Here, we investigated the E. odoratum complex, encompassing up to 10 recognized species in the Carpathians and western Balkans, to test its monophyletic origin, clarify species boundaries and elucidate the main drivers of diversification. We combined cytotype screening (chromosome counting and flow cytometry), morphometric analysis, and two high-throughput sequencing methods: RADseq, to resolve phylogenetic relationships as well as to detect fine-scale genetic structure and introgression; and target enrichment (Hyb-Seq), to elucidate polyploid origins. Our results demonstrate that the studied complex is polyphyletic, and we focused on the lineage comprising E. odoratum s.str. and Carpathian species. Phylogenomic data from the Carpathians contradict traditional taxonomy, which recognized up to four diploid endemics, and instead support a single species, E. witmannii with geographically structured genetic variation. Within E. odoratum s.str., we identified multiple polyploid cytotypes resulting from independent auto- and allopolyploidization events, although disentangling parental subgenomes, ancestral polymorphisms, and introgression remains difficult. We propose that diversification in this species complex has been driven by a combination of allopatric divergence and reticulate evolution (involving both introgression and allopolyploidy), further shaped by chromosomal dynamics such as dysploidy.