Hybrid zones provide excellent opportunities to study evolutionary processes linked to interspecific gene flow, including introgression, genetic erosion, polyploid establishment, and speciation. The genus Cardamine (Brassicaceae) serves as an excellent model for polyploid evolution, including one of the few well-documented neo-allopolyploid species that have evolved in the last 300 yr. Using a combination of flow cytometric screening of nuclear DNA content, next-generation restriction site-associated DNA sequencing, and genomic in situ hybridization, we uncovered an unprecedented case of extensive interspecific hybridization in Cardamine, involving four parental species and their predominantly triploid offspring. We demonstrate the recurrent and polytopic origins of both autotriploids and allotriploids, the latter integrating different parental genomes. Our findings highlight Cardamine rivularis as a central player in this system, likely producing unreduced female gametes at a high rate, which drives the formation of diverse triploids. However, this species may also face the risk of genetic swamping and ecological displacement. The substantial genetic variation of the hybrids, their high frequency, partial fertility, and efficient clonal spread suggest significant evolutionary potential. Overall, we propose that these hybrid zones provide a rare and valuable natural laboratory for studying the emergence of neo-allopolyploids and the mechanisms shaping polyploid evolution.

Central European Institute of Technology Masaryk University 625 00 Brno Czechia

Department of Botany Faculty of Science Charles University 128 01 Prague Czechia

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czechia

Institute of Botany Plant Science and Biodiversity Centre Slovak Academy of Sciences 845 23 Bratislava Slovak Republic

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