Open Access

Yannis Schöneberg, Sven Winter, Oscar Arribas, Matteo Riccardo Di Nicola, Maya Master, John Benjamin Owens, Michail Rovatsos, Wolfgang Wüster, Axel Janke, Uwe Fritz

• Highlights • Genomes for all five Natrix species, two represented by two distinct subspecies each, were sequenced. • Two genomes were de-novo assembled to their 1.7 Gb length with a contig N50 of 4.6 Mbp and 1.5 Mbp. • Evidence for interspecific hybridization, both between allopatric and widely sympatric species. • Fossil-calibrated molecular clock using genomes indicates that species are ancient several million-year-old lineages. • Our findings imply that speciation took place despite continued gene flow. Abstract Understanding speciation is one of the cornerstones of biological diversity research. Currently, speciation is often understood as a continuous process of divergence that continues until genetic or other incompatibilities minimize or prevent interbreeding. The Palearctic snake genus Natrix is an ideal group to study speciation, as it comprises taxa representing distinct stages of the speciation process, ranging from widely interbreeding parapatric taxa through parapatric species with very limited gene flow in narrow hybrid zones to widely sympatric species. To understand the evolution of reproductive isolation through time, we have sequenced the genomes of all five species within this genus and two additional subspecies. We used both long-read and short-read methods to sequence and de-novo-assemble two high-quality genomes (Natrix h. helvetica, Natrix n. natrix) to their 1.7 Gb length with a contig N50 of 4.6 Mbp and 1.5 Mbp, respectively, and used these as references to assemble the remaining short-read-based genomes. Our phylogenomic analyses yielded a well-supported dated phylogeny and evidence for a surprisingly complex history of interspecific gene flow, including between widely sympatric species. Furthermore, evidence for gene flow was also found for currently allopatric species pairs. Genetic exchange among these well-defined, distinct, and several million-year-old reptile species emphasizes that speciation and maintenance of species distinctness can occur despite continued genetic exchange.