Integrating morphometrics, k-mer–based genomic, and phylogenomics reveals a complex reticulate history of Cypripedium × ventricosum

Disentangling the relative contributions of hybridization and incomplete lineage sorting (ILS) in recently diverged plant groups remains challenging, particularly in those with large and complex genomes. Cypripedium × ventricosum has long been hypothesized to have a hybrid origin, yet its evolutionary history remains unresolved. Here, we integrate morphological data, flow cytometry, organellar phylogenomics, ribosomal DNA variation, formal hybridization tests (HyDe), and alignment-free genome-wide similarity analyses based on k-mers to better understand its evolutionary history. We detect both morphological intermediacy and transgressive variation, with C. × ventricosum occupying an intermediate position between the Macranthos and Calceolus clades in multivariate space. Genome-wide k-mer analyses and HyDe reveal pronounced signals of hybridization and genomic heterogeneity. Diagnostic k-mer analyses indicate strong genomic affinity to C. macranthos lineage, moderate contributions from C. calceolus , weaker but detectable signals from C. × microsaccos , and minimal signals from C. shanxiense relative to background levels. Cytonuclear discordance in some individuals further highlights mismatches among nuclear, organellar, and morphological patterns. Two triploid individuals were identified, whose genomic composition closely resembles that of diploid hybrids without evidence of additional parental contributions. Together, our results are consistent with the interpretation of C. × ventricosum as a heterogeneous reticulate complex rather than a single, uniform hybrid lineage. The observed patterns are consistent with a combination of hybridization, incomplete lineage sorting, and subsequent genomic interactions. More broadly, this study demonstrates that integrating genome-wide k-mer approaches with phylogenomic and morphological data provides an effective framework for resolving complex evolutionary histories in large-genome plant systems.