Integrative species delimitation of the <i>Cycas sexseminifera</i> complex (Cycadaceae) utilizing genomic and morphological data

Abstract The definition and boundary of a species is fundamental to various branches of biology, including conservation biology, biogeography, and evolutionary biology. This highlights the need for precise species delimitation, especially for those with conservation priority. Relying solely on morphological traits for species identification may pose challenges, whereas molecular data can reveal cryptic species, and its application in species delimitation research has become widespread. The karst mountains of southwestern China and adjacent areas harbors a high biodiversity, including Cycas , one of the most ancient seed plant lineages. A total of six morphologically similar Cycas species were described from the karst mountains, forming the C. sexseminifera complex, but their taxonomy is a matter of debate. In this study, by employing restriction site‐associated DNA sequencing (RAD‐seq) and morphological analysis, we aimed to resolve the classification of Cycas throughout the karst mountains of southwestern China. Our sampling covered the whole distribution of this complex, representing four of the six taxa, which are C. ferruginea , C. longisporophylla , C. sexseminifera , and C. spiniformis . We failed to include C. brevipinnata and C. septemsperma into the dataset, due to the absence of wild populations and cultivated individuals. Phylogenomic inference, admixture, Bayesian phylogenetics and phylogeography (BPP), and principal component analysis (PCA) based on genome‐wide data all support the four taxa to cluster into two clades: Clade I solely consists of C. ferruginea , while Clade II contains the remaining three taxa. Further statistical analyses based on six crucial morphological characteristics also support the results of molecular data. Under the integrative species concept (ISC), we redefine the complex as two species: C. ferruginea and a newly delineated C. sexseminifera , which includes C. sexseminifera , C. longisporophylla and C. spiniformis . We also offer detailed taxonomical treatments for the two species. This study not only lays the foundation of the taxonomy of the C. sexseminifera complex, but also demonstrates the power of ddRAD‐seq in resolving complex species classifications, particularly for large‐genome gymnosperms.