Integrating morphology, nuclear SSR, and chloroplast genomes to investigate hybrid origin and species delimitation in the Lycium barbarum L. and its varieties in China

The genus Lycium L. has a considerable economic and ecological value, yet species delimitation has long posed taxonomic challenges. The two varieties, L. barbarum var. auranticarpum K. F. Ching and L. barbarum var. implicatum T. Y. Chen & Xu L. Jiang, exhibit intermediate or overlapping morphological characteristics and are therefore speculated to be of hybrid origin involving L. barbarum var. barbarum L. and L. ruthenicum Murray. To test this hypothesis, we assessed their phylogenetic relationships using morphological traits, nuclear simple sequence repeat (SSR) markers, and chloroplast genomic data. Morphological examination confirmed that the individuals of both varieties exhibited intermediate traits between L. barbarum var. barbarum and L. ruthenicum or overlapping L. barbarum var. barbarum. Analysis of nuclear SSR data further indicated that all assessed individuals possessed a mixed genetic composition derived from the two putative parental species, supporting their hybrid status. Chloroplast phylogeny reconstructed using both the maximum likelihood (ML) and Bayesian inference (BI) methods yielded highly consistent topologies. These analyses strongly support (BS = 100%; PP = 1) a monophyletic clade containing L. barbarum var. auranticarpum, L. barbarum var. implicatum, and L. ruthenicum. Collectively, evidence from morphology, SSR markers, and chloroplast genome data indicate that L. barbarum var. auranticarpum and L. barbarum var. implicatum were hybrids derived from crosses between L. ruthenicum and L. barbarum var. barbarum. This study highlighted the value of integrating multiple data types to resolve complex taxonomic issues and provides a practical framework for future species delimitation studies. It also offers molecular tools and genetic insights that may facilitate the breeding of improved Lycium varieties with desirable agronomic traits.