Comparative Analysis of Chloroplast Genomes and New Insights into the Phylogenetic Relationships of Lycoris

The genus Lycoris, comprising approximately 25 species, has high medicinal and ornamental value. However, frequent interspecific hybridization and polyploidization have complicated species delimitation and germplasm identification. Complete chloroplast genomes are widely used for phylogenetic reconstruction and species identification. In this study, we sequenced and assembled the complete chloroplast genomes of L. haywardii, L. guangxiensis, L. chunxiaoensis, L. wulingensis, L. tsinlingensis, L. sp., L. caldwellii, and L. hubeiensis. The genome sizes ranged from 158,461 bp in L. wulingensis to 158,772 bp in L. guangxiensis. A total of 132 gene copies were annotated in each chloroplast genome, comprising 86 protein-coding, 38 tRNA and eight rRNA gene copies (78, 30 and four unique sequences, respectively, i.e., 112 unique genes). All eight genomes exhibited the typical quadripartite structure, with conserved genome organization and gene content. Size variation was mainly attributable to expansion and contraction at the IR/SC boundaries. Phylogenetic analysis of extant Lycoris species resolved four major clades, each containing one or two representative and widely recognized native species. Divergence-time estimation dated the crown of the sampled Lycoris plastid lineages to the late Miocene (6.65 Mya, 95% HPD 5.73–7.67 Mya), with the two deepest splits within the genus at 6.06 and 4.08 Mya and most terminal plastid divergences occurring during the Pleistocene (0.02–1.47 Mya). This pattern is consistent with previous studies suggesting that most Lycoris species are evolutionarily young and originated through hybridization and polyploidization. Among the newly sequenced taxa, the unidentified wild species (L. sp.) from Shaanxi Province is morphologically nearly identical to L. shaanxiensis, differing mainly in flower color, and plastome phylogenies place it in the same clade as L. shaanxiensis and its allies. The taxon may therefore represent a color variant derived from L. shaanxiensis, but this hypothesis cannot be tested with plastome data alone: recent divergence, shared maternal ancestry, chloroplast introgression, hybrid origin and intraspecific variation remain equally plausible, and nuclear or genome-wide markers with population-level sampling are needed to resolve its status.