Abstract
BACKGROUND: Accurate plant species circumscription is fundamental to biodiversity conservation, medicinal resource development, and ecological research. Yet challenges such as sample incompleteness and reliance on limited molecular markers often hinder precise species circumscription. A single identification method-molecular or morphological-is in most cases not sufficient to accurately recognize plant species. Notably, single-copy nuclear genes, despite their critical importance in resolving species circumscription through higher evolutionary rates and biparental inheritance, remain underexplored in current research. A critical next step is developing nuclear genes as DNA barcodes. In some cases, micro-morphological characteristics mirror molecular evidence and confirm species identification. Epimedium (Berberidaceae), well known for its medical and horticultural significance, remains poorly understood taxonomically due to its phenotypic diversity. It is an ideal taxon to explore integrative plant species circumscription combining molecular and micro-morphological data. RESULTS: Chloroplast genome structure analysis revealed that the variations near the IR/SC boundary, the unique trnQ-UUG gene rearrangement, and the repeat sequences in Epimedium hold significant evolutionary implications. It not only uncovered the conservation and specificity of genomic structures but also provided novel insights into the phylogeny and molecular evolution of this genus. We identified eight hypervariable regions in Epimedium species that emerged as strong candidates for potential DNA special barcodes. These regions and the whole chloroplast genome showed higher species discriminability compared to standard barcodes. Single-copy nuclear genes were more useful in species circumscription over chloroplast genomes. Furthermore, micro-morphological characteristics served as strong complementary evidence for species circumscription and could help distinguish species that were unresolved using only molecular or genomic analyses. CONCLUSIONS: Using Epimedium as a case study, we propose a Multilayer Precision Species Circumscription Approach (MPSCA), a diagnostic framework that combines standard barcodes, chloroplast genome, single-copy nuclear genes, and micro-morphological data.