Plastid super-barcoding in the pan-plastomic era: dense sampling of Pulsatilla patens plastomes exposes the limits of species delimitation in reticulate complexes

Background. Whole-plastome "super-barcoding" is widely applied to resolve taxonomic uncertainties in complex plant groups. In lineages prone to reticulate evolution, such as the tribe Anemoneae, sparse sampling can overestimate species-level diagnostic power. We evaluated the utility of complete chloroplast genomes for the identification of Pulsatilla patens by expanding the sampling to 33 plastomes within a broader genus-level dataset (49 plastomes), aiming to distinguish true species divergence from deep population structure. Results. We identified high genomic variability in P. patens , with a total of 2,127 mutations (1,360 indels and 767 SNPs). Variation was strongly clustered, with hotspots concentrated in ycf1 , rpoC2 , and ndhF . Despite this richness, dense sampling led to the collapse of taxonomic resolution. P. patens was resolved as paraphyletic, with P. vernalis specimens nested within its variation. After excluding introgressed individuals and divergent subspecies, the number of fixed diagnostic nucleotides for P. patens s.s. dropped to merely two. Phylogenetic structure showed a mosaic pattern (LMEM analysis) that reflected ancient Pleistocene connectivity rather than contemporary geography or habitat type. Conclusions. Our results demonstrate a "super-barcoding paradox" in Pulsatilla : increasing data volume combined with dense sampling eliminates apparent diagnostic markers due to pervasive historical introgression and shared ancestral polymorphism. In reticulate complexes, the plastome functions as a record of ancient connectivity (the "ghost" of the Mammoth Steppe) rather than a straightforward taxonomic barcode. Practical barcoding should target identified hotspots but must be integrated with nuclear markers to reflect contemporary isolation and guide effective conservation management.