The large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal species.

Having great medicinal values, species of "Fengdou" (DSFs) are a taxonomically complex group in genus including many closely related and recently diverged species. Traditionally used DNA markers have been proved to be insufficient in authenticating many species of this group. Here, we investigated 101 complete plastomes from 23 DSFs, comprising 72 newly sequenced and 29 documented, which all exhibited well-conserved genomic organization and gene order. Plastome-wide comparison showed the co-occurrence of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), which can be explained by both the repeat-associated and indel-associated mutation hypotheses. Moreover, guanine-cytosine (GC) content was found to be negatively correlated with the three divergence variables (SNPs, indels and repeats), indicating that GC content may reflect the level of the local sequence divergence. Our species authentication analyses revealed that the relaxed filtering strategies of sequence alignment had no negative impact on species identification. By assessing the maximum likelihood (ML) trees inferred from different datasets, we found that the complete plastome and large single-copy (LSC) datasets both successfully identified all 23 DSFs with the maximum bootstrap values. However, owing to the high efficiency of LSC in species identification, we recommend using LSC for accurate authentication of DSFs.