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Anemone  and  Gladiolus  are economically important geophyte genera due to their usage as ornamental plants. However, species identification is still under debate. Recently, DNA barcoding has become an efficient alternative tool for species identification. Here, candidate DNA barcode regions,  rbcL ,  rpoB , and  trnH-psbA  were analyzed as potential DNA barcodes in 248 accessions of these geophyte genera using pair-wise distance-based, sequence similarity (best match and best close match) and tree-based (neighbor joining, maximum likelihood, and Bayesian tree) methods. Among all barcoding single or combination loci,  trnH-psbA  had the highest interspecific genetic variation in  Anemone , and the region was the most promising candidate for barcoding gap analysis. However, none of the barcode regions showed a clear barcode gap for identification. The internal spacer,  trnH-psbA , also showed the highest correct identification rate in  Anemone , while  rbcL  showed the best performance in  Gladiolus  using the sequence similarity method. In the single barcode approach,  rpoB  had the best discrimination rate for both genera when using tree-based methods. Therefore, we recommend a tree-based method because it was clear that all tested loci had specific performance degrees depending on the methods. This is the first application of  rpoB  in terms of DNA barcoding criteria in geophyte taxa.

DNA barcoding evaluation of geophytes: Comparative efficiency of three barcode loci for Anemone (Ranunculaceae) and Gladiolus (Iridaceae)