Untangling Coelogyne: Efficacy of DNA Barcodes for Species and Genus Identification

<b>Background/Objectives</b>: While morphological similarity and incomplete specimens pose a challenge to the precise identification of <i>Coelogyne</i> orchids, accurate species and genus assignment is essential for conservation and CITES enforcement. This study evaluated the efficacy of five DNA barcode regions-<i>rbcL</i>, <i>matK</i>, <i>trnH-psbA</i>, <i>atpF-atpH</i>, and ITS2-and their combinations for species- and genus-level discrimination within the genus <i>Coelogyne</i>, aiming to develop a rapid and simple diagnostic tool for use by customs officers and trade inspectors. This is the first comprehensive comparative analysis of these five barcode regions specifically within <i>Coelogyne</i>, a genus underrepresented in molecular identification studies, and the first to propose multi-locus combinations for potential practical use. This study identified DNA barcode regions with high resolution and reliability, providing a solid basis for practical identification kits. Such tools will enhance CITES enforcement by enabling rapid detection of <i>Coelogyne</i> species in trade, directly supporting their conservation and contributing to the reduction in illegal orchid trade. <b>Methods</b>: Using a CTAB protocol, genomic DNA was extracted from leaf samples belonging to 19 <i>Coelogyne</i> species. Sanger sequencing was performed after PCR amplification using published primer sets for every barcode region. Sequences were modified in BioEdit, and BLASTn (accessed 15 June 2025) was used to compare them to GenBank (NCBI Nucleotide). Amplification efficiency was calculated per locus. Species and genus identification success rates were determined by the congruence of top BLAST hits with morphologically pre-identified taxa. Multi-barcode combinations (<i>matK</i> + <i>rbcL</i>, ITS2 + <i>matK</i>, <i>matK</i> + <i>trnH-psbA</i>, <i>rbcL</i> + <i>trnH-psbA</i>, and <i>matK</i> + <i>rbcL</i> + <i>trnH-psbA</i>) were also assessed. <b>Results</b>: With <i>rbcL</i>, <i>atpF-atpH</i>, and ITS2 yielding ≤11%, the highest single-locus species identification rates were for <i>trnH-psbA</i> (21%) and <i>matK</i> (16%). Among single-locus barcodes, <i>matK</i> showed the highest performance, with 84% genus assignment. ITS2 reached 27%, but genus-level resolution remained limited for the <i>rbcL</i>, <i>trnH-psbA</i> and <i>atpF-atpH</i> barcodes. Multi-barcode approaches maintained species resolution: <i>matK</i> + <i>rbcL</i> + <i>trnH-psbA</i>, <i>matK</i> + <i>rbcL</i>, and <i>matK</i> + <i>trnH-psbA</i> correctly identified 16% of species and achieved 74-79% genus assignment. <b>Conclusions</b>: No single locus achieves robust species discrimination in <i>Coelogyne</i>, but <i>trnH-psbA</i>, <i>matK</i> and <i>atpF-atpH</i> provide the best single-marker performance. Using the <i>matK</i> locus alone, in combination with either <i>trnH-psbA</i> or <i>rbcL</i>, or all three together ensures consistent genus-level identification and significantly improves taxonomic resolution. This study introduces a novel multi-locus barcode strategy tailored to <i>Coelogyne</i>, offering a practical solution for identification and enforcement. While promising, this approach represents a potential application that requires further validation before routine implementation.