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

## TL;DR

This study tests DNA barcodes to identify Coelogyne orchids, aiming to help conservation and CITES enforcement by finding the most effective genetic markers.

## Contribution

The first comprehensive analysis of DNA barcode regions for Coelogyne, proposing multi-locus combinations for practical identification.

## Key findings

- No single DNA barcode region effectively identifies Coelogyne species, but trnH-psbA, matK, and atpF-atpH perform best.
- Combining matK with trnH-psbA or rbcL significantly improves genus-level identification accuracy.
- Multi-locus approaches like matK + rbcL + trnH-psbA correctly identified 16% of species and achieved 74–79% genus assignment.

## Abstract

Background/Objectives: While morphological similarity and incomplete specimens pose a challenge to the precise identification of Coelogyne orchids, accurate species and genus assignment is essential for conservation and CITES enforcement. This study evaluated the efficacy of five DNA barcode regions—rbcL, matK, trnH-psbA, atpF-atpH, and ITS2—and their combinations for species- and genus-level discrimination within the genus Coelogyne, 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 Coelogyne, 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 Coelogyne species in trade, directly supporting their conservation and contributing to the reduction in illegal orchid trade. Methods: Using a CTAB protocol, genomic DNA was extracted from leaf samples belonging to 19 Coelogyne 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 (matK + rbcL, ITS2 + matK, matK + trnH-psbA, rbcL + trnH-psbA, and matK + rbcL + trnH-psbA) were also assessed. Results: With rbcL, atpF-atpH, and ITS2 yielding ≤11%, the highest single-locus species identification rates were for trnH-psbA (21%) and matK (16%). Among single-locus barcodes, matK showed the highest performance, with 84% genus assignment. ITS2 reached 27%, but genus-level resolution remained limited for the rbcL, trnH-psbA and atpF-atpH barcodes. Multi-barcode approaches maintained species resolution: matK + rbcL + trnH-psbA, matK + rbcL, and matK + trnH-psbA correctly identified 16% of species and achieved 74–79% genus assignment. Conclusions: No single locus achieves robust species discrimination in Coelogyne, but trnH-psbA, matK and atpF-atpH provide the best single-marker performance. Using the matK locus alone, in combination with either trnH-psbA or rbcL, 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 Coelogyne, offering a practical solution for identification and enforcement. While promising, this approach represents a potential application that requires further validation before routine implementation.