Phylogenetic relationship based on DNA barcodes and comparative analysis of phytochemical contents among Rhynchostylis orchids in Thailand

Abstract Rhynchostylis orchids are native to Thailand and have considerable commercial importance. However, species delimitation remains difficult because of overlapping morphology, environmental plasticity, short flowering periods, and extensive horticultural hybridization. In addition, comprehensive molecular and phytochemical data for the genus remain limited. This study examined phylogenetic relationships among nine Rhynchostylis accessions and one Aerides species using the internal transcribed spacer (ITS) and maturase K (matK) DNA-barcode regions, and further evaluated their phytochemical properties to support species identification, conservation, and superior genotype selection. The ITS and matK regions were amplified and sequenced, and phylogenetic trees were reconstructed using maximum likelihood (ML) and Neighbour-joining (NJ) approaches. These analyses generally recovered R. retusa and R. coelestis as distinct lineages from the R. gigantea complex. ITS showed slightly higher discriminatory power among closely related R. gigantea forms, whereas matK provided complementary phylogenetic structure. When interpreted together with diagnostic floral traits, these molecular data strengthened species delimitation within the genus. Phytochemical profiling of ethanolic leaf extracts revealed significant interspecific variation, with total phenolic content (TPC), total flavonoid content (TFC), and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity ranging from 2.30 ± 0.32 to 11.38 ± 0.01 mg GAE/g DW, 1.47 ± 0.62 to 18.14 ± 0.33 mg RE/g DW, and 12.91 ± 0.09 to 42.17 ± 0.33 mg TE/g DW, respectively. R. gigantea ‘Chang Phlai’ consistently exhibited the highest phenolic content and antioxidant capacity, highlighting its potential pharmacological relevance. Collectively, this study demonstrates that integrating DNA barcoding with floral morphology and phytochemical evidence provides a baseline molecular and chemical framework that helps clarify taxonomic interpretation in Rhynchostylis. It may also inform future conservation assessments and sustainable utilization efforts for these economically and medicinally important orchids.