Molecular Identification and Phylogenetic Assessment of Luisia tristis Using rbcL DNA Barcoding and Genetic Distance Analysis

AbstractMolecular methods are necessary because physical similarities and phenotypic flexibility can make it difficult toaccurately identify orchid species. The molecular identification and phylogenetic analysis of Luisiatristis were conducted in thisstudy using the chloroplast rbcL gene. A 344 bp segment of the rbcL gene was successfully amplified using certain primers.Under the accession number MZ666900, the acquired sequence was uploaded to the NCBI GenBank database.Neighbor-Joining(NJ) and Fast Minimum Evolution (FastME) were used to conduct phylogenetic studies based on genetic distance. Luisiatristis'smolecular distinctiveness was confirmed by both methods, which consistently identified it as a unique lineage among particularorchid taxa.L. tristis maintained its independent taxonomic position by remaining distinct from the main Habenaria clade despiteexhibiting close evolutionary ties with related taxa like Habenaria and Pecteilis. Platanthera, Dactylorhiza, Cymbidium, andHerminium were among the more distantly related genera that formed distinct clusters with higher genetic divergence.Highsequence quality and species-specific signatures were shown by the rbcL sequence's uniform nucleotide composition and lack of unclear sections in the DNA barcode display. Furthermore, conserved α-helices, β-sheets, and loop areas were identified by three-dimensional structural modelling of the rbcL protein, demonstrating both structural integrity and evolutionary conservation. All things considered, the study shows that the rbcL gene is a useful marker for molecular identification and phylogenetic analysis ofLuisia tristis within the Orchidaceae family.