DNA Barcoding of 11 Endemic Vanda Species of the Philippines Based on rbcL, matK, and ITS Sequences

DNA barcoding has gained great attention due to its potential for identifying, discriminating, and classifying plant species and revealing the phylogenetic relationships among closely related species. The efficacy of three barcode markers (rbcL, matK, and ITS) and their combinations were evaluated using BLASTn, genetic distance similarity, and tree-based methods on eleven endemic Vanda species of the Philippines. A total of 40 accessions representing 11 species of Vanda were successfully amplified and sequenced for each barcode region using universal primers. The number of variable sites in the aligned sequences was the highest in the ITS region (30%) – followed by the combined regions (3%), matK (2%), and rbcL (2%). BLASTn results revealed that most of the samples were correctly identified up to the genus level based on the available rbcL, matK, and ITS sequences in the NCBI database. Only two species – namely Vanda sanderiana and Vanda luzonica – were correctly identified at the species level based on the matK and ITS sequences, respectively, in the NCBI database. The computed genetic distance from the combination of the three regions ranged from 0.0000–0.01528. The genetic distance between Vanda aurantiaca and Vanda lamellata var remediosa was greatest (0.01528), which suggested that there was low genetic similarity between them. A phylogenetic tree was constructed for each gene sequence using maximum parsimony with a 1000-bootstrap replication test. Among the phylogenetic trees generated from the sequences of the rbcL, matK, and ITS regions, the phylogenetic tree produced from the ITS sequences completely distinguished the 11 Vanda species based on their current morphology-based sectional classification. The phylogenetic tree constructed based on the combined sequences of the three regions was similar to that constructed from the ITS region, except that the clusters were more strongly supported by the MP analysis. The existing morphological section of Vanda is supported by the molecular data presented in the MP tree. The single-barcode ITS is a suitable barcode for the Philippine Vanda species. Therefore, the ITS should be used in combination with the plant core barcode, rbcL, and matK for discriminating and classifying endemic Vanda species of the Philippines. This study provides a barcode database of the Vanda species in the Philippines that may significantly contribute to the conservation of this valuable genus in the orchid family.