Molecular Diversity and DNA Barcode Identification of Selected Philippine Endemic Hoya Species (Apocynaceae)
Although several Hoya species are endemic to the Philippines, their limited geographical distribution makes some species more vulnerable to extinction. Likewise, a number of species were also reported to exhibit phenotypic plasticity, which makes it difficult to distinguish them using morphological characters. DNA barcoding, together with morphological traits, may help in the proper identification and protection of this group of plants. Determination of molecular diversity, through analysis of the sequence variation of selected genes and identification of DNA barcodes for five Hoya species, was conducted using the recommended standard gene barcoding regions in the plant-maturase K gene (matK) and in the ribulose 1,5-bisphosphate carboxylase oxygenase large sub-unit gene (rbcL). Gene amplification was done using a single primer pair for each gene which generated product sizes of 535 bp and 809-815 bp for rbcL and matK, respectively. Phylogenetic trees were generated using the partial gene sequences. The Neighbour-Joining (NJ) method was likewise used with Maximum Likelihood as the measure of evolutionary distance. Distinctive clades with high bootstrap support (95–99%) were generated for three Hoya species using the matK sequences and for two species for rbcl with low support (63–64%). Pair-wise sequence analysis of rbcL sequences showed 0–1% level of sequence variability while matK sequences had 0–2%, indicating high sequence homology for both genes. Potential barcoding regions in the Hoya species were identified through haplotype construction. MatK sequences exhibited a higher number of variable sites, which is 21, while only two were observed in the rbcL sequences. The maximum number of haplotypes constructed for matK was four and three for rbcL. Greater resolution and discrimination among species was achieved when haplotypes from both genes were combined.
Key Words: DNA barcode, Hoya, matK, molecular diversity, rbcL