Evaluation of species limits of Hortonia by DNA barcoding

A study was carried out to determine the species limits of the endemic genus Hortonia by DNA barcoding with a view to establishing conclusive molecular evidence regarding the speciation of Hortonia in Sri Lanka. Total DNA was extracted from all three species and the purity of the extracted DNA was confirmed Internal transcribed spacer (ITS) region and the trnH-psbA region were first amplified by the polymerase chain reaction (PCR) with specific primers and ligated to a pBlueScript vector followed by plasmid purification. The purified plasmids containing the DNA of interest were subjected to sequencing. Sequence homology of ITS and trnH-psbA from all three species were compared using MacVector software. Between H. ovalifolia and H. floribunda, the ITS region showed a 2.37 % sequence divergence and the trnH-psbA region showed a 1.5 % sequence divergence. Between H. ovalifolia and H. angustifolia, the ITS region and trnH-psbA region showed a 3.36 % and 1.89 % sequence divergences, respectively. The percentage sequence divergence between ITS and trnH-psbA regions of H. floribunda and H. angustifolia were 3.36 % and 2.65 %, respectively. The high sequence divergence values clearly indicate that the genus Hortonia has three different species. Considering the percentage sequence divergence values, H. ovalifolia and H. floribunda are more closely related to each other than to H. angustifolia.