Distinguishing two morphologically similar Rhinacanthus species using Chloroplast barcoding regions

Abstract

Rhinacanthus flavovirens (Maha Anitta) belonging to the family Acanthaceae, is an endemic species to Sri Lanka. R. flavovirens is distributed in North central, Central, Southern and Western provinces in the country. R. flavovirens and R. nasutus possess a high level of morphological similarity, creating confusions in differentiating between the two species. DNA barcoding is a widely used molecular method for herbal plant identification and authentication, especially when morphological features are not sufficient. Chloroplast barcoding regions matK, rbcL and trnHpsbA have been used for this study. DNA was extracted and quantified using a Nanodrop spectrophotometer before amplification. The amplified fragment lengths for R. nasutus were 762 bp (matK), 369 bp (rbcL), and 333 bp (trnH-psbA), while those for R. flavovirens were 769 bp (matK), 370 bp (rbcL), and 364 bp (trnH-psbA). The Sanger sequencing showed that, among these three markers trnH-psbA showed the highest nucleotide variation (68.46%) whereas matK showed the lowest (1.3%). Pairwise genetic distance analysis showed that R. nasutus and R. flavovirens have closer genetic relatedness for matK region (0.0133) and higher divergence in trnH-psbA region (1.83). The MEME suite program identified a higher frequency of insertion/deletion events in trnH-psbA compared to matK and rbcL. The result of this study showed that DNA barcoding, particularly using variable region trnH-psbA, can effectively distinguish R. flavovirens from R. nasutus. Future studies incorporating whole chloroplast genome sequencing may provide enhanced resolution for species identification, authentication and phylogenetic analysis.