Identification of morphologically similar species of Tribulus (Zygophyllaceae) by employing DNA barcodes and rRNA secondary structures

Abstract

Abstract Tribulus terrestris is one of the essential constituents in traditional Indian systems of medicine and commonly used in several herbal preparations. It is adulterated with morphologically similar species, viz., T. subramanyamii, T. lanuginosus and T. alatus. T. terrestris is often misidentified for T. subramanyamii and T. lanuginosus. Hence, the error-free recognition and collection of this medicinal herb are important to ensure the drug’s effectiveness and biosafety. In the present study, DNA barcoding technique was employed to identify and to differentiate T. terrestris from its closely related species. Phylogenetic analysis was carried out for the three species of Tribulus using barcode candidates, such as, nuclear ribosomal DNA region ITS, chloroplast plastid gene psbA-trnH and rbcL. Sequence alignment revealed 7.5% polymorphic sites in ITS, with different specific regions showing the distribution of polymorphic sites as follows: ITS1 1.7%, ITS2 3.6% and the plastid intergenic spacer psbA-trnH 2.33%. The region ITS2 possesses a higher rate of transition/transversion ratio, percentage of variation and mean distance as compared to ITS1 and psbA-trnH. In addition, ITS2 has unique polymorphic loci which aid to differentiate T. terrestris from other species of Tribulus. Additionally we predicted the structure of ITS2 and 5.8S rRNA which distinguishes three phenotypically alike species based on the helical structure. The present study opens up new avenue for the identification of closely related species based on ITS2 region and 5.8S rRNA structure which could be employed for diversity studies