Challenges in using DNA barcodes for authentication of Sida species

The Indian system of medicine's "Ayurvedic pharmacopeia of India (API)" recommends the use of Sida cordifolia Linn (root), Sida cordata (Burm.f.) Borss.Waalk. (aerial part), Sida rhombifolia Linn. (root) and Abutilon indicum (Linn.) Sw. (root) in drug preparations of Bala, Nagabala, Mahabala and Atibala respectively. Moreover many Sida sp. are being used in China, South East Asia, Africa and South America in their traditional healthcare systems. It is a taxonomically complex genus often difficult to authenticate from dried/chopped herbal market samples. Many Sida sequences from the NCBI database, including published reports, were highly suspect and were redesignated into species groups during phylogenetic clustering. Among the four loci studied, ITS2 region was identified as the best for the Sida species identification followed by trnH-psbA. The trnH-psbA phylogeny however fails to differentiate between (1) S. beddomei and S. cordata, , (2) S. alnifolia and S. scabrida, , (3) S. cordifolia and S. fryxellii that formed monophyletic clusters. The average evolutionary divergence over Sequence Pairs within each species group for ITS2 locus ranged from 0.000 to 0.009 (Average=0.0021), =0.0021), while average Interspecific distance between species was 0.1175 making them ideal for authentication of Sida species. The matK and rbcL is recommended as a back-up loci for identifying intergeneric adulterants in case, the ITS2 or trnH-psbA amplification fails. The present study identified two market samples as adulterant species; (1) S. alnifolia and (2) a mixture of S. acuta and S. alnifolia/S.scabrida. . The study provides a roadmap for Ayurvedic/ herbal industry to utilize DNA barcoding for authentication of Sida species. At the same time the presence of "Unknown Sida group" highlights the need for further research to accurately classify and identify all Sida species at the phylogenetic level, utilizing the DNA barcode sequences to thoroughly understand the diversity and evolution of the Sida genus.