Prospects for discriminating Zingiberaceae species in India using DNA barcodes

We evaluated nine plastid (matK, rbcL, rpoC1, rpoB, rpl36-rps8, ndhJ, trnL-F, trnH-psbA, accD) and two nuclear (ITS and ITS2) barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India. Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction (PCR) amplicons in all the accessions tested. However, only 35 (58%) and 40 accessions (66%) yielded ITS and ITS2 sequences, respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species (75%) into monophyletic groups and five species into two paraphyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species (60%), and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence of natural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of ITS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling.