Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

Abstract

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia.

Peer Review #2 of "Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae (v0.1)"

“…Core barcoding regions, matK and rbcLa also had variable, species specific informative sites but performed relatively poor than that of ITS2. In consistence with prior studies (China Plant BOL group, 2011;Zhang et al, 2012;Li et al, 2016;Saadullah et al, 2016;Mishra et al, 2017), matK and rbcLa are recommended to be used as multi-locus barcodes (ITS2+matK, ITS2+rbcLa, ITS2+matK+rbcLa) as evident in figures 1 and 2 and table 3.…”

“…Phylogenetic trees of ITS2, ITS2+rbcLa, and ITS2+matK+rbcLa had similar percentage of nodes having 99% or more bootstrap support hence keeping the cost and time effectiveness into account, single barcode is preferred on multi-locus barcode specifically for small dataset (Feng et al, 2015;Braukmann et al, 2017;Mishra et al, 2017). This is in contrast to the study of Hilu & Liang (1997) and Hollingworth et al (2011) who have declared matK as the best analogue of CO1 animal barcode due to rapidly evolving plastid DNA region.…”

“…The BCM function offered more stringent criteria by keeping a threshold of 0.1-0.5% pairwise distance in pairwise summary function. The queries above the threshold value were classified as "no match" and the others that are below the threshold value were analyzed according to the criteria established in "best match" analysis (Meier et al, 2006;Giudicelli et al, 2015;Hartvig et al, 2015;Mishra et al, 2017).…”

Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae

“…The BCM function offered more stringent criteria by keeping a threshold of 0.1–0.5% pairwise distance in pairwise summary function. The queries above the threshold value were classified as “no match” and the others that are below the threshold value were analyzed according to the criteria established in “best match” analysis ( Meier et al, 2006 ; Giudicelli, Mäder & Freitas, 2015 ; Hartvig et al, 2015 ; Mishra et al, 2017 ).…”

Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae)

“…Hitherto, many researchers have evaluated the combination of several proposed plastid and nuclear regions to envisage the universal barcode in plants through their comprehensive studies in taxonomically complex groups 24 , 25 , 27 , 29 , 34 – 36 . Currently, the barcoding research is shifting beyond this evaluation phase.…”