Limited resolution of DNA barcodes and environmental influence on phytochemical diversity in Berberis integerrima (Berberidaceae)

Berberis has numerous applications in the food industry and traditional medicine because of its antioxidant, anti-inflammatory, antimicrobial, and cholagogic properties. In the current study, delimitation of four closely related species (i.e., B. crataegina, B. integerrima, B. orthobotrys and B. vulgaris; in total 96 accessions) and the genetic variation of the Iranian B. integerrima (59 accessions) were studied using DNA barcodes and a phytochemical approach. A total of 240 sequences of six nuclear and chloroplast DNA barcodes, including ITS, matK, ndhF, rbcL, trnH-psbA, and trnL-trnF intergenic spacers, were obtained and analyzed using ML, STRUCTURE, UPGMA, AMOVA, and PCoA approaches. Based on our findings, the evaluated DNA barcodes, either individually or in combination, do not provide sufficient resolution for discriminating the closely related species of Berberis. However, ITS and trnH-psbA did get better resolution compared to the other markers. In order to evaluate the genetic diversity of Iranian B. integerrima, we studied 14 wild populations to build the first local DNA barcode reference library. The results of STRUCTURE analysis suggested that B. integerrima accessions can be grouped into four subpopulations. In correlation with the results of STRUCTURE analysis, the studied accessions were resolved into four major clusters on the UPGMA dendrogram, where the accessions from the same or neighboring areas were mostly grouped in a common cluster. The AMOVA analysis revealed that 24.91% (nrDNA) and 34.91% (cpDNA) of the observed variances corresponded to differences among subpopulations, whereas 75.08% (nrDNA) and 65.09% (cpDNA) matched to differences within subpopulations. For the phytochemical analysis, six key traits (total phenolic content (TPC), total flavonoid content (TFC), anthocyanin content, and antioxidant activity using DPPH, FRAP, and ABTS⁺ assays) were evaluated across 25 populations of B. integerrima. Moreover, we observed significant variation in phytochemical content among B. integerrima populations. The accessions that grow at higher elevations have the most antioxidants (DPPH = 90.62%, FRAP = 4.62 mg GAE g⁻1 DW), anthocyanins (5.06 mg g⁻1 DW), TPC (36.38 mg GAE g⁻1 DW), and TFC (8.52 mg QE g⁻1 DW). We identified positive correlations between these phytochemicals and antioxidant activity. Cooler climates, higher latitudes, increased UV radiation, and lower rainfall enhanced bioactive compound levels, indicating environmental stress stimulates secondary metabolite production. These findings underscore the significant nutraceutical and medicinal potential of populations growing at higher altitudes. To our knowledge, this study represents the most comprehensive analysis of the genetic variation and population structure of barberry in Iran. The insights gained from this work offer valuable implications for germplasm collection, genetic improvement, and the systematic utilization of B. integerrima.