Background Acanthophyllum species are notable for their medicinal and industrial importance due to the presence of valuable bioactive compounds, especially saponins. Assessing genetic diversity is crucial for the strategic development and selection of superior ecotypes in Acanthophyllum species. The present study aimed to investigate eight different phytochemical traits of ten species of the Acanthophyllum genus, including Acanthophyllum bracteatum Boiss (7 ecotypes), Acanthophyllum diezianum Hand-Mazz (1 ecotype) Acanthophyllum glandulosum Bunge ex Bioss (22 ecotypes), Acanthophyllum heratense Schiman-Czeika (1 ecotype), Acanthophyllum korshinskyi Schischk (5 ecotypes), Acanthophyllum laxiusculum Schiman-Czeika (1 ecotype), Acanthophyllum microcephalum Boiss (12 ecotypes), Acanthophyllum sordidum Bunge ex Boiss (7 ecotypes), Acanthophyllum spinosum C.A.Mey (6 ecotypes) and Acanthophyllum squarrosum (6 ecotypes), collected from different natural habitats of Acanthophyllum in Iran. Results The investigated ecotypes have significant differences in terms of all the measured as revealed using analysis of variance (P <0.01). The highest levels of total phenolics (28.83 mg gallic acid equivalent g−1 dry weight), total flavonoids (9.08 mg quercetin g−1 DW), and total flavonols (5.8 mg quercetin g−1 DW) were recorded in ecotypes of A. bracteatum3, A. glandulosum11, and A. glandulosum4, respectively. The highest total tannins contents in roots (194.36 mg catechin equivalent g−1 DW) and leaves (94.97 mg tannic acid equivalent g−1 DW) were found in A. glandulosum21 and A. squarrosum2, respectively. The highest contents for total polysaccharide (15.14 mg glucose equivalent g−1 DW) and total saponins (158.75 mg saponin equivalent g−1 DW) in roots were observed in A. heratense1 and A. glandulosum17, respectively. The highest antioxidant activity, (2,2-diphenyl-1-picrylhydrazyl), was measured as 93.28% in A. microcephalum12 and A. bracteatum7 ecotypes. A significant and positive correlation (r = 0.37**), between total phenolics content and antioxidant activity was observed. Cluster analysis based on phytochemical traits grouped the ecotypes into three distinct clusters, demonstrating high genetic variability: group I (26 ecotypes), group II (20), and group III (22), which highlights the high potential for selecting advantageous traits according to their cluster classification. Conclusion These findings emphasize significant phytochemical diversity between and within Acanthophyllum species, positioning them as promising natural sources of antioxidants and bioactive compounds. A. squarrosum and A. laxiusculum demonstrated the greatest convergence in phytochemical traits, while A. glandulosum and A. spinosum showed the greatest divergence. Superior Acanthophyllum species and their associated ecotypes are promising candidates for breeding programs and future economic exploitation in different food industries and pharmaceutical aims.

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