Phenotypic, biochemical, and genetic diversity within varieties of Amaranthus tricolor L.: Implications of betalain biosynthesis as a taxonomic marker of speciation

• Betalains may serve as intraspecific chemotaxonomic markers. • Phenols and ascorbic acid vary in green and red Amaranthus tricolor varieties. • Polymorphisms of matK and rbcL can differentiate varieties of A. tricolor . • A. tricolor green and red varieties are paraphyletic and clearly diverged. • Biochemical and taxonomic markers can delineate varieties of betalainic plants. It is difficult to distinguish between different Amaranthus species or varieties within a species based solely on their morphological characteristics. This study aimed to establish the phenotypic, biochemical, and genetic basis for differentiating between red and green varieties of Amaranthus tricolor L., a nutritionally important leafy plant in tropical countries and beyond. Biochemical markers like betalains, which are present in the red variety while absent in the green one, and their biosynthesis-related metabolites (ascorbic acid higher in the green and tyrosine lower in the green) may serve as important intraspecific chemotaxonomic markers. Phenols and other metabolites also vary in the two varieties, possibly because of the pleiotropic effects of the loss/gain of betalain biosynthesis. For molecular taxonomy, Maximum likelihood (ML) trees, genetic diversity, and barcode gap analysis were used based on three regions of the chloroplast genome ( matK, rbcL, trnH-psbA ) and a nuclear-transcribed spacer (ITS). The highest polymorphism occurred in matK followed by rbcL . The interspecies distance is greater than the intraspecies distance, and there is a barcode gap in matK, rbcL , and trnH-psbA . Within A. tricolor, matK (1.59 %) and rbcL (1.03 %) sequences of the studied red and green varieties showed considerable genetic distance, indicating intraspecies variations pointing at possible speciation. Phylogenetic analysis also revealed that A. tricolor green and red varieties are paraphyletic and clearly diverged. These findings could form the basis for establishing biochemical and taxonomic markers for delineating the red and green varieties of various species of Amaranthus genus and other betalainic taxa.