The report of anthocyanins in the betalain-pigmented genus Hylocereus is not well evidenced and is not a strong basis to refute the mutual exclusion paradigm

Here we respond to the paper entitled “Contribution of anthocyanin pathways to fruit flesh coloration in pitayas” (Fan et al., BMC Plant Biol 20:361, 2020). In this paper Fan et al. 2020 propose that the anthocyanins can be detected in the betalain-pigmented genus Hylocereus, and suggest they are responsible for the colouration of the fruit flesh. We are open to the idea that, given the evolutionary maintenance of fully functional anthocyanin synthesis genes in betalain-pigmented species, anthocyanin pigmentation might co-occur with betalain pigments, as yet undetected, in some species. However, in absence of the LC-MS/MS spectra and co-elution/fragmentation of the authentic standard comparison, the findings of Fan et al. 2020 are not credible. Furthermore, our close examination of the paper, and re-analysis of datasets that have been made available, indicate numerous additional problems. Namely, the failure to detect betalains in an untargeted metabolite analysis, accumulation of reported anthocyanins that does not correlate with the colour of the fruit, absence of key anthocyanin synthesis genes from qPCR data, likely mis-identification of key anthocyanin genes, unreproducible patterns of correlated RNAseq data, lack of gene expression correlation with pigmentation accumulation, and putative transcription factors that are weak candidates for transcriptional up-regulation of the anthocyanin pathway.