Quantifying the roles of environmental, seasonal, and genome size variation in the expression of medicinally valuable compounds in Helichrysum kraussii Sch. Bip

Abstract

• Helichrysum kraussii populations show high metabolomic variability. • Population-season interaction drives Helichrysum kraussii chemical profiles. • Samples vary in their antibacterial activity against Cutibacterium acnes . • Relative genome size differs between more spatially isolated populations. Intraspecies phenotypic variation is driven by numerous factors, including spatial isolation, seasonality, and genome size variation. The importance of these drivers with regard to a species’ medicinal value is often overlooked, potentially compromising the robustness of inferences. This study’s primary aim was to illustrate the influence of these factors on the metabolomes and bioactivity of medicinal plants, using Helichrysum kraussii Sch. Bip. as a case study, with the expectation that significant differences in these variables would exist between populations, seasons and genome sizes. Ethanol extracts were prepared from 93 samples from nine populations in the South African provinces of North West and Gauteng over two seasons and analysed using high-performance thin-layer chromatography to elucidate secondary metabolite profiles. Select extracts were tested against Cutibacterium acnes to determine their half-maximal inhibitory concentration (IC 50 ) values and evaluate whether significant differences in antibacterial activity were present between the samples. Fresh plant material was analysed using flow cytometry to determine the relative genome sizes of select populations using Pisum sativum L. and Helianthus annuus L. as internal standards. Permutational analysis of variance using population, season, and their interaction indicated that these factors significantly influenced variation in secondary metabolite profiles, although these were not discernible in principal component analysis and agglomerative hierarchical clustering. A k-means analysis grouped samples into two robust clusters, but a Mantel test revealed a poor correlation between geographic distance and secondary metabolite profiles. The potential antibacterial activity of H. kraussii was evaluated against Cutibacterium acnes , where extracts exhibited significantly different IC 50 values, ranging between 17.10 ± 4.16 μg/mL and 391.16 ± 15.02 μg/mL. Finally, flow cytometry revealed that one population contained individuals with significantly smaller relative genome sizes, although this could not be associated with variability in metabolomes or bioactivity. The k-means clustering and the difficulty in identifying patterns based on population and season led to the hypothesis that two chemotypes not solely reliant on these variables exist within H. kraussii . Our results show that seasonal, population-level, and interactions between these variables drive variation in secondary metabolite profiles in Helichrysum kraussii . It is therefore strongly recommended that future research on medicinal plants prioritise intraspecific variation and attempt to characterise its sources.