Origin, morphological, genetic, and chemical variability, and traditional uses of Cannabis: basis for new applications

[eng] Cannabis sativa L. (hereafter referred to as Cannabis) is one of the earliest domesticated plants, with human use spanning over millennia for a variety of purposes. Given its widespread use and significance, Cannabis has been the focus of research in diverse scientific areas, from medicine, pharmacy, and agronomy to environmental and social sciences. However, most of the research has concentrated on modern hemp cultivars and drug strains, with limited attention given to wild-growing populations or traditional landraces across the plant's natural distribution area, leaving significant gaps in understanding their diversity. Similarly, while numerous publications have documented the traditional uses of Cannabis, this body of knowledge remains fragmented across various types of literature. This fragmentation, much like the limited research on wild-growing populations, complicates efforts to conduct comprehensive statistical analyses and gain a holistic understanding of the plant's diversity and cultural significance. This doctoral thesis aims to fill this gap by exploring the genetic, morphological, and phytochemical diversity of Cannabis and its traditional uses worldwide through seven interconnected studies. The research integrated various interdisciplinary methods, such as flow cytometry, Hyb-Seq genomic analysis, high-resolution geometric morphometrics, phytochemical analysis using UPLC-MS, and systematic ethnobotanical data collection, offering a comprehensive view of this versatile plant. In the first chapter we explored the genetic and cytogenetic diversity of Cannabis. Using flow cytometry, we examined genome size diversity across a wide range of wild-growing, landrace, and cultivated Cannabis accessions. The analysis revealed minimal variation in ploidy levels, with only one triploid individual identified, while the rest were diploid. A 1.189-fold variation in genome size was observed, but no strong correlation was found between genome size and geographical distribution or between wild-growing and cultivated plants. This suggests that the geographical expansion and domestication of Cannabis had a limited effect on its genome size. Notably, significant differences in genome size were observed between male and female plants. In another work of this chapter, we examined the phylogeographic and population genetics structure of wild-growing and landrace Cannabis populations, by employing a Hyb-Seq approach, combining shotgun sequencing and target capture with the Angiosperms353 enrichment panel. The results supported the classification of Cannabis as a monotypic genus, structured into three primary genetic groups: E Asia, which is sister to both Paleotropis group and Boreal group. Further subdivisions within these groups revealed phylogeographic patterns driven more by geographical distribution than by use-type. In the second chapter we focused on the morphological diversity of Cannabis. Due to significant changes in leaflet number during the plants’ development, a novel method was developed that allows, for the first-time, the application of a high-resolution geometric morphometrics approach on the entire leaf. By using polynomial models across more than 3,000 pseudo-landmarks, we modelled theoretical leaves with comparable leaflet numbers. This innovative approach enables the accurate prediction of genetic and developmental traits, providing new insights into Cannabis leaf morphology and uncovering heterochronic mechanisms responsible for changes in leaf shape during the development. Phytochemical diversity was the focus of the third chapter, which analysed cannabinoid composition in different Cannabis tissues, sexes, and across different phylogeographic groups identified in the first chapter. Significant differences in cannabinoid profiles were found between male and female inflorescences, but not between the leaves of either sex. While the cannabinoid profiles did not precisely correlate with phylogeographic groups, there was a clear differentiation between cultivated drug-type landraces and wild-growing populations, indicating the influence of selective breeding during domestication. The last, forth chapter explored traditional knowledge of Cannabis use. The first study in this chapter involved the development of the CANNUSE database, which consolidated 2,330 entries from 649 publications across 41 countries, providing a valuable resource for understanding traditional Cannabis uses. The second study analysed the standardized dataset from CANNUSE database and found that medicinal applications dominated, treating over 200 human ailments. Strong associations were observed between specific Cannabis plant parts and their corresponding uses and in treating different body systems. The third ethnobotanical study investigated the traditional knowledge of Cannabis use in Armenia through ethnobotanical surveys and bibliographic review. The results revealed a significant decline in medicinal and fibre uses but highlighted the continued importance of Cannabis seeds in alimentary uses, particularly in symbolic dishes, demonstrating the persistence of Cannabis as an important cultural and historical resource. In conclusion, the results of this thesis highlight the extraordinary genetic, morphological, and phytochemical diversity of Cannabis, particularly evident when focusing on wild-growing populations and traditional landraces. The findings support the classification of Cannabis as a monotypic genus, with marked intraspecific variability. The newly gathered comprehensive dataset of traditional uses provided novel insights into its rich traditional heritage, and revealed new potential applications rooted in traditional knowledge. These results underscore the importance of further research and the preservation of diversity within wild-growing populations of this versatile species.