OR WAIT null SECS
This article explores what we do and don’t know about the origins of Cannabis ssp. from a botanical perspective and outlines some of the hypotheses proposed to explain the “why” of cannabinoid production in plants and the approaches that researchers are taking to try and answer this elusive question.
In this final installment of "Back to the Root," we will be exploring the botanical import of cannabinoids. Cannabis has been cultivated for thousands of years and is arguably one of the first crops ever domesticated by humanity. This domestication, however, has proved problematic for understanding the role of cannabinoids in plant secondary metabolism. This piece begins by exploring what we do and don’t know about the origins of Cannabis ssp. from a botanical perspective. Then it outlines some of the hypotheses proposed to explain the “why” of cannabinoid production in plants and the approaches that researchers are taking to try
and answer this elusive question.
Welcome to the final installment of "Back to the Root." This article series was written with the goal of exploring the roles that botany and plant physiology play in cannabis testing and research. Previous articles spanned multiple industry relevant topics including mechanisms of plant heavy metal uptake, pesticide interactions with plant defense systems, and the genetic and environmental factors associated with plant terpene synthesis (1–3). In this final article, I intend to draw connections from cannabis’ agricultural history to the real world problems that researchers face in attempting to resolve its speciation, determine how or if various strains differ from one another, and generally quantify and organize the genetic diversity of the genus Cannabisas a whole. In particular, we will discuss the implications that this unresolved relationship has for our understanding of the botanical import of cannabinoids and why Cannabis ssp. produce them at all.
The domestication of cannabis occurred in ancient times, some estimates as distant as tens of thousands of years ago. It’s unclear exactly when humanity first began utilizing cannabis, but archeological and cultural evidence does exist to provide some insight. The first known use of cannabis to date was as a food source (4). Cannabis fruits (see note below), which are more commonly referred to as “seeds,” were found in a kitchen midden (that is, trash pile), in Japan roughly 9000 years ago (4). [Note: Botanically speaking a fruit is a seed-bearing structure that forms from the ovary where as a seed is an embryonic plant enclosed in a protective outer covering. Cannabis fruits are classified as achenes, a simple dry fruit that contains a single seed. Simply put, this is a conversation about layers wherein the additional dry fruit layer formed from the ovary that surrounds the seed defines how we classify it botanically.] Archeologists have long understood that useful information that has the potential to resolve burning historical questions can be obtained by literally digging through someone’s trash. Estimates of when fiber use of cannabis began are roughly around the same time as use in a food context (4). Ancient texts exist that indicate the cultivation of cannabis in China has been occurring for more than 6000 years (5). There is even evidence for the first use of cannabis as an inebriant; residues found inside a censor in the Pamirs mountains of central Asia were analyzed via gas chromatography–mass spectrometry (GC–MS) and shown to contain decarboxylated cannabinoids (6). Considering the long history of cultivation and domestication of cannabis, it begs the question: What impacts might historical and present day agricultural usage have on understanding the evolutionary relationships among cannabis chemotypes?
In trying to nail down the specific speciation of Cannabis (genus), researchers have come at the problem from a variety of angles with a range of approaches. The earliest attempt was also the most classical approach: a taxonomic evaluation. Small and Cronquist made inferences regarding the speciation of Cannabis by comparing morphological traits and geographic population distributions. Based on this methodology they found that the genus Cannabis was monospecific, or consisting of a single species, Cannabis sativa (7). Within C. sativa two subspecies, ssp. sativa and ssp. indica, were identified (7). C. sativa ssp. sativa encompasses a group of generally northern, minimally intoxicant plants that were influenced by breeding for characteristics such as fiber and oil production (7). Alternatively, C. sativa ssp. indica encompasses a group of generally southern plants, with significant intoxicant qualities that were influenced by selection for intoxicant properties (7). So, what exactly does this mean? The conclusion is that C. sativa ssp. sativa and C. sativa ssp. indica are equivalent to what are colloquially referred to as “Hemp” and any of the plethora of names associated with “drug-type” cannabis (that is, recreational, marijuana, high tetrahydrocannabidiol [THC]), respectively. Furthermore, these two chemotypes are of the same species and only distinguished on the subspecies level. While this study utilized morphological characteristics to classify groups—an approach that has lost favor in the age of genetic research—it was a critical first step utilizing the best methodology available at the time; the authors findings continue to be the most widely accepted classification of Cannabis sp. to date.
In the modern era of genetics-based research it seems plausible that we would have a clearer understanding of the true species level classification of Cannabis. Several studies have attempted to clarify the relationship among members of the genus Cannabis. Hillig performed a large scale study that looked at 157 populations of cannabis and examined the genetic diversity of its deoxyribonucleic acid (DNA) at 17 unique gene locations (5). In particular, Hillig looked for variation in alleles, or alternate forms of the same gene, to better understand Cannabis speciation. Hillig was the first to propose that the genus Cannabis contained three different species: C. sativa, C. indica, and C. ruderalis (5). While the author makes a compelling case for speciation between hemp and drug-type cannabis, the evidence for the existence of a third species (C. ruderalis) is less clear due to limited sample availability, as Hillig willingly admits (5). This study is pivotal in that it essentially opened the door to what is still a hotly contested debate among botanists, taxonomists, and geneticists.
In an attempt to approach the debate from an alternative genetic angle, Gilmore and colleagues conducted a study examining genetic variation in organelle DNA (8). Organelles are essentially tiny cellular “organs” that allow cells to organize and compartmentalize various biochemical processes. Unless otherwise specified, when we think of “DNA” what we are actually thinking of is the DNA found within the nucleus of the cell, or nuclear DNA. However, two of these organelles, specifically chloroplasts and mitochondria, are unique in that they possess their own independent DNA. [Note: As a quick Biology 101 refresher: mitochondria are present in both animal and plant cells where they function as the “powerhouses” of the cell, converting sugar molecules into usable energy for cellular processes, whereas chloroplasts are found only in plant cells and are the site where photosynthesis occurs.] This is an artifact of the evolutionary history of these organelles known as Endosymbiotic Theory, which I have referenced in case there is interest but will leave off explaining for the sake of brevity. By examining the DNA present in these endosymbionts-turned-organelles, researchers can generate complimentary evolutionary and biogeographic insights to nuclear DNA; particularly because these organelles are transferred matrilineally, or from the mother to any offspring, it makes them a useful tool for understanding evolutionary relationships.
The results of this study are interesting in that very little genetic variation was discovered across 12 different mitochondrial and chloroplast DNA gene locations in cannabis (8). Using what variation they did find, Gilmore and colleagues divided their genetic data into six distinct haplotypes, or groups of genes within an organism that are inherited together from a single parent, in this case from the mother. The haplotypes were then mapped to better understand the geographic distribution of genetic variation in Cannabis organelles. A distillation of the findings showed that genetic variation found within the organelles of cannabis was largely in support of a single species concept (1,8). Markedly, the lack of genetic variation between all populations lends strong support for defining Cannabis as a monospecific genus comprising of several subspecies. The implications of these findings are that we have conflicting genetic evidence for how speciation of the genus Cannabis is to be defined.
In the age of rapid DNA replication, whole-genome sequencing and other powerful genetics techniques, the speciation of Cannabis continues to be elusive. In particular, the role that domestication has played in obscuring the origins and speciation of cannabis is significant. In particular, the loss of genetic diversity through deliberate selection for desirable traits and incidental gene flow via escaped cultivars, has led some researchers to call for the conservation of the scarce remaining wildtypes of cannabis (4). McPartland and Small, both of whom began studying cannabis starting in the 1970s, in particular are at the forefront of this call. In a recent study that combined the morphological examination of more than 1100 herbarium specimens with a meta-analysis of phytochemical and genetic data, McPartland and Small demonstrated that Cannabis (genus) contains one species.
Further, they distinguished cannabis past the subspecies level, down to variants to make distinctions between domesticated and wildtype cannabis (4). Their analysis showed that natural selection, driven by differences in climate, initiated a divergence between wildtype variants that was reinforced by selection of the respective domesticates for desirable traits (4). Another way to say this is that differences in climate were causing the wildtype variants to diverge from one another and breeding for desirable characteristics such as THC content, reinforced the divide. However, over the last 50 years hybridization of the two respective domesticates, which are equivalent to “indica” and “sativa” within the recreational cannabis market, has so thoroughly remixed the gene pool that the two domestic variants are indistinguishable from each other (4). More simply put, due to hybridization there is no longer any discernable genetic difference between “indica” or “sativa” strains.
Beyond examining the genetic divergence and recombination of the more familiar cannabis domesticates, McPartland and Small were able to identify and distinguish four wildtypes of cannabis. They present a case for the conservation of these wildtypes as reservoirs of population level genetic diversity in the genus Cannabis, which are being threatened by cross-breeding, via the two avenues mentioned previously (4). Conserving these wildtypes is critical to conserving not only genetic diversity, but also to the study of the evolutionary history of cannabis. In turn, understanding the evolutionary history of cannabis, as we will discuss for the remainder of this article, is necessary for understanding the botanical function of novel phytochemicals, namely cannabinoids.
In looking over the body of genetic analyses we can see that artificial selection, combined with gene flow between populations that might have otherwise been isolated has reduced the population level genetic diversity of cannabis. This makes it very difficult to use modern genomics techniques to answer questions regarding speciation, or even to explain differences between recreational strains. You can think of this using a deck of cards as an analogy. In the beginning all the cards are mixed together in one deck (our total gene pool). As you deal them out to different individuals the cards are distributed among the players unequally (populations). As the game progresses the cards move between individuals (gene flow), but each individual is trying to build an ideal hand (natural selection) to win the game. But what if suddenly the dealer takes all the cards back and shuffles them? This is a highly simplified model, but it gets at what large scale hybridization has done; we’ve reshuffled the cards back together again such that we can no longer tell the difference between each player’s hand. This has downstream implications for understanding the evolutionary and ecological processes that determined how and why certain novel compounds such as cannabinoids might have arisen and their ecological role in plant secondary metabolism. However, in spite of this several researchers have attempted to address the “why” of cannabinoids. That is to say, why do cannabis plants produce cannabinoids?
To date, the most cited piece of literature regarding the role of secondary metabolites present in cannabis is “Chemical Ecology of Cannabis” by Pate in 1994 (9). This paper provides a suite of hypotheses for the botanical purposes of cannabinoids, which have been largely understudied outside of their intoxicant effects. This is due in part to the former and current illicit status of cannabis, which had many downstream effects for research. Until legalization of recreational cannabis opened the door to the scientific community at large in the United States, the University of Mississippi was the only federally authorized grower of cannabis and thus had a monopoly on cannabis research (10). Elsewhere in the world, cannabis research has overwhelmingly focused on the chemistry of cannabis, in particular metabolomics, medical research, and chemical analysis. To the best of my knowledge, very little research exists that examines the botanical function of cannabinoids.
The primary hypothesis for the “why" of cannabinoids is that they are produced in response to abiotic stress. Studies have shown that drought stress increased trichome density and cannabinoid content in pollen (9). In particular, THC content increased in plants that typically produced greater quantities of cannabidiol (CBD), when they were grown in a hot, dry climate (Sudan) rather than a temperate climate (England) (9). More recently, Caplan and colleagues demonstrated that controlled drought stress increases both inflorescence dry weight and cannabinoid content (11). However, it is unknown if cannabis chemotypes will all respond similarly to a controlled drought regime, meaning that more research needs to be done on a broader swath of chemotypes before this method would become a recommended practice.
While drought stress is probably the most studied variable, there are other abiotic factors that also might trigger an uptick in cannabinoid production. Temperature has been suggested as a possible stressor, but it seems most likely that any impacts on plant physiology would be through its association with drought stress (9). Soil nutrients also have the potential to impact cannabinoid production. In particular, soil potassium is negatively correlated to cannabinoid production whereas magnesium and iron both appear to have a positive effect (9). Ultraviolet (UV) radiation has also received a fair amount of scientific attention in the past. In particular, it has been hypothesized that greater exposure to UV radiation selected for higher production of THC compared to CBD in certain wildtypes, thus driving the evolution of cannabis (9). It has been demonstrated that UV exposure results in higher THC production, suggesting a light-protective phytochemical function for the plant (9). However, another study found only minor differences in UV-B light absorbency between THC and CBD, which appears to undermine the idea that THC is basically plant “sunscreen” (9). To date it remains unclear what functional benefit enhanced THC production might have for the plant on a physiological level. In order for us to say that natural selection for a given trait has resulted in evolution, population level studies linked to genetic evidence are needed, making the call to conserve wildtype cannabis variants all the more pressing. The extreme loss of population level genetic diversity within Cannabis (genus) due to hybridization could close the door on our ever understanding the evolutionary history of this fascinating plant.
In addition to abiotic responses, it has been hypothesized that cannabinoids might function in plant defense against biotic stressors. Certainly the stickiness of cannabinoids, which are excreted via glandular trichomes on the leaf surface, lend themselves to mechanical defense against herbivores and insects in particular (9). In fact, trichomes are a well-known mechanical defense to herbivory for a broad swath of plant species. However, that does not rule out the possibility that they also possess chemically defensive properties. In the wild, cannabis is subject to few natural enemies, which could be the result of cannabinoids acting as a chemical defense. But any grower knows that insect pests can wreak havoc on a harvest. So, what exactly might be going on? It is possible that in a wild system, where cannabis plants are only exposed to natural enemies cannabinoids are chemically defensive. However, once you change the system say to a greenhouse, you have effectively changed the environment that the plant now has to cope with. Adaptations are environment specific, so changing the abiotic (light, temperature, water regime, and so on) and biotic (insect pests) conditions can change the fitness of a particular trait. If the insects that thrive in a greenhouse are not the natural enemies of cannabis, then they are potentially inappropriate for understanding the chemical ecology of cannabinoids.
So, if cannabinoids aren’t keeping the bugs at bay, might they instead function as protection against microorganisms? There is certainly evidence that shows that cannabinoids possess antimicrobial properties (9). However, we once again run up against the same questions previously mentioned. And the answer is much the same; we cannot know exactly to what extent the pests and pathogens that blight greenhouse operations are natural enemies of cannabis. Is the necessarily artificial environment of the greenhouse confounding our understanding of the ecological and physiological function of cannabinoids? This is the age old ecological debate of laboratory versus field experiments; in reality to really get a complete picture you need both controlled and observational studies.
While some of these abiotic (drought, nutrients, UV light) and biotic stressors may impact cannabinoid production little is known about the impacts they might have on cannabis plant physiology, chemical ecology, and evolution. One persistent theme throughout this series is that research exploring the mechanisms driving these physiological responses need to be conducted in cannabis (1–3). So much variation in plant stress responses exist that we cannot necessarily assume any one mechanism is at play without strong supporting evidence. In 1994, Pate called for a more ecological approach to studying cannabis that combined our knowledge of cannabis phytochemicals with the study of their function in ecosystem processes (9). Studies that explore the chemical ecology and evolutionary history of cannabis are desperately needed, if we are to ever understand exactly how and why these interesting, intoxicating, and novel compounds are produced by cannabis plants.
GWEN BODE, B.S., is an aspiring doctoral candidate and botanist with a strong chemistry background. As an undergraduate at Eastern Washington University she investigated the vitamin content of a wild edible plant via HPLC. She has since worked at the front line of the cannabis testing industry, integrating her botanical knowledge with the practical aspects of analytical testing. Direct correspondence to: firstname.lastname@example.org
G. Bode, Cannabis Science and Technology 4(1), 46-50 (2021).