Experimental Endozoochory of Cannabis sativa Achenes

John M. McPartland and Steve G. Naraine

Medical Cannabis and Cannabinoids, 2018, 1, 96–103

DOI: 10.1159/000492971

https://www.karger.com/Article/FullText/492971

Abstract :
The mechanism by which Cannabis sativa dispersed from its center of origin remains an open question. The literature provides many hypotheses, which we review for the first time, but experiments are few. Darwin was interested in zoochory – the transport of plants by animals. He demonstrated endozoochory (transport of seeds via animal digestive systems) of C. sativa achenes (seeds) by carrier pigeons, but he did not quantify achene survival rates. We assessed mammalian endozoochory in a triplicate experiment: feeding C. sativa achenes into a simulated gastrointestinal system, a dog, and a human. The in vitro system subjected achenes to sequential digestive enzymes. Achenes were planted in potting soil and monitored for emergence under growroom conditions. The in vivo experiments added achenes to a normal morning meal (dog food or granola). Feces were collected for daily instillation into an outdoor
garden and monitored for seedling emergence for 16 days. Control achenes were planted directly into soil without ingestion. In the in vitro study, 34.7% of the digested achenes emerged as seedlings. The in vivo emergence rates were 10.3, 1.3, and 76.0% for the dog, human, and control conditions. The three groups differed significantly (χ2 = 1,264.93, p < 0.0001). Achene survival was greatest under in vitro conditions,
which lacked a mastication step, compared to dog (minimal chewing) and human (maximal chewing) conditions. Although C. sativa lacks evolutionary traits for classic endozoochory (i.e., a fleshy fruit), it seems well adapted to this manner of seed dispersal.

Keywords :
Cannabis sativa · Cannabis ruderalis · Evolution · Zoochory · Long-distance dispersal

Introduction
Understanding the origin of medicinal plants and their ecological selection pressures may offer insights into their evolution of secondary metabolites. Cannabis and her sister genus Humulus diverged 27.8 million years ago [1]. Cannabis sativa has a center of origin in Central Asia [2], or more specifically the northeastern Tibetan plateau [3]. A meta-analysis of fossil pollen studies suggests C. sativa had dispersed to Europe by 1.8 million years ago [4]. The European distribution of C. sativa expanded and contracted with glacial cycles, like that of many plants. During interstadials (warmer, wetter periods, like our present time), C. sativa pollen was limited to “refugia” – steppe landscapes that persisted in otherwise forested Europe.

Conventional wisdom states that differences between C. sativa subsp. sativa (in Europe) and C. sativa subsp. indica (in Asia) are due to human selection, and therefore they are not “natural” segregates. However, European C. sativa likely went through repeated genetic bottlenecks during interstadials, when the population shrank to small numbers during range contractions. Small populations experience genetic drift, where new genotypes arise randomly. Thus, differences between European and Asian C. sativa began with vicariance and genetic drift, and not human selection [3].

The species’ migration velocity during glacial cycles is suggestive of rapid biological dispersal. Biological dispersal refers to the movement of individuals away from the population into which they were born. Dispersal has consequences for individual fitness, gene flow, population genetics, and species distribution. Plants rely on passive transport of diaspores (e.g., seeds), by vectors such as wind (anemochory), water (hydrochory), and animals (zoochory). Animals transport seeds via their digestive systems (endozoochory) or via seeds externally attached to their bodies (epizoochory).

Ever since Darwin [5], biologists have studied the roles of zoochory in the biological dispersal of plants. Darwin focused upon long-distance dispersal (LDD) to distant oceanic islands, vectored by birds, but LDD has been defined to include distances as little as 100 m, with roles played by terrestrial mammals [6]. Plants adapted for endozoochory classically have seeds embedded in the fleshy pulp of an edible fruit or berry. The fruit provides a nutritional reward to the disperser.

Plants with dry nuts or achenes, such as C. sativa, have been considered “unspecialized” because they lack classic adaptations for dispersal [7]. Nevertheless, Ridley [8] collated empirical evidence of endozoochory amongst plants with dry nuts or achenes. His examples included many agricultural weeds that passed through cattle into excreta: “we can realise at once how very many small herbaceous plants with small dry fruits and seeds in capsules are so widely spread” [8]. Seeds excreted in a germinable state explain why agricultural fields manured with cattle dung may acquire mass infestations of weed species [9].

In plants adapted for classic endozoochory, the fruit pulp often contains germination inhibitors. Animals’ digestive processes, both mechanical and chemical, strip away the pulp from the seeds. Thus, in classic endozoochory, excreted seeds often show higher percentages of germination, and/or accelerated emergence, compared to noningested seeds [9, 10]. This is not the case with dry nuts or achenes, which usually show reduced germination rates compared to noningested seeds [8–11].

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