Origin, early expansion, domestication and anthropogenic diffusion of Cannabis, with emphasis on Europe and the Iberian Peninsula

Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 Available online 1 April 2022 1433-8319/© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Origin, early expansion, domestication and anthropogenic diffusion of Cannabis, with emphasis on Europe and the Iberian Peninsula Valentí Rull Botanic Institute of Barcelona (CSIC), Pg. del Migdia s/n, 08038 Barcelona, Spain ARTICLE INFO Keywords: Cannabis Taxonomy Biogeography Evolution Domestication Dispersal Pollen ABSTRACT Cannabis is among the oldest human domesticates and has been subjected to intensive artificial (human-mediated) selection throughout history to create a wide array of varieties and biotypes for diverse uses, including fiber, food, biofuel, medicine and drugs. This paper briefly reviews the available literature on the taxonomy, evolutionary origin and domestication of this plant, as well as its worldwide dispersal, in both its wild and cultivated forms. Emphasis is placed on Europe and especially on the Iberian Peninsula. Today, it is accepted that Cannabis is a monospecific genus with two subspecies, C. sativa subsp. sativa and C. sativa subsp. indica, originating in Europe and Asia, respectively, by allopatric differentiation after geographic isolation fostered by Pleistocene glacial-interglacial cycles. Palynological and phylogeographic evidence situates the Cannabis ancestor on the NE Tibetan Plateau during the mid-Oligocene. The timing and place of domestication is still a matter of debate between contrasting views that defend single or multiple Neolithic domestication centers situated in different parts of the Eurasian supercontinent, notably central/southeastern China and the Caucasus region. Recent meta-analyses have suggested that wild Cannabis may have already been spread across Europe in the Pleistocene, and its domestication could have occurred during the European Copper/Bronze ages. According to the available reviews and meta-analyses, pre-anthropic dispersal of Cannabis into the Iberian Peninsula seems to have occurred only in postglacial times, and the earlier signs of cultivation date to the Early Medieval Ages. However, the palynological and archeological evidence used to date is insufficient for a sound assessment, and the development of thorough Iberian databases to address further meta-analysis is essential for more robust conclusions. Some clues are provided for these achievements to be fulfilled. 1. Introduction Since ancient times, Cannabis has been extensively utilized by humans for a variety of uses, such as textiles, paper, food, medicine, biofuel or recreational drugs. Cannabis has been an integral part of human life since its domestication and continues into the present (e.g., Clarke and Merlin, 2013; Fike, 2016; Gray et al., 2016a; Grotenhermen and Müller-Vahl, 2016). However, research on the uses and applications of this plant significantly decreased and almost ceased in the mid-20th century when the species and its varieties were declared illegal in most Western countries, regardless of the concentrations of psychoactive compounds (Duvall, 2014; Warf, 2014). Therefore, one of the most ancient crops, long-valued for its multiple uses, became largely ignored during the last 50 years. Thus, compared with other crop plants, Cannabis has not fully benefited from modern scientific technologies, which has created a major knowledge gap that remains to be filled (Gray et al., 2016b). The last decade has witnessed a revival of interest in Cannabis research, especially in relation to the genetic and phytochemical features of the different varieties and to its evolutionary origin, domestication and further geographic diffusion (e.g., Clarke and Merlin, 2013; Small, 2015; Gray et al., 2016b; Vergara et al., 2016). For example, the use of modern methods of molecular analysis has helped clarify the taxonomy of the Cannabis complex and its genetic modifications over time as a result of artificial (human-mediated) selection during the domestication process (Clarke and Merlin, 2016). Palynological and archeological records have been used mostly to identify the geographical center of origin of Cannabis, its center of domestication and further human-driven diffusion patterns throughout the world. Modern global and regional databases and associated handling facilities, notably geographic information system (GIS) tools, have been instrumental in this type of research (McPartland, 2020). This paper briefly reviews the newly available information on the center of origin and the center of domestication of Cannabis and its E-mail address: [email protected]. Contents lists available at ScienceDirect Perspectives in Plant Ecology, Evolution and Systematics journal homepage: www.elsevier.com/locate/ppees https://doi.org/10.1016/j.ppees.2022.125670 Received 12 January 2022; Received in revised form 21 March 2022; Accepted 30 March 2022 Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 2 further worldwide dispersal, with a focus on Europe, where spatiotemporal diffusion patterns remain controversial, and the Iberian Peninsula, which represents a knowledge gap regarding the tempo and mode of Cannabis arrival and diffusion. Previous reviews on the subject date from roughly a decade ago (Clarke and Merlin, 2013; Duvall, 2014; Warf, 2014; Small, 2015). A review including the research developed on Cannabis during the last decade, which has been fundamental for setting the present standards of knowledge, is lacking. This is especially true for the meta-analyses and molecular phylogenetic investigations developed during the last five years. The present review represents an updated outlook on the evolution, domestication and worldwide diffusion of Cannabis that includes the latest developments on this topic. The review begins with a brief section regarding the current state of Cannabis taxonomy, ecology, phenology and human uses. The next section discusses the evolutionary center of origin and the time of appearance of the original wild Cannabis, followed by similar geographic and chronological considerations on Cannabis domestication and diffusion of its cultivated forms. The next section focuses on Europe, where domestication and diffusion patterns are still under discussion. The last section highlights the scarcity of information in regard to the Iberian Peninsula, in comparison with most European regions, evaluates the potential causes for this paucity and suggests how further research could contribute to bridging this knowledge gap. Time units used: Ma, million years before present; (k)yr BP, (kilo)years before present; CE/ BCE, Common Era/Before Common Era. 2. Present-day status 2.1. Species The genus Cannabis is in the family Cannabaceae, with 11 genera and approximately 170 species. Discussions on the existence of one (C. sativa) or two species (C. sativa and C. indica) started during the time of Linnaeus and Lamarck and have continued until very recently. Some morphological differences exist between the two taxa, as C. sativa is taller with a fibrous stalk, whereas C. indica is shorter with a woody stalk. There are also phytochemical divergences, as manifested in the tetrahydrocannabinol/cannabidiol (THC/CBD) ratio, which is higher in C. indica. The discontinuous geographic ranges – the sativa lineage in Europe and the indica lineage in Asia – would also support the occurrence of two different taxa within Cannabis. The question is whether these differences qualify for defining two separate species or two subspecific taxa. Using quantitative criteria based on key molecular DNA sequences (DNA barcode), McPartland (2018) concluded that the genetic differences are in the rank of subspecies and that the proper nomenclature is C. sativa subsp. sativa and C. sativa subsp. indica. Therefore, Cannabis is now considered a monospecific genus (Barcaccia et al., 2020; Kovalchuck et al., 2020). 2.2. Biotypes A number of new varieties have been obtained from these two original subspecies by artificial selection, which have different morphologies, phytochemical compositions, geographic distributions and uses (Clarke and Merlin, 2016; Lynch et al., 2016; Rahn et al., 2016). According to Small (2015), the consequences of artificial selection make it impossible to determine if unaltered primeval or ancestral populations still exist. This author recommends that C. sativa be recognized as a single species with a narcotic subspecies (drug) and a non-narcotic subspecies (hemp), each with domesticated and ruderal varieties. A similar approach is the definition of four major biotypes of cultivated Cannabis (NLH, BLH, NLD and BLD) with morphological and chemical differences submitted to different uses, either fiber/oil or drug production (see Table 1 for more information on these biotypes). There is also a hybrid between NLD and BLD, known as sinsemilla, which is highly psychoactive (high THC and low CBD) and, hence, is used mainly as a drug. The sinsemilla cultivars were developed in the New World and diffused worldwide. In contrast to the former biotypes, this hybrid is only cultivated, with no feral escapes or ruderal populations. The current distribution of these biotypes is shown in Fig. 1. 2.3. Ecology and phenology Cannabis sativa is a sun-loving (heliotropic) species that requires well-drained and nitrogen-rich soils, warmth and moisture. Therefore, most natural populations are found seasonally across accommodating northern temperate latitudes. This plant grows well along exposed riverbanks, lakesides, margins of agricultural lands and other areas disturbed by humans. Cannabis plants are annual and usually diecious, as determined by X and Y chromosomes, and anemophylous (windpollinated). The annual cycle extends from spring (germination) to summer (fast juvenile growing) and autumn (flowering). Male plants, which are slightly taller than female plants, die shortly before pollination. Female plants ripen viable seeds just before the arrival of winter killing frosts. Seed dissemination is carried out mostly by wind or feeding birds. During germination, seeds are surrounded by bracts with hairs that produce a resinous blend of cannabinoids and aromatic compounds as secondary metabolites, which are believed to protect seeds against pests and pathogens. Cannabinols may be psychoactive (THC) or not psychoactive (CBD) for humans (Small, 2015; Clarke and Merlin, 2016). 2.4. Uses Almost all parts of the Cannabis plants are utilized for a variety of uses (Table 2). For example, stem bark and fiber are used for cordage, woven textiles, building materials, paper, animal bedding and fuel. Seeds and seed oils are used for human food, animal feed, industrial feedstock and fuel. Female flowers and seeds are used for medicine or recreational drugs. All parts of the plant, primarily bark, seeds and female flowers, are used in ritual and social activities (healing and life cycle rituals, inebriation). Cannabis populations may also be used for environmentally related activities, such as the control of soil erosion and to increase CO 2 sequestration. Esthetic and educational applications include botanical gardens and the iconic character of the plant as a symbol of a very ancient crop deeply rooted in human culture (Gray et al., 2016b). An extensive database on the traditional uses of Cannabis (CANNUSE) is available at http://cannusedb.csic.es/. See Balant et al. (2021) for detailed explanations on this database. Table 1 The four biotypes of cultivated Cannabis as defined by Clarke and Merlin (2016), following the criteria of Small (2015). See Fig. 1 for the present geographical distribution of these biotypes. Biotype Name Psychoactive THC CBD Use Origin Diffusion NLH Narrow-leaf hemp Rarely Low High Fiber/oil Europe New World BLH Broad-leaf hemp Mildly Low/Moderate High Fiber/oil East Asia Europe, New World NLD Narrow-leaf drug Very High Low/absent Drug South Asia Africa, Europe, Middle East, New World BLD Broad-leaf drug Moderately Moderate/high Moderate/high Drug Afghanistan Europe, New World V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 3 3. Time and place of origin Early attempts to identify the place of origin of wild Cannabis prior to human contact were based on the geographic distribution of its wild, cultivated and ruderal populations, combined with the known ecological requirements and reproductive strategies. However, the distribution of this plant and its biotypes/varieties is closely associated with human settlements and trade routes, and therefore, the original native range is obscured (Clarke and Merlin, 2013). In spite of this, a broad area referred to as central Asia (presently China) was proposed as the center of origin of Cannabis (Schultes, 1969; Merlin, 1972). Another, less generally accepted, possibility mentioned was south Asia (presently India). Regarding timing, accepting that the central Asian steppes were colonized by humans by 35,000 years ago (Wells, 2002; Finlayson, 2005), it has been assumed that wild Cannabis could have originated earlier. These hypotheses, however, were based on circumstantial evidence, and robust empirical evidence was lacking. This empirical evidence was provided by the fossil record and the use of time-calibrated molecular DNA phylogenies. 3.1. Fossil record The macrofossil record of Cannabis is relatively scarce and consists of only a few leaf and fruit/seed impressions with ages ranging between the Oligocene and the late Miocene (McPartland et al., 2019). However, microfossils, specifically pollen, are abundant and widespread and have commonly been utilized to reliably reconstruct the history of Cannabis. Nevertheless, the identification of Cannabis pollen deserves special attention because of its similarity with other members of the family Cannabaceae, especially Humulus (hop), a sister genus that bears contrasting ecological requirements and cultural connotations. Therefore, inaccurate identification might lead to erroneous conclusions (Rull and Vegas-Vilarrúbia, 2014). This is why different authors have used broader taxonomic categories for this pollen type, such as Cannabis-type, Cannabis/Humulus or Cannabaceae. In addition, it is unclear whether pollen from wild and cultivated Cannabis may be distinguished morphologically. Several morphological details have been suggested to be useful to differentiate Cannabis and Humulus pollen. Godwin (1967) emphasized several differential characteristics of the pore complex. Further statistical studies revealed that Cannabis pollen is generally larger than Humulus, but this character alone was not sufficient to allow reliable separation (Whittington and Edwards, 1989; Whittington and Gordon, 1987). Pollen size also seems to be an unreliable parameter to separate wild from cultivated Cannabis (review in McPartland et al., 2018). The combination of pore complexes and size seems to provide a more reliable, yet not universally accepted, identification criterion to differentiate between Cannabis and Humulus pollen (Fleming and Clarke, 1998; Mercuri et al., 2002). However, even in the case of conclusive Cannabis pollen identifications, comparisons with studies referring to this pollen type as Cannabistype, Cannabis/Humulus or Cannabaceae remain problematic, which may be a handicap for the development of meta-analyses aimed at reconstructing past biogeographic and cultural patterns. Recently, some meta-analyses have been conducted using different criteria, such as considering the entire Cannabis/Humulus complex or taking into account Fig. 1. Worldwide distribution of the different Cannabis biotypes developed by humans (Table 1). Hemp biotypes are in green and drug biotypes in red. NLH, narrowleaf hemp; BLH, broad-leaf hemp; NLD, narrow-leaf drug; BLD, broad-leaf drug; NLD/BLD, hybrid between NLD and BLD; PA?, putative cannabis ancestor. Redrawn from Fig. 2 of Clarke and Merlin (2016). Table 2 Examples of the variety of uses of the different parts of the C. sativa plants. Modified from Clarke and Merlin (2016). Plant parts Use category Material type or benefits Stem bark Cordage Long cellulose fibers Stem fiber Cordage, woven textiles, building materials Long cellulose fibers, concrete reinforcement Wood/bark Paper, building materials, animal bedding, fuel Long/short cellulose fibers, chip board, concrete matrix, heat, light Female flowers/ seeds Medicinal Herbal remedies, pharmaceuticals, nutraceuticals Female flowers and associated resin glands Recreational drugs Marijuana, hashish Seeds (oil) Human food, industrial feedstock, fuel Proteins/essential fatty acids, paint/plastic manufacture Seeds (cake), foliage Animal feed Proteins and essential fatty acids All parts Ritual and social Healing, life cycle rituals, inebriation Plant-people interplay Esthetic Intrinsic beauty of the plant Genus Educational Iconic example of an economic plant and its ancient human relationships V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 4 only those studies that explicitly identified Cannabis pollen (Clarke and Merlin, 2013; Long et al., 2017). These approaches tend to overestimate or underestimate the actual pollen record of Cannabis. A different approach, called here the assemblage approach, has recently been proposed by McPartland et al. (2018). These authors noted that wild C. sativa is typical of open temperate steppe habitats dominated by grasses, chenopods and Artemisia, whereas Humulus is a vine plant that requires trees to climb and is common in temperate deciduous forests dominated by alder (Alnus), willow (Salix) and poplar (Populus). A third assemblage corresponds to cultivated Cannabis, which is usually found together with cultivated cereals such as Avena (oats), Hordeum (barley), Secale (rye) and Triticum (wheat), as well weeds such as Centaurea (cornflowers) or Scleranthus (knawels) species. Using these phytosociological affinities, McPartland et al. (2018) attributed the Cannabis-like pollen types recorded in the literature to wild Cannabis if this pollen occurred together with steppe assemblages; to cultivated Cannabis when it was part of crop assemblages; or to Humulus if the dominant pollen assemblage corresponded to temperate deciduous forests. Based on these premises, these authors developed a more complicated identification algorithm that also considers the relationship between arboreal (AP) and non-arboreal (NAP) pollen (Fig. 2). Using these criteria, the oldest known pollen compatible with Cannabis was found in 19.6 Ma-old (early Miocene) rocks from the NE Tibetan Plateau (presently Ningxia, China), which was proposed as the center of origin of Cannabis (McPartland et al., 2019). Interestingly, this proposal roughly coincides with the former hypothesis based on indirect biogeographic evidence. 3.2. Calibrated DNA phylogenies The use of DNA molecular phylogenies calibrated with fossils of related genera such as Humulus, Celtis, Morus and Ficus (Fig. 3) allowed to estimate the age of divergence of Cannabis and Humulus to 27.8 Ma (mid-Oligocene). Using the same DNA phylogeny and the associated molecular clock, the divergence between C. indica (or C. sativa subsp. indica) and C. sativa (C. sativa subsp. sativa) would have occurred in the Middle Pleistocene approximately 1 Ma (McPartland, 2018). These authors noted that there is a gap of ca. 8 million years between the age of origin estimated by the molecular clock and the first fossil pollen encountered. In spite of this, they favored the mid-Oligocene age for the origin of Cannabis on the NE Tibetan Plateau (Fig. 4), assuming that, as demonstrated by the presence of Artemisia and other steppe elements, the region was covered by this type of vegetation, which would have been particularly well suited for the development of Cannabis. A previous study using Bayesian calibration estimated the divergence between Cannabis and Humulus to have occurred 21 Ma (Zerega et al., 2005), which is closer to the age (19.6 Ma) of the first fossil pollen evidence mentioned above (McPartland et al., 2019). 3.3. Pre-anthropic expansion According to pollen and seed fossil records, Cannabis would have experienced some expansion from its center of origin to Europe and East Asia well before the evolutionary appearance of the genus Homo (Fig. 4). The suggested dispersal agents are water (hydrochory) and animals (zoochory) (McPartland, 2018; McPartland et al., 2019). The first expansions occurred to the west (eastern Europe) and the east (north-- eastern China) during the Miocene-Pliocene. Most parts of the Asian continent were colonized by Cannabis during the Pleistocene (the last 2.6 Ma), before the onset of the Neolithic, when humans domesticated the first plants. During the Pleistocene, glacial-interglacial recurrence could have contributed to Cannabis diversification without human intervention. It has been suggested that Cannabis underwent recurrent range contractions (glacials) and expansions (interglacials) that facilitated allopatric processes, possibly leading to the differentiation between the European (C. sativa subsp. sativa) and Asian (C. sativa subsp. indica) subspecies, which would have diverged nearly 1 Ma (Clarke and Merlin, 2013; McPartland, 2020). The first is considered the putative hemp ancestor (PHA), and the second is the putative drug ancestor (PDA) (Clarke and Merlin, 2013). 4. Domestication and diffusion In addition to fossils and DNA phylogenies, archeological evidence is of paramount importance to reconstruct domestication and anthropogenic diffusion trends within Cannabis. The main types of evidence of Cannabis from archeological sites are pollen, seeds, fibers, fiber/seed impressions, carbonized remains, phytoliths and chemical remains. During the historical period, written and graphic documents are also of fundamental help (Clarke and Merlin, 2013). Pollen identification has some additional clues related to its abundance in sediments, as Cannabis produces much more pollen than Humulus, which is usually underrepresented (Lewis et al., 1983). This is especially useful in cases of very high percentages of this pollen type, which are difficult to explain unless the sediments come from a former hemp-retting site. Indeed, when flowering male hemp plants are soaked in a retting pond to separate the fibers from the stalk, large quantities of pollen settle into pond sediments. In these situations, the percentages of Cannabis pollen in sediments may reach 80–90% of the total, but percentages over 15% or 25% have been considered sufficient to infer hemp retting (Peglar, 1993; Mercuri et al., 2002; Lavrieux et al., 2013; Demske et al., 2016). The anemophylous pollen of Cannabis may be transported long distances; therefore, the finding of a few grains or their scattered occurrence throughout a stratigraphic section is not necessarily evidence for the local presence of the parent plant. Recent developments in molecular DNA analytical methods have increased the probability of identifying Cannabis sativa in lake sediments and comparing these results with the abundance of Cannabis/Humulus pollen. For example, in lake sediments from the French Alps, Giguet-Covex et al. (2019) found a good agreement between DNA and pollen records for the period 1500–1000 yr BP but significant inconsistencies for the periods 2000–1500 yr BP and 500 yr BP-present. During these periods, the Cannabis/Humulus pollen attained values of 10–15% and DNA was undetectable, which could be attributed to the dominance of Humulus pollen in the pollen record. 4.1. Domestication center(s) The debate regarding the Cannabis center of domestication has paralleled the taxonomic controversy on Cannabis species that, as seen before, was already active by the time of Linnaeus and Lamarck. See, for example, Clarke and Merlin (2013, 2016) and Small (2015) for more details on this long-standing discussion. Whether single or multiple Fig. 2. Algorithm developed to differentiate between Cannabis and Humulus pollen in sedimentary records, using the assemblage approach. As explained in the text, crop pollen usually includes cereal and weed pollen. AP, arboreal pollen; NAP, non-arboreal pollen; PAC, steppe assemblage (Poaceae-ArtemisiaChenopodiaceae); ASP, forest assemblage (Alnus-Salix-Populus). Original figure based on Fig. 1 of McPartland et al. (2018). V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 5 centers of Cannabis domestication occurred has profound implications for the tempo and mode of Cannabis diffusion in Eurasia, where the plant evolved and was domesticated. Some authors have proposed that Cannabis was domesticated in Central Asia (presently Mongolia and northern China) by 12,000 BCE (ca. 10,000 yr BP), which would place Cannabis among the oldest human domesticates (Warf, 2014). A recent genome-wide phylogeographic study supports a unique center of domestication in east Asia (China), from which all biotypes would have emerged and dispersed throughout the world (Ren et al., 2021). According to this analysis, early domesticated ancestors of hemp and drug types diverged from wild Cannabis ca. 12,000 yr BP, which indicates that the species had already been domesticated by early Neolithic times. Among the defenders of the single center hypothesis, Small (2015) suggested that Cannabis was domesticated in the northern Caspian Sea and that the four different domesticated groups were transported to other parts of the world during the last millennium. These four groups coincide with the abovementioned biotypes NLH (Europe), BLH (E Asia), NLD (South-Central Asia) and BLD (Afghanistan). Other authors propose two centers of domestication, one in the Caucasus (between the Caspian and the Black Sea), where the ancestor of hemp fiber domesticated biotypes (PHA) evolved, and another in south-eastern China, the place where the precursor of narcotic domesticates (PDA) originated (Fig. 5). The idea of at least two domestication centers situated in Europe and Asia is supported by archaeobotanical studies using pollen, seeds and fibers (Long et al., 2017; McPartland et al., 2018). 4.2. Anthropogenic diffusion The human-mediated diffusion of Cannabis outside Eurasia occurred relatively recently and is well documented historically. After a thorough review of the available archeological and historical evidence, Clarke and Fig. 3. Time-calibrated molecular DNA phylogenetic tree used for the estimation of the divergence rates within the Cannabaceae and some related families (molecular clock). Red dots are the nodes used for calibrating the phylogenetic tree using Humulus, Celtis, Morus and Ficus fossils of known age. Numbers are ages in million years before present (Ma). Redrawn from Fig. 2 of McPartland (2018). Fig. 4. Center of origin (red dot) and pre-anthropic dispersal of Cannabis, based on fossil pollen data and the use of the assemblage algorithm explained above (Fig. 2). Dot colors indicate the age of first occurrences of Cannabis pollen (see legend), in million years before present (bold numbers) and in thousand years before present (normal numbers). Original figure based on raw data from McPartland et al. (2019). V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 6 Merlin (2013) subdivided anthropogenic diffusion of Cannabis dispersal beyond Eurasia into six phases: 1) primary dispersal across Eurasia (10, 000–2000 yr BP), 2) spread into Africa and SE Asia (2000–500 yr BP), 3) diffusion into the Americas from Europe (1545–1800 CE), 4) diffusion to the Americas from Europe and Asia (1800–1945), 5) expansion after World War II (1945–1990), and 6) proliferation of industrial hemp (1990 CE to present). In the first phase, Paleolithic nomadic peoples – who were expanding their range during the postglacial ice retreat – could have contributed, either consciously or accidentally, to the dispersal of the wild hemp (PHA) and drug (PDA) precursors from their corresponding distribution areas located in the Caucasus region and southeastern China, respectively (Fig. 5). This was the onset of differentiation of the four major cannabis biotypes. NLH evolved from the PHA via an intermediate form Fig. 5. The first two phases of human-mediated Cannabis diffusion. As in Fig. 1, hemp biotypes are indicated by green letters and drug biotypes are in red letters. Original figure based on Maps 10 and 11 from Clarke and Merlin (2013). V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 7 known as the NLH ancestor (NLHA), whereas NLD and BLD originated from the PDA through the intermediate ancestral form called the NLDA. BLH could also have evolved from isolated PDA populations in northeast China. The second phase coincided with the expansion of the Arab Empire into Africa and the Indian Empire into SE Asia. These cultures used Cannabis mainly as a drug and introduced the NLD biotype in their dominions, primarily through adventurers and traders. After the initial introductions, migrants and traders spread the NLD biotype across much of Africa and Asia, whereas the hemp biotype (NLH) remained restricted to Europe until the colonization of the Americas. Diffusion into the New World (phase 3) occurred during European colonization and consisted mainly of the production of hemp (NLH) for cordage, cloth and seed (Fig. 6). It is speculated whether Vikings could have carried hemp to North America in pre-Columbian times, but there is no evidence for a sound assessment. In addition to domestic uses, hemp cultivation was stimulated by European governments of the colonizing countries to provide their sailing ships with ropes and sails. During this phase (1545–1800 CE), drug biotypes remained in the Old World, where the African expansion of NLD continued. It was not until the middle 1800s CE (phase 4) that Asian hemp (BLH) was introduced in North America through the Pacific Ocean (Fig. 6) to replace the inferior European hemp (NLH) in terms of fiber quality. The first psychoactive biotypes (NLD) were introduced from India into the Americas (mainly Latin America) by indentured laborers following the abolition of slavery in 1834. After World War II (1945–1949), Cannabis cultivation and use were prohibited at the international level, which signified the end of legal diffusion of this plant and initiated the fifth spreading phase (Fig. 6). The illegal traffic of marijuana (NLD) for recreational use did not stop but has experienced a significant increase since the 1960s. Most producers of clandestine Cannabis were from Africa, Asia and Latin America, which inadvertently exported seeds to the main consuming centers from Europe and North America, where local cultivation attempts began. In the 1970 s, Asian varieties of BLD were introduced into North America and western Europe, where the cultivation of NLD ×BLD hybrids flourished. During the last three decades (phase 6), Cannabis prosecution has generated new cultivation and dispersal modes to avoid legal detection. At the same time, renewed economic interest in hemp fiber has been sparked, which has promoted the legal industrial cultivation of hemp. Medical applications have also increased by artificial selection toward varieties with increased THC and other cannabinoids. New varieties have been created by hybridization of NLH and BLH biotypes aimed at expanding hemp cultivation to equatorial areas where European NLH does not grow well. Regarding drugs, the production of NLD/BLD hybrids has also increased and disseminated worldwide, and vegetative indoor cultivation of these hybrids has proliferated. As a result, the global genetic diversity of both hemp and drug forms experienced a significant reduction in comparison with the 1970s and 1980s, when the different biotypes were cultivated by traditional farmers in isolated geographic habitats. Fig. 6. Phases three to six of anthropogenic cannabis expansion. As in Fig. 1, hemp biotypes are indicated by green letters and drug biotypes are in red letters. Original figure based on Maps 12, 13 and 14 from Clarke and Merlin (2013). V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 8 5. Cannabis in Europe According to the single center of domestication hypothesis, European Cannabis would have been carried by humans from Asia already in a cultivated form. However, the finding of Pleistocene Cannabis pollen across Europe suggests that this plant would had arrived to this continent before its domestication, and therefore, the plant could have also been domesticated in Europe. Using the assemblage approach for the identification of C. sativa pollen explained above (Fig. 2), McPartland et al. (2018) performed a meta-analysis on nearly 480 sites and demonstrated that this pollen was widespread across Europe during the Pleistocene prior to human agency (Fig. 7). According to the same authors, no evident signs of Cannabis cultivation have been found either in the pollen or in the archeological record for most of the European Neolithic. The first palynological evidence for potential Cannabis cultivation appeared in present-day Bulgaria during the late Neolithic-Copper age and expanded during the Bronze and Iron ages (Fig. 7). Cannabis cultivation expanded across the entire continent between 2000 and 800 yr BP during the Roman Empire and the Early Middle Ages. These results were supported by a further meta-analysis of archeological evidence (textiles, cordage, fiber, seeds, pottery impressions and phytoliths) from almost 140 sites (McPartland and Hegman, 2018). These authors concluded that Cannabis dispersed from Asia to Europe in its wild form during the Pleistocene and was domesticated in situ during the Copper and Bronze ages. This challenges the single-site domestication hypothesis, according to which Cannabis would have been domesticated in Asia and expanded worldwide as a cultivated plant. The possibility of an European domestication of Cannabis is consistent with the former view of Clarke and Merlin (2013), who proposed that the precursor of the European NLH biotype – the wild PHA, related to C. sativa subsp. sativa – would have been centered in the Caucasus region and expanded to western Eurasia and eastern Europe during the Pleistocene (compare Fig. 7 with Fig. 5, phase 1). Chronologically, this possibility also seems plausible, as C. sativa subsp. sativa would have diverged from its Asian sister C. sativa subsp. indica in the Middle Pleistocene, approximately 1 Ma (McPartland, 2018), and the European records of wild Cannabis are younger than this divergence. In addition, the ages of these wild European records show consistent westward and southward dispersal patterns during the Middle and Late Pleistocene, with younger postglacial records situated in the west and south (Fig. 7). Notably, the European domestication of Cannabis (7–5 kyr BP) estimated Fig. 7. Pleistocene (upper panel) and Holocene (lower panel) European pollen records consistent with Cannabis, according to the assemblage identification approach (Fig. 2). Note that, in the Pleistocene panel, post-glacial ages have been differentiated from the rest of Late Pleistocene ages. PHA is the precursor of the European Cannabis according to Clarke and Merlin (2013); compare with Fig. 5, phase 1. Ages in million years before present (bold numbers) and in thousand years before present (normal numbers). Original figure based on raw data from McPartland et al. (2018). V. Rull Perspectives in Plant Ecology, Evolution and Systematics 55 (2022) 125670 9 from pollen and archeological records (McPartland et al., 2018; McPartland and Hegman, 2018) occurred after the European Neolithic and much later than Asian domestication (12 kyr BP), as estimated by calibrated phylogenetic trees (Ren et al., 2021). 6. The Iberian Peninsula The Iberian Peninsula (hereafter IP) is a key biogeographic region due to its high biodiversity and endemism levels, along with its transitional character between contrasting biogeographical regions, which results in a very peculiar biota in the European context. The IP is one of the main centers of Mediterranean plant diversity, together with the Anatolian, the Balkan and the Italian peninsulas (M´ edail and Quetzel, 1997). These peninsulas have had a fundamental role not only as biodiversity cradles but also as glacial refugia, especially during the Last Glacial Maximum (LGM) (Hewitt, 1999). It has been estimated that roughly a quarter of the LGM refugia have been located in the IP and the Balearic Islands (M´ edail and Diadema, 2009). The present Iberian vascular flora is especially rich – 6276 species (739 non-native), distributed in 1278 genera and 189 families – and shows a high degree of specific endemism (~ 23% of the native flora). This represents more than 50% of the European flora. The main Iberian centers of diversity and endemism are situated in the mountain ranges and the richest groups are the Compositae, the Leguminosae and the Gramineae, whereas the Gymnosperms and the Pteridophytes are the less represented (Aedo et al., 2017). Traditionally, the IP has been subdivided into two major biogeographical domains characterized by contrasting bioclimatic features: the Mediterranean and the Eurosiberian regions. The Mediterranean region is characterized by the occurrence of Mediterranean macrobioclimates, with at least two consecutive arid months during the warmest season (summer), and dominates most of the IP (Fig. 8). The Eurosiberian region is restricted to the N and NW sectors of the peninsula (~ 20% of the total surface) and is characterized by wet Temperate macrobioclimates, without a period of two or more consecutive with summer aridity (Rivas-Martínez et al., 2017). The Mediterranean region is dominated by species distributed across southern Europe and northern Africa, whereas the Eurosiberian region is characterized by species distributed across central and northern Europe (Aedo et al., 2017). Land use is also strongly influenced by bioclimatic and biogeographic patterns. In the Eurosiberian region, land use is comparable to the rest of Atlantic Europe, where the traditional agrarian economy is based on cattle raising for milk and meat, combined with small-scale crops such as maize, potatoes and other vegetables, as well as fruits such as apples and chestnuts. The resulting landscape is a mosaic of small fields, woodlands and pastures, combined with larger areas of heathlands, forests and grasslands, especially in higher elevations. The Mediterranean Iberia is dominated by a totally different landscape resulting from land-use practices adapted to the occurrence of the climatic summer drought. In these conditions, crops not needing irrigation are favored. Annual crops, especially cereals, largely dominate the central IP, which is characterized by huge medium-elevation flat terrains. In the southern half, where climates are not so cold, typical Mediterranean crops such as vineyards and olive grows dominate the landscape. In the Mediterranean region, extensive crops are the norm and animal husbandry occupies a subordinate position, except in some low-mountain areas, where extensive parkland landscapes dominate (Loidi, 2017). The Mediterranean biome and its climatic features are better suited than its Eurosuberian counterpart for the growth of Cannabis (Section 2.3). The oldest human remains found in the Iberian Peninsula date from approximately 1.2 Ma (Early Pleistocene) and correspond to Homo antecessor, an autochthonous species related to H. erectus (Bermúdez de Castro et al., 1997). The Neanderthals entered the IP by 200 kyr BP (Early Paleolithic) and the modern humans did the same by 40 kyr BP. The Neolithization of the IP began at approximately 5500 BCE (~ 7500 yr BP), when the former hunter-gatherer nomadic societies were replaced by more stable agricultural-based cultures (García-Martínez de Lagr´ an, 2015). During the Bronze Age (2700–800 BCE) and the Iron Age (800–70 BCE), a diversity of cultures developed on the IP, which were influenced by Indo-European migrations, from the north (first millennium BCE) and the Phoenicians, arriving by the Mediterranean coasts during the 11th century BCE. The autochthonous Iberian culture fully developed during the 7th-6th centuries BCE but colonizations and invasions from the Mediterranean area continued with the Greeks (8th century BCE), the Carthaginians (5th century BCE) and the Romans, who arrived by 220 BCE and occupied the IP for almost 600 years. After a transitional phase in which the IP was occupied by the Germanic Fig. 8. Map of the Iberian Peninsula indicating the location of the sites included in the available reviews and meta-analyses (Clarke and Merlin, 2013; McPartland and Hegman, 2018; McPartland et al., 2018). The Eurosiberian bioclimatic region is in green and the Mediterranean region in yellow. The numbers in brackets above the site names are the age of the first appearance of the Cannabis/Humulus pollen, in kyr BP. Original figure based on raw data from the following references: Alcúdia (Burjachs et al., 1994); Andorra (Ejarque et al., 2010); Antas (Pantale´ on-Cano et al., 2003); Algendar (Yll et al., 1997); Banyoles (P´ erez-Obiol and Juli` a, 1994); Coll del Moro (Alonso and Juan, 1994); Estanya (Riera et al., 2004); La Roya (Allen et al., 1996); Montcort` es (Rull et al., 2011); Rascafría (Franco-Mújica et al., 1998); Somolinos (Curr´ as, 2012); Totana (L´ opez, 1988). V. Rull