cannabis rasta and confusion with cannabis taxonomy

Genetic evidence for speciation in Cannabis (Cannabaceae)
Karl W. Hillig
Department of Biology, Indiana University, Bloomington, IN, USA; Current address: 1010 Saratoga Road,
Ballston Lake, NY 12019, USA Received 7 January 2003; accepted in revised form 28 June 2003
Key words: Allozyme, Cannabis, Evolution, Genetics, Origin, Taxonomy

Abstract
Sample populations of 157 Cannabis accessions of diverse geographic origin were surveyed for allozyme variation at 17 gene loci. The frequencies of 52 alleles were subjected to principal components analysis. A scatter plot revealed two major groups of accessions. The sativa gene pool includes fiber/seed landraces from Europe, Asia Minor, and Central Asia, and ruderal populations from Eastern Europe. The indica gene pool includes fiber/seed landraces from eastern Asia, narrow-leafleted drug strains from southern Asia, Africa, and Latin America, wide-leafleted drug strains from Afghanistan and Pakistan, and feral populations from India and Nepal. A third putative gene pool includes ruderal populations from Central Asia. None of the previous taxonomic concepts that were tested adequately circumscribe the sativa and indica gene pools.
A polytypic concept of Cannabis is proposed, which recognizes three species, C. sativa, C. indica and
C. ruderalis, and seven putative taxa.
Abbreviations: PCA – principal components analysis

Introduction
Cannabis is believed to be one of humanity’s oldest cultivated crops, providing a source of fiber, food, oil, medicine, and inebriant since Neolithic times
(Chopra and Chopra 1957; Schultes 1973; Li 1974; Fleming and Clarke 1998). Cannabis is normally a dioecious, wind-pollinated, annual herb, although
plants may live for more than a year in subtropical regions (Cherniak 1982), and monoecious plants occur in some populations (Migal 1991). The indigenous range of Cannabis is believed to be in Central Asia, the northwest Himalayas, and possibly extending into China (de Candolle 1885; Vavilov 1926;
Zhukovsky 1964; Li 1974). The genus may have two centers of diversity, Hindustani and European–Siberian (Zeven and Zhukovsky 1975). Cannabis
retains the ability to escape from cultivation and return to a weedy growth habit, and is considered to be only semi-domesticated (Vavilov 1926;
Bredemann et al. 1956). Methods of Cannabis cultivation are described in the ancient literature of China, where it has been utilized continuously for at least six thousand years (Li 1974). The genus may have been introduced into Europe ca. 1500 B.C. by nomadic tribes from Central Asia (Schultes 1970). Arab traders may have introduced Cannabis into Africa, perhaps one to two thousand years ago (Du Toit 1980). The genus is now distributed worldwide from the equator to about 60
N latitude, and throughout much of the southern hemisphere. Cannabis cultivated for fiber and/or achenes (i.e., ‘seeds’) is herein referred to as ‘hemp.’ Cannabis breeders distinguish eastern Asian hemp from the common hemp of Europe (Bo´csa and Karus 1998;
de Meijer 1999). Russian botanists recognize four ‘eco-geographical’ groups of hemp: Northern, Genetic Resources and Crop Evolution 52: 161–180, 2005. # Springer 2005 Middle-Russian, Southern, and Far Eastern (Serebriakova and Sizov 1940; Davidyan 1972). The Northern hemp landraces are smaller in stature and earlier maturing than the landraces from more southerly latitudes, with a series of overlapping gradations in phenotypic traits between the Northern, Middle-Russian, and Southern types. The Far-east Asian hemp landraces are most similar to the Southern eco-geographical group (Dewey
1914). Two basic types of drug plant are commonly distinguished, in accord with the taxonomic concepts of Schultes et al. (1974) and Anderson (1980): the narrow-leafleted drug strains and the wide-leafleted drug strains (Cherniak 1982; Anonymous 1989; de Meijer 1999).
The taxonomic treatment of Cannabis is problematic. Linnaeus considered the genus to consist of a single undivided species, Cannabis sativa L.
Lamarck (1785) determined that Cannabis strains from India are distinct from the common hemp of Europe, and named the new species C. indica Lam. Distinguishing characteristics include more branching, a thinner cortex, narrower leaflets, and the general ability of C. indica to induce a state of
inebriation. Opinions differ whether Lamarck adequately differentiated C. indica from C. sativa, but they are both validly published species. Other species of Cannabis have been proposed (reviewed in Schultes et al. 1974; and Small and Cronquist 1976), including C. chinensis Delile, and C. ruderalis
Janisch. Vavilov (1926) considered C. ruderalis to be synonymous with his own concept of C. sativa L. var. spontanea Vav. He later recognized wild Cannabis populations in Afghanistan to be distinct from C. sativa var. spontanea, and named the new taxon C. indica Lam. var. kafiristanica Vav. (Vavilov and Bukinich 1929). Small and Cronquist (1976) proposed a monotypic treatment of Cannabis, which is a modification of the concepts of Lamarck and Vavilov. They reduced C. indica in rank to C. sativa L. subsp. indica (Lam.) Small and Cronq. and differentiated it from C. sativa L. subsp. sativa, primarily on the
basis of ‘intoxicant ability’ and purpose of cultivation. Small and Cronquist bifurcated both subspecies into ‘wild’ (sensu lato) and domesticated varieties on the basis of achene size, and other achene characteristics. This concept was challenged by other botanists, who used morphological traits to delimit three species: C. indica, C. sativa, and C. ruderalis (Anderson 1974, 1980; Emboden 1974; Schultes et al. 1974). Schultes et al. and Anderson narrowly circumscribed C. indica to include relatively short, densely branched, wide-leafleted strains from Afghanistan. The differences of opinion between taxonomists supporting monotypic and polytypic concepts of Cannabis have not been resolved (Emboden 1981).
Few studies of genetic variation in Cannabis have been reported. Lawi-Berger et al. (1982) studied seed protein variation in five fiber strains and five drug strains of Cannabis, and found no basis for discriminating these predetermined groups. de Meijer and Keizer (1996) conducted a more extensive investigation of protein variation in bulked seed lots of 147 Cannabis accessions, and on the basis of five variable proteins concluded that fiber cultivars, fiber landraces, drug strains, and wild or naturalized populations could not be discriminated. A method that shows greater promise for taxonomic investigation of Cannabis is random amplified polymorphic DNA (RAPD) analysis.
Using this technique, Cannabis strains from different geographic regions can be distinguished (Faeti et al. 1996; Jagadish et al. 1996; Siniscalco Gigliano
2001; Mandolino and Ranalli 2002), but the number and diversity of accessions that have been analyzed in these investigations are too small to provide a firm basis for drawing taxonomic inferences.
Allozyme analysis has proven useful in resolving difficult taxonomic issues in domesticated plants (Doebley 1989). Allozymes are enzyme variants that have arisen through the process of DNA mutation. The genetic markers (allozymes) that are commonly assayed are part of a plant’s primary metabolic pathways, and presumed neutral to the effects of human selection. Through allozyme analysis, it is possible to discern underlying patterns of variation that have been outwardly obscured by the process of domestication. Because these genetic markers are cryptic, it is necessary to associate allozyme frequencies with morphological differences in order to synthesize the genetic data into a
formal taxonomic treatment (Pickersgill 1988).
Other types of biosystematic data may be included in the synthesis as well.
The purpose of this research is (1) to elucidate underlying genetic relationships among Cannabis accessions of known geographic origin, and (2) to assess previous taxonomic concepts in light of the 162 genetic evidence. The research reported herein is part of a broader systematic investigation of morphological, chemotaxonomic, and genetic variation in Cannabis, which will be reported separately.

Materials and methods
The Cannabis germplasm collection
A diverse collection of 157 Cannabis accessions of known geographic origin was obtained from breeders, researchers, genebanks, and law enforcement
agencies (Table 1). Each accession consisted of an unspecified number of viable achenes. Many of the landraces that were studied are no longer cultivated, and exist only in germplasm repositories.
Sixty-nine accessions were from hemp landraces conserved at the N.I. Vavilov Institute of Plant Industry (VIR) in Russia (Lemeshev et al. 1994).
Ten accessions were from Small’s taxonomic investigation of Cannabis (Small and Beckstead 1973; Small et al. 1976). Thirty-three accessions were from de Meijer’s study of agronomic diversity in Cannabis (de Meijer and van Soest 1992; de Meijer 1994, 1995; de Meijer and Keizer 1996). The accessions from Afghanistan were obtained from Cannabis breeders in Holland, and at least three of these strains (Af-4, Af-5, Af-9) are inbred (Anonymous 1989). Six Asian accessions were collected from extant populations, including a drug
landrace from Pakistan (Pk-1), three feral populations from India (In-2, In-3, In-5), and fiber landraces from India (In-4) and China (Ch-4).
Accession Ch-4 was collected in Shandong Province from seed propagated on the island of Hunan (Clarke 1995). Five accessions from Central Asia were collected from roadsides and gardens in the Altai region of Russia, and identified by the provider as C. ruderalis. Several weedy accessions from Europe were identified as C. ruderalis, ‘ssp. ruderalis,’ or ‘var. spontanea.’

A priori grouping of accessions
The accessions were assigned to drug or hempplantuse groups, or ruderal (wild or naturalized) populations as shown in Table 1. They were also assigned
to putative taxa according to the concepts of Lamarck (1785), Delile (1849), Schultes et al. (1974) and Anderson (1980), and Small and Cronquist (1976), based on morphological differences, geographic origin, and presumed reason for cultivation. Not all of the accessions could be unambiguously assigned to a taxon for each concept. To depict the various groups of interest, bivariate density ellipses were drawn on the PC scatter plot. Aprobability value of 0.75 was chosen because at this value the ellipses encompass the majority of accessions in a given group, but not the outliers.

Allozyme analysis
An initial survey was conducted to identify enzymes that produce variable banding patterns in Cannabis that can be visualized and interpreted reliably(WendelandWeeden1989). Eleven enzymes encoded at 17 putative loci were selected for a genetic survey of the entire Cannabis germplasm collection. Previously published methods of starch gel electrophoresis and staining were employed (Shields et al. 1983; Soltis et al. 1983; Morden et al.
1987; Wendel and Weeden 1989; Kephart 1990). Gel/electrode buffer systems
Three gel/electrode buffer systems were utilized. A Tris–citrate buffer system (modified from Wendel and Weeden 1989) was used to resolve aconitase
(ACN), leucine aminopeptidase (LAP), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucoisomerase (PGI), phosphoglucomutase
(PGM), and shikimate dehydrogenase (SKDH). A lithium–borate buffer system
(modified from Soltis et al. 1983) was used to resolve hexokinase (HK) and triosephosphate isomerase (TPI). A morpholine–citrate buffer system (modified fromWendel and Weeden 1989) was used to resolve LAP, malate dehydrogenase (MDH), ME, PGI, PGM, and an unknown enzyme (UNK) that appeared on gels stained for isocitrate dehydrogenase (IDH). IDH could not be
interpreted reliably, and was not used in the analysis.
A phosphate buffer (modified from Soltis et al. 1983) was used for enzyme extraction.

Electrophoresis and staining
For both the Tris–citrate and morpholine–citrate buffer systems, 5-mm thick gels were held at 30 mA, 163
Table 1. Passport data for the 157 Cannabis accessions examined.
Origin ID n Region/name Use Parallel ID Source Taxon
Afghanistan Af-1 10 Drug 891383b CPRO C. ind.j; ind. ind.k
Afghanistan Af-2 12 Ghazni Drug 91-100c AMSRS C. ind.j; ind. ind.k
Afghanistan Af-3 15 ‘Afghani No. 1’ Drug AMSRS C. ind.j; ind. ind.k
Afghanistan Af-4 10 ‘G13’ Drug SB C. ind.j; ind. ind.k
Afghanistan Af-5 10 ‘Hash Plant’ Drug 921199b SB C. ind.j; ind. ind.k
Afghanistan Af-6 9 ‘Heavily High’ Drug M 40 SSSC C. ind.j; ind. ind.k
Afghanistan Af-7 10 Mazar i Sharif Drug 921200b SB C. ind.j; ind. ind.k
Afghanistan Af-8 10 Drug BPDIN C. ind.j; ind. ind.k
Afghanistan Af-9 10 ‘N. Lights 1’ Drug SB C. ind.j; ind. ind.k
Afghanistan Af-10 10 Afghan mix Drug SB C. ind.j; ind. ind.k
Armenia Ar-1 8 Hemp VIR 472d VIR C. sat.i,j; sat. sat.k
Armenia Ar-2 9 Hemp VIR 482d VIR C. sat.i,j; sat. sat.k
Belorus Br-1 10 Hemp VIR 296d VIR C. sat.i,j; sat. sat.k
Bulgaria Bg-1 10 ‘Lovrin 110’ Hemp 883173b CPRO C. sat.i,j; sat. sat.k
Bulgaria Bg-2 10 Silistrenski Hemp 901107b CPRO C. sat.i,j; sat. sat.k
Bulgaria Bg-3 9 Hemp VIR 73d VIR C. sat.i,j; sat. sat.k
Bulgaria Bg-4 7 Hemp VIR 335d VIR C. sat.i,j; sat. sat.k
Bulgaria Bg-5 4 Hemp VIR 369d VIR C. sat.i,j; sat. sat.k
Bulgaria Bg-6 4 Hemp VIR 370d VIR C. sat.i,j; sat. sat.k
Cambodia Cm-1 10 Drug No. 154a SMALL C. ind.i; C. sat.j; ind. ind.k
China Ch-1 10 Hemp 901078b CPRO C. chi.h; C. sat.j; sat. sat.k
China Ch-2 12 Rud. No. 338a, 921201b NJBG C. chi.h; C. sat.j; sat. spo.k
China Ch-3 10 Hemp NJBG C. chi.h; C. sat.j; sat. sat.k
China Ch-4 10 Shandong Hemp 921198b AMSRS C. chi.h; C. sat.j; sat. sat.k
China Ch-5 10 ‘Shun-Da’ Hemp 921051b, VIR 175d CPRO C. chi.h; C. sat.j; sat. sat.k
China Ch-6 12 ‘Tin-Yan’ Hemp 883249b, VIR 184d CPRO C. chi.h; C. sat.j; sat. sat.k
China Ch-7 15 ‘Shan-Va’ Hemp 921218b, VIR 185d VIR C. chi.h; C. sat.j; sat. sat.k
Colombia Cl-1 10 Drug BPDIN C. ind.i; C. sat.j; ind. ind.k
Colombia Cl-2 10 Drug BPDIN C. ind.i; C. sat.j; ind. ind.k
Gambia Gm-1 10 Drug AMSRS C. ind.i; C. sat.j; ind. ind.k
Germany Gr-1 10 var. spontanea Rud. 883141b CPRO C. sat.i,j; sat. spo.k
Hungary Hn-1 10 ‘Szegedi-9’ Hemp 883044b CPRO C. sat.i,j; sat. sat.k
Hungary Hn-2 10 Nyiregyha´za´ i Hemp 883050b CPRO C. sat.i,j; sat. sat.k
Hungary Hn-3 10 Leveleki Hemp 883051b CPRO C. sat.i,j; sat. sat.k
Hungary Hn-4 10 Kisszekeresi Hemp 883058b CPRO C. sat.i,j; sat. sat.k
Hungary Hn-5 10 var. spontanea Rud. 883113b CPRO C. sat.i,j; sat. spo.k
Hungary Hn-6 10 var. spontanea Rud. 883114b CPRO C. sat.i,j; sat. spo.k
Hungary Hn-7 12 C. ruderalis Rud. No. 316f HBIPM C. sat.i,j; sat. spo.k
Hungary Hn-8 8 Rud. No. 317f HBIPM C. sat.i,j; sat. spo.k
Hungary Hn-9 10 C. ruderalis Rud. No. 1247f HBIPM C. sat.i,j; sat. spo.k
India In-1 12 Munar, Kerala Drug 91-194c AMSRS C. ind.i; C. sat.j; ind. ind.k
India In-2 12 Almora Rud. NBPGR C. ind.i; C. sat.j; ind. kaf.k
India In-3 12 Delhi Rud. NBPGR C. ind.i; C. sat.j; ind. kaf.k
India In-4 12 Pauri, Garhwal Hemp 921207b INDBS C. chi.h; C. sat.j; sat. sat.k
India In-5 12 Saharanpur Rud. NBPGR C. ind.i; C. sat.j; ind. kaf.k
Italy It-1 10 ‘Kompolti’ Hemp 883048b CPRO C. sat.i,j; sat. sat.k
Italy It-2 10 Hemp MDCC C. sat.i,j; sat. sat.k
Italy It-3 12 Hemp VIR 106d VIR C. sat.i,j; sat. sat.k
Italy It-4 10 Hemp 921050b, VIR 112d CPRO C. sat.i,j; sat. sat.k
Italy It-5 8 Turin Hemp VIR 195d VIR C. sat.i,j; sat. sat.k
Italy It-6 7 Napoletana Hemp VIR 278d VIR C. sat.i,j; sat. sat.k
Italy It-7 4 Distr. di Fatza Hemp VIR 280d VIR C. sat.i,j; sat. sat.k
Italy It-8 9 Carmagnola Hemp VIR 282d VIR C. sat.i,j; sat. sat.k
Italy It-9 4 Hemp VIR 462d VIR C. sat.i,j; sat. sat.k
Jamaica Jm-1 10 Drug No. 66a, 921209b SMALL C. ind.i; C. sat.j; ind. ind.k
Japan Jp-1 14 No. 152a, 921208b SMALL C. chi.h; C. sat.j; sat. sat.k
Japan Jp-2 18 Kozuhara zairai Hemp 883213b CPRO C. chi.h; C. sat.j; sat. sat.k
Kazakhstan Kz-1 9 Hemp VIR 468d VIR C. sat.i,j; sat. sat.k
Kazakhstan Kz-2 9 Hemp VIR 469d VIR C. sat.i,j; sat. sat.k
164
Table 1. Continued.
Origin ID n Region/name Use Parallel ID Source Taxon
Kazakhstan Kz-3 8 Hemp VIR 470d VIR C. sat.i,j; sat. sat.k
Kazakhstan Kz-4 6 Alma Ata Hemp VIR 484d VIR C. sat.i,j; sat. sat.k
Lesotho Ls-1 10 Drug SAP C. ind.i; C. sat.j; ind. ind.k
Mexico Mx-1 12 Drug No. 24a, 921231b SMALL C. ind.i; C. sat.j; ind. ind.k
Mexico Mx-2 8 Drug No. 41a SMALL C. ind.i; C. sat.j; ind. ind.k
Mexico Mx-3 12 Drug No. 289a, 921232b SMALL C. ind.i; C. sat.j; ind. ind.k
Mexico Mx-4 10 Drug 921230b SHOY C. ind.i; C. sat.j; ind. ind.k
Moldavia Ml-1 5 Hemp VIR 116d VIR C. sat.i,j; sat. sat.k
Nepal Np-1 10 Kalopani Rud. 891192b CPRO C. ind.i; C. sat.j; ind. kaf.k
Nepal Np-2 10 Dana Hemp 891193b CPRO C. chi.h; C. sat.j; sat. sat.k
Nepal Np-3 10 Rud. 921233b SB C. ind.i; C. sat.j; ind. kaf.k
Nigeria Ng-1 10 Drug AMSRS C. ind.i; C. sat.j; ind. ind.k
Pakistan Pk-1 30 NW Frontier Drug PAKI C. ind.j; ind. ind.k
Poland Pl-1 7 C.s. ‘gigantea’ Hemp VIR 443d VIR C. sat.i,j; sat. sat.k
Poland Pl-2 10 Hemp VIR 474d VIR C. sat.i,j; sat. sat.k
Poland Pl-3 10 Hemp VIR 475d VIR C. sat.i,j; sat. sat.k
Poland Pl-4 8 Hemp VIR 476d VIR C. sat.i,j; sat. sat.k
Romania Rm-1 10 ssp. ruderalis Rud. 883154b CPRO C. sativai,j; sat. spo.k
Romania Rm-2 10 ssp. ruderalis Rud. 901047b CPRO C. sativai,j; sat. spo.k
Romania Rm-3 10 Hemp VIR 374d VIR C. sat.i,j; sat. sat.k
Russia Rs-1 6 Khakass Rud. N 38g CSBG C. sat.i; C. rud.j; sat. spo.k
Russia Rs-2 5 Novosibirsk Rud. N 77g CSBG C. sat.i; C. rud.j; sat. spo.k
Russia Rs-3 10 Altai Rud. N 79g CSBG C. sat.i; C. rud.j; sat. spo.k
Russia Rs-4 10 Gorno-Altay Rud. N 82g CSBG C. sat.i; C. rud.j; sat. spo.k
Russia Rs-5 4 Khakass Rud. N 102g CSBG C. sat.i; C. rud.j; sat. spo.k
Russia Rs-6 10 Dalnevostochnaya Hemp 921214b, VIR 58d VIR C. sat.i,j; sat. sat.k
Russia Rs-7 7 Altaiskaya Hemp VIR 90d VIR C. sat.i,j; sat. sat.k
Russia Rs-8 10 Altaiskaya Hemp 883248b, VIR 100d CPRO C. sat.i,j; sat. sat.k
Russia Rs-9 10 Altaiskaya Hemp VIR 107d VIR C. sat.i,j; sat. sat.k
Russia Rs-10 7 Altaiskaya Hemp VIR 141d VIR C. sat.i,j; sat. sat.k
Russia Rs-11 12 Novosibirskaya Hemp 921217b, VIR 142d VIR C. sat.i,j; sat. sat.k
Russia Rs-12 8 Ermakovskaya Hemp VIR 310d VIR C. sat.i,j; sat. sat.k
Russia Rs-13 10 Dalnevostochnaya Hemp VIR 387d VIR C. sat.i,j; sat. sat.k
Russia Rs-14 6 Trubchevskaya Hemp VIR 41d VIR C. sat.i,j; sat. sat.k
Russia Rs-15 12 Orlovskaya Hemp 883247b, VIR 48d CPRO C. sat.i,j; sat. sat.k
Russia Rs-16 8 Toguchinskaya Hemp VIR 77d VIR C. sat.i,j; sat. sat.k
Russia Rs-17 7 Tyumenskaya Hemp VIR 85d VIR C. sat.i,j; sat. sat.k
Russia Rs-18 4 Smolenskaya Hemp VIR 110d VIR C. sat.i,j; sat. sat.k
Russia Rs-19 8 Permskaya Hemp VIR 140d VIR C. sat.i,j; sat. sat.k
Russia Rs-20 7 Maryiskaya Hemp VIR 151d VIR C. sat.i,j; sat. sat.k
Russia Rs-21 7 Tatarskaya Hemp VIR 156d VIR C. sat.i,j; sat. sat.k
Russia Rs-22 12 Kirovskaya Hemp VIR 313d VIR C. sat.i,j; sat. sat.k
Russia Rs-23 10 Kirovskaya Hemp 883289b, VIR 315d CPRO C. sat.i,j; sat. sat.k
Russia Rs-24 10 Maryiskaya Hemp 891327b, VIR 349d CPRO C. sat.i,j; sat. sat.k
Russia Rs-25 14 Chuvashskaya Hemp 921223b, VIR 354d VIR C. sat.i,j; sat. sat.k
Russia Rs-26 14 Maryiskaya Hemp 921224b, VIR 356d VIR C. sat.i,j; sat. sat.k
Russia Rs-27 10 Arkhonskaya Hemp 921226b, VIR 405d VIR C. sat.i,j; sat. sat.k
Russia Rs-28 8 Tyumenskaya Hemp VIR 528d VIR C. sat.i,j; sat. sat.k
Sierra Leone SL-1 10 Drug No. 63a, 921236b SMALL C. ind.i; C. sat.j; ind. ind.k
Spain Sp-1 10 Hemp 880973b CPRO C. sat.i,j; sat. sat.k
Spain Sp-2 10 Hemp 891240b CPRO C. sat.i,j; sat. sat.k
Spain Sp-3 10 Hemp 921213b, VIR 57d VIR C. sat.i,j; sat. sat.k
Spain Sp-4 6 Hemp VIR 163d VIR C. sat.i,j; sat. sat.k
South Africa SA-1 12 Pietersburg Drug SAP C. ind.i; C. sat.j; ind. ind.k
South Africa SA-2 10 Transkei Drug SAP C. ind.i; C. sat.j; ind. ind.k
South Africa SA-3 4 Transkei Drug AMSRS C. ind.i; C. sat.j; ind. ind.k
South Africa SA-4 10 Drug 921235b DNHSA C. ind.i; C. sat.j; ind. ind.k
South Korea SK-1 12 Andong Hemp 901161b CPRO C. chi.h; C. sat.j; sat. sat.k
Continued on next page
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Table 1. Continued.
Origin ID n Region/name Use Parallel ID Source Taxon
South Korea SK-2 10 Bonghwa Hemp 901162b CPRO C. chi.h; C. sat.j; sat. sat.k
South Korea SK-3 10 Milyang Hemp 901163b CPRO C. chi.h; C. sat.j; sat. sat.k
South Korea SK-4 12 Chonnamjong Hemp RDASK C. chi.h; C. sat.j; sat. sat.k
South Korea SK-5 10 Kangwansong Hemp IT.180388e RDASK C. chi.h; C. sat.j; sat. sat.k
South Korea SK-6 12 Sunchangsong Hemp IT.180384e RDASK C. chi.h; C. sat.j; sat. sat.k
South Korea SK-7 12 Sungjusong Hemp IT.180386e RDASK C. chi.h; C. sat.j; sat. sat.k
Swaziland Sw-1 12 Drug SAP C. ind.i; C. sat.j; ind. ind.k
Syria Sy-1 10 Hemp VIR 397d VIR C. sat.i,j; sat. sat.k
Thailand Th-1 12 Drug No. 10a SMALL C. ind.i; C. sat.j; ind. ind.k
Thailand Th-2 10 Sakon Nokhon Drug 91-170c AMSRS C. ind.i; C. sat.j; ind. ind.k
Thailand Th-3 12 Drug 91-171c AMSRS C. ind.i; C. sat.j; ind. ind.k
Thailand Th-4 8 Drug 91-172.8c AMSRS C. ind.i; C. sat.j; ind. ind.k
Thailand Th-5 10 Drug 92-176c AMSRS C. ind.i; C. sat.j; ind. ind.k
Thailand Th-6 10 Drug AMSRS C. ind.i; C. sat.j; ind. ind.k
Thailand Th-7 10 Meao, THCVA Hemp 921237b SHOY C. chi.h; C. sat.j; sat. sat.k
Turkey Tk-1 10 Tokumu Hemp 883272b CPRO C. sat.i,j; sat. sat.k
Turkey Tk-2 12 Hemp 891088b CPRO C. sat.i,j; sat. sat.k
Turkey Tk-3 10 Hemp 891090b CPRO C. sat.i,j; sat. sat.k
Turkey Tk-4 10 Hemp 891093b CPRO C. sat.i,j; sat. sat.k
Turkey Tk-5 10 Kurdistan Hemp RBREN C. sat.i,j; sat. sat.k
Turkey Tk-6 7 Hemp VIR 52d VIR C. sat.i,j; sat. sat.k
Turkey Tk-7 10 Hemp VIR 54d VIR C. sat.i,j; sat. sat.k
Turkey Tk-8 7 Hemp VIR 464d VIR C. sat.i,j; sat. sat.k
Turkey Tk-9 9 Hemp VIR 465d VIR C. sat.i,j; sat. sat.k
Uganda Ug-1 10 Drug No. 76a SMALL C. ind.i; C. sat.j; ind. ind.k
Uganda Ug-2 10 Mbale district Drug 921239b KWNDA C. ind.i; C. sat.j; ind. ind.k
Ukraine Uk-1 9 Novgorod-Severskaya Hemp VIR 37d VIR C. sat.i,j; sat. sat.k
Ukraine Uk-2 12 Transcarpathian Hemp 921215b, VIR 125d VIR C. sat.i,j; sat. sat.k
Ukraine Uk-3 12 Transcarpathian Hemp 921216b, VIR 126d VIR C. sat.i,j; sat. sat.k
Ukraine Uk-4 4 Transcarpathian Hemp VIR 128d VIR C. sat.i,j; sat. sat.k
Ukraine Uk-5 7 Transcarpathian Hemp VIR 130d VIR C. sat.i,j; sat. sat.k
Ukraine Uk-6 12 Hemp 921219b, VIR 205d VIR C. sat.i,j; sat. sat.k
Uzbekistan Uz-1 5 Kokand Rud. AMSRS C. sat.i; C. rud.j; sat. spo.k
Yugoslavia Yg-1 12 Domaca local Hemp 921210b, VIR 11d VIR C. sat.i,j; sat. sat.k
Yugoslavia Yg-2 5 Nisca Hemp VIR 19d VIR C. sat.i,j; sat. sat.k
Yugoslavia Yg-3 10 Hemp 921211b, VIR 22d VIR C. sat.i,j; sat. sat.k
Yugoslavia Yg-4 10 Hemp 921212b, VIR 29d VIR C. sat.i,j; sat. sat.k
Yugoslavia Yg-5 7 Leskovacha Hemp VIR 377d VIR C. sat.i,j; sat. sat.k
Yugoslavia Yg-6 10 Novosadska Hemp VIR 442d VIR C. sat.i,j; sat. sat.k
Zimbabwe Zm-1 10 Drug No. 235a, 921234b SMALL C. ind.i; C. sat.j; ind. ind.k
Origin – country of origin; ID – accession code; n – approximate number of plants sampled for genetic analysis (varies with enzyme); Region/Name – region where achenes were originally collected (if known)/name (if a commercial cultivar); Use – a priori assignment to plant-use group: Drug, Hemp, or Rud. ¼ Ruderal (wild or naturalized); Parallel ID – parallel accession codes: aSMALL; bCPRO; cAMSRS; dVIR; eRDASK; fHBIPM; gCSBG.
Source: AMSRS – HortaPharm B.V., Amsterdam, Holland; BPDIN – Bloomington Police Department, Bloomington, IN, USA; CPRO – Centre for Plant Breeding and Reproduction Research, Wageningen, Holland; CSBG – Central Siberian Botanical Garden, Novosibirsk, Russia; DNHSA – Department of National Health, Pretoria, Republic of South Africa; HBIPM – Hortus Botanicus, Institui Plantarum Medicinalium, Budakalasz, Hungary; HBP – Hortus Botanicus Pekinensis, Instituti Botanici Academiae Sinicae, Beijing, China; INDBS –
Botanical Survey of India, Dehra Dun, India; KWNDA – Kawanda Research Station, Kampala, Uganda; MDCC – Museo Della Civilta Contadina, Bologna, Italy; NBPGR – National Bureau of Plant Genetic Resources, New Delhi, India; NJBG – Nanjing Botanical Garden, Mem. Sun Yat-Sen, Jiangsu, China; PAKI – Pakistan Narcotics Control, Islamabad, Pakistan; RBREN – Dr. Rudolph Brenneisen, Institute of Pharmacy, Berne, Switzerland; RDASK – Rural Development Administration, Suwon, South Korea; SAP – Forensic Science Laboratory, Pretoria, Republic of South Africa; SB – The Seed Bank, Ooy, Holland (commercial seed company); SHOY – Dr Y. Shoyama, Faculty of
Pharmaceutical Sciences, Kyushu University, Japan; SMALL – Dr E. Small, Biosystematics Research Institute, Ottawa, Canada; SSSC – Super Sativa Seed Club, Amsterdam, Holland (commercial seed company); VIR – N.I. Vavilov All-Union Institute of Plant Industry, St. Petersburg, Russia.
Taxon: a priori assignment of accessions to taxonomic concepts of hDelile; iLamarck; jSchultes et al. and Anderson; kSmall and Cronquist. Taxon
abbreviations: C. chi. –C. chinensis; C. ind. –C. indica; C. sat. –C. sativa; C. rud. –C. ruderalis; sat. sat. – C. sativa subsp. sativa var. sativa; sat. spo. –C. sativa subsp. sativa var. spontanea; ind. ind. –C. sativa subsp. indica var. indica; ind. kaf. –C. sativa subsp. indica var. kafiristanica.
166 and 10-mm thick gels at 45 mA throughout electrophoresis.
For the lithium–borate buffer system, only 5-mm thick gels were used. These were held at 50 mA for the first 10 min (after which the wicks were removed), and at 200 V subsequently. Current was applied for about 6 h to obtain good band separation. Staining recipes for all enzymes except HK were modified from Soltis et al. (1983). The HK recipe was modified from Morden et al. (1987).

Tissue sample collection
Sample populations of each accession were grown in two secure greenhouses at Indiana University, Bloomington, Indiana. Voucher specimens are deposited in the Deam Herbarium (IND) at Indiana University. About 10 plants of each accession were surveyed, except for accessions obtained late in the investigation. Thirty Cannabis plants were sampled for each gel. To make the gels easier to interpret, two lanes were left blank or loaded with a plant other than Cannabis. Tissue samples were collected the afternoon before extraction and electrophoresis, and stored overnight on moist filter paper in small Petri dishes, under refrigeration. Shoot tips generally produced the darkest bands,
although mature leaf tissue was better for visualizing PGM.

Multivariate analysis
Putative genotypes were inferred from the allozyme banding patterns, and allele frequencies were calculated for small populations of each accession
(Wendel and Weeden 1989). Allele frequencies were analyzed using JMP version 5.0 (SAS Institute 2002). Principal components analysis (PCA), commonly employed in numerical taxonomic investigations, was used to visualize the underlying pattern of genetic variation. The principal components were extracted from the correlation matrix of allele frequencies. Each PC axis is defined by a linear combination of the allele frequencies. PC axis 1 accounts for the largest amount of variance that can be attributed to a single multivariate axis, and each succeeding axis accounts for a progressively smaller proportion of the remaining variance. PC analysis simplifies the original
n-dimensional data set (n ¼ the number of alleles) by enabling the data to be plotted on a reduced number of orthogonal axes while minimizing the loss of information. The degree of similarity among the accessions can be inferred from their proximity in PC space (Wiley 1981; Hillig and Iezzoni 1988).
The average number of alleles per locus (A), number of alleles per polymorphic locus (Ap), and percent polymorphic loci (P) were calculated for each accession, and the expected heterozygosity (He) averaged over all loci was calculated using the mean allele frequencies of each sample population,
for the 11 enzymes that were assayed (Nei 1987; Doebley 1989).
Several industrial hemp strains developed in European breeding programs were genetically characterized, but excluded from the statistical analysis because of their possible hybrid origin (de Meijer and van Soest 1992; de Meijer 1995).
For the purpose of this investigation, an accession was considered hybrid if the parental strains came from more than one country. Nine Chinese accessions from the VIR collection were excluded because of suspected hybridization during seed regeneration. Only accessions analyzed in this
investigation are shown in Table 1.


Results
Gel interpretation
The allozyme banding patterns were interpreted as shown in Figure 1. Only diploid banding patterns were observed. When more than one set of bands appeared on a gel, the loci were numbered sequentially starting with the fastest migrating (most anodal) locus. Alleles at a given locus were lettered sequentially, starting with the fastest migrating band. Monomeric enzymes (ACN, HK, LAP, PGM, SKDH, UNK) showed a single band for homozygous individuals, and two bands for heterozygous individuals. Dimeric enzymes (6PGD, MDH, PGI, TPI) typically showed one band for homozygotes, and three bands for heterozygotes. Malic enzyme (ME) is tetrameric (Weeden and
Wendel 1989), and heterozygous individuals produced a five-banded pattern. Curiously, a pair of bands appeared at the bottom of gels stained for LAP due to cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) migrating into the gels (Figure 1e).
Cannabinoid data were not included in the statistical analysis.
A total of 65 alleles were detected for the 11 enzymes that were assayed. Thirteen of these were excluded from the analysis because they appeared
in just a single accession. Although they are not useful in this study for taxonomic discrimination, these alleles may indicate regions of high genetic
Figure 1. Starch gels stained for enzyme activity. The scale (cm) shows the distance of migration from the origin. (a) ACN; (b) HK; so-called ‘ghost’ bands are artifacts and can be ignored. (c) IDH (not used in analysis) and UNK; (d) PGM; (e) LAP; cannabinoids CBDA and THCA appear toward the bottom of the gel. (f) MDH; (g) 6PGD; (h) ME; (i) SKDH; (j) TPI; (k) PGI; (l) PGI; the twobanded pattern in lane 3 is attributed to the expression of a ‘silent’ allele (As).

168diversity. Ten of the 13 rare alleles were detected in accessions from southern and eastern Asia (India, Japan, Pakistan, South Korea), and just two were detected in accessions from Europe. The 52 alleles that were detected in more than one accession were included in the statistical analysis.

Principal components analysis
The Cannabis accessions were plotted on PC axis 1 (PC1) and PC axis 2 (PC2), which account for 12.3 and 7.3% of the total variance, respectively
(Figure 2). Two large clusters of accessions, as well as several outliers, are evident on a density contour overlay of the PC scatter plot (Figure 3).
A line separating the two major groups is arbitrarily drawn at PC1 ¼ 1. The geographic distribution of the accessions was visualized by drawing bivariate density ellipses (P ¼ 0.75) on the PC plot for the 19 countries of origin represented by three or more accessions (Figure 4). It can be seen in
Figure 4 that the ellipses cluster into the two major groups visualized in Figure 3.
Accessions with values of PC1 > 1 are mostly from Asian and African countries, including Afghanistan, Cambodia, China, India, Japan, Nepal, Pakistan, South Korea, Thailand, and Uzbekistan, as well as Gambia, Lesotho, Nigeria, Sierra Leone, South Africa, Swaziland, Uganda, and Zimbabwe.
Accessions from Colombia, Jamaica, and Mexico are also associated with this group. The other major group, with values of PC1 > 1, is comprised of accessions from Europe, Asia Minor, and Asiatic regions of the former Soviet Union, including Armenia, Belorus, Bulgaria, Germany, Hungary, Italy, Kazakhstan, Moldavia, Poland, Romania, Russia, Spain, Syria, Turkey, Ukraine,
and former Yugoslavia. Although the ellipses for Russia and former Yugoslavia extend into the neighboring cluster, none of the Yugoslavian accessions, and only two of the Russian accessions (Rs-1, Rs-3) had values of PC1<1. The ellipse for Russia is relatively large because of several outliers, including a group of five accessions (Rs-7, Rs-9, Rs-10, Rs-14, Rs-21), three of which are from the Altai region of Central Asia. Three ruderal accessions from the same region (Rs-1, Rs-4, Rs-5) are also outliers, but situated apart from the previous group. Two ruderal Romanian accessions (Rm-1, Rm-2) are outliers, resulting in an elongated ellipse that extends beyond the main cluster, and envelops five ruderal Hungarian accessions (Hn-5, Hn-6, Hn-7, Hn-8, Hn-9) as well.
For further analysis, accessions with values of PC1 < 1 were assigned to the indica gene pool, and those with values of PC1 > 1 were assigned to the sativa gene pool. The gene pools are so-named because they correspond (more or less) to the indica/sativa dichotomy perceived by Lamarck and others. A map showing the countries of origin of accessions from Eurasia and Africa is shaded to indicate the approximate geographic range of the indica and sativa gene pools on these continents
(Figure 5). A third ruderalis gene pool was hypothesized, to accommodate the six Central Asian ruderal accessions (Rs-1 through Rs-5, Uz-1) situated on the PC plot between the indica and sativa gene pools. The ruderalis accessions
correspond to Janischevsky’s (1924) description of C. ruderalis. The indigenous range of the putative ruderalis gene pool is believed to be in Central Asia.
A more detailed analysis of spontaneous Cannabis populations along the migratory routes of ancient nomadic people, ranging from Central Asia to the Carpathian Basin, may reveal further details regarding the ruderalis gene pool.
The frequencies ( f ) of 29 out of 52 alleles differed significantly (P  0.05) between accessions assigned to the indica and sativa gene pools (Table 2). The most common allele at each locus is the same for both gene pools, but their frequencies differed significantly for 10 of the 17 loci surveyed. The absolute values of the eigenvectors (Table 2) indicate the relative contribution of each allele to a given PC axis. Several alleles that account for much of the differentiation between the two major gene pools on PC1 (ACN1-F, LAP1-B, 6PGD2-A, PGM-B, SKDH-D, UNK-C) are relatively common ( f  0.10) in the sativa gene pool, and uncommon ( f  0.05) in the indica gene
pool. Four of these alleles (ACN1-F, 6PGD2-A, PGM-B, SKDH-D) are also common in the ruderalis gene pool. Several other alleles that largely contribute to the differentiation of accessions on PC2 (ACN1-A, LAP1-C,ME-C, UNK-A) are significantly more common in the ruderalis gene pool than in the indica or sativa gene pools. Only two alleles (ACN2-C, LAP1-D) were found that are common (f  0.10) in accessions assigned to the indica gene pool, and uncommon in accessions assigned to the sativa gene pool. However, several
less-common (0.05  f < 0.10) alleles in the indica gene pool were uncommon or rare ( f  0.03) in thesativa gene pool (PGI2-C, SKDH-A, SKDH-B, SKDH-F).
The ruderal accessions from Europe and Central Asia tend to group apart. Although Rs-5 is a distant outlier, plants of this accession appeared morphologically similar to others from the same region. The outlying position of Rs-5 may be partially due to sampling error, since only four viable achenes were obtained. Allele LAP2-A is common among the ruderal accessions from
Europe and Central Asia, but relatively uncommon among the other accessions in the collection, particularly those assigned to the indica gene pool.
The germplasm collection included two very early maturing Russian hemp accessions typical of the Northern eco-geographical group (Rs-22, Rs-23). These are situated on the PC plot with early maturing accessions from nearby regions (Rs-25, Rs-26), and with three ruderal accessions (Hn-7, Hn-9, Rs-2). However, accessions from more southerly latitudes in Europe also cluster nearby (Bg-4, Rm-3, Sp-3). No formal distinction was made in this investigation between theMiddle- Russian and Southern eco-geographic groups of hemp, or between fiber and seed accessions. There appears to be little basis for differentiating these groups on the PC scatter plot.

MDH2-C was detected in four of the five Russian outliers situated toward the right side of the PC scatter plot (Rs-7, Rs-9, Rs-14, Rs-21). This allele was not found in any of the other accessions. The taxonomic significance of this group, if any, is unknown.
The fiber/seed accessions assigned to the indica gene pool are genetically diverse. All but six of the 57 alleles detected in the indica gene pool were
present in this group, including seven rare alleles that were detected in just a single accession. The outliers in the upper left corner of the PC scatter plot are mostly hemp landraces from eastern Asia that had allele frequencies outside the normal range, which sets them apart from the other indica accessions.
The narrow-leafleted drug accessions are relatively devoid of genetic variation, compared to the other conceptual groups recognized in this study. Even so, geographic patterns of genetic variation are apparent within this group. The 12 African accessions are from three regions: western Africa (Nigeria, Gambia, Sierra Leone), eastcentral Africa (Uganda) and southern Africa (South Africa, Swaziland, Lesotho, Zimbabwe). Sample populations of the two Ugandan accessions (Ug-1, Ug-2) consisted entirely of monoecious
plants devoid of detectable allozyme variation. The position of these two accessions on the PC scatter plot represents a region of low genetic variation, with drug accessions from southern Africa and Southeast Asia situated nearby. A rare allele (SKDH-A) was found in all seven southern
African accessions, but in only two other accessions, from Nigeria and Colombia. For the African accessions, an allele (SKDH-C) that was commonly
found in most other accessions was not detected. The wide-leafleted drug accessions from Afghanistan and Pakistan (Af-1 thru Af-10, Pk-1) cluster with the other accessions assigned to the indica gene pool. Allele HK-B was found in nine of the 11 wide-leafleted drug accessions, and in a few hemp accessions from China and South Korea, but not in any of the narrow-leafleted drug accessions or feral indica accessions. HK-B is common in the sativa gene pool, being found in 60 of the 89 accessions assigned to that group. However, several other alleles that are common in the sativa gene
pool (ACN1-F, LAP1-B, 6PGD2-A, PGM-B, TPI1-A, UNK-C) were rare or undetected in the wide-leafleted drug accessions.

Taxonomic interpretation
One objective of this study is to assess previous taxonomic concepts in light of the genetic evidence. Cannabis is commonly divided into drug and hemp plant-use groups, and a third group of ruderal (wild or naturalized) populations. The density ellipse for the drug accessions (Figure 6a) overlies
the indica gene pool, while the ellipse for the hemp accessions overlies both major gene pools, as does the ellipse for the ruderal accessions. Delile’s (1849) concept of C. chinensis is given consideration, because hemp accessions from southern and eastern Asia group separately from those assigned to the sativa gene pool, and Delile was the first taxonomist to describe a separate taxon of eastern Asian hemp. The density ellipse
for accessions assigned to C. chinensis (Figure 6b) shows that they comprise a subset of the indica gene pool.
Lamarck’s (1785) taxonomic concept differentiates the narrow-leafleted C. indica drug accessions from C. sativa, but it is ambiguous how he would have classified the wide-leafleted drug accessions, or the eastern Asian hemp accessions. Figure 6c shows good separation of the two species proposed
by Lamarck, but his concept of C. indica does not circumscribe all of the accessions assigned to the indica gene pool.
Schultes et al. (1974) and Anderson (1980) narrowly circumscribed C. indica to include wideleafleted strains from Afghanistan. The narrowleafleted drug strains, together with hemp strains from all locations are circumscribed under
C. sativa. The density ellipse for C. indica shows that the accessions assigned to this concept comprise a subset of the indica gene pool (Figure 6d), while the ellipse for C. sativa includes accessions assigned to both the indica and sativa gene pools. Schultes et al. and Anderson also recognized C. ruderalis, and emphasized that it only exists in regions where Cannabis is indigenous. The ellipse for the six Central Asian accessions assigned to C. ruderalis lies between and overlaps both the indica and sativa gene pools.
Small and Cronquist (1976) proposed two subspecies and four varieties of C. sativa. Their circumscription of C. sativa L. subsp. sativa var. sativa includes hemp strains from all regions, and the resulting ellipse overlaps the indica and sativa gene pools (Figure 6e). C. sativa L. subsp. sativa var. spontanea (Vav.) Small and Cronq. includes ruderal accessions from both Europe and Central Asia. The resulting ellipse encompasses most of the sativa gene pool and a portion of the indica gene pool, although only two accessions assigned to var. spontanea (Rs-1, Rs-3) had values of PC1 < 1.
The density ellipses for C. sativa L. subsp. indica Lam. var. indica (Lam.) Wehmer, and for C. sativa L. subsp. indica Lam. var. kafiristanica (Vav.)
Small and Cronq. encompass different subsets of the indica gene pool.
The author’s concept is illustrated by density ellipses for the indica, sativa, and ruderalis gene pools (Figure 6f ). The ellipses for accessions assigned to the indica and sativa gene pools overlay the two major clusters of accessions, while the ellipse for the ruderalis accessions is intermediate, and overlaps the other two. Since the existence of a separate ruderalis gene pool is less certain, it is indicated with a dotted line.

Genetic diversity statistics
Genetic diversity statistics for gene pools and putative taxa of Cannabis are given in Table 3. The taxa listed in Table 3 circumscribe different subsets of
the indica and sativa gene pools. C. ruderalis is also included here. The circumscriptions of C. sativa subsp. sativa var. sativa and C. sativa subsp. sativa var. spontanea exclude accessions assigned to C. chinensis and C. ruderalis, respectively, while C. indica sensu Lamarck excludes accessions
assigned to C. sativa subsp. indica var. kafiristanica.
In general, the sativa accessions exhibited greater genetic diversity than the indica accessions (including C. sativa subsp. indica var. kafiristanica
and C. chinensis), and the ruderalis accessions were intermediate. Within the indica gene pool, the accessions assigned to C. chinensis exhibited the greatest genetic diversity, and the narrow-leafleted drug accessions (C. indica sensu Lamarck) exhibited the least. Within the sativa gene pool, the
cultivated (var. sativa) and weedy (var. spontanea) accessions exhibited virtually identical levels of genetic diversity.

Discussion
The allozyme data show that the Cannabis accessions studied in this investigation were derived from two major gene pools, ruling out the hypothesis of a single undivided species. The genetic divergence of the cultivated accessions approximates the indica/sativa split perceived by previous investigators.
However, none of the earlier taxonomic treatments of Cannabis adequately represent the underlying relationships discovered in the present study.
The allozyme data, in conjunction with the different geographic ranges of the indica and sativa gene pools and previous investigations that demonstrate
significant morphological and chemotaxonomic differences between these two taxa (Small and Beckstead 1973; Small et al. 1976), support the formal recognition of C. sativa, C. indica, and possibly C. ruderalis as separate species. This opinion represents a synthesis of the species concepts of Lamarck, Delile, Janischevsky, Vavilov, Schultes et al. and Anderson. It rejects the singlespecies concepts of Linnaeus, and Small and Cronquist, because the genetic data demonstrate a fundamental split within the Cannabis gene pool. It is more ‘practical and natural’ to assign the indica
and sativa gene pools to separate species, and to leave the ranks of subspecies and variety available for further classification of the putative taxa recognized herein.
The C. sativa gene pool includes hemp landraces from Europe, Asia Minor and Central Asia, as well as weedy populations from Eastern Europe. The C. indica gene pool is more diverse than Lamarck originally conceived. Besides the narrow-leafleted drug strains, the C. indica gene pool includes wide-leafleted drug strains from Afghanistan and Pakistan, hemp landraces from southern and
eastern Asia, and feral populations from India and Nepal. C. ruderalis, assumed to be indigenous to Central Asia, is delimited to exclude naturalized C. sativa populations occurring in regions where Cannabis is not native. The existence of a separate C. ruderalis gene pool is less certain, since only six accessions of this type were available for study. The first two PC axes account for a relatively small proportion of the total variance (19.6%), compared with a typical PC analysis of morphological data. Morphological data sets often
have a high degree of ‘concomitant character variation,’ such as the size correlation between different plant parts (Small 1979). As a result, the first
few PC axes often account for a relatively large proportion of the variance. This type of ‘biological correlation’ was absent from the data set of allele
frequencies. Although the less common alleles are of taxonomic importance, the common alleles largely determined the outcome of the PC analysis. When only the most frequent allele at each locus was entered into the analysis, the first two PC axes accounted for 25.8% of the total variance, and the C. indica and C. sativa gene pools were nearly as well discriminated.
The role of human selection in the divergence of the C. indica and C. sativa gene pools is uncertain. Small (1979) presumed the dichotomy to be largely a result of selection for drug production in the case of the indica taxon, and selection for fiber/seed production in the case of sativa. The genetic evidence challenges this assumption, since the fiber/ seed accessions from India, China, Japan, South Korea, Nepal, and Thailand all cluster with the C. indica gene pool. An alternate hypothesis is that the C. indica and C. sativa hemp landraces were derived from different primordial gene pools and independently domesticated, and that the drug strains were derived from the same primordial
gene pool as the C. indica hemp landraces. It is assumed that, in general, when humans introduced Cannabis into a region where it did not previously
exist, the gene pool of the original introduction largely determined the genetic make-up of the Cannabis populations inhabiting the region thereafter.
It remains to be determined whether the C. indica and C. sativa gene pools diverged before, or after the beginning of human intervention in the evolution of Cannabis.
The amount of genetic variation in Cannabis is similar to levels reported for other crop plants (Doebley 1989).Hamrick (1989) compiled data from different sources that show relatively high levels of genetic variation within out-crossed and windpollinated populations, and low levels of variation within weedy populations. Differentiation between populations is relatively low for dioecious and out-crossed populations, and high for annuals and plants (such as Cannabis) with gravity-dispersed seeds. Hamrick reported the within-population means of 74 dicot taxa. The number of alleles per locus (1.46), percentage of polymorphic loci (31.2%) and mean heterozygosity (0.113) are within the ranges estimated for the putative taxa of Cannabis. The extensive overlap of the density ellipses for the countries of origin of accessions assigned to the C. sativa gene pool (Figure 4) suggests that this group is relatively homogeneous throughout its range. In comparison, the ellipses for the C. indica gene pool do not all overlap, suggesting that regional differences within this gene pool are more distinct.
Divergence in allele frequencies between populations (gene pools) can occur in two principle ways (Witter, cited in Crawford 1989). Initially, a founder population can diverge partly or wholly by genetic drift. The second process, which presumably takes much longer, involves the accumulation of new mutations in the two populations. Both of these processes may help to explain the patterns of genetic variation present in Cannabis, albeit on a larger scale. The alleles that differentiate C. indica from C. sativa on PC1 are common in the C. sativa gene pool and uncommon in the C. indica gene pool, which suggests that a founder event may have narrowed the genetic base of C. indica.
However, a considerable number of mutations appear to have subsequently accumulated in both gene pools, indicating that the indica/sativa split may be quite ancient. The assumption that the alleles that were surveyed in this study are selectively neutral does not imply that humans have not affected allele frequencies in Cannabis. It only means that these genetic markers are ‘cryptic’ and not subject to deliberate manipulation. Humans have undoubtedly been instrumental in both the divergence and mixing of the Cannabis gene pools. For example, the commercial hemp strain ‘Kompolti Hybrid TC’ takes
advantage of heterosis (hybrid vigor) in a cross between a European hemp strain corresponding to C. sativa, and a Chinese ‘unisexual’ hemp strain corresponding to C. indica (Bo´csa 1999). Evidence of gene flow from eastern Asian hemp to cultivated C. sativa is provided by certain alleles (e.g., LAP1-
D, PGI2-C, SKDH-B, SKDH-F) that occur in low frequency in the C. sativa gene pool, and are significantly more common among the hemp accessions
assigned to C. indica. There is also limited evidence of gene flow in the reverse direction; allele PGM-B, which is common in accessions assigned
to C. sativa, was detected at low frequency in a few of the hemp accessions assigned to C. indica. Some of the accessions in the collection encompass
little genetic variation, which may be a result of inbreeding, genetic drift, or sampling error (e.g., the achenes may have been collected from a single
plant). In general, the accessions cultivated for drug use, particularly the narrow-leafleted drug accessions, show more signs of inbreeding than those cultivated for fiber or seed. The absence of allele PGM-B in the gene pool of narrow-leafleted drug accessions indicates a lack of gene flow from C. sativa. Although it is possible that the entire gene pool of narrow-leafleted drug strains passed through a ‘genetic bottleneck,’ the low genetic diversity of this group may also be a result of the way these plants are often cultivated. It is not unusual for growers to select seeds from the few best plants in the current year’s crop to sow the following year, thereby reducing the genetic diversity of the initial population. Since staminate plants are often culled before flowering, the number of pollinators may also be extremely limited.
The gene pool of a cultivated taxon is expected to contain a subset of the alleles present in the ancestral gene pool (Doebley 1989). In the case of
Cannabis, the available evidence is insufficient to make an accurate determination of progenitor– derivative relationships. Aboriginal populations
may have migrated from Central Asia into Europe as ‘camp followers,’ along with the cultivated landraces (Vavilov 1926). If so, then the weedy populations of Europe may represent the aboriginal gene pool into which individuals that have escaped from cultivation have merged.
Although fewer alleles were detected in the ruderal accessions from Central Asia and Europe than in the cultivated C. sativa gene pool, this result is preliminary given the relatively small number of ruderal accessions available for study. Similarly, the feral C. indica accessions from India and Nepal do not encompass as much genetic variation as the cultivated accessions of C. indica, but again this result is based on insufficient data to draw firm conclusions.
Even so, both results suggest that ruderal (feral) populations are secondary to the domesticated ones. From the evidence at hand, it appears that the feral C. indica accessions could represent the ancestral source of the narrow-leafleted drug accessions, but perhaps not of the wide-leafleted
drug accessions, since allele HK-B was found in nine of the 11 wide-leafleted drug accessions, but not in any of the ruderal C. indica, or narrowleafleted
drug accessions. Vavilov and Bukinich (1929) reported finding wild Cannabis populations in eastern Afghanistan (C. indica Lam. f. afghanica Vav.), which could represent the progenitor of the wide-leafleted drug strains. Unfortunately, wild populations from Afghanistan were not represented in the present study.

Conclusion
This investigation substantiates the existence of a fundamental split within the Cannabis gene pool.
A synthesis of previous taxonomic concepts best describes the underlying patterns of variation. The progenitor–derivative relationships within Cannabis are not well understood, and will require more extensive sampling and additional genetic analyses to further resolve. A revised circumscription of the infraspecific taxonomic groups is warranted, in conjunction with analyses of morphological and chemotaxonomic variation within the germplasm collection under study.

Acknowledgements
I am grateful to Professor Paul G. Mahlberg for
facilitating this investigation. Thanks also to
Professor Gerald Gastony and Valerie Savage for
technical assistance, and to Dr Etienne de Meijer,
David Watson and the others who donated germplasm
for this study. I appreciate the help of
Drs Beth andWilliam Hillig, Dr John McPartland,
Dr Paul Mahlberg, and two anonymous referees in
reviewing this manuscript. This research was
supported by a grant from HortaPharm B.V.,
The Netherlands.
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Errata
Figure 1b. HK lane 13: genotype = AC
Figure 1e. LAP1 lane 11: genotype = CD
Figure 1f. MDH3 lanes 1, 2, 4, 5, 8–14: genotype = CC
lane 3: genotype = AC
lane 6: genotype = CE

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zamalito said:

I'd also like to refer you to the opium poppy and how the latitude of the region of origin affects the morphine/codeine ratio I feel this is info applies very much to cannabis and thc/cbd ratios

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