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Genetic evidence for speciation in Cannabis (Cannabaceae)
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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 (e-mail: khillig@bio.indiana.edu)
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
theNorthern, 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. A
probability 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
165
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
167
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).
168
diversity. 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,
Figure 1. Continued.
169
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
Figure 2. Scatter plot of 156 Cannabis accessions on PC axis 1 and PC axis 2. Accession codes are given in Table 1. Rs-5, a distant outlier,
is not shown.
170
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 the
sativa 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. The large ellipse for
Russia (Figure 4) envelops accessions assigned
to both the sativa and ruderalis gene pools. Allele
Figure 3. Density contour overlay of the PC scatter plot. The two large clusters of accessions are separated by a line drawn at PC1¼1.
Several outlying accessions are evident, including Rs-5, not shown in Figure 2. Density contours are in 10% increments, with 0.7 kernel
sizes for both axes.
171
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
Figure 4. Density ellipses (P ¼ 0.75) are drawn on the PC scatter plot for the countries of origin of the various accessions. Ellipses were
only generated for countries represented by a minimum of three accessions.
172
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
Figure 5. Map showing the countries of origin of accessions assigned to the indica and sativa gene pools. The arrows suggest humanvectored
dispersal from the presumed origin of Cannabis in Central Asia.
173
Table 2. Mean allele frequencies for accessions assigned to the indica, sativa and ruderalis gene pools. For a given allele, means (in rows)
not connected by the same letter are significantly different using Student’s t-test (P ¼ 0.05). The most common allele at each locus is
shown in bold. n ¼ number of accessions assigned to each group. Also shown are the Eigenvectors for the first two principal component
axes (PC1 and PC2).
Allele
indica sativa ruderalis
Eigenvector
n ¼ 62 Mean n ¼ 89 Mean n ¼ 6 Mean PC1 PC2
ACN1-A 0.02 b 0.01 b 0.11 a 0.039 0.280
ACN1-B 0.95 a 0.89 b 0.79 b 0.082 0.183
ACN1-D 0.02 a 0.00 a 0.02 a 0.023 0.039
ACN1-E 0.01 a 0.00 a 0.00 a 0.045 0.067
ACN1-F 0.00 b 0.10 a 0.09 a 0.161 0.025
ACN2-B 0.90 b 0.99 a 0.80 b 0.105 0.342
ACN2-C 0.10 a 0.01 b 0.20 a 0.104 0.341
HK-A 0.92 a 0.85 b 0.82 ab 0.080 0.189
HK-B 0.08 b 0.15 a 0.18 ab 0.080 0.187
LAP1-A 0.00 a 0.01 a 0.00 a 0.095 0.082
LAP1-B 0.03 b 0.33 a 0.00 b 0.231 0.154
LAP1-C 0.68 b 0.64 b 0.93 a 0.037 0.288
LAP1-D 0.30 a 0.03 b 0.07 b 0.190 0.189
LAP2-A 0.01 b 0.07 a 0.20 a 0.126 0.178
LAP2-B 0.99 a 0.92 b 0.81 b 0.154 0.175
LAP2-C 0.00 a 0.02 a 0.00 a 0.140 0.030
MDH1-A 0.01 a 0.00 a 0.00 a 0.017 0.017
MDH1-B 0.99 a 0.94 b 0.93 ab 0.218 0.132
MDH1-C 0.00 b 0.06 a 0.07 ab 0.237 0.133
MDH2-B 1.00 a 0.99 a 1.00 a 0.154 0.059
MDH2-C 0.00 a 0.01 a 0.00 a 0.156 0.060
MDH3-A 0.00 a 0.00 a 0.00 a 0.030 0.067
MDH3-C 0.99 a 0.98 a 0.97 a 0.045 0.092
MDH3-E 0.00 b 0.02 a 0.03 a 0.077 0.041
ME-B 0.99 a 0.99 a 0.93 b 0.011 0.160
ME-C 0.01 b 0.01 b 0.07 a 0.004 0.168
6PGD1-A 0.00 a 0.00 a 0.00 a 0.047 0.040
6PGD1-B 0.99 a 1.00 a 1.00 a 0.038 0.143
6PGD2-A 0.02 b 0.17 a 0.15 a 0.252 0.045
6PGD2-B 0.98 a 0.82 b 0.85 b 0.249 0.044
6PGD2-C 0.00 a 0.00 a 0.00 a 0.022 0.011
PGI2-A 0.08 b 0.21 a 0.00 b 0.143 0.066
PGI2-As 0.01 a 0.00 a 0.00 a 0.036 0.111
PGI2-B 0.86 a 0.79 b 0.98 a 0.095 0.033
PGI2-C 0.05 a 0.00 b 0.02 ab 0.083 0.025
PGM-B 0.01 c 0.34 a 0.20 b 0.294 0.011
PGM-C 0.98 a 0.66 c 0.80 b 0.291 0.009
PGM-D 0.01 a 0.00 b 0.00 ab 0.035 0.040
SKDH-A 0.05 a 0.00 b 0.00 ab 0.124 0.123
SKDH-B 0.09 a 0.02 b 0.00 ab 0.104 0.058
SKDH-C 0.31 a 0.37 a 0.04 b 0.083 0.132
SKDH-D 0.05 b 0.14 a 0.20 a 0.137 0.105
SKDH-E 0.42 b 0.43 ab 0.63 a 0.036 0.068
SKDH-F 0.08 a 0.03 b 0.13 a 0.098 0.239
TPI1-A 0.05 b 0.10 a 0.11 ab 0.098 0.097
TPI1-B 0.95 a 0.90 b 0.89 ab 0.096 0.097
TPI2-A 0.01 a 0.01 a 0.00 a 0.023 0.034
TPI2-B 0.99 a 0.99 a 1.00 a 0.019 0.013
TPI2-C 0.00 a 0.00 a 0.00 a 0.005 0.049
UNK-A 0.00 b 0.00 b 0.03 a 0.029 0.219
UNK-B 0.99 a 0.60 b 0.97 a 0.305 0.114
UNK-C 0.01 b 0.39 a 0.00 b 0.304 0.126
174
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
Figure 6. The PC scatter plot, with density ellipses (P ¼ 0.75) showing how well various conceptual groups coincide with the genetic
data. The accessions were sorted according to the following concepts: (a) plant-use group; (b) Delile; (c) Lamarck; (d) Schultes et al. and
Anderson; (e) Small and Cronquist; (f) author’s concept.
175
(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
Table 3. Means for the number of alleles per locus (A), number of alleles per polymorphic locus (Ap), percentage of polymorphic loci (P)
and average expected heterozygosity (He) for gene pools and putative taxa of Cannabis. Means (in columns) not connected by the same
letter are significantly different using Student’s t-test (P ¼ 0.05). The gene pools and putative taxa were tested separately. n ¼ number of
accessions.
n A Ap P He
Gene pool
sativa 89 1.60 a 2.20 b 48.3 a 0.17 a
indica 62 1.35 b 2.39 a 22.2 c 0.08 c
ruderalis 6 1.39 b 2.13 b 34.0 b 0.13 b
Putative taxon
C. sativa subsp. sativa var. sativaa Small and Cronq. 81 1.60 a 2.20 bc 48.4 a 0.17 a
C. sativa subsp. sativa var. spontaneab Small and Cronq. 8 1.59 ab 2.19 bc 47.0 a 0.17 a
C. sativa subsp. indica var. kafiristanica Small and Cronq. 5 1.44 bc 2.38 ab 22.4 cde 0.09 cd
C. indica Lam.c 27 1.19 d 2.43 a 12.8 e 0.05 e
C. indica sensu Schultes et al. and Anderson 11 1.29 c 2.21 bc 22.1 d 0.07 d
C. chinensis Delile 19 1.59 a 2.44 a 35.6 b 0.12 bc
C. ruderalis Janisch. 6 1.39 c 2.13 c 34.0 bc 0.13 b
aExcluding accessions assigned to C. chinensis.
bExcluding accessions assigned to C. ruderalis.
cExcluding accessions assigned to C. sativa subsp. indica var. kafiristanica.
176
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) compileddatafrom
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
177
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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tables from p. 163
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Karl W. Hillig
Department of Biology, Indiana University, Bloomington, IN, USA; Current address: 1010 Saratoga Road,
Ballston Lake, NY 12019, USA (e-mail: khillig@bio.indiana.edu)
Received 7 January 2003; accepted in revised form 28 June 2003
Key words: Allozyme, Cannabis, Evolution, Genetics, Origin, TaxonomyDepartment of Biology, Indiana University, Bloomington, IN, USA; Current address: 1010 Saratoga Road,
Ballston Lake, NY 12019, USA (e-mail: khillig@bio.indiana.edu)
Received 7 January 2003; accepted in revised form 28 June 2003
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THIS IS THE MOST UP TO DAY INFO :thumbs:
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 (e-mail: khillig@bio.indiana.edu)
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
theNorthern, 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. A
probability 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
165
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
167
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).
168
diversity. 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,
Figure 1. Continued.
169
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
Figure 2. Scatter plot of 156 Cannabis accessions on PC axis 1 and PC axis 2. Accession codes are given in Table 1. Rs-5, a distant outlier,
is not shown.
170
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 the
sativa 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. The large ellipse for
Russia (Figure 4) envelops accessions assigned
to both the sativa and ruderalis gene pools. Allele
Figure 3. Density contour overlay of the PC scatter plot. The two large clusters of accessions are separated by a line drawn at PC1¼1.
Several outlying accessions are evident, including Rs-5, not shown in Figure 2. Density contours are in 10% increments, with 0.7 kernel
sizes for both axes.
171
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
Figure 4. Density ellipses (P ¼ 0.75) are drawn on the PC scatter plot for the countries of origin of the various accessions. Ellipses were
only generated for countries represented by a minimum of three accessions.
172
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
Figure 5. Map showing the countries of origin of accessions assigned to the indica and sativa gene pools. The arrows suggest humanvectored
dispersal from the presumed origin of Cannabis in Central Asia.
173
Table 2. Mean allele frequencies for accessions assigned to the indica, sativa and ruderalis gene pools. For a given allele, means (in rows)
not connected by the same letter are significantly different using Student’s t-test (P ¼ 0.05). The most common allele at each locus is
shown in bold. n ¼ number of accessions assigned to each group. Also shown are the Eigenvectors for the first two principal component
axes (PC1 and PC2).
Allele
indica sativa ruderalis
Eigenvector
n ¼ 62 Mean n ¼ 89 Mean n ¼ 6 Mean PC1 PC2
ACN1-A 0.02 b 0.01 b 0.11 a 0.039 0.280
ACN1-B 0.95 a 0.89 b 0.79 b 0.082 0.183
ACN1-D 0.02 a 0.00 a 0.02 a 0.023 0.039
ACN1-E 0.01 a 0.00 a 0.00 a 0.045 0.067
ACN1-F 0.00 b 0.10 a 0.09 a 0.161 0.025
ACN2-B 0.90 b 0.99 a 0.80 b 0.105 0.342
ACN2-C 0.10 a 0.01 b 0.20 a 0.104 0.341
HK-A 0.92 a 0.85 b 0.82 ab 0.080 0.189
HK-B 0.08 b 0.15 a 0.18 ab 0.080 0.187
LAP1-A 0.00 a 0.01 a 0.00 a 0.095 0.082
LAP1-B 0.03 b 0.33 a 0.00 b 0.231 0.154
LAP1-C 0.68 b 0.64 b 0.93 a 0.037 0.288
LAP1-D 0.30 a 0.03 b 0.07 b 0.190 0.189
LAP2-A 0.01 b 0.07 a 0.20 a 0.126 0.178
LAP2-B 0.99 a 0.92 b 0.81 b 0.154 0.175
LAP2-C 0.00 a 0.02 a 0.00 a 0.140 0.030
MDH1-A 0.01 a 0.00 a 0.00 a 0.017 0.017
MDH1-B 0.99 a 0.94 b 0.93 ab 0.218 0.132
MDH1-C 0.00 b 0.06 a 0.07 ab 0.237 0.133
MDH2-B 1.00 a 0.99 a 1.00 a 0.154 0.059
MDH2-C 0.00 a 0.01 a 0.00 a 0.156 0.060
MDH3-A 0.00 a 0.00 a 0.00 a 0.030 0.067
MDH3-C 0.99 a 0.98 a 0.97 a 0.045 0.092
MDH3-E 0.00 b 0.02 a 0.03 a 0.077 0.041
ME-B 0.99 a 0.99 a 0.93 b 0.011 0.160
ME-C 0.01 b 0.01 b 0.07 a 0.004 0.168
6PGD1-A 0.00 a 0.00 a 0.00 a 0.047 0.040
6PGD1-B 0.99 a 1.00 a 1.00 a 0.038 0.143
6PGD2-A 0.02 b 0.17 a 0.15 a 0.252 0.045
6PGD2-B 0.98 a 0.82 b 0.85 b 0.249 0.044
6PGD2-C 0.00 a 0.00 a 0.00 a 0.022 0.011
PGI2-A 0.08 b 0.21 a 0.00 b 0.143 0.066
PGI2-As 0.01 a 0.00 a 0.00 a 0.036 0.111
PGI2-B 0.86 a 0.79 b 0.98 a 0.095 0.033
PGI2-C 0.05 a 0.00 b 0.02 ab 0.083 0.025
PGM-B 0.01 c 0.34 a 0.20 b 0.294 0.011
PGM-C 0.98 a 0.66 c 0.80 b 0.291 0.009
PGM-D 0.01 a 0.00 b 0.00 ab 0.035 0.040
SKDH-A 0.05 a 0.00 b 0.00 ab 0.124 0.123
SKDH-B 0.09 a 0.02 b 0.00 ab 0.104 0.058
SKDH-C 0.31 a 0.37 a 0.04 b 0.083 0.132
SKDH-D 0.05 b 0.14 a 0.20 a 0.137 0.105
SKDH-E 0.42 b 0.43 ab 0.63 a 0.036 0.068
SKDH-F 0.08 a 0.03 b 0.13 a 0.098 0.239
TPI1-A 0.05 b 0.10 a 0.11 ab 0.098 0.097
TPI1-B 0.95 a 0.90 b 0.89 ab 0.096 0.097
TPI2-A 0.01 a 0.01 a 0.00 a 0.023 0.034
TPI2-B 0.99 a 0.99 a 1.00 a 0.019 0.013
TPI2-C 0.00 a 0.00 a 0.00 a 0.005 0.049
UNK-A 0.00 b 0.00 b 0.03 a 0.029 0.219
UNK-B 0.99 a 0.60 b 0.97 a 0.305 0.114
UNK-C 0.01 b 0.39 a 0.00 b 0.304 0.126
174
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
Figure 6. The PC scatter plot, with density ellipses (P ¼ 0.75) showing how well various conceptual groups coincide with the genetic
data. The accessions were sorted according to the following concepts: (a) plant-use group; (b) Delile; (c) Lamarck; (d) Schultes et al. and
Anderson; (e) Small and Cronquist; (f) author’s concept.
175
(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
Table 3. Means for the number of alleles per locus (A), number of alleles per polymorphic locus (Ap), percentage of polymorphic loci (P)
and average expected heterozygosity (He) for gene pools and putative taxa of Cannabis. Means (in columns) not connected by the same
letter are significantly different using Student’s t-test (P ¼ 0.05). The gene pools and putative taxa were tested separately. n ¼ number of
accessions.
n A Ap P He
Gene pool
sativa 89 1.60 a 2.20 b 48.3 a 0.17 a
indica 62 1.35 b 2.39 a 22.2 c 0.08 c
ruderalis 6 1.39 b 2.13 b 34.0 b 0.13 b
Putative taxon
C. sativa subsp. sativa var. sativaa Small and Cronq. 81 1.60 a 2.20 bc 48.4 a 0.17 a
C. sativa subsp. sativa var. spontaneab Small and Cronq. 8 1.59 ab 2.19 bc 47.0 a 0.17 a
C. sativa subsp. indica var. kafiristanica Small and Cronq. 5 1.44 bc 2.38 ab 22.4 cde 0.09 cd
C. indica Lam.c 27 1.19 d 2.43 a 12.8 e 0.05 e
C. indica sensu Schultes et al. and Anderson 11 1.29 c 2.21 bc 22.1 d 0.07 d
C. chinensis Delile 19 1.59 a 2.44 a 35.6 b 0.12 bc
C. ruderalis Janisch. 6 1.39 c 2.13 c 34.0 bc 0.13 b
aExcluding accessions assigned to C. chinensis.
bExcluding accessions assigned to C. ruderalis.
cExcluding accessions assigned to C. sativa subsp. indica var. kafiristanica.
176
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) compileddatafrom
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
177
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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