Cannabis sativa and Cannabis indica versus “Sativa” and “Indica”

Abstract

The formal botanical taxonomy of Cannabis sativa Linnaeus and C. indica Lamarck has become entangled and subsumed by a new vernacular taxonomy of “Sativa” and “Indica.” The original protologues (descriptions, synonymies, and herbarium specimens) by Linnaeus and Lamarck are reviewed. The roots of the vernacular taxonomy are traced back to Vavilov and Schultes, who departed from the original concepts of Linnaeus and Lamarck. The modified concepts by Vavilov and Schultes were further remodeled by underground Cannabis breeders in the 1980s and 1990s. “Sativa” refers to plants of Indian heritage, in addition to their descendants carried in a diaspora to Southeast Asia, South- and East Africa, and even the Americas. “Indica” refers to Afghani landraces, together with their descendants in parts of Pakistan (the northwest, bordering Afghanistan). Phytochemical and genetic research supports the separation of “Sativa” and “Indica.” But their nomenclature does not align with formal botanical C. sativa and C. indica based on the protologues of Linnaeus and Lamarck. Furthermore, distinguishing between “Sativa” and “Indica” has become nearly impossible because of extensive cross-breeding in the past 40 years. Traditional landraces of “Sativa” and “Indica” are becoming extinct through introgressive hybridization. Solutions for reconciling the formal and vernacular taxonomies are proposed.

Full text

Chapter 4
Cannabis sativa and Cannabis indica
versus “Sativa”and “Indica”
John M. McPartland
Abstract The formal botanical taxonomy of Cannabis sativa Linnaeus and C.
indica Lamarck has become entangled and subsumed by a new vernacular taxon-
omy of “Sativa”and “Indica.”The original protologues (descriptions, synonymies,
and herbarium specimens) by Linnaeus and Lamarck are reviewed. The roots of the
vernacular taxonomy are traced back to Vavilov and Schultes, who departed from
the original concepts of Linnaeus and Lamarck. The modified concepts by Vavilov
and Schultes were further remodeled by underground Cannabis breeders in the
1980s and 1990s. “Sativa”refers to plants of Indian heritage, in addition to their
descendants carried in a diaspora to Southeast Asia, South- and East Africa, and
even the Americas. “Indica”refers to Afghani landraces, together with their
descendants in parts of Pakistan (the northwest, bordering Afghanistan).
Phytochemical and genetic research supports the separation of “Sativa”and
“Indica.”But their nomenclature does not align with formal botanical C. sativa and
C. indica based on the protologues of Linnaeus and Lamarck. Furthermore, dis-
tinguishing between “Sativa”and “Indica”has become nearly impossible because
of extensive cross-breeding in the past 40 years. Traditional landraces of “Sativa”
and “Indica”are becoming extinct through introgressive hybridization. Solutions
for reconciling the formal and vernacular taxonomies are proposed.
4.1 Introduction
Taxonomy includes classification (the identification and categorization of organ-
isms) and nomenclature (the naming and describing of organisms). The formal
botanical taxonomy of Cannabis by Small and Cronquist (1976) recognizes two
subspecies: C. sativa subsp. sativa, and C. sativa subsp. indica. They are considered
different species, C. sativa and C. indica, by some botanists (e.g., Hillig and
Mahlberg 2004; Clarke and Merlin 2013).
J.M. McPartland (&)
GW Pharmaceuticals Place, 1 Cavendish Place, London W1G 0QF, UK
e-mail: mcpruitt@myfairpoint.net
©Springer International Publishing AG 2017
S. Chandra et al. (eds.), Cannabis sativa L. - Botany and Biotechnology,
DOI 10.1007/978-3-319-54564-6_4
101

In the worlds of recreational and medicinal cannabis, everyone seems to be
talking about “Sativa”and “Indica.”This vernacular taxonomy of drug-type
Cannabis has gone viral. Enter “Sativa versus Indica”into Google, and the search
returns 45,000 hits. Please stay alert to the fact that “Sativa”and “Indica”in
quotation marks are not the same as C. sativa and C. indica written in italics.
“Sativa”and “Indica”have become sources of confusion (Small 2007; Erkelens and
Hazekamp 2014; McPartland 2014; Russo 2016). Hazekamp and Fischedick (2012)
call for an alternative approach, “from cultivar to chemovar,”where plants are
identified by their chemical fingerprint, rather than a whimsical name.
The goals for this chapter are four-fold: (1) review the formal botanical taxon-
omy of C. sativa and C. indica; (2) trace the history of vernacular “Sativa”and
“Indica”and their misalignment with C. sativa and C. indica; (3) recognize dif-
ferences between “Sativa”and “Indica”in phytochemistry and genetics; (4) align
the vernacular taxonomy with the formal botanical taxonomy.
4.2 Formal Botanical Nomenclature: C. sativa
Linnaeus named C. sativa in Species Plantarum, the starting point for botanical
nomenclature (Linnaeus 1753). C. sativa in the strict sense, sensu stricto, is
demarcated by Linnaeus’s protologue. The International Code of Nomenclature
(ICN) defines a protologue as everything associated with a taxonomic name at its
first valid publication. It includes the species’s description, synonymy, and
herbarium specimens (McNeill 2012).
Linnaeus’s protologue of C. sativa is described in full for the first time by
McPartland and Guy (2017). It is abstracted here: Linnaeus’s description was
exceptionally brief: a generic account of flower parts, which applies equally to any
plant ever describe in the genus Cannabis (Linnaeus 1753,1754). Linnaeus listed
four synonyms: C. foliis digitatis, C. mas, C. erratica, C. femina; and five authors
who used those names: himself, Dalibard, van Royen, d’Aléchamps, and Bauhin.
The authors and their synonyms delimit C. sativa to plants from northern Europe.
His herbarium specimens also came from northern Europe. Linnaeus’s type
specimen of C. sativa is stored at the Linnaeus herbarium (Fig. 4.1). The seeded
pistillate plant’s morphology is consistent with a northern European fiber-type
landrace. Its inflorescences are loose, not dense; subtending floral leaves have a
sparse covering of sessile glandular trichomes; perigonal bracts that enclose ach-
enes (seeds) have a relatively sparse covering of capitate stalked glandular
(CSG) trichomes. Evidence by Stern (1974) indicates that Linnaeus collected the
specimen in Sweden. Other C. sativa specimens collected by Linnaeus and stored at
the British Museum are consistent with “the old cultivated hemp stock of northern
Europe”(Stern 1974).
Linnaeus notably excluded Asian plants from the C. sativa protologue. He
certainly knew about Asian Cannabis. Sixteen years earlier, Linnaeus (1737) cited
six authors who assigned names to psychoactive Asian Cannabis: C. Bauhin
102 J.M. McPartland

(Cannabi similis exotica), J. Bauhin and Cherler (Bangue cannabi simile), Ray
(Bangue cannabi), Rheede (Kalengi cansjava and Tsjeru cansjava), Morison
(Cannabis peregrina gemmis fructuum longioribus), and Kaempfer (Ba and Ma). In
summary, Linnaeus’sC. sativa taxon represents “rope, not dope”(McPartland et al.
2000). It does not align with vernacular “Sativa,”known for its potent
psychoactivity.
4.3 Formal Botanical Nomenclature: C. indica
Lamarck (1785) coined C. indica for plants of Indian provenance and their
descendants in Southeast Asia and South Africa. For a full account of his proto-
logue see McPartland and Guy (2017). The description of C. indica differed from
that of C. sativa by eight “very distinct”morphological characters, in stalks,
branching habitus, leaflets, and flowers. Lamarck noted fine details in C. indica,
“female flowers have a vellous calyx and long styles.”In other words, the perigonal
bract (“calyx”) is velvety (“vellous”), due to a dense pubescence of CSG trichomes.
Nearly 230 years passed before others noted long styles in C. indica (Small and
Naraine 2015a). Lamarck also described chemotaxonomic differences: C. indica
produced a strong odor, and caused intoxication when smoked in a pipe.
Lamarck’s type specimen at the Paris herbarium was collected by Pierre
Sonnerat, probably around Pondicherry. Lamarck’s specimen shows denser growth
and more compact branching than Linnaeus’s specimen (Fig. 4.1). Its inflorescences
Fig. 4.1 Herbarium type specimens of C. sativa L. (left), and C. indica Lam. (right), photographs
courtesy of McPartland and Guy (2017)
4Cannabis sativa and Cannabis indica …103

are somewhat dense; subtending floral leaves have an abundant covering of sessile
glandular trichomes; perigonal bracts express a moderate density of CSG trichomes.
The styles and stigmas are prominent, agglutinized, and light brown in color.
Lamarck listed six synonyms: Cannabi similis exotica Bauhin (who cited da
Orta and Acosta in Goa, India); Kalengi-cansjava and Tsjeru-cansjava Rheede
(plants from Kochi, India); Cannabis peregrina gemmis fructuum longioribus
Morison (who cited Bauhin and Rheede); C. indica Rumph (plants from Indonesia);
and Dakka ou Bangua Prévost (who cited Kolb in South Africa). In summary,
Lamarck delimited C. indica to plants from southern India and their descendants in
Indonesia and South Africa.
4.4 The Slide from Formal to Vernacular
How did the species names C. sativa and C. indica reappear inaccurately as
“Sativa”and “Indica”? We traced a path through Afghanistan by Nikolai Ivanovich
Vavilov and Richard Evans Schultes. Vavilov traveled there in 1924, where he
encountered Afghani farmers who cultivated Cannabis for gashisha (hashīsh). He
assigned these plants to C. sativa (Vavilov and Bukinich 1929). This departed from
Linnaeus’s concept of C. sativa as a European fiber-type plant. Vavilov also
encountered wild-type and feral plants, which he named, respectively, C. indica
var. kafiristanica and C. indica f. afghanica. His student Tatiana Yakovlevna
Serebriakova assigned Afghani plants to C. sativa, and Indian plants to C. indica
(Serebriakova and Sizov 1940).
Schultes travelled to Afghanistan in 1971. Schultes et al. (1974) narrowly typ-
ified C. indica to plants in Afghanistan, with broad, oblanceolate leaflets, densely
branched, with very dense inflorescences, more or less conical in shape, and very
short (<1.3 m). This departed from the original taxonomic concept of Lamarck,
who was entirely unfamiliar with Afghani Cannabis. Lamarck’sindica designates
Cannabis from India—relatively tall, laxly branched, with narrow leaflets.
Anderson (1980) echoed Schultes and assigned Afghani plants to C. indica—
short, conical, densely branched, with broad leaflets. He assigned plants from India
to C. sativa—relatively tall, laxly branched, with narrow leaflets—plants that
Lamarck would have called C. indica. Anderson illustrated these concepts in a line
drawing (Fig. 4.2) that now appears everywhere on the internet.
Clarke (1981) referred to plants from Afghanistan “as type examples for
Cannabis indica.”Cherniak (1982) assigned “cannabis sativa”to plants of South
Asian heritage (Nepal, Burma, Thailand), and their descendants in Columbia,
Jamaica, and Mexico. He applied the name “cannabis indica”to plants from
Afghanistan. His classification gets a bit muddled, because he also categorizes
plants from India as “cannabis indica.”The earliest consistent use of “Sativa”and
“Indica”appears in a Dutch seed catalog (Watson 1985).
104 J.M. McPartland

Meijer and van Soest (1992) brought attention to this vernacular taxonomy in
peer-reviewed literature: “Indica”refers to plants with broad leaflets, compact habit,
and early maturation, typified by plants from Afghanistan. “Sativa”refers to plants
with narrow leaflets, slender and tall habit, and late maturation, typified by plants
from India and their descendants in Thailand, South and East Africa, Colombia, and
Mexico.
Clinical descriptions of “Sativa”and “Indica”are barely a decade old (Corral
2001; Black and Capler 2003): “Sativa”plants produce much more ∆
9
-tetra-
hydrocannabinol (THC) than cannabidiol (CBD), and produce a terpenoid profile
that smells “herbal”or “sweet.”“Sativa”imparts a stimulating, uplifting, and
energizing psychoactivity, and is recommended for treating depression, headaches,
nausea, and loss of appetite. “Indica”plants produce a nearly equal THC-to-CBD
ratio, and a terpenoid profile that imparts an acrid or “skunky”aroma. “Indica”
induces relaxing, sedating, and pain-reducing effects, and is suggested for treating
insomnia, pain, inflammation, muscle spasms, epilepsy, and glaucoma.
McPartland et al. (2000) separated “Sativa”and “Indica”from European hemp,
and provisionally named the three populations C. indica, C. afghanica, and C.
sativa, respectively. Small (2007) noted that “Sativa”and “Indica”were “quite
inconsistent”with formal nomenclature, because C. sativa subsp. sativa applied to
non-intoxicant plants.
Hillig (2004a,b,2005a,b) avoided formal/vernacular conflicts by applying the
name “narrow-leaflet drug (NLD) biotype”to plants corresponding with Lamarck’s
C. indica. He assigned “wide-leaflet drug (WLD) biotype”to plants corresponding
with Vavilov’safghanica (i.e., Schultes’sC. indica and vernacular “Indica”). This
nomenclature has gained traction (e.g., McPartland and Guy 2004; Russo 2007;
Lynch et al. 2015).
“Indica”
“Sativa”
Fig. 4.2 Cannabis
vernacular taxonomy, image
adapted from Anderson
(1980)
4Cannabis sativa and Cannabis indica …105

McPartland and Guy (2004) and McPartland (2014) proposed reconciling
“Sativa”and “Indica”with C. sativa and C. indica by correcting the vernacular
nomenclature: “Sativa”is really indica, and “Indica”is actually afghanica, and
“Ruderalis”is usually sativa. The initial reaction to this proposition by recreational
users was negative. An editorial in High Times characterized the corrected
nomenclature as “undoubtedly a little kooky”(Sirius 2015).
Researchers, however, are starting to take it on board (e.g, Henry 2015). Clarke
and Merlin (2016) published a vernacular correction nearly identical to McPartland
(2014), although they did not cite the precedent publication. Two table headings in
their respective taxonomic tables are exampled:
•McPartland (2014): Indica (formerly “Sativa”)
•Clarke and Merlin (2016): Indica—Wrongly called “sativa”.
The title of their article is adapted from other antecedents, also uncited
(Tejkalováand Hazekamp 2014; Piomelli and Russo 2016). If these phrases were
botanical names, a taxonomist would invoke the principle of priority. For example,
Clarke and Merlin (2013) erected a new biotype name, “BLD”(broad leaf drug).
They objected to Hillig’s names of biotypes based on leaf shape. Nevertheless,
invoking priority, BLD is a later synonym of Hillig’s WLD. Similarly, Clarke and
Merlin (2015) strenuously rejected Small’s taxonomic character “intoxicant.”They
replaced it with “psychoactive.”
Erkelens and Hazekamp (2014) outlined the history of “Indica”and they
emphasized taxonomical conflicts between monotypic and polytypic views of
Cannabis. For the rest of this chapter, “Sativa”refers to the NLD biotype, or plants
of Indian heritage (including their putative descendants in Southeast Asia, Africa,
and the Americas). “Indica”refers to the WLD biotype, or Afghani landraces
(including related populations in northwestern Pakistan bordering Afghanistan, and
possibly neighboring Turkestan—Uzbekistan and Xīnjiāng).
4.5 The Hybridization Impasse
Selective cross-breeding of drug-type Cannabis accelerated in the 1970s.
Germplasm from Afghanistan was smuggled into California in the early 1970s (D.
Watson, pers. commun. 1984), or the late 1970s (Clarke 1987). During the 1980s at
least seven Cannabis breeders sold exotic germplasm in Holland. They crossed
plants of Indian heritage (“sweet but late maturing”) with Afghani landraces, valued
for rapid maturation, cold-tolerance, short stature, and dense, tightly-packed flower
clusters. By the late 1980s, nearly all drug-type Cannabis grown in the USA,
Canada, and Europe had been hybridized. Unadulterated plants of Indian heritage
and Afghani landraces became difficult to obtain (Clarke 1987).
Alarmingly, foreign germplasm has corrupted Indian and Afghani landraces in
their former centers of diversity. Peterson (2009) deplored the importation of
106 J.M. McPartland

“Skunk #1”into South Africa around 1984, which “destabilized”the genepool.
Jamaicans have replaced gañjāof Indian origin with Afghani hybrids (J. McP, pers.
observ. 2013). Beisler (2006) boasted of importing and growing “Mexican Gold”in
Afghanistan around 1972. Pietri (2009) stated that Beisler crossed “Acapulco Gold”
with Afghani landraces. Turner et al. (1979b) analyzed 12 accessions collected in
northwest India, and some plants in Punjab expressed low THC/CBD profiles
suggestive of Afghani landraces.
Ubiquitous hybridization of “Sativa”and “Indica”has rendered their distinctions
almost meaningless. Most hybrids are characterized as “Sativa-dominant”or
“Indica-dominant.”The arbitrariness of these designations is illustrated by
“AK-47,”a hybrid that won “Best Sativa”in the 1999 Cannabis Cup, and won
“Best Indica”four years later. Hybrids have been assigned “strain”names. The
desire for unique weed has led to an explosion of new strain names. At the dawn of
this era, Watson (1985) offered 10 strains for sale. Fifteen years later, Clarke (2001)
estimated that Dutch seed companies offered 150 strains for sale, and 80% of them
contained hybridized ancestry from Watson’s original strains. A decade later the
number of named strains reached 900 (Cannabis Strain Database 2010). Leafly
(2015) listed 1535 strain names, and Seedfinder (2015) listed 6510 strain names.
Doyle (2007) called the strain names “ganjanyms.”
In today’s largely illicit market, strain names are swapped and counterfeited, and
generally unreliable (Lee 2013; Sawler et al. 2015; Pierson 2016). Unrecognized
hybrids have plagued recent taxonomic studies of “Sativa”and “Indica.”
Unrecognized hybrids assigned to C. sativa or C. indica dampen signal in any
taxonomic methodology. Widespread crossbreeding and introgression make it
challenging to meet the third goal of this book chapter: identifying differences
between the NLD biotype (“Sativa”) and the WLD biotype (“Indica”). The biotypes
show differences in cannabinoids, terpenoids, and genetics.
In the next couple sections of this chapter, analytical studies that measured
cannabinoids and terpenoids in NLD and WLD biotypes will be compared. This
comparison is hampered by the fact that different studies used different analytical
methods (e.g., gas chromatography versus high performance liquid chromatogra-
phy). These analytical methods may vary in their yields of cannabinoids and ter-
penoids (Wheals and Smith 1975; Hazekamp et al. 2005; Giese et al. 2015).
Cannabinoid and terpenoid content is best measured in a common garden experi-
ment (CGE), where plants from different places are grown in a single location,
under identical environmental conditions, and uniformly processed.
4.6 Cannabinoids
Cannabinoid content differs in terms of quantity and quality; these differ in their
modes of inheritance (Hillig 2002). Cannabinoid quantity (dry weight percentage)
is polygenic and influenced by environmental factors. Cannabinoid quality (the
THC/CBD ratio, known as the cannabinoid profile or chemotype) is largely genetic
4Cannabis sativa and Cannabis indica …107

—possibly monogenic. For more information regarding the differences between
quantity and quality, see this book’s chapter by Grassi and McPartland. The
THC/CBD ratio, a dimentionless ratio, cancels two quantities (THC%, CBD%), and
provides a more valid comparison between plants cultivated in different
environments.
Hillig and Mahlberg (2004) published an exemplary CGE: 157 Cannabis
accessions from around the world, with passport data regarding provenance.
Accessions were classified into seven biotypes, with a priori segregation based on
geographic origins and a genetic analysis (Hillig 2005a). We will focus on two
populations in their study: the WLD biotype (n = 12 from Afghanistan, the
North-West Frontier Province of Pakistan, and Uzbekistan) and the NLD biotype
(n = 27 from India, Thailand, Cambodia, Mexico, Colombia, Jamaica, South- and
East Africa).
They prepared a voucher specimen of each accession, deposited in a herbarium.
Voucher specimens are critical for authenticating the identification of a taxon;
vouchers allow other researchers to retrospectively analyse accessions (Culley
2013). Hillig and Mahlberg made a great effort to exclude hybrids. For example,
“Not everything from Afghanistan is Afghani”(Hillig, pers. commun., 2006). An
examination of their vouchers reveals that a few hybrids snuck into their analysis.
They cultivated plants in a glasshouse under natural and supplemental light, and
staggered the harvest to sample each accession at peak, uniform maturity.
Cannabinoids were measured in individual plants, rather than in bulked samples.
Results obtained from NLD and WLD biotypes are presented in Table 4.1.
Hillig and Mahlberg reported a statistical difference in CBD%, but no statistical
difference in THC% (Table 4.1). They depicted cannabinoid profiles in graphs
(histograms and Cartesian graphs), but they did not present actual numerical data.
We calculated cannabinoid profiles of NLD and WLD biotypes from their data in
Table 4.1, as the quotient of THC/CBD. NLD biotype = 5.48/0.02 = 274.0, WLD
biotype = 6.49/1.21 = 5.4. The order of magnitude difference between 274.0 and
5.4 is significant, although statistical inferences cannot be calculated for n = 2.
The WLD biotype produced a much greater concentration of cannabinoids
(represented by THC% + CBD% in Table 4.1). This is likely due to the WLD
biotype’s greater density of perigonal bracts, and greater expression of CSG tri-
chomes on floral leaves, compared to NLD plants. The size of resin heads (gland
heads) may also differ. Small and Naraine (2015b) measured resin head size in ten
strains of “high-THC medical marijuana”(WLD-NLD hybrids), which averaged
129 µm in diameter. Seven cultivars of low-THC industrial hemp averaged 81 µm.
Table 4.1 Cannabinoid content (mean ±standard deviation) in two Cannabis biotypes, data
from Hillig and Mahlberg (2004)
THC% CBD%
a
THC% + CBD%
a
THCV% + CBDV%
a
NLD biotype 5.48 ±2.41 0.02 ±0.02 5.50 ±2.42 0.25 ±0.40
WLD biotype 6.49 ±4.09 1.21 ±2.78 7.70 ±3.45 0.14 ±0.30
a
Means in this column are statistically different using Student’s pairwise ttest (p!0.05)
108 J.M. McPartland

Previous studies of CSG trichome density did not include Afghani plants (Small
et al. 1976; Turner et al. 1977).
Hillig and Mahlberg measured cannabigerol (CBG), cannabigerol-
monomethylether (CBGM), and cannabichromene (CBC), with no statistical dif-
ferences between NLD and WLD biotypes. NLD biotypes produced more
tetrahydrocannabivarin (THCV) and cannabidivarin (CBDV) than WLD biotypes.
THCV and CBDV are the short-tailed C
19
analogs of THC and CBD, respectively.
This trend can be seen in data reported by Turner et al. (1973). Some researchers
include C
19
analogs in the calculation of cannabinoid profiles, as THC
+THCV/CBD+CBDV (Turner et al. 1979a; Onofri et al. 2015; Welling et al. 2016).
Prior to Hillig and Mahlberg, few CGEs studied Afghani landraces. Holley et al.
(1975) analyzed a worldwide collection of females, males, mixtures of females and
males, immature plants, and cross-pollinated hybrids. Here are some of their results,
in nonhybridized females, as THC%, CBD%, and the quotient of THC/CBD:
Afghanistan: 0.59/1.26 = 0.47, India A: 1.78/0.03 = 59.3, India E:
3.31/0.02 = 165.5, Nepal: 2.75/0.02 = 137.5, Pakistan: 1.32/0.01 = 132, South
Africa D: 1.84/0.01 = 184, South Africa E: 0.62/0.06 = 10.3, South Africa F:
0.33/0.01 = 33, and Brazil: 2.16/0.01 = 216. In summary, the results by Holley and
colleagues are similar to those of Hillig and Mahlberg: the THC/CBD ratio in
Afghani landraces was much lower than that of NDL landraces.
Meijer et al. (1992) analyzed 97 accessions, many of uncertain provenance (e.g.,
“Nederwiet”), or hybridized material, such as “Skunk #1.”They included three
accessions from Afghanistan: “Rjaf 1”: 1.15/1.60 = 0.720; “Afghanistan”:
1.69/0.25 = 6.76; and “Afgaan”: 2.00/1.18 = 1.69. No samples from India were
included in the study. Two early CGEs that lacked Afghani plants were never-
theless instructive, because they analyzed plants of Indian heritage prior to the era
of widespread hybridization. Fetterman et al. (1971) measured cannabinoid ratio,
including cannabinol (CBN), as THC+CBN/CBD, in samples from Mexico’68: 1.0
+0.55/0.075 = 20.5, Mexico’69: 1.4+0.073/0.12 = 12.3, Thailand A:
2.2 + trace/0.16 = 13.8, Thailand B: 1.3 + trace/0.11 = 11.8.
Small and Beckstead (1973) analyzed 350 accessions from around the world.
Many accessions came from botanical gardens, of questionable provenance (e.g.,
three indica accessions with no measurable THC). Here are some of their results—
accessions of Indian heritage with solid passport data, presented as THC%, CBD%,
and THC/CBD. India: 1.58/0.15 = 10.5, Malawi A: 1.44/0.5 = 28.8, Malawi B:
1.92/0.11 = 17.45, Malawi C: 0.90/0.07 = 12.86, South Africa: 1.34/0.09 = 14.89,
Rhodesia: 0.73/0.06 = 12.17, Cambodia: 1.03/0.12 = 8.5, Uganda: 2.56/0.34 =
7.53, Mauritius: 1.90/0.26 = 7.31, Mexico: 1.52/0.23 = 6.61, Jamaica: 1.19/0.3 =
3.97. No Afghani landraces were included in the study.
Comparisons of police-confiscated samples lack the accuracy of CGEs.
However, some studies are instructive because they predate the era of widespread
hybridization. Marshman et al. (1976) tested 36 samples from Jamaica, with a mean
of 3.03/0.10 = 29.5 (two samples reached 99.0 and 104.4). Jenkins and Patterson
(1973) measured THC, CBN, and CBD in herb and hashīsh seizures. Means were
calculated from their raw data: Afghanistan (n = 4): 52.0 + 12.0/36.1 = 1.77,
4Cannabis sativa and Cannabis indica …109

Pakistan (n = 19): 35.7 + 16.1/48.2 = 1.07, South Africa (n = 6): 75.6 +
16.0/8.5 = 10.78, Jamaica (n = 7): 77.5 + 13.4/9.1 = 9.99, Burma (n = 5):
15.7 + 67.9/16.34 = 5.12.
Mobarak et al. (1978) analyzed hashīsh from “Kandeh in Petschtal,”a.k.a.
Kandai in Pech River valley—just 55 km from where Vavilov collected C. indica
var. afghanica. They report THC + CBN/CBD as 8 + 14.4/11.6 = 1.93. Martone
et al. (1990) analyzed THC+CBN/CBD in hashīsh seizures, including Afghanistan:
4.45 + 0.36/1.73 = 2.78 and India: 4.48 + 0.40/1.59 = 3.07.
Researchers after Hillig and Mahlberg faced greater difficulties parsing hybrids
from their studies. Mahlberg and Hillig collected germplasm during the 1970s–
1990s. Since then, unadulterated landraces have become needles in haystacks. For
example, de Meijer and colleagues recently reported Afghani plants with extremely
high THC/CBD quotients (e.g., 683.7 and 516.6, Onofri et al. 2015), or extremely
low THC/CBD quotients (e.g., 0.04, Meijer et al. 2009). These results depart from
an earlier study by Meijer et al. (1992), where THC/CBD quotients for Afghani
plants averaged around 3.1 (Meijer et al. 1992).
Researchers in Holland analyzed 11 strains in a non-CGE study (Fischedick
et al. 2010). No provenance was provided, partially due to proprietary rights. Also,
the operational gray-zone of Dutch coffeeshops (“legal front door, illegal back
door”) impedes information transfer regarding passport data and provenance. Six
strains were considered nonhybridized “Indicas”:“AD,”“AF,”“AM,”“AN,”
“AO,”and “Bedropuur.”All six were essentially devoid of CBD. This was a major
departure from studies of Afghani landraces collected in the 1970s–1990s, which
had significant CBD levels. The lack of CBD in 21st century “Indicas”is incon-
sistent with Afghani landraces from the 1970s–1990s.
The same group (Tejkalováand Hazekamp 2014; Tejkalová2015) conducted an
enlarged study of “typical representatives”of “Sativa”(n = 44) and “Indica”
(n = 77). They obtained samples from Dutch coffeeshops and proprietary sources
(Bedrocan BV, HempFlax BV), with limited information regarding provenance.
They used a multivariate clustering method, Principal Component Analysis (PCA).
The PCA scatterplot clearly discriminated “Sativa”samples from “Indica”samples,
but THC and CBD did not provide discriminatory value (i.e., the PCA weights or
eigenvector values for THC and CBD did not discriminate between “Sativa”and
“Indica”).
Hazekamp et al. (2016) adjusted their sample size to “Sativa”(n = 68) and
“Indica”(n = 63), obtaining samples from the same sources. This time they pre-
sented a PCA scatterplot as well as numerical means. They found no significant
differences between “Sativa”and “Indica”in either THC or CBD content. “Sativa”
THC/CBD means 12.74/0.38 = 33.5; “Indica”THC/CBD means 13.71/0.30 =
47.7.
Elzinga et al. (2015) analyzed 35 strains obtained from “chemotypical medicinal
cannabis dispensaries.”They assigned strains to “Indica,”“Sativa,”or “Hybrid”
based on reports by the Leafly website. Instead of THC/CBD ratios, they presented
“average THCmax%”for each strain. “Indica”(n = 13) averaged 17.30%, and
“Sativa”(n = 5) averaged 13.84%. For CBD they offered only summary statistics
110 J.M. McPartland

for all 35 strains: mean 0.6%, median 0.3%. Only one strain produced >1.49%
CBD. Based on such low CBD levels, it can be deduced that all but one of their
strains were high THC hybrids. They say so in a roundabout way, “previous papers
used samples collected worldwide, and based upon their reported cannabinoid
levels, are not representative of the cannabi[s] currently available in the United
States to patients and recreational users.”Not surprisingly, their PCA analysis “does
not support the classification between indica and sativa as it is commonly pre-
sented”(i.e., classification by Leafly).
Lynch et al. (2015) concatenated databases for a genotype-chemotype study,
which may explain their unique results. They filtered two large databases of strain
sequences, and found 195 strains with common polymorphic sites (see genetics
section). Cluster analysis of polymorphic sites sorted the 195 strains into groups
named WLD and NLD biotypes. The Strain Fingerprint™database, developed by
Steep Hill Labs (and displayed by Leafly on its website) included chemotype
information for 54 of the 195 strains. Lynch and colleagues presented histograms of
mean THC% and CBD%, from which cannabinoid ratios can be estimated: WLD:
16.5/0.2 = 82.5, NLD: 14.2/2.2 = 6.45. Thus the latest THC/CBD ratios of 21st
century ganjanyms shows a stunning reversal of THC/CBD ratios compared to their
corresponding 1970s–1990s landraces.
4.7 Terpenoid Studies
Terpenoids include simple terpenes (isoprenes) and modified terpenes—where
methyl groups have been moved or removed, or oxygen added as alcohols, esters,
aldehydes, or ketones. The characteristic odor of Cannabis comes from its unique
blend of monoterpenoids (C
10
H
16
templates) and sesquiterpenoids (C
15
H
24
tem-
plates). Terpenoids provide a key distinguishing feature between “skunky”Afghanis
and “herbal-sweet”plants of Indian heritage (Black and Capler 2003). Despite this
key diagnostic feature, few terpenoid studies have included Afghani landraces.
Hood and Barry (1978) analyzed “headspace”—the odor given off by plants, rather
than contents of glandular trichomes. Headspace favors the detection of monoter-
penoids over less-volatile sesquiterpenoids. Hood and Barry quantified 17 terpenoids in
14 accessions, including plants from Afghanistan and Pakistan (n = 3) and plants from
India and Mexico (n = 5). Running statistics on their raw data revealed some terpenoids
with statistical differences: Hood and Barry reported more limonene in Af/Pak plants
(mean 16.5% ±1.66 SD) than Indi-Mex plants (6.5% ±1.01, p< 0.001), and more
b-farnesene in Indi-Mex (0.44% ±0.13) than Af/Pak (0.10% ±0.05, p= 0.10).
Differences in three other terpenoids fell a little short of statistical significance: more b-
caryophyllene in Indi/Mex (3.0% ±0.39) than Af/Pak (1.9% ±0.52, p= 0.16), more
a-humulene in Indi/Mex (0.76% ±0.20) than Af/Pak (0.53% ±0.15, p= 0.20), and
more b-myrcene in Af/Pak (10.0% ±0.53) than Indi/Mex (7.6% ±1.3, p= 0.21).
Hillig (2004b) identified 21 terpenoids in a subset of the Cannabis collection that
he analyzed for cannabinoids. He compared terpenoid profiles in WLD biotypes
4Cannabis sativa and Cannabis indica …111

(n = 9) and NLD biotypes (n = 21), using a PCA analysis. The PCA scatterplot
clearly discriminated WLD plants from NLD plants. Four terpenoids with the
greatest discriminatory value (i.e., greatest PCA weight or eigenvector value) were
sesquiterpene alcohols: guaiol, c-eudesmol, b-eudesmol, and a peak tentatively
identified as a-eudesmol. All significant differences (p< 0.05) are presented in
Table 4.2. Regarding b-myrcene, Hillig reported the same trend as Hood and Barry:
WLD = 9.0%, NLD = 5.8%, falling short of statistical significance.
Fischedick et al. (2010) analyzed 23 terpenoids in six strains considered non-
hybridized “Indicas”:“AD,”“AF,”“AM,”“AN,”“AO,”and “Bedropuur.”As
mentioned above, the “Indica”strains had no quantifiable CBD, so they likely were
unrecognized hybrids. They also analyzed five strains classified as hybrids: “AE”
(“mostly Sativa”), “AG”(“Indica/Sativa”), “Ai94”(“mostly Sativa”), “Bediol”
(“Indica/Sativa/Ruderalis”), “Bedrocan”(“Indica/Sativa”). (“Indica dominant”).
They made an interesting discovery: three “Indicas”(i.e, unrecognized hybrids
(“Bedropuur,”“AO,”and “AF”) expressed measurable levels of guaiol, c-eudes-
mol, and b-eudesmol. These sesquiterpene alcohols are unique to Afghani landraces
(Hillig 2004b). Furthermore, the same three strains contained higher levels of
limonene than the other accessions, results consistent with Hillig. Thus hybridized
“Indicas,”despite selection for elevated THC/CBD ratios, retained unique ter-
penoids in common with their landrace ancestors.
Hazekamp and Fischedick (2012) identified terpenoids in more hybrids, two
“Sativa dominant”samples (“Amnesia,”“Bedrobinal”) and two “Indica dominant”
samples (“White Widow,”“Bedica”). Once again, only “Indica dominant”hybrids
contained guaiol, c-eudesmol, and b-eudesmol.
The aforementioned study of samples from Dutch coffeeshops and proprietary
sources (Tejkalováand Hazekamp 2014; Tejkalová2015) analyzed 21 monoter-
penoids and 19 sesquiterpenoids. Multivariate clustering with PCA produced a
scatterplot that segregated “Sativa”and “Indica”into distinct clusters, with some
Table 4.2 Terpenoid
concentration
(mean ±standard deviation)
in NDL and WLD biotypes,
reported as statistically
different by Hillig (2004b)
NDL WLD
Limonene 1.3% ±1.2 4.0% ±4.3
c-terpinene 0.2% ±0.2 0.1% ±0.2
b-fenchol 0.2% ±0.2 0.8% ±0.9
Terpinoline 4.4% ±8.0 1.0% ±2.9
b-caryophyllene 15.7% ±7.2 9.7% ±6.2
a-guaiene 1.0% ±1.3 0.4% ±0.7
Trans b-farnesene 7.6% ±4.4 4.1% ±3.3
Caryophyllene oxide 8.9% ±7.9 4.2% ±4.2
Guaiol 0.2% ±0.4 3.5% ±1.8
c-eudesmol 0.6% ±0.6 4.8% ±2.1
b-eudesmol 0.8% ±0.6 7.4% ±4.0
a-eudesmol (peak 41) 0.1% ±0.3 1.4% ±1.4
Percentages are ratios of individual peak areas relative to the total
area of all 48 terpenoid peaks
112 J.M. McPartland

overlap and outliers. “Indica”strains produced more guaiol, c-eudesmol, and b-
eudesmol, as well as another sesquiterpene alcohol—a-bisabolol, plus three mon-
terpene alcohols: a-terpineol, b-fenchol, and linalool. “Sativa”strains leaned
towards unoxygenated sesquiterpenes: a-humulene, trans-b-caryophyllene, a-
guaiene, and trans-a-bergamotene.
Hazekamp et al. (2016) adjusted their sample size to “Sativa”(n = 68) and
“Indica”(n = 63), and analyzed 17 monoterpenoids and 19 sesquiterpenoids. Once
again, “Indica”samples produced more sesquiterpene alcohols than “Sativa”
samples (guaiol, c-eudesmol, b-eudesmol, and a-bisabolol), as well as more
monterpene alcohols (a-terpineol, b-fenchol, and linalool). This time they also
report two more monterpene alcohols (cis-sabinene hydrate, borneol) in “Indica.”
They concluded that hydroxylated terpenoids in general, not just sesquiterpene
alcohols, distinguished “Indica”strains. They also report significantly more limo-
nene and myrcene in “Indica,”consistent with Hillig’s Afghani landraces (although
they erroneously state that Hillig found less myrcene in Afghani landraces).
Mansouri et al. (2011) analyzed terpenoids in Iranian plants, which expressed
significant amounts of b-eudesmol and c-eudesmol, like plants of Afghani heritage.
Casano et al. (2011) compared 16 unnamed hybrid accessions, characterized as
“mostly Indica”or “mostly Sativa.”“Mostly Indica”plants produced significantly
higher levels of limonene, b-myrcene, and camphene. “Mostly Sativa”produced
significantly higher levels of sabinene, ∆-3-carene, a-phellandrene, 1,8-cineole, cis-
b-ocimene, trans-b-ocimene, and a-terpinolene.
Elzinga et al. (2015) assigned strains to “Sativa”or “Indica”according to the
Leafly database, as described earlier. They noted that strains named Kush,“char-
acteristic of the wide leaflet drug type strains originating from Hindus Kush region
of Afghanistan and Pakistan,”contained higher levels of guaiol, b-eudesmol, b-
myrcene, trans-ocimene, and b-pinene.
Lynch et al. (2015) concatenated databases for a genotype-chemotype study, see
explanation. They reported seven terpenoids in strains assigned to the NLD biotype
(n = 35) or the WLD biotype (n = 17). NLDs produced greater levels of b-myrcene
and a-terpinolene (0.48% and 0.16%, respectively) than did WLDs (0.35% and
0.09%). WLDs produced greater levels of linalool (0.08%) than did NLDs (0.02%).
No statistically significant differences were seen in limonene, a-pinene, b-car-
yophyllene, and caryophyllene oxide. No sesquiterpene alcohols were measured.
Terpenoids modulate the effects of THC (McPartland and Pruitt 1999;
McPartland and Russo 2001). Two terpenoids in particular have gained attention.
Anonymous (2006) claimed that b-myrcene added to THC made the drug sensation
more “physical, mellow, sleepy,”whereas limonene added to THC made the drug
sensation more “cerebral and euphoric.”Russo (2011) attributed the sedative
“couch-lock”of “Indica”to b-myrcene, and Russo (2016) attributed the uplifting
effects of “Sativa”to limonene. Chemotype studies do not entirely support these
observations. Regarding limonene, earlier studies showed greater amounts in
Afghani landraces than in plants of Indian heritage (Hood and Barry 1978; Hillig
2004b). This trend was seen in some recent studies of “mostly Indica”
4Cannabis sativa and Cannabis indica …113

(i.e., Afghani), versus “mostly Sativa”(i.e., plants of Indian heritage) (Fischedick
et al. 2010; Casano et al. 2011; Hazekamp et al. 2016). Other studies of 21st century
ganjanyms show no differences in limonene between “Indica”and “Sativa”
(Elzinga et al. 2015; Lynch et al. 2015).
Regarding b-myrcene, earlier studies showed greater amounts in Afghani lan-
draces than in plants of Indian heritage, albeit short of statistical significance (Hood
and Barry 1978; Hillig 2004b). This trend continued in four recent studies of
“Indica”versus “Sativa”(Fischedick et al. 2010; Casano et al. 2011; Elzinga et al.
2015; Hazekamp et al. 2016), although other studies show no differences
(Hazekamp and Fischedick 2012), or even a reversal of earlier results (Lynch et al.
2015).
4.8 Genetic Studies
Hillig (2005a) analyzed allozyme variation in the same Cannabis collection tested
for cannabinoids. Samples were evaluated for variation at 17 gene loci, and fre-
quencies of 52 alleles were subjected to PCA. The PCA scatterplot segregated
drug-type plants and fiber-type plants into distinct clusters, but the WLD ellipse and
NLD ellipse substantially overlapped.
Gilmore et al. (2007) examined 76 Cannabis accessions for five polymorphic loci
sequenced from chloroplast and mitochondrial DNA. The study’sflaws are manifold,
but parsimony analysis recovered three clades. Clade A comprised a majority of
fiber-type plants. Clade B included Afghani landraces along with most drug strains—
hybrids and police seizures. Clade C was the most interesting—nothing but classic
“Sativas”: 12 landraces from India, Nepal, Thailand, Jamaica, Mexico, and Africa.
Gilmore (2005) gave the name C. sativa rasta to plants in Clade C.
Knight et al. (2010) tested six seized plants, identified by their morphology as
“Sativa”(n = 2) or “Indica”(n = 4). Five short tandem repeat (STR) loci, analyzed
with PCA, clearly segregated “Sativa”plants from three of the “Indica”plants. The
fourth “Indica”exhibited a unique genotype suggestive of a polyploid condition.
Piluzza et al. (2013) compared 19 accessions: one Afghani, five of Indian her-
itage, three “Skunk”hybrids, and an assortment of fiber-type plants from Europe
and East Asia. Six RAPD primers detected DNA polymorphisms, with haplotypes
clustered using a neighbor-joining algorithm. Plants of Afghani and Indian heritage
fell into separate clusters. Each shared interesting clade-mates. The Afghani lan-
drace was sister to a cluster of fiber-type plants. The cluster of Indian heritage plants
was sister to the “Skunk”cluster.
Onofri et al. (2015) searched for single nucleotide polymorphisms (SNPs) in
THCA-S sequences. They found nine unique THCA-S sequences amongst 18
accessions of fiber- and drug-type plants. Two accessions were Afghani plants, and
they expressed three polymorphic sequences between them. One sequence was
shared by plants of Indian heritage, and the other two sequences were unique to
Afghani plants.
114 J.M. McPartland

Next-Gen sequencing (high-throughput sequencing) has generated a plethora of
genetic information. Van Bakel et al. (2011) used a whole genome shotgun
(WGS) method with Illumina technology to sequence “Purple Kush”and two hemp
cultivars, ‘Finola’and ‘USO-31.’Van Bakel and colleagues also obtained tran-
scriptomes (cDNA libraries) from different tissues in these plants. Soon two other
Cannabis genomes were sequenced with WGS/Illumina machines, “Chemdawg”
and “LA Confidential”(Medicinal Genomics Corporation 2011).
Tejkalová(2015) utilized Cannabis genomes (van Bakel et al. 2011) for
SNP-calling and genotyping with the KASP/SNPline platform. Haplotypes based
on 57 SNP positions for 44 samples of “Sativa”and 77 of “Indica”were analyzed
with STRUCTURE. This probabilistic software identifies the optimal number of
clusters (K) to divide a population, based on allele frequencies. Testing K values
from one to nine, the haplotype data best fit K = 2 (two populations), but
STRUCTURE’s assignment of individuals into “Sativa”and “Indica”matched
poorly with their a priori identification.
Sawler et al. (2015) used genotyping-by-sequencing (GBS), which utilizes
restriction enzymes to break the genome into short reads (WGS uses random
ligation). They coupled ApeKI enzymes with Illumina machines for SNP discovery
and genotyping in fiber-type and drug-type samples. GBS identified 14,031 SNPs
for analysis, after quality filtering. Drug-type strains were classified along a gradient
of ancestry proportions (percent “Sativa”vs. percent “Indica”) reported in online
strain databases.
Their PCA analysis of genetic structure (SNP variations) using PLINK 1.9
clearly segregated 43 fiber-type samples from 81 drug-type samples. The clusters of
“Sativa”and “Indica”partially overlapped. Proportional ancestry in each sample
correlated moderately (r
2=
0.36) with the principle component (PC axis 1) of
genetic structure. Similar results were obtained with fastSTRUCTURE, where data
from all 124 samples best fit K = 2. The inability to separate “Sativa”and “Indica”
and the poor correlation of report ancestry was due, in part, to counterfeit strain
names: In a comparison of 17 paired samples with the same strain name, six pairs
(35%) were dissimilar, and shared more genetic similarity with other strain names.
Sawler calculated the fixation index (F
ST
) between subgroups based on
identity-by-state (IBS, implemented in PLINK). F
ST
values range from 0 to 1; a
zero value indicates the subgroups interbreeding freely; a 1 value indicates the
subgroups are completely isolated from one another. The average F
ST
between
fiber- and drug-type plants was 0.156, which is similar to the degree of genetic
differentiation in humans between Europeans and East Asians. Average F
ST
between fiber-type plants and “100% Sativa”was 0.161; F
ST
between fiber-type
plants and “100% Indica”was 0.136; no comparison was made between “Sativa”
and “Indica.”
Medicinal Genomics Corporation (2015) used Reduced Representation Shotgun
(RRS) sequencing to identify 100,000–200,000 SNPs per strain. These data were
used to generate a nearest-neighbor tree with “Purple Kush,”‘Finola,’‘USO-31,’
and 50 ganjanym strains. Henry (2015) utilized open-access RRS data to evaluate
28 strains, using Adegenet 2.0. K-partition optimized at K = 1. PCA clustering with
4Cannabis sativa and Cannabis indica …115

a subset of 42 most-informative SNPs, however, clearly segregated three clusters:
“Sativa”(n = 17) “Indica”(n = 9), and two fiber-type strains. These results were
confirmed with a neighbor-joining algorithm.
Lynch et al. (2015) sequenced 60 accessions using WGS, and added to this
dataset seven previous WGS reads (Van Bakel et al. 2011, Medicinal Genomics
Corporation 2011). For SNP-calling they aligned sequences with the draft genome
(Van Bakel et al. 2011). Then they sequenced 182 accessions using GBS, with
ECoRI and MseI restriction enzymes, for SNP-calling. A subset of 195 accessions
from WGS and GBS shared 2894 SNPs for analysis.
Two algorithms were used to K-partition the 195 accessions. FLOCK recognized
K = 3 groups, and fast STRUCTURE optimized the data at K = 2. The authors
went with FLOCK, because of perceived shortcomings in fast STRUCTURE,
although these perceived differences are contentious (Anderson and Barry 2015).
The K = 3 groups were recognized as WLD biotypes (e.g., “Afghan Kush,”
“Chemdawg”), NLD biotypes (e.g., “Durban Poison,”“Easy Sativa”), and a
polyphyletic “hemp”group (e.g., ‘Finola,’“AC/DC,”Chinese hemp, Dagestan
plants).
Lynch and colleagues found no evidence for admixture (hybridization) in these
populations, based on results with the f
3
statistic and TreeMix. This seems unlikely,
given historical evidence of hybridization going back to the 1970s. TreeMix and the
f
3
statistic were developed with animal models; they may fall short with plants
having complicated histories of hybridization. TreeMix analyzes data with a
maximum of only 10 admixture (“migration”) events. The f
3
statistic must compare
three populations, so it was applied to the disputed FLOCK results.
They used vcftools to calculate F
ST
between each FLOCK population.
F
ST
= 0.099 between “hemp”and combined NLD + WLD, and F
ST
= 0.036
between WLDs and NLDs. More genetic heterozygosity existed within drug-type
plants (0.31%) than within fiber-type plants (0.22%, significant p< 0.001), which
they attributed to widespread hybridization of drug strains—an incongruous
hypothesis, given the previous paragraph.
Phylogenetic relationships between the 195 accessions were visualized in an
unrooted neighbor-joining network—a phylogenetic tree with reticulation (diver-
gence and hybridization among ancestral lineages). The network revealed aspects of
ancestry not captured by a simple bifurcating tree, such as genetic admixtures
between Chinese hemp and feral hemp plants in the USA.
Next they pooled WGS data with GBS data from Sawler et al. (2015), with 4105
SNPs in common, and generated a neighbor-joining network with 210 accessions.
These data revealed a second NLD biotype clade, consisting of Indian, Southeast
Asian, and South African populations, along with various “Haze”hybrids. This
clade may represent accessions of Indian heritage with minimal admixture from
WLD biotypes. Lastly they pooled WGS data with both GBS datasets, a total of
289 accessions, filtered for overlapping SNPs (only 45 SNPs in common—the two
GBS datasets were generated with different restriction enzymes), and used MEGA6
to generate a neighbor-joining tree.
116 J.M. McPartland

4.9 Conclusions
Research supports the classification of “Sativa”and “Indica,”but not their
nomenclature. “Sativa”(consistent with Lamarck’sindica, the NLD biotype) differs
chemically and genetically from “Indica”(consistent with Vavilov’safghanica, the
WLD biotype). The systematics of these populations remains an open question.
Systematics adds the element of time to taxonomy. “Sativa”and “Indica”pre-
sumably diverged from a common ancestor—but when, and under what selection
pressures? One population evolved in low-warm-and-wet India, and the other in
high-cool-and-dry Afghanistan. Natural selection likely drove their initial
divergence.
Good (1964) and Takhtajan (1986) divided the world into “floristic regions”
based on the distribution of distinctive (endemic) plant populations. The borders
between these floristic regions were delimited by natural barriers (geographic and
climatic) that prevented natural plant dispersal. Most of India lies in the Indian
Region. Afghanistan is part of the Irano-Turanian Region (Takhtajan’s term; Good
called it the Western and Central Asiatic Region). Another floristic region lies
between them, which includes most of Pakistan—the Sudano-Zambezian Region
(Takhtajan’s term), a.k.a. the North African-Indian Desert Region (Good’s term).
Then humans took over with artificial selection. In India, unpollinated females
were processed individually. Intentional selection of potent, high-THC individuals
was a straightforward process. In contrast, Afghani plants were processed in bulk,
with no selection of potent, high-THC individuals. Thus a millennium of selecting
different products—gañjāversus hashīsh—unintentionally drove divergence in
THC/CBD ratios. David Watson (pers. commun. 2012) stated that Afghani hashīsh
producers preferred certain terpenoids for aroma, and for physicochemical effects
on sifted hashīsh (e.g., the condensability of sesquiterpene alcohols). Consistent
with this, Hooper (1908) found the perceived quality and cost of three hashīsh
specimens from Kāšḡar correlated with their percentage of essential oil (i.e., ter-
penoids), and not with their percentage of resin (i.e., cannabinoids): Grade No. 1:
essential oil 12.7% and resin 40.2%; Grade No. 2: essential oil 12.4% and resin
40.9%; Grade No. 3: essential oil 12.0% and resin 48.1%.
Extensive cross-breeding between “Sativa”and “Indica”in the past 40 years has
rendered their distinctions almost meaningless in today’s marketplace. Plants
should be identified by their chemical fingerprint, rather than characterizations such
as “Sativa-dominant,”“Indica-dominant,”or a whimsical strain name (Hazekamp
and Fischedick (2012); Hazekamp et al. 2016). Several analytical laboratories have
moved “from cultivar to chemovar,”and identify plants by their cannabinoid and
terpenoid content. These services include Strain Fingerprint™by Steep Hill Labs,
PhytoFacts™by Napro Research, Profile Testing by Werc Shop, and Know Your
Medicine by SC Labs.
However, as documented here, phytochemical and genetic research supports the
separation of NLD and WLD biotypes. Old landraces of Indian and Afghani
4Cannabis sativa and Cannabis indica …117

heritage face extinction through introgressive hybridization. We need to recognize
this biodiversity and conserve it—for future breeding efforts, at the very least.
Acknowledgements Arno Hazekamp is thanked for helpful discussions regarding this
manuscript.
References
Anderson LC (1980) Leaf variation among Cannabis species from a controlled garden. Harvard
Univ Bot Mus Leaflets 28(1):61–69
Anderson EC, Barry PD (2015) Interpreting the FLOCK algorithm from a statistical perspective.
Mol Ecol Resour 15:1020–1030
Anonymous (2006) Studying the effects of terpenes. O’Shaughnessy’s Spring 2
Beisler J (2006) The bandit of Kabul. Regent Press, Oakland, CA
Black H, Capler R (2003) Operational standards for the distribution of medicinal cannabis. British
Columbia Compassion Club Society, Vancouver, BC
Cannabis Strain Database (2010) Available at http://weed-forums.com/strain-bank/10861-
cannabis-strain-database.html
Casano S, Grassi G, Martini V, Michelozzi M (2011) Variations in terpene profiles of different
strains of Cannabis sativa L. Acta Hortic 925:115–121
Cherniak L (1982) The great books of Cannabis, Vol. I: Book II. Cherniak/Damele Publishing Co.,
Oakland CA
Clarke RC (1981) Marijuana botany. And/Or Press, Berkeley, CA
Clarke RC (1987) Cannabis evolution. MS thesis, Indiana University, Bloomington, IN
Clarke RC (2001) Sinsemilla heritage—what’s in a name. In: King J (ed). The Cannabible. Ten
Speed Press, Berkeley CA
Clarke RC, Merlin MD (2013) Cannabis evolution and ethnobotany. University of California
Press, Berkeley
Clarke RC, Merlin MD (2015) Letter to the editor: Small, Ernest. 2015. Evolution and
classification of Cannabis sativa (marijuana, hemp) in relation to human utilization. Bot Rev
81:295–305
Clarke RC, Merlin MD (2016) Cannabis taxonomy: the ‘sativa’vs. ‘indica’debate. HerbalGram
110:44–49
Corral VL (2001) Differential effects of medical marijuana based on strain and route of
administration: a three-year observational study. J Cannabis Ther 1(3/4):43–59
Culley TM (2013) Why vouchers matter in botanical research. Appl Plant Sci 1(11): apps.1300076
Doyle R (2007) The transgenic involution”. In: Kac E (ed) Signs of life. Massachussetts Institute
of Technology, Cambridge, MA, pp 70–82
Elzinga S, Fishchedick J, Podkolinski R, Raber JC (2015) Cannabinoids and terpenes as
chemotaxonomic markers in Cannabis. Nat Prod Chem Res 3:4
Erkelens JL, Hazekamp A (2014) That which we call indica, by any other name would smell as
sweet. Cannabinoids 9(1):9–15
Fetterman PS, Seith ES, Waller CW, Guerrero O, Doorenbos NJ, Quimby MW (1971)
Mississippi-grown Cannabis sativa L.: preliminary observation on chemical definition of
phenotype and variations in tetrahydrocannabinol content versus age, sex, and plant
part. J Pharm Sci 60:1246–1249
Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R (2010) Metabolic fingerprinting
of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug
standardization purposes. Phytochemistry 71:2058–2073
118 J.M. McPartland

Giese MW, Lewis MA, Giese L, Smith KM (2015) Development and validation of a reliable and
robust metod for the analysis of cannabinoids and terpenes in Cannabis. J AOAC Int 98:1503–
1522
Gilmore S (2005) Rasta lends its name to a third ‘race’of cannabis. New Sci 187 (Sep 17-Sep 23): 12
Gilmore S, Peakall R, Robertson J (2007) Organelle DNA haplotypes reflect crop-use
characteristics and geographic origins of Cannabis sativa. Forensic Sci Int 172:179–190
Good R (1964) The geography of the flowering plants, 3rd edn. John Wiley, NY
Hazekamp A, Fischedick JT (2012) Cannabis—from cultivar to chemovar. Drug Testing and
Analysis 4:660–667
Hazekamp A, Giroud C, Peltenburg A, Verpoorte R (2005) Chromatographic and spectroscopic
data of cannabinoids from Cannabis sativa L. J Liq Chromatogr Relat Technol 28:2361–2382
Hazekamp A, TejkalováK, Papadimitriou S (2016) Cannabis: from cultivar to chemovar II—a
metabolomics approach to Cannabis classification. Cannabis Cannabinoid Res 1(1):202–215
Henry P (2015) Genome-wide analyses reveal clustering in Cannabis cultivars: the ancient
domestication trilogy of a panacea. PeerJ PrePrints https://peerj.com/preprints/1553.pdf
Hillig KW (2002) Letter to the editor. J Ind Hemp 7(1):5–7
Hillig KW (2004a) A multivariate analysis of allozyme variation in 93 Cannabis accessions from
the VIR germplasm collection. J Ind Hemp 9(2):5–22
Hillig KW (2004b) A chemotaxonomic analysis of terpenoid variation in Cannabis. Biochem Syst
Ecol 32:875–891
Hillig KW (2005a) Genetic evidence for speciation in Cannabis (Cannabaceae). Genet Resour
Crop Evol 52:161–180
Hillig KH (2005b) A systematic investigation of Cannabis. Doctoral Dissertation, Department of
Biology, Indiana University, Bloomington, IN
Hillig KW, Mahlberg PG (2004) A chemotaxonomic analysis of cannabinoid variation in
Cannabis (Cannabaceae). Am J Bot 91(6):966–975
Holley JH, Hadley KW, Turner CE (1975) Constituents of Cannabis sativa L. XI: cannabidiol and
cannabichromene in smaples of known geographical origin. J Pharm Sci 64:892–895
Hood LVS, Barry GT (1978) Headspace volatiles of marihuana and hashish: gas chromatographic
analysis of samples of different geographic origin. J Chromatogr 166:499–506
Hooper D (1908) Charas of Indian hemp. Year-Book of Pharmacy 1908:435–444
Jenkins RW, Patterson DA (1973) The relationship between chemical composition and
geographical origin of Cannabis. Foresnsic Sci 2:59–66
Knight G, Hansen S, Conner M, Poulsen H, McGovern C, Stacy J (2010) The result of an
experimental indoor hyroponic Cannabis growing study using the ‘screen of green’(ScOG)
method: yield, tetrahyrocannabinol (THC) and DNA analysis. Forensic Sci Int 202:36–44
Lamarck JB (1785) Encyclopédie Méthodique 1(2): 695. Panckoucke, Paris
Leafly (2015) Leafly online database. Available at: https://www.leafly.com
Lee MA (2013) Project CBD update: the tango of supply and demand. O’Shaughnessy’s
Winter/Spring 2013:22–23
Linnaeus C (1737) Hortus cliffortianus. George Clifford, Amsterdam
Linnaeus C (1753) Species Plantarum 2: 1057. Laurentii Salvii, Stockholm
Linnaeus C (1754) Genera Plantarum, 5th ed. Laurentii Salvii, Stockholm
Lynch RC, Vergara D, Tittes S, White K, Schwartz CJ, Gibbs MJ, Ruthenburg TC, deCesare K,
Land DP, Kane NC (2015) Genomic and chemical diversity in Cannabis. BioRxiv. doi:10.
1101/034314
Mansouri H, Asrar Z, Amarowicz R (2011) The response of terpenoids to exogenous gibberellic
acid in Cannabis sativa at flowering stage. Acta Physiologiae Plantarum 33:1085–1091
Marshman JA, Popham RE, Yawney CD (1976) A note on the cannabinoid content of Jamaican
ganja. Bull Narc 28:63–68
Martone G, Della Casa E (1990) Analysis of the ageing processes in hashish samples from
different geographic origin. Forensic Sci Int 47:147–155
Medicinal Genomics Corporation (2011) The cannabis sativa genome. Available at: https://aws.
amazon.com/datasets/the-cannabis-sativa-genome/
4Cannabis sativa and Cannabis indica …119

Medicinal Genomics Corporation (2015) KannaPedia. The distributed consensus on cannabis
genetics. Available at: www.medicinalgenomics.com/kannapedia/
McNeill J, (ed) (2012) International Code of Nomenclature for Algae, Fungi, and Plants
(Melbourne Code). Koeltz Scientific Books, Königstein, Germany
McPartland JM (2014) Corrected vernacular nomenclature: indica, afghanica, sativa.
O’Shaughnessy’s (J Calif Cannabis Res Med Group) Autumn 2014, p. 1
McPartland JM, Guy G (2004) The evolution of Cannabis and coevolution with the cannabinoid
receptor–a hypothesis. In: Guy G, Robson R, Strong K, Whittle B (eds) The medicinal use of
cannabis. Royal Society of Pharmacists, London, pp 71–102
McPartland JM, Guy GW (2014) A question of rank: using DNA barcodes to classifyCannabis
sativa and Cannabis indica. Proceedings of the 24th Annual Symposium on theCannabinoids.
International Cannabinoid Research Society, Research Triangle Park, NC, p 54
McPartland JM, Guy G (2017) Models of Cannabis taxonomy: a systematic review. Manuscript
under review, The Botanical Review
McPartland JM, Pruitt PL (1999) Side effects of pharmaceuticals not elicited by comparable herbal
medicines: the case of tetrahydrocannabinol and marijuana. Altern Ther Health Med 5:57–62
McPartland JM, Russo EB (2001) Cannabis and cannabis extracts: Greater than the sum of their
parts? J Cannabis Ther 1(3–4):103–132
McPartland JM, Clarke RC, Watson DP (2000) Hemp Diseases and Pests - Management and
Biological Control. CABI Publ, Wallingford, UK, p 251
de Meijer EPM (1999) “Cannabis germplasm resources.”In: Ranalli P, ed. Advances in Hemp
Research. Haworth Press New York. pp 133-151
Meijer EPM de, van Soest LJM (1992) The CPRS Cannabis germplasm collection. Euphytica
62:201–211
Meijer EPM de, van der Kamp HJ, van Eeuwijk FA (1992) Characterization of Cannabis
accessions with regard to cannabinoid content in relation to other plant characters. Euphytica
62:187–200
Meijer EPM de, Hammond KM, Micheler M (2009) The inheritance of chemical phenotype in
Cannabis sativa L. (III): variation in cannabichromene production. Euphytica 165:293–311
Mobarak Z, Bieniek D, Korte F (1978) Some chromatographic aspects of hashish analysis II.
Forensic Sci 11:189–193
Omar J, Olivares M, Alzaga M, Etxebarria N (2013) Optimisation and characterisation of
marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction
and retention time locking GC-MS. J Sep Sci 36:1397–1401
Onofri C, de Meijer EPM, Mandolino G (2015) Sequence heterogeneity of cannabidiolic- and
tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical
phenotype. Phytochemistry 116:57–68
Peterson C (2009) Prohibition and resistance: a socio-political exploration of the changing
dynamics of the southern African cannabis trade, c. 1850-the present. Masters thesis, Rhodes
University, Grahamstown, South Africa
Pierson D (2016) Why chocolope? To sell marijuana, you need a clever name. Los Angeles Times,
Feb. 9, 2016.
Pietri JR (2009) Who is Dr. Frankenbeanstein. Treat Yourself Mag 17:23–29
Piluzza G, Delogu G, Cabras A, Marceddu S, Bullitta S (2013) Differentiation between fiber and
drug types of hemp (Cannabis sativa L.) from a collection of wild and domesticated
accessions. Genet Resour Crop Evol 60:2331–2342
Piomelli D, Russo EB (2016) The Cannabis sativa versus Cannabis indica debate: an interview
with Ethan Russo. Cannabis Cannabinoid Res 1(1):44–46
Russo EB (2007) History of Cannabis and its preparations in saga, science, and sobriquet. Chem
Biodivers 4:1614–1648
Russo EB (2011) Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid
entourage effects. Br J Pharmacol 163:1344–1364
Russo EB (2016) The Cannabis sativa versus Cannabis indica debate: an interview with Ethan
Russo, MD. Cannabis Cannabinoid Res 1(1):44–46
120 J.M. McPartland

Sawler J, Stout JM, Gardner KM, Hudson D, Vidmar J, Butler L, Page JE, Myles S (2015) The
genetic structure of marijuana and hemp. PLoS ONE 10(8):e0133292
Schultes RE, Klein WM, Plowman T, Lockwood TE (1974) Cannabis: an example of taxonomic
neglect. Harvard University Botanical Museum Leaflets 23:337–367
Serebriakova TY, Sizov IA (1940) “Cannabinaceae,”In: Vavilov NI, Vul′f EV, eds., Культурная
флора СССР, Tom V (Cultural Flora of the USSR, Vol. 5). State Publishing House of
Collective-farm and State−farm Literture, Moscow-Leningrad. pp 1−53
Seedfinder (2015) Seedfinder online database. Available at: http://en.seedfinder.eu/
Sirius J (2015) A scientific approach: redefining weed. High Times Februrary 18, 2015
Small E (2007) Cannabis as a source of medicinals, nutraceuticals, and functional foods. Adv Med
Plant Res 2007:1–39
Small E, Beckstead HD (1973) Common cannabinoid phenotypes in 350 stocks of Cannabis.
Lloydia 36: 144–116
Small E, Naraine SGU (2015a) Expansion of female sex organs in response to prolonged virginity
in Cannabis sativa (marijuana). Genet Resour Crop Evol 63:339–348
Small E, Naraine SGU (2015b) Size matters: evolution of large drug-secreting resin glands in elite
pharmaceutical strains of Cannabis sativa (marijuana). Genet Resour Crop Evol 63:349–359
Small E, Jui P, Lefkovitch LP (1976) A numerical taxonomic analysis of Cannabis with special
reference to species delimitation. Syst Bot 1:67–84
Stern WT (1974) Typification of Cannabis sativa L. Harvard University Botanical Museum
Leaflets 23(9):325–336
Takhtajan AL (Crovello TJ transl, Cronquist A, ed.) (1986) Floristic regions of the world.
University of California Press, Berkeley
TejkalováK (2015) Cannabis sativa—objasněnívztahu mezi domnělými poddruhy. Master’s
thesis, Faculty of Natural Science, Masaryk University, Brno, Czech Republic
TejkalováK, Hazekamp A (2014) “The sativa-indica dilemma,”pg. P3-13. In: 24th Annual
Symposium on the Cannabinoids. International Cannabinoid Research Society, Research
Triangle Park, NC, USA
Turner CE, Hadley K, Fetterman PS (1973) Constituents of Cannabis sativa L. VI. Propyl
homologs in samples of known geographical origin. J Pharm Sci 62:1739–1741
Turner CE, Elsohly MA, Cheng PC, Lewis G (1979a) Constituents of Cannabis sativa L.; XIV:
intrinsic problems in classifying Cannabis based on a single cannabinoid analysis. J Nat Prod
42:317–319
Turner CE, Cheng PC, Lewis GS, Russell MH, Sharma GK (1979b) Constituents of Cannabis
sativa. XV: botanical and chemical profile of Indian variants. Planta Med 37:217–225
Turner JC, Hemphill JK, Mahlberg PG (1977) Gland distribution and cannabinoid content in
clones of Cannabis sativa. Am J Bot 64:687–693
Turner CE, ElSohly MA, Boeren EG (1980) Constituents of Cannabis sativa L. XVII. A review of
the natural constituents. Journal of Natural Products 43: 169-234
Turner JC, Hemphill J, Mahlberg PG (1980) Trichomes and cannabinoid content of developing
leaves and bracts of Cannabis sativa L. (Cannabaceae). Am J Bot 67:1397–1406
Van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, Page JE (2011) The draft
genome and transcriptome of Cannabis sativa. Genome Biol 12(10):R102
Vavilov NI, Bukinich DD (1929) Konopli. Trudy po Prikladnoi Botanike, Genetike i Selektsii 33
(Suppl.): 380–382
Watson DP (1985) Cultivator’s choice catalog #4. Self-published, Amsterdam, Holland
Welling MT, Liu L, Shapter T, Raymond CA, King GJ (2016) Characterisation of cannabinoid
composition in a diverse Cannabis sativa L. germplasm collection. Euphytica, epub before
print
Wheals BB, Smith RN (1975) Comparative cannabis analysis: A comparison of high-pressure
liquid chromatography with other chromatographic techniques. J Chromatogr A 105:396–400
4Cannabis sativa and Cannabis indica …121