Syst. Biol. 60(1):45–59, 2011
Published by Oxford University Press on behalf of the Society of Systematic Biologists 2010.
Advance Access publication on November 17, 2010
Species Delimitation under the General Lineage Concept: An Empirical Example Using
Wild North American Hops (Cannabaceae: Humulus lupulus)
PATRICK A. REEVES AND CHRISTOPHER M. RICHARDS∗
United States Department of Agriculture, Agricultural Research Service, National Center for Genetic Resources Preservation,
1111 South Mason Street, Fort Collins, CO 80521, USA;
∗Correspondence to be sent to: United States Department of Agriculture, Agricultural Research Service, National Center for Genetic Resources
Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA; E-mail: email@example.com.
Received 13 April 2009; reviews returned 1 October 2009; accepted 4 June 2010
Associate Editor: Mark Fishbein
Abstract.—There is an emerging consensus that the intent of most species concepts is to identify evolutionarily distinct
lineages. However, the criteria used to identify lineages differ among concepts depending on the perceived importance of
various attributes of evolving populations. We have examined five different species criteria to ask whether the three taxo-
nomic varieties of Humulus lupulus (hops) native to North America are distinct lineages. Three criteria (monophyly, absence
of genetic intermediates, and diagnosability) focus on evolutionary patterns and two (intrinsic reproductive isolation and
niche specialization) consider evolutionary processes. Phylogenetic analysis of amplified fragment length polymorphism
(AFLP) data under a relaxed molecular clock, a stochastic Dollo substitution model, and parsimony identified all varieties
as monophyletic, thus they satisfy the monophyly criterion for species delimitation. Principal coordinate analysis and a
Bayesian assignment procedure revealed deep genetic subdivisions and little admixture between varieties, indicating an
absence of genetic intermediates and compliance with the genotypic cluster species criterion. Diagnostic morphological
and AFLP characters were found for all varieties, thus they meet the diagnosability criterion. Natural history informa-
tion suggests that reproductive isolating barriers may have evolved in var. pubescens, potentially qualifying it as a species
under a criterion of intrinsic reproductive isolation. Environmental niche modeling showed that the preferred habitat of
var. neomexicanus is climatically unique, suggesting niche specialization and thus compliance with an ecological species
criterion. Isolation by distance coupled with imperfect sampling can lead to erroneous lineage identification using some
species criteria. Compliance with complementary pattern- and process-oriented criteria provides powerful corroboration
for a species hypothesis and mitigates the necessity for comprehensive sampling of the entire species range, a practical
impossibility in many systems. We hypothesize that var. pubescens maintains its genetic identity, despite substantial niche
overlap with var. lupuloides, via the evolution of partial reproductive isolating mechanisms. Variety neomexicanus, con-
versely, will likely persist as a distinct lineage, regardless of limited gene flow with vars. lupuloides and pubescens because
of ecological isolation—adaptation to the unique conditions of the Rocky Mountain cordillera. Thus, we support recogni-
tion of vars. neomexicanus and pubescens as species, but delay making a recommendation for var. lupuloides until sampling
of genetic variation is complete or a stable biological process can be identified to explain its observed genetic divergence.
[AFLP; Cannabaceae; hops; niche model; phylogeography; species delimitation.]
“...how entirely vague and arbitrary is the
distinction between species and varieties.”
(Darwin 1859, p. 48)
Historically, the two fundamental goals of systematic
biology have been the discovery of new species and the
reconstruction of organismal relationships. The need
for species discovery and description (i.e., species de-
limitation) has only increased in past decades due to
the extinction crisis (Pimm et al. 1995; Thomas et al.
2004). However, until recently, the attention of systema-
tists has been focused on developing and applying new
methods for building phylogenies. Important advances,
beginning with the conceptualization of species, have
fostered the reemergence of species delimitation as a
major focus of systematic biology (Wiens 2007).
de Queiroz (1998, 2007) has argued that at the root of
all modern species concepts, there is general agreement
on the fundamental nature of species: species are sep-
arately evolving metapopulation lineages. What differs
between concepts are the criteria, or lines of evidence,
used to identify such lineages. For example, intrinsic
reproductive isolation (Mayr 1942) and the existence of
1990) are both properties of evolutionarily distinct lin-
eages. The observation of either property would suggest
that the group in question is a species and that it may
warrant taxonomic recognition as such. But, the order in
which properties of lineages appear during cladogene-
sis, or whether they appear, cannot always be predicted,
thus the application of several different criteria may
be necessary. The perspective that species are lineages,
and that multiple criteria may be used to identify them,
has been termed the general lineage concept of species
(de Queiroz 1998).
Numerous criteria for the identification of lineages
have been proposed, some of which are integral to pop-
ular species concepts (de Queiroz 1998, 2007), whereas
others are ad hoc (e.g., Pons et al. 2006; Knowles and
Carstens 2007) (reviewed by Sites and Marshall 2003,
2004). The relative performance of some of these criteria
under different evolutionary scenarios has been inves-
tigated (Hudson and Coyne 2002; Wiens and Penkrot
2002; Dettman et al. 2003; Marshall et al. 2006; Reeves
and Richards 2007). It is reasonable to propose, as has de
Queiroz (2007), that the greater the number of species
criteria satisfied by a group, the more likely it becomes
that the group is a distinct lineage—an identifiable bi-
ological entity on an independent evolutionary trajec-
tory. At a minimum, as more criteria are satisfied, the
at DigiTop USDA's Digital Desktop Library on January 18, 2011
proposal that a group merits recognition as a species
should become less contentious. In spite of offering a
resolution to the “species problem” (de Queiroz 2005)
and the potential for stable noncontroversial species
circumscriptions, the general lineage concept of species
has rarely been applied, although the practice is increas-
ing (e.g., Marshall et al. 2006; Alstr¨ om et al. 2008; Light
et al. 2008; Padial and De La Riva 2009).
Under the general lineage concept, the only neces-
sary property of a species is existence as a separately
evolving metapopulation lineage (de Queiroz 2005).
Thus, the concept itself is egalitarian in its treatment of
species delimitation criteria: any criterion that identifies
lineages identifies species. However, the tests that are
employed to determine whether groups meet criteria
are not necessarily equal in their abilities. For example,
the commonly implemented test for monophyly, inspec-
tion of a reconstructed phylogenetic tree, may perform
poorly for identifying lineages in the presence of gene
flow (Reeves and Richards 2007) or due to errors asso-
ciated with randomly sampling few individuals from a
complex underlying genealogy (Rosenberg 2007). Ad-
ditionally, in the case of clinal variation or isolation
by distance, imperfect geographical sampling can lead
to erroneous conclusions under some species criteria,
most notably those reliant on tests of character diver-
gence (see, e.g., Rosenberg et al. 2005; Schwartz and
McKelvey 2009). Therefore, although all species criteria
identify valid properties of lineages, the particular tests
used to evaluate compliance with those criteria may
vary in their efficacy.
We note two different categories of species delimita-
tion criteria: those that seek to discern patterns in data
consistent with evolution along lineages and those that
attempt to identify evolutionary processes capable of
maintaining distinct lineages. For example, monophyly
(Donoghue 1985; de Queiroz and Donoghue 1988), fixed
character state differences (i.e., diagnosability; Cracraft
1983; Nixon and Wheeler 1990), exclusive coalescence
of alleles (Baum and Shaw 1995), and formation of a
distinct genotypic cluster (i.e., absence of genetic in-
termediates; Mallet 1995) are pattern-oriented criteria,
whereas intrinsic reproductive isolation (Mayr 1942),
shared specific mate recognition (Paterson 1985), and
occupation of a distinct niche (which implies adap-
tation; Van Valen 1976) are process-oriented criteria.
One set of criteria proposes an evolutionary cause for
the persistence of a lineage; the other reflects the ef-
fect of having existed as a lineage for some period of
time. Without specific knowledge of the relative perfor-
mance of the various tests for compliance with species
criteria, affirmation from tests of complementary pat-
tern and process-oriented species criteria would seem
to provide strong corroborating evidence for the ex-
istence of a lineage and, hence, the delimitation of a
Our study organism, Humulus lupulus, or hops, is a
perennial, dioecious, wind-pollinated vine in the fam-
ily Cannabaceae. Wild populations are typically ripar-
ian, climbing to heights in excess of 6 m, supported
by deciduous trees. In recognition of differences be-
tween wild North American Humulus and Old World
congeners, Nuttall (1848) described a single North
American taxon, H. americanus, distributed through-
out the United States. The specific epithet americanus
was never uniformly used. Examination of distinctive
specimens from New Mexico led to the proposal of the
subspecific taxon H. lupulus variety neomexicanus, the
first recognition of geographical differentiation within
New World Humulus (Nelson and Cockerell 1903). Sub-
sequently, var. neomexicanus was treated as a species,
H. neomexicanus, by Rydberg (1917). Most recently and
authoritatively, H. lupulus was divided into five tax-
onomic varieties based on morphology and geogra-
phy (Small 1978, 1981). These varieties include 1) var.
lupulus, which has a native distribution in Europe and
Asia but has naturalized in many regions of the world
following escape from commercial hopyards (female
inflorescences of var. lupulus are used to impart char-
acteristic aromatic and bitterness qualities to beer), 2)
var. cordifolius, restricted to east Asia and Japan, 3) var.
neomexicanus, a native of the arid western United States,
4) var. pubescens, from the Midwestern United States,
and 5) var. lupuloides, distributed from the northern
Great Plains eastward.
The three North American varieties together form
a diagnosable monophyletic group distinct from Eu-
ropean and Asian varieties (Pillay and Kenny 1996;
Murakami et al. 2006a, 2006b). The geographic distribu-
tions of the varieties overlap at their peripheries (Fig. 1)
suggesting the possibility for gene flow. Although
numerous studies have described genetic variation in
lus varieties (based on Small 1978). Shaded ovals indicate regions
where DNA samples were taken from individuals conforming to each
variety. Variety lupuloides (“l”) was sampled from a region where it
cooccurs with var. neomexicanus, var. pubescens (“p”) was sampled
from a region where it cooccurs with var. lupuloides, and var. neomexi-
canus (“n”) was sampled in allopatry.
Approximate ranges of North American Humulus lupu-
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REEVES AND RICHARDS—SPECIES DELIMITATION IN WILD HOPS
cultivated hops (Small 1981; Brady et al. 1996; Hartl
and Seefelder 1998;ˇSuˇ star-Vozliˇ c and Javornik 1999;
Seefelder et al. 2000; Jakˇ se et al. 2001; Patzak 2001;
Stajner et al. 2008) and its wild progenitors (Small 1978;
Stevens et al. 2000; Henning et al. 2004; Murakami et al.
2006a, 2006b; Townsend and Henning 2009), the ex-
tent and mechanisms of evolutionary isolation among
H. lupulus varieties in North America have not been
Wild North American hops cannot be directly used
in brewing because of undesirable chemical proper-
ties that produce excessive bitterness and objectionable
aromas. However, many modern hop cultivars with
desirable pathogen resistance and elevated bittering
properties trace a portion of their pedigree to crosses
between European cultivars (var. lupulus) and a wild in-
dividual (var. lupuloides) from Manitoba (Salmon 1934;
Neve 1991). North American H. lupulus continues to
be explored as a source for agronomically desirable
traits such as disease, drought, and insect resistance
(Hampton et al. 2001).
We are interested in determining whether the three
named taxonomic varieties of hops native to North
America form evolutionarily distinct lineages. North
American H. lupulus shares with many temperate plant
species factors that complicate species delimitation such
as broad geographic distribution, incomplete reproduc-
tive isolation, and distributional overlap among groups.
Wild hops thus represent an intriguing case for apply-
ing the principles of the general lineage concept to the
problem of species delimitation. Moreover, the identifi-
cation of discrete evolutionary lineages among relatives
of crop species should facilitate the use of wild genetic
resources in crop improvement programs, a strategy
receiving increased attention (Tanksley and McCouch
1997; Gur and Zamir 2004). Genes and gene complexes
responsible for adaptation to evolutionary challenges
should occur at high frequency in lineages persisting
lenge. Thus, in our view, the accurate identification of
distinct evolutionary lineages has economic as well as
In this study, we apply the following five criteria
for species delimitation to DNA polymorphism, nat-
ural history, and distributional data from wild North
American H. lupulus:
1. monophyly (Donoghue 1985; de Queiroz and
2. diagnosability, that is, the appearance of fixed dif-
ferences (Cracraft 1983; Nixon and Wheeler 1990)
3. absence of genetic intermediates (Mallet 1995)
4. intrinsic reproductive isolation (Mayr 1942), and
5. niche specialization (Van Valen 1976).
Whether varieties merit taxonomic recognition as
species is discussed in light of the plurality of evi-
dence in favor of their existence as distinct evolutionary
MATERIALS AND METHODS
DNA Sampling and Amplified Fragment Length
Leaf tissue was collected from 131 H. lupulus var. lupu-
loides individuals from 29 wild populations in the Great
Plains of southern Canada and the northern United
eas and river terraces along the Souris, Qu’Appelle, and
Assiniboine rivers of the Red River drainage, and the
Missouri, Knife, Little Knife, and White Earth rivers of
the Mississippi River drainage. Wild var. lupuloides pop-
ulations from this region of Manitoba, Saskatchewan,
and North Dakota may be an important source of genes
for crop improvement (Hampton et al. 2001). Seven-
teen H. lupulus var. pubescens individuals were sampled
from four wild populations along the Missouri River in
southeastern Nebraska. Two additional individuals of
var. pubescens and 9 var. neomexicanus individuals were
sampled from herbarium specimens (Table 2).
Genomic DNA was extracted from fresh leaves (pre-
served in vapor phase of liquid nitrogen) or herbarium
tissue using the DNeasy 96 Plant Kit (Qiagen). Ampli-
fied fragment length polymorphism (AFLP) reactions
were performed using the method of Vos et al. (1995)
as modified by Marques et al. (1998) and Myburg et al.
(2001). Ligation reactions were diluted 10-fold prior
to preamplification. Preamplification reactions were
diluted 1:40 prior to selective amplification. Eighteen
duplexed primer pair combinations, chosen as optimal
from a preliminary screen of 32, were used for selec-
tive amplification (see online Appendix 1, http://www
.sysbio.oxfordjournals.org). AFLPs were visualized on a
model 4200 LI-COR automated DNA sequencer follow-
ing Myburg et al. (2001). Polymorphic loci from 50 to
500 bp were manually assigned binary scores (present =
1, absent = 0) using SAGA MX software (version 2.1;
LI-COR). AFLP quality and comparability were ensured
by confirming that monomorphic bands observed in
samples derived from fresh material were also found in
herbarium samples, following Lambertini et al. (2008).
To establish scoring error rates, DNA from three indi-
viduals was subjected to the experimental protocol in
duplicate, from DNA extraction through scoring.
To test for monophyly of varieties, two approaches
were used. First, the data set containing 159 individ-
uals and 555 AFLP characters was subjected to parsi-
mony analysis using PAUP 4.0b10 (Swofford 1999). The
heuristic search option and tree bisection-reconnection
(TBR) branch swapping were used with the MULTREES
setting “on,” and characters were treated as equally
weighted following the suggestions of Koopman (2005).
Maximum parsimony searches were conducted using
an idle-time distributed computing cluster (Reeves
et al. 2005). Jobs were run until >3000 random-order
taxon addition replicates had been completed (3681 in
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