Extreme differences in population structure and genetic diversity for
three invasive congeners: knotweeds in western North America
Gaskin, J. F., Schwarzländer, M., Grevstad, F. S., Haverhals, M. A., Bourchier, R.
S., & Miller, T. W. (2014). Extreme differences in population structure and genetic
diversity for three invasive congeners: knotweeds in western North America.
Biological Invasions, 16(10). doi:10.1007/s10530-014-0652-y
10.1007/s10530-014-0652-y
Springer
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
Biol Invasions (2014) 16:2127–2136
DOI 10.1007/s10530-014-0652-y
ORIGINAL PAPER
Extreme differences in population structure and genetic
diversity for three invasive congeners: knotweeds in western
North America
John F. Gaskin • Mark Schwarzländer •
Fritzi S. Grevstad • Marijka A. Haverhals
Robert S. Bourchier • Timothy W. Miller
•
Received: 4 March 2013 / Accepted: 4 February 2014 / Published online: 15 February 2014
Ó Springer International Publishing Switzerland (outside the USA) 2014
Abstract Japanese, giant, and the hybrid Bohemian
knotweeds (Fallopia japonica, F. sachalinensis and
F. 9 bohemica) have invaded the western USA and
Canada, as well as other regions of the world. The
distribution of these taxa in western North America,
and their mode of invasion, is relatively unresolved.
Using amplified fragment length polymorphisms of
858 plants from 131 populations from British Columbia to California to South Dakota, we determined that
Bohemian knotweed was the most common taxon
(71 % of all plants). This result is in contrast to earlier
reports of F. 9 bohemica being uncommon or nonexistent in the USA, and also differs from the
European invasion where it is rarer. Japanese knotweed was monotypic, while giant knotweed and
Bohemian knotweed were genetically diverse. Our
genetic data suggest that Japanese knotweed in
western North America spreads exclusively by vegetative reproduction. Giant knotweed populations were
mostly monotypic, with most containing distinct
genotypes, suggesting local spread by vegetative
propagules, whereas Bohemian knotweed spreads by
both seed and vegetative propagules, over both long
and short distances. The high relative abundance and
genetic diversity of Bohemian knotweed make it a
priority for control in North America.
Keywords AFLP Bohemian knotweed Fallopia Giant knotweed Invasion Japanese
knotweed
Introduction
Electronic supplementary material The online version of
this article (doi:10.1007/s10530-014-0652-y) contains supplementary material, which is available to authorized users.
Closely related invasive species can vary greatly in
many aspects that affect their invasion dynamics.
There may be significant dissimilarities in genetic
J. F. Gaskin (&)
USDA Agricultural Research Service, 1500 N. Central
Avenue, Sidney, MT 59270, USA
e-mail: john.gaskin@ars.usda.gov
R. S. Bourchier
Agriculture and AgriFood Canada-Lethbridge Research
Centre, 5403-1st Avenue South, Lethbridge, AB T1J 4B1,
Canada
M. Schwarzländer M. A. Haverhals
Department of Plant, Soil and Entomological Sciences,
University of Idaho, Moscow, ID 83844, USA
T. W. Miller
Northwestern Washington Research and Extension Center,
Washington State University, 16650 State Route 536,
Mount Vernon, WA 98273, USA
F. S. Grevstad
Department of Botany and Plant Pathology, Oregon State
University, Corvallis, OR 97331, USA
123
2128
diversity, mode of reproduction, allocation of
resources, level of competitive ability, or resistance
or tolerance to control methods which can result in
each species requiring a distinct management program. Gene flow between closely related invasive
species adds a layer of complexity to invasion
dynamics of each species, as well as to their control.
Specifically, hybridization and introgression increase
the chances of taxonomic confusion, and this misidentification can lead to improper control methods or
accidental control of desired plants (e.g. Saltonstall
2002; Moody and Les 2007). Increases in genotypic
diversity and novel genotypes created by hybridization can expand the ability of invasives to adapt to new
selective forces (Ellstrand and Schierenbeck 2000).
Classical biological control, in particular, because of
the requirement for highly host-specific control
agents, may benefit from knowledge of the diversity
and distribution of hybrids and intraspecific genotypes
if invasive genotypes vary in their susceptibility to
control (Müller-Schärer et al. 2004). Also, variability
in reproductive strategies of invasive species may
drive the choice of control agent guild. For many plant
invasions, these topics have not been adequately
addressed.
Japanese, giant and Bohemian knotweeds are
herbaceous rhizomatous perennial Asian taxa considered invasive in the USA and Canada, as well as other
parts of the world (Weber 2003; Global Invasive
Species Database [GISD] 2005); Fallopia japonica
(Houttuyn) Ronse Decraene, F. sachalinensis (F.
Schmidt) Ronse Decraene and F. 9 bohemica (Chrtek
and Chrtková) J. P. Bailey, respectively; family
Polygonaceae. Morphological identification of Japanese and Bohemian knotweeds is at times difficult,
especially in New England, due to production of
hybrid swarms that may include backcrossed and later
generation hybrids (Gammon et al. 2007). There are
also ploidal differences within and between the taxa
(Japanese: 2n = 44, 52, 88; giant: 2n = 44, 66, 102;
Bohemian: 2n = 44, 66, 88; Bailey and Stace 1992;
Bailey et al. 1996). Taxonomic confusion in the field
has led to many requests from land managers to
identify specimens using molecular techniques. The
root of the confusion may be the conflict between field
identifications and published distribution lists. For
example, Bohemian knotweed has only recently been
recognized as common in parts of North America by
Zika and Jacobson (2003) and the Plants Database
123
J. F. Gaskin et al.
(USDA, NRCS 2012) still indicates that Bohemian
knotweed is not present in the USA, only Canada.
Population structure and identification of invasive
knotweeds have been investigated using chromosomal
and molecular markers in Europe (e.g. Bailey and
Stace 1992; Bailey et al. 2009; Krebs et al. 2010;
Mandák et al. 2005; Tiébré et al. 2007a, b), and the
studies have shown that the nature of the invasions
differ across regions. The UK is invaded primarily by a
single clone of Japanese knotweed (Hollingsworth and
Bailey 2000), although Bohemian knotweed also
appears to be present (Bailey and Wisskirchen
2006). On mainland Europe the abundance of the
three Fallopia taxa varies across regions (Bailey and
Wisskirchen 2006), and molecular data suggest that
Japanese knotweed is the most common taxon in
Germany/Switzerland (Krebs et al. 2010). In North
America the situation is less clear. The three taxa are
primarily listed as noxious on the coastal states, with
lower densities in most mid-continental states (USDA,
NRCS 2012). Earlier studies of diversity and distribution were primarily performed on Japanese knotweed and collections focused on the east coast, with
few west coast collections (Grimsby and Kesseli 2010,
n = 19 western North American plants; Gammon and
Kesseli 2010, n = 4; Grimsby et al. 2007, n = 8). One
molecular intra-populational study has been performed in North America, on three populations of
Japanese knotweed in Massachusetts (Grimsby et al.
2007), but still, little is known about the diversity and
distribution of knotweed genotypes in western North
America (Bailey et al. 2009), which may be very
different from what has been found in eastern North
America and Europe (Grimsby et al. 2007, p. 962).
The different taxa of knotweeds are already known
to respond differentially to various control practices
(Bı́mová et al. 2001; Bailey et al. 2009), and this also
holds true for classical biological control. A psyllid,
Aphalara itadori Shinji has been released in the
United Kingdom as a biological control agent for
Japanese knotweed (Shaw et al. 2009). The two strains
of the psyllid that are being considered for use in North
America have contrasting survival and impact on
Japanese, giant and Bohemian knotweed (Grevstad
et al. 2013). The psyllid also shows differential
development on different Bohemian knotweed collections (Bourchier and Grevstad pers. obs.). Thus,
knowledge of the distribution of each taxon, and
intraspecific phenotypes with differing biological
Extreme differences in population structure
traits, will be essential to the future management of
knotweeds.
Knotweeds typically spread through movement of
rhizomes in rivers or by humans (Bailey et al. 2009).
They can reproduce sexually, but in the invaded
ranges Japanese knotweed contains male sterile flowers, and requires other knotweed species to obtain
pollen and produce seed (Hollingsworth and Bailey
2000; Forman and Kesseli 2003; Grimsby et al. 2007;
Engler et al. 2011). Moreover, the extent to which
sexual reproduction occurs in the field is unclear as
seedling establishment appears to be a rare event
(Forman and Kesseli 2003; Engler et al. 2011; but see
Bzde˛ga et al. 2012). Genotypic data can determine if
dispersal is via vegetative material (all plants are
identical genotypes) or sexually produced seed (most
plants are varying genotypes) giving insight to how the
weed species spreads; information that is useful to the
integrated management of these invasive plants.
The goal of this study is to compare invasion
processes across closely related invasive taxa using
molecular data [amplified fragment length polymorphisms (AFLPs)] and discuss how this information can
be applied towards management of a multispecies
invasion. Our four objectives are to (1) determine the
diversity and distribution of taxa and genotypes in the
western North American knotweed invasion, (2)
compare and contrast the western North American
invasion with eastern USA and European invasions,
(3) determine if invasive spread is primarily by
vegetative material or seed, and (4) determine if field
identifications of knotweeds by land managers are
accurate to the species/hybrid level.
2129
a
b
Materials and methods
We obtained leaf material from 867 knotweed plants,
including 25 plants from biological control agent hostspecificity tests conducted by Agriculture and AgriFood Canada (Lethbridge, AB), nine plants from
biological control release sites in the UK provided by
R. Shaw (CABI-UK), and 833 plants from 131
geographically distinct, naturalized populations of
Japanese, giant and Bohemian knotweeds in the
western USA and south-western Canada provided by
land managers responding to a request for samples
(Fig. 1a, b; ‘‘Appendix 1’’ in Supplementary material). These populations typically represented large
Fig. 1 a Map of knotweed collections from western North
America. Plant taxa indicated by symbols. b Close-up view of a,
focusing on watersheds in Washington and Oregon, USA.
Populations included in study of riverways are marked by river
names
continuous patches with transect collections made
every 5 m. We requested invasive knotweed collections in general from our many collectors, not specific
species or hybrids. Morphological identifications of
selected plants were performed using the key in Zika
and Jacobson (2003) which emphasizes differences in
leaf shape and the presence, shape and length of
123
2130
trichomes on the abaxial leaf surface. Along some
rivers in Washington and Oregon we collected from
multiple populations up and downstream (Fig. 1b),
and used these collections to investigate movement of
genotypes within a riverway.
Leaf material was collected on site and stored in
silica gel at room temperature. Genomic DNA was
extracted from approximately 20 mg of silica-dried
material using a modified CTAB method (Hillis et al.
1996). The AFLP method followed Vos et al. (1995)
with modifications as in Gaskin and Kazmer (2009).
All 15 selective primer combinations of MseI ? CAA,
CAC, CAT, CTA, or CTA and EcoRI ? AAG, ACC,
or ACT were pre-screened for PCR product quality
and number of variable loci using eight samples, and
the two most polymorphic primer pairs were chosen
(MseI ? CAC/EcoRI ? AAG and MseI ? CAC/
EcoRI ? ACC). Reliable AFLP bands (loci) were
determined as in Ley and Hardy (2013). Briefly, we
generated all AFLP data on an Applied Biosystems
(ABI, Foster City, CA, USA) 3130 Genetic Analyzer,
including 192 repeats (22 % of North America
samples), and omitted any individuals that did not
produce a typical electropherogram pattern (i.e. noise
[20 relative fluorescence units (rfu) or failure to
produce sufficient number of peaks). All repeats and
their matching original .fas files were placed in
PeakScanner Software 2 (ABI) and analyzed at a
minimum peak height of 20 rfu. The resultant .csv file
from the PeakScanner sizing table was prepared with a
header for tinyFLP v1.22 (Arthofer 2010) automatic
peak scoring and selection using settings of: minimum
peak height = 20, maximum peak width = 2, minimum size = 50, maximum size = 500, size tolerance
range = 0.5, minimum peak–peak distance = 1, peak
height difference = 0, minimum frequency = 0.3,
maximum frequency 99.7. The tinyFLP output file
was prepared for SPAGeDi v1.4 (Hardy and Vekemans 2002) to test for reproducibility of peaks, using
broad sense heritability (H2) and its significance,
calculated as Fst of Weir and Cockerham (1984).
Peaks (loci) with an H2 value of[0 and P \ 0.05 were
considered heritable for this study (significant H2 was
always [0.18).
NTSYS-pc ver. 2.1 software (Rohlf 1994) was used
to calculate the Dice (1945) similarity coefficient: 2a/
(2a ? b?c) where a = number of bands present in
both samples, b and c = number of bands present in
only one or the other sample, respectively. Principal
123
J. F. Gaskin et al.
coordinates analysis (PCoA) was performed on Dice
similarity coefficients using the DCENTER and
EIGEN modules of NTSYS. A unweighted pair group
method with arithmetic mean (UPGMA) of the 858
plants and bootstrap values from 1,000 repetitions
were created using PAUP v4.0b3 (Swofford 2000).
We used the UPGMA bootstrap support to determine
if genetic clusters correlated with morphological
identification of taxa.
Number of genotypes (G) was calculated using the
software GenoType v1.2 (Meirmans and Van Tienderen 2004). We used the G/N (N = number of plants)
of each population in a Mann–Whitney test to determine if differences in G/N were statistically significant
between taxa. Simpson’s diversity index (D) corrected
for finite sample size (Pielou 1969) was calculated with
D = 1 - R ni(ni - 1)/N(N - 1) for i = 1 to G,
where ni is the number of plants that share genotype.
Values of D can range from 0 to 1, with higher values
corresponding to greater genetic diversity. Genotypic
evenness (E) was calculated, with E = (D - Dmin)/
(Dmax - Dmin), where Dmin = (G - 1)(2 N - G)/
N(N - 1) and Dmax = N(G - 1)/G(N - 1). Values
of E can range from 0 to 1, with lower values indicating
that a certain genotype is common in the collection,
and higher values indicating that the number of plants
representing each genotype is more evenly distributed.
Proportion of Loci that are Polymorphic (PLP) was
calculated using the function Diversity in AFLPdat
(Ehrich 2006).
Analysis of molecular variance (AMOVA), as
implemented in Arlequin v3.5.1.2 (Excoffier et al.
2005), was used to examine the distribution of genetic
variation of AFLP genotypes among and within
populations for each taxon separately. Only populations with five or more plants were included in the
AMOVA analyses. Our assumption of similar mating
pattern across all populations of a taxon may lead to an
underestimation of levels of genetic diversity.
Results
Screening of repeatability of loci described above
provided 208 significantly heritable, variable loci for
MseI ? CAC/EcoRI ? AAG and MseI ? CAC/EcoRI ?
ACC combined. After manual elimination of loci that
were not discontinuous for rfu’s between 0 and
minimum peak height (see Figure in ‘‘Appendix 2’’
Extreme differences in population structure
2131
0.27
C
n = 609
Dim-2 (22%)
0.12
No morphological ID performed
Morphological ID = Japanese (n = 22)
Morphological ID = Giant (n = 29)
Morphological ID = Bohemian (n = 174)
-0.02
B
n = 116
-0.17
D
Population 68, n = 3
South Dakota, USA
A
n = 130
-0.31
-0.70
-0.44
-0.19
0.06
0.32
Dim-1 (36%)
Fig. 2 Principal coordinates analysis of knotweed plants from
western North America (n = 858). Squares plants morphologically identified as Bohemian knotweed, diamonds morphologically identified as giant knotweed, triangles morphologically
identified as Japanese knotweed, circles plants that were not
morphologically identified. Four genetic clusters each with
100 % bootstrap support in a UPGMA analysis are identified by
closed curves marked as A, B, C and D
in Supplementary material) we scored 63 and 72 (total
135) unambiguous, polymorphic loci for the two
primer pairs, respectively.
A PCoA is presented in Fig. 2, with the first and
second axes accounting for 36 and 22 % of the overall
variation, respectively. Forty-four populations, representing 225 plants, were morphologically identified in
the field as Japanese, giant, Bohemian or mixed taxa
populations of knotweeds prior to genotyping (see
‘‘Appendix 3’’ in Supplementary material). Genotypes
of the morphologically identified plants are shown as
solid black shapes in the PCoA (Fig. 2), along with
genotypes of plants that were not morphologically
identified (empty circles), and the combined set of
genotypes were found to separate into four clusters,
each supported by 100 % UPGMA bootstrap values
(not shown). We assumed that all morphologically
unidentified plants in a PCoA/UPGMA cluster were
the same taxon as the morphologically identified
plants in that cluster, thus in Fig. 2: A = Japanese
knotweed, B = giant knotweed, C = Bohemian knotweed and D = one population that was identified as
Bohemian knotweed from South Dakota.
Overall we found 155 genotypes, with some of these
being very common (‘‘Appendix 3’’ in Supplementary
material). For example, all Japanese knotweeds,
27/116 giant knotweeds, and 256/621 Bohemian
knotweeds represented the three most common genotypes for each taxon.
Japanese knotweed had the lowest proportion of
loci that were polymorphic (PLP) and was significantly less diverse for G/N than the other knotweeds,
while Bohemian knotweed had the highest PLP,
highest number of genotypes and the lowest percentage of monotypic populations (Table 1). Giant knotweed was highest in Simpson’s Diversity (D) and
Evenness (E). Our one Japanese knotweed genotype
(n = 130 plants) was genetically identical to all eight
invasive Japanese knotweed specimens sent from the
UK (‘‘Appendix 3’’ in Supplementary material). For
all taxa combined the majority of populations were
monotypic (62/105 populations with n C 5), however,
some populations contained many genotypes of a
taxon. Especially notable for diversity were a giant
knotweed population (124, Washington) with nine
genotypes in ten plants, and three Bohemian knotweed
populations (37, n = 5; 42, n = 5; 126, n = 6), with
each plant being a different genotype (population
locations in ‘‘Appendix 1’’ in Supplementary
material).
123
2132
J. F. Gaskin et al.
Table 1 Genetic diversity of invasive knotweeds from western North America
Taxon
N
PLP
Japanese
130
0.00
G/N1
D
E
No. and (%) of
monotypic populations2
1
0.01a
0
na
15/15, 100
b
0.89
0.83
9/13, 69
0.81
0.71
38/77, 49
G
Giant
116
0.42
32
0.28
Bohemian
612
0.88
122
0.20b
Total
858
155
N number of plants, PLP proportion of loci that are polymorphic, G number of genotypes, D Simpson’s diversity, E evenness
1
G/N calculated from all collections of a species pooled. To test significance of G/N between species, we calculated G/N for each
population with N C 5. Mean of G/N values of populations between species with different superscript letters are significantly
different (P [ 0.05) as determined by Mann–Whitney test
2
Includes only populations with N C 5
Table 2 Genetic diversity of knotweed from riverways in Oregon and Washington states
River (listed from North
to South)
Taxa
present
No. of
populations
No. of
plants
Nooksack
Japanese
6
Bohemian
21
G
No. of G shared with other
riverways
(%) G shared with other
riverways
PLP
1
1
100
0.00
13
1
8
0.45
Combined
6
27
14
2
14
0.51
Samish
Bohemian
5
25
1
1
100
0.00
Skagit
Bohemian
5
25
2
1
50
0.01
Snoqualmie
Japanese
1
1
1
100
0.00
0.33
Bohemian
28
5
1
20
Combined
6
29
6
2
33
0.44
Cedar
Bohemian
5
25
17
0
0
0.38
Salmon
Bohemian
3
12
1
0
0
0.00
Lower Nehalem
Bohemian
7
34
3
1
33
0.08
Trask
Japanese
6
1
1
100
0.00
Bohemian
4
1
0
0
0.00
Combined
4
10
2
1
50
0.28
Little Nestucca
Luckiamute
Japanese
Bohemian
3
5
15
49
1
9
1
1
100
11
0.00
0.21
Big Creek
Giant
5
25
2
0
0
0.01
54
276
Total
G number of genotypes, PLP proportion of loci that are polymorphic
In the AMOVA analyses, Giant knotweed contained most of its genetic variation among populations
(90 %, P \ 0.0001), and Bohemian knotweed had
83 % (P \ 0.0001) variation among populations. For
the monotypic Japanese knotweed, there was no
variation within or between populations.
For some invaded rivers in Washington and Oregon
we collected plant samples from multiple populations
up and downstream, providing an opportunity to gain
insight on how invasive knotweed taxa move within
riverways. On these 11 rivers we collected 276 plants
123
from 54 populations (Table 2; Fig. 1b; ‘‘Appendix 3’’
in Supplementary material). Four riverways contained
Japanese knotweed, one of these with only Japanese
knotweed, and it was monotypic. One riverway
contained giant knotweed, and had two genotypes.
The most common taxon, found in nine out of 11
riverways, was Bohemian knotweed (223/276 plants;
81 %), represented by 47 genotypes. Of the nine
riverways that contained Bohemian knotweed, six of
them contained the most common Bohemian genotype, representing 153/223 Bohemian plants. This was
Extreme differences in population structure
also the only Bohemian knotweed genotype shared
between riverways.
Discussion
The tight correlation between our morphological
identifications and genetic clusters in the PCoA
suggests that accurate field identifications of knotweed
taxa are possible in western North America using the
Zika and Jacobson (2003) key. Even in populations
from which both Japanese and Bohemian knotweeds
were morphologically identified, the genetic cluster
and morphological identification corresponded in all
cases (populations 35 and 45, Washington). Population 68 (South Dakota) formed its own bootstrap
supported cluster (D), and stands out as a collection
that deserves further taxonomic and cytological
research. Researching the ploidal levels would also
help elucidate further sub-structuring within Bohemian knotweed.
Japanese and giant knotweeds were found primarily
west of the Cascade Mountains, with Bohemian
knotweed dominant inland (Fig. 1a), suggesting that
different regions may require different management
techniques tailored to the predominant taxon. No prior
studies, including ours, have done appropriate random
sampling to compare proportions of these taxa in
invaded regions, but in the UK, the ratio of Japanese to
Bohemian knotweed was approximately 30:1 (Bailey
and Wisskirchen 2006), in stark contrast to our ratio of
1:5 (Table 1). Studies in Belgium, Germany-Switzerland and the Czech Republic had ratios of approximately 1:1, 3:1 and 1:1, respectively (Tiébré et al.
2007a; Krebs et al. 2010; Mandák et al. 2005). A
primarily eastern USA survey (Grimsby and Kesseli
2010), that also contained 19 plants from the Pacific
Northwest and Alaska, USA, found that the ‘‘British
clone’’ (our Japanese knotweed genotype) comprised
47 % of their collections, much higher than the 15 %
we found in our study. In their 19 western USA plants
they found 13 (68 %) Bohemian knotweeds; a ratio
very similar to what we found in our 858 western
North American plants (72 %; Table 1). In summary,
these studies suggest that the dominance of Bohemian
knotweed over other taxa is unique to western North
America.
Giant knotweed was our most genetically diverse
taxon. Similarly, in Germany/Switzerland, where one
2133
plant per patch was genotyped, Giant knotweed was
highly diverse with each plant being a distinct
randomly amplified polymorphic DNA (RAPD) genotype (Krebs et al. 2010), and in the Czech Republic
there were 16 inter-simple sequence repeat (ISSR)
genotypes in 50 plants while in the eastern USA, two
giant knotweed genotypes were found in four plants
(Gammon and Kesseli 2010). We found one Japanese
knotweed genotype, as was found in Germany/Switzerland (using RAPDs; Krebs et al. 2010) and the
Czech Republic (using ISSRs; Mandák et al. 2005).
Eight chloroplast DNA sequence haplotypes were
found in the eastern USA in only 54 Japanese
knotweed plant samples, suggesting that the knotweed
invasions in the eastern USA were founded by a larger
number of genotypes, or that mutations to that region
of DNA sequence have occurred post-introduction
(Gammon and Kesseli 2010). Our Bohemian knotweed had 122 genotypes in 612 plants (G/N = 0.20),
while Germany/Switzerland had a higher ratio of 24
RAPD genotypes in 32 plants (G/N = 0.75; Krebs
et al. 2010), probably due to differences in sampling
scheme (one plant per patch in Germany/Switzerland,
compared to our transect collections in patches). The
Czech Republic had G/N = 0.375, although these
were also typically collected as one plant per patch
(Mandák et al. 2005). Despite the variation in collecting methods, Bohemian and giant knotweeds are
consistently more diverse than Japanese knotweed
across Europe and North America.
In studies from Germany/Switzerland and Belgium
(Krebs et al. 2010; Tiébré et al. 2007a), Bayesian
analysis using STRUCTURE (Pritchard et al. 2000)
suggested three distinct genotypic clusters for Japanese, giant and Bohemian knotweed. Variation in
ploidal level within and between taxa, and presence of
clonal reproduction likely violate assumptions of
STRUCTURE (Falush et al. 2007), thus we did not
perform that analysis, though our UPGMA and PCoA
(Fig. 2) also defined three major genetic clusters
corresponding to morphological identifications. In
Krebs et al. (2010), the Bayesian analysis assigned
Bohemian knotweeds to their own cluster, instead of
an admixture of Japanese and giant knotweeds, and
thus they suggest that most Bohemian knotweeds are
not recent hybrids, but were introduced post-hybridization (or alternatively hybridization has led to rapid
changes in genome structure since introduction of the
parental taxa).
123
2134
The existence of only one Japanese knotweed
genotype in our study suggests no movement of this
species by seed, within or between riverways or even
within populations. This result is similar to what was
found in the UK and Belgium (Hollingsworth and
Bailey 2000, Tiébré et al. 2007a), and is considered the
result of there being no male-fertile flowers present in
the invasive range. Sexual reproduction does occur,
but with other taxa such as Bohemian and giant
knotweed supplying the pollen (Bailey et al. 2009).
The ability of a single genotype to become highly
invasive across a wide geographic range suggests high
levels of phenotypic plasticity (Sultan 1995; Sexton
et al. 2002; Geng et al. 2007; Richards et al. 2008)
perhaps mediated through epigenetic differentiation
between identical genotypes, as was investigated in
knotweeds by Richards et al. (2012).
Giant knotweed populations were overwhelmingly
monotypic (9/13, 69 %), and almost all genetic
variation (90 %, AMOVA) was among, not within,
populations. Across the whole study, only two giant
knotweed genotypes were shared between populations; one found in four populations (82, 83, 87, 88) on
Vancouver Island, British Columbia, Canada, and one
found in five populations along Big Creek, Oregon,
USA (populations 6–10). This species is self-incompatible (Bailey 1989, 1990), thus monotypic populations must be formed by vegetative reproduction.
Long distance movement of vegetative material seems
rare and seed appears to be the mode for long distance
travel, as almost each geographically distant population is a distinct genotype. Alternatively, and perhaps
more likely, multiple introductions of giant knotweed
genotypes, originally to nurseries or gardens, may be
responsible for the genetic diversity among populations, followed by local spread via vegetative
reproduction.
Bohemian knotweed appears to spread both by seed
and vegetatively. This taxon is also self-incompatible
(Bailey 1989) and the presence of the common
genotype (n = 256/612) in 46/77 of all populations
surveyed, with some riverways monotypic for this
genotype, suggests short and long distance movement
of reproductive vegetative plants parts throughout
western North America. Other less common genotypes
were also shared between riverways and among distant
populations. For example, another genotype was found
in 13 populations in Idaho, Montana, Oregon, Washington, and Wyoming. In some Bohemian knotweed
123
J. F. Gaskin et al.
populations (37, 42, 126) each sampled shoot, collected every five meters along a transect, was a distinct
genotype; evidence of reproduction by seed. The
existence of novel Bohemian genotypes in many
populations suggests that spread by seed is common.
In all, Bohemian knotweed appears to spread easily
through both vegetative material and seed, over both
long and short distances. There are multiple ploidal
levels (aneuploid and tetraploid to octoploid; Bailey
et al. 1996; Tiébré et al. 2007b) within Bohemian
knotweed, which we did not investigate, and any
variation in ability to produce viable seed among
cytotypes may influence their spread and abundance.
Since the three taxa react differently to digging,
chemical (glyphosate: Bı́mová et al. 2001) and
biological control methods (Grevstad et al. 2013),
taxon identification will be important for land managers. Our results demonstrate the three taxa can be
accurately identified using morphology, even in mixed
populations, which suggests that managers should
confidently utilize current identification keys (Zika
and Jacobson 2003; Wilson 2007), and that this
information should help update distribution lists (e.g.
Plants Database; USDA, NRCS 2012). The high
proportion of Bohemian knotweed places special
emphasis on its control priority. It is also the most
genetically diverse taxon found in our study in terms
of overall number of genotypes. Though genetic
diversity is only one consideration in development of
classical biological control, the differences found
between our taxa suggest that Bohemian knotweed
will present the most complex project, with perhaps a
subset of the continuum of genotypes required for
impact and host-specificity testing. The presence of
different genotypes of giant knotweed in almost each
population suggests that land managers should be alert
for variation among populations in efficacy of a
control strategy. Alternatively, Japanese knotweed,
with one genotype, simplifies the management plan,
though control will not necessarily be any more
successful than with the other taxa. Only one Japanese
knotweed genotype needs to be tested against candidate biological control agents or herbicides, due to the
lack of genetic diversity in this species found
throughout the invasion.
The high level of sexual reproduction in Bohemian
knotweed suggests an increased potential for evolving
resistance or tolerance to chemical or biological
control programs compared to Japanese knotweed,
Extreme differences in population structure
which has no genetic diversity upon which natural
selection can act. Certainly, each invasive knotweed
taxon presents its own unique challenges to control,
but the high level of gene flow between these three
taxa exacerbates the problem and points toward the
utility of genetic studies to help understand the
dynamics of a multi-taxon invasion. The use of
molecular markers to determine that there are common
genotypes, as well as significant genetic differences
across invasive ranges will facilitate sharing of
management information across continents.
Acknowledgments This research was made possible in part
by funding from the Bureau of Land Management (Montana,
South and North Dakota) Billings Office. Thanks to K. Mann
and J. Lassey for generating AFLP data. Thanks to our many
collectors (listed in ‘‘Appendix 1’’ in Supplementary material)
for sending in population collections of knotweed, to J. Andreas
for assisting in recruitment of collectors in Oregon and
Washington, and two anonymous reviewers for their helpful
suggestions.
References
Arthofer W (2010) tinyFLP and tinyCAT: software for automatic peak selection and scoring of AFLP data tables. Mol
Ecol Res 10:385–388
Bailey JP (1989) Cytology and breeding behaviour of giant alien
Polygonum species in Britain. Ph.D. Thesis, University of
Leicester, UK
Bailey JP (1990) Breeding behaviour and seed production in
alien giant knotweed in the British Isles. In: Anon (ed) The
biology of invasive plants; a BES industrial ecology group
symposium, pp 110–130. Richards, Moorhead and Laing,
Ruthin, Clwyd, UK
Bailey JP, Stace CA (1992) Chromosome number, morphology,
pairing, and DNA values of species and hybrids in the
genus Fallopia; (Polygonaceae). Plant Syst Evol
180:29–52
Bailey J, Wisskirchen R (2006) The distribution and origins of
Fallopia 9 bohemica (Polygonaceae) in Europe. Nord J
Bot 24:173–199
Bailey JP, Child LE, Conolly AP (1996) A survey of the distribution of Fallopia 9 bohemica (Chrtek & Chrtkova) J.
Bailey (Polygonaceae) in the British Isles. Watsonia
21:187–198
Bailey J, Bı́mová K, Mandák B (2009) Asexual spread versus
sexual reproduction and evolution in Japanese Knotweed
s.l. sets the stage for the ‘‘Battle of the Clones’’. Biol
Invasions 11:1189–1203
Bı́mová K, Mandák B, Pyšek P (2001) Experimental control of
Reynoutria congeners: a comparative study of a hybrid and
its parents. In: Brundu G, Brock J, Camarda I, Child L,
Wade M (eds) Plant invasion: species ecology and
2135
ecosystem management. Backuys Publishing, Leiden,
pp 283–290
Bzde˛ga K, Janiak A, Tarłowska S, Kurowskab M, TokarskaGuzika B, Szarejkob I (2012) Unexpected genetic diversity
of Fallopia japonica from Central Europe revealed after
AFLP analysis. Flora 207:636–645
Dice L (1945) Measures of the amount of ecologic association
between species. Ecology 26:297–302
Ehrich D (2006) AFLPDAT: a collection of r functions for
convenient handling of AFLP data. Mol Ecol Notes
6:603–604
Ellstrand NC, Schierenbeck KA (2000) Hybridization as a
stimulus for the evolution of invasiveness in plants? Proc
Natl Acad Sci USA 97:7043–7050
Engler J, Abt K, Buhk C (2011) Seed characteristics and germination limitations in the highly invasive Fallopia
japonica s.l. (Polygonaceae). Ecol Res 26:555–562
Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: An
integrated software package for population genetics data
analysis. Evol Bioinforma 1:47–50
Falush D, Stephens M, Pritchard JK (2007) Inference of population structure using multilocus genotype data: dominant
markers and null alleles. Mol Ecol Notes 7:574–578
Forman J, Kesseli RV (2003) Sexual reproduction in the invasive species Fallopia japonica (Polygonaceae). Am J Bot
90:586–592
Gammon M, Kesseli R (2010) Haplotypes of Fallopia introduced into the US. Biol Invasions 12:421–427
Gammon MA, Grimsby JL, Tsirelson D, Kesseli R (2007)
Molecular and morphological evidence reveals introgression in swarms of the invasive taxa Fallopia japonica, F.
sachalinensis, and F. 9 bohemica (Polygonaceae) in the
United States. Am J Bot 94:948–956
Gaskin J, Kazmer D (2009) Introgression between invasive
saltcedars (Tamarix chinensis and T. ramosissima) in the
USA. Biol Invasions 11:1121–1130
Geng Y-P, Pan X-Y, Xu C-Y, Zhang W-J, Li B, Chen J-K, Lu
B-R, Song Z-P (2007) Phenotypic plasticity rather than
locally adapted ecotypes allows the invasive alligator weed
to colonize a wide range of habitats. Biol Invasions
9:245–256
GISD (2005) Polygonum cuspidatum. http://www.issg.org/
database/species/ecology.asp?si=91&fr=1&sts=sss&lang=
EN. Accessed 2012
Grevstad F, Shaw R, Bourchier R, Sanguankeo P, Cortat G,
Reardon R (2013) Efficacy and host specificity compared
between two populations of the psyllid Aphalara itadori,
candidates for biological control of invasive knotweeds in
North America. Biol Control 65:53–62
Grimsby J, Kesseli R (2010) Genetic composition of invasive
Japanese knotweed s.l. in the United States. Biol Invasions
12:1943–1946
Grimsby JL, Tsirelson D, Gammon MA, Kesseli R (2007)
Genetic diversity and clonal vs. sexual reproduction in
Fallopia spp. (Polygonaceae). Am J Bot 94:957–964
Hardy OJ, Vekemans X (2002) SPAGeDi: a versatile computer
program to analyse spatial genetic structure at the individual or population levels. Mol Ecol Notes 2:618–620
Hillis DM, Mable BK, Larson A, Davis SK, Zimmer EA (1996)
Molecular systematics. Sinauer, Sunderland
123
2136
Hollingsworth ML, Bailey JP (2000) Evidence for massive
clonal growth in the invasive weed Fallopia japonica
(Japanese Knotweed). Bot J Linn Soc 133:463–472
Krebs C, Mahy G, Matthies D, Schaffner U, Tiébré M-S, Bizoux
J-P (2010) Taxa distribution and RAPD markers indicate
different origin and regional differentiation of hybrids in
the invasive Fallopia complex in central-western Europe.
Plant Biol 12:215–223
Ley AC, Hardy OJ (2013) Improving AFLP analysis of largescale patterns of genetic variation–a case study with the
Central African lianas Haumania spp (Marantaceae)
showing interspecific gene flow. Mol Ecol 22:1984–1997
Mandák B, Bimova K, Pysek P, Stepanek J, Plackova I (2005)
Isoenzyme diversity in Reynoutria (Polygonaceae) taxa:
escape from sterility by hybridization. Plant Syst Evol
253:219–230
Meirmans PG, Van Tienderen PH (2004) GENOTYPE and
GENODIVE: two programs for the analysis of genetic
diversity of asexual organisms. Mol Ecol Notes 4:792–794
Moody M, Les D (2007) Geographic distribution and genotypic
composition of invasive hybrid watermilfoil (Myriophyllum spicatum 9 M. sibiricum) populations in North
America. Biol Invasions 9:559–570
Müller-Schärer H, Schaffner U, Steinger T (2004) Evolution in
invasive plants: implications for biological control. Trends
Ecol Evol 19:417–422
Pielou EC (1969) An introduction to mathematical ecology.
Wiley Interscience, New York
Pritchard JK, Stephens M, Donnelly P (2000) Inference of
population structure using multilocus genotype data.
Genetics 155:945–959
Richards CL, Walls RL, Bailey JP, Parameswaran R, George T,
Pigliucci M (2008) Plasticity in salt tolerance traits allows
for invasion of novel habitat by Japanese knotweed s. l.
(Fallopia japonica and F. 9 bohemica, Polygonaceae).
Am J Bot 95:931–942
Richards CL, Schrey AW, Pigliucci M (2012) Invasion of
diverse habitats by few Japanese knotweed genotypes is
correlated with epigenetic differentiation. Ecol Lett
15:1016–1025
Rohlf FJ (1994) NTSYS-pc: numerical taxonomy and multivariate analysis system. Exeter Software, Setauket
123
J. F. Gaskin et al.
Saltonstall K (2002) Cryptic invasion by a non-native genotype
of the common reed, Phragmites australis, into North
America. Proc Natl Acad Sci USA 99:2445–2449
Sexton JP, McKay JK, Sala A (2002) Plasticity and genetic
diversity may allow saltcedar to invade cold climates in
North America. Ecol Appl 12:1652–1660
Shaw RH, Bryner S, Tanner R (2009) The life history and host
range of the Japanese knotweed psyllid, Aphalara itadori
Shinji: potentially the first classical biological weed control
agent for the European union. Biol Control 49:105–113
Sultan SE (1995) Phenotypic plasticity and plant adaptation.
Acta Bot Neerl 44:363–383
Swofford DL (2000) PAUP*. Phylogenetic analysis using parsimony (* and Other Methods). Sunderland, Massachusetts: Sinauer Associates
Tiébré M-S, Bizoux J-P, Hardy OJ, Bailey JP, Mahy G (2007a)
Hybridization and morphogenetic variation in the invasive
alien I (Polygonaceae) complex in Belgium. Am J Bot
94:1900–1910
Tiébré MS, Vanderhoeven S, Saad L, Mahy G (2007b)
Hybridization and sexual reproduction in the invasive alien
I (Polygonaceae) complex in Belgium. Ann Bot
99:193–203
USDA, NRCS (2012) The PLANTS Database. (http://plants.
usda.gov). National Plant Data Team, Greensboro, NC
27401-4901 USA. Accessed Jan 2013
Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M,
Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995)
AFLP—a new technique for DNA-fingerprinting. Nucl
Acids Res 23:4407–4414
Weber E (2003) Invasive plants in the world: a reference guide
to environmental weeds. CABI-Publishing, London
Weir BS, Cockerham CC (1984) Estimating F-statistics for the
analysis of population structure. Evolution 38:1358–1370
Wilson LM (2007). Key to identification of invasive knotweeds
in British Columbia. B.C. Min. For. and Range, For. Prac.
Br., Kamloops, B.C. http://www.for.gov.bc.ca/hra/
Publications/invasive_plants/Knotweed_key_BC_2007.pdf.
Accessed 28 Jan 2013
Zika P, Jacobson A (2003) An overlooked hybrid Japanese
knotweed (Polygonum cuspidatum 9 sachalinense; Polygonaceae) in North America. Rhodora 105:143–152