DISCUSSION
Genomic-based epidemiology using markers specific to a characterized
resistant haplotype of EPSPS duplication from the Central Great
Plains identified three unique EPSPS haplotypes in GR kochia
populations from across western North America. Haplotypes 1 and 2 both
showed increased copy numbers of the MGE previously identified, but
haplotype 2 does not have insertion of the MGE next to EPSPS at
the same position as identified in haplotype 1. EPSPS duplication
in haplotype 2 may have occurred through a similar genetic mechanism as
haplotype 1 involving insertion of an active mobile genetic element next
to EPSPS followed by tandem duplication. Resequencing and
assembly of the EPSPS locus in haplotype 2 will be needed to
determine the precise duplication mechanism that occurred and to
determine whether the MGE, which had increased copy number in haplotype
2, is associated with the EPSPS duplication. We consider
haplotype 3 to represent a third independent origin because it had
increased EPSPS copy number and no increase in MGE, indicating
that EPSPS duplication may also have occurred through yet another
mechanism such as a double-strand break initiated by a different MGE.
These haplotypes, based on either the presence or absence of type I and
type II elements and ratio of MGE, provide evidence that glyphosate
resistance evolved multiple times in geographically distinct locations,
with three independent origins supported by the data.
Haplotype 3 was found in populations from OR, ID, and WY. From the
neighbor-joining tree, population WY1R (Northern Plains) was closest to
OR5R (Pacific Northwest) with support of 41% (Figure 2), but this pair
was distant from the other Pacific Northwest populations and not
clustered with Pacific Northwest populations in the STRUCTURE plot
(Figure 3). The shared haplotype 3 between geographically isolated
northern Wyoming and the Pacific Northwest could indicate two separate
origins of haplotype 3 (increased-EPSPS without increased MGE),
or it could indicate lack of resolution in the population genetics data
to resolve gene flow from independent origins of resistance. Further
characterization of the duplicated resistant locus is needed to
determine whether populations from the Northern Plains (WY1R) and the
Pacific Northwest have a shared or separate origin of duplicatedEPSPS genes. Other Northern Plains populations (WY and southern
MT) contained individuals with either haplotype 2 or haplotype 3,
indicating gene flow via migration between populations and/or dynamic
MGE changes in copy number over time.
We predicted that population genetics analysis would show clear
geographical structure if the three glyphosate resistance evolutionary
events occurred and were followed by rapid dispersal and introgression
within a region. Aside from a large grouping of Central Great Plains
populations and a second grouping of Pacific Northwest populations, we
were not able to identify clear geographical structure for the three
regions corresponding to the three EPSPS haplotypes. The
STRUCTURE analysis supported K=3, although populations were not
consistently assigned to three groups corresponding geographically to
the regions containing the three EPSPS haplotypes and most
populations contained some presence of all three groups. Some strong
signals of relatedness were detected between populations from
geographically isolated locations, such as OR4R and KS10R (Figures 2,
3). While we consider it to be unlikely that the same duplication
mechanism is evolving independently multiple times within a region on
different genetic backgrounds, the SSR data may have insufficient
resolution to identify population genetic relationships and extensive
long-distance gene flow via seeds may make regional differences harder
to detect. This aligns with previous population genetics studies in
kochia that have found high genetic diversity within individuals and
little population structure (Friesen et al., 2009; Kumar et al., 2019;
Mengistu & Messersmith, 2002).
Haplotype distribution was consistent with division by mountainous
geographic barriers, with haplotype 1 distributed across the Central
Great Plains associated with the earliest report of glyphosate
resistance in kochia, 2007, in Kansas (Waite et al., 2013); haplotype 2
located within the Northern Plains including northern Montana and
Alberta; and haplotype 3 located in sugar beet fields in the Pacific
Northwest and north-central Wyoming (Figure 1). Since glyphosate
resistance evolved recently, the geographic patterns of haplotype
distribution (via seed dispersal) also occurred more recently and appear
to be introgressing at a more regional scale into diverse genetic
backgrounds (via pollen flow).
The presence of more than one EPSPS haplotype within populations
provides strong evidence for gene flow among populations. This was
observed in some northern Colorado populations, with some haplotype 2
individuals present in populations that were mostly haplotype 1 (e.g.,
CO3R from Cope, CO showing high EPSPS , no Type I or II, and high
MGE, like samples from northern Montana and Alberta), and some haplotype
3 individuals present in populations mostly containing haplotype 1
(Eaton, CO) (Figure 1, Table S1). These two populations showing
admixture in CO indicates gene flow has occurred in this area.
A curious result in the population relatedness data was that several
populations from Texas (TX3R, TXR4, TX5R), a population from Oregon
(OR1R), and the furthest north population from Alberta, Canada (AB1R)
were grouped with relatively low (>20%) bootstrap support
in the phylogeny tree (Figure 2). Further research will be needed to
define the relationship of these populations and to determine whether
long-distance gene flow is occurring in kochia, either through natural
tumbling dispersal or through human-mediated seed migration. Some other
weed species also demonstrate little population structure. Palmer
amaranth (Amaranthus palmeri ), an obligate outcrossing weed
species subjected to widespread glyphosate selection pressure, also
maintains high levels of genetic diversity and little population
structure (Küpper et al., 2018; Molin, Patterson, & Saski, 2020a).
Kochia has protogynous flowers and pollen that is both wind and insect
dispersed (Blackwell & Powell, 1981), facilitating high outcrossing
levels. Kochia is also one of the primary tumbleweed species of the
western US and Canada. This uncommon mode of seed dispersal may serve to
further homogenize genetic diversity across long geographic distances.
Single origins of herbicide resistance followed by substantial
geographical distribution by pollen and seed mediated gene flow is known
to have a major contribution to resistance frequency in multiple weed
species (Beckie et al., 2019). Multiple independent origins of
glyphosate resistance with little population structure was found in
glyphosate-resistant populations of common morning glory (Ipomoea
purpurea ) (Kuester, Chang, & Baucom, 2015). A genomics-based approach
in the same species found evidence for parallel genetic responses in
genomic regions encoding herbicide detoxification in, while other
genomic regions showed divergent patterns of selection (Van Etten, Lee,
Chang, & Baucom, 2020). The high sequence similarity of the
extrachromosomal DNA containing the EPSPS gene in Palmer amaranth
(Koo et al., 2018; Molin, Yaguchi, Blenner, & Saski, 2020b) across
widespread populations supports the hypothesis of a single origin
followed by dispersal (Molin et al., 2020a; Molin, Wright, Lawton-Rauh,
& Saski, 2017; Molin et al., 2018). Glyphosate-resistant populations of
flaxleaf fleabane from across multiple Australian states were highly
related, supporting a high frequency of seed movement (Minati, Preston,
& Malone, 2020). Multiple independent origins of glyphosate resistance
were detected in horseweed in California, with localized movement of
resistant individuals accounting for spread on regional levels
correlating with groundwater regulations that encouraged more glyphosate
use and less use of other herbicides (Okada et al., 2013).
In summary, we used genomic-based epidemiology to track the mutations
underlying one specific origin of glyphosate resistance in kochia and
showed that at least three independent origins of glyphosate resistance
have evolved in kochia, followed by substantial regional gene flow to
spread the resistance alleles to new genetic backgrounds. Due to the
tumbling dispersal of kochia, intercepting seed movement across the
landscape has high potential to mitigate the negative impact of
herbicide resistance spreading from an initial origin. With the kochia
reference genome now available (Patterson et al., 2019), the population
genomics approach used by Kreiner et al. (2019) can be used in kochia to
study population divergence and origins of resistance (Martin et al.,
2019). Sequencing and assembly of the duplicated region from haplotypes
2 and 3 would provide insights and new markers to further investigate
the evolutionary dynamics of the EPSPS tandem duplication in
kochia across western North America.