Evolutionary Applications - 2010 - Muller - The Origin and Evolution of A Recent Agricultural Weed Population Genetic Sunflower

Evolutionary Applications ISSN 1752-4571
ORIGINAL ARTICLE
The origin and evolution of a recent agricultural weed:

population genetic diversity of weedy populations of
sunflower (Helianthus annuus L.) in Spain and France
Marie-Hélène Muller,1 Muriel Latreille2 and Christine Tollon2
1 INRA UMR 1097 DIA PC, Domaine de Melgueil, Mauguio, France
2 INRA UMR 1097 DIA PC, Montpellier Cedex 1, France
Keywords Abstract
crop-wild complexes, genetic diversity,
invasive species, sunflower, weediness. The recurrent evolution of crop-related weeds during agricultural history raises
serious economic problems and challenging scientific questions. Weedy forms
Correspondence of sunflower, a species native from America, have been reported in European
Marie-Hélène Muller, INRA UMR 1097 DIA sunflower fields for a few decades. In order to understand their origin, we anal-
PC, Domaine de Melgueil, 34130 Mauguio,
ysed the genetic diversity of a sample of weedy populations from France and
France.
Tel.: +33 4 67 29 06 39;
Spain, and of conventional and ornamental varieties. A crop-specific maternally
fax: +33 4 67 29 39 90; inherited marker was present in all weeds. At 16 microsatellite loci, the weedy
e-mail: Marie-Helene.Muller@supagro.inra.fr populations shared most of their diversity with the conventional varieties. But
they showed a large number of additional alleles absent from the cultivated
Received: 16 August 2010 pool. European weedy populations thus most probably originated from the
Accepted: 19 September 2010 unintentional pollination of maternal lines in seed production fields by wild
First published online: 26 October 2010
plants growing nearby, resulting in the introduction of crop-wild hybrids into
doi:10.1111/j.1752-4571.2010.00163.x
the farmers’ fields. The wide diversity and the low population structure
detected were indicative of a multiplicity of introductions events rather than of
field-to-field propagation. Further studies are required to understand the local
evolutionary dynamics of a weedy population, and especially the respective
roles of crop-to-weed gene flow and selection in the fate of an initial source of
crop-wild hybrids.
with the crop and to lead to more or less severe yield

Introduction
losses (Basu et al. 2004). Crop-related weeds have evolved
Crop plants stem from wild species through the process through different kinds of processes (Londo and Schaal
of domestication, the adaptation to cultivation and use by 2007): colonization of cultivated fields by plants from
humans (Zeder et al. 2006). During agricultural history, wild populations (e.g. wild rice taxa in Asia, Ellstrand
the continuous adaptation of the crop to new environ- 2003), crop-wild hybridization (Arnold 2004; Boudry
ments, new uses and new cultural practices, the constant et al. 1993), reversion of cultivated plants to wild habits
evolution of the wild populations, as well as gene flow (e.g. some weedy rice, Vaughan et al. 2005). Among these
between crops and their wild relatives led to the constitu- processes, crop-wild and crop-weed hybridization is rec-
tion of a continuum between typical ancestral wild plants ognized as very important in generating more noxious
and current elite crop varieties (i.e. crop-wild-weed com- weeds (Arnold 2004; Campbell et al. 2006). Understand-
plexes, Barnaud et al. 2009; Van Raamsdonk and Van der ing how crop-related weeds originate and spread, and
Maesen 1996). Some crop-related populations have how they adapt to the agro-ecosystem constraints is a
adapted in an undesired way to the new environment fundamental topic of research, as an example of recent
constituted by agricultural fields, the agro-ecosystem, giv- and rapid evolution (Kane and Rieseberg 2008). It may
ing rise to agricultural weeds (Arnold 2004). Weeds can also help prevent the evolution of new weeds as well as
be defined as plants that have the capacity to compete design appropriate management methods.
ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 499

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Origin and evolution of European weedy sunflowers Muller et al.
Common sunflower (Helianthus annuus L.) is an Different scenarios can account for the origin of these
annual self-incompatible insect-pollinated plant. It is weedy populations. First, seeds of wild forms could have
native to North America where it has been domesticated been introduced unintentionally by travellers, or have
at least 4000 years ago (Harter et al. 2004). Nowadays escaped from research nurseries (S1). Second, during the
cultivated sunflower markedly differs from typical wild seed production process, pollination of seed bearers by
sunflower on a set of characters: absence of anthocyan wild sunflowers could have introduced crop-wild hybrids
pigmentation in stem and head, absence of seed dor- in the seed lots sold to farmers (S2, as described for weed
mancy and seed shattering, self-compatibility, absence of beets, Boudry et al. 1993). Third, weedy sunflower could
branching, bigger heads and seeds (Burke et al. 2002). result from the spontaneous evolution of volunteer popu-
Wild and domesticated sunflower belong to the same spe- lations (S3). Finally, ornamental sunflowers commonly
cies and are interfertile (Snow et al. 1998). When unhar- grown in gardens and showing traits such as branching
vested seeds of the crop germinate, they give rise to and anthocyan pigmentation could have escaped and/or
volunteers: such plants grow within fields, or in fallows pollinated volunteer populations (S4).
and field margins; they display the morphological traits of To discriminate among these hypothesis, we analysed
the cultivated form, except for the segregation of a reces- the genetic diversity of a sample of French and Spanish
sive branching trait (Reagon and Snow 2006; Rojas-Barros weedy populations, volunteer populations, conventional
et al. 2008) and have never been reported to constitute and ornamental varieties, with 16 microsatellite loci and a
self-perpetuating populations nor to raise serious agro- mitochondrial crop-specific DNA marker (Rieseberg et al.
nomic problems (Ostrowski et al. 2010). By contrast, in 1994). Under scenarios S2 and S3, weeds are expected to
America, weedy forms of sunflower strongly affect the all carry the crop-specific maternally inherited marker,
yield of crops such as soybean and corn (Kane and Riese- whereas it should be absent for scenario S1. Because
berg 2008). Sunflower is even listed as noxious in some microsatellite diversity is known to be greatly reduced in
states and has evolved resistance to herbicide (Massinga the cultivated pool compared to the wild populations
et al. 2003). Based on an analysis of microsatellite diver- (Tang and Knapp 2003), the scenarios S1 and S2 imply
sity, Kane and Rieseberg (2008) showed that weedy sun- that a large diversity is observed in weedy populations,
flower populations are genetically close to the natural compared to scenario S3 where the diversity of weeds
wild populations occurring in the same region, suggesting is expected to mirror the diversity of the conventional
that weediness can evolve multiple times independently varieties. The outcome of S4 depends on the pattern of
from wild H. annuus. No phenotypic traits distinguishing diversity within the ornamentals, for which no data is
weedy and wild forms have been described in published available yet.
studies. Based on our results, we compare the validity of the
In Europe, where H. annuus is not native, volunteers scenarios and discuss the contribution of different factors
are also commonly observed in cultivated areas (Ostrow- to the further evolution of weedy sunflowers: recurrent
ski et al. 2010). Weedy sunflower populations have been introductions, dispersal from one field to another, and
reported since the 1970s (Faure et al. 2002; Holec et al. gene flow from the crop towards the weedy populations.
2005; Vischi et al. 2006), and have been more precisely
described through field surveys in France and Spain
Material and methods
(Muller et al. 2009): they show typical wild traits (e.g.
pigmentation, seed dormancy, strong branching), in com- Plant material
bination with domesticated traits. Weedy populations are A summary of the sample analysed in this study is given
thus made of a wide diversity of phenotypes, constituting in Table 1. We collected 38 weedy populations in three
a continuum between wild and cultivated morphotypes. regions: Andalusia in Spain, and two French regions, Poi-
French and Spanish weedy sunflowers are mostly tou-Charentes and Lauragais (Fig. 1). Spanish populations
observed within sunflower fields, and more rarely in other were sampled in summer 2005 during a survey of the
summer crops such as soybean and sorghum; in Spain, occurrence and distribution of weedy sunflowers (Muller
they sometimes occur on roadsides and ditches. Around et al. 2009). Populations located within sunflower fields
20% of sunflower fields are affected in the surveyed areas; and roadside populations were sampled. French popula-
in 1–4% of the fields, local density can reach 15 plants tions were collected during three separate surveys, in
per m2, which strongly impedes the yield and the harvest 2004, 2005 and 2006. Populations sampled in 2005 and
by farmers. Achenes can persist in the seed banks for sev- 2006 were described in Muller et al. (2009). One popula-
eral years (Muller et al. 2009). The only management tion was sampled both in 2004 and in 2006 (Odars, sam-
technique currently available is the manual eradication of ples further denoted Odars04 and Odars06). Sampled
the weeds, as long as the infestation is not too strong. French populations were all located in moderately to
500 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

Muller et al. Origin and evolution of European weedy sunflowers
Table 1. Summary of the sample analysed in the present study. Table 2. Name, producer and sample size of the conventional and
ornamental varieties.
Total Average
Number of sample sample size Seed Sample
populations size per population Name company/producer size
Conventional F1-hybrids 18 86 4.8 Conventional varieties

Ornamental varieties 6 31 5.2 Albena Euralis 5
Volunteers 3 64 21.3 All stars Euralis 6
Weedy populations 38 308 Atomic Euralis 5
Spain 14 58 4.1 Aurasol Monsanto 4
Lauragais A 18 71 3.9 Boogy Syngenta 5
Lauragais B 6 179 29.8 Country Syngenta 3
Charentes 3 7 2.3 Filia RAGT 4
Wild American populations 4 12 3 Heliasol Semences de France 7
LG5665M Limagrain 3
Italics: details for the sample of weedy populations (see text). Melody Syngenta 5
Pegasol Monsanto 4
PR64H41 Pioneer 6
Prodisol Monsanto 4
Rigasol Monsanto 5
Salsa Euralis 4
Santiago Syngenta 4
Tekny Syngenta 7
Tellia RAGT 5
Ornamental varieties
Beaute d’automne Royalfleur 4
Melange Kokopelli 9
Oiseaux Jardiland 5
Ring flowers Royalfleur 3
Soleilsimple Royalfleur 5
Sunrich F1 Royalfleur 5
Molecular data
We randomly chose around five progenies per natural
population, except for six French weedy populations
(subsample denoted Lauragais B in Table 1) and one vol-
Figure 1 Location of the regions where volunteer and weedy popula- unteer population (FR001, Ostrowski et al. 2010) where
tions have been sampled. at least 25 progenies per population were sampled. One
seed per progeny was germinated. Between 3 and 10 seeds
highly infested sunflower fields. We collected three volun- per variety, and between two and four seeds per Ameri-
teer populations in 2004. One population was located in can population were germinated (Table 2). As a whole,
the Gard (described in Ostrowski et al. 2010) and the two 501 individuals were included in the DNA analysis. As a
others in the Lauragais. Weedy and volunteer populations result of extraction or amplification failures, the sample
were mapped using global positioning system. They have sizes per population varied between 1 and 44.
in common to grow spontaneously, without having been DNA was isolated from about 100 mg of plant leaves
sown, and will be referred to as ‘the natural populations’ according to the Dneasy Plant Mini kit (Qiagen, GmbH,
in the following. In all situations, independent, open-pol- Hilden, Germany) with the following modification: 1% of
linated, progenies of seeds were collected. Polyvinylpyrrolidone (PVP 40 000) was added to buffer
We constituted a sample of 18 conventional F1-hybrids AP1.
including some of the main varieties cultivated in the past
years. Six ornamental varieties including commercial F1-
Microsatellites
hybrid and populations as well as seeds sold for bird feed-
ing were obtained from garden centres (Table 2). Finally, Sixteen microsatellite loci were selected from Tang et al.
we included four American wild H. annuus populations (2002). Care was taken to choose loci with no previous
stored in the laboratory as an external reference. evidence of null allele (Table 3, Tang et al. 2003; Tang

Table 3. Microsatellite loci, linkage group, core motif, fluorescent dye, allele size range and summary diversity statistics over the whole sample
(excluding wild American populations).
Locus Linkage group Position (cM) Core motif Fluorescent dye Allele size range No. of alleles He
ORS297 17 29.1 GT FAM 214–237 18 0.709

ORS309 4 75.5 A FAM 116–130 8 0.526
ORS337 4 62.2 AC FAM 165–197 15 0.394
ORS342 2 65 GT NED 305–361 26 0.665
ORS344 15 71.4 AC FAM 205–241 15 0.375
ORS371 1 44.2 GT HEX 234–264 17 0.666
ORS380 10 69.7 GT NED 380–434 25 0.721
ORS432 3 42.3 AC HEX 155–167 7 0.602
ORS610 1 3.4 AG NED 128–167 19 0.642
ORS620 4 57.1 AG FAM 224–266 18 0.659
ORS656 16 26.1 CT NED 181–254 23 0.792
ORS674 4 100.8 CT NED 331–372 23 0.805
ORS735 17 62.6 AG HEX 352–385 16 0.698
ORS788 16 46.2 AG FAM 252–296 25 0.628
ORS887 9 38.4 AC HEX 224–249 16 0.639
ORS925 2 5.9 AC FAM 165–251 22 0.834
He: Nei’s genetic diversity.
and Knapp 2003). The amplification reaction consisted of detect the occurrence of PET1 in our sample. Our ampli-
50 ng DNA, 4 pmol of unlabelled reverse primer, 2 pmol fication procedure consisted of 10 ng DNA, 10 pmol of
of forward primer, fluorescently labelled with NED, HEX each primer, 1· reaction buffer, 2 mm MgCl2, 100 lm
or FAM, 1· reaction buffer, 2 mm MgCl2, 200 lm dNTP, dNTP, 0.5 U Taq DNA polymerase, in a total volume of
0.25 U Taq DNA polymerase, in a total volume of 25 lL. 25 lL. The amplification method was as follows: 95C for
The amplification method was as follows: 95C for 2 min, 4 min, 36 cycles of 94C for 45 s, Tx for 45 s (Tx is ini-
36 cycles of 94C for 30 s, Tx for 30 s (Tx is initially 63C tially 65C and decreases of 1C per cycle for the 12 first
and decreases of 1C per cycle for the six first cycles, until cycles, until it reaches 56C), and 72C for 2 min and
it reaches 57C), and 72C for 45 s, followed by a final 30 s. Amplification products were separated by electro-
extension for 20 min at 72C. Electrophoresis was per- phoresis on 1.5% agarose gels, stained with ethidium bro-
formed on an ABI 3130xl Genetic Analyser (Applied Bio- mide and photographed under UV light. This procedure
systems, Foster City, CA, USA). Samples were prepared was applied to the whole sample of varieties, wild and
by adding 3 lL of diluted PCR products to 6.875 lL weedy populations, except for a part of weedy population
formamide and 0.125 lL of GenScan 400HD Rox size F06bis, for which DNA was not available anymore. Only
standard. The GENEMAPPER software (Applied Biosys- eight volunteer individuals were included.
tems) was used to analyse the DNA fragments and to
score the genotypes.
Data analysis
Genotypic disequlibrium
Mitochondrial marker
We analysed genotypic disequilibrium for all pairs of loci
The quasi totality of the varieties cultivated in Europe are in each sufficiently sampled population of weedy sunflow-
F1-hybrids, which result from the cross between two ers and volunteers (seven populations, see Molecular data)
inbred lines. The maternal lines carry a cytoplasm confer- with exact tests with GENEPOP (Raymond and Rousset
ring male-sterility, and until now, a single cytotype, 1995) and applied a sequential Bonferroni correction
named CMS89 or PET1, has been used. Because of its (Rice 1989).
maternal inheritance, this cytotype is thus present in all
commercial F1-hybrid varieties (Horn 2002). Using a Group level analysis and distinction between different classes
PCR-based strategy, Rieseberg et al. (1994) showed that of alleles
PET1 was absent from wild H. annuus populations, sug- Standard statistics of diversity were computed using
gesting that it is diagnostic of the maternal lines used for GENETIX (Belkhir et al. 2001) and FSTAT (Goudet 2001):
hybrid-seed production. We used the combination of the Nei’s expected heterozygosity (He, Nei 1987), the number
three PCR-primers described in Rieseberg et al. (1994) to of different alleles and the allelic richness standardized for

similar sample sizes (Ra, Petit et al. 1998). Because the corresponds to 33 weedy populations, three volunteer
genetic structures of the different groups were highly het- populations, three wild H. annuus populations, and all
erogeneous – F1-hybrids theoretically genetically homoge- conventional and ornamental varieties. The sample
neous, ornamental varieties of unknown genetic structure, Odars04 was not included, except for the neighbor-join-
mixture of natural populations of different sample sizes, ing tree (see below). Population statistics (He, Ra and
we first computed these statistics at the group level forig) were computed for each weedy and volunteer popu-
(Table 1). The significance of the differences in genetic lation. Overall FST values and their significance were com-
diversity statistics was tested using Wilcoxon signed-rank puted using GENETIX. AMOVA between groups was
tests, comparing values for the same loci in different performed with Arlequin (Excoffier et al. 2005).
groups using R (R Development Core Team 2008). A neighbour-joining tree (Saitou and Nei 1987) based
We denoted as ‘cultivated’ alleles (C), the alleles that on pairwise Cavalli-Sforza chord distances (Dc, Cavalli-
were observed in the conventional varieties or in the vol- Sforza and Edwards 1967) was built using PHYLIP ver-
unteer populations. All the other alleles were called ‘origi- sion 3.65 (phylogenetic inference package, Felsenstein
nal’ alleles (O). For each pool, we computed the 2005). The confidence of the tree was assessed through
frequencies of original and cultivated alleles (forig and 1000 bootstraps of allele frequency data: the majority rule
fcult), separately for each locus and over loci. The differ- tree and the bootstrap values were obtained with CON-
ence on forig between groups was tested using Wilcoxon SENSE.
signed-rank test as described earlier. To determine whether weedy population genetic struc-
Rarefaction curves were built for conventional varieties, ture followed a pattern of isolation by distance, Dc genetic
ornamental varieties and weedy populations to assess to distance matrices were correlated with geographical dis-
what extent our sample caught the allelic diversity exist- tance matrices using a Mantel test in FSTAT version
ing in each group. This especially allows assessing if the 2.9.3.2. Tests were performed for two subsets of the data:
O alleles are original because of an incomplete sampling Spanish weedy populations and Lauragais weedy popula-
of the conventional varieties or because of a true original- tions.
ity of the weedy populations. For a given group compris- For the seven natural populations (six weedy and one
ing N varieties (or populations), and for each value of k volunteer) for which more than 25 genotypes were avail-
varying between 2 and N ) 1, we constituted 200 random able, FIS values were computed and their significance was
subsamples of k varieties. We computed the total number assessed by 1000 permutations of alleles among individu-
of alleles observed over loci for each sample, and on aver- als with GENETIX version 4.05.2. The significance of the
age over the 200 subsamples of size k. For k = 1, we used differences of He, Ra and forig between populations was
the N observed values. For the ornamental group (six assessed as above for group-level comparisons. forig was
varieties), all combinations of k varieties among six were also computed at the individual level.
made as it was computationally feasible. For population
Odars, only the sample Odars06 was included in the com- Population structure on individual alleles
putations. We used permutations to look for a geographical struc-
All C alleles were shared between weedy populations ture of cultivated or original alleles, i.e. to detect if some
and conventional varieties (see Results). In order to com- alleles were more geographically clustered than expected
pare their pattern of allele frequency in these two groups, at random. First, for each allele, we computed the average
we computed for each locus allelic frequencies within the distance of an allele copy to the barycentre of all copies
‘C allele pool’ of the weedy populations, i.e. excluding O of that allele: this value was called the dispersion of an
alleles. We computed average within-population frequen- allele. We computed the average over loci and/or over
cies over all weedy populations, and separately over Span- allelic class (O or C) of the dispersion values. Singletons
ish and Lauragais weedy populations. Using R, we then were excluded from the data set.
performed a linear regression of C allele frequencies in Then for each locus, two kinds of permutations were
the weedy pools on C allele frequencies in the pool of performed: first, 1000 permutations of allele copies among
conventional varieties. Additionally, we used an analysis all geographical locations; second, 1000 permutations of
of covariance to include the loci as a cofactor.pAllele fre-
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi populations among geographical locations. Permutations
quencies were transformed by the function arcsinðxÞ of alleles allow detecting a geographical clustering which
prior to analysis. can be due both to differentiation between populations
and to resemblance between geographically closed popu-
Population-level analysis: diversity and differentiation lations; permutations of populations allow detecting
Population-level analyses were conducted only for popu- specifically the contribution of geographical distance
lations with a sample size of at least three individuals: this between populations to the clustering of alleles. For each

permutation, the dispersion values and their averages were Table 4. Population genetic statistics in the different groups of varie-
computed. The significance of observed values was ties, wild and weedy sunflower.
obtained by computing the proportion of simulated values No. of
that were lower than the observed one. Significance was original
assessed at the allelic level (for the individual allelic values), A Ra He alleles forig
at the locus level (for the averages over alleles at a locus), at
Conventional varieties 4.19 4.05 b 0.506 a 0 0.000
the allele class level (for the averages over O and C alleles) Ornemental varieties 6.44 6.31 c 0.701 bc 43 0.224 ab
and crossing the locus and allele class levels. Permutations Volunteer populations 3.63 3.30 a 0.475 a 0 0.000
were programmed in R. Weeds
Spain 13.81 10.66 e 0.753 c 152 0.261 b
Genetic clustering and admixture analysis France 14.00 8.25 d 0.665 b 151 0.147 a
To strengthen our descriptive results relying on the dis- Overall 17.75 9.04 0.687 211 0.168
Wild Helianthus 8.56 – 0.833 93 0.584
tinction between C and O alleles, we used STRUCTURE
annuus
version 2.3.3 (Falush et al. 2003; Hubisz et al. 2009). This
software uses multilocus genotypes to infer clusters of A: Average number of alleles per locus.
genetically similar individuals and to estimate admixture Ra: Average allelic richness per locus, standardized for a sample size
proportion of individuals between clusters. We ran the of 26 diploid individuals.
He: Nei’s genetic diversity over loci.
program for values of K = 1 to K = 10 clusters. Each run
The number of original alleles per group is the sum over loci.
consisted of a burn-in of 100 000 steps followed by Values with the same letters are not significantly different (P < 0.05).
100 000 steps of data collection and was repeated five ORS337 is included; including it or not does not change the signifi-
times. We used the model of correlated allele frequencies cance of the tests. Wild populations were not included in the statisti-
between clusters. We incorporated sampling locations as cal tests.
priors in the clustering (Hubisz et al. 2009): we defined
as locations every natural population, American popula-
tion and ornamental variety but grouped all conventional effect of the presence or absence of ORS337 was assessed
varieties as a single location. Following Evanno et al. in the statistical tests to avoid any misconclusion.
(2005), we calculated DK, the second-order change of the Summary statistics are given in Table 3 (sample-wide
likelihood function divided by the standard deviation of values for individual locus) and Table 4 (group level val-
the likelihood, to discuss the optimal value of K. Results ues). Between 7 and 26 alleles per locus were scored over
were visualized using the program DISTRUCT (Rosen- the whole sample. Conventional varieties and volunteer
berg 2004). populations showed the lowest diversity statistics, whereas
ornamental varieties, French and Spanish weedy popula-
tions were always significantly more diverse. Spanish pop-
Results
ulations showed a greater genetic diversity than French
Cytoplasmic analysis populations. Ornamental varieties were intermediate
Results were clear-cut. For conventional varieties, for vol- either between conventional varieties and weedy popula-
unteer populations and for one ornamental variety (Sun- tions with respect of allelic richness, and between French
rich F1, which is an F1-hybrid), all plants analysed and Spanish weedy population with respect of total
showed as expected the PET1 cytotype. By contrast, this genetic diversity. American wild populations exhibited
cytotype was absent from all other ornamental varieties very high levels of diversity, despite a very small sample
and all wild individuals. Finally, all weedy individuals size.
showed the PET1 cytotype. Conventional varieties and ornamental varieties showed
intravarietal genotypic diversity. This was unexpected for
conventional varieties as they are supposedly composed of
Nuclear diversity
a single genotype. However, this diversity was almost
Among 840 tests for genotypic disequilibrium between all caused by differences involving between one and four
pairs of loci, 63 (7.5%) were significant at the level of loci, and the number of alleles per loci never exceeded
P < 0.05, 19 of which concerning the volunteer popula- three, except for one seed lot where a mixing may have
tion FR001. Overall, only three tests were significant when occurred (Tekny). The alleles added by the outlier geno-
applying Bonferroni correction. Two of these tests types were never original relative to the other varieties,
involved ORS337 and respectively ORS620 (linked on i.e. were always present in an other variety. Seed lot con-
LG4, Table 3) and ORS735. Although linkage does not tamination was probably involved in this polymorphism.
seem to strongly affect genotypic disequilibrium, the Polymorphism was not unexpected in the ornamental

0.7
ORS 297 Ornamentals
0.6 Conventional varieties
Weeds
0.5 Volunteers
Allele frequency
0.4
0.3
0.2
0.1
0
214 215 216 217 219 220 222 223 224 225 226 228 230 232 233 234 235 237
1
ORS 344
0.9
0.8
0.7
Allele frequency
0.6
0.5
0.4
0.3
0.2
0.1
0
205 208 209 211 213 215 217 219 221 223 224 226 228 239 241
0.7
ORS 620
0.6
0.5
Allele frequency
0.4
0.3
0.2
0.1
0
233 235 237 239 241 242 243 245 247 249 251 253 255 257 259 262 264 266
Microsatellite size (bp)
Figure 2 Allele frequency distribution in the different groups for three representative loci.
varieties, as their genetic structure was not supposed to In the following, we will distinguish two classes of
be homogenous. alleles: the ‘cultivated’ ones (C) are those observed either
The volunteer populations shared all their alleles with in the conventional varieties or in the volunteer popula-
the conventional varieties except for six rare ones tions. They represent the diversity of the European culti-
among a total of 58. Over all loci, the cultivated pool vated pool. The ‘cultivated’ alleles were all shared with the
showed 67 different alleles, 73 when including the vol- weedy populations (Fig. 2 for three representative loci).
unteer populations that were supposedly derived from All the other alleles will be referred to as the ‘original’
cultivated fields. alleles (O).

Two hundred and eleven O alleles occurred in the

weedy pool (Table 4), 209 when we considered only the
250
33 weedy populations retained for further analysis. This
value was high compared to the 73 cultivated alleles. But
these O alleles were always rare relative to the C alleles.
200
Number of alleles
Fifty-six of these alleles were observed only once, and 90
were present in a single population. On average over loci,
150
O alleles represented 16.8% of the diversity of the weedy
pool. This was reflected in the patterns of allelic frequen-
cies in the different pools (Fig. 2). The main alleles in the
100
weedy populations were always C alleles. The ornamental
varieties, although less diverse, also displayed O alleles,
50
some absent from the weedy populations. The main Conventional varieties
Weeds
Weeds Spain
alleles in the ornamentals are not always C ones. Weeds France
Ornamentals
C alleles not only represent the largest part of the

0 5 10 15 20 25 30 35
diversity of the weedy populations, they also have similar
frequencies in the weedy and the cultivated pool. Linear k
regressions of C allele frequencies in the weedy pools on Figure 3 Total number of alleles over the 16 loci for subsamples of
frequencies in the cultivated pool were always highly sig- different sizes (k) within each group. Each point is the average over
nificant. The locus effect was not significant, e.g. no dif- 200 random subsamples, except for the ornamentals where all combi-
ference on the slope or on the y-intercept was detected. nations of subsamples (<200) have been constructed. Dashed lines
The best fit was obtained with the y-intercept set to zero show the minimum and maximum values for the weeds and for the
(R2 = 0.93, P < 0.0001 at the level of the whole weedy conventional varieties.
pool). For each locus, the most common alleles in the

cultivated pool are always the most common alleles in the
weedy populations (Fig. 2). of He and Ra markedly distinguished the volunteers from
Because we analysed only respectively 6 and 18 orna- the weedy populations (Fig. 4).
mental and conventional varieties, we assessed if our A similar level of variation was observed between the
sample was representative of the diversity of the culti- seven intensively sampled natural populations (Table 5).
vated pool. As shown in Fig. 3, the total number of The frequency of O alleles per individual showed a wide
alleles scored in k conventional varieties reached a pla- range of variation from individuals carrying only C alleles
teau: for k = 18 varieties, an added variety hardly added to individuals showing more than 50% of O alleles, espe-
new allele to the sample. We can then assume that we cially in Villefranche, the most diverse and most original
sampled almost if not all the diversity of the pool of population (not shown). Significant FIS values and high
conventional varieties at the genotyped loci, and that, to variance of these values among locus were detected. The
a large extent, we did not falsely qualify alleles as origi- number of C alleles per population exceeded what is
nal. By contrast, the number of alleles in the ornamental observed in the volunteer populations, and the number of
sample did not reach a plateau showing that we did not different O alleles reached 5 for some locus in three pop-
catch a significant part of the whole diversity of orna- ulations (Table 5).
mentals. The curves of the weedy populations did not Significant FST values were detected within each group
reach a plateau either; they are always higher than the of natural populations: Spanish weeds (FST = 0.120;
two others curves, which suggests that neither cultivated P < 0.001), French weeds (FST = 0.080; P < 0.001) and
nor ornamental varieties can account for the alleles volunteer populations (FST = 0.167; P < 0.001). AMOVA
observed in the weedy populations: these alleles must revealed a significant differentiation between these groups,
have a different origin. even if much of the variation lies within groups and popu-
lations (Table 6). Apart from this, no clear pattern of
geographical differentiation between populations arose:
Population variability and structure
evidence for isolation by distance was only detected in the
There was a wide variability among natural populations Lauragais (R2 = 33.16; P = 0.0005), but it was almost only
for diversity statistics (Fig. 4). Spanish weedy populations because of F09 (R2 = 4.24; P = 0.017 without F09) a weedy
were among the most diverse and those showing the population distant from the other populations.
highest frequency of O alleles, but a large overlap exist Based on Cavalli-Sforza chord distance (Fig. 5), only
with French weedy populations. By contrast, low values wild populations clustered notably together (i.e. with boot-

9 weedy populations clustered, one corresponding to the two

Spain
8 samples of population Odars. Drawing a NJ tree of individ-
France
7
uals did not show clear clustering patterns except for wild
Number of populations
Volunteers
populations and did not provide further information on
6
weedy populations’ relationships (not shown).
5
Permutations of allele copies between populations
4 showed that observed dispersion values are smaller than
3 expected, on average for C and O alleles, as well as for
2 the average locus values, when distinguishing O and C.
1
This shows that populations are differentiated signifi-
cantly from each other when considering separately O
0
1.8 2 2.2 2.4 2.6 2.8 3
and C alleles (Table 7). By contrast, permutations of pop-
Allelic richness ulations yielded significant results only for cultivated
9 alleles, and mostly for Lauragais populations. This is not
only because of a lack of power of our sampling design as
8
there are also less loci for which the observed values were
7
lower than the average of permutations. As a whole, this
6 suggests that the geographical location of populations did
5 not explain much of the differentiation observed.
4
3 Genetic clustering and admixture analysis
2
The variation of the likelihood of the data and the statis-
1
tic DK supported the number of clusters K = 2 as the
0 most probable (Fig. 6). For this value, a clear distinction
0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8
between conventional varieties and wild American popu-
He
8
lations was observed. Volunteer populations clustered
with the conventional varieties (Fig. 7), confirming that
7
they arose from the escape of cultivated plants. Weedy
6 and ornamental individuals showed varying levels of

5 admixture between the two clusters. Interestingly, for
weedy populations, the proportion of ancestry to the wild
4
cluster is strongly correlated with the frequency of origi-
3 nal alleles, at the population level (R2 = 0.8955) and at
2 the individual level (R2 = 0.8063). Increasing K did not
give rise to clear and strongly repeatable results; the pat-
1
terns observed revealed mainly (i) subdivisions within the
0 cluster of conventional varieties and (ii) strong admixture
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
f orig within almost all weedy populations: in some of them
appeared a new cluster, which was absent or extremely
Figure 4 Distribution of diversity statistics across weedy populations rare from the wild American populations and from all
from France and Spain and volunteer populations. Allelic richness is conventional and ornamental varieties (not shown).
the mean allelic richness over loci, standardized for two diploid indi-
viduals per population.
Discussion
strap values >50%). Volunteer, weedy populations, con- Origin of weedy populations
ventional and ornamental varieties were intermixed. There We analysed the genetic diversity and population struc-
was some clustering of varieties, Spanish and Lauragais ture of weedy sunflowers in Europe, in reference to culti-
weedy populations, but few bootstrap values exceeded vated varieties. According to our results, we can conclude
50%. Two clusters included varieties from the same seed that the most probable origin of these weedy populations
company (V1 for Monsanto, and V2 for Syngenta, Fig. 5). is the unintentional pollination of the maternal lines of
Interestingly, each volunteer population clustered signifi- F1-hybrid seed by wild plants growing nearby and the
cantly with a conventional variety. Finally, three pairs of resulting introduction of crop-wild hybrids into the

Table 5. Diversity statistics within the intensively sampled natural populations.
He Ra forig FIS O allele per locus C allele per locus
Baziege 0.540 cd 3.96 c 0.061 c 0.156*** 0.81 [0,2] 3.81 [2,6]

Fourquevaux 0.612 bc 4.94 b 0.134 bc 0.192*** 2.19 [0,5] 3.75 [1,6]
F06bis 0.597 bc 4.07 c 0.103 bc 0.311*** 1.06 [0,3] 3.37 [1,6]
Gardouch 0.640 ab 4.86 b 0.170 ab 0.257*** 1.87 [0,5] 3.94 [1,7]
Odars 06 0.640 b 5.05 b 0.160 b 0.233*** 2.44 [0,4] 3.69 [2,7]
Villefranche 0.698 a 5.86 a 0.239 a 0.152*** 3.44 [1,5] 3.75 [2,7]
FR001 0.450 d 2.53 d 0 0.252*** 0 2.87 [2,5]
Values with the same letters are not significantly different from each other.
***P < 0.0001 O (resp. C) alleles per locus gives the average over loci of the number of different original (resp. C) alleles within the population,
with its minimum and maximum values. FR001 is a volunteer population.
Table 6. AMOVA and hierarchical analysis. whole have been detected. Even if previously grown land-
races are expected to be more diverse than elite inbred
France/Spain Weeds/volunteers
lines and might explain the occurrence of some original
Source of variation (%) alleles, the number of scored O alleles is still too big to
Among groups 2.5 5.4 be accounted for by the diversity of the cultivated pool.
Among populations 10.1 11.8 Ornamental varieties shared O alleles with the weedy
within groups
populations, but also displayed alleles absent both from
Within populations 87.4 82.8
Fixation indices
the weeds and from the conventional varieties. Even if
FST 0.126*** 0.176*** Fig. 3 suggests that we missed alleles from the ornamental
FSC 0.104*** 0.124*** varieties and even it cannot be excluded that they have
FCT 0.025*** 0.054* contributed to some weedy populations, it seems that
they represent a distinct gene pool that again cannot
*P < 0.05, ***P < 0.001.
account for the extraordinary diversity of alleles present
in the weeds (Fig. 2). The position of ornamental varieties
cultivated fields. First, all weedy plants carry the mito- in breeding schemes relative to conventional varieties,
chondrial DNA diagnostic for the maternal line used in wild forms and even related Helianthus species is unclear.
hybrid seed production. This is in accordance with the A more thorough investigation of the diversity of the
observation of male-sterile weeds during field surveys ornamentals would be illuminating first in this respect,
(Muller et al. 2009) and discards the hypothesis of wild and as a by-product may bring some light on their actual
seeds being a source for weedy populations (scenario S1) role in the evolution of weedy populations.
Second, the main part of the diversity of the weeds is Our small sample of four wild American populations
made of the alleles present in the conventional varieties. illustrates the enormous allelic richness occurring in the
The frequencies of these alleles within the weedy pool are area of origin of H. annuus. Some O alleles are found in
strongly correlated with their frequencies within the pool this sample, and a preliminary analysis of a bigger data
of conventional varieties: it suggests that the main part of set including 77 wild populations shows that grossly all C
these ‘cultivated’ alleles come from the varieties, either and O alleles are present in the wild. A wild origin of
through the initial crop-wild hybrids introduced in the these alleles is thus a reasonable hypothesis. Helianthus
field or from gene flow from the varieties cultivated in annuus is a bee-pollinated outcrosser, and pollination dis-
the field towards the weedy populations. tance can reach at least 1000 m (Arias and Rieseberg
Last, at nuclear microsatellite loci, weedy populations 1994) which is more than the isolation distance required
display a large diversity of original alleles (denoted as O for commercial seed production (500 m in the European
alleles), absent from our sample of conventional varieties. Community). In America, off-type plants showing wild-
Although this sample is not exhaustive, the curves in type traits and supposedly derived from wild pollen con-
Fig. 3 showed that it probably caught the vast majority of tamination during seed production are found in most
cultivated alleles. Moreover, Tang and Knapp (2003), sunflower fields (Reagon and Snow 2006). In Europe, F1-
Tang et al. (2003) and Zhang et al. (2005) analysed hybrid seeds have been introduced from America in the
respectively 19, 24 and 124 inbred lines at microsatellite 1970s, in the beginning of F1-hybrid seed development.
loci: For the fourteen loci that we have in common with These importations have been suggested as the probable
at least one of these studies, only three more alleles as a origin for weeds occurring in Spain and Italy (Faure et al.

E01
E30
E57
La Benatre
E53
All stars
F05 Filia
Heliasol V2
F06bis
L363
Villefranche Gardouch Atomic
LG5665M Fourquevaux
Santiago Country
Rigasol Baziège 100 Melody
E20 L365
Prodisol Tekny
V1 100
72
FR006
Boogy Albena
FR007
Aurasol 58
74 L126
Pegasol 53
94 99 FR001
Soleil simple
Tellia
PR64 E50 Auxence
E34
54
L366 54
52 E05
L315
Odars06 E08
L360 E41
Odars04 79
L345 L336
L343
L344
L349
E13
91
Salsa E61
Mélange
Oiseaux
E27
Ringflowers
SunrichF1 Beauted’automne W435
W446 W998
Wild populations
0.1
Figure 5 Consensus neighbour-joining tree depicting the relationships between sampled natural populations and varieties, based on Cavalli-
Sforza’s genetic distance. Percentages on each branch indicate the proportion of bootstrap replicates in which the two sets separated by that
branch appear. Only values over 50 are reported. Italics: ornamental varieties. Underlined: conventional varieties. Shadowed: volunteer popu-
lations. Bold: American wild populations. Other: weedy populations. V1, V2: cluster of varieties sold by the same seed company (see text).

Table 7. Summary of the results of the permutations.
Permutations of alleles Permutations of populations
Allele type General Lauragais Spain General Lauragais Spain
P-value of average dispersion values per allele type

C <0.001 0.001 0.004 0.002 0.028 0.221
O 0.288 <0.001 <0.001 0.177 0.084 0.277
Significant loci (loci with lower than expected dispersion value)
C 6 (11) 8 (13)* 2 (13) 14 (16) 9 (16) 0 (13)
O 3 (6)** 14 (15) 5 (12) 4 (9)* 3 (13) 1 (8)
P-value of average dispersion values per allele type: proportion of simulated values of dispersion that were lower than the observed ones. P-values
less than 0.05 denotes a significantly lower than expected dispersion of alleles copy within a given allele type.
Significant loci: number of loci for which the observed average dispersion values were significantly lower than the simulated ones (P-value < 0.05).
The number of loci for which observed dispersion values were lower than the average of simulated values, significantly or not, is given between
parentheses.
*,**One or two loci for which the average dispersion value was significantly higher than expected (i.e. P-value > 0.95).
Only 15 loci available.
Including or not ORS337 in the computation did not change the P-values.
2002), although no studies have been conducted to ascer- exhaustive sample of wild American populations and
tain this hypothesis. Nowadays, commercial seeds are still ornamental varieties is to be constituted.
imported from America, although it is difficult to say
from import and export data if these seeds are sown in
Evolutionary history of the weedy populations: multiple
France or re-exported to other countries (C. Lascrombes,
introduction events, dispersal and crop-weed gene flow
personal communication).
Analysis of the population structure existing in Ameri- The structure of genetic diversity within and between
can populations (Harter et al. 2004) could potentially allow weedy populations results from the interactions between
identifying a geographical origin of the contaminants. different factors: (i) the initial introduction event in a
However, as weedy plants are now present in Europe, they field, involving a given seed lot or a given contaminating
can serve as pollen contaminants in seed production fields wild population. The source can be shared or not
located in Europe. In France, no specific area is devoted to between populations. (ii) Dispersal from an infested field,
seed production; seed production fields occur in the giving rise to a new weedy population; and (iii) gene flow
regions where weedy populations have been observed from the varieties cultivated in the field towards the
(http://www.gnis.fr), even if no survey has been conducted weedy populations.
to assess precisely their proximity. The rate of impurity The outcome of such history is expected to be complex
allowed in certified seed is 5%: with a seed density of and difficult to interpret. As an illustration, within popu-
60 000 plants per hectare, even 1% of contaminants will lation, the number of different cultivated and original
result in 600 crop-wild hybrids sown in a field. In Laurag- alleles can reach 7 for C alleles and 5 for O alleles
ais, we saw every now and then, off-type plants, showing (Table 5). Even if the status and origin of cultivated and
wild traits growing on the rows of otherwise uninfested original alleles is not clear, this pattern at least suggests a
sunflower fields. Even if the fate of these sparse plants is diversity of contributors for a given weedy population: an
not certain, it suggests that new weedy populations might unknown number of initial crop-wild hybrids introduced
now arise from European crop-weed hybrids and blur the in the field, a possible multiplicity of introductions, and
information on the original introductions. This, in addi- different varieties further cultivated in this field. By con-
tion to the rarity and the wide diversity of original alleles, trast, the volunteer populations have a simpler genetic
makes rather unrealistic a full elucidation of the scenario of constitution and cluster each with a single conventional
introduction and diffusion of these weeds. variety in Fig. 5; this variety may be very close to the one
Our Bayesian analysis validated our conclusions based or the one itself from which this volunteer population
on the analyses on C and O alleles. It additionally high- originated.
lighted the complexity of the genetic structure of weedy Despite this complexity, our results can still bring a
populations and of ornamental varieties, which appear as few insights in two respects: the dynamics of new infesta-
strongly admixed even when increasing the number of tions, and the occurrence of crop to weed gene flow
clusters K. Such analysis looks promising if a more within infested fields.

(A) K P-C
0 1 2 3 4 5 6 7 8 9 10
–19 000
Spain
–20 000
–21 000
Ln P(D)
–22 000
–23 000
–24 000
(B)
80
70
Weedy
Lauragais
populations
60
50
ΔK
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10
K
Figure 6 (A) Average and standard deviation of the likelihood of the

data vs. the number of clusters (K) assumed under the software
STRUCTURE. Standard deviations are plotted when they are larger
than the symbol used for the average. (B) Second-order rate of
change in K (DK) vs. K.
Volunteer
populations
Population structure and introduction events
Weedy populations were significantly differentiated from
Wild
each other, with a small but significant differentiation populations
between France and Spain (Table 6). But apart from this,
no clear geographical structure was detected: no signifi- Ornamental
varieties
cant clustering in the neighbour-joining tree (Fig. 5) and
no evidence of isolation by distance. On the assumption
that the original alleles, contrary to the cultivated ones, Conventional
could carry the footprints of an introduction event, and varieties
of its dispersal, we conducted separate analysis on the
geographical structure of these alleles only: permutations
of alleles between populations showed that these alleles
Figure 7 Bayesian analysis of population structure and admixture in
were significantly more clustered than expected at ran- our data set, for a number of K = 2 clusters. Each individual is rep-
dom. This could result from differentiation of single pop- resented by a thin horizontal line, which is partitioned into two col-
ulations, because of different introduction events and/or oured segments that indicate the individual’s ancestry into the two
from strong genetic drift since the introduction. By clusters.

contrast, permutation of populations led to much less sig- demonstrated for the origin and evolution of weed beets
nificant results, showing that original alleles are not invading sugar beet producing fields in Northern Europe
shared by geographically close populations, but are rather (Boudry et al. 1993) and for some American weedy rice
randomly distributed in space. populations (Londo and Schaal 2007). Wild carrots are
Although our sampling design is not sufficiently power- also known to pollinate seed plants in seed production
ful to demonstrate any detailed history (original alleles are fields (Magnussen and Hauser 2007). Nowadays, as new
all rare and population sample sizes are small), our results contaminations seem to be more rapid than seed dispersal
are rather compatible with multiple, uncorrelated intro- across the agro-ecosystem, attention has to be paid to the
ductions of the weedy populations. Dispersal of weeds isolation of seed producing fields. For instance, in the
from infested fields to neighbouring fields seems rare, case of weed beets, a shift of the seed production area,
which is compatible with field observations (Muller et al. together with an increased care taken to the elimination
2009): strongly infested fields often neighboured fields of wild beets flowering around seed production fields has
without any trace of weeds. New apparitions of infested helped to reduce the contamination rate to 0.04%
fields through contaminated seed lots could be more (Arnaud et al. 2009).
frequent than new apparitions through seed dispersal. Further studies are now needed especially at the field
level to disentangle the interacting factors that may (or
may not) lead from an introduction event (a few crop-
Crop to weed gene flow
wild hybrids) to a strongly infested field: size of the initial
Crop to weed gene flow is possible, as the weeds and the source, importance of crop-weed flowering overlap, mat-
crop belong to the same species, grow in the same loca- ting patterns and selective pressures acting on wild vs.
tion and overlap partially in flowering time (Muller et al. domesticated traits. This is of interest to understand the
2009). Weedy populations showed variable proportions of evolution of invasiveness in presence of gene flow. A
original alleles (Fig. 4, Table 5). Phenotypic data is avail- major question is especially on how gene flow from the
able on the populations sampled in 2006. Interestingly, crop may or not promote adaptive divergence of the
the populations showing the highest frequencies of origi- weedy populations, and further the evolution of more
nal alleles are those that show the highest frequencies of aggressive or less controllable weeds. The interaction
wild phenotypic traits and reciprocally (anthocyan pig- between selection and gene flow is a basic field of
mentation, self-incompatibility, seed dormancy, Muller research (Räsänen and Hendry 2008) as well as a key
et al. 2009), suggesting original alleles are indeed and issue when investigating the spread of crop alleles into
grossly indicative of a variable proportion of wild gen- wild populations (Chapman and Burke 2006). More par-
ome. Significant FIS values as well as their high variance ticularly, such studies could help predict the impact of
across loci denoted nonrandom mating within the popu- new varieties carrying an herbicide resistance recently
lation: partial selfing and gene flow variable in space and released in Europe (Sala et al. 2008).
time. Therefore, the observed variation across populations
of the frequencies of original alleles depends (i) on the Acknowledgements
actual rates of crossing and actual flowering time of
the sampled heads at the present generation and (ii) on We thank Florent Pourageaux for contribution to molec-
the evolutionary history within each field in the preceding ular analysis and Marie Roumet and Laurène Gay for
generations, including the age and size of the initial intro- careful reading of the manuscript. We thank the reviewers
duction event and recurrent gene flow from the cultivated for their helpful comments. This work was funded by the
varieties. Both factors could also explain the differences Bureau des Ressources Génétiques and by the Centre
observed at the country level: Spanish weedy populations technique interprofessionnel des oléagineux métropoli-
indeed showed a higher allelic richness, a higher fre- tains (CETIOM).
quency of original alleles and a higher level of admixture
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Evolutionary Applications - 2010 - Muller - The Origin and Evolution of A Recent Agricultural Weed Population Genetic Sunflower

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Evolutionary Applications ISSN 1752-4571

The origin and evolution of a recent agricultural weed:

with the crop and to lead to more or less severe yield

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 499

500 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

Conventional F1-hybrids 18 86 4.8 Conventional varieties

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ORS297 17 29.1 GT FAM 214–237 18 0.709

He: Nei’s genetic diversity.

502 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 503

504 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

Microsatellite size (bp)

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 505

Two hundred and eleven O alleles occurred in the

C alleles not only represent the largest part of the

pool). For each locus, the most common alleles in the

506 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

9 weedy populations clustered, one corresponding to the two

6 and ornamental individuals showed varying levels of

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 507

Table 5. Diversity statistics within the intensively sampled natural populations.

He Ra forig FIS O allele per locus C allele per locus

Baziege 0.540 cd 3.96 c 0.061 c 0.156*** 0.81 [0,2] 3.81 [2,6]

508 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

SunrichF1 Beauted’automne W435

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 509

Table 7. Summary of the results of the permutations.

Permutations of alleles Permutations of populations

Allele type General Lauragais Spain General Lauragais Spain

P-value of average dispersion values per allele type

510 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

Figure 6 (A) Average and standard deviation of the likelihood of the

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 511

512 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514 513

514 ª 2010 Blackwell Publishing Ltd 4 (2011) 499–514

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