On the Origin of Sunflowers: Fossils, Genes, Genomes, and Hybridization
Loren H. Rieseberg1, Benjamin K. Blackman2, Moira Scascitelli1, and Nolan C. Kane1
1University of British Columbia, 3529-6270 University Blvd., Vancouver, B.C. V6T 1Z4, Canada,
lriesebe@mail.ubc.ca
2Department of Biology, Duke University, Durham, NC 27701, USA, bkb7@duke.edu
ABSTRACT
 The domestication of plants and animals by prehistoric humans was perhaps the most far-reaching
cultural development in human history. Not only were domesticated organisms crucial to the rise of
modern civilization, but their widespread use has dramatically altered the ecology and evolutionary
history of numerous other species. As a consequence, there is considerable interest in determining
the geographic origins, timing, and genetic bases of domestication.
 Here we review how molecular data has shed light on the origin of domesticated sunflowers, thereby
resolving a debate about whether agriculture arose wholly independently in Eastern North America.
Analyses of variation at microsatellite loci indicates that wild populations from the East-Central
USA are the most likely ancestral source for all domesticated sunflower landraces. Likewise, for
two of three candidate genes, domesticated alleles occur in wild populations from eastern North
America but are absent from Mexican wild populations. Thus, all extant cultivated sunflowers
appear to have arisen from a single domestication event in eastern North America.
 Ongoing studies are employing genome scan approaches to (1) identify large numbers of
domestication genes, (2) study the geographic distribution of domestication alleles, (3) determine the
targets of selection by early farmers, and (4) establish the role of hybridization in sunflower
domestication and improvement. In the future, we hope to extend these genome scan approaches to
include archaeological sunflower remains, which should allow us to estimate the timing and strength
of selection on domestication alleles.
Keywords - archaeological evidence - domestication genes - microsatellites - selective sweeps sunflower origins
INTRODUCTION
In 1951, Charley Heiser showed that the cultivated sunflower most likely was domesticated by
Native Americans in the East-Central USA (Heiser, 1951). This finding, which was based on analyses of
both extant sunflowers and archaeological remains, was of considerable general significance because it
implied that agriculture had arisen independently in eastern North America. Over the next 50 years,
Heiser’s conclusions were corroborated by additional findings of domesticated achenes from
archaeological sites in eastern North America (Crites, 1993) and by the failure to find sunflower remains
outside of this region.
These conclusions were revisited recently due to the discovery of a fossilized seed and achene,
tentatively identified as belonging to domesticated sunflowers, at the San Andrés archaeological site in
Tabasco, Mexico (Lentz et al., 2001; Pope et al., 2001). Using accelerator mass spectrometry the authors
dated the San Andrés remains to before 2000 B.C. The size of the Mexican achenes exceeded those found
in archaeological sites in eastern North America from the same time period. These data were interpreted
as evidence of an independent origin of the domesticated sunflower in southern Mexico that predated and
possibly influenced the later domestication of sunflower in eastern North America (Lentz et al., 2001).
This interpretation received further support from the discovery of three putative domesticated sunflower
achenes from the Cueva del Gallo site in Morelos, Mexico, one of which dated to 290 B.C. (Lentz et al.,
2008).
However, several scholars have taken issue with these findings. For example, Smith (2006) noted
that that the San Andrés specimens lack morphological characters diagnostic for Helianthus, and Heiser
(2008) documented the striking similarity of the San Andrés achene with bottle gourd seeds, which are
common at the site. Smith (2008) further commented that unlike the San Andrés material, the Cueva del
Gallo achenes ‘fall within the size range of the Marble Bluff (Arkansas) sunflower assemblage (n = 260),
which predates Gallo by >1,000 years.’ Thus, he argued that the Cueva del Gallo specimens (if shown to
be sunflower) probably represent an introduction from eastern North America.
Given these disagreements over the validity and interpretation of the fossil data, several studies have
been conducted using putatively neutral molecular markers and/or candidate domestication genes to
determine the number and geographic location(s) of domesticated sunflower origins. Here we briefly
review the findings from these studies and also describe some new analyses of microsatellite loci that
provide further support for a single origin of the domesticated sunflower in eastern North America.
NEUTRAL MARKER STUDIES
Several early studies examined relationships among cultivated and wild sunflowers using allozymes
(Rieseberg and Seiler, 1990), chloroplast DNA restriction site variation (Rieseberg and Seiler, 1990),
random amplified polymorphic DNA (Arias and Rieseberg, 1995), and nuclear microsatellite loci (Tang
and Knapp, 2003). However, these earlier studies failed to include significant sampling from Mexico,
and thus were not well designed to determine the geographic origin(s) of the domesticated sunflower. A
more comprehensive sampling strategy was employed by Harter et al. (2004), who analyzed variation at
18 microsatellite loci in 21 wild populations from throughout the native range of H. annuus, including
eight populations from Mexico. In addition to the wild population samples, Harter et al. assayed seven
landraces cultivated by Native American groups in the USA, two domesticated landraces from Mexico,
and one modern cultivar. Analyses of genetic relationships showed that all cultivars employed in the
study had a single origin in eastern North America. This result was corroborated by a subsequent analysis
of chloroplast DNA variation across a geographically broad sample of wild and domesticated accessions,
which pointed towards a single domestication ‘somewhere outside of Mexico’ (Willis and Burke, 2006).
While it initially appeared that the Harter et al. (2004) and Willis and Burke (2006) studies had
settled the sunflower origins debate in favour of a single origin in eastern North America, concerns were
expressed about the limited number of Mexican landraces included in these studies (Lentz et al. 2008).
Therefore, the microsatellite study of Harter et al. (2004) was extended to include five additional Mexican
landraces (Blackman et al., 2011). An assignment test using the computer program STRUCTURE
confirmed the previous conclusions of Harter et al. (2004): all seven of the Mexican landraces clustered
with eastern North American wild samples and not with the Mexican wild samples. Ancestry analyses
further revealed that > 96% of the alleles found in the Mexican cultivars derived from eastern North
American (ENA) wild populations.
Here we further investigated the assignment of these domesticated samples and their genetic
composition on a more detailed scale, using the four main wild clusters identified by Harter et al. (2004)
as potential ancestral sources. These four groups correspond to: East-Central USA (ENA), US Great
Plains, East-Central Mexico (plus Arizona) and West Mexico. We performed ten independent simulations
using a Bayesian clustering method, as implemented in Structure v2.3.3 (Pritchard et al., 2000). The
East-Central USA cluster was the most likely ancestral source for all the cultivated groups, including all
the seven Mexican landraces (Fig. 1), with an average ancestry coefficient of 0.99 for the eastern North
America cultivars and 0.98 for the Mexican (MX) cultivars.
Fig. 1. Estimated ancestry of domesticated H. annuus from North America (ENA Domesticated) and
Mexico (MX Domesticated). Each vertical bar represents an individual’s genome and grey shading
represents the proportion of the estimated ancestry of wild populations from the East-Central USA (USA,
ENA), USA Great Plains, eastern Mexico plus Arizona, and western Mexico.
We also performed a neighbour-joining (NJ) tree analysis with the extended sample set of Blackman
et al. (2011). The NJ tree was based on a matrix of Nei’s genetic distance (DA; Takezaki and Nei, 1996)
generated between pairs of populations or landraces, with 1000 bootstrap replicates. We then obtained a
consensus NJ tree with an extended Majority-Rule method using the programs neighbor and consense,
available in the package Phylip v3.69 (Felsenstein, 1989). This distance based method (Fig. 2) supports
the findings of the Bayesian clustering assignment analysis (Fig. 1): all the cultivars (ENA and MX)
cluster most closely with wild ENA populations and away from any of the wild Mexican groups. Taken
together, these results indicate all the extant cultigens, including all the indigenous Mexican landraces
currently analyzed, are derived wild sunflowers from the East-Central United States.
Fig. 2. Neighbor-joining tree for wild and domesticated sunflowers based on microsatellite genetic
distances. Wild populations are indicated by squares (grey-shading within squares matches potential
ancestral clusters used in Figure 1. ENA and Mexican landraces are visualized with empty or filled black
triangles, respectively. Two modern cultivars, Mammoth and USDA, are also included in the NJ tree.
CANDIDATE GENE STUDIES
The origin of extant domesticated sunflowers in eastern North America is also supported by sequence
variation in genes that have undergone selective sweeps during sunflower domestication. These
“domestication genes” are especially useful for elucidating the history of domestication because sequence
variation in positively selected genes is more likely to accurately reflect phylogenetic relationships than is
variation in neutral genes.
To further test for the possibility of a second origin of the domesticated sunflower in Mexico,
Blackman et al. (2011) analyzed sequence variation in three candidate domestication genes in an extended
sample of North American wild populations, Mexican wild populations, and indigenous Mexican
landraces (Blackman et al., 2011). The three candidate genes employed were: (1) c4973, a chorismate
synthase homolog involved in aromatic amino acid synthesis (Chapman et al., 2008); HaFT1, a homolog
of the floral inducer FLOWERING LOCUS T (Blackman et al., 2010); and (3) HaGA2ox, a Gibberellin 2oxidase homolog (Blackman et al., 2011). All three genes exhibit significantly reduced sequence
diversity in domesticated sunflowers when compared to neutral loci. HaFT1 has been shown to underlie a
major flowering time QTL in domesticated sunflowers, and selection for later flowering may be
responsible for the putative selected sweep observed for this gene. The selection pressures responsible for
the apparent sweeps at c4973 and HaGA2ox are unknown. However, gibberellin 2-oxidases are known to
be involved in the regulation of seed germination, and selection for reduced seed dormancy represents
one possible explanation for the selective sweep observed at HaGA2ox (Blackman et al., 2011).
The relative and absolute frequencies of the haplotypes found in wild and cultivated sunflowers from
USA/Canada or Mexico (Blackman et al., 2011) for the three genes are summarized in Figure 3. For two
of the three genes (HaFT1 and HaGa2ox), the domesticated landraces from Mexico were fixed (or nearly
fixed) for the same ‘domesticated’ allele found in North American cultivars. North American wild
populations carried the domesticated alleles at low frequencies, whereas these alleles were absent in
Mexican wild samples, as expected under the hypothesis of a single domestication event in eastern North
America. The fact that the domesticated haplotypes are at low frequency in wild USA populations is
consistent with a selective sweep, in which the domesticated allele increased in frequency during the
domestication process.
Fig. 3. Frequencies of domesticated and wild alleles in North American and Mexican landraces (ENA
and MX Domest., respectively) and in American and Mexican wild populations (ENA or MX Wild,
respectively). The number of haplotype sequences per allele class is reported inside each pie chart. The
three genes analyzed (HaFT1, HaGa2ox and c4973) appeared to have undergone selective sweeps during
early domestication in sunflowers (Chapman et al., 2008; Blackman et al., 2011).
Two domesticated haplotypes were found for the third gene, (c4973), both of which were present in
ENA and MX wild samples (Blackman et al., 2011). Thus, it was not possible to distinguish between
hypotheses of a single origin of the domesticated sunflower in eastern North America versus multiple
independent origins in both eastern North America and Mexico. However, as noted by Blackman et al.
(2011), the higher frequency of the domesticated haplotypes in wild populations from the East-Central
USA supports the former hypothesis.
In conclusion, all of the molecular data support the hypothesis originally put forward by Heiser
(1951) that cultivated sunflowers derive from a single domestication event in eastern North America. An
important caveat is that these molecular analyses have only sampled from extant sunflower cultivars.
Thus, the possibility of independent domestication and subsequent extinction of Mexican sunflower
cultivars cannot be ruled out.
FUTURE DIRECTIONS
While it might seem that the issue of domesticated sunflower origins is fully solved, at least with
respect to what molecular data can contribute, we feel that much more can and should be done. For
example, genome-wide scans are underway to identify a much larger fraction of candidate domestication
genes. Analyses of the geographic distribution of a larger number of domesticated alleles should allow us
to more precisely identify the ancestral germplasm that gave rise to the domesticated sunflower and better
determine the targets of selection by early farmers. Extension of the genome scan studies to include
archaeological sunflower remains will make it possible to estimate the timing and strength of selection on
domestication alleles, as well as to determine the ancestry of the Mexican fossils. Lastly, as sequence
data becomes available for other sunflower species, it will become increasingly possible to assess the role
of hybridization in sunflower domestication and improvement.
ACKNOWLEDGEMENTS
We thank Charley Heiser for inspiring the work described in this paper, David Lentz and Robert Bye
for their extensive collections of wild and domesticated sunflowers from Mexico, Abby Harter for access
to her microsatellite data set, and Bruce Smith for helpful discussions about sunflower origins.
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