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Supporting Information
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Supporting Fig S1 . Helianthus phylogeny reprinted with permission from (Timme, Simpson &
Linder 2007).
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Supporting Methods S1. Methods for unpublished studies.
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Studies A and B). Phylogenetically explicit leaf trait common garden study.
Unpublished. For comparison of leaf traits, Phoebanthus tenuifolius and fourteen Helianthus
species were grown in a greenhouse common garden in the summer of 2012. Seeds were either
collected from wild populations of each species or obtained from the USDA Germplasm
Resources Information Network. Three populations per species were selected for inclusion from
across the range of each species, with a total of eight replicate plants per population averaged
to obtain population means for LMA and leaf percent N. Seeds were germinated on wet filter
paper in petri dishes and planted in a 3:1 river sand:Turface (Profile Products, Buffalo Grove, IL
USA)) soil mix in pots arranged in a randomized complete block design. In order to provide nonlimiting growth conditions, plants were watered daily to field capacity and fertilized with 2 tsp
Osmocote Plus slow-release fertilizer [15-9-12] with micronutrients (Scotts, Inc., Marysville,
OH).
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Leaf trait measurements were standardized by ontogenetic stage (as recommended by
(Mason, McGaughey & Donovan 2013)). Because of large differences in growth rates among
species, measurements could not be made simultaneously on all species. As such, leaves were
harvested for measurement on four dates early in the growing season, once all of the members
of a given species had reached a standardized minimum size, always before budding. The mostrecently-fully-expanded leaf was harvested from each plant at dawn, scanned with a digital
scanner to obtain fresh leaf area, and then dried and weighed to obtain leaf dry mass. From
these, leaf mass per area (LMA) was calculated. The dried leaf was then ground into a fine
powder with a ball mill and analyzed for percent nitrogen at the University of Georgia Analytical
Chemistry Lab with Micro-Dumas Combustion (NA1500, Carlo Erba Strumentazione, Milan,
Italy).
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For comparison of root traits, the same seed sources used for fourteen of the fifteen
species and a subset of the populations used in the leaf trait study (Fig. 2) were grown in a
parallel common garden study experiment. Between three and eight replicates per population
were germinated on wet filter paper, planted in 3:1 river sand:turface soil mix, and arranged in
a randomized complete block design in the UGA greenhouses. Water and fertilization regimes
were the same as those in the leaf trait comparison. To avoid root-binding inside the pots, root
trait measurements were conducted on all plants at seven weeks after sowing, on four
consecutive days. Each day, a different spatial block was harvested, and the order of harvesting
individuals was completed randomly. All plants were still in the juvenile (pre-reproductive)
stage at harvest. Fine roots were collected from each plant based on branching order to ensure
comparison of functionally analogous material across species (Pregitzer et al. 2002). A sample
of first- and second-order roots from individual plants was collected from each plant, gently
rinsed clean with tap water, and placed in individual plastic bags in a cooler until analysis in the
lab.
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For analysis, root samples were first stained for 5 minutes with Toluidine Blue (0.01%
w/v) and rinsed with deionized water. Individual samples were then spread out in a thin layer of
water in a transparent plastic tray to minimize overlap, and scanned on a flatbed Epson
Expression STD1600 scanner at a resolution of 400 dpi. The program WinRHIZO (v. 2002c,
Regent Instruments, Quebec) was used to determine the total root length of the scanned
samples with automatic threshold detection for each sample (Bouma, Nielsen & Koutstaal
2000). Fine root samples were then dried and weighed. Specific root length (SRL) was calculated
as the total root length of a sample divided by the mass of that sample. The dried root samples
were then ground into a fine powder with a ball mill and analyzed for percent nitrogen at the
University of Georgia Analytical Chemistry Lab with Micro-Dumas Combustion (NA1500, Carlo
Erba Strumentazione, Milan)..
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Continuous maximum likelihood ancestral state reconstruction was performed with the
contmap function in the R package phytools (Revell 2012), which uses maximum likelihood
(Schluter et al. 1997) and assumes a Brownian motion model for trait evolution (Cavalli-Sforza
& Edwards 1967; Felsenstein 1973; Felsenstein 1985). The analysis was done on a modification
of the Helianthus phylogeny of Timme et al. (Fig. S1) (Timme, Simpson & Linder 2007). The
Timme phylogeny was pruned to remove species not of interest, and for species with multiple
accessions included in the tree, all but one accession was pruned, with the accession with the
longest branch length retained for consistency. This resulted in a species-level tree. The
species-level tree was then modified by adding a trichotomy to each species tip, resulting in a
new tip for each population of each species. The intraspecific branch lengths for these
trichotomies were set to 0.003, which is the calculated average of branch lengths among
accessions within monophyletic species within the original Timme et al. phylogeny. Trees were
then converted to ultrametric trees using the chronopl function (lambda=0.1) in the R package
ape (Paradis, Claude & Strimmer 2004). Both the species-level and population-level ultrametric
trees were used for ancestral state reconstructions.
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Blomberg’s K (Blomberg, Garland & Ives 2003) was calculated for LMA, Leaf N, SRL, and
Root N using both the original pruned species-level tree and the ultrametric version with the
phylosig function in the R package phytools (Revell 2012). For all four traits, regardless of tree
used, all values of K were not significantly different from 1, which indicates no difference from a
Brownian motion model of evolution.
Trait
Original Tree
Ultrametric Tree
K
p-value
K
p-value
LMA
0.767
0.172
1.035
0.056
Leaf N
0.470
0.655
0.662
0.663
SRL
0.972
0.058
1.047
0.064
Root N
0.599
0.474
0.754
0.490
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Study C) Qst-Fst Study for H. anomalus (Brouillette et al. 2006)
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Study D) Cultivated Sunflower Differentiation, H. annuus. Unpublished. Achenes from
two hundred eighty-eight accessions of cultivated H. annuus were obtained from the Burke Lab
(UGA) as well as from the USDA Germplasm Resources Information Network (GRIN). These lines
represent the “Core 288” association mapping population (Mandel et al. 2011) that
encompasses 90% of the allelic variation in the cultivated H. annuus germplasm. Seeds for each
accession were scarified and germinated on moistened filter paper. Seedlings were planted in 1
inch diameter Ray Leach Cone-tainers (Stuewe and Sons, OR, USA) containing sand then grown
under greenhouse common garden conditions in a randomized complete block design. The
plants were fertilized with a solution of Jack’s 20-10-20 General Purpose liquid fertilizer [N-P-K]
(JR PETERS, INC, PA, USA) and watered twice daily by hand. Due to poor germination rates, only
205 accessions germinated successfully and were used in this experiment (1-3 replicates per
accession).
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Leaf traits were measured at juvenile stage, 5 weeks after planting. The pair of most
recently fully expanded leaves was harvested. Fresh leaf surface area and dry mass were used
to calculate leaf mass per area (LMA). The dried leaf was then ground into a fine powder and
analyzed for percent nitrogen at the University of Georgia Analytical Chemistry Lab with MicroDumas Combustion.
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Study E) Response to nutrients/water for H. porteri (Gevaert 2011).
Study F) Response to Heavy Metals for several Helianthus species .Unpublished.
Achenes for wild H. annuus (nine populations), H. argophyllus (nine populations), and H. exilis
(twelve populations) were obtained from the USDA Germplasm Resources Information
Network. Wild populations were selected to represent the span of the geographic distribution
for each species. Seeds for each accession were germinated on moistened filter paper and
planted in seedling trays in a growth chamber. Three weeks after germination, seedlings were
moved to a greenhouse where they were randomly assigned and transplanted to 10-inch pots
containing a 3:1 sand/turface mixture and one tablespoon Osmocote® fertilizer [15-9-12]. The
pots were randomized and divided into three treatment groups: 1) non-amended control soil,
2) soil amended to 30 mg/kg (dry weight) each of arsenic, cadmium, chromium, and nickel, and
3) soil amended to 30 mg/kg each of lead, selenium, and zinc. Plants were watered twice daily
with automated drippers. Plastic saucers were placed below each pot to minimize leaching of
soil metals. Each population was replicated three times within each treatment (total number of
plants = 270). Leaf traits were measured at juvenile stage. One most recently fully expanded
leaf was harvested from each plant once the plant reached 6-8 fully expanded leaf pairs. Fresh
leaf surface area and dry mass were used to calculated leaf mass per area (LMA). The dried leaf
was then ground into a fine powder and analyzed for percent nitrogen at the University of
Georgia Analytical Chemistry Lab with Micro-Dumas Combustion.
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Study G) Ontogenetic changes in leaf traits. Mason, McGaughey & Donovan 2013
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Study H) H. anomalus vs. ancestral parent comparison 1 (Schwarzbach, Donovan &
Rieseberg 2001)
Study I) H. anomalus vs. ancestral parent comparison 2 (Rosenthal et al. 2002)
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Study J) Relative Growth Rate of H. anomalus vs. ancestral parents (Brouillette &
Donovan 2011);
Study K) UT Native Populations of H. anomalus (Donovan et al. 2007; Donovan et al.
2009)
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Study L ) UT Resource Manipulations for H. anomalus (Ludwig, Jewitt & Donovan 2006)
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Study M) UT Common Garden (Ludwig et al. 2004; Donovan et al. 2009; Donovan et al.
2010)
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Supporting References:
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Blomberg, S.P., Garland, T. & Ives, A.R. (2003) Testing for phylogenetic signal in comparative data:
Behavioral traits are more labile. Evolution, 57, 717-745.
Bouma, T.J., Nielsen, K.L. & Koutstaal, B. (2000) Sample preparation and scanning protocol for
computerised analysis of root length and diameter. Plant and Soil, 218, 185-196.
Brouillette, L.C. & Donovan, L.A. (2011) Relative growth rate and functional traits of a hybrid species
reflect adaptation to a low-fertility habitat. International Journal of Plant Sciences, 172, 509-520.
Brouillette, L.C., Gebremedhin, M., Rosenthal, D.M. & Donovan, L.A. (2006) Testing hypothesized
evolutionary shifts toward stress tolerance in hybrid Helianthus species. Western North
American Naturalist, 66, 409-419.
Cavalli-Sforza, L.L. & Edwards, A.W.F. (1967) Phylogenetic analysis models and estimation procedures.
American Journal of Human Genetics, 19, 233-&.
Donovan, L.A., Dudley, S.A., Rosenthal, D.M. & Ludwig, F. (2007) Phenotypic selection on leaf water use
efficiency and related ecophysiological traits for natural populations of desert sunflowers.
Oecologia, 152, 13-25.
Donovan, L.A., Ludwig, F., Rosenthal, D.M., Rieseberg, L.H. & Dudley, S.A. (2009) Phenotypic selection on
leaf ecophysiological traits in Helianthus. New Phytologist, 183, 868-879.
Donovan, L.A., Rosenthal, D.M., Sanchez-Velenosi, M., Rieseberg, L.H. & Ludwig, F. (2010) Are hybrid
species more fit than ancestral parent species in the current hybrid species habitats? Journal of
Evolutionary Biology, 23, 805-816.
Felsenstein, J. (1973) Maximum-Likelihood Estimation of Evolutionary Trees from Continuous
Characters. American Journal of Human Genetics, 25, 471-492.
Felsenstein, J. (1985) Phylogenies and the Comparative Method. American Naturalist, 125, 1-15.
Gevaert, S.D. (2011) Exploring the potential for adaptive differentiation in the granite outcrop plant,
Helianthus porteri. Ph.D., University of Goergia.
Ludwig, F., Jewitt, R.A. & Donovan, L.A. (2006) Nutrient and water addition effects on day- and nighttime conductance and transpiration in a C-3 desert annual. Oecologia, 148, 219-225.
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Ludwig, F., Rosenthal, D.M., Johnston, J.A., Kane, N., Gross, B.L., Lexer, C., Dudley, S.A., Rieseberg, L.H. &
Donovan, L.A. (2004) Selection on leaf ecophysiological traits in a desert hybrid Helianthus
species and early-generation hybrids. Evolution, 58, 2682-2692.
Mandel, J.R., Dechaine, J.M., Marek, L.F. & Burke, J.M. (2011) Genetic diversity and population structure
in cultivated sunflower and a comparison to its wild progenitor, Helianthus annuus L. Theoretical
Applied Genetics, 123, 693-704.
Mason, C.M., McGaughey, S.E. & Donovan, L.A. (2013) Ontogeny strongly and differentially alters leaf
economic and other key traits in three diverse Helianthus species. Journal of Experimental
Botany, In Press.
Paradis, E., Claude, J. & Strimmer, K. (2004) APE: Analyses of phylogenetics and evolution in R language.
Bioinformatics, 20, 289-290.
Pregitzer, K.S., DeForest, J.L., Burton, A.J., Allen, M.F., Ruess, R.W. & Hendrick, R.L. (2002) Fine root
architecture of nine North American trees. Ecological Monographs, 72, 293-309.
Revell, L.J. (2012) phytools: an R package for phylogenetic comparative biology (and other things).
Methods in Ecology and Evolution, 3, 217-223.
Rosenthal, D.M., Schwarzbach, A.E., Donovan, L.A., Raymond, O. & Rieseberg, L.H. (2002) Phenotypic
differentiation between three ancient hybrid taxa and their parental species. International
Journal of Plant Sciences, 163, 387-398.
Schluter, D., Price, T., Mooers, A.O. & Ludwig, D. (1997) Likelihood of ancestor states in adaptive
radiation. Evolution, 51, 1699-1711.
Schwarzbach, A., Donovan, L. & Rieseberg, L. (2001) Transgressive character expression in a hybid
sunflower species. American Journal of Botany, 88, 270-277.
Timme, R.E., Simpson, B.B. & Linder, C.R. (2007) High-resolution phylogeny for Helianthus (Asteraceae)
using the 18s-26s ribosomal DNA external transcribed spacer. American Journal of Botany, 94,
1837-1852.
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