Supporting Information Methods S1 & Tables S1–S3
Adaptive differentiation of traits related to resource use in a desert annual along a resource
gradient
Larry C. Brouillette, Chase M. Mason, Rebecca Y. Shirk, and Lisa A. Donovan
Supporting Information Methods S1 for Additional Plant Traits Not in Main Text
Height relative growth rate (RGR) was calculated as the slope of the regression line
through the first four height measures made on 21 January, 23 January, 4 February and 11
February. The gas exchange leaf image was assessed for leaf shape (Lfshp) with ImageJ
freeware (National Institutes of Health, Bethesda, MD). The gas exchange leaf wet mass, dry
mass, and area (Lfarea) were also used to calculate leaf succulence (Lfsucc) as fresh mass minus
dry leaf mass divided by Lfarea. Leaf dry matter content (LDMC) was calculated as leaf dry leaf
mass divided by leaf wet mass. The dried leaf tissue assessed for Nmass was also assessed for leaf
carbon (C) and carbon to nitrogen ration (CNrat). Photosynthetic nitrogen use efficiency (PNUE)
was calculated after Field & Mooney (Field & Mooney, 1986).
Leaf toughness (Lftough) and leaf hair density (Lfhair) were measured on the most
recently fully expanded leaf on 3 April 2008. Lfhair was estimated by counting the number of
leaf hairs on three regularly-spaced, 1/3 cm2 areas on the adaxial surface of the leaf. The density
of the leaf hairs per cm2 was calculated as the sum of the three counts. Lftough was measured as
the force required to penetrate the leaf (E-DFE-002, Chatillon/Ametek, Largo, FL). Seven
measurements were taken on the same leaf, and the mean of those was used as the estimate of
1
Lftough for the plant. Leaf nitrogen resorption proficiency (ResProf) was estimated as
percentage of nitrogen remaining in the leaf followed for leaf lifetime (LL) after it had senesced
(turned completely brown). Resorption efficiency (ResEff) was calculated as the difference
between nitrogen concentration of the green gas exchange leaf (Nmass) and the senescent LL leaf
(ResProf), divided by Nmass, all multiplied by 100.
The date of the emergence of a terminal flower bud (DFB) was recorded. After all flower
ligules had expanded, floral metrics were estimated on the first flower: flower petiole length
(Petlen), phyllary shape (Phylshp), number of phyllaries (Phyllaries), ligule length (Liglen),
ligule width (Ligwid), number of ligules (Ligules), and flower disk diameter (Diskdi). Bulk
pollen was collected from and reapplied to open flowers three times weekly between 3 March
and 4 April 2008. When all ligules were shed, pollinated inflorescences were bagged with nylon
mesh to prevent loss of seeds. Mature seeds were extracted from bagged seed heads, counted,
dried, and weighed. Seeds were scanned at 600 dpi on a flatbed scanner and images were
imported into Tomato Analyzer (Brewer et al. 2006) to estimate the seed shape and seed color
parameters (Seedshp and Seedlm, respectively). Average seed mass (Seedmg) was calculated as
the mass of the dried seeds divided by the number of seeds.
Plants were harvested 26 April – 1 May 2008. Each plant was partitioned into leaves,
stem, reproductive, and root components. Inflorescences were categorized as buds, flowers, or
seed heads. Total reproductive units (TotalRepro) was the sum of buds, flowers and seed heads
at harvest. The reproductive ratios (ReproRatio) was calculated as the sum of flowers and seed
heads divided by the total reproductive units, and serves as a measure of reproductive phenology.
Roots were removed from the soil within two weeks of harvest, and a subsample of the fine roots
less than 1mm in diameter were collected and stored in water for estimation of specific root
2
length (SRL). The root subsample was stained with Toluidine Blue O, and the total length of the
root sample was estimated using WinRhizo (Regent Instruments, Quebec, QC, Canada). All
biomass was dried at 60°C, weighed, and summed to estimate total plant biomass (Biomass). The
ratio of root biomass over total biomass (root, leaves, stems, and reproductive biomass) was
designated as RMR-1. The ratio of root biomass to total vegetative biomass (roots, leaves, stems)
was designated as RMR-2.
Leaf tissue from the harvest was coarsely ground (Wiley mill, Thomas Scientific,
Swedesboro, NJ) and ashed to estimate foliar concentrations of boron (B), calcium (Ca) ,
magnesium (Mg), manganese (Mn), phosphorus (P), potassium (K), and sodium (Na) (Enviro 36
Inductively Coupled Argon Plasma, Thermo Jarrell Ash Corp., Franklin, MA).
Brewer MT, Lang L, Fujimura K, Dujmovic N, Gray S, van der Knaap E. 2006.
Development of a controlled vocabulary and software application to analyze fruit shape
variation in tomato and other species. Plant Physiology 141: 15–25.
Field C, Mooney HA 1986. The photosynthesis-nitrogen relationship in wild plants. In: Givnish
TJ ed. On the economy of plant form and function. New York: Cambridge University
Press, 25-55.
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. 2005. Very high resolution
interpolated climate surfaces for global land areas. International Journal of Climatology
25: 1965-1978.
3
Supporting Information Table S1 Helianthus anomalus population information for the greenhouse common garden study:
abbreviations, source site characteristics (GPS coordinates, Mean Annual Temperature (MAT), Mean Annual Precipitation (MAP),
and soil C N, P and K), number of half-sibling families and individuals sampled per population, and population means (± SE) for
plant traits sampled (see Table S2 for explanations of trait abbreviations).
Population
Abbreviation
GPS N
GPS W
MAT (C)
MAP (mm)
Soil organic
content (%)
Soil N (%)
Soil P (ppm)
Soil K (ppm)
Families
Individuals
Amass (nmol g-1 s-1)
Aarea (μmol m-2 s-1)
Nmass (%)
Narea (mmol m-2)
LMA (g m-2)
LL (months)
LWC (g g-1)
δ13C (‰)
DFF (days)
Hanksville
airport
AIR
38
110 39.383
11.2
165
0.26
Goblin
Valley
GOB
38
110 34.606
10.5
180
0.19
Hall’s
Crossing
HAL
37
110 26.864
12.5
185
0.27
Junction
Jericho
JCT
38
110 22.975
11.5
167
0.21
0.003
13.37
11.38
14
44
903.83
36.03
5.97±0.07
170.19±3.13
39.94±0.58
1.12±0.04
11.58±0.17
-31.68±0.08
51.64±0.71
0.003
14.90
16.54
14
45
891.45
36.88
5.75±0.06
170.38±3.01
41.46±0.57
1.08±0.04
11.35±0.17
-31.56±0.08
60.36±0.70
0.004
6.10
11.99
12
40
1001.25
37.52
6.62±0.07
180.67±3.34
38.18±0.62
1.06±0.04
11.80±0.18
-31.33±0.09
48.21±0.76
0.003
11.00
17.33
13
41
880.61
35.93
5.72±0.07
167.01±3.21
40.98±0.59
1.10±0.04
11.57±0.17
-31.68±0.08
57.65±0.73
4
South of
Hanksville
SOU
38
110 33.642
12.4
161
0.24
White Sands
JER
39
112 22.175
9.9
264
0.48
North of
Hanksville
NTH
38
110 35.520
10.6
180
0.17
0.003
17.59
19.67
8
15
867.77
34.13
5.80±0.14
163.87±6.76
39.61±1.25
1.00±0.08
10.27±0.36
-31.68±0.17
58.75±1.54
0.003
13.11
12.82
14
43
876.10
35.88
5.78±0.07
169.70±3.18
41.17±0.59
1.06±0.04
11.57±0.17
-31.63±0.08
60.77±0.72
0.003
10.40
17.06
13
38
919.81
34.81
6.21±0.07
168.51±3.58
38.07±0.66
1.13±0.04
11.99±0.19
-31.49±0.09
53.70±0.81
0.003
11.97
24.66
9
22
834.92
33.61
5.72±0.11
164.32±5.12
40.66±0.95
1.16±0.06
10.26±0.27
-31.93±0.13
53.12±1.16
WHS
39
112 19.153
9.6
292
0.39
Supporting Information Table S2 Species means and SE for plant traits (n = 8 populations)
with traits ordered as they appear in Supporting Information Fig. S1, except the final three traits
for which QST was undefined (traits in bold are analyzed and discussed further in main text).
Variable
Abbrev.
Units
Mean
SE
Leaf Intercellular CO2
Ci
ppm
276.3
0.6
Leaf Potassium
K
ppm 2650.5
63.3
Leaf Calcium
Ca
ppm 2187.1
46.8
Root Mass Ratio-Total
RMR-1
g g-1
0.099
0.003
Leaf Magnesium
Mg
ppm
412.0
10.1
Leaf carbon isotopic ratio
δ13C
‰
-31.62
0.06
Leaf Boron
B
ppm
5.4
0.2
Leaf Manganese
Mn
ppm
11.4
1.0
Height Relative Growth Rate
RGR
cm day-1
0.17
0.01
Total Reproductive Units TotalRepro
count
73.4
3.2
Flower Petiole length
Petlen
cm
11.23
1.03
Root Mass Ratio-Vegetative
RMR-2
g g-1
0.127
0.005
Photosynthetic Rate, Area Basis
Aarea
μmol m-2 s-1
35.60
0.47
Leaf Mass Per Area
LMA
g m-2
40.01
0.46
Phyllary Shape
Phylshp
mm mm-1
7.40
0.44
Stomatal Conductance
g
molCO2 m-2 s-1
1.88
0.07
Seed Mass
Seedmg
mg
10.18
0.61
5
Flower Disk Diameter
Diskdi
mm
14.3
0.4
Specific Root Length
SRL
m g-1
111.2
3.2
Number of Phyllaries
Phyllaries
count
17.7
0.6
Total biomass at harvest
Biomass
g
49.99
3.92
Leaf Succulence
Lfsucc
gH20 cm-2
0.045
0.001
Leaf Area
Lfarea
cm2
20.8
1.6
Ligule Length
Liglen
mm
29.0
0.9
Leaf Phosphorus
P
ppm
157.8
8.6
Ligule Width
Ligwid
mm
13.4
0.4
Leaf water content
LWC
g g-1
11.3
0.2
Seed Luminosity
Seedlm
relative scale
92.3
2.2
Leaf Dry Matter Content
LDMC
mg g-1
82.0
1.7
Leaf Shape
Lfshp
cm2 cm-1
0.84
0.05
Photosynthetic Rate, Mass Basis
Amass
nmol g-1 s-1
897.0
17.36
Ratio Reproductive Units ReproRatio
count count-1
0.56
0.02
Leaf Hair Density
Lfhair
count cm-1
22.9
4.7
Number of Ligules
Ligules
count
10.8
0.4
Leaf Toughness
Lftough
G
56.0
2.5
Days to First Bud
DFB
days
36.9
1.1
Days to First Flower
DFF
days
55.5
1.6
Seed Shape
Seedshp
cm2 cm-1
0.06
0.01
Leaf Nitrogen, Mass Basis
Nmass
%
5.95
0.11
6
Leaf Carbon-to-Nitrogen Ratio
CNrat
% %-1
6.0
0.1
Leaf Carbon
C
%
35.0
0.3
Leaf Nitrogen, Area Basis
Narea
mmol m-2
169.3
1.8
Leaf Sodium
Na
ppm
2.5
0.1
Leaf N Resorption Proficiency
ResProf
%
0.99
0.04
Leaf Lifetime
LL
months
1.09
0.02
Photosynthetic Nitrogen Use
Efficiency
PNUE
μmolCO2 molN -1 s-1
212.0
1.3
Leaf Resorption Efficiency
ResEff
%
83.5
0.6
7
Supporting Information Table S3 Helianthus anomalus population sample size for number of
maternal families sampled for SSR markers (1 plant per maternal family) (N), number of alleles
(A), expected heterozygosity (He), and observed heterozygosity (Ho) based on SSR markers.
Population
AIR
GOB
HAL
JCT
JER
NTH
SOU
WHS
N
14
14
12
13
8
14
13
9
Ho
0.382
0.342
0.378
0.346
0.292
0.336
0.258
0.331
He
0.514
0.511
0.655
0.544
0.300
0.482
0.463
0.378
8