Appendix S1. Supplementary methods and results.
Graphical vector analysis (GVA):
To construct the GVA plots in Fig. 4A and B, we converted the individual foliar chemical
content, concentration, and biomass of offspring of each taxon into values relative to
Fremont, by dividing the Fremont value by the taxon value. Analogously, for Fig. 4C
and D, we converted the individual foliar chemical content, concentration, and biomass
of offspring that experienced maternal herbivory into values that are relative to the
maternal control values by dividing the maternal control value by the maternal herbivory
value within specific taxa (Haase & Rose 1995; Koricheva 1999). We constructed each
of the graphs by first plotting the control point, where relative content (X-axis) and
concentration (Y-axis) equals one. This point represents Fremont in Fig. 4A and B, and
the maternal control in Fig. 4C and D. We then extended a diagonal line with a slope of
one (i.e., relative content = relative concentration) from the graph origin to represent the
control reference for individual foliar biomass; treatment vectors falling on the left or
right side of the diagonal line represent decreased or increased leaf biomass, respectively
(Veteli et al. 2007). Seedling traits in taxon, relative to those of Fremont (Fig. 4A,B), or
seedling responses to the maternal herbivory treatment, relative to the maternal control
(Fig. 4C,D), are then illustrated by plotting phytochemical content against concentration.
Molecular analysis:
We used FAM, NED or HEX 5′-labeled oligonucleotide primers (Integrated DNA
Technologies, Inc., San Diego, CA, USA) for the genotyping. Reaction mixtures
contained 20 ng of DNA, 50 ng of each SSR primer, 200 µmol/L dNTPs, 0.5 U Taq DNA
polymerase/µL (Omega Bio-tek Inc., Norcross, GA, USA), 10 µmol/L Tris–HCl (pH
8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl2, and 0.1 mg bovine serum albumin/mL. We
used the following ‘touchdown’ amplification conditions: initial denaturation step at 95
ºC for 5 minutes, followed by 10 cycles of 95 ºC for 15 seconds, 58 ºC for 15 seconds
(decreasing by 1 ºC each cycle), and 72 ºC for 30 seconds, followed by an additional 20
cycles of 95 ºC for 15 seconds, 50 ºC for 15 seconds and 72 ºC for 30 seconds, and final
extension at 72 °C for 5 minutes. We diluted reaction products up to 1:20, denatured in
1
HiDi formamide containing a 600-bp LIZ standard (Applied Biosystems, Foster City,
CA, USA). Finally, we visualized samples on an ABI 3730xl genetic analyzer and
analyzed the data using ABI GeneMapper 4.0 (Applied Biosystems).
Supplementary results and discussion, Molecular Analysis:
The seven SSR genetic markers were highly polymorphic (Table S1) and resulted in a
high cumulative exclusion probability of 0.9995. The majority of the offspring from the
four F1 families were successfully assigned to a taxon (149 out of 165; 90.3%) based on
NewHybrids assignments. One hundred and eight (65.5%) offspring appear to be first
generation backcross to narrowleaf and 41 (24.8%) offspring were F2 hybrids, which
resulted from matings between two F1 parents. No successful matings between F1
mothers and Fremont cottonwoods were observed in the study. Specific paternity was
determined for 60% of the offspring and these displayed very similar results to the
offspring taxon assignments (χ2 = 101.285; df = 1; p < 0.0001).
The fairly frequent backcross events that we observed between F1 hybrids and
narrowleaf, relative to the few successful matings between two F1 hybrids and no
observed backcrosses between F1 hybrids and Fremont are consistent with the
unidirectional introgression of genes (from Fremont to narrowleaf) that has been
documented in previous studies in the cottonwood system (Keim 1989; Martinsen et al.
2001). If the genotype x maternal herbivory interactions that we found in the F1 hybrids
for phytochemical defense production are due to epistasis, it is possible that favorable
gene combinations for transgenerational plasticity that arise in the F1 due to
recombination could introgress into narrowleaf (but not into Fremont). The likelihood of
this introgression occurring is, of course, dependent on a number of factors, including the
genetic architecture behind transgenerational plasticity.
2
Table S1. Simple sequence repeat (SSR) genetic markers employed in this study,
including linkage group and the number of alleles amplified.
Locus
GCPM_25a
GCPM_2900a
GCPM_3681a
ORPM_202b
ORPM_206b
ORPM_220b
WPMS_15c
Linkage Group
VII
III
VIII
VIII
XIX
IV
V
No. Alleles
11
13
7
5
10
5
4
a
from http://www.ornl.gov/sci/ipgc/ssr_resource.htm
from Tuskan et al. 2004
c
Smulders et al. 2001
b
3
Table S2. A comparison of relative phenolic glycoside concentrations and condensed tannin
concentrations in different developmental stages and among taxa in cottonwood.
Developmental
stage
Seedling
Juvenile ramet
Adult juvenile
developmental
zone
Adult mature
developmental
zone
Phenolic
glycoside foliar
concentration
High
High
Moderate/High
Low
Condensed
tannin foliar
concentration
Very low
Low
Low
Rank order of
taxa
References
NL > F1 > Fre
NL > F1 *
NL > F1 > Fre
or
F1 > NL > Fre
Rehill et al.,
2006;
F1 > NL >/= Fre
Holeski et al.,
in review
(current ms)
Holeski et al.,
2012;
High
4
Table S3. Results of GLM ANOVAs for total phenolic glycoside content (A and B) and
concentration (C and D) in seedlings with F1 hybrid mothers. Factors in bold print had a
significant effect on the particular variable of interest. Factors in bold, grey print have a
marginally significant effect on the particular variable of interest. We could not calculate a
maternal genotype x paternal taxon x maternal herbivory interaction, as low representation
of the F1 paternal taxon led to a highly unbalanced design. Parts B and D eliminate the
possible effect of that interaction by considering only seedlings with narrowleaf fathers;
these represent the majority (more than 2/3) of the seedlings with F1 mothers.
Response
variable
A. Total
phenolic
glycoside
content
B. Total
phenolic
glycoside
content
C. Total
phenolic
glycoside
concentration
D. Total
phenolic
glycoside
concentration
Factor
Foliar dry leaf weight
Paternal taxon
Maternal genotype
Maternal herbivory
Maternal genotype x maternal
herbivory
Foliar dry leaf weight
Maternal genotype
Maternal herbivory
Maternal genotype x maternal
herbivory
Paternal taxon
Maternal genotype
Maternal herbivory
Maternal genotype x maternal
herbivory
Maternal genotype
Maternal herbivory
Maternal genotype x maternal
herbivory
5
F
statistic
389.27
10.98
0.24
0.26
1.37
df
1,135
1,135
3,133
1,135
3,135
pvalue
<0.001
0.001
0.867
0.640
0.254
332.63
0.16
0.02
2.36
1,95
3,95
1,95
3,95
<0.001
0.918
0.891
0.076
6.97
1.09
0.17
3.90
1,137
3,137
1,137
3,137
0.009
0.356
0.679
0.010
0.17
0.09
5.01
3,97
1,97
3,97
0.908
0.779
0.003
Figure S1. Construction and interpretation of the directional shifts of vectors describing
changes in foliar phytochemical concentrations, foliar phytochemical content, and
individual foliar biomass relative to control references. Dotted lines represent no change
in phytochemical content (X-axis) or concentration (Y-axis) relative to control. Dashed
line represents relative foliar biomass for the control, vector endpoints falling to the left
or right of the dashed line represent decreased or increased foliar biomass, respectively.
Interpretations of the six more common vector endpoints are shown. Figure is replicated
from Couture 2011.
6
Parent:
Control
Damaged
25
A
20
(% dw)
Total phenolic glycosides
Figure S2. Total phenolic glycoside concentrations (% dry weight (%dw)) in foliage of
seedlings from maternal herbivory or maternal control trees, averaged over genotypes
within each taxon. NL is narrowleaf. Error bars represent +/- 1 standard error (SE) from
the mean.
B
15
C
10
5
0
NL
F1
Fremont
7
Figure S3. Condensed tannin concentrations (% dw) in seedlings of maternal herbivory
versus control trees within each taxon. Error bars represent 1 SE from the mean. Note:
Fremont is shown for visual comparison only, and is not included in the statistical
analysis (see details in Materials and Methods section).
1.00
Control
Herbivory
A
A
0.75
(% dw)
Condensed tannins
Maternal:
0.50
B
0.25
0.00
NL
F1
Fremont
8