Supporting Information
Table S1. Voucher information.
Specimen
C. amoena ssp. huntiana (Jepson) H. Lewis
& M. E. Lewis*
C. arcuata (Kellogg) A. Nelson & J. F.
Macbride*
C. gracilis ssp. albicaulis (Jepson) H.
Lewis & M. E. Lewis
Voucher/Location
LDG 8872
C. gracilis ssp. gracilis (Piper) A. Nelson
& J. F. Macbride
TRM 039 (from population 8908, collected
by L. Gottlieb)
C. gracilis ssp. sonomensis (C. L.
Hitchcock) H. Lewis & M. E. Lewis
TRM 041 (population 8513 in Sonoma
County, CA, same population used in
Gottlieb and Ford, 1988)
C. gracilis ssp. tracyi (Jepson) AbdelHameed & Snow
TRM 042 (Highway 128 and Chiles Pope
Valley Rd., collected by B. Barringer)
C. franciscana H. Lewis and P. H. Raven*
C8 EC Oakland Hills
LDG 8812
TRM 040 (Centerville Road, 1.1 miles S of
Nimshew Rd, Butte County, CA, collected
by B. Barringer)
C. lassenensis (Eastwood) H. Lewis & M.
NFW 152
E. Lewis *
C. rubicunda (Lindley) H. Lewis & M. E. LDG 8870
Lewis*
C. breweri (A. Gray) Greene
NFW 103
* DNA kindly provided by Ken Sytsma (UW-Madison, Botany Department)
Table S2. Primers used for quantitative (qPCR) and qualitative (RT) gene expression
assays. * Primers designed and kindly provided by V. Eckhart.
Gene
F3h
Primer
(V. Eckhart)*
(V. Eckhart)*
F3’h 1
qF3’H1 F
qF3’H1 R
F3’5’h 1A F3'5'H1a-q3F
F3’5’H-q2R
F3’5’h 1B F3'5'H1b-q3F
F3’5’H-q1R
Dfr1 A
DFR1-q2F
DFR1a-q2R
Dfr1 B
DFR1-q2F
DFR1b-q2R
Dfr2
DFR2-q1F
DFR2-q2R
Dfr3
DFR3-q1F
DFR2-q2R
Ans
(V. Eckhart)*
(V. Eckhart)*
Actin
ACT-q4F
ACT qR
Sequence (5’-3’)
TCTCATACCCGATCCGAAAC
GTCAGAGCCTCCTTGTCGAG
TTATTTCTCTCACTGATGAC
CCGGAAGGTCTCCTTGATTA
AAGGCGGCATGAAGAAACTACAC
TTTCGGCTAGGGACCATTCGATG
AAGGTGGTATGAAGAAGCTACAT
CCATTCGATGATGCTTGATG
CACTAG CAGAACAAGCTG
TCGGATGCTCGTATAGGTAA
CACTAGCAGAACAAGCTG
TCGGATGCTCGTAAAGATAT
AGCTCGTCTTCACGTCATCA
TAGGGTGGGTATGATACTGATC
CGTTCAGAAACCCGTCTAT
TAGGGTGGGTATGATACTGATC
AGCTGTTCTACGAGGGCAAA
GACAGCCCAAGAAATCCTGA
CATGTATGTTGCGATCCAG
TGAACATGTAACCTCTCTCRGT
Assay
RT
RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
qPCR, RT
RT
RT
qPCR, RT
qPCR, RT
Table S3. Primers used in Dfr2 genotyping assay of F2 plants from the P cross and
expected fragment sizes. For Dfr2-A, alleles were differentiated by running fluorescently
labeled samples alongside a 500 bp ladder in a ABI 3730XL DNA Analyzer. Primers
amplify portions of exon 2 and intron 2. Length polymorphism is due to indel in intron.
For Dfr2-B, PCR products were digested with AseI and products were run on an agarose
gel. Primers amplify a portion of intron 2.
Gene
Method
Dfr2-A
FLA
Allele A
Allele B
Dfr2-B
Allele A
Allele B
variation
(cut sight
bold)
Forward primer (5’-3’)
Reverse Primer (5’-3’)
FAMATAACATGAAGAAGGTG
AAGCA
CATTTTTGGATTGGTCCT
TGC
Expected
sizes (bp)
424
434
RDAseI
AAACACCCATTCCCGTGA
GAGA
CTAGCTCCTTGTGTACGT
GGTG
AAAATTA
ATTGA
AAAATTA
TTTGA
298, 75
391
Table S4. Primers used in Dfr2 genotyping assay of F2 plants from the I cross and
expected fragment sizes. Fragment lengths were determined by running fluorescently
labeled samples alongside a 500 bp ladder in an ABI 3730XL DNA Analyzer. Primers
amplify intron 3 and partial sequences of exons 3 and 4. Length variation is due to indels
present in the intron.
Gene
Dfr2-A
Forward primer (5’-3’)
FAMAGCTCGTCTTCACGTCATCA
Reverse Primer (5’-3’)
TAGGGTGGGTATGATACTGATC
Allele A
Allele B
Dfr2-B
Allele A
Allele B
Expected
sizes (bp)
299
288
FAMAGCTCGTCTTCACGTCATCA
TAGGGTGGGTATGATACTGATC
300
304
Figure S1. Petal sections corresponding to top (T), center (C) and base (B) of the mature
petal used in HPLC analyses for unspotted, central spotted, basal spotted, and double
spotted plants, from left to right.
Figure S2. Age of petals for basal, central, and double spotted flowers when dissections
were performed for qualitative RT-PCR assays. Gray lines indicate proximate site of
dissections. T, C, and B refer to top, center, and base, respectively.
Figure S3. Stages of C. gracilis petal development. Double-spotted individual shown.
Central spot appears before basal spot, and both spots appear before background color.
Petals are shown to scale.
B B A A
P P I I =
B B P A
P P II =
C C A A
P P I I
C C P A
P P II =
C C P P
P P II
C B A A
P P I I
B B P P
P P II =
C B P A
C B P P
P P II
P P II =
=
Figure S4. Genotypes at the P and I loci and expected phenotypes.
Figure S5. Phylogenetic trees of anthocyanin pathway genes in section Rhodanthos.
C. gracilis homeologs are indicated as (A) or (B). Note that absence of a species from a
particular cluster does not imply that a species does not have a gene corresponding to that
cluster because we did not exhaustively search for genes in species other than C. gracilis.
C. gracilis sequences represent those from all four subspecies. Values above branches
show bootstrap support (100 replicates). All trees obtained using the GTR model of
nucleotide evolution as implemented in ML analyses in PAUP*. A single optimal
maximum likelihood tree was recovered for each gene. a, F3h (-lnL= 407.19313, 120 bp
included characters—sites111-125 and 174-183 in alignment were excluded); b, F3’h (lnL= 1473.04082, 287 bp included characters); c, F3’5’h (-lnL= 860.47633, 288bp
included characters); d, Ans (-lnL= 307.13268, 141bp included characters); e, Dfr (-lnL=
2377.47274, 559bp included characters). F3h x labeled as such because it is unclear if the
two divergent sequences are homologous.  denotes sequences with stop codons and/or
frameshift mutations. Intraspecific allelic variants were combined into a single sequence
and polymorphisms were coded as ambiguous characters. All sequences were obtained
from genomic DNA with the exception of all Dfr sequences from C. gracilis, Dfr2 from
C. lassenensis and C. franciscana, and F3’h1-A from C. gracilis. Arabidopsis thaliana
sequences used are AT5G07990 (F3’h), AT4G22880 (Ans), and AT5G42800 (Dfr).
albic. son_uns son_spo trac.
T C B
T C B
T C B
T C B
Dfr2-A/B
Actin
Figure S6. Dfr2-A/B expression and Actin control in C. gracilis plants derived from seeds
collected in natural populations. albic., son_uns, son_spo, trac, refer to C.g. albicaulis (3
replicates), C.g. sonomensis (unspotted plant, 3 replicates), C.g. sonomensis (spotted, 2
replicates), and C.g. tracyi (1 replicate). Spot phenotypes are indicated in cartoon, petal
base is on the right.
Figure S7. Alignment of all Dfr sequences from C. gracilis. Boxed-in region shows AA
translation for active site. * denotes AA133, which has been repeatedly implicated in
changes in substrate specificity.
See attached txt file (Notes S1) for alignments used for phylogenetic analyses. Note that
alignments only show characters included in phylogenetic analyses. Sequences isolated
in this study are found under Genbank accession numbers KC019211-KC019249.