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Title: Conservation and canalization of gene expression during angiosperm diversification accompany the origin and evolution of the flower
Series Title: Chanderbali, A.S. et al. 2010. Conservation and canalization of gene expression during angiosperm diversification accompany the origin and evolution of the flower. Proceedings of the National Academy of Sciences 107: 22570 -22575.
Physical Description: Journal Article
Creator: Chanderbali, A. S.
Yoo, M.-J.
Zahn, L. M.
Brockington, S. F.
Wall, P. K.
Gitzendanner, M. A.
Albert, V. A.
Leebens-Mack, J.
Altman, N. S.
Ma, H.
dePamphilis, C. W.
Soltis, D. E.
Soltis, P. S.
Publisher: Proceedings of the National Academy of Sciences
Publication Date: 12/28/2010
Spatial Coverage:
Abstract: The origin and rapid diversification of the angiosperms (Darwin's “Abominable Mystery”) has engaged generations of researchers. Here, we examine the floral genetic programs of phylogenetically pivotal angiosperms (water lily, avocado, California poppy, and Arabidopsis) and a nonflowering seed plant (a cycad) to obtain insight into the origin and subsequent evolution of the flower. Transcriptional cascades with broadly overlapping spatial domains, resembling the hypothesized ancestral gymnosperm program, are deployed across morphologically intergrading organs in water lily and avocado flowers. In contrast, spatially discrete transcriptional programs in distinct floral organs characterize the more recently derived angiosperm lineages represented by California poppy and Arabidopsis. Deep evolutionary conservation in the genetic programs of putatively homologous floral organs traces to those operating in gymnosperm reproductive cones. Female gymnosperm cones and angiosperm carpels share conserved genetic features, which may be associated with the ovule developmental program common to both organs. However, male gymnosperm cones share genetic features with both perianth (sterile attractive and protective) organs and stamens, supporting the evolutionary origin of the floral perianth from the male genetic program of seed plants.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Matthew Gitzendanner.
Publication Status: Published
Funding: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund.
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Conservation and canalization of gene expression

during angiosperm diversification accompany the

origin and evolution of the flower

Andre S. Chanderbalia"b1, Mi-Jeong YOOa,2, Laura M. Zahnc,3, Samuel F. Brockingtona"4, P. Kerr Wallc'5,
Matthew A. Gitzendannera, Victor A. Albertd, James Leebens-Macke, Naomi S. Altmanf, Hong Macg,
Claude W. dePamphilisc, Douglas E. Soltisa, and Pamela S. Soltisb

aDepartment of Biology and bFlorida Museum of Natural History, University of Florida, Gainesville, FL 32611; Departments of cBiology and fStatistics,
Pennsylvania State University, University Park, PA 16802; dDepartment of Biological Sciences, State University of New York, Buffalo, NY 14260; eDepartment
of Plant Biology, University of Georgia, Athens, GA 30602; and gState Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life
Sciences, Center of Evolutionary Biology, Fudan University, Shanghai 200433, China
Edited* by David L. Dilcher, University of Florida, Gainesville, FL, and approved November 9, 2010 (received for review September 8, 2010)

The origin and rapid diversification of the angiosperms (Darwin's
"Abominable Mystery") has engaged generations of researchers.
Here, we examine the floral genetic programs of phylogenetically
pivotal angiosperms (water lily, avocado, California poppy, and
Arabidopsis) and a nonflowering seed plant (a cycad) to obtain
insight into the origin and subsequent evolution of the flower.
Transcriptional cascades with broadly overlapping spatial do-
mains, resembling the hypothesized ancestral gymnosperm pro-
gram, are deployed across morphologically intergrading organs
in water lily and avocado flowers. In contrast, spatially discrete
transcriptional programs in distinct floral organs characterize the
more recently derived angiosperm lineages represented by Cali-
fornia poppy and Arabidopsis. Deep evolutionary conservation in
the genetic programs of putatively homologous floral organs
traces to those operating in gymnosperm reproductive cones. Fe-
male gymnosperm cones and angiosperm carpels share conserved
genetic features, which may be associated with the ovule devel-
opmental program common to both organs. However, male gym-
nosperm cones share genetic features with both perianth (sterile
attractive and protective) organs and stamens, supporting the
evolutionary origin of the floral perianth from the male genetic
program of seed plants.

ABCE model fading borders floral evolution floral origin
transcriptional profiling

The evolutionary origin of flowering plants, or angiosperms,
remains one of the greatest unsolved biological mysteries.
The presence of a diverse assemblage of floral forms shortly after
the sudden appearance of angiosperm fossils in early Cretaceous
deposits ca. 130 Mya suggests that a rapid radiation established
most of the modern lineages within a few million years (1). Fa-
mously declared an "abominable mystery" over a century ago by
Charles Darwin (in a letter to J.D. Hooker in 1879) (2), the
origin and subsequent diversification of flowering plants have
captured the imagination of generations of researchers in wide-
ranging botanical disciplines. Essential to any explanation has
been the origin of the flower itself. Hypotheses on this topic,
whether based on reconstructions from morphological features
(3) or developmental genetics (4, 5), all attempt to resolve the
evolution of floral organs from preexisting structures in non-
flowering seed plants (gymnosperms).
Flowers typically are composed of a perianth of leaf-like sepals
and colorful petals surrounding stamens (the male reproductive
organs) and carpels (the female reproductive organs). Angiosperm
stamens and carpels are widely regarded as homologous with their
functional counterparts in the simple strobili (cones) of gymno-
sperms, microsporophylls, and megasporophylls, respectively (see
ref. 6 for alternative views), but sepals and petals are unique to
flowers and therefore, lack clear evolutionary precursors. However,

22570-22575 I PNAS I December 28, 2010 I vol. 107 I no. 52

extensive research on the genetic control of flower development
in Arabidopsis has demonstrated that floral organs are cross-
transformable into one another and even can be modified into
leaves through genetic manipulation of certain key developmental
regulators (7-9). These insights are encapsulated in the ABCE
model for the genetic control of floral organ identity (Fig. 1,
reviewed in refs. 5, 10, and 11). Most of what we know about the
regulation of floral development has been discovered through ge-
netic manipulation of derived eudicot model systems, but com-
parative analyses suggest core components of the ABCE genetic
program are conserved across angiosperms (12) and may trace to
an original BC program that operated in the common ancestor of
all seed plants (5). However, evolutionary dynamism in the spatial
deployment of ABCE function, and of B function in particular,
may underlie fundamental changes in floral organization during
angiosperm diversification (13).
Here, we conduct comparative analyses of global transcriptome
data which encompass the properties of whole developmental
systems (e.g., 14, 15) to investigate floral developmental genetics
beyond the action of candidate regulatory genes. Specifically, we
analyze transcriptome data for the water lily Nuphar advena
(Nymphaeales) representing the sister lineage of all extant flow-
ering plants exceptAmborella (16), the magnoliidPersea americana
(avocado; Laurales), the eudicots Eschscholzia califorica (Cal-
ifornia poppy; Ranunculales) and Arabidopsis thaliana (Brassi-
cales), and the gymnosperm Zamia vazquezii (Cycadales) to help
reconstruct the origin and evolution of flowers. Our analyses ex-
tend previous comparisons of floral transcriptional programs (17-
19) to several additional phylogenetically significant taxa, and we
present insights into the genetic relationships among individual
floral organs and gymnosperm reproductive cones.

Author contributions: A.S.C., V.A.A., J.L.-M., H.M., C.W.d., D.E.S., and P.S.S. designed research;
A.S.C., M.-S.C., MY., L.M.Z., and S.F.B. performed research; A.S.C., P.K.W., M.A., and N.S.A.
analyzed data; and A.S.C., V.A.A., J.L.-M., H.M., C.W.d., D.E.S., and P.S.S. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail:
2Present address: Department of Ecology, Evolution, and Organismal Biology, Iowa State
University, Ames, IA 50011.
3Present address: American Association for the Advancement of Science, Washington, DC
4Present address: Department of Plant Sciences, University of Cambridge, Cambridge CB2
3EA, United Kingdom.
'Present address: BASF Plant Science, Research Triangle Park, NC 27709.
This article contains supporting information online at 0.
1073/pnas.1 013395108/-/DCSupplemental.


Stamen Stamen




Fig. 1. The ABCE model of floral organ identity. Sepals are produced where
A function acts alone, petals where A and B functions overlap, stamens
where B and C functions combine, and carpels where C function acts alone.
In the eudicot genetic model Arabidopsis thaliana, APETALA1 (AP1) and
APETALA2 (AP2) are the A-function genes, APETALA3 (AP3) and PISTILLATA
(PI) together specify B function, C function is specified by AGAMOUS (AG),
and multiple SEPALLATA genes provide E function (7-9).

Global gene-expression data are rich in patterns of transcript
abundances that we have explored through comparative cross-
species analyses for evolutionary insights. We conducted a series of
comparisons to examine the spatial distribution of florally biased
expression in each species (Fig. 2). Manual ranking of positive log2
floral organ/leaf expression ratios by floral organ of primary ex-
pression indicates that genes participating in the transcriptional
programs of Nuphar and Persea flowers are deployed in broad
domains across adjacent whorls and beyond, whereas those in
Arabidopsis and Eschscholzia are more tightly constrained to in-


dividual floral organ categories (Fig. 2B). Likewise, pairwise
comparisons of the gene-expression profiles of adjacent floral
organ categories translated into substantially greater correlations
in Nuphar and Persea than in the two eudicots (Fig. 2C).
Next, comparisons of organwise transcriptional profiles based
on relative abundance (RA) scores (20) of all putatively ho-
mologous genes were conducted to assess process homology (i.e.,
sharing a genetic program that is potentially, although not nec-
essarily, inherited from a common ancestor (21) among floral
organs. We found strong evidence that carpels are process ho-
mologous, as are stamens, across angiosperms (Fig. 3). Petals of
Arabidopsis and Eschscholzia also appear to be process homol-
ogous, as are sepals of these two eudicots. In contrast to the
eudicots, the outer and inner perianth organs of Nuphar and
Persea are not differentiated into distinct sepals and petals [this
traditional distinction in Nuphar may be spurious (22)] but are
morphologically similar and are termed "tepals." We found that
Nuphar and Persea tepals are genetically most similar to each
other, within their respective flowers, and collectively are more
process homologous with eudicot sepals than with any other
floral organs. Deeper in the cluster hierarchy, the sepals/tepals
transcriptional program was closest to that operating in stamens,
whereas eudicot petals were more similar to carpels. This hier-
archy of organ relationships is strongly supported by random
resampling analyses (Fig. 3) and also is robust to directed
resampling in three modified data sets that exclude genes not
sampled in all species, exclude data for Nuphar and Persea, or
exclude data for Arabidopsis (Fig. Si).
The potential biological significance of the gene clusters sup-
porting these organ groupings was estimated through Gene
Ontology (GO) annotation (Table S1 and Figs. S2-S7) and
transcription factor binding sites (TFBS) enrichment analyses for
their Arabidopsis members (Table S2). Genes coexpressed in
sepals and tepals are involved in biological processes related to
photosynthesis and defense (Fig. S2), and their promoters share
various related G-box promoter elements associated with light
responses (23-25), as well as the Unfolded Protein Response
(UPR) motif associated with stress responses (26). Genes of the

Fig. 2. Canalization of floral organ transcriptional
programs during angiosperm diversification. (A)
Flowers of Nuphar and Persea bear an un-
differentiated perianth of petaloid organs (tepals),
whereas in Eschscholzia and Arabidopsis flowers
the perianth is differentiated into leaf-like outer
sepals and colorful inner petals. (B) Log2 floral or-
gan/leaf gene expression ratios ranked by organs of
peak expression contrast the blurred boundaries
between adjacent floral organs in Nuphar and
Persea versus the sharp boundaries in Arabidopsis
and Eschscholzia. Stamen-preferential genes gener-
ally are more broadly expressed, but more so in
Nuphar and Persea than in the eudicots, whereas
carpel-preferential genes generally are more spatially
restricted. Car, carpels; Itp, inner tepals; Otp, outer
tepals; Pet, petals; Sep, sepals; Stm, stamens. The scale
of log2 ratios ranges from saturated yellow (1 and
higher = at least twofold up-regulated) to black (0
and lower = no change or down-regulated). (C) Scat-
ter plots of log2 floral organ/leaf ratios and Pearson
correlations indicate that transcriptional profiles of
adjacent floral organs are more strongly correlated
in Nuphar and Persea than in eudicots. Analyses are
based on 4,588 Nuphar, 4,508 Persea, 5,468 Eschschol-
zia, and 12,785 Arabidopsis genes up-regulated in their
respective flowers relative to leaves.

PNAS I December 28, 2010 I vol. 107 I no. 52 I 22571

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Chanderbali et al.

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Fig. 3. Hierarchy of genetic relationships among floral organs supports the process homology of stamens, carpels, sepals, and petals, respectively, and places
tepals with sepals in a group collectively closest to stamens. (A) RA scores of expression levels across floral organs within their respective flowers were
clustered and subsequently mean centered for visual effect. The color scale ranges from 0.25 (yellow) to -0.25 (blue). (B) Bootstrap (red) and approximately

unbiased (green) support values indicate strong stability for all clusters.

stamens cluster also participate in stress responses (Fig. S3) but
additionally participate in cellular growth and differentiation and
pollination (probably, pollen production). G-box and UPR
motifs, as well the CArG binding site targeted by MADS-box
genes, are enriched in their promoters. The cluster of genes
coexpressed in sepals, tepals, and stamens is associated with
stress responses, general energy-related processes (Fig. S4), and
the G-box, UPR, and Low Temperature Responsive Element
(LTRE) motifs. GO enrichment for petal genes highlights cel-
lular growth and differentiation, morphogenesis, and response to
stimuli (Fig. S5), and TFBS enrichment identifies the MYB1
binding site and two of the G-box elements that were found
among the sepal, tepal, and stamen genes. Enrichment statistics
for carpel genes indicate significant activity related to the regu-
lation of gene expression, including both activation and silencing,
along with several developmental processes (Fig. S6), and
identify the TELO-box and E2F motifs that previously have been
associated with rapid growth (27). Genes expressed in both
petals and carpels share enrichment for the MYB1 binding site,
the biological processes of petals, the cell cycle and floral de-
velopment processes of carpels, and the stress responses of
sepals, tepals, and stamens (Fig. S7).
Because the reproductive cones of gymnosperms are the likely
evolutionary precursors for flowers (5, 28, 29), we compared the
expression profiles of male and female cones of the cycad Zamia
vazquezii with those of angiosperm floral organs. Cluster analyses
retrieved the organwise topology for floral organs described
above with Zamia male cones placed next to the sepals/tepals
cluster and female cones closest to angiosperm carpels (Fig. 4).
Genes in the sepals/tepals cluster were unequally divided be-
tween expression primarily in male and female cones. Enrich-

ment statistics identify photosynthesis and defense-related pro-
cesses in both subsets (Table Sl), but most enriched binding sites
are associated with the subset expressed in male cones (Table
S2). Genes coexpressed in stamens and male cones share sig-
nificant enrichment for response to external stimuli and various
metabolic processes (Table Sl) and the suite of G-box, UPR and
CArG binding sites of the stamen genes. Genes coexpressed in
carpels and female cones regulate gene expression and share the
TELO-box and E2F motifs (Table S2), as did carpels considered
alone. Small gene clusters involving genes coexpressed in petals
and either of the Zamia reproductive cones lack significant en-
richment for GO annotation terms or TFBS.

Evolutionary reconstructions of expression patterns of key floral
transcription factors show progressively more spatially restricted
deployment throughout angiosperm evolution, from across the
floral meristem inAmborella, Nuphar, and Persea, for example, to
specific organs in Arabidopsis and other eudicots (12, 30, 31).
Likewise, the transcriptional cascades that are broadly deployed
in Nuphar and Persea have been tightly constrained spatially
within organ-specific boundaries in Eschscholzia andArabidopsis.
The molecular mechanisms responsible for this distinction likely
lie within the machinery of the ABCE model itself. Especially
relevant is the "fading borders" modification of the ABCE model
(32), in which floral transcriptional regulators are broadly
arrayed across the flower with gradually fading gradients of in-
fluence from focal to peripheral organ categories imparting
intergrading morphologies across floral organs (Fig. 5).
Our observations that transcriptional programs operate
broadly across adjacent floral organs in Nuphar and Persea ex-

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Chanderbali et al.

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Fig. 4. (A) Expression profiles of Zamia reproductive cones share moderate similarity with angiosperm functional counterparts. Male cones are genetically
closest to the angiosperm sepals/tepals cluster in the larger cluster with stamens, whereas female cones are genetically closest to carpels. Cluster analyses are
based on previously analyzed RA expression data (Fig. 3) and an additional 825 Arabidopsis genes tagged as putative homologs in the Zamia data set. RA
values for Zamia were mean centered across male and female cones and then halved to adjust their color range to that of the angiosperms; color scale ranges
from 0.25 (yellow) to -0.25 (blue). (B) Bootstrap (red) and approximately unbiased (green) support values indicate strong support for the position of Zamia
male cones, but the placement of female cones with carpels is not well supported.

tend the "fading borders" model from the action of specific
regulatory genes to downstream transcriptional cascades in floral
development (see also ref. 17) and support the inference that this
model is the ancestral regulatory program for flowers. A shared
transcriptional program across the perianth organs and stamens of
Aquilegia (Ranunculales) (33) suggests that some form of "fading
borders" operates in some basal members of the eudicot clade
as well. Moreover, extrapolating the trajectory of floral tran-
scriptome evolution to its likely origin leads to an ancestrally
uniform program in which separate components (if any) overlap
fully, resembling the genetic program operating in unisexual
gymnosperm cones (Fig. 5). This gymnosperm program would
have been progressively compartmentalized during floral evolu-
tion, with flowers developing through the "fading borders" pro-
gram before the strict ABCE scheme. Autoregulatory feed-back
loops (5) and negative regulators that maintain strict spatial
domains of ABCE function, at least inArabidopsis (34; see ref. 35
for a comprehensive review), may have contributed to the sharp-
ening of transcriptional boundaries, but the broadly overlapping
floral transcriptional cascades evident in Nuphar and Persea sug-
gest that adjacent organ identity functions are not separated fully
from each other in these flowers (Fig. 5). Homologs of organ-
identity genes also are broadly expressed during the initial stages of
Nuphar and Persea floral development (12, 30), when organ
identity is thought to be specified, suggesting that the transcrip-

tional patterns we find in their late-stage flowers also may char-
acterize their early developmental programs.
Despite broader spatial deployment of organwise transcriptional
programs in Nuphar and Persea, we found substantial conservation
in the genetic profiles of carpels and stamens, respectively, across
angiosperms. The hierarchy of genetic relationships between these
reproductive organs and sepals, petals, tepals, and gymnosperm
reproductive cones, together with supporting data from GO an-
notation and TFBS enrichment analyses, provide unprecedented
insights into the deep history of floral organ evolution.
The placement of Zamia female reproductive cones with
carpels (Fig. 3), despite an evolutionary distance spanning per-
haps 300 million years (36), may be a remarkable testament to
the antiquity encapsulated in the ovule, the defining innovation
of seed plants. Because both angiosperms and gymnosperms
bear ovules, the transcriptional cascade contributed to carpels by
ovules may be their most ancient component. Indeed, the reg-
ulation of gene expression and developmental processes that are
dominant in both angiosperm carpels and female gymnosperm
cones (Table S1) may relate to ovule development, which in
carpels occurs during late floral development. The association of
Arabidopsis and Eschscholzia petals with carpels is robust to
random and directed resampling analyses (Fig. S1) but conflicts
with the proposed recent origin of eudicot petals from stamens,
sepals, or tepals (37). However, enrichment statistics indicate

PNAS I December 28, 2010 I vol. 107 I no. 52 I 22573

Chanderbali et al.

- Arabidopsis

- Eschscholzia

Carpel Strict ABCE Model
Stamen Stamen

pal Sepal


Negative regulation

Carpel Fading Borders
Stamen Stamen
Inner tepal Inner tepal

Outer tepal 1.^Z h? Outer tepal


- Persea

I Nuphar


ABc aBC abC 3aC ABc


BC system


Fig. 5. The genetic regulation of floral development may
have evolved from the BC system of gymnosperm cones
into a "fading borders" program that later was shaped into
the strict ABCE scheme. In the "fading borders" program,
floral organs are influenced by transcriptional cascades
regulated by "ABc," "aBC," and "abC" activities, where
lowercase font indicates lower functional influence. These
cascades promote the development of morphologically
intergrading petaloid organs (tepals), stamens, and carpels,
respectively. In the strict ABCE scheme, the sepal/petal dis-
tinction is maintained by strictly controlled B-function ex-
pression, although both perianth organs are influenced by
A function. Strict regulation of the B-function domain also
maintains a distinct boundary between stamens and car-
pels, but both are simultaneously influenced by C function.
Similarly, repression of C function in petals would separate
their developmental program from stamens, but both
organs are influenced by B function. ABC functions extend
beyond these organ boundaries and promote broadly
overlapping transcriptional cascades in the "fading bor-
ders" program.

associations between carpel-specific genes and genes coex-
pressed in both petals and carpels (Table S1 and Figs. S6 and
S7). Specifically, cell growth and differentiation and MYB1
binding sites are common to the two organs. Perhaps, in the
evolution of petals from their ancestral organs, these compo-
nents of the carpel development program were recruited for
petal development, as has been envisioned previously on the
basis of some key developmental regulators (38).
Less evolutionary conservation in male reproductive genetics
is suggested by the position of Zamia male cones next to sepals/
tepals rather than stamens (Fig. 4). However, this genetic re-
lationship could be related to the pollen-producing program of
the developmentally mature stamens in our data set, as GO
annotations indicate, separating the stamens from the effectively
sterile premeiotic male cones. The origin of the floral perianth
from sterile male organs, an implicit corollary of evolutionary
scenarios for the origin of the flower, also is consistent with their
genetic relationships with premeiotic gymnosperm male cones.
Notably, homologs of both B- and C-function genes are expressed
in the tepals of Persea (17, 30), Nuphar (18), and other Nym-
phaeales (31), supporting the link between male reproductive
genetics and perianth organs in the flowers of these species. The
appearance of the evolutionarily derived eudicot sepals within the
tepals cluster is compatible with an intercalationn" hypothesis for
the origin of "true" leafy sepals via restriction of petaloidy from

the outer perianth in core eudicot flowers (38, 39). Accordingly,
the ancestral, tepaloid, angiosperm perianth may have been ge-
netically programmed to function both as a protective envelope
for the reproductive organs and to attract floral visitors for pol-
lination purposes.
TFBS add to the commonalities across the genetic profiles of
angiosperm sepals, tepals, and stamens. Whether organ-specific
or shared across organs, the sets of genes expressed in these
organs are significantly enriched for a common suite of binding
sites, some of which are shared with the petal genes (Table S2).
Shared binding sites are anticipated features of coexpressed
genes, indicating their likely coregulation (e.g., 40). Therefore,
these genomic features of genes with distinct expression patterns
in floral organs may represent vestiges of an ancestral set of
coexpressed genes. This scenario gains credibility in the context
of hypotheses of floral origin from a single gymnosperm re-
productive cone, in which case the preponderance of binding
sites shared between Zamia male cones and angiosperm sepals,
tepals, petals, and stamens (Table S2) supports the "Mostly
Male" (29) and "Out of Male" (5) hypotheses. An alternative
scenario in which these binding sites evolved de novo in the
distinct sets of genes expressed in sepals/tepals/male cones, in
stamens, and in petals is less parsimonious.
Our results establish connections between organwise tran-
scriptome evolution and the morphological evolution of flowers.


Chanderbali et al.

Ancestral female genetic programs inherited from gymnosperms
are moderately conserved in angiosperm carpels, whereas male
gymnosperm reproductive programs are dispersed among sta-
mens and perianth organs. Canalization of ancestrally over-
lapping "fading borders" transcriptional cascades to produce
organ-specific patterns of expression in angiosperm flowers may
trace to the origin of the strict ABCE scheme characteristic of
the eudicot angiosperms, although an earlier canalization that

includes the monocots and/or multiple independent canaliza-
OU tions cannot be discounted.

S Methods
Microarray expression data for leaves and mature floral organs were
extracted from data sets for Eschscholzia [Gene Expressio Omnibus (GEO)
accession no. GSE24237], Arabidopsis (GEO accession no. GSE5632), Persea
(GEO accession no. GSE13737), and Nuphar (GEO accession no. GSE23082).
Cross-species analyses were conducted on RA measures of gene expression
among floral organs within species to remove systemic biases (i.e., normal-
ized) across the species-specific datasets (20). RA scores were assembled into
a multispecies expression matrix composed of 6,584 Arabidopsis, 4,006
Nuphar, 3,725 Persea, and 4,568 Eschscholzia genes identified as likely
homologs through best reciprocal tBLASTx (National Center for Bio-
technology Information) E-scores <10-5. A total of 125,502 transcripts was
collected by 454 transcriptome sequencing of nonnormalized cDNA libraries
made from immature (likely premeiotic) male and female Zamia cones [Se-
quence Read Archive (SRA) accession nos. SRX019097 and SRX019098, re-

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spectively]; 78,843 Zamia transcripts assembled into unigenes potentially
homologous with 6,920 Arabidopsis genes (tBLASTx E-score <10-5). Tag
counts per unigene per library were summarized on the basis of putative
homology with Arabidopsis genes and normalized to the total number of
tags in the respective libraries. RA measures of Zamia gene expression across
cones were calculated by dividing normalized tag counts by their sum across
cones and were appended to the angiosperm data (together with data
for 825 additional Arabidopsis genes not tagged in Nuphar, Persea, or
Eschscholzia) on the basis of putative homology. Cluster analyses are based
on Pearson correlation scores with the average-linkage clustering algorithm
implemented in Cluster 3.0 (41), and the results were visualized with Java
TreeView (42). Support values for organwise clusters were estimated with
the same clustering parameters using the R package Pvclust (43) with 10,000
replicate runs. GO annotation enrichment analyses were conducted with
Benjamin and Hochberg false discovery rate correction and significance set
at P < 0.05 using the Cytoscape (44) plug-in BiNGO (45). TBFS enrichment
analyses were conducted with Bonferroni correction and significance set at
P < 0.05 using Athena (46).

ACKNOWLEDGMENTS. We thank Dennis Stevenson and Christian Schultz
(New York Botanical Garden, Bronx, NY) for Zamia tissues. Oligonucleotide
probes for the Persea, Nuphar, and Eschscholzia arrays were designed by
Raad Gharaibeh and Cynthia Gibas (University of North Carolina-Charlotte).
This study was supported by National Science Foundation Grants PRG-
0115684 for the Floral Genome Project and PGR-0638595 for the Ancestral
Angiosperm Genome Project. Publication of this article was funded in part
by the University of Florida Open Access Publishing Fund.

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PNAS I December 28, 2010 I vol. 107 I no. 52 I 22575

Chanderbali et al.

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