MBE Advance Access published August 11, 2013
Article
Discoveries section
RH: SEP gene evolution in Zingiberales
Roxana Yockteng1,2, Ana M.R. Almeida1, Kelsie Morioka1, Elena R. Alvarez-Buylla1,3 and
Chelsea D. Specht1
1
Department of Plant and Microbial Biology, Department of Integrative Biology and the
University and Jepson Herbaria. University of California, Berkeley. CA 94720
2
Origine, Structure et Evolution de la Diversité (UMR 7205 CNRS, Muséum National
d’Histoire Naturelle, CP39, 16 rue Buffon, 75231 Paris Cedex 05, France
3
Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Dpto. de Ecología
Funcional, Instituto de Ecología, UNAM, Tercer Circuito Exterior, Junto al Jardín Botánico,
México DF 04510
Corresponding address:
111 Koshland Hall MC3102
University of California
Berkeley, CA 94720
[email protected]
© The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and
Evolution. All rights reserved. For permissions, please e-mail: [email protected]
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Molecular evolution and patterns of duplication in the SEP/AGL6-like lineage of the
Zingiberales: a proposed mechanism for floral diversification.
ABSTRACT
The diversity of floral forms in the plant order Zingiberales has evolved through alterations in
floral organ morphology. One striking alteration is the shift from fertile, filamentous stamens to
sterile, laminar (“petaloid”) organs in the stamen whorls, attributed to specific pollination
syndromes. Here we examine the role of the SEPALLATA (SEP) genes, known to be important in
regulatory networks underlying floral development and organ identity, in the evolution of
development of the diverse floral organs phenotypes in the Zingiberales. Phylogenetic analyses
copies. Selection tests on the SEP-like genes indicate that the two copies of SEP3 have mostly
evolved under balancing selection, probably due to strong functional restrictions as a result of
their critical role in floral organ specification. In contrast, the two LOFSEP copies have
undergone differential positive selection, indicating neofunctionalization. RT-PCR, gene
expression from RNA-seq data and in situ hybridization analyses show that the recovered genes
have differential expression patterns across the various whorls and organ types found in the
Zingiberales. Our data also suggest that AGL6, sister to the SEP-like genes, may play an
important role in stamen morphology in the Zingiberales. Thus, the SEP-like genes are likely to
be involved in some of the unique morphogenetic patterns of floral organ development found
among this diverse order of tropical monocots. This work contributes to a growing body of
knowledge focused on understanding the role of gene duplications and the evolution of entire
gene networks in the evolution of flower development.
KEYWORDS: Flower Development, Plant Evolution, SEPALLATA, SEP-like, SEP3, LOFSEP,
AGL6, gene duplication, subfunctionalization, gene expression, Zingiberales, Musa, Zingiber.
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show that the SEP-like genes have undergone several duplication events giving rise to multiple
INTRODUCTION
Gene duplication and the subsequent reorganization of gene regulatory networks underlying
developmental processes plays a critical role in the patterns of evolution of morphological and
functional diversity (Teichmann and Babu 2004). Duplicated genes can arise by local events
such as tandem duplications, or by larger genomic events such as the duplication of chromosome
regions, of entire chromosomes, or of whole genomes (Lynch and Force 2000). Following
selection on the duplicate genes. Initial genetic redundancy confers robustness against
deleterious mutations (Crow et al. 2006; Gu 2003; Seoighe et al. 2003; Wagner et al. 2003),
allowing the potential for evolutionary divergence of the two copies. Ancestral function can be
divided between duplicate copies (subfunctionalization) or one of the paralogs may take on a
novel function (neofunctionalization) (Lynch and Force 2000). Duplicated genes may thus be
preferentially retained, either due to increased gene dosage or due to functional divergence (and
neofunctionalization) (Cornell et al. 2007). Duplication events in various gene lineages of
flowering plants, especially the MADS-box genes, have profound effects in shaping regulatory
networks that influence floral development and flowering time (Freeling et al. 2008; Shan et al.
2009).
In Angiosperms, members of the MADS-box gene family of DNA-binding transcriptional
regulators are responsible for essential functions that range from the formation of the floral
meristem, initiation and development of the flower, the establishment of the identity of floral
organs including reproductive structures, and the control of flowering time (Alvarez-Buylla et al.
2000a; Becker et al. 2000; Coen and Meyerowitz 1991). The ABC model of flower development,
based on genetic data from Arabidopsis thaliana and Antirrhinum majus, described three
functional classes (A, B, and C) of MADS-box genes that are necessary for the specification of
floral organ identity in sepals, petals, stamens and carpels (Coen and Meyerowitz 1991). Later
studies demonstrated that the A-, B-, and C-class genes are necessary but not sufficient for
recovering floral organ determination (Ditta et al. 2004; Goto et al. 2001; Pelaz et al. 2000; Pelaz
et al. 2001). The SEPALLATA (SEP) lineage of MADS-box genes were shown to be expressed
across the entire floral meristem and during early floral development, and together with A-, B-
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duplication, the two paralogs are initially redundant in function enabling a period of relaxed
and C- class genes they play a crucial role in floral organ identity and organ determinacy (Ditta
et al. 2004; Goto et al. 2001; Pelaz et al. 2000; Pelaz et al. 2001). Quaternary protein complexes
that include the SEP genes are now known to be required for the formation of each floral organ
type in Arabidopsis; Class A and SEP genes are required to specify sepals; A, B and SEP, petals;
B, C and SEP, stamens; and C and SEP, carpels (Ditta et al. 2004; Theissen and Saedler 2001).
SEP genes are thus required for the proper initiation and development of each of the four types
of floral organs via their protein interactions with the A, B, and C class MADS-box genes.
diversification of extant angiosperms (Zahn et al. 2005). Members of the SEP subfamily have
been identified in all angiosperms investigated to date, including in several basal angiosperm
lineages, but they have not been recovered from gymnosperms (Zahn et al. 2005). Genes forming
the sister clade of the SEP genes, the AGL6 genes, are present in both gymnosperms and
angiosperms, suggesting that the SEP subfamily arose from an AGL6 duplication that occurred in
the ancestor of extant angiosperms (Zahn et al. 2005), and that the resulting SEP gene lineage
may have played a critical role in the origin and development of the angiosperm flower
(Malcomber and Kellogg 2005; Zahn et al. 2005). A second duplication event predating the
diversification of extant angiosperms divided the SEP subfamily in two clades; LOFSEP and
SEP3 (Malcomber and Kellogg 2005; Zahn et al. 2005). The entire SEP subfamily has since
undergone many independent, lineage-specific duplication events across flowering plants; the
resulting variability in copy number, point mutations and expression patterns combined with the
critical role of these genes in floral development indicates that the SEP genes are well-positioned
to have been involved in the evolution and diversification of floral morphology (Agrawal et al.
2005; Cui et al. 2010; Ditta et al. 2004; Kobayashi et al. 2010; Pelaz et al. 2000; Vandenbussche
et al. 2003). Evidence that critical residues within these genes may have been fixed in taxa with
altered floral organ morphologies could be used to infer their role in the development of organspecific morphogenic patterns.
In Arabidopsis there are four SEP gene lineages. SEPALLATA 3 (SEP3) is regarded as the glue
for the MADS-box transcription factor protein complex that binds DNA, and is essential for
proper floral organ initiation and formation (Immink et al. 2009). SEP3 acts by facilitating the
protein-DNA interaction and potentially enhancing transcriptional activity within the multimeric
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The SEP subfamily of MADS-box genes arose following an early duplication predating the
transcription factor complexes (Goto et al. 2001). In Arabidopsis, SEP3 enables both B and C
function by forming complexes with AP3 and AGAMOUS (AG) genes (Castillejo et al. 2005).
The presence of SEP3 is required for the function of several of the DNA-binding complexes: for
example the B-class complex APETALA3/PISTALLATA is not able to transform cauline leaves
into petals without SEP3 (Honma and Goto 2001; Pelaz et al. 2001).
The remaining three Arabidopsis SEP subfamily genes have been placed in the LOFSEP clade, a
lineage that has undergone various independent, lineage-specific duplication events across
the eudicot lineage gave rise to three characterized LOFSEP genes: AGL3, FB9 and AGL2/4.
Arabidopsis SEP1 (formerly known as AGL2) and SEP2 (AGL4) are part of the AGL2/4 clade
and SEP4 (AGL3) is part of the AGL3 clade; other species such as Petunia also maintain the
three copies (Malcomber and Kellogg 2005; Zahn et al. 2005). In monocots, two duplication
events have been detected, the first giving rise to the OsMADS34 (PAP2) clade and the second to
clades LHS1 (OsMADS 1) and OsMADS5.
In comparison to LOFSEP, the SEP3 gene lineage (formerly known as AGL9) appears to have
either undergone fewer duplication events or fewer duplicate copies have been retained; only a
single SEP3 copy has been characterized from the eudicots. In grasses, a single duplication event
resulted in OsMADS8 and OsMADS7 clades (Malcomber and Kellogg 2005; Zahn et al. 2005)
but this duplication appears to be restricted to just the grasses (Poaceae).
While only a few studies have investigated SEP-like genes in non-grass monocots (Chang et al.
2009; Kanno et al. 2006; Tzeng et al. 2003), they demonstrate a potential for lineage-specific
changes in SEP-like gene copy number: lineage-specific duplication events are thought to have
occurred in both SEP (e.g. Elaeis guineensis in LOFSEP and SEP3) and AGL6 (e.g. Crocus)
subfamilies (Shan et al. 2009; Viaene et al. 2010). Due to their important role during floral
development, duplication events in the SEPALLATA genes during lineage-specific diversification
could have been important for the appearance of novel flower morphologies and the evolution of
floral organ form.
The monocot order Zingiberales contains approximately 2500 species, among them
commercially important food crops [banana and plantains (Musaceae)], spices [ginger,
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eudicots and monocots (Malcomber and Kellogg 2005; Zahn et al. 2005). Two duplications in
cardamom, turmeric (Zingiberaceae)], and ornamentals [heliconias (Heliconiaceae), cannas
(Cannaceae), bird-of-paradise (Strelitziaceae)]. The order comprises eight families divided in
two groups based largely on overall floral form; the banana families (Musaceae, Heliconiaceae,
Orchidantha and Lowiaceae) and the derived and monophyletic ginger families (Zingiberaceae,
Costaceae, Marantaceae and Cannaceae). Flowers of the Zingiberales are very diverse
morphologically, particularly in perianth and stamen whorls. This floral diversity has been
associated with the formation of specialized pollination syndromes (Classen-Bockhoff and Heller
pollination syndrome are correlated with an increase in rate of diversification across the order
(Specht et al. 2012) and in specific families (Kay et al. 2005; Specht 2005; Specht 2006; Specht
and Stevenson 2006).
The Zingiberales display a series of lineage-specific morphological innovations in floral pattern
that are the result of discrete evolutionary changes during flower development. Thus, a detailed
study of SEP gene duplication and evolution during the morphological diversification of the
Zingiberalean flower provides an ideal study system for addressing the specific role of gene
duplications in morphological innovation and evolution. In this paper, we examine the evolution
of the SEPALLATA genes across the Zingiberales using a comparative phylogenetic framework.
The phylogenetic reconstruction enabled us to assess the number of SEPALLATA genes present
in the Zingiberales and map any lineage-specific duplication events occurring within the order.
We further examined the role of selection in the diversification of the SEPALLATA genes in the
Zingiberales and test if shifts in selection in specific lineages correspond with changes in
morphology and expression, indicating functional diversification and the potential for
neofunctionalization. Hence, patterns of duplication, selection and expression are analyzed to
examine the role of SEP-like genes in floral development and test the role of SEP evolution and
diversification in observed evolutionary changes of floral morphology.
RESULTS
We obtained sequences for SEPALLATA-like genes from taxa across the monocot order
Zingiberales to study their evolution and to investigate the potential role of these genes in the
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2008; Kress 1990; Ley and Classen-Bockhoff 2011), and there is evidence that shifts in
development and evolution of flower morphology. All sequences were blasted against NCBI’s
genetic sequence database (GenBank) using BlastN to confirm their identity and similarity to
annotated SEP genes. Several cloned sequences were recovered per taxon. Only sequences with
more than 1.0% of divergence (number of different nucleotides/number of total of nucleotides)
were used in the phylogenetic analyses. Sequences from the SEP gene family, including
LOFSEP and SEP3 subfamilies, were obtained for at least one species of each of the eight
families of Zingiberales (Table 1). Sequences for AGL6, the lineage characterized as being sister
of the AGL6/SEP-like clade (Zahn et al. 2005) were also cloned from Zingiberales taxa.
Sequences from Zingiberales were aligned and analyzed with SEP sequences from other
monocots, eudicots, basal angiosperms, and from a few gymnosperm taxa (Malcomber and
Kellogg 2005; Reinheimer and Kellogg 2009; Shan et al. 2009; Viaene et al. 2010; Zahn et al.
2005).
Evolution of SEPALLATA genes across Zingiberales
Phylogenetic analyses were conducted to estimate the evolution of SEPALLATA genes across
monocots using Bayesian Inference (MrBayes) and Maximum Likelihood (PhyML). The total
alignment contains 386 sequences of which 152 are from Zingiberales taxa (Table 1). The total
length of the nucleotide alignment is 501 bp (supplemental material). In order to conduct a
phylogenetic analysis of the entire dataset (AGL6 + SEP), regions were excluded that could not
be aligned without ambiguity, in particular the 3’ end where the highly-variable C-domain is
located. We also excluded from the analyses one region of 13 amino acids in the intervening (I)
domain, recognized as a weakly conserved region (Riechmann and Meyerowitz 1997), as this
region is highly variable among the species sampled and between different copies obtained.
Topologies obtained using Bayesian and Likelihood methods are very similar, however support
values (bootstrap) for the likelihood topology are lower than those obtained with Bayesian
analyses (Figure 1). As in previous phylogenetic studies (Malcomber and Kellogg 2005; Shan et
al. 2009; Zahn et al. 2005), the AGL6 clade is recovered as monophyletic and appears as the
sister clade of the SEP genes when rooted with AP1/SQUA, and the SEP clade itself is divided
into two well-supported clades each one containing SEP3-like and LOFSEP-like genes.
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to the SEP subfamily (Zahn et al. 2005), and for AP1/SQUA, considered to be the sister lineage
As shown in previous studies (Malcomber and Kellogg 2005; Reinheimer and Kellogg 2009;
Shan et al. 2009; Viaene et al. 2010; Zahn et al. 2005), the monocot and eudicot AGL6 and SEP
form separate monophyletic lineages within each gene family. Zingiberales sequences, as
expected, fall within a well-supported monocot clade (Figure 1). A single clade of Zingiberales
AGL6-like genes was recovered, nested within a well-supported monocot clade and sister to
palms (Arecales) and Tradescantia (Commelinales) (Figure 1; ZinAGL6). The Zingiberales
SEP3-like genes are divided into two separate clades, each containing sequences from all
taxa (Figure 1; ZinSEP3-1), while the other appears as sister to a clade containing palm and
orchid sequences (Figure 1; ZinSEP3-2). Within the LOFSEP clade, all Zingiberales sequences
form a single clade; however the Zingiberales sequences are divided into two well-supported
lineages, each lineage containing sequences from taxa across the eight Zingiberales families
(Figure 1; LOFSEP-1 and LOFSEP-2).
To analyze sequence evolution within the individual gene lineages, we realigned closely related
sequences and reconstructed individual phylogenies of the monocot sequences using eudicot
sequences as outgroups to avoid the need to remove ambiguous regions that emerge in
alignments across all gene subfamilies. Bayesian Inference (BI) and Maximum Likelihood (ML)
analyses were used to reconstruct gene trees for each of the three individual datasets separately
(AGL6; SEP3; LOFSEP). As the topologies obtained by the two methods were very similar for
each of the three individual datasets, only the Bayesian Inference trees are shown for each gene
with the corresponding posterior probabilities values and bootstrap likelihood support indicated
at each node (Figures 2-4).
AGL6 phylogeny
The final length of the AGL6 aligned dataset is 825 bp with 100 sequences (Supplemental
material), of which 26 are from Zingiberales taxa. Genes recovered from two gymnosperm
species (Gnetum and Pinus) were used to root the trees. In the BI and ML phylogenetic
reconstructions (Figure 2), AGL6 sequences from monocots, eudicots (core eudicots + proteales
+ ranunculales) and magnoliids form three separate unresolved yet well-supported monophyletic
groups. AGL6 sequences from basal angiosperms Amborella and the Nymphaeales (Nymphaea +
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members of the order (Figure 1; SEP3). One clade is recovered as sister to the sampled Poales
Nuphar) form a grade at the base of the sampled Angiosperms. The AGL6 tree recovers the
expected duplication events described by Viaene et al (2010) and Reinheimer and Kellogg
(2009): One duplication event predates the core eudicot divergence (Figure 2, red arrow)
resulting in two copies, one of which is found in the majority of the eudicot species while the
other is retained in only a few species (Viaene et al 2010). In Magnoliids, the two copies
recovered in Persea and Magnolia indicate that a duplication event may have occurred in the
Magnoliids after their divergence from a common ancestor with monocots (Figure 2).
“Poales”), sister to the remaining monocot sequences. We recover the previously described
Oryza-specific duplication event and demonstrate that one copy, OsMADS6, is nested within
other Poaceae AGL6 sequences, while the OsMADS17 copy is only found in Oryza and is
recovered as sister to all represented Poales s.str. (Joinvillea + Poaceae; Figure 2). Reinheimer
and Kellogg (2009) suggested that this duplication event is likely explained by an Oryza specific
whole genome duplication for several reasons; (1) after extensive screening of related grasses, no
orthologous sequence of OsMADS17 has been found and (2) the two copies are located in
recognized duplicated genomic regions.
Within the monocots, the recovered gene tree is relatively consistent with our current
understanding of monocot evolutionary relationships. The Orchidaceae AGL6 sequences are at
the base of the monocot clade (Figure 2), with the Asparagales + Liliales forming a clade that is
sister to the remaining sampled monocots. The Zingiberales sequences are sister to Arecaceae
(represented by the palm genus Elaeis), and together are sister to a clade of Commelinales +
Poales sequences (Figure 2; Monocots).
The Zingiberales AGL6 (Zin-AGL6) sequences form a single, well-supported monophyletic
lineage (PP=1.0) that is sister to the palm AGL6 (Elaeis guineensis; Arecaceae), indicating the
presence of a single copy of AGL6 in the Zingiberales (Figure 2; “Zingiberales”). The Zin-AGL6
sequences form two well-supported clades that roughly subdivide the order into the Banana
families (PP=0.96) and the Ginger families (PP=0.99), with the exception of the Marantaceae
sequences falling out with the Banana families (Figure 2; sister to Musa) although in a position
that is not well supported and on a relatively long branch. Long branch attraction may explain
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The grass sequences form a well-supported clade (posterior probability (PP)=1.0; Figure 2
the unexpected position of the Marantaceae; the calculated divergence of the three Marantaceae
sequences from their apparent sister (Musa) is the highest among Zingiberales-AGL6. In
addition, constraining the Marantaceae sequences to form part of the ginger clade does not
significantly decrease the likelihood score for the overall topology, as tested using the KishinoHasegawa test (Kishino and Hasegawa 1989) and the Shimodaira-Hasegawa test (Shimodaira
2002) implemented in PAUP 4.0 (Swofford 2003). Blasting the AGL6 sequence from Musa
acuminata (gb: EU869308) against the fully sequenced Musa genome sequence (D'hont et al.
our PCR-cloning results that only one AGL6 gene is present in Zingiberales.
LOFSEP phylogeny
The total length of the aligned LOFSEP dataset is 424 bp with a total of 135 sequences (28
Zingiberales) (Supplemental material). Our phylogenetic analyses recover the general LOFSEP
topology obtained by other authors (Christensen and Malcomber 2012; Malcomber and Kellogg
2005; Shan et al. 2006; Shan et al. 2009; Zahn et al. 2005) with separate monocot, magnoliid,
eudicot and basal angiosperm clades (Figure 3).
In the LOFSEP lineage, several duplication events are recognized both in monocots and in the
eudicots. Inside the core eudicots, the LOFSEP gene underwent two sequential duplication
events producing three duplicates (Figure 3; red arrows). The first duplication occurred after the
divergence of the core eudicots from the basal eudicots, explaining the single copy found in the
basal eudicots (here represented by Aquilegia and Eschscholtzia). The single copy in these basal
eudicots is most similar to the AGL3 lineage of the core eudicots, forming a well-supported clade
(PP=0.98) in our phylogeny. These results indicate that this copy may retain the ancestral
function. Our result differs from the phylogeny obtained from Shan et al (2009) and Zahn et al
(2005) in which the eudicots LOFSEP sequences formed a clade.
A second duplication event during the diversification of the core eudicots occurred prior to the
divergence of the rosids and asterids, giving rise to the FLORAL BINDING PROTEIN9 (FBP9)
and AGL2/4 gene lineages (Figure 3). Arabidopsis SEP4 is part of the AGL3 clade, while
Arabidopsis SEP1 and SEP2, which originated in an ancient polyploidy event exclusive to the
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2012) retrieves one single genomic region of chromosome 2 (emb: CAIC01023292) supporting
Brassicaceae (Schranza and Mitchell-Olds 2006), correspond to the AGL2/4 clade (Figure 3).
Arabidopsis does not appear to contain any orthologous FBP9 gene (Shan et al. 2009).
Several duplication events can be identified within the monocot LOFSEP clade, especially in the
Poales, as reported previously (Christensen and Malcomber 2012; Malcomber and Kellogg 2005;
Shan et al. 2009; Zahn et al. 2005). Two separate duplication events occurring since the
divergence of the Poales resulted in three different copies of LOFSEP genes within the Poales
(Figure 3; red arrows). The first duplication event resulted in the PANICLE PHYTOMER 2
event gave rise to the clades containing OsMADS5 and Leafy Hull Sterile 1 (LHS1). Based on
species composition in these clades, both duplication events likely occurred early during Poales
diversification with the second duplication possibly occurring after the divergence of Joinvillea
(Joinvilleaceae: Poales), Streptochaeta (Anomochlooideae: Poaceae) and Pharus (Pharoideae:
Poaceae) from the remaining Poaceae (Figure 3). All three copies play important and separate
roles in the development and evolution of the grass spikelet (Kobayashi et al. 2010; Malcomber
and Kellogg 2004).
The Zingiberales LOFSEP sequences form a well-supported clade (PP=0.99) that is sister to the
LOFSEP from palms (Elaies guineensis) and Poales (Figure 3), forming a Commelinid monocot
clade (PP=0.91). Our analyses indicate that a single Zingiberales-specific duplication event
occurred early in the diversification of the Zingiberales, resulting in two retained LOFSEP copies
in all species of Zingiberales sampled (Figure 3; ZinLOFSEP-1 and ZinLOFSEP-2) with the
exception of Orchidantha siamensis (Lowiaceae). This could be due to a loss of this copy in
Orchidantha, or due to insufficient screening. Blasting the two LOFSEP sequences (LOFSEP-1
and LOFSEP-2) cloned from Musa acuminata against the Musa genome results in the
identification of two regions: Musa acuminata ZinLOFSEP-1 is highly similar to a region on
chromosome 7 (emb: CAIC01021865.1) and ZinLOFSEP-2 recovers a region on chromosome 8
(emb: CAIC01022551.1). This is consistent with our cloning results yielding two copies in
Musaceae and our hypothesis of an early Zingiberales-specific duplication event.
The other monocot LOFSEP sequences including those from Asparagales (Asparagus,
Dendrobium and Allium), Liliales (Lilium) and Acorales (Acorus) form a paraphyly at the base of
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(PAP2) clade, also known as OsMADS34 (after Oryza sativa MADS34); the second duplication
the Commilinid monocot clade. Several of these (Acorus and Asparagus) have two LOFSEP
genes; however the two copies cluster together within their taxonomic group. It is possible that
several of these taxa have also experienced a lineage-specific duplication; however we cannot
differentiate between species-specific and higher order duplication events given our sampling
outside the Zingiberales. LOFSEP sequences from Magnoliids form a clade sister to the wellsupported monocot clade (PP=0.99).
SEP3 phylogeny
material). The overall topology (Figure 4) supports the known phylogeny of angiosperms, with a
strongly supported monophyletic monocot clade (PP=0.99), nested within a paraphyletic
Magnoliid group. The Magnoliid + Monocot clade is recovered as sister to a well-supported core
eudicot clade (PP=0.98) with the exception of Gerbera, an asteroid (core eudicot) that is
recovered as sister to Houttuynia (Piperales) but on a long branch.
The SEP3 gene appears to have diverged more in the monocots than in the core eudicots.
Although several core eudicot species (e.g. Anthirrhinum, Prunus, Petunia) have several copies,
they form species-specific clades (and thus may represent allelic variation) and are included as
part of a single well-supported core eudicot SEP3 clade (PP=0.98). Arabidopsis appears to have
a single SEP3 gene.
In monocots, the SEP3 lineage has undergone several duplication events (Figure 4; red arrows).
One duplication event appears to have occurred after divergence of Poales from remaining
monocots, resulting in duplicates OsMADS7 and OsMADs8. We do not have sufficient sampling
to determine if this duplication is specific to the Poaceae or the entire Poales s.l. Several
duplication events appear to have occurred within the Asparagales (Figure 4; Asparagales), our
results indicate that these events are specific to particular lineages and perhaps even species (eg.
Crocus and Asparagus Figure 4), however this could be a result of the limited sampling in this
clade. Sequences from Orchidaceae also indicate potential lineage-specific duplications (e.g.
Dendrobium), however their phylogenetic position falls outside the Asparagales clade. In the
case of Arecales, only data from Elaies guineensis is available so it is difficult to say when
exactly the duplication leading to the three palm SEP3 genes occurred..
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The final SEP3 alignment contains 140 taxa with an aligned length of 567 bp (supplemental
SEP3 sequences from the Zingiberales are recovered in two separate and well-supported clades,
indicating the presence of two copies of SEP3 within the order: ZinSEP3-1 (PP= 1.0) and
ZinSEP3-2 (PP=1.0) (Figure 4). According to our phylogeny, ZinSEP3-1 is sister to SEP3
sequences from the Poaceae (PP=1.0), and together ZinSEP3-1 and Poaceae SEP3 are wellsupported (PP=0.99) as sister to Asparagales SEP3 sequences excluding the orchid sequences,
which do not form a monophyletic lineage with remaining Asparagales sequences (Figure 4).
ZinSEP3-2 appears more related to the SEP3 of Arecaceae (Elaeis guineensis) and Orchidaceae
is a gap of three amino acids in the ZinSEP3-2 copy that is located in the second α-helix of the K
domain. In MADS box genes, the a-helices are important for protein-protein interactions
(Alvarez-Buylla et al. 2000b; West et al. 1997).
Although only two copies of ZinSEP3 are found across all members of the eight Zingiberales
families, it is possible that copy-specific duplication events have occurred during the
diversification of the Zingiberales order. The majority of Zingiberales species sampled have
more than one sequence recovered in the two SEP3 clades with a within species divergence
greater than 5%. To independently test for the number of duplicates, we blasted the SEP3
sequences from Musa acuminata (MADS1:EU869307, MADS2:EU869306, MADS3:AY941800,
MADS3:EU869308, MADS4:EU869309) against the full genome sequence (D'hont et al. 2012) to
confirm that each sequence recovered corresponded to a separate and single region in the
genome. The three ZinSEP3-2 of Musa acuminata matched two different regions of its genome:
sequences AY941800 and EU869309 matched a region on chromosome 6 (emb CAIC01024381)
and sequence EU869306 matched a region on chromosome 9 (emb CAIC01018285.1). The
ZinSEP3-1 sequence from Musa acuminata (EU869307) matched one single region on
chromosome 11 (emb CAIC01023165.1). These results suggest that, in addition to the
duplication event that gave rise to the ZinSEP3-1 and ZinSEP3-2 lineages, an additional
Zingiberales-specific duplication event occurred in the SEP3-2 gene lineage.
Expression of SEP genes
The diversification of the SEP genes could have a functional importance in the evolution of
flower morphology across the Zingiberales. Using expression analyses, we attempted to assess if
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(Aranda and Dendrobium). The main difference between ZinSEP3-1 and ZinSEP3-2 sequences
the different SEP copies differ in expression patterns across the floral organs, suggesting
divergent roles in protein complex formation and organ development.
Results from RT-PCR (Figure 5 and Figure S1) show that gene expression patterns of the three
SEP-like gene lineages differ across the five whorls, in different organ types, and in different
species of Zingiberales. AGL6, with a single copy found in all organisms sampled, is expressed
in all organs of Strelitzia sp. (Strelitziaceae) and Musa acuminata (Musaceae) with weak
expression in Musa stamens. Interestingly, AGL6 does not appear to be expressed in the infertile
officinale (Zingiberaceae), indicating a possible association of this gene with the loss of fertility
in these organs. AGL6 is only expressed in the sepals of Zingiber officinale (Figure 5, blue).
These results are corroborated by RNA-seq based expression analyses (eXpress; Roberts and
Pachter 2013) using transcriptome libraries generated from petals, labellum and fertile stamen
(filament and theca) from Costus spicatus and from free petal and fertile stamens (theca and
filament) from Musa basjoo (Figure 6, Table S2). In Costus, expression of AGL6 is reduced in
the labellum as compared to expression in petal and stamen (filament and theca). In Musa, AGL6
is expressed in petals, with reduced expression in filament and theca.
In comparison, the two copies of SEP3 (ZinSEP3-1 and ZinSEP3-2) are relatively consistently
expressed across all taxa and across all organ types, as evaluated by RT-PCR. Both SEP3 genes
are expressed in all floral organs of Costus spicatus, Canna indica, Strelitzia sp., Musa
acuminata and Musa basjoo, with the exception of SEP3-1, which is not expressed in the fertile
stamen of Zingiber (Figure 5, dark and light purple; Figure S1). In situ hybridizations using
young flowers of Costus spicatus indicate that SEP3-1 and SEP3-2 are expressed in the labellum
(fused petaloid staminodes) and in the thecae of the petaloid fertile stamen (Figure S2). In the
organ-specific transcriptomes for Costus spicatus and Musa basjoo, SEP3-1 and SEP3-2 are
expressed in the petals, labellum and fertile stamen (filament and theca) of Costus and in the free
petal and stamens (theca and filament) of Musa basjoo. These results also show that SEP3-2 has
at least 2-fold greater expression than the SEP3-1 copy in both species (Figure 6).
In contrast, the expression of LOFSEP genes is highly variable across organs and species. In
Musa, LOFSEP-1 is expressed only in stamens, however in Strelitzia it is expressed in all organs
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stamens (staminodes) of Costus spicatus (Costaceae), Canna indica (Cannaceae) and Zingiber
except sepals (Figure 5, dark green). In Costus spicatus, the RT-PCR results for LOFSEP1
indicate it is not expressed in sepals, labellum or gynoecium. In Canna, it is not expressed in the
petaloid staminodes, and in Zingiber LOFSEP1 is not expressed in petals. LOFSEP2 is equally
variable in its expression pattern: In Strelitzia, the expression of LOFSEP2 is limited to the inner
floral whorls (stamens and gynoecium) while in Musa acuminata the expression is further
limited to gynoecium. In contrast, LOFSEP2 (Figure 5, light green) is more universally
expressed in the ginger clade, where our results indicate evidence of expression in all of the
exception of the sepals of Zingiber. RNA-Seq results show that the expression of the LOFSEP
genes across the floral organs is very low in comparison with the expression of the SEP3 genes
(Figure 6 and Table S2). In general, the LOFSEP expression results are consistent with the RTPCR results.
Ages of divergence and Selection in SEP genes
We estimated the ages of divergence and performed tests of selection on the three different gene
subfamilies. We used Beast (Drummond et al. 2012) to estimate the ages of divergence of the
three genes and mapped the estimated ages (Figure 7). The duplication event separating the
AGL6 and the SEP-like genes was estimated to occur around 198.09-207.03 MYA, while the
duplication event, which produced the LOFSEP and SEP3 clades, has been estimated to have
occurred approximately 198 MYA. LOFSEP copies for the different lineages would be older
than SEP3 and AGL6 under our estimation; these age estimates are likely influenced by the
independent and divergent rates of evolution of each gene.
Tests of selection (on sites and branches) were conducted specifically to test if changes in
selection pressure can be detected in particular branches corresponding to the Zingiberales
sequences compared to the Poales and eudicot sequences. Using the branch model in PAML, the
rates of non-synonymous/synonymous substitution (ω) for all of the genes is higher than 1.0,
however significant changes in selection among the Zingiberales branches compared with other
taxonomic lineages are detected in SEP3 and LOFSEP. Selection tests were done using the
alignments obtained for phylogenetic analyses. In figure 7, we represent these alignments and
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floral organ types in Costus spicatus, in Canna indica and in Zingiber officinale, with the
indicate detected sites under positive selection. Because most of the Zingiberales sequences do
not have the C-domain, we do not represent the complete sequence of the outgroup taxa.
In SEP3, the Zingiberales sequences more closely related to the Poales (i.e. ZinSEP3-1) shows a
higher rate of non-synonymous/synonymous substitution (ω=0.164) as compared with the second
SEP3 clade of Zingiberales (ZinSEP3-2 (ω=0.0504)), Poales SEP3 (ω=0.067) and eudicot SEP3
(ω=0.049). This result indicates that the ZinSEP3-1 lineage is subject to selection pressures that
are significantly different from those experienced by the same genes in related lineages and from
To check if the changes in selection pressure in the Zingiberales were due to the presence of sites
under positive selection, we used the branch site model (Model A) implemented in PAML.
Under this model, the sites are more frequently under negative selection. The test does not reject
the null hypothesis that the clades evolved under neutral selection. However, the FEL test
implemented in HYPHY suggests that some sites evolved under positive selection in the
Zingiberales lineages and in other lineages tested, such as Poales and eudicots (Figure 7). These
sites are located in the intervening (I) and the keratin (K) domains. Sites 132 and 139 of
ZinSEP3-1 and site 142 of ZinSEP3-2 are detected as having been fixed by positive selection.
These sites are located in the second a-helix of the K domain. Sites 61, 82, 85, 89 and 109 for
Poales and sites 107, 164 and 176 for eudicots were inferred to have been fixed by positive
selection. Changes in the K-domain can affect the protein function as it generates an interaction
surface that consists in amphipathic a-helices that mediates the interaction between MIKC-type
proteins (Zachgo et al 1995). The I-domain influences the specificity of the DNA-binding dimer
formation (Riechmann et al 1996). In general, sites under positive selection do not correspond to
a change in charge or polarity of the amino acid, with the exception of site 109 in Eudicots and
site 107 in Poales, in which there is a change from a non-polar to a polar amino-acid.
For LOFSEP, selection pressure for the Zingiberales clade (ω=0.41) was found to be twice that
of the Poales (ω=0.192) and eudicots (ω=0.156). We found that the values for the two copies
present in the Zingiberales clades are significantly higher (ω=0.336 and ω=0.458 for
ZinLOFSEP1 and ZinLOFSEP2 respectively), indicating that they evolved under different
selection pressures in comparison to genes outside the Zingiberales. Within the duplicates of
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the ancestral sequence.
LOFSEP in eudicots and in Poales, selection is also variable. In Poales, PAP2 shows a higher w
(ω=0.264), while the derived copies have lower values (ω= 0.148 for LHS1 and ω=0.186 for
OsMADS5). In eudicots, SEP4 has a value of ω=0.188, TM29 of ω=0.117 and FBP9 of
ω=0.226.
Model A testing for positive selection in certain branches does not reject the hypothesis that the
various gene lineages are evolving neutrally, except for the OsMADS5 copy of Poales
(p=0.00048) that seems to have evolved under positive selection. The FEL test in HYPHY
in the variable I domain and in the K domain; only few sites under positive selection are located
in the conserved MADS domain (Figure 7). Sites 45 and 126 of ZinLOFSEP-1 and site 41 of
ZinLOFSEP-2 seem to have been fixed by positive selection; sites 41 and 45 are located in the
MADS-domain. Site 45 is more specifically located in the β−strand that is a hydrophobic patch
essential for dimerization (Sharrocks et al. 1993).
Using the program Selecton (Stern et al. 2007) four additional sites (26,78, 82 and 85) of
ZinLOFSEP2 are detected as having been fixed by positive selection (Figure 7). Three of the
sites are located in the K domain and one site is in the alpha helix of the MADS domain. The
three copies of LOFSEP of Poales have four sites detected as under positive selection, located in
the MADS domain and the I-domain. Site 60 of the I-domain also seems to have been fixed by
positive selection for all three genes (PAP2, LHS1 and OsMADS5). The three copies in the
eudicots also contain four sites under positive selection that are located in the first a-helix of the
K-domain (site 111 shared by the three genes, site 94 and 109 in TM29 and site 81 in FBP9
(Figure 7)). The positive selected sites do not correspond to amino-acid changes in polarity or
charge in any of the taxa.
For AGL6, we tested for differences in selection between the Banana families and the Ginger
clade sequences (Zingiberales) and other lineages. The LRT test is statistically significant
indicating that the AGL6 for eudicots, Poales and Zingiberales evolved under different selection
pressures compared to the ancestor of these clades (Figure 2). However, the LRT test does not
reject the hypothesis that both Zingiberales clades evolved under similar selection (w=0.107 for
banana families and ω=0.099 for ginger families (Figure 2)).
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indicates that several sites may have been fixed by positive selection and they are located mainly
The proportion of sites under positive selection found using codeml under the model A is null for
the clades tested (Zingiberales, eudicots and Poales); the majority of the sites are under purifying
selection and a few are consistent with neutral evolution. However, the FEL test in HYPHY
identified several sites under positive selection, mostly in the C domain for the different clades
(Figure 7). Sites 93, 218 and 221 for the Banana lineages and site 224 for the Ginger clade are
detected as being under positive selection. Site 245 is also under positive selection for both
taxonomic groups. The C-terminal region is the least conserved region of the MIKC genes, and
one site (223) is under positive selection in the Poales AGL6 (site 223; located in the C-domain)
while four sites are under positive selection in the eudicot AGL6 (sites 130 and 137 located in the
first a-helix of the K-domain and sites 247 and 272 located in the C-domain (Figure 7)).
DISCUSSION
Across monocots, MADS-box genes have been implicated in the evolution of novel floral organ
specification and morphogenetic patterns of organ development. Changes in copy number,
expression patterns, and/or functional changes to domains important for protein-protein
interactions have been investigated as means by which the evolution of MADS-box genes may
influence flower development. In Tulipa, which has a monomorphic perianth (tepals), B-class
genes are expressed in both the first and second whorls and appear to contribute to development
of the petaloid tepals that occupy those whorls (Kanno et al. 2003). In Lancadonia, B-class gene
expression is displaced to the center of the floral meristem, promoting the formation of stamens
in the center part of the homeotic flower (Alvarez-Buylla et al. 2010). In the Zingiberales, it has
been hypothesized that duplications followed by differential expression in B-class gene lineages
may provide opportunities for the evolution of novel organ form, such as the petaloid labellum in
the stamen whorl (Almeida et al. 2013; Bartlett and Specht 2010). In Orchids, duplication events
of the class B gene DEFICIENS are also implicated in the formation of the lip or labellum in the
petal whorl (Mondragon-Palomino and Theissen 2008).
In the Zingiberales, the diversity of floral patterns depends on variations in the morphogenetic
patterns of the perianth organs and the two stamen whorls. In the genus Musa (family
Musaceae), the perianth is essentially monomorphic with 3 sepals and 2 petals fusing to form a
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the role is to enhance and stabilize interactions mediated by the K-domain (Fan et al 1997). Only
floral tube. One petal remains free, but is similar in morphology and anatomy to the fused
perianth parts. The dimorphism of the perianth (sepals and petals) increases from the
plesiomorphic to the more derived members of the order (Bartlett and Specht 2011), while the
number of fertile stamens decreases (Almeida et al. 2013). Musa contains five fertile stamens
and one aborted staminode, but in the derived families the number of fertile stamens is reduced
to one in Costaceae and Zingiberaceae and to one-half in Cannaceae and Marantaceae. The
infertile stamens, or staminodes, form laminar petal-like structures in the derived families that
Zingiberaceae and Costaceae, 2-5 petaloid staminodes fuse to form a novel petal-like structure
called the labellum. The fertile stamen in many of the derived families is petaloid or laminar as
well, while in Musa the fertile stamens have radial filaments.
Duplication events are common in the MIKC type MADS-box transcription factors and they play
an important role in shaping the regulatory networks generating new interactions between
proteins (Shan et al. 2009). Based on detailed phylogenetic analyses of various MADS-box
transcription factors, it is clear that many of these genes have undergone one or more duplication
events during the evolution of angiosperms and that these events are coincident across the gene
subfamilies (Shan et al. 2009). This correlation of copy number is likely to be due to constraints
derived from protein interactions required for gene function (for review see Shan et al 2009).
Relaxed selection is detected in certain subfamilies, indicating the potential for functional
divergence to be a driving force in the increased complexity of the interaction network among
these genes (Shan et al. 2009). Given that SEP-like and AGL6 genes are known to be important
regulators of protein interactions and the DNA binding function of protein complexes that are
key to the formation of floral organs, an investigation of the evolution of SEP-like and AGL6
genes and their patterns of expression across Zingiberales provides an ideal model system to
investigate the role of gene duplication and divergence in floral diversification.
Evolution and diversification of SEPALLATA: the LOFSEP clade
Multiple large-scale and lineage-specific duplication events have been detected during the
evolution of the SEPALLATA gene family (Shan et al. 2009; Zahn et al. 2005). Early in the
evolution of angiosperms, a genomic duplication event resulted in the separation of the
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have coloration patterns and epidermal cell types associated with the attraction of pollinators. In
SEPALLATA subfamily from the ancestral AGL6 gene lineage. Our analyses estimate that this
duplication event occurred around 207-198 MYA (Figure 7). A second duplication event later
generated the two main clades in the SEPALLATA subfamily, SEP3 and LOFSEP (Figure 1,
Figure 7). Our estimates of the age of divergence show that this duplication event has occurred
around 198 MYA. Independently in both monocots and in the core eudicots, duplication events
have increased the number of retained copies of these genes within each lineage.
The LOFSEP clade is the most divergent within the SEPALLATA subfamily; multiple duplication
eudicots, an initial duplication event estimated at 152.56 MYA separated the SEP4(AGL3) gene,
and a second duplication event occurring ~130 MYA generated TM29(AGL2/4) and FBP9 gene
lineages. The single mutants for LOFSEP genes of Arabidopsis thaliana and Petunia hybrida do
not present homeotic alterations, suggesting a redundant function of these genes (Ditta et al.
2004; Pelaz et al. 2000; Vandenbussche et al. 2003). The flowers of triple mutants
(sep1sep2sep3) of Arabidopsis consist only of sepal-like organs, while the flowers of the
quadruple mutant sep1sep2sep3sep4 comprise only leaf-like structures (Ditta et al. 2004; Pelaz et
al. 2000), revealing an important function of SEP4 in sepal development.
In Monocots, the LOFSEP clade diverged ~146 MYA, and independent lineage-specific
duplication events have occurred in both Poaceae and in Zingiberales. The family Poaceae
maintains three copies in the LOFSEP clade, indicating two family-specific duplication events.
Our age estimations indicate that the first duplication event occurred 118 MYA producing the
gene OsMADS34 (PAP2) and the second duplication occurred 82 MYA producing the genes
OsMADS5 and OsMADS1 (LHS1) (Christensen and Malcomber 2012). LOFSEP sequences from
Zingiberales form a single well-supported clade that is divided into paralogous lineages,
indicating a Zingiberales-specific duplication event, which is estimated to have occurred
approximately 102 MYA; this age is consistent with the hypothesis that the ancestral
ZinLOFSEP duplicated in the common ancestor of all Zingiberales, with the potential loss of a
second copy in Orchidantha.
LOFSEP genes are expressed differentially in floral whorls of the various monocot species in
which they have been examined (Christensen and Malcomber 2012; Malcomber and Kellogg
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events have been detected across angiosperms (Zahn et al. 2005, Shan et al. 2009). In the core
2004; 2005). In the grasses, LHS1 orthologs are expressed in the lemma and palea, organs of the
most external floral whorls, and it has a role in regulating the morphology of these two floral
organs; LHS1 orthologs are never expressed in glumes (Malcomber and Kellogg 2004; Prasad et
al. 2005; Reinheimer et al. 2006). The mutants of the gene PAP2 in Oryza were documented to
have a disorganized arrangement of branches and spikelets and elongated glumes and sterile
lemmas, however no effect was found in the internal floral whorls (Kobayashi et al. 2010).
OsMADS5 in rice is expressed mostly in the floral meristem and in the palea, and mutants of this
2010). In the non-grass Poales taxon Joinvillea ascendens, LHS1 and OsMADS5 are expressed in
tepals, with reduced expression in the filaments of stamen and the pistil apex (Preston et al.
2009). LHS1 directly regulates transcription factor genes such as OsHB4, OsBLH1,
OsKANADI2, OsKANADI4, and OsETTIN2 indicating a role in meristem maintenance,
determinacy and lateral organ development (Khanday et al. 2013).
Likewise, the expression of the LOFSEP genes is variable across Zingiberales. One of the copies
(ZinLOFSEP2) is more consistently expressed in all floral whorls in the species of the ginger
families, while the expression of the other copy (ZinLOFSEP1) is more variable. In Musa
acuminata, the expression of the two genes is almost complementary in that ZinLOFSEP1 is
expressed in carpels, while ZinLOFSEP2 is expressed in stamens and floral tube (Figure 5). The
variable expression pattern of LOFSEP in Zingiberales indicates that these genes duplicates may
have undergone functional divergence following duplication, and thus may underlie some of the
diversity of developmental patterns and final morphology of the different floral organs across the
order. This hypothesis is consistent with ZinLOFSEP1 having a more highly conserved sequence
across the order, indicating conservation of ancestral function, while ZinLOFSEP2 demonstrates
a relatively rapid rate of diversification with a number of sites detected to be under positive
selection (Figure 7), signatures consistent with ZinLOFSEP2 having undergone
neofunctionalization following duplication (see below).
Evolution and diversification of SEPALLATA: the SEP3 clade
The SEP3 clade has fewer copies than the LOFSEP clade for all taxonomic groups studied to
date, either due to fewer duplication events in the SEP3 lineage or due to greater levels of SEP3
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gene produce lodicules attached to the palea and lemma (Agrawal et al. 2005; Kobayashi et al.
gene loss post-duplication. We estimate that the SEP3 clade in Angiosperms diverged from
LOFSEP around 149 MYA. The monocot SEP3 lineage has first diverged approximately 115
MYA, while the eudicot SEP3 began to diversify around 77 MYA.
Two SEP3 clades appear to have diverged from one another sometime during the monocot
lineage diversification. However, estimating the exact timing of the duplication and subsequent
divergence is complicated, as the SEP3 gene tree does not follow the established phylogeny for
the monocots. The grasses, for example, maintain a pair of duplicates, OsMADS7 and OsMADS8,
occurred around 58.24 MYA. In Zingiberales, the SEP3 lineage has also undergone a single
duplication event. In our phylogeny, however, one of the copies, ZinSEP3-1, is more closely
related to the Poaceae SEP3 and the second copy, ZinSEP3-2, is resolved as sister to the SEP3
genes from Arecales (palms) and orchids. Two hypotheses can explain the location of the two
ZinSEP3 clades. The first is that the duplication event predated the diversification of Monocots
and the duplicate more similar to the ZinSEP3-1 was lost in palms (represented by Elaeis) and in
orchids (represented by Dendrobium and Aranda), while the ZinSEP3-2 was lost in the Poaceae
and in non-orchid Asparagales (i.e. after the divergence of the Orchidaceae from remaining
Asparagales). A second hypothesis is that the duplication event happened in the ancestor of
Zingiberales creating two Zingiberales-specific clades. In this case, our gene tree would indicate
that ZinSEP3-1 sequence converged with the SEP3 sequences of the Poaceae, and ZinSEP3-2
converged with the SEP3 sequences Arecales and orchids resulting in a topology influenced by
homoplasy. In this latter case, the single duplication event yielding the two ZinSEP3 clades
would have occurred 86.57-91.15 MYA based on our estimation. Additional screening of
monocot taxa in addition to functional characterization and detection of selection on these copies
could help resolve these two potential scenarios.
Expression analyses for SEP3 genes (Figures 5, 6) show that both copies are expressed across all
the floral whorls in Zingiberales, indicating a potentially redundant function of the two copies in
these organs. Expression and function across all whorls seems to be a conserved feature of SEP3
in monocots. In Oryza sativa, knockdown of SEP3-like genes OsMADS7 and OsMADS8
produces changes in the three innermost whorls (Cui et al. 2010): lodicules are transformed to
palea/lemma structures, filaments and anther of stamens are thinner, the number of stamens is
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which are in single clade indicating a Poaceae-specific duplication event estimated to have
variable, the stamens are infertile and the carpels are aberrant (Cui et al. 2010). In the palm
Elaeis guineensis, the two external floral whorls mutants of AGL2-1 (the SEP3-like gene),
produce only leaf-like structures (Adam et al. 2007). The SEP3 gene product plays an essential
role in the formation of a multimeric protein complex (Immink et al. 2009) and is likely to
regulate a large number of downstream genes that confer organ identity and morphology
(Kaufmann et al. 2009). The complete loss of SEP3 has been shown to cause severe defects in
floral morphology in monocots (Adam et al. 2007), thus the maintenance of redundant SEP3
fitness effect of deleterious mutations (Gu et al. 2003).
Evolution and potential role of AGL6 in floral diversification
Unlike LOFSEP and SEP3, AGL6 has undergone fewer duplication events across angiosperm
evolution. AGL6 was estimated to have diversified in angiosperms around 137MYA, with
diversification in the eudicots occurring between 82.9-93.6 MYA and in monocots around 82.9
MYA. In eudicots, only one duplication event has been documented, occurring in the family
Actinidiaceae (Viaene et al. 2010). However, species-specific duplication events have been
documented in various taxa, for example in Arabidopsis (Viaene et al. 2010) and Oryza
(Reinheimer and Kellogg 2009). Reinheimer and Kellogg (2009) showed that ancestral AGL6 is
expressed in all floral whorls with the exception of whorl 1. In other grasses, AGL6 is expressed
in the palea, but expression is not documented in the stamens for many grasses species with the
exception of Oryza (Reinheimer and Kellogg 2009). In Oncidium (Orchidaceae), knocking-down
the expression of AGL6-like gene results in all floral organs converted to carpeloid sepals (Chang
et al. 2009), demonstrating the importance of this gene in the formation of all floral organs.
In Zingiberales, we found only one copy of AGL6, consistent with findings for other non-grass
monocot lineages (Reinheimer and Kellogg 2009). The AGL6 in Zingiberales is estimated to
have diversified around 66 MYA (Figure 1), appearing to be younger than the genes of the
Zingiberales SEPALLATA clades. This result is likely an artifact of a reduced rate of evolution of
this gene in comparison with the genes of the SEP3 and LOFSEP clades. ZinAGL6 is expressed
across all floral organs with a few notable exceptions (Figure 5). Notably, in Zingiber officinale,
there is no AGL6 expression in the labellum and in Costus spicatus is very reduced (the stamen
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copies in Zingiberales could contribute to the robustness of the genetic network by reducing the
whorl organ comprised of fused staminodes); in Canna there is no expression in the petaloid
staminodes. In both Costus and Canna, AGL6 is expressed in the single fertile stamen, however.
In Musa, with 5 fertile and radially symmetrical stamens, AGL6, is weakly expressed in the
stamen whorls. There is thus a correlation between the loss of expression of this gene and a loss
of fertility in the organs of the androecial whorl; thus AGL6 may have a function in the
development of fertility of stamens across the Zingiberales. These results are consistent with the
results from Oryza, in which the AGL6-like gene OsMADS6 up-regulates the expression of an
al. 2011). Furthermore, flowers of the double mutant mads6-1 mads3-4 present a reduced
number of fertile stamens and an increased number of lodicule-like structures (Li et al. 2011).
Absence of this gene may also promote petaloidy in the stamen whorl, although this is harder to
demonstrate as both Costus and Canna fertile stamens are also petaloid, although less so than the
infertile stamens (staminodes). This hypothesis is, however, supported by the reports of the
replacement of fertile stamens with lodicule-like staminodes in the Oryza mads6-1 mads3-4
double mutant (Li et al. 2011). The reduced expression of the AGL6 gene in the staminodes of
the Ginger clade (Costus, Zingiber and Canna) could be explained by changes in promotor
function or activity that occur during the evolution of the order.
Duplication, Selection and Functional Divergence among the SEPALLATA genes
The maintenance of gene copies following a duplication event can be justified by subfunctionalization or neo-functionalization processes (Lynch and Force 2000). Positive selection
acting on one copy relative to its paralog could indicate neo-functionalization, while balancing
selection on both copies is an indication of sub-functionalization when both copies are under
stabilizing selection to maintain a particular function. Our hypothesis is that the duplicates of
SEP3 in Zingiberales evolve under balancing selection (indicating subfunctionalization) while
the LOFSEP genes in Zingiberales have undergone differential positive selection (indicating
neofunctionalization).
In general, the majority of the sites in the three genes (AGL6-like, SEP3-like and LOFSEP-like)
for the lineages analyzed Zingiberales, Poales and eudicots are under balancing selection
(proportion of sites obtained in codeML and Selecton). The branch site Model A implemented in
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Agamous-like gene OsMADS3, which is important for stamen identity (including fertility) (Li et
CodeML failed to detect branches under positive selection except for the LOFSEP gene PAP2 in
Poaceae. Detecting positive selection is generally difficult because it often acts on a few sites and
in a short period of evolutionary time at particular divergence events, and the signal may be
swamped by the ubiquitous purifying selection (Zhang et al. 2005). Another explanation to the
low level of detected positive selection in most of our genes is that our alignments (except for
AGL6) do not include the C domain considered the most variable domain with the highest rate of
evolution across the MADS-box domains (Kaufmann et al. 2005), while the regions considered
leucine zipper-like motifs, which are leucine residues repeated at intervals of seven amino acids
residues, are found in the K domains of AGL6 and the two SEPALLATA clades in our alignments
(Figure 7). These motifs are shown to be important for protein-protein interactions (Lim et al.
2000 ; Moon et al. 1999).
Therefore, we decided to perform the FEL test in HYPHY that estimates synonymous and nonsynonymous rates directly at each site instead from a distribution of rates. Interestingly, this test
was able to detect some sites that could have been fixed by positive selection (Figure 7). These
sites are located mostly in the K and C domain of AGL6 (Figure 7) and in the I and K domains of
SEP3 and LOFSEP (Figure 7). These regions are recognized to be essential for protein-protein
interactions. In particular, the second helix of K domain (K2) and the inter-helical region
between of K1 and K2 are reported to be essential for the interactions between the class B gene
PISTILLATA (PI) and SEP3, and PI and SEP1 (TM29 gene of Arabidopsis) (Yang and Jack
2004). Most of the sites that were found to have been fixed by positive selection in the two
copies of SEP3 in Zingiberales and in the SEP3 of Eudicots are located in the K2 helices of the
K domain. Such sites could be important for the formation of novel interactions and different
protein complexes.
In LOFSEP, we found some sites located in the MADS domain that could have been fixed by
positive selection. This domain is critical for DNA binding at and hence is under strong
functional constraint, so it is possible that the specific sites that seem to have been fixed by
positive selection could affect the binding of the LOFSEP genes to different targets in
Zingiberales and Poales (Figure 8). Selecton results showed that the majority of the sites in these
three genes in the different lineages (eudicots, Poales and Zingiberales) seem to have been fixed
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in this study could be under more strict functional constraints. Conserved sites such as the
by purifying selection and some seem to have evolved under relaxed selection. However, only
the second copy of LOFSEP in Zingiberales (ZinLOFSEP2) has sites under positive selection
located in the K domain. The branch model implemented in CodeML showed a change in the
selective pressure in the LOFSEP clade in Zingiberales, in particular in the LOFSEP2 clade
(ω=0.458), indicating that this clade is evolving under relaxed selection as compared with the
other Zingiberales copy and other lineages. While ZinLOFSEP2 is expressed across all floral
organs in the species of Zingiberales examined (Figure 5), ZinLOFSEP1 demonstrates variability
in expression in the floral organs could be an indication of neofunctionalization of the LOFSEP
copies in Zingiberales with the more highly conserved copy (ZinLOFSEP1) retaining the
ancestral function.
In contrast to the results from LOFSEP showing great variability in patterns of selection and
expression, the two copies of SEP3-like genes are expressed across all floral organs of
Zingiberales. These genes do not follow a pattern expected to be indicative of either a
subfunctionalization (subdivision of ancestral expression) or neofunctionalization (acquisition of
novel expression) hypothesis. An explanation is that subfunctionalization has occurred at the
level of the different domains of the two SEP3 copies. In this case the domains of each copy
would have a complementary function. However, Duarte et al. (2006) showed that several pairs
of duplicates of the type II MADS-box genes in Arabidopsis do not have regulatory
subfunctionalization; instead they found reduction of expression to be more frequent than a total
loss of expression with 1/3 of the paralogous pairs sampled showing two to three-fold less
expression in one member than the other, a phenomenon termed hypofunctionalization (Duarte et
al. 2006). When we examine the expression data obtained from sequencing the transcriptomes of
Costus spicatus and Musa basjoo, ZinSEP3-2 has greater than 2 fold higher expression than
ZinSEP3-1 (Figure 6). A hypothesis of hypofunctionalization with different amounts of gene
expression can explain the redundant expression pattern observed, and may contribute to the
robustness of the genetic network in Zingiberales (Gu et al. 2003).
CONCLUSIONS
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in expression. The differences in rate of molecular evolution, in specific sites under selection and
The SEP-like genes have diversified in the order Zingiberales, showing a pattern of gene
duplication events leading to differential sequence divergence and diversification of expression
patterns. Here we characterize two SEP3 copies of Zingiberales, orthologues of SEP3 genes of
Araceae/Orchidaceae and Poaceae. These genes appear to have evolved under balancing
selection, and their expression patterns correspond with a hypothesis of hypofunctionalization of
the SEP3-1 copy, providing robustness to the functional role of SEP3 across the Zingiberales.
We also found two LOFSEP copies in the Zingiberales, formed from a duplication event that
under a model of relaxed selection, presenting several sites under positive selection in the
interacting domains and showing variable expression across the Zingiberales floral organs,
suggesting neofunctionalization of the LOFSEP genes and indicating their possible role in the
morphological novelties of some Zingiberales flowers. Finally, only one AGL6 copy was found
in the Zingiberales. These genes are mostly under purifying selection, probably due to
restrictions imposed by the gene network regulating flower development in which they
participate. The sites under positive selection are mostly located in the K domain, in particular in
the region of the second helix reported to be critical for interacting with other proteins. This
result indicates that novel protein-protein interactions may have evolved in the Zingiberales.
Some of these interactions are likely to play a role in stamen fertility and perhaps petaloidy, as
AGL6 expression was either undetected in the laminar staminodes of Costaceae, Zingiberaceae
and Cannaceae.
MATERIALS AND METHODS
Amplification of SEP and AGL-6 homologs.
In order to amplify SEP-like genes from the Zingiberales, mRNA was extracted from whole
flowers either fresh or preserved in RNA-later. RNA was extracted with Plant RNA Purification
Reagent (Invitrogen, Carlsbad, CA) following the manufacture’s protocol and first-strand cDNA
was prepared from 2 µg of extracted RNA using the iScript select cDNA synthesis Kit (Bio-Rad,
Hercules, CA, USA). Genomic DNA contamination and success of the reverse transcription
reaction were assessed with ß-actin using the primers F: 5’GGA CGA ACA ACT GGT ATC
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occurred after the Zingiberales diverged from the rest of the Monocots. These genes evolved
GTG CTG3’ and R: GAT GGA TCC TCC AAT CCA GAC ACT GTA3’ (Bartlett and Specht
2010).
Species used span the phylogeny of the Zingiberales and include at least one species per family
(Table 1). Degenerate primers were designed to amplify SEP and AGL-6 homologs (Table 2).
One ml of cDNA diluted 1:10 in water was used in 10 µl PCR reactions with 0.1µl Phire Hot
Start II DNA Polymerase (Finnzymes, Finland), 0.3 µmol each of the forward and reverse
primers, 0.2 mmol dNTPs and 1mmol MgCl2 with the following 3-step thermocycling protocol:
for 20s) and a one minute final extension at 72˚C.
PCR products were visualized in a 2% agarose gel and 1ml successful PCR products were
directly cloned into the pJET1.2 vector (Fermentas) following manufacture protocols. Eight
colonies were picked per cloning reaction and resuspended in 50µl of water. PCR reactions using
3ml of the resuspended clone and pJet vector-specific primers were direct sequenced. Cyclesequencing reactions with BigDye v3.1 (Applied Biosystems, Foster City, CA, USA) followed
manufacture instructions and sequencing reactions were visualized with an ABI 3100 Genetic
Analyzer (Applied Biosystems, Foster City, CA, USA). All sequences have been deposited in
GenBank (Accession No. KC815330-KC815467).
Multiple sequence alignment and phylogenetic analyses
Sequences of SEP-like and AGL-6-like genes from monocots, eudicots, basal angiosperms, and
from a few gymnosperm taxa (Malcomber and Kellogg 2005; Reinheimer and Kellogg 2009;
Shan et al. 2009; Viaene et al. 2010; Zahn et al. 2005) were retrieved from GenBank (Table S1)
and included in multiple sequence alignments along with newly generated Zingiberales
sequences (Table 1). We generated four different alignments, one for each gene subfamily
(LOFSEP, SEP3, AGL6) and one that includes all three subfamilies with SQUA/AP1. The
alignments were constructed with MUSCLE (Edgar 2004) and final alignments were edited by
eye using Geneious 5.5.6 (Drummond et al. 2011). Final alignments are included as
supplemental data.
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98˚C for 5min followed by 35 cycles (98˚C for 5s, 58-64˚C (depending on primers) for 5s, 72˚C
Outgroups for the full SEP/AGL6 phylogenetic analysis included sequences of SQUAMOSA/AP1
genes that were generated from Zingiberales and retrieved from GenBank (Table 1). Sequences
from Arabidopsis thaliana SEP-like genes were used to root lineage-specific phylogenies
focused on inferring the evolution of monocot SEP-like genes. Model selection for each
alignment was tested in jModeltest 0.1.1 (Posada 2008) using the Bayesian Information Criterion
(BIC). The best-fit model of evolution for the SEP3 dataset was TrN+G; SYM+G for LOFSEP;
TIM3+I+G for the AGL6 dataset was and TIM1ef+I+G for the full dataset including SQUA/AP1.
performed in MrBayes (Ronquist and Huelsenbeck 2003) and implemented in the CIPRES
Science Gateway (www.phylo.org) under the models specified above. Two Bayesian analyses
were performed simultaneously with distribution posterior probability (pp) of the generated trees
approximated using Metropolis-Coupled Markov Chain Monte Carlo (MCMCMC) algorithm
with four incrementally heated chains. Data were further analyzed to ensure convergence with
Tracer v1.5 (http://beast.bio.ed.ac.uk/). SumTrees v.3.0.0 using the DendroPy Phylogenetic
Computing Library v3.7.1 (Sukumaran and Holder 2010) was used to combine the trees from
both runs, calculate the burn-in and remove the appropriate trees saved prior to stationarity, and
to assemble a 50% majority rule tree from the remaining trees.
Maximum likelihood bootstrap trees were constructed using PhyML 3.0 (Guindon et al. 2010)
implemented on the ATGC South of France bioinformatics platform (http://www.atgcmontpellier.fr/phyml/) for 1000 bootstrap replicates.
Semiquantitative RT-PCR
Floral organ tissues from Canna indica, Costus spicatus, Musa acuminata, Musa basjoo,
Strelitzia sp. and Zingiber officinale were dissected shortly before anthesis and frozen in liquid
nitrogen. RNA was extracted and cDNA prepared as for whole flowers (above).
For Costus spicatus and Zingiber officinale, cDNA was prepared from five floral organ types:
sepal, petal, labellum (fused petaloid staminodes), fertile stamen and gynoecium. For Musa
basjoo and Musa acuminata we prepared cDNA from the floral tube (fused sepals (3) and petals
(2)), free petal, stamen, gynoecium and the single aborted staminode. For Canna indica, we used
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Bayesian inference was used to infer phylogenetic hypotheses for the four aligned datasets, as
cDNA from sepal, petal, petaloid staminode, petaloid part of the fertile stamen, the theca of the
fertile stamen, and gynoecium. For Strelitzia sp. cDNA was prepared from sepal, petal, theca,
filament and gynoecium.
Primers were designed to flank introns and to amplify a product of approx. 200 bp for the two
copies of SEP3, the two copies of LOFSEP and the one copy of AGL-6 for each of the species
sampled (Table 2). PCR reactions were performed as described above and b-actin was amplified
from all tissues as a reference with the primers described above. Products were visualized in
experiments was cloned and multiple colonies sequenced to verify that the bands represented
single sequences with no sequence variation.
Illumina sequencing and transcriptome analyses
Floral organs of Musa basjoo and Costus spicatus were dissected and immediately flash frozen
in liquid nitrogen. RNA from each floral organ was extracted as described above. Illumina
libraries were prepared using the TruSeq RNA sample prep kit v2. Two libraries of the free petal,
filament and theca from Musa basjoo and petal, labellum and filament and theca from Costus
spicatus were prepared from the extracted RNA and were multiplexed using barcoding set A.
Samples were run in a HiSeq2000 at IIGB HT Sequencing Facility at the University of
California, Riverside.
Raw reads were trimmed to remove adapters and regions of poor quality with cutadapt (Martin
2011). The clean reads were submitted in fastq format to Sequence Read Archive from NCBI.
The clean reads were aligned to the sequences of SEP3-1, SEP3-2, LOFSEP-1, LOFSEP-2 and
AGL6 of Musa acuminata (EU869307, EU869306, KC815372, KC815371 and EU869308
respectively) and Costus spicatus (KC815390, KC815421, KC815335, KC815361, KC815360
and KC815335 respectively) using gsnap/gmap (Wu and Watanabe 2005). The sam output files
from gsnap were converted and sorted in bam files using samtools (Li et al. 2009)..The
expression in FPKM was estimated using eXpress (Roberts and Pachter 2013), using as input
files the sorted bam files obtained from samtools. We normalized the FPKM results dividing by
the FPKM results of actin, used as endogenous gene control (Table S2).
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agarose gels stained with GelRed (Biotium). One PCR product from each of the RT-PCR
RNA in situ hybridization
Expression of SEP3-1 and SEP3-2 was assessed in Costus spicatus flowers prior to anthesis.
Inflorescences were removed from greenhouse from grown plants and quickly dissected by hand,
removing bracts to expose developing flowers. Flowers were dissected, fixed and photographed
as previously described (Bartlett et al. 2008). Probes for SEP3 genes were labeled by in vitro
transcription with T7 polymerase using a DIG RNA labeling kit (Roche) and RNA in situ
hybridizations were performed in duplicate as described previously (Bartlett et al. 2008). The
Estimating ages of divergence and detecting selection
The ages of divergence of the whole SEP/AGL6 phylogeny were calculated using the Bayesian
Evolutionary Analysis Sampling Trees (BEAST), a program for Bayesian Markov Chain Monte
Carlo analysis of molecular data (Drummond et al 2012). Two fossil calibrations were taken into
account as prior assumptions on divergence times: the age of divergence of the Monocot clade
(131 MYA +/- 10) and the Zingiberales clade (99.5 MYA +/-14.5) estimated by Janssen and
Bremer (2004). The ages of divergence were generated using a general-time-reversible
substitution model with gamma distribution and invariable rates among sites following the model
obtained by jmodel test and with a relaxed uncorrected lognormal molecular clock with Markov
Chain Monte Carlo runs of 10,000,000 steps sampled every 1,000 steps and analyzed with
Tracer. The program TreeAnnotator, component of the Beast package, was used to construct the
consensus tree using a burnin of 90000.
To detect differential selection in certain lineages of SEP3, LOFSEP and AGL6 we used the
fixed effects likelihood (FEL) model in HyPhy 2.0 (Pond et al. 2005) to estimate non-neutral
evolution for specific branches for the Bayesian 50% majority rule consensus tree. For each
lineage, a two-rate analysis was used to allow adjustment of dN and dS across sites, models
determined by jModeltest were specified for the nucleotide model of evolution, and dN/dS was
estimated from the data with branch corrections. As suggested by the HyPhy program, p-values <
0.10 were considered to be significant. To confirm the HYPHY results we used the online
program Selecton v2.2 (Stern et al. 2007) using the M8 model from Yang et al. that assumes that
w values come from a mixture of a discrete beta distribution and an additional category ω >1
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reverse complement sequence of SEP3-1 was used as a negative control.
(positive selection). Each w of each site is estimated reporting either positive selection (ω>1),
neutral evolution (ω= 1) or purifying selection (ω<1).
CodeML (PAML v.4.4) was used to estimate different dN/dS values (ω) among branches of the
bayesian tree and sites of the sequence alignment. We first calculated the LRT for variable
ω ratios in different branches of the three genes to test if some lineages have evolved under
different selection pressures (model= 2 and NSsites = 0) (tested branches indicated in Figures 2,
the optimized branch-site model A (model = 2 and NSsites = 2) (Yang and Nielsen 2002) to test
for positive selection in these particular branches . Branch-site models A and A1 (Yang and
Nielsen 2002) test the occurrence of positive selection on individual codons along specific
branch groups. Model A assumes that the branches in the phylogeny are divided a priori in
foreground and background clades, where only the former may have experienced positive
selection. For each analysis, a branch of interest was selected as the foreground branch and
remaining branches served as background. In the alternative model for the branch-site test of
positive selection (MA), the distribution of ω = dN/dS was set to 0 in background branches and
w was estimated in the foreground branch. In the null model (MA1), the distribution of w was set
to 1 in both foreground and background branches. A likelihood ratio test (LRT) was used to
estimate significance (p-value). Two times the difference in log-likelihood values for alternative
and null models was compared to the chi-square distribution with degrees of freedom (df) equal
to the difference of number of parameters for the models. Sites predicted by Bayes Empirical
Bayes (BEB) were included if pp ³ 0.90 and results were considered significant when p < 0.05 in
the LRT when Bonferroni when corrected for multiple testing. Using CodeML, we analyzed the
occurrence of positive selection along codon sites by comparing the model M8 with model M7
and the model M1a with model M2a (Yang et al. 2000).
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3 and 4). If some branches showed significant differences in selection in the first test, we used
ACKNOWLEDGEMENTS
We thank Thiago Andre for his help implementing the Beast analyses and Tatiana Giraud for
reading and commenting on the paper. We also thank Miranda Sun and Crystal Sun for their help
in RNA prep and laboratory experiments. This work was supported by the National Science
Foundation (CAREER IOS 0845641 to C.D.S.; DEB 1110461 to C.D.S and A.M.R.A.), the
Hellman Family Faculty Fund, the Prytanean Alumni Society, and a UC Berkeley College of
Natural Resources student-initiated Sponsored Projects for Undergraduate Research (SPUR)
University of California, Berkeley, California, USA.
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award to K.M. ERAB was supported by the Miller Institute for Basic Research in Science,
Legends
Figure 1. Maximum Likelihood reconstruction of the SEPALLATA-like genes with AP1/SQUA
as the outgroup. Posterior probabilities and ML bootstrap support values higher than 0.95 or
95%, respectively, are indicated with thicker branches. Sequences from species of the order
Zingiberales are colored according to family, as indicated by the legend. Red arrows indicate
duplication events.
Figure 3. Bayesian LOFSEP-like gene tree. Posterior probabilities (pp) >0.5 and ML bootstrap
support values > 50% are indicated (++=1.00 or 100%, -- <0.5). Sequences from species of the
order Zingiberales are colored according to family, as indicated by the legend. Putative gene
duplication events are marked with red arrows.
Figure 4. Bayesian SEP3-like gene tree. Posterior probabilities (pp) >0.5 and ML bootstrap
support values > 50% are indicated (++=1.00 or 100%, -- <0.5). Figure 4. Bayesian LOFSEPlike gene tree. Posterior probabilities (pp) >0.5 and ML bootstrap support values > 50% are
indicated (++=1.00 or 100%, -- <0.5). Sequences from species of the order Zingiberales are
colored according to family, as indicated by the color legend. Putative gene duplication events
are marked with red arrows.
Figure 5. Summary of semiquantitative RT-PCR results of Canna sp., Costus spicatus, Musa
acuminata, Strelitzia sp. and Zingiber officinale mapped on the Zingiberales phylogeny. Gene
expression for AGL6 (blue), SEP3 (purple), and LOFSEP (green) indicated for each whorl by
colored boxes. Organs in each whorl represented by cartoon indicating sepal-like (green), petallike (orange), and fertile stamen (yellow).
Figure 6. Mean normalized FPKM values of the SEP-like and AGL6-like genes in the floral
organ transcriptomes for Costus spicatus and Musa basjoo.
Figure 7. Illustration summarizing the duplication events and selection on genes in lineages of
monocots and eudicots. Illustrations of the MADS-box genes represent the alignment used for
the selection tests. The alignments do not include the complete coding sequences thus we could
not represent the complete MIKC domains. Sites detected as being under positive selection are
marked with triangles. Colors represent sites under positive selection for the different copies in a
lineage. Ages of divergence are indicated at nodes. Rates of non-synonymous/synonymous
substitution (w) for each lineage tested are indicated by the color gradient.
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Figure 2. Bayesian AGL6-like gene tree. Posterior probabilities (pp) >0.5 and ML bootstrap
support values > 50% are indicated (++=1.00 or 100%, -- <0.5). Sequences from species of the
order Zingiberales are colored according to family, as indicated by the legend. Putative gene
duplication events are marked with red arrows.
Table 1. List of species of Zingiberales used in this study
Species
Canna jaegeriana Urb.
Location
(HLA)
Costus productus Gleason ex Maas
2009.0525
(UCBG)
Costus spicatus (Jacq.) Sw.
1994-725 (UCBG)
L-91.0308 (Lyon)
SEP3-1-1: KC815407
AGL6-1: KC815353, LOFSEP-1: KC815383,
LOFSEP-2: KC815382, SEP3-1-1: KC815417,
SEP3-1-2: KC815418, SEP3-2-1: KC815465
LOFSEP-1: KC815367, SEP3-2-1: KC815441,
SEP3-2-2: KC815440
AGL6-1: KC815345, AP1: KC977558, LOFSEP1: KC815368, SEP3-1-1: KC815397, SEP3-1-2:
KC815396, SEP3-1-3: KC815398, SEP3-2-1:
KC815442, SEP3-2-2: KC815443
AGL6-1: KC815346, LOFSEP-1: KC815369,
LOFSEP-2: KC815370, SEP3-1-1: KC815402,
SEP3-1-2: KC815403, SEP3-1-3: KC815400,
SEP3-1-4: KC815401, SEP3-1-5: KC815399,
SEP3-1-6: KC815404, SEP3-2-1: KC815444
AGL6-1: KC815347, LOFSEP-1: KC815375,
SEP3-1-1: KC815413, SEP3-1-2: KC815414,
SEP3-2-1: KC815450, SEP3-2-2: KC815451,
SEP3-2-3: KC815452, SEP3-2-4: KC815453,
SEP3-2-1: KC815454
L-83.0894 (Lyon)
LOFSEP-2: KC815363, SEP3-2-1: KC815428,
SEP3-2-2: KC815429, SEP3-2-3: KC815427
2003.0185 (Lyon)
AGL6-1: KC815344, LOFSEP-2: KC815358,
SEP3-2-1: KC815438, SEP3-2-2: KC815439
L-80.0376 (Lyon)
SEP3-1-1: KC815405, SEP3-1-2: KC815406,
SEP3-2-1: KC815445, SEP3-2-2: KC815446
Tapeinochilos solomonensis Gideon
Heliconia lennartiana W.J. Kress
2003.0170 (Lyon)
0611775-005
(McBryde)
Heliconia metallica Planch. & Linden ex Hook.
266002
(MacBryde)
Heliconia pendula Wawra
711003-003
(McBryde)
Orchidantha siamensis K. Larsen
Donax grandis (Miq.) Ridl.
Halopegia azurea K. Schum.
Marantochloa leucantha (K. Schum.) MilneRedh.
Phrynium oliganthum Merr.
N/A *
LOFSEP-1: KC815378, SEP3-2-1: KC815457,
SEP3-2-2: KC815458, SEP3-2-3: KC815459
AGL6-1: KC815349, AGL6-2: KC815350,
LOFSEP-1: KC815379, SEP3-2-1: KC815460,
SEP3-2-2: KC815461, SEP3-2-3: KC815462
LOFSEP-1: KC815372, LOFSEP-2: KC815371,
SEP3-1-1: KC815409, SEP3-1-2: KC815408,
SEP3-2-1: KC815447
MADS1: EU869307, MADS2: EU869306,
MADS3: AY941800, MADS3: EU869308,
MADS4: EU869309
89.0873 (UCBG)
LOFSEP-1: KC815374, LOFSEP-2: KC815373
L-96.0226 (Lyon)
Schumannianthus virgatus (Roxb.) Rolfe
L-83.0899 (Lyon)
Musa acuminata Colla
02-075 (NMNH)
Musa basjoo Siebold
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2002-127 (NMNH)
Monocostus uniflorus (Poepp. ex Petersen) Maas
NCBI accession per gene
AGL6-1: KC815332; LOFSEP-1: KC815356;
LOFSEP-2: KC815357; SEP3-1-1: KC815389;
SEP3-2-1: KC815419
AGL6-1: KC815333, AGL6-2: KC815334, AP1-1:
KC977556, AP1-2: KC977557, LOFSEP-1:
KC815359, SEP3-1-1: KC815392, SEP3-2-1:
KC815420, SEP3-2-2: KC815422
AGL6-1: KC815335, LOFSEP-1: KC815361,
LOFSEP-2: KC815360, SEP3-1-1: KC815390,
SEP3-1-2: KC815391, SEP3-1-3: KC815393,
SEP3-2-1: KC815421, SEP3-2-2: KC815423
Musa velutina H. Wendl. & Drude
Aframomum angustifolium (Sonn.) K. Schum.
67.1096 (HLA)
AGL6-1: KC815330, LOFSEP-1: KC815355
Alpinia hainanensis K. Schum.
Alpinia pinetorum (Ridl.) Loes.
N/A*
87.0665 (HLA)
Curcuma sp. L.
1999-178 (UCGH)
Elettariopsis smithiae Kam
L-93.0137 (Lyon)
Ettlingera corneri Mood & Ibrahim
L-91.0443 (Lyon)
Globba laeta K. Larsen
L-92.0182 (Lyon)
Zingiber officinale Roscoe
MB0876 (UC)
L-67.0284 (Lyon)
Phenakospermum guyannense (Rich.) Endl.
McBryde
Strelitzia sp. Aiton
N/A *
SEP3-like: FJ861327, AP1like-FL1: EF521814,
AP1like-FL2: EF521816, AP1like-FL3: EF5218
AGL6-1: KC815331, SEP3-1-1: KC815385,
SEP3-1-2: KC815386, SEP3-1-3: KC815387,
SEP3-1-4: KC815388
AGL6-1: KC815336, AGL6-2: KC815337,
LOFSEP-2: KC815362, SEP3-2-1: KC815424,
SEP3-2-2: KC815425, SEP3-2-3: KC815426
AGL6-1: KC815339, AGL6-2: KC815338,
LOFSEP-2: KC815364, SEP3-1-1: KC815394,
SEP3-2-1: KC815430, SEP3-2-2: KC815431
AGL6-1: KC815340, AGL6-2: KC815341, AP1-1:
KC977555, LOFSEP-2: KC815365, SEP3-1-1:
KC815395, SEP3-2-1: KC815432
AGL6-1: KC815342, AGL6-2: KC815343,
LOFSEP-1: KC815366, SEP3-2-1: KC815434,
SEP3-2-2: KC815435, SEP3-2-3: KC815436,
SEP3-2-4: KC815437, SEP3-2-5: KC815433
AGL6-1: KC815354, LOFSEP-1: KC977554,
SEP3-2-1: KC815467, SEP3-2-2: KC815466
SEP3: DY344923
Location of live accessions or herbarium sheets: Lyon Arboretum, Oahu, Hawaii, USA (HLA); McBryde Botanical Garden,
Kauai, Hawaii,USA; University of California Botanical Garden (UCBG); University of California Berkeley Herbarium (UC),
Smithsonian Greenhouses (NMNH).
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
UC
SEP3-1-1: KC815412, SEP3-1-2: KC815410,
SEP3-1-3: KC815411, SEP3-2-1: KC815448,
SEP3-2-2: KC815449
SEP3-1-1: KC815412, SEP3-1-2: KC815410,
SEP3-1-3: KC815411, SEP3-2-1: KC815448,
SEP3-2-2: KC815449
AGL6-1: KC815351, AGL6-2: KC815352,
LOFSEP-1: KC815381, LOFSEP-2: KC815380,
SEP3-1-1: KC815416, SEP3-2-1: KC815463,
SEP3-2-2: KC815464
Table 2. Primer sequences used to amplify SEPALLATA genes and AGL6 genes (F in the names
indicates forward and R reverse primers)
Gene
SEP3-like
LOFSEPlike
LOFSEP
copy specific
Taxa
Zingiberales
Zingiberales
Zingiberales
Zingiberales
Zingiberales
Zingiberales
Zingiberales
Sequence (5'->3')
Canna-SEP3-1-F
Canna-SEP3-1-R
Canna-SEP3-2-F
Canna-SEP3-2-R
Costus-SEP3-1-F
Costus-SEP3-1-R
Costus-SEP3-2-F
Costus-SEP3-2-R
Strelitzia-SEP3-1-F
Strelitzia-SEP3-1-R
Strelitzia-SEP3-2-F
Strelitzia-SEP3-2-R
Zingiber-SEP3-1-F
Zingiber-SEP3-1-R
Zingiber-SEP3-2-F
Zingiber-SEP3-2-R
Musa-SEP3-1-R
Musa-SEP3-1-F
Musa-SEP3-2-F
Musa-SEP3-2-R
Canna
Canna
Canna
Canna
Costus
Costus
Costus
Costus
Strelitzia
Strelitzia
Strelitzia
Strelitzia
Zingiber
Zingiber
Zingiber
Zingiber
Musa
Musa
Musa
Musa
TGC AGC TAT GGA GGA TCA GA
TGG TCC AGC ATT TGT TGT GT
TGC AGC AGC TCC AGT ATG AT
TGC TAA GTG GTC CCA AAT CC
AAG AAG GCA TAC GAG CTC TCC
AGC TGA TTC TCC TTG GCT TGT AAG
ACT CAG ACC AGT CAG CAG GAG TA
TTG ATC AAG CAT GTA TTG TGT CC
GGA GAA TCA GTT GGT TCA GAG
TGG TCC AGC ATT TGT TGT GT
TAT CAA GGG AGA CTC AGA CCA G
CAT CAT GTT GTC GTT CAA GTT GT
GTC CAG AGC AGT CGT CAA GAG TA
TCC TTC AGT GAC ATA TCA AGT GG
ATC AAG GGA GAC TCA AAC TAG TCA A
ACA TCA AGT TGT CGC TCC AG
CAG TTG GTT CAA AGT AGT CG
GCA TTT GTT GTG TCC TTG TG
GGA GAC TCA GAG TAG TCA GCA AGA G
GCA TTT GTT GTG TCC TTG TG
Zin-LOFSEP-F
Zin-LOFSEP-R
Zingiberales
Zingiberales
AGG GTG GAG CTG AAG AGG AT
TTG KGT CTT TGT TGA TCT GAT
Canna-LOFSEP-1F
Canna-LOFSEP-1R
Canna-LOFSEP-2F
Canna-LOFSEP-2R
LOFSEP-1F
Canna
Canna
Canna
Canna
Costus, Zingiber, Musa
GAA CCA CCT ATG GTT TCC TCA TA
TTC ACC AAG GAG GTT TCT CTG
ATG CAG ATA CCA TGT CAC AAA TA
TTG ATC CAG CTC CTT GAC ATT
GCC ATA ATG TAT CAG AAC CAC CT
GCT GAA GMG GAT HGA GAA CAA
TGG TTD CTT TCH TCC AAC CTT
GAA GCG GAT CGA GAA CAA GA
TTC AGB TGG TCC ATG TAR GC
CST CAT CGT CTT CTC CAR CC
AGC AYT TGY TCC CKT CTT TG
CCS TCA TCG TCT TCT CCA RC
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
SEP3 copy
specific
Primer name
Zin-SEP3a-F
Zin-SEP3a-R
Zin-SEP3b-F
Zin-SEP3b-R
Zin-SEP3c/d-F
Zin-SEP3c-R
Zin-SEP3d-R
Costus, Zingiber, Musa
Costus
Costus
Musa
Musa
Strelitzia
Strelitzia
Strelitzia
Strelitzia
Zingiber
Zingiber
GAT CCT TAA CAT TTA GGG CAT CTA
GAA GGT GCA CAG ATA CTA ATT CAC A
TGG GTC TCA AGT TGT TCC AAC
CAG AAC CTG GAC TTC CAT CAA AT
TCT ACT TGG TTC TCA AGC TGC TC
AGG AAT GGC TTG CTG AAG AA
CTA GAT CTT CGC CAA GGA GGT
ATG TGG AGG TGG CGC TTA T
CCC TAA CAC TTA ATG GCT CCA G
AAA GCT ACA ACT TCA GCA AAT GA
TGG TTC TCT AGC TGC TCT AGC TC
Zin-AGL6-F
Zin-AGL6-R
Zingiberales
Zingiberales
CGC ACT CAT CAT CTT CTC CA
GCA GCT TCA GGA GGA ACA AA
Canna-AGL6-F
Canna-AGL6-R
Ginger-AGL6-F
Ginger-AGL6-R
Musa-AGL6-F
Musa-AGL6-R
Strelitzia-AGL6-F
Strelitzia-AGL6-R
Canna
Canna
Costus, Zingiber
Costus, Zingiber
Musa
Musa
Strelitzia
Strelitzia
GGA ATC CCT ACA ACG CTC AC
GAT GCT TGG ACC TGA AGA GC
ACC TTG GTC CCT TGA GTG TG
CAG TGG CTG CAC AGA GAA AG
CAA GAG ACA CAG GCA CGA AA
TGT GGT CCA GCA TCA GTT GT
ATG TCC AAG TTG AAG GCA AAG T
CGA AGT TCT TCC ATC TGG TC
AGL6-like
AGL6 copy
specific
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
LOFSEP-1R
Costus-LOFSEP-2F
Costus-LOFSEP-2R
Musa-LOFSEP-2F
Musa-LOFSEP-2R
Strelitzia-LOFSEP-1F
Strelitzia-LOFSEP-1R
Strelitzia-LOFSEP-2F
Strelitzia-LOFSEP-2R
Zingiber-LOFSEP-2F
Zingiber-LOFSEP-2R
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Supplemental materials
Figure S1. Semiquantitative RT-PCR results for Canna sp., Costus spicatus, Musa basjoo, Musa
acuminata, Strelitzia sp. and Zingiber officinale of AGL6, SEP3-1, SEP3-2, LOFSEP-1 and
LOFSEP-2. ft = floral tube; f = total flower; fi = filament; fp = free petal; gy = gynoecium; lab =
staminodial labellum; p = petal; pa = petaloid appendage of fertile stamen; s = sepal; st = fertile
stamen; sta = staminode; th = theca.
Figure S2. SEP3-1 and SEP3-2 in situ hybridization showing organ specific expression in
Costus spicatus (Costaceae). All images are presented with the adaxial side of the flower
uppermost in transverse sections (a) Sense control in SEP3-1 Costus spicatus flowers. B) In situ
hybridization of SEP3-1 transverse section of C. spicatus floral meristems. C) In situ
hybridization of SEP3-2 transverse section of C. spicatus floral meristems. gy, gynoecium; lab,
staminodial labellum; p, petal; pa petaloid appendage of fertile stamen; s, sepal; st, fertile
stamen.
Figure S3. Alignments of AGL6, LOFSEP and SEP3 sequences
Table S1. List of sequences from non Zingiberales taxa retrieved from NCBI and used in the
phylogenetic analyses
Table S2. Expression in FPKM of the SEPALLATA genes and actin for Costus spicatus and
Musa basjooo
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
Prunus_STK_EF602037
Musa velutina KC815449 (MU)
Heliconia metallica KC815443 (HE)
Heliconia metallica KC815442 (HE)
Costus productus KC815422 (CO)
Musa velutina KC815448 (MU)
Musa acuminata EU869306 (MU)
Phrynium oliganthum KC815459 (MA)
Donax grandis KC815429 (MA)
Schumannianthus virgatus KC815461 (MA)
Globba laeta KC815434 (ZI)
Marantochloa leucantha KC815446 (MA)
Canna jaegeriana KC815419 9CA)
Orchidantha sp. KC815454 (LO)
Heliconia lennartiana KC815441 (HE)
Orchidantha siamensis KC815453 (LO)
Costus spicatus KC815421 (CO)
Musa acuminata KC815447 (MU)
Musa acuminata EU869309 (MU)
Musa acuminata AY941800 (MU)
Schumannianthus virgatus KC815462 (MA)
Schumannianthus virgatus KC815460 (MA)
Phenakospermum guyannense KC815455 (ST)
Halopegia azurea KC815438 (MA)
Orchidantha siamensis KC815452 (LO)
Heliconia pendula KC815444 (HE)
Costus productus KC815420 (CO)
Heliconia lennartiana KC815440 (HE)
Donax grandis KC815428 (MA)
Donax grandis KC815427 (MA)
Phrynium oliganthum KC815458 (MA)
Phrynium oliganthum KC815457 (MA)
Marantochloa leucantha KC815445 (MA)
Zingiber officinale KC815467 (ZI)
Zingiber officinale KC815466 (ZI)
Phenakospermum guyannense KC815456 (ST)
Globba laeta KC815433 (ZI)
Curcuma sp. KC815426 (ZI)
Orchidantha siamensis KC815451 (LO)
Orchidantha siamensis KC815450 (LO)
Elettariopsis smithiae KC815431 (ZI)
Globba laeta KC815435 (ZI)
Halopegia azurea KC815439 (MA)
Tapeinochilos solomonensis KC815465 (CO)
Strelitzia sp. KC815464 (ST)
Strelitzia sp. KC815463 (ST)
Globba laeta KC815437 (ZI)
Costus spicatus KC815423 (CO)
Curcuma sp. KC815425 (ZI)
Ettlingera corneri KC815432 (ZI)
Elettariopsis smithiae KC815430 (ZI)
Globba laeta KC815436 (ZI)
Curcuma sp. KC815424 (ZI)
Elaeis guineensis AF411845
Elaeis guineensis AF411844
Elaeis guineensis AF411843
Dendrobium grex AF198174
Dendrobium crumenatum DQ119842
Aranda deborah X69107
Costus productus KC815392 (CO)
Musa acuminata EU869307 (MU)
Tapeinochilus solomonensis KC815418 (CO)
Tapeinochilus solomonensis KC815417 (CO)
Heliconia metallica KC815396 (HE)
Musa velutina KC815412 (MU)
Phenakospermum guyannense KC815415 (ST)
Heliconia metallica KC815397 (HE)
Alpinia pinetorum KC815386 (ZI)
Musa velutina KC815411 (MU)
Alpinia pinetorum KC815385 (ZI)
Strelitzia sp. KC815416 (ST)
Musa velutina KC815410 (MU)
Canna jaegeriana KC815389 (CA)
Heliconia pendula KC815400 (HE)
Alpinia pinetorum KC815387 (ZI)
Alpinia pinetorum KC815388 (ZI)
Orchidantha siamensis KC815414 (LO)
Musa acuminata KC815409 (MU)
Heliconia pendula KC815401 (HE)
Musa acuminata KC815408 (MU)
Marantochloa leucantha KC815406 (MA)
Marantochloa leucantha KC815405 (MA)
Zingiber officinale DY344923 (ZI)
Heliconia pendula KC815399 (HE)
Heliconia metallica KC815398 (HE)
Monocostus uniflorus KC815407 (CO)
Heliconia pendula KC815404 (HE)
Elettariopsis smithiae KC815394 (ZI)
Alpinia hainanensis FJ861327 (ZI)
Ettlingera corneri KC815395 (ZI)
Orchidantha siamensis KC815413 (LO)
Costus spicatus KC815393 (CO)
Costus spicatus KC815390 (CO)
Heliconia pendula KC815403 (HE)
Heliconia pendula KC815402 (HE)
Costus spicatus KC815391 (CO)
Triticum aestivum DQ512348
Lolium perenne AY198330
Oryza sativa U78891
Zea mays NM001111683
Zea mays NM001112055
Triticum aestivum AM502875
Triticum aestivum AF543316
Hordeum vulgare EU557048
Lolium perenne AY198333
Dendrocalamus latiflorus AY599758
Oryza sativa U78892
Pharus virescens AY827469
Crocus sativus EU424139
Crocus sativus EU424140
Crocus sativus EU424138
Crocus sativus EU424137
Asparagus officinalis AY383560
Asparagus virgatus DQ344499
Allium cepa CF450049
Asparagus virgatus DQ344500
Asparagus officinalis DQ344503
Zostera japonica AB474185
Liriodendron tulipifera AY850182
Persea americana AY850186
Persea americana AY850185
Magnolia grandiflora AY821782
Eschscholzia californica AY850180
Aquilegia coerulea JX680247
Arabidopsis thaliana NM102272
Gossypium hirsutum JF271886
Cannaceae (CA)
Costaceae (CO)
Heliconiaceae (HE)
Lowiaceae (LO)
Marantaceae (MA)
Musaceae (MU)
Strelitziaceae (ST)
Zingiberaceae (ZI)
ZinSEP3-2
ZINGIBERALES
ARECACEAE
ORCHIDACEAE
SEP3
ZINGIBERALES
ZinSEP3-1
OsMADS7
POACEAE
OsMADS8
ASPARAGALES
MAGNOLIIDS
RANUNCULALES
Prunus persica EF440351
Petunia x hybrida AY306171
Petunia x hybrida M91666
Lycopersicon esculentum NM001247455
Chrysanthemum x morifolium AY173057
Eustoma grandiflorum EF569230
Antirrhinum majus X95468
Antirrhinum majus AY306141
Antirrhinum majus X95469
Chrysanthemum x morifolium AY173058
Silene latifolia AB162020
Houttuynia cordata AB089157
Amborella trichopoda AY850178
Houttuynia cordata AB089159
Houttuynia cordata AB089158
Strelitzia sp. KC815380 (ST)
Musa basjoo KC815373 (MU)
Heliconia pendula KC815370 (HE)
Phenakospermum guyannense KC815377 (ST)
Musa acuminata KC815371 (MU)
Tapeinochilos solomonensis KC815382 (CO)
Curcuma sp. KC815336 (ZI)
Ettlingera corneri KC815365 (ZI)
Elettariopsis smithiae KC815364 (ZI)
Donax grandis KC815363 (MA)
Halopegia azurea KC815358 (MA)
Canna jaegeriana KC815357 (CA)
Costus spicatus KC815360 (CO)
Zingiber officinale KC977554 (ZI)
Costus spicatus KC815361 (CO)
Tapeinochilos solomonensis KC815383 (CO)
Costus productus KC815359 (CO)
Globba laeta KC815366 (ZI)
Schumannianthus virgatus KC815379 (MA)
Musa basjoo KC815374 (MU)
Phrynium oliganthum KC815378 (MA)
Aframomum angustifolium KC815355 (ZI)
Heliconia lennartiana KC815367 (HE)
Canna jaegeriana KC815356 (CA)
Heliconia metallica KC815368 (HE)
Heliconia pendula KC815369 (HE)
Strelitzia sp. KC815381 (ST)
Phenakospermum guyannense KC815376 (ST)
Musa acuminata KC815372 (MU)
Orchidantha siamensis KC815375 (LO)
Joinvillea ascendens JN661610
Pharus latifolius JN661616
Eleusine coracana JN661609
Aristida purpurea JN661605
Danthonia spicata JN661607
Zea mays NM001111679
Zea mays NM001111680
Setaria italica JN661617
Cenchrus americanus JN661615
Panicum miliaceum JN661614
Lithachne humilis JN661612
CORE EUDICOTS
MAGNOLIIDS
AMBORELLALES
MAGNOLIIDS
ZinLOFSEP-2
ZINGIBERALES
ZinLOFSEP-1
PAP2/OsMADS34
Triticum monococcum JN661618
Triticum aestivum DQ512344
Lolium perenne AY198332
Avena sativa JN661613
Ehrharta erecta JN661608
Leersia virginica JN661611
Oryza sativa AK100227
Streptochaeta angustifolia AY827470
Oryza meridionalis JN661627
Oryza sativa AK070981
Oryza glaberrima JN661628
Oryza barthii JN661629
Leersia virginica AY597517
Ehrharta erecta AY597515
Sorghum bicolor AY597522
Miscanthus sinensis JN661626
Zea mays AJ005338
Zea mays Y09303
Setaria italica AY597521
Pennisetum glaucum AY597520
Panicum miliaceum AY597519
Panicum maximum DQ315475
Eleusine indica DQ315476
Eleusine coracana AY597516
Danthonia spicata AY597514
Aristida longiseta AY597511
Lithachne humilis AY597518
Dendrocalamus latiflorus AY599750
Hordeum vulgare AJ249145
Triticum aestivum DQ512356
Triticum aestivum DQ512341
Triticum aestivum AJ577373
Lolium perenne AY198334
Avena sativa AY597512
Pharus latifolius JN661630
Dendrocalamus latiflorus AY599753
Ehrharta erecta JN661599
Oryza sativa OSU78890
Leersia virginica JN661602
POALES
LHS1
LOFSEP
Triticum aestivum DQ512362
Triticum aestivum DQ512370
Triticum aestivum DQ512347
Hordeum vulgare JN661604
Lolium perenne AY198331
Avena sativa JN661598
Eleusine coracana JN661600
Setaria italica JN661603
Pennisetum glaucum AY827473
Cenchrus americanus JN661597
Zea mays Y09301
Sorghum bicolor AY827471
Joinvillea ascendens JN661624
Elaeis guineensis AF411847
Elaeis guineensis AF411846
Allium cepa CF436727
Dendrobium grex AF198176
Lilium longiflorum AY826062
Asparagus virgatus DQ344501
Asparagus officinalis DQ344504
Amborella trichopoda AY850179
Malus domestica U78950
Petunia x hybrida AF335234
Lycopersicon esculentum AF448522
Gossypium hirsutum JF271885
Arabidopsis thaliana NM179599
Eschscholzia californica AY850181
Silene latifolia AB162019
Aquilegia coerulea JX680245
Aquilegia coerulea JX680244
Vitis vinifera XM002281446
Rosa rugosa AB099876
Prunus persica EF440352
Malus domestica AJ001682
Malus domestica U78947
Gossypium hirsutum JF271884
Antirrhinum majus X95467
Solanum lycopersicum NM001246982
Lycopersicon esculentum AJ302015
Petunia x hybrida AY370527
Petunia x hybrida AF335235
Gerbera hybrida AJ784156
Arabidopsis thaliana NM111098
Arabidopsis thaliana AT5G15800
Prunus persica DQ102369
Malus domestica U78949
Malus domestica AJ000760
Petunia x hybrida AF335236
Petunia x hybrida AF335241
Lycopersicon esculentum AY294329
Magnolia grandiflora AY821781
Acorus americanus AY850184
OsMADS5
ARECALES
ASPARAGALES
ORCHIDACEAE
LILIALES
ASPARAGALES
SEP4 (AGL3)
TM29 (AGL2/4)
FBP9
MAGNOLIIDS
ACORALES
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
Oncidium hybrid HM140845
Cymbidium goeringii HM208533
Cymbidium goeringii GQ265900
Cymbidium faberi HM208534
Lilium tigrinum GQ496626
Asparagus officinalis AY383559
Agapanthus africanus GQ496627
Hyacinthus orientalis AY591333
Crocus sativus EF041506
Crocus sativus EF041505
Streptochaeta angustifolia GQ496633
Oryza glaberrima GQ496629
Oryza barthii GQ496630
Oryza sativa FJ668596
Oryza meridionalis GQ496628
Pharus sp. GQ496632
Tripsacum dactyloides GQ496653
Tripsacum dactyloides GQ496652
Zea mays L46398
Sorghum bicolor GQ496651
Coix_sp_AGL6_GQ496654
Setaria italica GQ496658
Pennisetum glaucum GQ496657
Panicum miliaceum GQ496656
Eragrostis tef GQ496649
Eragrostis pilosa GQ496650
Eleusine indica GQ496648
Chasmanthium latifolium GQ496647
Dendrocalamus latiflorus AY599755
Dendrocalamus latiflorus AY599754
Triticum aestivum AB007505
Hordeum vulgare GQ496642
Lolium perenne AY198329
Lolium temulentum GQ496646
Brachypodium distachyon GQ496641
Phalaris canariensis GQ496640
Avena strigosa GQ496644
Poa annua AF372840
Lithachne humilis GQ496639
Leersia sp. GQ496638
Oryza meridionalis GQ496636
Oryza glaberrima GQ496635
Oryza barthii GQ496637
Oryza sativa U78782
Joinvillea ascendens GQ496631
Zingiber officinale KC815354 (ZI)
Costus spicatus KC815335 (CO)
Costus productus KC815334 (CO)
Aframomum angustifolium KC815330 (ZI)
Tapeinochilus solomonensis KC815353 (CO)
Costus productus KC815334 (CO)
Globba laeta KC815343 (ZI)
Ettlingera corneri KC815341 (ZI)
Ettlingera corneri KC815340 (ZI)
Curcuma sp. KC815337 (ZI)
Curcuma sp. KC815336 (ZI)
Globba laeta KC8153421 (ZI)
Elatteriopsis smithiae KC815339 (ZI)
Alpinia pinetorum KC815331 (ZI)
Elatteriopsis smithiae KC815338 (ZI)
Canna jaegeriana KC815332 (CA)
Strelitzia sp. KC815352 (ST)
Strelitzia sp. KC815351 (ST)
Orchidantha siamensis KC815347 (LO)
Phenakospermum guyannene KC815348 (ST)
Heliconia pendula KC815440 (HE)
Heliconia metallica KC815345 (HE)
Musa acuminata EU869308 (MU)
Schumannianthus virgatus KC815350 (MA)
Schumannianthus virgatus KC815349 (MA)
Halopegia azurea KC815344 (MA)
Elaeis guineensis AY739701
Tradescantia virginiana GQ496625
Nymphaea odorata GU048659
Nymphaea hybrid AB495345
Nuphar advena GU048649
Oncidium hybrid HM140843
Amborella trichopoda AY936234
Chimonanthus praecox FJ807387
Saurauia zahlbruckneri HM121973
Actinidia chinensis HM121974
Coffea arabica GU265824
Saurauia zahlbruckneri HM121979
Actinidia chinensis HM121980
Gustavia brasiliensis HM121977
Diospyros digyna HM121976
Philadelphus pubescens HM121982
Alangium platanifolium HM121981
Arabidopsis thaliana NM115976
Arabidopsis thaliana NM130127
Aquilegia coerulea JX680248
Epimedium sagittatum JN590217
Nelumbo nucifera GU048640
Persea americana DQ660396
Persea americana DQ660395
Magnolia grandiflora AY936233
Magnolia praecocissima AB050645
Magnolia praecocissima AB050645
Pinus radiata U42400
Gnetum gnemon AJ132215
Arabidopsis thaliana NM125484
Elaeis guineensis AF411842
Elaeis guineensis AF411840
Alpinia oblongifolia EF521814 (ZI)
Ettlingera corneri KC977555 (ZI)
Costus productus KC977556 (CO)
Costus productus KC977557 (CO)
Heliconia metallica KC977558 (HE)
Alpinia oblongifolia EF521815 (ZI)
Alpinia oblongifolia EF521816 (ZI)
ZinAGL6
ZINGIBERALES
0.2
CORE EUDICOTS
ORCHIDACEAE
LILIALES
ASPARAGALES
POALES
AGL6
ZINGIBERALES
ARECALES
COMMELINALES
NYMPHAEALES
MAGNOLIIDS
CORE EUDICOTS
RANUNCULALES
PROTEALES
MAGNOLIIDS
AP1/SQUA
++/++
Amborella trichopoda AY936234
AMBORELLALES
Nuphar
advena
GU048649
++/++
NYMPHAEALES
Nymphaea hybrid AB495345
++/++
Nymphaea odorata GU048659
Oncidium hybrid HM140843
Oncidium hybrid HM140845
++/++
Cymbidium goeringii HM208533
ORCHIDACEAE
Cymbidium faberi HM208534
++/99
0.7/-- Cymbidium goeringii GQ265900
0.84/-Lilium tigrinum GQ496626
LILIALES
++/95
Hyacinthus orientalis AY591333
Agapanthus africanus GQ496627
++/85 0.57/-0.56
Asparagus officinalis AY383559
ASPARAGALES
Crocus sativus EF041505
++/++
Crocus sativus EF041506
0.96
Elaeis guineensis AY739701
ARECACEAE
++/++ Heliconia metallica KC815345 (HE)
Heliconia pendula KC815440 (HE)
0.94
Phenakospermum guyannense KC815348 (ST)
Strelitzia sp. KC815352 (ST)
++
++/83
Strelitzia sp. KC815351 (ST)
++
Orchidantha siamensis KC815347 (LO)
++
Musa acuminata EU869308 (MU)
0.98
0.65
Halopegia azurea KC815344 (MA)
0.97/-++/++
Schumannianthus virgatus KC815349 (MA)
++/++
Schumannianthus virgatus KC815350 (MA)
++/99 ω
=0.099
Globba laeta KC815343 (ZI)
++/94
Alpinia pinetorum KC815331 (ZI)
Elettariopsis smithiae KC815338 (ZI)
0.85/-++/99
Elettariopsis smithiae KC815339 (ZI)
Curcuma sp. KC815336 (ZI)
++/++
Curcuma
sp. KC815337 (ZI)
0.78/-0.95/++
Globba laeta KC815342 (ZI)
0.99/70
Ettlingera corneri KC815340 (ZI)
0.52/73
0.87/51
++/++ Ettlingera corneri KC815341 (ZI)
Canna jaegeriana KC815332 (CA)
Costus productus KC815333 (CO)
0.55/-Tapeinochilus solomonensis KC815353 (CO)
++/97
Zingiber officinale KC815354 (ZI)
++/97
Aframomum angustifolium KC815330 (ZI)
++/84
Costus productus KC815334 (CO)
0.61/-++/87
Costus spicatus KC815335 (CO)
COMMELINALES
Tradescantia virginiana GQ496625
++/++ Oryza barthii GQ496630
Oryza glaberrima GQ496629
++/++
0.75
Oryza meridionalis GQ496628
0.56
Oryza sativa FJ668596
++/99
Joinvillea ascendens GQ496631
ω=0.105
Streptochaeta angustifolia GQ496633
Pharus sp. GQ496632
0.96/71
Chasmanthium latifolium GQ496647
Panicum miliaceum GQ496656
++/89
Tripsacum dactyloides GQ496652
++/96
++/97
Zea mays L46398
++/82 ++/68
Sorghum bicolor GQ496651
Coix sp. GQ496654
0.98/67
0.89/68
0.64/-- Tripsacum dactyloides GQ496653
Pennisetum glaucum GQ496657
++/82
++/98
Setaria italica GQ496658
Eleusine indica GQ496648
0.97/-Eragrostis pilosa GQ496650
++/98 ++/69
++/93
Eragrostis tef GQ496649
Leersia sp. GQ496638
++/98
Oryza sativa U78782
Oryza barthii GQ496637
++/99
0.97/88 Oryza glaberrima GQ496635
0.89/82 Oryza meridionalis GQ496636
Brachypodium distachyon GQ496641
0.99/79 ++/97 ++/99 Hordeum vulgare GQ496642
Triticum aestivum AB007505
Poa
annua AF372840
0.98/97
++/99
Lolium perenne AY198329
0.58
Lolium temulentum GQ496646
0.93/59
0.85/-Avena strigosa GQ496644
0.72/-Phalaris canariensis GQ496640
Lithachne humilis GQ496639
Dendrocalamus latiflorus AY599754
0.51/51
++/++ Dendrocalamus latiflorus AY599755
Persea americana DQ660395
0.7/55
Magnolia praecocissima AB050645
++/++
Magnolia grandiflora AY936233
0.66/-Magnolia praecocissima AB050645
Chimonanthus praecox FJ807387
0.81/-++/90
Persea americana DQ660396
Nelumbo nucifera GU048640
PROTEALES
Epimedium sagittatum JN590217
RANUNCULALES
++/83
Aquilegia coerulea JX680248
Arabidopsis thaliana NM130127
++/++
Arabidopsis thaliana NM115976
++/56 ω=0.147
Coffea arabica GU265824
++/73
Alangium
platanifolium
HM121981
++/++
Philadelphus pubescens HM121982
0.54/74
Diospyros digyna HM121976
Gustavia brasiliensis HM121977
0.97/88
Actinidia
chinensis HM121980
0.94/56
Saurauia zahlbruckneri HM121979
++/99
Actinidia chinensis HM121974
++/++
Saurauia zahlbruckneri HM121973
Gnetum gnemon AJ132215
Pinus radiata U42400
Cannaceae (CA)
Costaceae (CO)
Heliconiaceae (HE)
Lowiaceae (LO)
Marantaceae (MA)
Musaceae (MU)
Strelitziaceae (ST)
Zingiberaceae (ZI)
ZINGIBERALES
MONOCOTS
POALES
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
MAGNOLIIDS
CORE EUDICOTS
++/++
0.2
GYMNOSPERMS
Vitis vinifera XM002281446
Gossypium hirsutum JF271884
Silene latifolia AB162019
0.84/-Arabidopsis thaliana AT5G15800
++/99
0.9/-Arabidopsis thaliana NM111098
ω=0.117
Antirrhinum majus X95467
0.99/55
++/80
++/84
Petunia x hybrida AF335235
++/94
Petunia x hybrida AY370527
++/92
++/99 Lycopersicon esculentum AJ302015
Solanum lycopersicum NM001246982
Rosa rugosa AB099876
0.99/52
Prunus persica EF440352
0.99/88
Malus domestica U78947
0.97/68
++/92
Malus domestica AJ001682
Prunus persica DQ102369
++/97
Malus domestica U78949
0.99
++/81
Malus domestica AJ000760
++/76
Petunia x hybrida AF335236
ω=0.226 ++/96
Lycopersicon esculentum AY294329
++/98
Petunia x hybrida AF335241
Arabidopsis thaliana NM179599
0.99/61
Lycopersicon esculentum AF448522
++/98
++/58
Petunia x hybrida AF335234
Gossypium hirsutum JF271885
0.7/-Malus domestica U78950
0.98/-Eschscholzia californica AY850181
ω=0.188
Aquilegia coerulea JX680245
0.86/-0.92/53
Aquilegia coerulea JX680244
Magnolia grandiflora AY821781
0.88/-Houttuynia cordata AB089158
++/++
Houttuynia cordata AB089159
Dendrobium grex AF198176
0.81/31
Lilium longiflorum AY826062
Asparagus officinalis DQ344504
++/++
Asparagus virgatus DQ344501
Orchidantha siamensis KC815375 (LO)
Costus productus KC815359 (CO)
Costus spicatus KC815361 (CO)
Globba laeta KC815366 (ZI)
ω=0.336**
Heliconia lennartiana KC815367 (HE)
++/99
0.71/-Musa basjoo KC815374 (MU)
++/++
Phrynium oliganthum KC815378 (MA)
Schumannianthus virgatus KC815379 (MA)
Tapeinochilos solomonensis KC815383 (CO)
Aframomum angustifolium KC815355 (ZI)
++/-- Canna jaegeriana KC815356 (CA)
0.94/-Zingiber officinale KC977554 (ZI)
Heliconia metallica KC815368 (HE)
0.99/61
0.66/-Heliconia pendula KC815369 (HE)
0.5/-Musa acuminata KC815372 (MU)
Phenakospermum guyannense KC815376 (ST)
0.91/50
++/92
Strelitzia sp. KC815381 (ST)
Musa acuminata KC815371 (MU)
Phenakospermum guyannense KC815377 (ST)
++/++
Heliconia pendula KC815370 (HE)
++/96
Musa basjoo KC815373 (MU)
++/97
Strelitzia sp. KC815380 (ST)
ω=0.458**
Canna jaegeriana KC815357 (CA)
++/89
0.83/56
Halopegia azurea KC815358 (MA)
++/95
Donax grandis KC815363 (MA)
0.94/71
Costus spicatus KC815360 (CO)
Elettariopsis smithiae KC815364 (ZI)
++/92
0.87/55
Ettlingera corneri KC815365 (ZI)
++/++
Curcuma sp. KC815336 (ZI)
++/94 Tapeinochilos solomonensis KC815382 (CO)
Elaeis guineensis AF411846
0.68/-0.91/-Elaeis guineensis AF411847
Avena sativa JN661598
0.87/62
Lolium perenne AY198331
++/92
Hordeum vulgare JN661604
Triticum aestivum DQ512362
0.5/-- ++/98
Triticum aestivum DQ512347
0.86/-++/99
Triticum aestivum DQ512370
Sorghum bicolor AY827471
++/92
Zea mays Y09301
Eleusine coracana JN661600
++/69
0.99/79
Setaria italica JN661603
0.72/54
ω=0.186
Cenchrus americanus JN661597
++/++
++/68 Pennisetum glaucum AY827473
Leersia virginica JN661602
++/83
Oryza sativa OSU78890
++/98
0.84/50
Dendrocalamus latiflorus AY599753
0.69/-Ehrharta erecta JN661599
0.68/-Joinvillea ascendens JN661624
Streptochaeta angustifolia AY827470
Pharus latifolius JN661630
ω=0.1480.7/-Triticum aestivum AJ577373
0.99/-0.99/69
Hordeum vulgare AJ249145
0.98/63
Triticum aestivum DQ512341
0.97/79
++/93
++/98
Triticum aestivum DQ512356
Avena sativa AY597512
0.66/52
0.96/60
Lolium perenne AY198334
Ehrharta erecta AY597515
++/74
Leersia virginica AY597517
Oryza meridionalis JN661627
++/88
Oryza barthii JN661629
++/99
Oryza glaberrima JN661628
0.99/86
++/71
Oryza sativa AK070981
0.57/-Aristida longiseta AY597511
OsMADS1
Zea mays AJ005338
++/99
++/89
Zea mays Y09303
Miscanthus sinensis JN661626
0.96/60
0.75/71 Sorghum bicolor AY597522
++/93
0.99/90 Panicum maximum DQ315475
Panicum miliaceum AY597519
++/94
Pennisetum glaucum AY597520
0.88/58
0.79/68
Setaria italica AY597521
0.62/-Danthonia spicata AY597514
Eleusine coracana AY597516
++/81
++/99
Eleusine indica DQ315476
Dendrocalamus latiflorus AY599750
0.81/-Lithachne humilis AY597518
Pharus latifolius JN661616
Danthonia spicata JN661607
0.72/-Zea mays NM001111680
++/99
Zea mays NM001111679
++/++
++/76
Panicum miliaceum JN661614
0.87/-Cenchrus americanus JN661615
++/83
ω=0.263
++/66
Setaria italica JN661617
Aristida purpurea JN661605
0.57/-Eleusine coracana JN661609
0.51/-Joinvillea ascendens JN661610
OsMADS34
Triticum aestivum DQ512344
++/98
0.99/--
TM29 (AGL2/4)
EUDICOTS
FBP9
SEP4 (AGL3)
RANUNCULALES
MAGNOLIIDS
ORCHIDS
LILIALES
ASPARAGALES
ZinLOFSEP1
Zingiberales
ZinLOFSEP2
ARECACEAE
MONOCOTS
OsMADS5
LHS1
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
0.67/--
++/99
0.65/--
++/72
0.52/-0.99/79
++/82
Allium cepa CF436727
0.77/--
Acorus americanus AY850184
Amborella trichopoda AY850179
0.2
POACEAE
PAP2
Cannaceae (CA)
Triticum monococcum JN661618
Costaceae (CO)
Avena sativa JN661613
Lolium perenne AY198332
Lithachne humilis JN661612
Ehrharta erecta JN661608
Leersia virginica JN661611
Oryza sativa AK100227
Heliconiaceae (HE)
Lowiaceae (LO)
Marantaceae (MA)
ASPARAGALES
ACORALES
AMBORELLALES
Musaceae (MU)
Strelitziaceae (ST)
Zingiberaceae (ZI)
RANUNCULALES
Eschscholzia californica AY850180
0.98/--
0.98/61
++/99
ω=0.049
0.75
0.96/64
0.88/71
0.63/-0.79/-0.77
0.84
0.86/--
0.97/85
Silene latifolia AB162020
Eustoma grandiflorum EF569230
Antirrhinum majus X95469
Antirrhinum majus AY306141
Antirrhinum majus X95468
Chrysanthemum x morifolium AY173057
Lycopersicon esculentum NM001247455
Petunia x hybrida M91666
++/++
Petunia x hybrida AY306171
Gossypium hirsutum JF271886
Arabidopsis thaliana NM102272
Chrysanthemum x morifolium AY173058
CORE EUDICOTS
Prunus persica EF440351
++/93
0.95/--
Magnolia grandiflora AY821782
Liriodendron tulipifera AY850182
Persea americana AY850185
Persea americana AY850186
MAGNOLIIDS
Houttuynia cordata AB089157
Zostera japonica AB474185
Musa acuminata KC815447 (MU)
Musa velutina KC815449 (MU)
0.73/-Marantochloa leucantha KC815446 (MA)
++/92
Globba laeta KC815434 (ZI)
++/98
Schumannianthus virgatus KC815461 (MA)
++/80
Donax grandis KC815429 (MA)
0.56/-++/87
Phrynium oliganthum KC815459 (MA)
ω=0.0503*
Canna jaegeriana KC815419 (CA)
++/96 Heliconia lennartiana KC815441 (HE)
Orchidantha sp. KC815454 (LO)
++/98
Costus spicatus KC815421 (CO)
Orchidantha siamensis KC815453 (LO)
Musa acuminata AY941800 (MU)
++/++
Musa acuminata EU869309 (MU)
0.98
Curcuma sp. KC815424 (ZI)
++/96
Globba laeta KC815436 (ZI)
0.52
Globba
laeta KC815433 (ZI)
++/84
Curcuma sp. KC815426 (ZI)
++/96
Zingiber officinale KC815467 (ZI)
0.57/-++/99 0.79/52
Phenakospermum guyannense KC815456 (ST)
++/78 Zingiber officinale KC815466 (ZI)
Elettariopsis smithiae KC815431 (ZI)
Orchidantha siamensis KC815450 (LO)
++/81
++/99 Orchidantha siamensis KC815451 (LO)
++/87
0.99/84
Elettariopsis smithiae KC815430 (ZI)
Ettlingera
corneri KC815432 (ZI)
++/99
Curcuma sp. KC815425 (ZI)
Globba
laeta KC815435 (ZI)
0.99/65
0.8/70
Halopegia
azurea KC815439 (MA)
0.99/76
Tapeinochilos solomonensis KC815465 (CO)
++/60
0.53/-Costus spicatus KC815423 (CO)
0.95/76
0.99/50
Globba laeta KC815437 (ZI)
0.93/69
Strelitzia sp. KC815463 (ST)
0.97/65 Strelitzia sp. KC815464 (ST)
Marantochloa leucantha KC815445 (MA)
Schumannianthus virgatus KC815460 (MA)
++/95
Schumannianthus virgatus KC815462 (MA)
++/96 ++/94
Halopegia azurea KC815438 (MA)
Phenakospermum guyannense KC815455 (ST)
0.67/-0.97/78
Costus productus KC815420 (CO)
Heliconia lennartiana KC815440 (HE)
0.77/63 0.99/59 Heliconia pendula KC815444 (HE)
Orchidantha siamensis KC815452 (LO)
Phrynium oliganthum KC815457 (MA)
Phrynium oliganthum KC815458 (MA)
++/98
Donax grandis KC815427 (MA)
0.53/-0.98/62
Donax grandis KC815428 (MA)
Musa acuminata EU869306 (MU)
++/99
Musa velutina KC815448 (MU)
Costus productus KC815422 (CO)
0.81/-++/99
Heliconia metallica KC815442 (HE)
Heliconia metallica KC815443 (HE)
Elaeis
guineensis AF411845
++/97
Elaeis guineensis AF411843
++/99
Elaeis guineensis AF411844
0.81/-Dendrobium grex AF198174
++/78
Aranda deborah X69107
++/99
Dendrobium crumenatum DQ119842
Costus productus KC815392 (CO)
Musa acuminata EU869307 (MU)
++/96
Alpinia pinetorum KC815385 (ZI)
Alpinia pinetorum KC815386 (ZI)
0.97/61 Musa velutina KC815411 (MU)
0.99/56
Heliconia metallica KC815396 (HE)
Tapeinochilus solomonensis KC815417 (CO)
0.98/80
Musa velutina KC815412 (MU)
Tapeinochilus solomonensis KC815418 (CO)
0.8/-Heliconia metallica KC815397 (HE)
0.95/65 Phenakospermum guyannense KC815415 (ST)
Alpinia pinetorum KC815388 (ZI)
++/99
Musa acuminata KC815408 (MU)
Alpinia pinetorum KC815387 (ZI)
ω=0.165**
++/90 Heliconia pendula KC815400 (HE)
0.67/53 Canna jaegeriana KC815389 (CA)
0.97/74 Musa velutina KC815410 (MU)
0.96/50 Heliconia pendula KC815401 (HE)
0.94/53 Musa acuminata KC815409 (MU)
Orchidantha siamensis KC815414 (LO)
0.64/53 Strelitzia sp. KC815416 (ST)
Orchidantha siamensis KC815413 (LO)
++/++
Alpinia hainanensis FJ861327 (ZI)
0.6/-Elettariopsis smithiae KC815394 (ZI)
Zingiber officinale DY344923 (ZI)
++/98
0.89/72
Ettlingera corneri KC815395 (ZI)
Heliconia
pendula KC815404 (HE)
0.73/-Heliconia pendula KC815399 (HE)
Heliconia metallica KC815398 (HE)
0.8/53
0.57/-- Monocostus uniflorus KC815407 (CO)
0.88/66
Marantochloa leucantha KC815405 (MA)
++/++
Marantochloa leucantha KC815406 (MA)
++/++ Costus spicatus KC815391 (CO)
Heliconia pendula KC815402 (HE)
++/93 0.74/70
Heliconia pendula KC815403 (HE)
Costus spicatus KC815390 (CO)
++/91
Costus spicatus KC815393 (CO)
Oryza sativa U78892
++/++
Pharus virescens AY827469
Dendrocalamus
latiflorus AY599758
0.95/61
ω=0.067 0.82/60
Zea mays NM001112055
Cannaceae (CA)
Zea mays NM001111683
0.99/62
++
Oryza sativa U78891
Costaceae (CO)
Lolium
perenne AY198330
0.98/54
++/99
++/98
Triticum aestivum DQ512348
Heliconiaceae (HE)
Lolium perenne AY198333
Hordeum vulgare EU557048
0.93/62 ++/99
Lowiaceae (LO)
Triticum aestivum AF543316
++/70
Triticum aestivum AM502875
Crocus sativus EU424139
++/99
Marantaceae (MA)
++/83
Crocus sativus EU424140
Crocus sativus EU424137
++/99
Musaceae (MU)
Crocus sativus EU424138
++/71
++/99
Asparagus officinalis DQ344503
Strelitziaceae (ST)
Asparagus virgatus DQ344500
Allium cepa CF450049
++/87
Zingiberaceae (ZI)
Asparagus
officinalis
AY383560
0.72
0.91
Asparagus virgatus DQ344499
Amborella trichopoda AY850178
Aquilegia coerulea JX680247
PIPERALES
ALISMATALES
ZINGIBERALES
ZinSEP3-2
ARECACEAE
ORCHIDACEAE
ZINGIBERALES
ZinSEP3-1
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
0.75
0.2
POACEAE
ASPARAGALES
AMBORELLALES
RANUNCULALES
MONOCOTS
Sepals
Petals
Staminodes
Petaloid
stamen
Carpels
Cannaceae
1 fertile petaloid
stamen
Canna indica
Marantaceae
5 petaloid
staminodes
Sepals
Petals
Labellum
Petaloid
stamen
Carpels
Costaceae
Costus spicatus
1 petaloid
staminode
Sepals
Petals
Labellum
Petaloid
stamen
Carpels
Zingiberaceae
1/2 fertile
petaloid stamen
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
Dimorphic
perianth
Zingiber officinale
Heliconiaceae
Sepals
Petals
Carpels
Stamens
Strelitziaceae
Strelitzia sp.
Lowiaceae
Floral tube
Carpels
Stamens
Musaceae
Musa acuminata
SEP3-1
SEP3-2
LOFSEP-2
AGL-6
alifornia, Berkeley on August 22, 2013
Mean FPKM normalized to actin
25
20
15
AGL6
LOFSEP-1
LOFSEP-2
SEP3-1
SEP3-2
10
5
0
Petal
Labellum
Filament
Costus spicatus
Theca
Free Petal
Filament
Musa basjoo
Theca
2
ZinSEP3-2
93.5
57
MADS
75
I
K

Arecaceae
115.1
Orchidaceae
Asparagales
2
ZinSEP3-1
91.7
149.6
187
58.2
Poaceae OsMADS7
2
Poaceae OsMADS8
57
MADS
57
MADS
75
I

I
K

75

SEP3
187
SEP3
Ranunculales
2
Core Eudicots
57
MADS
Eudicots TM29 (AGL2/4)
Eudicots SEP4 (AGL3) 
2
57
MADS

75
I
Eudicots FBP9
130
187
K
Magnoliids
197.9

K


  
75
I
 
187
142
K
Magnoliids
Orchidaceae
175-187
Asparagales
Poales PAP2
118
207-198
82
2
Poales LHS1


 
57
MADS
LOFSEP

75
I
142
K
149.6
91.79
93.50
Downloaded from http://mbe.oxfordjournals.org/ at University of California, Berkeley on August 22, 2013
PoalesOsMADS5 
Arecales
102
2
ZinLOFSEP1
2
ZinLOFSEP2

57
 
57
MADS
MADS
75
I
K

I
ZinAGL6
124.2
79.4
137.6


Ginger
Banana
Oryza OsMADS17
2
Poales OsMADS6
MADS
MADS
60
60
I
I
86
142
142
75
Arecales
2

K

86
K
K
185

185

C
C


272
272
AGL6
Magnoliids
Proteales
Ranunculales
2
Core Eudicots
60
I
86

K
ω selection pressure
0 - 0.1
0.1 - 0.2
0.2 - 0.3
0.3 - 0.4
0.4 - 0.5
Duplication event
Positive selected site
β-strand
α-helix
185

C

272