MBE Advance Access published August 11, Article

Transcription

1 MBE Advance Access published August 11, 2013 RH: SEP gene evolution in Zingiberales Article Discoveries section Molecular evolution and patterns of duplication in the SEP/AGL6-like lineage of the Zingiberales: a proposed mechanism for floral diversification. Roxana Yockteng 1,2, Ana M.R. Almeida 1, Kelsie Morioka 1, Elena R. Alvarez-Buylla 1,3 and Chelsea D. Specht 1 1 Department of Plant and Microbial Biology, Department of Integrative Biology and the University and Jepson Herbaria. University of California, Berkeley. CA Origine, Structure et Evolution de la Diversité (UMR 7205 CNRS, Muséum National d Histoire Naturelle, CP39, 16 rue Buffon, 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 Corresponding address: 111 Koshland Hall MC3102 University of California Berkeley, CA ryockteng@berkeley.edu The Author Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please journals.permissions@oup.com

2 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 show that the SEP-like genes have undergone several duplication events giving rise to multiple 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.

3 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 duplication, the two paralogs are initially redundant in function enabling a period of relaxed 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-

4 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. The SEP subfamily of MADS-box genes arose following an early duplication predating the 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

5 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 eudicots and monocots (Malcomber and Kellogg 2005; Zahn et al. 2005). Two duplications in 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,

6 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 2008; Kress 1990; Ley and Classen-Bockhoff 2011), and there is evidence that shifts in 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

7 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 to the SEP subfamily (Zahn et al. 2005), and for AP1/SQUA, considered to be the sister lineage 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.

8 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 members of the order (Figure 1; SEP3). One clade is recovered as sister to the sampled Poales 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 +

9 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). The grass sequences form a well-supported clade (posterior probability (PP)=1.0; 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

10 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 Kishino- Hasegawa 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. 2012) retrieves one single genomic region of chromosome 2 (emb: CAIC ) supporting 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

11 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 (PAP2) clade, also known as OsMADS34 (after Oryza sativa MADS34); the second duplication 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: CAIC ) and ZinLOFSEP-2 recovers a region on chromosome 8 (emb: CAIC ). 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

12 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 The final SEP3 alignment contains 140 taxa with an aligned length of 567 bp (supplemental 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..

13 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 (Aranda and Dendrobium). The main difference between ZinSEP3-1 and ZinSEP3-2 sequences 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 AY and EU matched a region on chromosome 6 (emb CAIC ) and sequence EU matched a region on chromosome 9 (emb CAIC ). The ZinSEP3-1 sequence from Musa acuminata (EU869307) matched one single region on chromosome 11 (emb CAIC ). 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

14 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 stamens (staminodes) of Costus spicatus (Costaceae), Canna indica (Cannaceae) and Zingiber 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

15 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 floral organ types in Costus spicatus, in Canna indica and in Zingiber officinale, with 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 RT- PCR 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 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

16 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 the ancestral sequence. 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

17 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= 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= 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= ) that seems to have evolved under positive selection. The FEL test in HYPHY indicates that several sites may have been fixed by positive selection and they are located mainly 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)).

18 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 the role is to enhance and stabilize interactions mediated by the K-domain (Fan et al 1997). Only 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

19 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 have coloration patterns and epidermal cell types associated with the attraction of pollinators. In 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

20 SEPALLATA subfamily from the ancestral AGL6 gene lineage. Our analyses estimate that this duplication event occurred around 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 events have been detected across angiosperms (Zahn et al. 2005, Shan et al. 2009). In the core eudicots, an initial duplication event estimated at 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

21 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 gene produce lodicules attached to the palea and lemma (Agrawal et al. 2005; Kobayashi et al. 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

22 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, which are in single clade indicating a Poaceae-specific duplication event estimated to have occurred around 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 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

23 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 copies in Zingiberales could contribute to the robustness of the genetic network by reducing the 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 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

24 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 Agamous-like gene OsMADS3, which is important for stamen identity (including fertility) (Li et 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

25 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 in this study could be under more strict functional constraints. Conserved sites such as the 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 ; 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

26 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. The differences in rate of molecular evolution, in specific sites under selection and 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

27 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 occurred after the Zingiberales diverged from the rest of the Monocots. These genes evolved 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

28 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 MgCl 2 with the following 3-step thermocycling protocol: 98 C for 5min followed by 35 cycles (98 C for 5s, C (depending on primers) for 5s, 72 C 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. KC 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 (Drummond et al. 2011). Final alignments are included as supplemental data.

29 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 (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. Bayesian inference was used to infer phylogenetic hypotheses for the four aligned datasets, as performed in MrBayes (Ronquist and Huelsenbeck 2003) and implemented in the CIPRES Science Gateway ( 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 ( SumTrees v 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 ( 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

30 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 agarose gels stained with GelRed (Biotium). One PCR product from each of the RT-PCR 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, KC and EU respectively) and Costus spicatus (KC815390, KC815421, KC815335, KC815361, KC and KC 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).

31 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 reverse complement sequence of SEP3-1 was used as a negative control. 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 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

32 (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, 3 and 4). If some branches showed significant differences in selection in the first test, we used 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).

33 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 to C.D.S.; DEB 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) award to K.M. ERAB was supported by the Miller Institute for Basic Research in Science, University of California, Berkeley, California, USA.

34 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 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. 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.

35 Table 1. List of species of Zingiberales used in this study Species Location NCBI accession per gene Canna jaegeriana Urb. AGL6-1: KC815332; LOFSEP-1: KC815356; (HLA) LOFSEP-2: KC815357; SEP3-1-1: KC815389; SEP3-2-1: KC Costus productus Gleason ex Maas AGL6-1: KC815333, AGL6-2: KC815334, AP1-1: (UCBG) KC977556, AP1-2: KC977557, LOFSEP-1: KC815359, SEP3-1-1: KC815392, SEP3-2-1: KC815420, SEP3-2-2: KC Costus spicatus (Jacq.) Sw. AGL6-1: KC815335, LOFSEP-1: KC815361, (NMNH) LOFSEP-2: KC815360, SEP3-1-1: KC815390, SEP3-1-2: KC815391, SEP3-1-3: KC815393, SEP3-2-1: KC815421, SEP3-2-2: KC Monocostus uniflorus (Poepp. ex Petersen) Maas (UCBG) SEP3-1-1: KC Tapeinochilos solomonensis Gideon (Lyon) Heliconia lennartiana W.J. Kress (McBryde) Heliconia metallica Planch. & Linden ex Hook. Heliconia pendula Wawra Orchidantha siamensis K. Larsen Donax grandis (Miq.) Ridl. Halopegia azurea K. Schum. Marantochloa leucantha (K. Schum.) Milne- Redh (MacBryde) (McBryde) L (Lyon) L (Lyon) (Lyon) AGL6-1: KC815353, LOFSEP-1: KC815383, LOFSEP-2: KC815382, SEP3-1-1: KC815417, SEP3-1-2: KC815418, SEP3-2-1: KC LOFSEP-1: KC815367, SEP3-2-1: KC815441, SEP3-2-2: KC AGL6-1: KC815345, AP1: KC977558, LOFSEP- 1: KC815368, SEP3-1-1: KC815397, SEP3-1-2: KC815396, SEP3-1-3: KC815398, SEP3-2-1: KC815442, SEP3-2-2: KC 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: KC 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: KC LOFSEP-2: KC815363, SEP3-2-1: KC815428, SEP3-2-2: KC815429, SEP3-2-3: KC AGL6-1: KC815344, LOFSEP-2: KC815358, SEP3-2-1: KC815438, SEP3-2-2: KC SEP3-1-1: KC815405, SEP3-1-2: KC815406, SEP3-2-1: KC815445, SEP3-2-2: KC L (Lyon) Phrynium oliganthum Merr. LOFSEP-1: KC815378, SEP3-2-1: KC815457, L (Lyon) SEP3-2-2: KC815458, SEP3-2-3: KC Schumannianthus virgatus (Roxb.) Rolfe AGL6-1: KC815349, AGL6-2: KC815350, LOFSEP-1: KC815379, SEP3-2-1: KC815460, L (Lyon) SEP3-2-2: KC815461, SEP3-2-3: KC Musa acuminata Colla LOFSEP-1: KC815372, LOFSEP-2: KC815371, SEP3-1-1: KC815409, SEP3-1-2: KC815408, (NMNH) SEP3-2-1: KC MADS1: EU869307, MADS2: EU869306, MADS3: AY941800, MADS3: EU869308, N/A * MADS4: EU Musa basjoo Siebold (UCBG) LOFSEP-1: KC815374, LOFSEP-2: KC815373

36 Musa velutina H. Wendl. & Drude SEP3-1-1: KC815412, SEP3-1-2: KC815410, SEP3-1-3: KC815411, SEP3-2-1: KC815448, L (Lyon) SEP3-2-2: KC Phenakospermum guyannense (Rich.) Endl. SEP3-1-1: KC815412, SEP3-1-2: KC815410, SEP3-1-3: KC815411, SEP3-2-1: KC815448, McBryde SEP3-2-2: KC Strelitzia sp. Aiton AGL6-1: KC815351, AGL6-2: KC815352, LOFSEP-1: KC815381, LOFSEP-2: KC815380, SEP3-1-1: KC815416, SEP3-2-1: KC815463, UC SEP3-2-2: KC Aframomum angustifolium (Sonn.) K. Schum (HLA) AGL6-1: KC815330, LOFSEP-1: KC Alpinia hainanensis K. Schum. N/A* SEP3-like: FJ861327, AP1like-FL1: EF521814, AP1like-FL2: EF521816, AP1like-FL3: EF5218 Alpinia pinetorum (Ridl.) Loes (HLA) AGL6-1: KC815331, SEP3-1-1: KC815385, SEP3-1-2: KC815386, SEP3-1-3: KC815387, SEP3-1-4: KC Curcuma sp. L (UCGH) AGL6-1: KC815336, AGL6-2: KC815337, LOFSEP-2: KC815362, SEP3-2-1: KC815424, SEP3-2-2: KC815425, SEP3-2-3: KC Elettariopsis smithiae Kam L (Lyon) AGL6-1: KC815339, AGL6-2: KC815338, LOFSEP-2: KC815364, SEP3-1-1: KC815394, SEP3-2-1: KC815430, SEP3-2-2: KC Ettlingera corneri Mood & Ibrahim L (Lyon) AGL6-1: KC815340, AGL6-2: KC815341, AP1-1: KC977555, LOFSEP-2: KC815365, SEP3-1-1: KC815395, SEP3-2-1: KC Globba laeta K. Larsen L (Lyon) 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: KC Zingiber officinale Roscoe MB0876 (UC) N/A * AGL6-1: KC815354, LOFSEP-1: KC977554, SEP3-2-1: KC815467, SEP3-2-2: KC SEP3: DY 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).

37 Table 2. Primer sequences used to amplify SEPALLATA genes and AGL6 genes (F in the names indicates forward and R reverse primers) Gene Primer name Taxa Sequence (5'->3') SEP3-like Zin-SEP3a-F Zingiberales GCT GAA GMG GAT HGA GAA CAA Zin-SEP3a-R Zingiberales TGG TTD CTT TCH TCC AAC CTT Zin-SEP3b-F Zingiberales GAA GCG GAT CGA GAA CAA GA Zin-SEP3b-R Zingiberales TTC AGB TGG TCC ATG TAR GC Zin-SEP3c/d-F Zingiberales CST CAT CGT CTT CTC CAR CC Zin-SEP3c-R Zingiberales AGC AYT TGY TCC CKT CTT TG Zin-SEP3d-R Zingiberales CCS TCA TCG TCT TCT CCA RC SEP3 copy specific Canna-SEP3-1-F Canna TGC AGC TAT GGA GGA TCA GA Canna-SEP3-1-R Canna TGG TCC AGC ATT TGT TGT GT Canna-SEP3-2-F Canna TGC AGC AGC TCC AGT ATG AT Canna-SEP3-2-R Canna TGC TAA GTG GTC CCA AAT CC Costus-SEP3-1-F Costus AAG AAG GCA TAC GAG CTC TCC Costus-SEP3-1-R Costus AGC TGA TTC TCC TTG GCT TGT AAG Costus-SEP3-2-F Costus ACT CAG ACC AGT CAG CAG GAG TA Costus-SEP3-2-R Costus TTG ATC AAG CAT GTA TTG TGT CC Strelitzia-SEP3-1-F Strelitzia GGA GAA TCA GTT GGT TCA GAG Strelitzia-SEP3-1-R Strelitzia TGG TCC AGC ATT TGT TGT GT Strelitzia-SEP3-2-F Strelitzia TAT CAA GGG AGA CTC AGA CCA G Strelitzia-SEP3-2-R Strelitzia CAT CAT GTT GTC GTT CAA GTT GT Zingiber-SEP3-1-F Zingiber GTC CAG AGC AGT CGT CAA GAG TA Zingiber-SEP3-1-R Zingiber TCC TTC AGT GAC ATA TCA AGT GG Zingiber-SEP3-2-F Zingiber ATC AAG GGA GAC TCA AAC TAG TCA A Zingiber-SEP3-2-R Zingiber ACA TCA AGT TGT CGC TCC AG Musa-SEP3-1-R Musa CAG TTG GTT CAA AGT AGT CG Musa-SEP3-1-F Musa GCA TTT GTT GTG TCC TTG TG Musa-SEP3-2-F Musa GGA GAC TCA GAG TAG TCA GCA AGA G Musa-SEP3-2-R Musa GCA TTT GTT GTG TCC TTG TG LOFSEPlike Zin-LOFSEP-F Zingiberales AGG GTG GAG CTG AAG AGG AT Zin-LOFSEP-R Zingiberales TTG KGT CTT TGT TGA TCT GAT LOFSEP copy specific Canna-LOFSEP-1F Canna GAA CCA CCT ATG GTT TCC TCA TA Canna-LOFSEP-1R Canna TTC ACC AAG GAG GTT TCT CTG Canna-LOFSEP-2F Canna ATG CAG ATA CCA TGT CAC AAA TA Canna-LOFSEP-2R Canna TTG ATC CAG CTC CTT GAC ATT LOFSEP-1F Costus, Zingiber, Musa GCC ATA ATG TAT CAG AAC CAC CT

38 LOFSEP-1R Costus, Zingiber, Musa GAT CCT TAA CAT TTA GGG CAT CTA Costus-LOFSEP-2F Costus GAA GGT GCA CAG ATA CTA ATT CAC A Costus-LOFSEP-2R Costus TGG GTC TCA AGT TGT TCC AAC Musa-LOFSEP-2F Musa CAG AAC CTG GAC TTC CAT CAA AT Musa-LOFSEP-2R Musa TCT ACT TGG TTC TCA AGC TGC TC Strelitzia-LOFSEP-1F Strelitzia AGG AAT GGC TTG CTG AAG AA Strelitzia-LOFSEP-1R Strelitzia CTA GAT CTT CGC CAA GGA GGT Strelitzia-LOFSEP-2F Strelitzia ATG TGG AGG TGG CGC TTA T Strelitzia-LOFSEP-2R Strelitzia CCC TAA CAC TTA ATG GCT CCA G Zingiber-LOFSEP-2F Zingiber AAA GCT ACA ACT TCA GCA AAT GA Zingiber-LOFSEP-2R Zingiber TGG TTC TCT AGC TGC TCT AGC TC AGL6-like Zin-AGL6-F Zingiberales CGC ACT CAT CAT CTT CTC CA Zin-AGL6-R Zingiberales GCA GCT TCA GGA GGA ACA AA AGL6 copy specific Canna-AGL6-F Canna GGA ATC CCT ACA ACG CTC AC Canna-AGL6-R Canna GAT GCT TGG ACC TGA AGA GC Ginger-AGL6-F Costus, Zingiber ACC TTG GTC CCT TGA GTG TG Ginger-AGL6-R Costus, Zingiber CAG TGG CTG CAC AGA GAA AG Musa-AGL6-F Musa CAA GAG ACA CAG GCA CGA AA Musa-AGL6-R Musa TGT GGT CCA GCA TCA GTT GT Strelitzia-AGL6-F Strelitzia ATG TCC AAG TTG AAG GCA AAG T Strelitzia-AGL6-R Strelitzia CGA AGT TCT TCC ATC TGG TC

39 References Adam H, Jouannic S, Orieux Y, Morcillo F, Richaud F, Duval Y, Tregear JW Functional characterization of MADS box genes involved in the determination of oil palm flower structure. J Exp Bot 58: Agrawal G, Abe K, Yamazaki M, Miyao A, Hirochika H Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene. Plant Mol Biol 59: Almeida AMR, Brown A, Specht CD Tracking the Development of the Petaloid Fertile Stamen in Canna indica: Insights into the origin of androecial petaloidy in the Zingiberales. AoB PLANTS 5: plt009. Alvarez-Buylla ER, Ambrose BA, Flores-Sandoval E et al. (10 co-authors) B-Function Expression in the Flower Center Underlies the Homeotic Phenotype of Lacandonia schismatica (Triuridaceae). Plant Cell 22: Alvarez-Buylla ER, Liljegren SJ, Pelaz S, Gold SE, Burgeff C, Ditta GS, Vergara-Silva F, Yanofsky MF. 2000a. MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. Plant J 24: Alvarez-Buylla ER, Pelaz S, Liljegren SJ, Gold SE, Burgeff C, Ditta GS, de Pouplana LR, Martinez-Castilla L, Yanofsky MF. 2000b. An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. P Natl Acad Sci USA 97: Bartlett ME, Kirchoff BK, Specht CD Epi-illumination microscopy coupled to in situ hybridization and its utility in the study of evolution and development in non-model species. Dev Genes Evol 218: Bartlett ME, Specht CD Changes in expression pattern of the teosinte branched1-like genes in the Zingiberales provide a mechanism for evolutionary shifts in symmetry across the order. Am J Bot 98: Bartlett ME, Specht CD Evidence for the involvement of GLOBOSA-like gene duplications and expression divergence in the evolution of floral morphology in the Zingiberales. New Phytol 187: Becker A, Winter KU, Meyer B, Saedler H, Theissen G MADS-box gene diversity in seed plants 300 million years ago. Molecular Biology and Evolution 17: Castillejo C, Romera-Branchat M, Pelaz S A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression. Plant J 43: Chang YY, Chiu YF, Wu JW, Yang CH Four Orchid (Oncidium Gower Ramsey) AP1/AGL9-like MADS Box Genes Show Novel Expression Patterns and Cause Different Effects on Floral Transition and Formation in Arabidopsis thaliana. Plant Cell Physiol 50:

40 Christensen AR, Malcomber ST Duplication and diversification of the LEAFY HULL STERILE1 and Oryza sativa MADS5 SEPALLATA lineages in graminoid Poales. EvoDevo 3: 4. Classen-Bockhoff R, Heller A Floral synorganization and secondary pollen presentation in four Marantaceae from Costa Rica. Int J Plant Sci 169: Coen ES, Meyerowitz EM The War of the Whorls - Genetic Interactions Controlling Flower Development. Nature 353: Cornell MJ, Alam I, Soanes DM et al. (# co-authors) Comparative genome analysis across a kingdom of eukaryotic organisms: Specialization and diversification in the Fungi. Genome Res 17: Crow KD, Stadler PF, Lynch VJ, Amemiya C, Wagner GP The "fish-specific" Hox cluster duplication is coincident with the origin of teleosts. Molecular Biology and Evolution 23: Cui RF, Han JK, Zhao SZ et al. (7 co-authors) Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa). Plant J 61: D'hont A, Denoeud F, Aury JM et al. (61 co-authors) The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature 488: Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky MF The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Curr Biol 14: Drummond A, Ashton B, Buxton S et al. (12 co-authors) Geneious v5.4, Available from Drummond AJ, Suchard MA, Xie D, Rambaut A Bayesian Phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29: Duarte JM, Cui LY, Wall PK, Zhang Q, Zhang XH, Leebens-Mack J, Ma H, Altman N, depamphilis CW Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol Biol Evol 23: Edgar RC MUSCLE: a multiple sequence alignment method with reduced time and space complexity. Bmc Bioinformatics 5: Freeling M, Lyons E, Pedersen B, Alam M, Ming R, Lisch D Many or most genes in Arabidopsis transposed after the origin of the order Brassicales. Genome Res 18: Goto K, Kyozuka J, Bowman JL Turning floral organs into leaves, leaves into floral organs. Curr Opin Genet Dev 11:

41 Gu X Evolution of duplicate genes versus genetic robustness against null mutations. Trends in Genetics 19: Gu ZL, Steinmetz LM, Gu X, Scharfe C, Davis RW, Li WH Role of duplicate genes in genetic robustness against null mutations. Nature 421: Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst Biol 59: Honma T, Goto K Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409: Immink RGH, Tonaco IAN, de Folter S, Shchennikova A, van Dijk ADJ, Busscher-Lange J, Borst JW, Angenent GC SEPALLATA3: the 'glue' for MADS box transcription factor complex formation. Genome Biol 10. Janssen T, Bremer K The age of major monocot groups inferred from 800+rbcL sequences. Bot J Linn Soc 146: Kanno A, Hienuki H, Ito T et al. (9 co-authors) The structure and expression of SEPALLATA-like genes in Asparagus species (Asparagaceae). Sex Plant Reprod 19: Kanno A, Saeki H, Kameya T, Saedler H, Theissen G Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana). Plant Mol Biol 52: Kaufmann K, Melzer R, Theissen G MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene 347: Kaufmann K, Muino JM, Jauregui R, Airoldi CA, Smaczniak C, Krajewski P, Angenent GC Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower. Plos Biol 7: Kay KM, Reeves PA, Olmstead RG, Schemske DW Rapid speciation and the evolution of hummingbird pollination in neotropical Costus subgenus Costus (Costaceae): evidence from nrdna ITS and ETS sequences. Am J Bot 92: Khanday I, Yadav SR, Vijayraghavan U Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways. Plant Physiol: Kishino H, Hasegawa M Evaluation of the Maximum-Likelihood Estimate of the Evolutionary Tree Topologies from DNA-Sequence Data, and the Branching Order in Hominoidea. J Mol Evol 29:

42 Kobayashi K, Maekawa M, Miyao A, Hirochika H, Kyozuka J PANICLE PHYTOMER2 (PAP2), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet meristem identity in rice. Plant Cell Physiol 51: Kress WJ The Phylogeny and Classification of the Zingiberales. Ann Mo Bot Gard 77: Ley AC, Classen-Bockhoff R Evolution in African Marantaceae - Evidence from Phylogenetic, Ecological and Morphological Studies. Syst Bot 36: Li H, Handsaker B, Wysoker A et al. (7 co-authors) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25: Li HF, Liang WQ, Hu Y, Zhu L, Yin CS, Xu J, Dreni L, Kater MM, Zhang DB Rice MADS6 Interacts with the Floral Homeotic Genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in Specifying Floral Organ Identities and Meristem Fate. Plant Cell 23: Lim J, Moon YH, An G, Jang SK Two rice MADS domain proteins interact with OsMADS1. Plant Mol Biol 44: Lynch M, Force A The probability of duplicate gene preservation by subfunctionalization. Genetics 154: Malcomber ST, Kellogg EA Heterogeneous expression patterns and separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in Grasses. Plant Cell 16: Malcomber ST, Kellogg EA SEPALLATA gene diversification: brave new whorls. Trends Plant Sci 10: Martin M Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, North America 17. Mondragon-Palomino M, Theissen G MADS about the evolution of orchid flowers. Trends Plant Sci 13: Moon YH, Kang HG, Jung JY, Jeon JS, Sung SK, An G Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system. Plant Physiol 120: Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405: Pelaz S, Tapia-Lopez R, Alvarez-Buylla ER, Yanofsky MF Conversion of leaves into petals in Arabidopsis. Curr Biol 11: Pond SLK, Frost SDW, Muse SV HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:

43 Posada D jmodeltest: Phylogenetic model averaging. Mol Biol Evol 25: Prasad K, Parameswaran S, Vijayraghavan U OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs. The Plant Journal 43: Preston JC, Christensen A, Malcomber ST, Kellogg EA MADS-box gene expression and implications for developmental origins of the grass spikelet. Am J Bot 96: Reinheimer R, Kellogg EA Evolution of AGL6-like MADS Box Genes in Grasses (Poaceae): Ovule Expression Is Ancient and Palea Expression Is New. Plant Cell 21: Reinheimer R, Malcomber ST, Kellogg EA Evidence for distinct roles of the SEPALLATA gene LEAFY HULL STERILE1 in Eleusine indica and Megathyrsus maximus (Poaceae). Evol Dev 8: Riechmann JL, Meyerowitz EM MADS domain proteins in plant development. Biol Chem 378: Roberts A, Pachter L Streaming fragment assignment for real-time analysis of sequencing experiments. Nature Methods 10: 71-U99. Ronquist F, Huelsenbeck JP MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: Seoighe C, Johnston CR, Shields DC Significantly different patterns of amino acid replacement after gene duplication as compared to after speciation. Molecular Biology and Evolution 20: Shan HY, Su KM, Lu WL, Kong HZ, Chen ZD, Meng Z Conservation and divergence of candidate class B genes in Akebia trifoliata (Lardizabalaceae). Dev Genes Evol 216: Shan HY, Zahn L, Guindon S, Wall PK, Kong HZ, Ma H, depamphilis CW, Leebens-Mack J Evolution of Plant MADS Box Transcription Factors: Evidence for Shifts in Selection Associated with Early Angiosperm Diversification and Concerted Gene Duplications. Mol Biol Evol 26: Sharrocks AD, Gille H, Shaw PE Identification of Amino-Acids Essential for DNA- Binding and Dimerization in P67srf - Implications for a Novel DNA-Binding Motif. Mol Cell Biol 13: Shimodaira H An approximately unbiased test of phylogenetic tree selection. Syst Biol 51: Specht CD Phylogenetics, Floral Evolution, and Rapid Radiation in the Tropical Monocot Family Costaceae (Zingiberales). In: Sharma AKSaA, editor. Plant Genome: Biodiversity and Evolution. Enfield, NH: Science Publishers, Inc. p

44 Specht CD Systematics and evolution of the tropical monocot family Costaceae (Zingiberales): A multiple dataset approach. Syst Bot 31: Specht CD, Stevenson DW A new phylogeny-based generic classification of Costaceae (Zingiberales). Taxon 55: Specht CD, Yockteng R, Almeida AMR, Kirchoff BK, Kress JW Homoplasy, Pollination, and Emerging Complexity During the Evolution of Floral Development in the Tropical Gingers (Zingiberales). Botanical Review 78: Stern A, Doron-Faigenboim A, Erez E, Martz E, Bacharach E, Pupko T Selecton 2007: advanced models for detecting positive and purifying selection using a Bayesian inference approach. Nucleic Acids Res 35: W506-W511. Sukumaran J, Holder MT DendroPy: a Python library for phylogenetic computing. Bioinformatics 26: Swofford DL PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods).. Version 4.0b10. Sunderland, Massachusetts: Sinauer Associates. Teichmann S, Babu MM Gene regulatory network growth by duplication. Nat Genet 36: Theissen G, Saedler H Plant biology - Floral quartets. Nature 409: Tzeng TY, Hsiao CC, Chi PJ, Yang CH Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis. Plant Physiol 133: Vandenbussche M, Zethof J, Souer E, Koes R, Tornielli GB, Pezzotti M, Ferrario S, Angenent GC, Gerats T Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia. Plant Cell 15: Viaene T, Vekemans D, Becker A, Melzer S, Geuten K Expression divergence of the AGL6 MADS domain transcription factor lineage after a core eudicot duplication suggests functional diversification. Bmc Plant Biol 10. Wagner GP, Amemiya C, Ruddle F Hox cluster duplications and the opportunity for evolutionary novelties. Proceedings of the National Academy of Sciences of the United States of America 100: West AG, Shore P, Sharrocks AD DNA binding by MADS-box transcription factors: A molecular mechanism for differential DNA bending. Mol Cell Biol 17: Wu TD, Watanabe CK GMAP: a genomic mapping and alignment program for mrna and EST sequences. Bioinformatics 21:

45 Yang YZ, Jack T Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins. Plant Mol Biol 55: Yang ZH, Nielsen R Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol 19: Yang ZH, Nielsen R, Goldman N, Pedersen AMK Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155: Zahn LM, King HZ, Leebens-Mack JH, Kim S, Soltis PS, Landherr LL, Soltis DE, depamphilis CW, Ma H The evolution of the SEPALLATA subfamily of MADS-Box genes: A preangiosperm origin with multiple duplications throughout angiosperm history. Genetics 169: Zhang JZ, Nielsen R, Yang ZH Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22:

46 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

47 Cannaceae (CA) Costaceae (CO) Heliconiaceae (HE) Lowiaceae (LO) Marantaceae (MA) Musaceae (MU) Strelitziaceae (ST) Zingiberaceae (ZI) Prunus_STK_EF Musa velutina KC (MU) Heliconia metallica KC (HE) Heliconia metallica KC (HE) Costus productus KC (CO) Musa velutina KC (MU) Musa acuminata EU (MU) Phrynium oliganthum KC (MA) Donax grandis KC (MA) Schumannianthus virgatus KC (MA) Globba laeta KC (ZI) Marantochloa leucantha KC (MA) Canna jaegeriana KC CA) Orchidantha sp. KC (LO) Heliconia lennartiana KC (HE) Orchidantha siamensis KC (LO) Costus spicatus KC (CO) Musa acuminata KC (MU) Musa acuminata EU (MU) Musa acuminata AY (MU) Schumannianthus virgatus KC (MA) Schumannianthus virgatus KC (MA) Phenakospermum guyannense KC (ST) Halopegia azurea KC (MA) Orchidantha siamensis KC (LO) Heliconia pendula KC (HE) Costus productus KC (CO) Heliconia lennartiana KC (HE) Donax grandis KC (MA) ZinSEP3-2 Donax grandis KC (MA) Phrynium oliganthum KC (MA) Phrynium oliganthum KC (MA) Marantochloa leucantha KC (MA) Zingiber officinale KC (ZI) Zingiber officinale KC (ZI) Phenakospermum guyannense KC (ST) Globba laeta KC (ZI) Curcuma sp. KC (ZI) Orchidantha siamensis KC (LO) Orchidantha siamensis KC (LO) Elettariopsis smithiae KC (ZI) Globba laeta KC (ZI) Halopegia azurea KC (MA) Tapeinochilos solomonensis KC (CO) Strelitzia sp. KC (ST) Strelitzia sp. KC (ST) Globba laeta KC (ZI) Costus spicatus KC (CO) Curcuma sp. KC (ZI) Ettlingera corneri KC (ZI) Elettariopsis smithiae KC (ZI) Globba laeta KC (ZI) Curcuma sp. KC (ZI) Elaeis guineensis AF Elaeis guineensis AF Elaeis guineensis AF Dendrobium grex AF Dendrobium crumenatum DQ Aranda deborah X69107 Costus productus KC (CO) Musa acuminata EU (MU) Tapeinochilus solomonensis KC (CO) Tapeinochilus solomonensis KC (CO) Heliconia metallica KC (HE) Musa velutina KC (MU) Phenakospermum guyannense KC (ST) Heliconia metallica KC (HE) Alpinia pinetorum KC (ZI) Musa velutina KC (MU) Alpinia pinetorum KC (ZI) Strelitzia sp. KC (ST) Musa velutina KC (MU) Canna jaegeriana KC (CA) Heliconia pendula KC (HE) Alpinia pinetorum KC (ZI) Alpinia pinetorum KC (ZI) Orchidantha siamensis KC (LO) Musa acuminata KC (MU) Heliconia pendula KC (HE) ZinSEP3-1 Musa acuminata KC (MU) Marantochloa leucantha KC (MA) Marantochloa leucantha KC (MA) Zingiber officinale DY (ZI) Heliconia pendula KC (HE) Heliconia metallica KC (HE) Monocostus uniflorus KC (CO) Heliconia pendula KC (HE) Elettariopsis smithiae KC (ZI) Alpinia hainanensis FJ (ZI) Ettlingera corneri KC (ZI) Orchidantha siamensis KC (LO) Costus spicatus KC (CO) Costus spicatus KC (CO) Heliconia pendula KC (HE) Heliconia pendula KC (HE) Costus spicatus KC (CO) Triticum aestivum DQ Lolium perenne AY Oryza sativa U78891 Zea mays NM Zea mays NM Triticum aestivum AM Triticum aestivum AF Hordeum vulgare EU Lolium perenne AY Dendrocalamus latiflorus AY Oryza sativa U78892 Pharus virescens AY Crocus sativus EU Crocus sativus EU Crocus sativus EU Crocus sativus EU Asparagus officinalis AY Asparagus virgatus DQ Allium cepa CF Asparagus virgatus DQ Asparagus officinalis DQ Zostera japonica AB Liriodendron tulipifera AY Persea americana AY Persea americana AY Magnolia grandiflora AY Eschscholzia californica AY Aquilegia coerulea JX Arabidopsis thaliana NM Gossypium hirsutum JF Prunus persica EF Triticum monococcum JN Triticum aestivum DQ Lolium perenne AY Avena sativa JN Ehrharta erecta JN Leersia virginica JN Oryza sativa AK Streptochaeta angustifolia AY Oryza meridionalis JN Oryza sativa AK Oryza glaberrima JN Oryza barthii JN Leersia virginica AY Ehrharta erecta AY Sorghum bicolor AY Miscanthus sinensis JN Zea mays AJ Zea mays Y09303 Setaria italica AY Pennisetum glaucum AY59752 Panicum miliaceum AY Panicum maximum DQ Eleusine indica DQ Eleusine coracana AY Danthonia spicata AY Aristida longiseta AY Lithachne humilis AY Dendrocalamus latiflorus AY Hordeum vulgare AJ Triticum aestivum DQ Triticum aestivum DQ Triticum aestivum AJ Lolium perenne AY Avena sativa AY Pharus latifolius JN Dendrocalamus latiflorus AY Ehrharta erecta JN Oryza sativa OSU78890 Leersia virginica JN Triticum aestivum DQ Triticum aestivum DQ Triticum aestivum DQ Hordeum vulgare JN Lolium perenne AY Avena sativa JN Eleusine coracana JN Setaria italica JN Pennisetum glaucum AY Cenchrus americanus JN661 Zea mays Y09301 Sorghum bicolor AY Joinvillea ascendens JN Elaeis guineensis AF Elaeis guineensis AF Allium cepa CF43672 Dendrobium grex AF Lilium longiflorum AY Asparagus virgatus DQ Asparagus officinalis DQ Amborella trichopoda AY Malus domestica U78950 Petunia x hybrida AF Lycopersicon esculentum AF Gossypium hirsutum JF Arabidopsis thaliana NM Eschscholzia californica AY Silene latifolia AB Aquilegia coerulea JX Aquilegia coerulea JX Vitis vinifera XM Rosa rugosa AB Prunus persica EF Malus domestica AJ Malus domestica U78947 Gossypium hirsutum JF Antirrhinum majus X95467 Solanum lycopersicum NM Lycopersicon esculentum AJ Petunia x hybrida AY Petunia x hybrida AF Gerbera hybrida AJ Arabidopsis thaliana NM Arabidopsis thaliana AT5G15800 Prunus persica DQ Malus domestica U78949 Malus domestica AJ Petunia x hybrida AF Petunia x hybrida AF Lycopersicon esculentum AY Magnolia grandiflora AY Acorus americanus AY Oncidium hybrid HM Cymbidium goeringii HM Cymbidium goeringii GQ Cymbidium faberi HM Lilium tigrinum GQ Asparagus officinalis AY Agapanthus africanus GQ Hyacinthus orientalis AY Crocus sativus EF Crocus sativus EF Streptochaeta angustifolia GQ Oryza glaberrima GQ Oryza barthii GQ Oryza sativa FJ Oryza meridionalis GQ Pharus sp. GQ Tripsacum dactyloides GQ Tripsacum dactyloides GQ Zea mays L46398 Sorghum bicolor GQ Coix_sp_AGL6_GQ Setaria italica GQ Pennisetum glaucum GQ Panicum miliaceum GQ Eragrostis tef GQ Eragrostis pilosa GQ Eleusine indica GQ Chasmanthium latifolium GQ Dendrocalamus latiflorus AY Dendrocalamus latiflorus AY Triticum aestivum AB Hordeum vulgare GQ Lolium perenne AY Lolium temulentum GQ Brachypodium distachyon GQ Phalaris canariensis GQ Avena strigosa GQ Poa annua AF Lithachne humilis GQ Leersia sp. GQ Oryza meridionalis GQ Oryza glaberrima GQ Oryza barthii GQ Oryza sativa U78782 Joinvillea ascendens GQ Zingiber officinale KC (ZI) Costus spicatus KC (CO) Costus productus KC (CO) Aframomum angustifolium KC (ZI) Tapeinochilus solomonensis KC (CO) Costus productus KC (CO) Globba laeta KC (ZI) Ettlingera corneri KC (ZI) Ettlingera corneri KC (ZI) Curcuma sp. KC (ZI) Curcuma sp. KC (ZI) Globba laeta KC (ZI) Elatteriopsis smithiae KC (ZI) Alpinia pinetorum KC (ZI) Elatteriopsis smithiae KC (ZI) Canna jaegeriana KC (CA) Strelitzia sp. KC (ST) Strelitzia sp. KC (ST) ZinAGL6 Orchidantha siamensis KC (LO) Phenakospermum guyannene KC (ST) Heliconia pendula KC (HE) Heliconia metallica KC (HE) Musa acuminata EU (MU) Schumannianthus virgatus KC (MA) Schumannianthus virgatus KC (MA) Halopegia azurea KC (MA) Elaeis guineensis AY Tradescantia virginiana GQ Nymphaea odorata GU Nymphaea hybrid AB Nuphar advena GU Oncidium hybrid HM ZINGIBERALES ZINGIBERALES ZINGIBERALES OsMADS7 OsMADS8 Petunia x hybrida AY Petunia x hybrida M91666 Lycopersicon esculentum NM Chrysanthemum x morifolium AY Eustoma grandiflorum EF Antirrhinum majus X95468 Antirrhinum majus AY Antirrhinum majus X95469 Chrysanthemum x morifolium AY Silene latifolia AB Houttuynia cordata AB Amborella trichopoda AY Houttuynia cordata AB Houttuynia cordata AB Strelitzia sp. KC (ST) Musa basjoo KC (MU) Heliconia pendula KC (HE) Phenakospermum guyannense KC (ST) Musa acuminata KC (MU) Tapeinochilos solomonensis KC (CO) Curcuma sp. KC (ZI) Ettlingera corneri KC (ZI) ZinLOFSEP-2 Elettariopsis smithiae KC (ZI) Donax grandis KC (MA) Halopegia azurea KC (MA) Canna jaegeriana KC (CA) Costus spicatus KC (CO) Zingiber officinale KC (ZI) Costus spicatus KC (CO) Tapeinochilos solomonensis KC (CO) Costus productus KC (CO) Globba laeta KC (ZI) Schumannianthus virgatus KC (MA) Musa basjoo KC (MU) Phrynium oliganthum KC (MA) Aframomum angustifolium KC (ZI) ZinLOFSEP-1 Heliconia lennartiana KC (HE) Canna jaegeriana KC (CA) Heliconia metallica KC (HE) Heliconia pendula KC (HE) Strelitzia sp. KC (ST) Phenakospermum guyannense KC (ST) Musa acuminata KC (MU) Orchidantha siamensis KC (LO) Joinvillea ascendens JN Pharus latifolius JN Eleusine coracana JN Aristida purpurea JN Danthonia spicata JN Zea mays NM Zea mays NM Setaria italica JN Cenchrus americanus JN661 Panicum miliaceum JN Lithachne humilis JN PAP2/OsMADS34 LHS1 OsMADS5 SEP4 (AGL3) TM29 (AGL2/4) FBP9 ZINGIBERALES Amborella trichopoda AY Chimonanthus praecox FJ Saurauia zahlbruckneri HM Actinidia chinensis HM Coffea arabica GU Saurauia zahlbruckneri HM Actinidia chinensis HM Gustavia brasiliensis HM Diospyros digyna HM Philadelphus pubescens HM Alangium platanifolium HM Arabidopsis thaliana NM Arabidopsis thaliana NM Aquilegia coerulea JX Epimedium sagittatum JN Nelumbo nucifera GU Persea americana DQ Persea americana DQ Magnolia grandiflora AY Magnolia praecocissima AB Magnolia praecocissima AB Pinus radiata U42400 Gnetum gnemon AJ Arabidopsis thaliana NM Elaeis guineensis AF Elaeis guineensis AF Alpinia oblongifolia EF (ZI) Ettlingera corneri KC (ZI) Costus productus KC (CO) Costus productus KC (CO) Heliconia metallica KC (HE) Alpinia oblongifolia EF (ZI) Alpinia oblongifolia EF (ZI) ARECALES COMMELINALES NYMPHAEALES MAGNOLIIDS CORE EUDICOTS RANUNCULALES PROTEALES MAGNOLIIDS ARECACEAE ORCHIDACEAE POACEAE ASPARAGALES MAGNOLIIDS RANUNCULALES CORE EUDICOTS MAGNOLIIDS AMBORELLALES MAGNOLIIDS ZINGIBERALES ARECALES ASPARAGALES ORCHIDACEAE LILIALES ASPARAGALES ACORALES ORCHIDACEAE LILIALES POALES CORE EUDICOTS MAGNOLIIDS ASPARAGALES POALES SEP3 LOFSEP AGL6 AP1/SQUA 0.2

48 0.2 ++/++ ++/ / / /-- 0.7/55 ++/73 ++/++ Amborella trichopoda AY Nuphar advena GU /++ Nymphaea hybrid AB Nymphaea odorata GU Oncidium hybrid HM /++ Oncidium hybrid HM Cymbidium goeringii HM /99 Cymbidium faberi HM /-- Cymbidium goeringii GQ /95 Lilium tigrinum GQ Hyacinthus orientalis AY / /-- Agapanthus africanus GQ Asparagus officinalis AY /++ Crocus sativus EF Crocus sativus EF Elaeis guineensis AY /++ Heliconia metallica KC (HE) 0.94 Heliconia pendula KC (HE) Phenakospermum guyannense KC (ST) Strelitzia sp. KC (ST) ++ ++/83 Strelitzia sp. KC (ST) ++ Orchidantha siamensis KC (LO) ++ Musa acuminata EU (MU) /-- Halopegia azurea KC (MA) ++/++ Schumannianthus virgatus KC (MA) ++/99 ++/++ Schumannianthus virgatus KC (MA) ω=0.099 Globba laeta KC (ZI) ++/94 Alpinia pinetorum KC (ZI) 0.85/-- ++/99 Elettariopsis smithiae KC (ZI) Elettariopsis smithiae KC (ZI) Curcuma sp. KC (ZI) ++/ /++ Curcuma sp. KC (ZI) Globba laeta KC (ZI) 0.99/ /73 Ettlingera corneri KC (ZI) 0.87/51 ++/++ Ettlingera corneri KC (ZI) Canna jaegeriana KC (CA) Costus productus KC (CO) 0.55/-- Tapeinochilus solomonensis KC (CO) ++/97 Zingiber officinale KC (ZI) ++/97 Aframomum angustifolium KC (ZI) ++/ /-- Costus productus KC (CO) ++/87 Costus spicatus KC (CO) Tradescantia virginiana GQ /++ Oryza barthii GQ /++ Oryza glaberrima GQ Oryza meridionalis GQ Oryza sativa FJ /99 Joinvillea ascendens GQ ω=0.105 Streptochaeta angustifolia GQ /71 Pharus sp. GQ Chasmanthium latifolium GQ Panicum miliaceum GQ /89 ++/96 Tripsacum dactyloides GQ /97 Zea mays L /82 ++/68 Sorghum bicolor GQ / /68 Coix sp. GQ /-- Tripsacum dactyloides GQ /82 Pennisetum glaucum GQ /98 Setaria italica GQ Eleusine indica GQ /98 ++/69 Eragrostis pilosa GQ /93 Eragrostis tef GQ /98 Leersia sp. GQ Oryza sativa U /99 Oryza barthii GQ /88 Oryza glaberrima GQ /82 Oryza meridionalis GQ Brachypodium distachyon GQ /79 ++/97 ++/99 Hordeum vulgare GQ Triticum aestivum AB /97 Poa annua AF /99 Lolium perenne AY Lolium temulentum GQ / /-- Avena strigosa GQ /-- Phalaris canariensis GQ Lithachne humilis GQ /51 Dendrocalamus latiflorus AY /++ Dendrocalamus latiflorus AY Persea americana DQ /++ Magnolia praecocissima AB Magnolia grandiflora AY Magnolia praecocissima AB Chimonanthus praecox FJ Persea americana DQ Nelumbo nucifera GU Epimedium sagittatum JN Aquilegia coerulea JX /++ Arabidopsis thaliana NM Arabidopsis thaliana NM /56 ω=0.147 Coffea arabica GU /++ Alangium platanifolium HM Philadelphus pubescens HM /74 Diospyros digyna HM Gustavia brasiliensis HM / /56 Actinidia chinensis HM /99 Saurauia zahlbruckneri HM /++ Actinidia chinensis HM Saurauia zahlbruckneri HM Gnetum gnemon AJ Pinus radiata U / /-- ++/90 ++/83 AMBORELLALES NYMPHAEALES ORCHIDACEAE LILIALES ASPARAGALES ARECACEAE ZINGIBERALES COMMELINALES POALES MAGNOLIIDS PROTEALES RANUNCULALES CORE EUDICOTS Cannaceae (CA) Costaceae (CO) Heliconiaceae (HE) Lowiaceae (LO) Marantaceae (MA) Musaceae (MU) Strelitziaceae (ST) Zingiberaceae (ZI) MONOCOTS GYMNOSPERMS

49 Vitis vinifera XM /-- Gossypium hirsutum JF Silene latifolia AB /-- ++/99 Arabidopsis thaliana AT5G /-- Arabidopsis thaliana NM ω= /55 Antirrhinum majus X /80 ++/84 Petunia x hybrida AF /94 Petunia x hybrida AY /92 ++/99 Lycopersicon esculentum AJ Solanum lycopersicum NM Rosa rugosa AB / /88 Prunus persica EF /68 Malus domestica U /92 Malus domestica AJ Prunus persica DQ /97 Malus domestica U / Malus domestica AJ /76 ω= /96 Petunia x hybrida AF /98 Lycopersicon esculentum AY Petunia x hybrida AF Arabidopsis thaliana NM /61 ++/98 Lycopersicon esculentum AF /58 Petunia x hybrida AF Gossypium hirsutum JF /-- ω= /-- Malus domestica U78950 Eschscholzia californica AY /-- Aquilegia coerulea JX /53 Aquilegia coerulea JX /-- Magnolia grandiflora AY /++ Houttuynia cordata AB Houttuynia cordata AB /31 Dendrobium grex AF Lilium longiflorum AY /++ Asparagus officinalis DQ Asparagus virgatus DQ Orchidantha siamensis KC (LO) Costus productus KC (CO) Costus spicatus KC (CO) Globba laeta KC (ZI) ω=0.336** Heliconia lennartiana KC (HE) 0.71/-- ++/99 Musa basjoo KC (MU) ++/++ Phrynium oliganthum KC (MA) Schumannianthus virgatus KC (MA) Tapeinochilos solomonensis KC (CO) Aframomum angustifolium KC (ZI) 0.94/-- ++/-- Canna jaegeriana KC (CA) Zingiber officinale KC (ZI) 0.99/ /-- Heliconia metallica KC (HE) Heliconia pendula KC (HE) 0.5/-- Musa acuminata KC (MU) 0.91/50 Phenakospermum guyannense KC (ST) ++/ / / /-- Amborella trichopoda AY Musa acuminata KC (MU) Phenakospermum guyannense KC (ST) ++/++ Heliconia pendula KC (HE) ++/96 Musa basjoo KC (MU) ++/97 ω=0.458** Strelitzia sp. KC (ST) ++/89 Canna jaegeriana KC (CA) ++/95 Halopegia azurea KC (MA) Donax grandis KC (MA) 0.94/71 Costus spicatus KC (CO) ++/92 Elettariopsis smithiae KC (ZI) 0.87/55 ++/++ Ettlingera corneri KC (ZI) Curcuma sp. KC (ZI) ++/94 Tapeinochilos solomonensis KC (CO) 0.91/ /-- Elaeis guineensis AF Elaeis guineensis AF /62 Avena sativa JN /92 Lolium perenne AY Hordeum vulgare JN /-- Triticum aestivum DQ /98 Triticum aestivum DQ /-- ++/99 Triticum aestivum DQ /92 Sorghum bicolor AY Zea mays Y /69 Eleusine coracana JN / /54 Setaria italica JN ω= /++ Cenchrus americanus JN /68 Pennisetum glaucum AY /83 Leersia virginica JN /98 Oryza sativa OSU /50 Dendrocalamus latiflorus AY / /-- Ehrharta erecta JN Joinvillea ascendens JN Streptochaeta angustifolia AY ω=0.148 Pharus latifolius JN /-- Triticum aestivum AJ /69 Hordeum vulgare AJ / /79 Triticum aestivum DQ /98 ++/93 Triticum aestivum DQ Avena sativa AY / /52 Lolium perenne AY Ehrharta erecta AY /74 Leersia virginica AY Oryza meridionalis JN /88 Oryza barthii JN /99 Oryza glaberrima JN / /86 Oryza sativa AK /-- Aristida longiseta AY /99 Zea mays AJ /89 Zea mays Y /60 Miscanthus sinensis JN /71 ++/93 Sorghum bicolor AY /90 Panicum maximum DQ Panicum miliaceum AY /94 Pennisetum glaucum AY / /68 Setaria italica AY /-- Danthonia spicata AY /81 Eleusine coracana AY /99 Eleusine indica DQ Dendrocalamus latiflorus AY /-- Lithachne humilis AY Pharus latifolius JN Danthonia spicata JN /-- ++/99 Zea mays NM Zea mays NM /++ ++/76 Panicum miliaceum JN /-- ω= /83 Cenchrus americanus JN /66 Setaria italica JN Aristida purpurea JN /-- Eleusine coracana JN /-- Joinvillea ascendens JN /98 Triticum aestivum DQ Acorus americanus AY Strelitzia sp. KC (ST) 0.67/ / /-- ++/99 Allium cepa CF / /79 ++/82 Triticum monococcum JN Avena sativa JN Lolium perenne AY Lithachne humilis JN Ehrharta erecta JN Leersia virginica JN Oryza sativa AK TM29 (AGL2/4) FBP9 SEP4 (AGL3) ZinLOFSEP1 ZinLOFSEP2 OsMADS5 LHS1 OsMADS1 PAP2 OsMADS34 RANUNCULALES MAGNOLIIDS ORCHIDS LILIALES ASPARAGALES Zingiberales ARECACEAE POACEAE ASPARAGALES ACORALES AMBORELLALES EUDICOTS MONOCOTS Cannaceae (CA) Costaceae (CO) Heliconiaceae (HE) Lowiaceae (LO) Marantaceae (MA) Musaceae (MU) Strelitziaceae (ST) Zingiberaceae (ZI) 0.2

50 /-- ω= /-- Eschscholzia californica AY / / / /-- ++/ / /85 Silene latifolia AB Eustoma grandiflorum EF Antirrhinum majus X95469 Antirrhinum majus AY Antirrhinum majus X95468 Chrysanthemum x morifolium AY Lycopersicon esculentum NM /++ Petunia x hybrida M91666 Petunia x hybrida AY Gossypium hirsutum JF Arabidopsis thaliana NM Prunus persica EF Chrysanthemum x morifolium AY RANUNCULALES CORE EUDICOTS / /-- Cannaceae (CA) Costaceae (CO) Heliconiaceae (HE) Lowiaceae (LO) Marantaceae (MA) Musaceae (MU) Strelitziaceae (ST) Zingiberaceae (ZI) ++/93 Magnolia grandiflora AY Liriodendron tulipifera AY Persea americana AY Persea americana AY Houttuynia cordata AB Zostera japonica AB Musa acuminata KC (MU) Musa velutina KC (MU) Marantochloa leucantha KC (MA) ++/92 ++/98 Globba laeta KC (ZI) ++/80 Schumannianthus virgatus KC (MA) 0.56/-- Donax grandis KC (MA) ++/87 ω=0.0503* Phrynium oliganthum KC (MA) Canna jaegeriana KC (CA) ++/96 Heliconia lennartiana KC (HE) Orchidantha sp. KC (LO) ++/98 Costus spicatus KC (CO) Orchidantha siamensis KC (LO) ++/++ Musa acuminata AY (MU) 0.98 Musa acuminata EU (MU) ++/96 Curcuma sp. KC (ZI) 0.52 Globba laeta KC (ZI) ++/84 Globba laeta KC (ZI) Curcuma sp. KC (ZI) ++/ /-- Zingiber officinale KC (ZI) ++/ /52 Phenakospermum guyannense KC (ST) ++/78 Zingiber officinale KC (ZI) Elettariopsis smithiae KC (ZI) ++/81 Orchidantha siamensis KC (LO) ++/87 ++/99 Orchidantha siamensis KC (LO) 0.99/84 Elettariopsis smithiae KC (ZI) ++/99 Ettlingera corneri KC (ZI) Curcuma sp. KC (ZI) 0.99/65 Globba laeta KC (ZI) 0.8/ /76 Halopegia azurea KC (MA) ++/60 Tapeinochilos solomonensis KC (CO) 0.53/-- Costus spicatus KC (CO) 0.99/ /76 Globba laeta KC (ZI) 0.93/69 Strelitzia sp. KC (ST) 0.97/65 Strelitzia sp. KC (ST) Marantochloa leucantha KC (MA) ++/95 Schumannianthus virgatus KC (MA) ++/96 ++/94 Schumannianthus virgatus KC (MA) Halopegia azurea KC (MA) Phenakospermum guyannense KC (ST) 0.67/ /78 Costus productus KC (CO) Heliconia lennartiana KC (HE) 0.77/ /59 Heliconia pendula KC (HE) Orchidantha siamensis KC (LO) Phrynium oliganthum KC (MA) ++/98 Phrynium oliganthum KC (MA) 0.53/-- Donax grandis KC (MA) 0.98/62 Donax grandis KC (MA) ++/99 Musa acuminata EU (MU) Musa velutina KC (MU) 0.81/-- Costus productus KC (CO) ++/99 Heliconia metallica KC (HE) Heliconia metallica KC (HE) ++/97 Elaeis guineensis AF /99 Elaeis guineensis AF /-- ++/78 Elaeis guineensis AF Dendrobium grex AF /99 Aranda deborah X69107 Dendrobium crumenatum DQ Costus productus KC (CO) ++/96 Musa acuminata EU (MU) Alpinia pinetorum KC (ZI) Alpinia pinetorum KC (ZI) 0.99/ /61 Musa velutina KC (MU) Heliconia metallica KC (HE) Tapeinochilus solomonensis KC (CO) 0.98/80 Musa velutina KC (MU) 0.8/-- Tapeinochilus solomonensis KC (CO) Heliconia metallica KC (HE) 0.95/65 Phenakospermum guyannense KC (ST) ++/99 Alpinia pinetorum KC (ZI) ω=0.165** Musa acuminata KC (MU) Alpinia pinetorum KC (ZI) ++/90 Heliconia pendula KC (HE) 0.67/53 Canna jaegeriana KC (CA) 0.97/74 Musa velutina KC (MU) 0.96/50 Heliconia pendula KC (HE) 0.94/53 Musa acuminata KC (MU) Orchidantha siamensis KC (LO) 0.64/53 Strelitzia sp. KC (ST) Orchidantha siamensis KC (LO) 0.6/-- ++/++ Alpinia hainanensis FJ (ZI) Elettariopsis smithiae KC (ZI) ++/ /72 Zingiber officinale DY (ZI) Ettlingera corneri KC (ZI) 0.73/-- Heliconia pendula KC (HE) Heliconia pendula KC (HE) 0.8/53 Heliconia metallica KC (HE) 0.57/-- Monocostus uniflorus KC (CO) 0.88/66 ++/++ Marantochloa leucantha KC (MA) Marantochloa leucantha KC (MA) ++/++ Costus spicatus KC (CO) ++/ /70 Heliconia pendula KC (HE) Heliconia pendula KC (HE) Costus spicatus KC (CO) ++/91 Costus spicatus KC (CO) ++/++ Oryza sativa U78892 Pharus virescens AY /61 ω= /60 Dendrocalamus latiflorus AY Zea mays NM /62 ++ Zea mays NM Oryza sativa U /54 ++/99 Lolium perenne AY /98 Triticum aestivum DQ Lolium perenne AY /62 ++/99 Hordeum vulgare EU Triticum aestivum AF /70 Triticum aestivum AM /99 Crocus sativus EU /83 Crocus sativus EU /99 Crocus sativus EU /71 Crocus sativus EU /99 Asparagus officinalis DQ Asparagus virgatus DQ /87 Allium cepa CF Asparagus officinalis AY Asparagus virgatus DQ Amborella trichopoda AY Aquilegia coerulea JX ZINGIBERALES ZinSEP3-2 ZINGIBERALES ZinSEP3-1 MAGNOLIIDS PIPERALES ALISMATALES ARECACEAE ORCHIDACEAE POACEAE ASPARAGALES AMBORELLALES RANUNCULALES MONOCOTS 0.2

51 1 fertile petaloid stamen Petaloid Sepals Petals Staminodes stamen Carpels Cannaceae Canna indica 5 petaloid staminodes Marantaceae Sepals Petals Labellum Petaloid stamen Carpels Costaceae Costus spicatus 1 petaloid staminode 1/2 fertile petaloid stamen Sepals Petals Labellum Petaloid stamen Carpels Zingiberaceae Zingiber officinale Dimorphic perianth Heliconiaceae Sepals Petals Lowiaceae Floral tube Stamens Stamens Carpels Carpels Strelitziaceae Strelitzia sp. Musaceae Musa acuminata SEP3-1 SEP3-2 LOFSEP-2 AGL-6