Copyright Ó 2006 by the Genetics Society of America DOI: 10.1534/genetics.106.057125
Reconstructing the Evolutionary History of Paralogous APETALA1/FRUITFULL-Like Genes in Grasses (Poaceae) Jill C. Preston1 and Elizabeth A. Kellogg Department of Biology, University of Missouri, Saint Louis, Missouri 63121 Manuscript received February 13, 2006 Accepted for publication June 23, 2006 ABSTRACT Gene duplication is an important mechanism for the generation of evolutionary novelty. Paralogous genes that are not silenced may evolve new functions (neofunctionalization) that will alter the developmental outcome of preexisting genetic pathways, partition ancestral functions (subfunctionalization) into divergent developmental modules, or function redundantly. Functional divergence can occur by changes in the spatio-temporal patterns of gene expression and/or by changes in the activities of their protein products. We reconstructed the evolutionary history of two paralogous monocot MADS-box transcription factors, FUL1 and FUL2, and determined the evolution of sequence and gene expression in grass AP1/FUL-like genes. Monocot AP1/FUL-like genes duplicated at the base of Poaceae and codon substitutions occurred under relaxed selection mostly along the branch leading to FUL2. Following the duplication, FUL1 was apparently lost from early diverging taxa, a pattern consistent with major changes in grass floral morphology. Overlapping gene expression patterns in leaves and spikelets indicate that FUL1 and FUL2 probably share some redundant functions, but that FUL2 may have become temporally restricted under partial subfunctionalization to particular stages of floret development. These data have allowed us to reconstruct the history of AP1/FUL-like genes in Poaceae and to hypothesize a role for this gene duplication in the evolution of the grass spikelet.
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ENE duplications have been implicated in the origin of evolutionary novelty through creation of paralogous genes that may be coopted for new or altered functions (Ohno 1970; Force et al. 1999; Hughes 1999). Classic models of gene duplications generally predict the silencing and loss of one descendant gene by accumulation of deleterious mutations (nonfunctionalization) (Nei and Roychoudhury 1973; Li 1980) or acquisition of a novel function by one descendent gene following periods of positive Darwinian selection (Ohno 1970; Hughes 1994). Changes in the nucleotide sequence of a duplicated gene can take place either in the coding region (Ohno 1970; Walsh 1995) or in associated cis-regulatory elements (Li and Noll 1994; Doebley and Lukens 1998). Recent authors have suggested other possible fates for duplicated genes, including maintenance of the ancestral function by both copies (redundancy) or partitioning of the ancestral role between copies (subfunctionalization) following a period of relaxed selection (Force et al. 1999; Lynch et al. 2001). Despite nonfunctionalization being a more likely evolutionary outcome, the existence of ancient multigene families is evidence that duplicated genes can be maintained in the genome for long
Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under accession nos. DQ792945–DQ792980. 1 Corresponding author: Department of Biology, R223, University of Missouri, One University Boulevard, Saint Louis, MO 63121. E-mail:
[email protected] Genetics 174: 421–437 (September 2006)
periods of time (Lynch and Force 2000; De Bodt et al. 2003). Since duplication and subsequent diversification of genes have been implicated in the evolution of morphological diversity (Li and noll 1994; Force et al. 1999; Hughes 1999; Garcia-Fernandez 2005), studies aimed at understanding the fate of duplicated genes may be key to establishing a causal link between molecular evolution and the evolution of novel form. Morphological novelty occurs through the creation or modification of developmental pathways (Carroll et al. 2001). These pathways involve multiple interacting proteins, many of which are transcription factors. In plants, some of the best-studied transcription factors are members of the MIKC-type (denoting the MADS intervening keratin-like and C-terminal domains) MADS-box family (Purugganan et al. 1995; Becker and Theissen 2003; Kofuji et al. 2003; Martinez-Castilla and AlvarezBuylla 2003; Robles and Pelaz 2005). These proteins share a highly conserved DNA binding domain (the MADS domain) at or near the N terminus, a less-conserved intervening (I) domain, a keratin-like (K) domain, and finally a highly variable C terminus (Riechmann et al. 1996); the primary sequence of the latter often contains motifs specific to a particular subtype of MADS-box proteins (e.g., Lamb and Irish 2003). Although MADSbox proteins function at many developmental stages, they are best characterized for their role in inflorescence and floral development. According to the classic ABC model of floral development in Arabidopsis, floral
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Figure 1.—Simplified comparison between flowers of grasses, typical non-grass monocots, and the core eudicot Arabidopsis. (A) Arabidopsis flower and associated inflorescence branches. (B) Generalized monocot flower with inflorescence branches. Each branch is subtended by a bract and gives rise to a prophyll (leaf-like structure). (C) Generalized grass spikelet composed of a single floret, within which is a single flower. (D) Modified spikelet or spikelet equivalent of Streptochaeta, composed of several bracts and a terminal flower. Shaded structures are homologous between diagrams. Solid structures are nonhomologous between diagrams. BRA, bract; PRO, prophyll; GLU, glume; LEM, lemma; PAL, palea; TEP, tepal; SEP, sepal; PET, petal; LOD, lodicule; STA, stamen; GYN, gynoecium. Arrowheads indicate indeterminate growth.
homeotic genes function combinatorially to determine the number and identity of each of the four concentric whorls (Bowman et al. 1989; Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). A-class genes [APETALA1 (AP1) and APETALA2 (AP2)] specify the identity of sepals (Mandel et al. 1992; Jofuku et al. 1994); A and B class [PISTILLATA (PI) and APETALA3 (AP3)] (Krizek and Meyerowitz, 1996), petals; B and C class [AGAMOUS (AG)] (Yanofsky et al. 1990), stamens; and C class, carpels (Bowman et al. 1989, 1991). The products of Arabidopsis E-class genes [SEPALLATA1/2/3 (SEP1/2/3)] are thought to further participate by forming multiprotein complexes with ABC proteins to specify petals, stamens, and carpels (Pelaz et al. 2000, 2001; Honma and Goto 2001), although this has yet to be demonstrated in planta. Except for AP2, all these genes encode MADS-box transcription factors. B and C functions are largely conserved among homologous genes across the angiosperms (Ambrose et al. 2000; Ma and Depamphilis 2000; Whipple et al. 2004; but see Kramer et al. 2003; Lamb and Irish 2003), but similar expression analyses and reverse genetics data on the AP1 gene family (e.g., Huijser et al. 1992; Rosin et al. 2003) show that A function (specification of sepal and petal identity) may be derived in the angiosperms, evolving some time during the diversification of the core eudicots (Theissen et al. 2000; Litt and Irish 2003). Arabidopsis has three AP1-like genes, AP1, CAULIFLOWER (CAL), and FRUITFULL (FUL), all of which act redundantly in the transition from vegetative to inflorescence development by specifying the identity of the floral meristems that give rise to flowers (Kempin et al. 1995; Mandel and Yanofsky 1995; Ferra´ndiz et al.
2000) (Figure 1A). Unlike CAL, AP1 and FUL also act nonredundantly, with FUL being involved in the proper development of both fruit and leaves (Gu et al. 1998; Liljegren et al. 2004). Sequence analyses of MADS-box genes spanning both angiosperms and gymnosperms show that all members of the AP1/FUL, AGL6, and SEP gene subfamilies, except for the core eudicot AP1 clade, share a conserved hydrophobic motif in the C-terminal domain (Litt and Irish 2003; Zahn et al. 2005). This observation suggests that the ancestral AP1/FUL gene may have been functionally closer to FUL than AP1, with a conserved role in meristem identity and possibly fruit development within the monocots and basal angiosperms (Litt and Irish 2003). Mutations in both CAL and AP1 of Arabidopsis result in the proliferation of inflorescence meristems in positions that would normally develop flowers. Unlike ap1/cal double mutants, ap1/cal/ful triple mutants never produce flowers but rather continue to reiterate the development of leafy shoots (Ferra´ndiz et al. 2000). Similar mutations in the AP1 homolog SQUAMOSA of Antirrhinum result in replacement of flowers with shoots subtended by bracts (Carpenter et al. 1995). As in ap1 cal double mutants, squa mutants occasionally produce flowers, but these are often misshapen and are subtended by prophyll-like structures (Huijser et al. 1992). In wild-type plants, interactions between AP1/ FUL and other genes (see Muller et al. 2001; Yu et al. 2004) suppress the development of nonfloral meristems, allowing subsequent development of flowers. The grass family (Poaceae) comprises an estimated 10,000 species diverging from two successive sister families, Ecdeicoleaceae and Joinvilleaceae, 55–70 million
Evolution of AP1/FUL-Like Genes
Figure 2.—Simplified grass phylogeny showing subfamilial relationships. Lowercase names are grass subfamilies. Uppercase names are sister families to grasses. Names in parentheses are examples of genera within subfamilies. BEP, Bambusoideae–Ehrhartoideae–Pooideae clade; PACCAD, Panicoideae– Arundinoideae–Centothecoideae–Chloridoideae–Aristidoideae-Danthoniodeae clade. Based on Grass Phylogeny Working Group (2001).
years ago (MYA) (Kellogg 2001; Bremer 2002). Most species can be placed into two large clades, BEP and PACCAD, each representing two major radiations 40– 50 MYA (Bremer 2002) (Figure 2). Grasses are morphologically unique in having highly modified flowers, each developing within a larger floral structure termed the grass spikelet (Cheng et al. 1983; Clifford 1987; Ikeda et al. 2004) (Figure 1C). Each spikelet is generally composed of one to two basal bracts (glumes) enclosing one or more flowers, each of which is subtended by two more bracts, the palea and lemma. Spikelet literally means ‘‘little spike,’’ referring to its similarity to an indeterminate branching inflorescence developing within the larger inflorescence. Depending on the species, spikelet meristems will produce one to several floral meristems; these will then develop flowers. Grass flowers can be bisexual, unisexual, or reduced. Bisexual flowers contain an outer whorl of lodicules (modified perianth), an inner whorl of stamens, and a central gynoecium. Unlike lodicules, stamens, and carpels, the lemmas, paleae, glumes, and the spikelet as a whole have no obvious counterpart within flowering plants (Figure 1). The role of ABCDE gene homologs in grass spikelet development is starting to be elucidated (reviewed in Bommert et al. 2005; Whipple and Schmidt 2006). Genetic studies in rice and maize suggest both functional conservation and diversification of related genes between Arabidopsis and grasses. Rice has two AG-like genes (OsMADS58 and OsMADS3), one of which is involved in stamen identity (OsMADS58) and the other in floral meristem and carpel identity (OsMADS3) (Yamaguchi et al. 2006). Likewise, in both maize and rice, AP3-like orthologs Silky1 and SUPERWOMAN1,
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respectively, play roles in stamen and lodicule identity (Ambrose et al. 2000; Nagasawa et al. 2003) and PI-like proteins function in stamen identity (Whipple et al. 2004). In contrast to B- and C-class-related genes, the rice SEPALLATA-like gene LHS1 is involved in specifying the identity of floral organs unique to grasses. Mutant lhs1 plants have leafy lemmas and paleae ( Jeon et al. 2000; Prasad et al. 2001), and expression analyses indicate a conservation of this role across grasses (Malcomber and Kellogg 2004). In Arabidopsis, AP1/FUL-like genes specify floral meristem identity and outer whorl organogenesis (Kempin et al. 1995; Mandel and Yanofsky 1995; Ferra´ndiz et al. 2000) but their function remains largely unknown in grasses (but see Trevaskis et al. 2003; Yanet al. 2003; Petersen et al. 2004). It is therefore of great interest to determine the role of grass AP1/FULlike homologs in the novel developmental pathway of the spikelet. A crucial step in this endeavor is to reconstruct the evolutionary history of these genes within grasses. Phylogenetic studies that include grass AP1/FUL genes have shown that diploid members of Poaceae possess three copies derived from two duplication events (Litt and Irish 2003). The first duplication apparently occurred around the base of the monocots, giving rise to the FUL3 clade. The other occurred somewhat later, at some time during the divergence of the Commelinid clade (Arecales 1 Commelinales 1 Poales 1 Zingiberales) (Graham et al. 2006), giving rise to the FUL1 and FUL2 clades (Litt and Irish 2003). Studies of expression of the latter two genes within spikelets of maize (Zea mays), barley (Hordeum vulgare), ryegrass (Lolium temulentum), and rice (Oryza sativa) have shown heterogeneous spatio-temporal patterns, between both orthologs in different taxa (Mena et al. 1995; Schmitz et al. 2000; Gocal et al. 2001; Pelucchi et al. 2002) and paralogs in the same taxa (Kyozuka et al. 2000; Pelucchi et al. 2002). In addition, reports of differences in the timing of expression within meristems (Schmitz et al. 2000) and the absence of FUL2 RNA in leaves (Mena et al. 1995; Schmitz et al. 2000; Gocal et al. 2001) suggest a possible diversification of roles following the origin of FUL1 and FUL2. Upregulation of FUL1 gene expression in shoot apical meristems and leaves of barley (Schmitz et al. 2000; Danyluk et al. 2003; Trevaskis et al. 2003; Von Zitzewitz et al. 2005), ryegrass (Petersen et al. 2004, 2006), and wheat (Triticum monococcum and T. aestivum) (Danyluk et al. 2003; Trevaskis et al. 2003; Yan et al. 2003) has been correlated with the transition to flowering, indicating a role for these genes in competency to flower. It has also been suggested that grass AP1/FUL genes may play a general role in floral meristem identity and/or a more specific role in spikelet organ identity (Gocal et al. 2001; Cooper et al. 2003). Few studies have focused on the evolution of AP1/FUL genes in plant families outside those of the model species of core eudicots. One exception is the study of
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AP1/FUL genes within a few grass species (e.g., Mena et al. 1995; Gocal et al. 2001; Pelucchi et al. 2002; Yan et al. 2003). We would like to broaden our understanding of AP1/FUL gene diversification in grasses by concentrating on four questions. First, did the duplication event that gave rise to FUL1 and FUL2 occur before or after the origin of grasses? By determining the timing of this event we can both assess whether gene duplication is correlated with the origin of the grass spikelet and establish patterns of molecular evolution before and after the duplication event. Second, do patterns of codon evolution following the duplication event suggest different fates of either paralog? To test for evidence of neofunctionalization under positive Darwinian selection, we use codon models specifying different selective pressures along branches immediately following the duplication event and determine bias in codon substitutions across the phylogenetic tree. Third, are patterns of gene expression across grasses consistent with a role for AP1/FUL genes in spikelet development? If both genes have a general role in spikelet development their expression would be expected in glumes and florets across a wide range of grasses. Finally, are patterns of gene expression across grasses consistent with functional diversification of the genes? Organ-specific patterns of expression in taxa containing genes similar to the ancestral gene (before the duplication event) and taxa containing the descendent genes (after the duplication event) can illuminate changes in expression consistent with redundancy, subfunctionalization, or neofunctionalization.
MATERIALS AND METHODS Study species: We included sequence data on 22 species of Poaceae in this study, of which 17 are newly sampled here. For 14 of these we also obtained gene expression data. Two species of Poaceae represent early diverging lineages within the family and the rest span the two major remaining lineages. Withingroup choices were based on both availability and, more importantly, variation in spikelet morphology, particularly in number and position of fertile and reduced florets. We also generated new sequence data on three non-grass Poales, two of which were included in the gene expression analysis. Finally, we included seven monocots outside the Poales, two of which are newly sampled here. For three of these we obtained gene expression data. Seeds were obtained either from the United States Department of Agriculture or from preexisting seed stocks, and plants were grown at the University of Missouri– St. Louis or the Missouri Botanical Garden. The GenBank accession numbers for each AP1/FUL-like sequence included in this study are listed in Table 1. Isolation and sequencing of AP1/FUL genes: Total RNA was extracted from inflorescence tissue using RNAwiz solution (Ambion, Austin, TX) according to the manufacturer’s instructions. A pool of cDNA including all MADS-box genes was generated from the extracted RNA using Superscript OneStep RT–PCR with Platinum Taq (Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions, using a degenerate primer designed to bind at the 59 end of the MADS box (MADS1F; 59-ATG GGT MGS GGS AAG GTG GAG CTG AAG
CGG-39) (Malcomber and Kellogg 2004) and a polyT primer with adaptor (59-CCG GAT CCT CTA GAG CGG CCG CTT TTT TTT TTT TTT TTT-39). Each reaction was run for 30 cycles with an annealing temperature of 52° on an MJ Research PTC-200 thermocycler (GMI, Ramsey, MN). AP1/ FUL homologs from grasses and non-grass Poales were PCR amplified from the pooled MADS-box cDNAs using a forward primer designed to bind at the 39 end of the MADS box (ZAP4F; 59-ATC TCC GTS CTC TGY GA-39) and a reverse primer at the 39 end of the C-terminal domain (ZAP1oneR; 59GAR GKK GCT CAG CAT CCA T-39). Because of problems with the 39 primer, in some species we used a primer at the 59 end of the C-terminal domain (ZAP1fourR; 59-AGR RYC TTG TTC TCC TCC TG-39), producing a slightly truncated PCR product. AP1/FUL homologs from monocots outside Poales were amplified using the 59 MADS-box primer MADS1F and one of the primers shown in supplemental Table 1 (http://www. genetics.org/supplemental/). Each reaction was run for 30 cycles with an annealing temperature of 55°. PCR products were gel purified through a QIAquick spin column (QIAGEN, Valencia, CA) and subcloned into the pGEM-T easy vector (Promega, Madison, WI). Plasmid DNA for 10 clones per ligation was isolated through a QIAprep spin miniprep column (QIAGEN), and sequenced using the Big Dye 3.1 terminator cycle sequencing protocol (Applied Biosystems, Foster City, CA) with the plasmid primers T7 and SP6. Sequencing reactions were analyzed on an ABI377 automated DNA sequencer (Applied Biosystems). Doublestranded sequences were aligned and edited in SeqManII (DNASTAR, Madison, WI), and base callings with Phred scores (Ewing et al. 1998) ,20 were resequenced. Genes were named according to the clade to which they belong (FUL1 or FUL2), plus the first letters of the genus and species name, respectively. Thus the FUL1 gene from Chasmanthium latifolium is ClFUL1, from Eleusine indica, EiFUL1, etc. Phylogenetic analyses: Nucleotide sequences were translated into amino acids and manually aligned using MacClade 4 (Maddison and Maddison 2003) (supplemental Figure 1 at http://www.genetics.org/supplemental/) with amino acid sequences derived from previously characterized GenBank sequences. After alignment, nucleotide sequences were analyzed with PAUP version 4.0b10 (Swofford 2001) and MrBayes 3.0 (Huelsenbeck and Ronquist 2001). A maximum parsimony (MP) heuristic search was conducted using 1000 random addition sequences with TBR branch swapping and gaps treated as missing data. Full heuristic bootstrap analyses (Felsenstein 1985) were conducted using 500 replicates. Parameters for the maximum-likelihood (ML) and Bayesian analyses were estimated using MrModelTest 2.2 (Nylander 2004). The ML analysis was performed with 10 random addition sequences and Bayesian phylogenetic estimates were run for 3 3 106 generations on a Beowulf cluster (University of Missouri, St. Louis). Both ML and Bayesian analyses implemented the GTR 1 I 1 G model of evolution on the basis of the results from MrModelTest 2.2. A Shimodaira–Hasegawa (SH) test (Goldman et al. 2000) was undertaken to test whether we could statistically reject alternative hypotheses for the timing of the AP1/FUL duplication. A priori constraint trees were created in MacClade. Each topology differed in the placement of Joinvillea (Joinvilleaceae) and Xyris (Xyridaceae) FUL sequences and the basal grasses Streptochaeta (Anomochlooideae) and Pharus (Pharidoideae) FUL2 sequences as follows: (1) Joinvillea JaFUL sister to all FUL1 sequences, (2) Joinvillea JaFUL sister to all FUL2 sequences, (3) Streptochaeta SaFUL2 sister to both FUL1 and FUL2 clades, (4) Streptochaeta SaFUL2 and Pharus PsFUL2 sister to both FUL1 and FUL2 clades, (5) Xyris XsFUL and Joinvillea sister to all FUL1 sequences, and (6) Xyris XsFUL and
Evolution of AP1/FUL-Like Genes
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TABLE 1 AP1/FUL homologs included in this study Family/subfamily
Genea
Accession no.
Dendrobium grex Crocus sativus Lilium lancifolium Allium sp. Agapanthus africanus Elaeis guineensis Tradescantia virginiana Cyperus longus Xyris sp. Joinvillea ascendens Streptochaeta angustifolia Pharus sp. Dendrocalamus latiflorus Lithachne humilis Oryza sativa
Orchidaceae/— Iridaceae/— Liliaceae/— Alliaceae/— Agapanthaceae/— Arecaceae/— Commelinaceae/— Cyperaceae/Cyperoideae Xyridaceae/Xyridoideae Joinvilleaceae/— Poaceae/Anomochlooideae Poaceae/Pharoideae Poaceae/Bambusoideae Poaceae/Bambusoideae Poaceae/Ehrhartoideae
Ehrharta erecta
Poaceae/Ehrhartoideae
Leersia virginica
Poaceae/Ehrhartoideae
Brachypodium distachyon
Poaceae/Pooideae
Triticum aestivum Triticum monococcum
Poaceae/Pooideae Poaceae/Pooideae
Hordeum vulgare
Poaceae/Pooideae
Lolium temulentum
Poaceae/Pooideae
Phalaris canariensis
Poaceae/Pooideae
Avena strigosa Avena sativa
Poaceae/Pooideae Poaceae/Pooideae
Eleusine indica
Poaceae/Chloridoideae
Eragrostis pilosa
Poaceae/ Chloridoideae
Chasmanthium latifolium
Poaceae/Centothecoideae
Setaria viridis Setaria italica
Poaceae/Panicoideae Poaceae/Panicoideae
Sorghum bicolor Zea mays
Poaceae/Panicoideae Poaceae/Panicoideae Poaceae/Panicoideae
Pennisetum glaucum
Poaceae/Panicoideae
DOMADS2 Kcap1b LisFUL AlFL AgsFUL PAS1 TvFL1 CylFUL XsFUL JaFUL SaFUL2 PsFUL2 DlMADS1 LhFUL2 OsMADS14 OsMADS15 OsMADS18 EeFUL1 EeFUL2 LvFUL1 LvFUL2 BdFUL1 BdFUL2a BdFUL2b TaMADS11 TmFUL2a TmFUL2b HvMADS3 HvMADS5 HvMADS8 LtMADS1 LtMADS2 PcFUL1 PcFUL2 AstFUL1 AsFUL1a AsFUL1b AsFUL2 EiFUL1a EiFUL1b EiFUL2 EpFUL1 EpFUL2 ClFUL1 ClFUL2 SvFUL1 SiFUL1 SiFUL2 SbFUL1 SbMADS2 ZmMADS4 ZmMADS15 ZmMADS3 ZAP1 PgFUL1a PgFUL1b
AF198175 AY337929 DQ792945 AY306138 DQ792946 AF411840 AY306190 DQ792947 DQ792948 DQ792949 DQ792950 DQ792951 AY395714 DQ792952 AF058697 AF058698 AF091458 DQ792953 DQ792954 DQ792955 DQ792956 DQ792957 DQ792958 DQ792959 AB007504 DQ792960 DQ792961 AJ249143 AJ249144 AJ249146 AF035378 AF035379 DQ792962 DQ792963 DQ792964 DQ792965 DQ792966 DQ792967 DQ792968 DQ792969 DQ792970 DQ792971 DQ792972 DQ792973 DQ792974 DQ792975 DQ792976 DQ792977 DQ792978 U32110 AJ430641 AJ430632 AF112150 L46400 DQ792979 DQ792980
Taxon
Voucher information for each sequence is included in GenBank. Newly generated sequences are underlined. a Gene names of new sequences correspond to the species’ initials and the putative phylogenetic position: prior to the duplication event (FUL), following the duplication event in the OsMADS14 clade (FUL1), or following the duplication event in the OsMADS15 clade (FUL2). Distinct alleles are designated a or b.
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Joinvillea JaFUL sister to all FUL2 sequences. Each constraint tree was tested against the unconstrained ML phylogeny. Maximum parsimony ancestral state reconstructions were estimated in MacClade 4 (Maddison and Maddison 2003) after supplementation with previously reported sequence and expression data for Crocus (Iridaceae) (Tsaftaris et al. 2004) and Dendrobium (Orchidaceae) (Yu and Goh 2000). Southern blot hybridization: Total DNA was extracted from 250 mg of leaf tissue from H. vulgare, Sorghum bicolor, Pharus sp., and Streptochaeta angustifolia, using a modified protocol of Doyle and Doyle (1987). Approximately 10 mg of total DNA was digested with the enzymes BamHI, EcoRI, or HindIII, and digested DNA was run on a 0.8% agarose gel overnight. Digested DNA was blotted onto a nylon membrane (Amersham Biosciences, Piscataway, NJ) and hybridized at moderate stringency (16 hr at 60°) with a 32P-dCTP-labeled FUL2 genespecific probe spanning the KC domains and the 39-UTR. To remove nonspecific binding of the probes each membrane was washed twice with 23 SSC/1% SDS and twice with 13 SSC/1% SDS at 65°, following the protocol of Laurie et al. (1993). Tests for positive selection: If natural selection has driven multiple adaptive changes in amino acids in a protein, theory predicts that the number of mutations leading to amino acid changes (nonsynonymous mutations, or dN) should be significantly larger than the number of mutations at synonymous sites (dS) (Miyata and Yasunaga 1980; Li 1997). Conversely, if selection consistently weeds out amino acid changes, then dN should be significantly smaller than dS. Thus the nature of selection can be assessed by the ratio of dN/dS; for convenience, dN/dS is commonly abbreviated by the symbol v. If the ratio is much .1, then adaptive (positive) selection is inferred; if ,1, then negative selection; and if the ratio is 1, then the sequence is inferred to be evolving neutrally. To test for evidence of positive selection following the AP1/FUL duplication, the best-supported tree obtained from the larger phylogenetic analysis was pruned in MacClade (as indicated in Figure 3) and evaluated using the program codeml from the PAML package, version 3.14 (Yang 1997). The pruned tree only included sequences that spanned all four MIKC domains as follows: CylFUL, XsFUL, OsMADS14, TaMADS11, HvMADS5, LtMADS1, AsFUL1, EiFUL1, ClFUL1, ZmMADS4, ZmMADS15, OsMADS15, HvMADS8, LtMADS2, AsFUL2, EiFUL2, ZmMADS3, ZAP1, SbMADS2, SvFUL2, PsFUL2, and SaFUL2. PAML calculates the likelihood of the data given the tree under a set of increasingly complex models of evolution. The likelihood values are then tested statistically to determine whether the complex models fit the data significantly better than the simple ones, and thus if there is significant evidence of positive selection. We tested three models, all of which assume that the entire coding region is evolving the same way over the entire tree. These models were M0, the one-ratio test assuming equal k (ratio of transition to transversion rates), codon usage, and v (ratio of nonsynonymous to synonymous substitutions or dN/ dS) over all sites and branches; M1a, a model of nearly neutral evolution (0 , v1 , 1) averaged over all sites and branches; and M2a, a discrete model assuming positive selection (v1 . 1) averaged over all branches and sites (Yang et al. 2000). We would infer positive selection if M2a proved to be significantly better than either M0 or M1a. It is unlikely that all residues in a protein, and therefore all sites in a coding sequence, are subject to the same selection pressures. We therefore tested three models that allow the evolutionary rate to vary among sites but keep the rate the same for a given site over all branches in the tree. These differ in their assumptions about the distribution of sites and in the possibility of positive selection: M3 (discrete distribution), M7 (b distribution), and M8 (b distribution and positive selection) (Bielawski and Yang 2003). We would infer positive
selection if M8 proved to be significantly better than either M3 or M7. The ML-based method can produce different estimates of v at a given codon site, depending on the input v-value, due to multiple local maxima of the likelihood function (Suzuki and Nei 2001; Wong et al. 2004). For this reason, we reran models 2, 3, 7, and 8 using 0.4, 3.14, and 4 as the input v-values. Only the results with the highest log-likelihood [InL] values are presented. We wished to test explicitly for increased positive selection along all branches subsequent to the duplication event (two ratio) and increased followed by decreased positive selection after the duplication event (three ratio) (Yang 1998). To do this we used models that could incorporate heterogeneity over both sites and branches by specifying two v-ratios. Model A assumes 0 , v0 , 1 (estimated) and v1 ¼ 1, while model B determines both v0 and v1 as free parameters to be estimated from the data. In each case v1 was specified as the branch immediately following the duplication event and leading to clade FUL1 or FUL2, respectively (Bielawski and Yang 2003). Nested models were compared against a x2 distribution of trees using a likelihood ratio test (LRT). M0 was compared with M3 and M7 with M8 to test for uniformity of parameters over different sites. M1a was compared with M2a to examine whether v ¼ 1 or .1. To test for increased v following the duplication event, M0 was compared with the two-ratio model, and the two- with the three-ratio model. Finally, to incorporate heterogeneity of parameters over both branches and sites, M1a and null model A with v2 fixed at 1 were compared with model A, and null model B with only two site categories was compared with model B. To identify amino acid sites distinguishing clade FUL1 from FUL2, codon transitions were mapped onto the AP1/FUL gene tree in MacClade 4 (Maddison and Maddison 2003). The binomial test (Pr ¼ pk) (Sokal and Rolf 2001) was used to determine if amino acid replacements were significantly more prevalent within FUL1 or FUL2 gene lineages prior to divergence of the two main grass lineages [i.e., the rice-containing BEP clade and the maize-containing PACCAD clade (Figure 2)]. Pr is the probability that the difference between replacement substitutions in the two lineages is at least as extreme as observed, p is the probability of change, and k is the number of codon synapomorphies discriminating either clade. The parameter p was set at 0.5 and thus assumed an equal time available for change of either gene following the duplication event. Reverse transcriptase (RT)–PCR characterization of expression: Total RNA was extracted separately from stems, leaves, outer tepals, inner tepals, stamens and carpels of nongrass monocots, and roots, stems, leaves, glumes, and florets of grasses as described above. For each species and each organ, RNA was extracted at least twice. To detect even low abundance AP1/FUL-like gene transcripts, RT–PCR used a nested PCR approach. We preferred this approach because of its reliability in determining presence or absence of transcripts, even though it prevents assessment of their relative abundance. RNA was first reverse transcribed into MADS-boxspecific cDNA using the primer MADS1F (at the translation start site) and polyT-anchor in a one-step RT–PCR as described above. PCR products were then diluted 1:100 and 1-ml template was used in the nested PCR. For the non-grass monocot species forward or reverse gene-specific primers (supplemental Table 1 at http://www.genetics.org/supplemental/) were designed according to published sequences and used in combination with the reverse primer corresponding to the C terminus (ZAP1oneR) or the forward primer corresponding to the translation start (MADS1F), respectively, to determine gene expression within each organ type. To identify
Evolution of AP1/FUL-Like Genes paralogous copies from each grass subfamily, forward primers (supplemental Table 1 at http://www.genetics.org/supplemental/) were designed on the basis of aligned sequences used in the phylogenetic analysis. Each forward primer was used in combination with ZAP1oneR, which spans the conserved hydrophobic motif of the C-terminal domain, to amplify copy- and cDNA-specific products. As a positive control, the constitutively expressed actin gene was amplified using the primers ACT-59F (59-AGG CTG GTT TCG CTG GGG ATG ATG-39) and ACTIN-764R (59-GGA CCT CGG GGC ACC TGA ACC TCT-39). Each reaction was run for 30 cycles with an annealing temperature of 52°–55°. Two independent experiments were carried out for each organ type. When no expression was detected, each experiment was repeated two more times using the same RNA. To verify results from the nested RT–PCR experiment and to assess approximately relative levels of transcripts, semiquantitative RT–PCR was carried out for Oryza, Sorghum, Chasmanthium, and Eleusine. Gene-specific primers were designed in the reverse orientation and used in combination with forward primers described above (supplemental Table 1 at http://www.genetics.org/supplemental/). To control for DNA contamination, amplified products spanned intron– exon boundaries within the C terminus and 39-UTR. For each RNA sample, actin was amplified for 25 and 30 cycles with an annealing temperature of 55° as below. Bands were resolved on agarose gels and their relative intensities determined by densitometric analysis in ImageJ (Abramoff et al. 2004). PCR conditions for each target gene were one ‘‘hot start’’ cycle at 94° for 5 min, followed by 25–35 cycles of 94° for 30 sec, 55°– 58° for 30 sec and 72° for 1 min, followed by a final extension step at 72° for 10 min. Different numbers of cycles were carried out to confirm linearity of each amplification (He et al. 1995; Freeman et al. 1999). Target bands were quantified in ImageJ (Abramoff et al. 2004) and standardized against actin. Unlike the nested RT–PCR approach, semiquantitative PCR is likely to miss low abundance RNAs.
RESULTS
Phylogenetic analyses of AP1/FUL genes: We generated AP1/FUL sequences for 22 monocot taxa. Of these newly generated sequences, most spanned the entire coding region of the gene minus the first 100–150 bp of the MADS-box domain. In the case of eight sequences, sequence coverage was less because either the reverse primer ZAP1fourR was used to give a truncated product ending at the 59 end of the C terminus or a portion of the sequence had Phred scores ,20: EeFUL1 (C domain only), PcFUL2 (K–C domains), LvFUL2 (K–C domains), ClFUL2 (M–K domains), JaFUL (M–K domains), AgsFUL (M–K), SbFUL1 (M–K), and LisFUL (M–I). Nucleotide sequence divergence between the IKC domains of paralogous genes was 23%, with the C domain showing the highest pairwise divergence of 33%. Distinct AP1/ FUL alleles sharing .95% nucleotide identity were sequenced for AsFUL1, BdFUL2, EiFUL1, PgFUL1, and TmFUL2. All loci for which C-terminal sequence was obtained had the FUL-motif (L/MPPWML/V) (Litt and Irish 2003). No premature stop codons were found, suggesting that all genes sequenced could be functional. Only one FUL2-like sequence was retrieved
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for Streptochaeta and for Pharus; despite multiple attempts at PCR and sequencing of multiple clones, we did not detect a FUL1-like sequence in these species. ML analyses gave one tree with a likelihood score of 14,118 (Figure 3). Tree topologies from each of the MP, ML, and Bayesian analyses showed no significant differences, except for the arrangement of the non-grass Poales genes from Xyris, Cyperus, and Joinvillea. Each tree was largely similar to those found in previous molecular phylogenetic analyses (Grass Phylogeny Working Group 2001) except for the placement of genes from three ehrhartoid species (Oryza, Leersia, and Ehrharta). Phylogenetic analyses strongly support the existence of three distinct grass AP1/FUL clades, FUL1, FUL2, and FUL3. Placement of Joinvillea, Xyris, and Cyperus genes as sister to the grass FUL1 and FUL2 clades indicates that the duplication event that gave rise to FUL1 and FUL2 occurred near the base of Poaceae. This relationship is supported by both the MP bootstrap (84%) and Bayesian analyses (99%). Placement of the Streptochaeta sequence within the FUL2 clade is also strongly supported by both bootstrap (99%) and Bayesian (100%) analyses, indicating that the duplication event occurred prior to the divergence of the most basally placed grasses (Figure 3). The SH test rejects, at the P , 0.05 level, all topologies that explicitly test for alternative origins of the duplication event (data not shown). In agreement with the nonparametric bootstrap and Bayesian posterior probabilities, these data strongly support the hypothesis that the AP1/FUL duplication event occurred prior to the common ancestor of extant grasses (Figure 3). AP1/FUL gene copy number: PCR identified only one FUL sequence in Streptochaeta and Pharus. To test whether we had failed to amplify a second sequence, we undertook Southern blot hybridization of probes spanning the KC domains and 39-UTR of FUL2 from each species as well as from two diploid grasses representing the two major grass radiations, Sorghum (Panicoideae) and Hordeum (Pooideae). The latter two possess two and three similar copies of AP1/FUL-like genes, respectively (data not shown), consistent with reports from other studies on Hordeum AP1/FUL-like genes demonstrating two copies of HvMADS5 (FUL1) within the barley genome (Schmitz et al. 2000; Von Zitzewitz et al. 2005). In contrast to these findings, only one band was identified on the blots of Pharus and Streptochaeta (data not shown), consistent with our PCR results. This supports the hypothesis of two independent losses of FUL1 within early-diverging lineages of grasses. Banding patterns for Sorghum, Hordeum, and Streptochaeta were confirmed using gene-specific probes hybridized under high stringency conditions (data not shown). Tests of selection: Data from the pruned AP1/FUL tree could not reject a nearly neutral model (M1a) (2D‘ ¼ 0.00) of evolution, indicating no evidence of positive selection averaged across the entire tree.
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Figure 3.—Maximum-likelihood tree using GTR 1 I 1 G model of evolution. Bayesian posterior probabilities .90 shown above the branches, MP nonparametric bootstrap values .70 shown below branches. Solid circle, inferred duplication event; circle with x, sequences included in the PAML analysis; A, foreground branch 1 in branch and branch-site models; B, foreground branch 2 in branch and branch-site models. Arrows indicate origin of the grass spikelet. Shaded boxes indicate taxa lacking a spikelet.
However, sites are not evolving homogeneously across the coding region of the gene. The more complex discrete model (M3), which allows two or more unrestricted site classes, was significantly better at explaining the data (2D‘ ¼ 199.26, P , 0.01) than the oneratio (M0) model. Codons fall into different classes with v-values ranging from 0.01531 (near purifying selection) to 0.89122 (relaxed selection) (Table 2). There is no significant signature of positive selection on sites following the duplication event, indicating that any nonsynonymous substitutions along these branches are probably the result of relaxed selection following functional redundancy. A model allowing an additional site class under positive selection (M8) has a likelihood score not significantly different from the model restricting all v-values to #1 (M7) (2D‘ ¼ 0.9) (Table 2), meaning that positive selection is not necessary to explain the data. Similarly, under the branch models
(two ratios and three ratios) that specify a different evolutionary rate on branches following the duplication event, likelihood scores are not significantly better than the most simple branch model specifying a single v-value for all branches (M0) (2D‘ ¼ 0.34 and 0.1). Indeed, despite strong evidence that different codons are experiencing different selection pressures, branchsite models (A and B) specifying branch FUL1 or FUL2 as the foreground branch were not significantly better at explaining the data than the nested site models [M1a (2D‘ ¼ 0.00 and 0.98) and null model A (2D‘ ¼ 0.00 and 0.00), and null model B (2D‘ ¼ 0.00 and 0.00), respectively]. Multiple codon substitutions within the FUL2 lineage prior to diversification of the BEP/PACCAD clades: Twenty-one codon substitutions were found in the FUL2 lineage prior to diversification of the BEP and PACCAD clades (8 before the divergence of SaFUL2, 7 before
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TABLE 2 Codon model parameter estimates for AP1/FUL paralogs Model
p
‘
M0: one ratio
1
3714.44
v ¼ 0.1181
None
Branch-specific models Two ratios Three ratios
2 3
3714.27 3714.22
1 ¼ 0.1525 v 0 ¼ 0.1167, v v 0 ¼ 0.1223, v 1 ¼ 0.1527 v 2 ¼ 0.1147
None None
Site-specific models M1a: near neutral M2a: selection
2 4
3635.68 3635.68
Not allowed None
M3: discrete (K ¼ 3)
3
3614.81
M7: b M8: b and v
2 4
3618.86 3619.31
p0 ¼ 0.87366, (p1 ¼ 0.12634) p0 ¼ 0.87368, p1 ¼ 0.12632 (p2 ¼ 0.00000), v 2 ¼ ‘ p0 ¼ 0.52591, (p1 ¼ 0.39457), p2 ¼ 0.07953, v 0 ¼ 0.01531 v 1 ¼ 0.18259, v 2 ¼ 0.89122 p ¼ 0.32839, q ¼ 1.74796 p0 ¼ 0.99707, p ¼ 0.32866 q ¼ 1.75003, (p1 ¼ 0.00293) v ¼ 118.19
Branch-site models Model A: FUL1 (¼ null Model A)
4
3635.68
Model B: FUL1 (¼ null Model B)
5
3626.84
Model A: FUL2 (¼ null Model A)
4
3635.19
Model B: FUL2 (¼ null Model B)
5
3626.84
Estimates of parameters
p0 ¼ 0.87366, p1 ¼ 0.12634, (p2a 1 p2b ¼ 0.000) v 0 ¼ 0.0729 v 2a ¼ 1.00000, v 2b ¼ 1.00000 p0 ¼ 0.80202, p1 ¼ 0.19798, (p2 1 p3 ¼ 0.00), v 0 ¼ 0.05027 v 1 ¼ 0.52984, v 2 ¼ ‘ p0 ¼ 0.77157, p1 ¼ 0.11071 (p2a 1 p2b ¼ 0.11773) v 0 ¼ 0.0714, v 2a ¼ 1.00000 v 2b ¼ 1.00000 p0 ¼ 0.80202, p1 ¼ 0.19798, (p2 1 p3 ¼ 0.00), v 0 ¼ 0.05027 v 1 ¼ 0.52984, v 2 ¼ ‘
Positively selected sites
None
Not allowed 167V (P ¼ 0.86) 207T (P ¼ 0.60) 208T (P ¼ 0.73) 215L (P ¼ 0.68)
None
None 45L (P ¼ 0.69) 50T (P ¼ 0.62) 145Q (P ¼ 0.61) 206D (P ¼ 0.51)
Parameter estimates that are underlined indicate positive selection; those in parentheses are not free parameters (p). Putative sites under positive selection are based on the coding region of rice OsMADS14. Estimates of k ranged between 1.74 and 1.9. ‘, log likelihood; p, number of parameters; q, free parameter.
divergence of PsFUL2, and 6 before divergence of the ehrhartoids), in contrast to 0 codon substitutions in the corresponding branch leading to the FUL1 lineage (Figure 4, first number below each branch). Six of these 21 substitutions are unique to the entire tree (not shown). Of the 8 substitutions that occurred before the divergence of Streptochaeta, 4 are conserved for the entire FUL2 clade (Figure 4, boxed number above branch): one each within the MADS box (M) (26), intervening (I) (79), keratin-like (K) (145), and C-terminal (C) (175) domains (Figure 5). Of the 7 codon substitutions occurring after the Streptochaeta divergence and before that of Pharus, 5 (47, 88, 102, 109, 159) are conserved. Of the 6 substitutions that occurred after the divergence of Pharus, 4 (182, 233, 240, 266) of these changes are conserved, respectively (Figures 4 and 5). Thus early in grass diversification, the FUL2 genes
accumulated 21 nonsynonymous mutations, of which 13 have remained unchanged for 50 million years. These codon changes are not biased to any particular functional domain within the gene. The trend of nonsynonymous substitutions within the FUL2 lineage immediately following AP1/FUL-like gene duplication is highly significant and is consistent with a hypothesis of neofunctionalization in the FUL2 clade. MP reconstructions of ancestral AP1/FUL amino acid sequences estimate that no codon changes occurred within the FUL1 lineage prior to BEP-PACCAD clade diversification (Figure 4). According to the binomial test, the probability that all thirteen conserved codon substitutions occurred by chance only in the FUL2 lineage prior to BEP-PACCAD diversification (Pr) is 0.0001. We tested whether the difference simply reflects an increase in the rate of evolution in the FUL2 clade by
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Figure 4.—Inferred history of codon substitutions within grass FUL1 and FUL2 clades based on maximum parsimony ancestral reconstructions. Numbers below the branches indicate total number of nonsynonymous substitutions and number of unique nonsynonymous substitutions, respectively. Branches with thick lines represent the period of time between gene duplication and diversification of the BEP/PACCAD clades; one branch in the FUL1 lineage is equal to three branches in the FUL2 lineage since Streptochaeta and Pharus have lost their FUL1 copy. Numbers in boxes above these branches denote the number of unreversed nonsynonymous substitutions.
comparing the number of nonsynonymous substitutions after the common ancestor of Zea and Oryza for FUL1 and FUL2. The average number is slightly higher in the FUL1 clade, but the difference between the two clades is not significant. We conclude that the elevated rate of amino acid substitution in FUL2 reflects a change in the selection pressure on the gene soon after its origin by duplication. Analysis of AP1/FUL gene expression in vegetative and floral organs: Expression patterns of AP1/FUL genes in non-grass monocots varied across the five taxa sampled (Figure 6). Expression patterns in Lilium (Liliaceae) (not included in phylogenetic analysis due to short sequence) and Tradescantia (Commelinaceae) are identical, with AP1/FUL transcripts being detected in all vegetative (stem and leaves) and floral organs (outer and inner tepals, stamens, and carpel) sampled. In contrast, in Agapanthus (Agapanthaceae) (not included in phylogenetic analysis due to short sequence) expression is not detected in vegetative tissues but is found in each of the four floral whorls. Interestingly, no transcript is detected in floral bracts of Agapanthus or Cyperus (Cyperaceae) despite multiple independent RT–PCR experiments, indicating that AsFUL and CpFUL expression are both restricted to flowers. Expression of XsFUL in Xyris (Xyridaceae) is the most divergent, with
transcripts being detected in both vegetative (stem) and reproductive organs (carpels), but absent from tepals. The common expression of AP1/FUL genes in carpels is consistent with the hypothesis that monocot AP1/FUL genes may be functionally more similar to Arabidopsis FRUITFULL than APETALA1. Expression patterns of AP1/FUL genes in grasses are generally conserved throughout vegetative tissues and glumes, but show variation in florets (Figure 7). Gene expression in glumes is completely conserved, with both genes being detected in glumes of all taxa examined. In S. angustifolia, a species that lacks a true spikelet (Figure 1D), SaFUL2 is expressed in bracts I–V, VI, and VII–XII, the latter surrounding the stamens and carpel. Within vegetative tissues, FUL1 and FUL2 transcripts are detected in roots, stems, and leaves of most taxa sampled. However, in Streptochaeta (Anomochlooideae) SaFUL2 was never detected in leaves, in Chasmanthium (Centothecoideae) ClFUL1/ClFUL2 were never detected in roots, and in maize (Z. mays: Panicoideae) ZmMADS15/ ZmMADS4/ZmMADS3/ZAP1 were never detected in roots or stems and ZmMADS3/ZAP1 were never detected in leaves. Nested RT–PCR gives a clear picture of presence/ absence of transcripts because the initial step of creating a pool of MADS-box RNAs makes it more likely that rare
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Figure 5.—Aligned AP1/FUL codon sequences from Xyris (XsFUL), Joinvillea (JaFUL), Oryza [OsMADS14 (FUL1) and OsMADS15 (FUL2)], Zea [ZmMADS15 (FUL1) and ZAP1 (FUL2)], Pharus (PsFUL2) and Streptochaeta (SaFUL2). Solid arrowheads indicate conserved codons distinguishing all members of the FUL1 and FUL2 clades in the larger data set. Shaded arrowheads indicate codon changes that are not conserved in all subsequent clades. All changes occurred along the branch leading to the FUL2 clade, and six of the conserved changes resulted in a new class of amino acid.
transcripts will be detected. However, nested RT–PCR provides a misleading picture of relative amounts of transcripts of different loci. Conversely, semiquantitative RT–PCR provides good information on relative transcript abundance but is considerably less sensitive than the nested approach and so will miss low abundance RNAs. In contrast to the nested RT–PCR results (Figure 7), semiquantitative RT–PCR was unable to detect Sorghum SbFUL1/SbFUL2, Chasmanthium ClFUL2, Eleusine EiFUL2, and Oryza OsMADS15 expression in leaves (Figure 8). Follow-up nested RT–PCR on these samples verified that RNA of these genes is present, but at levels too low to detect without first limiting the RNA pool to MADS-box transcripts. Conversely, all transcripts that were detected by the semiquantitative approach were also detected by nested RT–PCR. The semiquantitative results suggest FUL2 transcription in leaves is generally much lower than transcription of FUL1. All grass taxa included in this study bear both fertile and reduced florets except for Streptochaeta, which has
a single fertile floret surrounded by bracts (Figure 1D), and Lithachne (Bambusoideae), Pharus (Pharoideae), and Zea, all of which have reduced staminate or pistillate florets borne singly (Lithachne, Pharus) or in pairs (Zea) within the spikelet. With the exception of Streptochaeta, all fertile florets can be considered homologous. In contrast, reduced florets vary from sterile lemmas (e.g., Oryza) to florets lacking fully developed reproductive organs (e.g., Zea). Reduced florets may be borne below (Oryza, Phalaris, Chasmanthium, Sorghum, Setaria, Zea) or above (Chasmanthium, Eleusine, Eragrostis) the fertile ones, or in a separate spikelet entirely (Pharus, Lithachne, Hordeum). The reduced floret may be underdeveloped (Phalaris, Hordeum, Triticum, Avena, Lolium, Chasmanthium distal), staminate (Pharus, Lithachne, Setaria, Zea tassel), pistillate (Pharus, Lithachne, Eleusine, Eragrostis, Zea ear), or reduced to a sterile lemma (Oryza, Chasmanthium proximal, Sorghum). The mechanisms that cause florets to become variously reduced are unknown and are likely to be different in
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Figure 6.—Non-grass monocot gene-specific RT–PCR products amplified from RNA of stems (ST), leaves (LF), floral bracts (FB), fertile florets (FF), outer tepals (T1), inner tepals (T2), stamens (S), and carpels (C). RNA was extracted from early through mid-stage buds. Actin was used as a positive control.
different taxa. In addition, reduced florets have evolved multiple times within the grasses. The morphological and developmental differences among reduced florets, combined with their independent appearance in evolutionary time, all suggest that reduced florets are nonhomologous; they are comparable only in that they lack one or more floral organs.
Although gene expression varied among species and among florets within spikelets we could find no clear pattern of gene expression that was consistent with floral fertility, position, or number, or taxonomic relatedness (Figures 7 and 8). In reduced florets Zea FUL1 transcripts (ZmMADS4 and ZmMADS15) are detected, whereas Pharus PsFUL2 and Zea ZAP1/ZmMADS3 transcripts are never detected. In fertile florets FUL1 transcripts are detected in all taxa sampled, whereas FUL2 transcripts are never detected in fertile florets of Streptochaeta or Chasmanthium despite repeated attempts. Furthermore, semiquantitative RT–PCR was unable to detect Eleusine EiFUL2 and Oryza OsMADS15 in fertile florets (Figure 8). This suggests an overall lower level of FUL2 gene expression in florets of these taxa compared to expression of FUL1. Reconstruction of ancestral AP1/FUL expression before and after gene duplication: MP reconstructions of ancestral AP1/FUL expression patterns estimate that expression in all four floral whorls is ancestral for all monocot clades included in this study. Expression in leaves is equivocal at the base of the monocots, but is estimated as present in the ancestor of the commelinid clade. This hypothesis suggests that expression in leaves was lost twice within the FUL2 clade, once in Streptochaeta (SaFUL2) and once in Zea (ZmMADS3 and ZAP1). Expression in floral bracts is optimized as absent at the base of monocots but present around the time of gene duplication at the base of grasses. Similarly, AP1/FUL expression in fertile grass florets is inferred as ancestral
Figure 7.—Grass gene-specific RT–PCR products amplified from RNA of roots (RT), stems (ST), leaves (LF), glumes (GL) or bracts (I–V, VI, X–XII; numbered sequentially from outer to inner), fertile florets (FF), and reduced florets (RF). The type of reduced floret and its location within the spikelet is indicated below the RF lane: sterile florets at the base (BS), staminate florets at the base (BST), pistillate florets at the apex (AP), staminate florets from male inflorescence (S), sterile florets in different spikelets from fertile florets (SS), and not applicable (NA). Dashed lines represent missing data. RNA was extracted from a variety of developmental stages except for florets, where RNA was extracted only at mid to late stages of development. Actin was used as a positive control.
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Figure 8.—Semiquantitative RT–PCR of FUL1 and FUL2 amplified from RNA of stems (ST), leaves (LF), glumes (GL), fertile florets (FF), and reduced florets (RF). Histograms show relative expression profiles for FUL1 and FUL2 of Sorghum (A), Chasmanthium (B), Eleusine (C), and Oryza (D) standardized against actin expression. Gel photos below histograms show FUL1 (top), FUL2 (middle), and actin (bottom) bands used for quantification.
for grasses, suggesting that gene expression was again lost in the FUL2 clade, within the lineages leading to Streptochaeta (Anomochlooideae) and Chasmanthium (Centothecoideae). AP1/FUL gene expression in reduced florets was not optimized on the phylogeny because reduced florets are nonhomologous across grasses, and thus comparisons are not meaningful.
DISCUSSION
Gene duplications may lead to novel forms by partitioning of ancestral functions (subfunctionalization) that can then be integrated into divergent developmental modules (Force et al. 1999; Lynch et al. 2001) or by evolution of novel functions (neofunctionalization) that can alter the developmental outcome of preexisting genetic pathways (Ohno 1970; Hughes 1994). Changes in the spatio-temporal patterns of gene expression and/or interactions of their protein products are two ways in which functional divergence can occur (Doebley and Lukens 1998; Becker and Theissen 2003). We have reconstructed the evolutionary history of two paralogous monocot MADS-box transcription factors, FUL1 and FUL2, as a framework to determine
patterns of sequence and gene expression evolution that may be implicated in evolution and/or diversification of the grass spikelet. AP1/FUL genes duplicated prior to evolution of the grass spikelet: Phylogenetic analyses of monocot AP1/ FUL genes support the hypothesis that the gene duplication event giving rise to FUL1 and FUL2 occurred before the common ancestor of extant grasses. Support values from bootstrap and Bayesian analyses, and the SH test, suggest duplication after the divergence of Joinvilleaceae, one of the sister families of Poaceae, but before the split of Anomochlooideae, the earliest diverging grass lineage. These data suggest that the FUL1 and FUL2 paralogs have been maintained in the genome of Poaceae for at least 55–65 million years (Bremer 2002) and thus implicate selection for retention of both gene activities throughout grass evolution (Monson 2003). Other studies have also found genes duplicated at the base of grasses [e.g., Adh1/Adh2 (Gaut et al. 1999)], often associated with changes in expression and/or function. Interestingly, only a single AP1/FUL copy was found in Streptochaeta and Pharus. Streptochaeta is unique in that it lacks a true spikelet, but has a flower-bearing structure surrounded by multiple bracts ( Judziewicz
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and Soderstrom 1997) (Figure 1D). In contrast, Pharus has unisexual single-flowered spikelets that are conventional in structure except for the absence of lodicules in all mature florets of most species ( Judziewicz et al. 1999). The single copy of AP1/FUL in Streptochaeta could be due to either the duplication occurring after the split of Anomochlooideae from the rest of the grasses or, as supported in our phylogenetic analyses, a loss or rapid divergence of FUL1 following the gene duplication event. Loss or rapid divergence of FUL1 is also supported for Pharus by both the SH test and phylogenetic reconstructions, suggesting that FUL1 may have been under relaxed or positive selection independently on at least two occasions. Taken together, these findings suggest a complex history of duplication and divergence and/or loss of AP1/FUL genes at the base of Poaceae, a pattern that correlates with major changes in grass floral morphology. Evidence for a period of relaxed selection followed by stabilizing selection on the branch leading to the FUL2 clade: Codon-based models of evolution suggest that selection on monocot AP1/FUL genes is not homogeneous, but rather varies across sites and lineages. This finding is consistent with the modular structure of MIKC MADS-box genes, where the N-terminal MADS domain is under extreme purifying selection and the C-terminal domain evolves rapidly under a combination of relaxed and positive selection (Becker and Theissen 2003). Comparison of site models testing for evidence of nearly neutral vs. positive selection did not allow rejection of the nearly neutral model. This result does not preclude adaptive amino acid replacements, as the models testing for v . 1.0 may simply lack power to detect a limited number of isolated adaptive events (Wong et al. 2004). Rather, this result suggests that, on average, codon sites are evolving under a combination of stabilizing and relaxed selection. The inferred pattern of codon substitutions suggests that FUL1 and FUL2 genes may have been subject to quite different selective pressures. Our data provide no evidence for positive selection along branches immediately following the gene duplication, but do suggest that sites with elevated nonsynonymous substitutions could have evolved under relaxed selection. Twenty-one and 0 codon substitutions occurred within the FUL2 and FUL1 gene lineages, respectively, prior to BEP/PACCAD diversification (Figure 4). One-quarter of these codon substitutions change the biochemical class of the amino acid. The fact that 13 of these codon substitutions have been maintained, at least since the estimated split of rice and maize 50 MYA (Gaut 2002), suggests that these sites may have been preserved under purifying selection. This pattern is inconsistent with that expected under subfunctionalization. According to the subfunctionalization model, degenerative mutations have no fitness value and are thus not conserved over long periods of time (Force et al. 1999; Lynch et al. 2001). In addition,
subfunctionalization in modular genes generally predicts degeneration within different domains of either duplicate gene (Force et al. 2004). Complex AP1/FUL gene expression patterns are consistent with a combination of incomplete subfunctionalization and redundancy following gene duplication: Recent studies on vernalization (Yan et al. 2003; Jensen et al. 2005; Fu et al. 2005) and photoperiod (Danyluk et al. 2003) responses of cereal cultivars have implicated FUL1 as a possible integrator of grass flowering-time pathways. This putative function is supported by the early flowering phenotypes of rice ( Jeon et al. 2000) and maize (Danilevskaya et al. 2005) ectopically expressing FUL1. In addition, when the AP1/FUL gene from Dendrobium was expressed in Arabidopsis, the resulting plants exhibited an early flowering phenotype (Yu and Goh 2000). In contrast to other studies (Mena et al. 1995; Kyozuka et al. 2000; Gocal et al. 2001; but see Petersen et al. 2004) we found expression of FUL2 in leaves of both seedlings (data not shown) and mature plants. Since quantitative changes in FUL1 leaf expression have been positively correlated with competency to flower (Yanet al. 2003), the expression of both FUL1 and FUL2 in leaves may suggest redundancy of function between these genes with respect to flowering time. Furthermore, the general expression of AP1/FUL genes of non-grass monocots may suggest a conserved role for these genes in flowering time across monocots. Compared to flowering time, the role of AP1/FULlike genes in grass spikelet development is less well known. RNA interference mutants of VRN-1 (FUL1) in T. aestivum (Loukoianov et al. 2005) and ZmMADS3 (FUL2) in Z. mays (Heuer et al. 2001) exhibit normal spikelet development and morphology. Nonetheless both genes are consistently expressed in the spikelet in all grasses investigated to date, suggesting that the genes have a functional role there. Due to the lack of mutant phenotypes, determination of function has required extrapolation from expression analysis; these types of analyses have been carried out to varying degrees for rice (Moon et al. 1999; Kyozuka et al. 2000; Pelucchi et al. 2002), ryegrass (Gocal et al. 2001), wheat (Murai et al. 2003), barley (Schmitz et al. 2000), sorghum (Greco et al. 1997), and maize (Mena et al. 1995). We have found conserved expression of both FUL1 and FUL2 in glumes, confirming and extending published results from rice and ryegrass in which both FUL1 and FUL2 gene transcripts have been found in glume primordia (Kyozuka et al. 2000; Gocal et al. 2001). The consistent expression pattern suggests a redundant role for these genes in glume identity. As predicted on the basis of previous studies (see above), we found expression patterns in grass florets to be more diverse than in glumes. However, we found no clear pattern of gene expression that was consistent with floral fertility, position, number, or taxonomic relatedness. Prior to the gene duplication event (i.e.,
Evolution of AP1/FUL-Like Genes
in non-grass monocots), AP1/FUL genes are consistently expressed in carpels, indicating a possible ancestral role in carpel development. In grasses, the ancestral state of expression for both genes was inferred as present in fertile florets, but no analysis could be done for reduced florets since they are nonhomologous across taxa. FUL2 transcripts were detected in florets of most, but not all, grass taxa. Within Pooideae, expression patterns for ryegrass, barley, and wheat matched those previously reported (Schmitz et al. 2000; Gocal et al. 2001; Murai et al. 2003), i.e., both genes were expressed in fertile florets, and our new data on TmFUL2 were identical to those of wheat AP1 (WAP1). In Ehrhartoideae, expression patterns of rice also confirmed those previously reported in fertile florets (Moon et al. 1999; Pelucchi et al. 2002), but no expression of either gene was detected in sterile lemmas. Finally, in maize, gene expression did not completely correspond to a previous report (Mena et al. 1995), which found ZAP1 expression in male florets using Northern blot analysis. We were unable to detect it in these florets using RT–PCR. This discrepancy may reflect different developmental stages used in the two studies. Variation in gene expression patterns within and between grasses indicates a combination of incomplete subfunctionalization and redundancy following gene duplication. Further studies are needed to determine the biochemical function and developmental role of FUL1 and FUL2, and how these are partitioned between paralogs across grasses. This study illustrates the complex history of AP1/FULlike genes in grasses and their relatives, and has generated testable hypotheses regarding the role of these genes in spikelet evolution and flowering time. We have shown that immediately following duplication at the base of the grasses nonsynonymous changes occurred solely within the FUL2 lineage prior to BEP/PACCAD diversification. Expression analyses across grasses further demonstrate restriction of FUL2 transcription within spikelets of unrelated taxa: Streptochaeta, Pharus, Oryza, Chasmanthium, Sorghum, and Zea. We hypothesize that heterologous gene expression found in this and other studies indicates a combination of incomplete subfunctionalization and redundancy for FUL1 and FUL2 genes, possibly in specifying floral organ identity. Finally, we suggest that AP1/FUL genes play a general role in regulating flowering time in monocots and that the FUL1 and FUL2 genes of grasses may be redundant regarding their role in vernalization response in cereals. To further elucidate these roles we are continuing to investigate the spatio-temporal pattern of FUL1/FUL2 expression across Poaceae and their relatives. We thank the Missouri Botanical Garden and the United States Department of Agriculture for access to plant materials, Jessica Kossuth for help with RT–PCR, and Mark Beilstein, Andrew Doust, Iva´n Jime´nez, Simon Malcomber, Robert Marquis, Robert Schmidt,
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Peter Stevens, and Patrick Sweeney for helpful comments. This work was supported by National Science Foundation grant DBI-0110189 to E.A.K.
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