Patterns of Gene Duplication and Functional Evolution During the Diversification of the AGAMOUS Subfamily of MADS Box Genes in Angiosperms

Corresponding author: Elena M. Kramer, Harvard University, 16 Divinity Ave., Cambridge, MA 02138., ekramer{at}oeb.harvard.edu (E-mail)

Communicating editor: D. WEIGEL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Members of the AGAMOUS (AG) subfamily of MIKC-type MADS-box genes appear to control the development of reproductive organs in both gymnosperms and angiosperms. To understand the evolution of this subfamily in the flowering plants, we have identified 26 new AG-like genes from 15 diverse angiosperm species. Phylogenetic analyses of these genes within a large data set of AG-like sequences show that ancient gene duplications were critical in shaping the evolution of the subfamily. Before the radiation of extant angiosperms, one event produced the ovule-specific D lineage and the well-characterized C lineage, whose members typically promote stamen and carpel identity as well as floral meristem determinacy. Subsequent duplications in the C lineage resulted in independent instances of paralog subfunctionalization and maintained functional redundancy. Most notably, the functional homologs AG from Arabidopsis and PLENA (PLE) from Antirrhinum are shown to be representatives of separate paralogous lineages rather than simple genetic orthologs. The multiple subfunctionalization events that have occurred in this subfamily highlight the potential for gene duplication to lead to dissociation among genetic modules, thereby allowing an increase in morphological diversity.

THE production of reproductive organs is arguably the most important process in the development of any organism, particularly from an evolutionary standpoint. In the angiosperm model species Arabidopsis thaliana, the MADS-box gene AGAMOUS (AG) is critical to the formation of sex organs in the developing flower (BOWMAN et al. 1989 ). This function is a component of what is known as the ABC model of organ identity determination. The ABC model describes the combinatorial activities of three classes of genes, termed A, B, and C, which function in overlapping domains to encode the identity of organ primordia that arise from the floral meristem (COEN and MEYEROWITZ 1991 ) (Fig 1). In Arabidopsis, AG is the primary C class gene, APETALA1 (AP1) and APETALA2 (AP2) are the A class genes, and APETALA3 (AP3) and PISTILLATA (PI) represent the B class (BOWMAN et al. 1991 , BOWMAN et al. 1993 ). With the exception of AP2, all of these genes are representatives of the pan-eukaryotic MADS-box family of transcription factors (reviewed in SHORE and SHARROCKS 1995 ; THEISSEN et al. 2000 ). More specifically, they are classified as type II (ALVAREZ-BUYLLA et al. 2000 ) or MIKC-type (MUNSTER et al. 1997 ), a plant-specific group within the MADS-box gene family. The MIKC abbreviation reflects a conserved structure composed of four domains: the MADS (M) domain, responsible for DNA binding and dimerization (RIECHMANN et al. 1996B ); the intervening (I) and keratin-like (K) domains, which mediate dimerization between different MIKC-type proteins (RIECHMANN et al. 1996A ); and the variable C-terminal (C) domain, which appears to promote higher-order protein interactions (EGEA-CORTINES et al. 1999 ). Further investigations into the functions of other florally acting MIKC-type MADS-box genes have led to modifications of the ABC model (Fig 1). On the basis of analysis of the FBP7 and FBP11 genes from Petunia, D function was proposed as responsible for the establishment of ovule identity (COLOMBO et al. 1995 ). Recently, E class genes (THEISSEN and SAEDLER 2001 ), represented in Arabidopsis by SEPALLATA1–3 (SEP1–3), have been identified as critical facilitators of B and C class function (PELAZ et al. 2000 ). Our current understanding of the biochemical interactions among the A, B, C, and E class proteins is that dimerization between individual proteins is mediated by the M, I, and K domains while the interaction of dimers to form higher-order complexes is controlled by the C domain (EGEA-CORTINES et al. 1999 ; HONMA and GOTO 2001 ).

View larger version (79K): In this window In a new window Download PPT slide   Figure 1. ABC model with modifications as suggested in THEISSEN et al. 2002 . SEP, sepals; PET, petals; STA, stamens; CAR, carpels; ov, ovules.

Given their critical roles in controlling floral development, the diversification of the MADS-box gene family has been cited as an important factor in the radiation of the land plants (THEISSEN et al. 2002 ). This connection between gene duplication, functional diversification, and the evolution of complexity has a relatively long history, having been most notably outlined by OHNO 1970 . Early studies of the phenomenon focused on two major pathways of paralog evolution: pseudogene formation vs. the acquisition of novel gene function (neofunctionalization). As we have gained a better understanding of the complex nature of gene functions, however, the process of subfunctionalization, as suggested by HUGHES 1999 and elaborated by Force and Lynch (FORCE et al. 1999 ; LYNCH and FORCE 2000 ), has come to the forefront. Under this model, multiple ancestral functions of a gene lineage may become partitioned between paralogs, causing the duplicates to be selectively maintained without neofunctionalization. Longterm, however, subfunctionalization may result in some degree of divergence as the paralogs specialize or eventually acquire additional functions (HUGHES 1999 ). It has also become clear that functional redundancy can be maintained for surprisingly long periods (HUGHES and HUGHES 1993 ), possibly because of an advantage conferred by genetic buffering (ZHANG 2003 ).

Due to the fairly large amount of functional data that are available for AG homologs, this subfamily of MADS-box genes is well suited for an analysis of patterns of functional evolution. In addition to promoting stamen and carpel identity, AG function includes repression of AP1 expression in the third and fourth whorls (GUSTAFSON-BROWN et al. 1994 ) and establishment of the determinate nature of the floral meristem (BOWMAN et al. 1989 ). Analyses of AG homologs from both core eudicots and monocots indicate that these functions are broadly conserved, but gene duplications have introduced variation (BRADLEY et al. 1993 ; KEMPIN et al. 1993 ; PNUELI et al. 1994 ; KANG et al. 1998 ; YU et al. 1999 ; KAPOOR et al. 2002 ; KYOZUKA and SHIMAMOTO 2002 ). For example, the Antirrhinum gene PLENA (PLE) functions very similarly to AG (CARPENTER and COEN 1990 ), but some aspects of stamen identity are mediated by the closely related gene FARINELLI (FAR; DAVIES et al. 1999 ). Parsing of function is also thought to have occurred in Zea mays, where the paralogs ZAG1 and ZMM2 appear to be subfunctionalized into carpel- and stamen-specific paralogs, respectively (MENA et al. 1996 ). In other instances, neofunctionalization has followed gene duplication, as in the case of the SHATTERPROOF (SHP1 and 2) genes, which are AG-like genes from Arabidopsis (LILJEGREN et al. 2000 ). One aspect of their function is to specify tissues that are unique to the silique fruit of the Brassicaceae, indicating that this activity may have been acquired relatively recently (THEISSEN 2000 ). However, other components of SHP1/2 function are redundant with both AG and the FBP7-like gene SEEDSTICK (STK; FAVARO et al. 2003 ; PINYOPICH et al. 2003 ). AG homologs have been identified in all the major gymnosperm lineages (TANDRE et al. 1995 ; RUTLEDGE et al. 1998 ; WINTER et al. 1999 ; JAGER et al. 2003 ) and analyses of expression in Gnetum and Picea suggest that members of the AG subfamily play a deeply conserved role in the production of reproductive tissue. In contrast, no clear AG-like genes have been recovered in studies of lower vascular plants (MUNSTER et al. 1997 ; HASEBE et al. 1998 ; SVENSSON and ENGSTROM 2002 ) or mosses (KROGEN and ASHTON 2000 ; HENSCHEL et al. 2002 ).

Despite these extensive comparative studies, many aspects of the evolution of the AG subfamily remain unclear. In particular, the timing of various gene duplication events and the ensuing patterns of molecular and functional evolution are not well defined. In this study, we have sought to obtain better resolution of ortholog/paralog relationships within the phylogeny of AG-like genes. To these ends, 26 new AG homologs have been identified from 15 angiosperm taxa spanning the core eudicots, magnoliid dicots, and basal ANITA grade (the earliest branching lineages of the angiosperms). Phylogenetic analyses of the expanded AG data set have clarified the evolution of the separate C and D gene lineages and revealed both ancient and recent gene duplications. Most notably, we have found that PLE and AG are not simple genetic orthologs but represent relatively ancient paralogous lineages. This confirms a previous, more limited analysis, which suggested that AG and FAR are orthologous (DAVIES et al. 1999 ). The implications of these findings for the evolution of gene function within the AG subfamily are discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
A broad developmental range of floral tissue was obtained from the following taxa: Saxifraga caryana (Saxifragaceae), Phytolacca americana (Phytolaccaceae), Ranunculus ficaria (Ranunculaceae), Helleborus orientalis (Ranunculaceae), Clematis integrifolia (Ranunculaceae), Aquilegia alpina (Ranunculaceae), Thalictrum dioicum (Ranunculaceae), Berberis gilgiana (Berberidaceae), Akebia quinata (Lardizabalaceae), Sanguinaria canadensis (Papaveraceae), Meliosma dilleniifolia (Sabiaceae), Houttuynia cordata (Saururaceae), Chloranthus spicatus (Chloranthaceae), Saruma henryii (Aristolochiaceae), and Nymphaea sp. (Nymphaeaceae). Voucher information for all of these species is available in supplemental Table 1 at http://www.genetics.org/supplemental/.

Cloning and characterization of AG homologs:
Isolation of AG homologs was performed using RT-PCR in a manner similar to that described in KRAMER et al. 1998 . Initial amplification of first-strand cDNA used a degenerate forward primer (5'-GGIMGIGGIAARATIGARATIAARMGIAT) designed to the highly conserved first 10 amino acids of the MADS domain with a poly(T) reverse primer, 5'-CCGGATCTCTAGACGGCCGC(T)₁₇. The products of the primary PCR reaction were cleaned with the QIAquick PCR purification kit (QIAGEN, Valencia, CA), diluted 1:100, and used as template in a PCR reaction with a second degenerate primer, 5'-ACIAAYMGICARGTIACITTYTG, and the same anchored poly(T) reverse primer. This second forward primer is designed to the highly conserved MADS-box sequence TNRQVTFC, in which the C-terminal cysteine represents a synapomorphy for the AG subfamily (THEISSEN et al. 1996 ). All PCR amplifications were performed in 100 µl of PCR buffer (200 mM Tris-HCl, pH 8.4; 500 mM KCl; 50 mM MgCl₂) containing 50 and 10 pmol of 5' and 3' primer, respectively, 25 µmol of each dNTP, and 2 units of PlatinumTaq polymerase (Invitrogen, Carlsbad, CA). The amplification program began with a 12-min activation step at 95°, followed by a 1-min incubation step at 95°, a 30-sec annealing step at temperatures ranging from 50° to 65°, and a 1-min extension at 72°. The program was repeated for 37 cycles and was terminated by a 10-min incubation step at 72°. The amplified PCR products were cloned using the TOPO TA cloning kit (Invitrogen) per manufacturer's instructions. For each taxon, 50–200 clones of >650 bp were characterized by sequencing (BigDye Terminator v3.0, ABI prism 3100, Applied Bioscience, Foster City, CA) and/or restriction analysis. At least 5 independent clones were sequenced for every putative locus. All cDNA sequences have been deposited in GenBank (for accession numbers, see supplemental data available online at http://www.genetics.org/supplemental/). ScAG, CsAG1, and CsAG2 were identified in the context of an earlier screen (KRAMER and IRISH 2000 ), but are being reported here for the first time.

5' rapid amplification of cDNA ends (RACE) was performed on MdAG1, SrhAG, and NymAG1 using the SMART cDNA RACE kit (BD Biosciences Clontech, Palo Alto, CA). Reverse primers for each locus are as follows: MdAG1, 5'-ACTATTGTTTGCATATTCATAAAGCCGGCCGCGAGT; SrhAG, 5'-TGTGACATAACCTCATACCCTCCCCCACCTG; and NymAG1, 5'-TTCACTGACACCTTCGCCTAGCATTTGCC.

Phylogenetic analyses:
Additional AG-like sequences were identified on the basis of previously published analyses and BLAST searches (ALTSCHUL et al. 1997 ; for references and accession numbers, see Table S1 available online at http://www.genetics.org/supplemental/). In cases in which the database contained nearly identical sequences from the same taxon, only one representative was included. Full-length amino acid and nucleotide alignments of the 26 new AG homologs with 66 previously released AG-like sequences were initially compiled using ClustalW. ClustalW multiple-alignment parameters were gap penalty 8 and gap extension penalty 2, using the PAM protein weight matrix for the amino acid alignment with transitions weighted for the nucleotide. The alignments were then refined by hand using MacClade 4.0 (MADDISON and MADDISON 2000 ), and final amino acid and nucleotide alignments were adjusted so that they were identical (for NEXUS files, see supplementary data available at http://www.genetics.org/supplemental/). The N-terminal extensions present in many AG-like genes were excluded from the alignments. The nucleotide alignment was used for phylogenetic analyses while the equivalent amino acid alignment was used only to identify shared sequence characters and generally conserved motifs.

Although the C domain tends to show much lower conservation than the other three regions, alignment is typically possible within subfamilies (KRAMER et al. 1998 ; JOHANSEN et al. 2002 ; TZENG et al. 2002 ). In the case of the AG lineage, the generally higher degree of sequence conservation further facilitates the alignment of the C domain. The majority of the indels in this region are due to expansions in repetitive sequences, rather than to a large number of nonsynonymous changes. Several particularly long repetitive stretches (more than five amino acids) present in the C domains of the grass AG-like genes were condensed to one or two amino acids to facilitate alignment (see Fig 2). Analyses of a data set lacking the C domain produced phylogenies very similar to those obtained with the full-length alignment, but with less resolution at recent nodes and generally lower bootstrap support (data not shown).

View larger version (129K): In this window In a new window Download PPT slide   Figure 2. Alignment of C-terminal regions of predicted amino acid sequences for select representatives of the C and D lineages and gymnosperm (Gymno) AG-like genes. Colored vertical bars on the left correspond to the phylogenetic positions of the adjacent genes (see Fig 3). Sequences shown in boldface type were identified in this study. Two highly conserved regions, AG motif I and AG motif II, are boxed. Residues that show chemical conservation with the C-lineage consensus sequence are shown in boldface type and shaded. Red arrows in P0408G07.14 indicate the position of a stretch of seven alanines that were removed from the alignment. Consensus sequences for both motifs are from each of the three major lineages in the AG subfamily.

Maximum-parsimony (MP) trees were generated through heuristic searches of 1000 random stepwise additions, with tree bisection-reconnection branch swapping and saving of multiple parsimonious trees (MulTrees on). Gaps were encoded as missing data and third positions were excluded. Bootstrap support was estimated by performing 1000 heuristic searches with 10 additional sequence replicates per bootstrap, using the same criteria as in the original search. Wilcoxon sign-rank (also called a Templeton test; TEMPLETON 1983 ) and Kishino-Hasegawa (KISHINO and HASEGAWA 1989 ) tests were conducted on the MP trees to explore topologies that would suggest alternative patterns of gene duplication.

Bayesian phylogenetic analyses were conducted on the nucleotide alignments, including all positions using the program MRBAYES v3.0 (HUELSENBECK and RONQUIST 2001 ). The best model of evolution was determined using Modeltest v3.06 (POSADA and CRANDALL 1998 ). The model of DNA substitution selected was GTR + I + , which assumes general time reversibility (GTR), a certain proportion of invariable sites (I), and a gamma approximation of the rate variation among sites (). The option "codon" was used for the nucleotide substitution model, following the probabilistic model of codon evolution by MUSE and GAUT 1994 . We ran four chains of the Markov chain Monte Carlo, sampling 1 tree every 100 generations for 1,000,000 generations starting with a random tree. The search reached stationarity after 23,000 generations. The first 23,000 generations were considered the "burn-in" period and were not included in generating the consensus phylogeny.

Cloning and characterization of intron 8 region of Nymphaea AG homologs:
Nymphaea sp. genomic DNA was prepared from leaf tissue using the DNeasy plant mini kit (QIAGEN). To obtain fragments of the NymAG1 genomic locus, the DNA was amplified using a specific forward primer, NymAG1F 5'-CAGCACATCAATCTAATGGAATCCTCCCACCAC with a specific reverse primer, NymAG1R 5'-TGGACCCAACATATTCATGTTACTAATGCTGCTGAT. The primers were designed to regions of the NymAG1 cDNA predicted to fall within exon 7 for NymAG1F and exon 8 for NymAG1R. PCR amplification was performed using a BD Advantage Genomic PCR kit (BD Biosciences Clontech) per manufacturer's instructions. The amplification program began with a 1-min activation step at 94°, followed by a 15-sec denaturing step at 94°, a 20-sec annealing step at 50°–60°, and a 3-min extension step at 68°, repeated for 30 cycles. The resulting genomic fragments, of 1.8 kb in length, were cloned using the TOPO TA cloning kit (Invitrogen). Approximately 30 clones were screened for size and 6 clones were sequenced as described above. The resulting consensus genomic sequence was aligned to the NymAG1 cDNA to determine exon/intron boundaries. The NymAG3 genomic fragment was similarly obtained and analyzed using a forward primer, 5'-CTGGAACTACAAAGTGATAATATGTATCTTCGA, designed to fall within exon 6, and a reverse primer, 5'-CAGACAACACCATAGCATATTGTGCGGTA, designed to bind within the last exon of the cDNA.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Characterization of AG homologs and phylogenetic analysis—AG homologs show a high degree of conservation:
Twenty-six AG-like cDNAs were identified in 15 taxa from the core eudicots, magnoliid dicots, and ANITA group. Alignment of the predicted amino acid sequences of the new loci with those of previously identified AG homologs reveals a high degree of conservation throughout the M, I, and K regions, with many positions nearly invariant throughout the seed plants (for amino acid alignment, see supplementary data at http://www.genetics.org/supplemental/). Beyond the traditionally defined K domain (MA et al. 1991 ), positions 95–165 in our alignment, a fairly high level of identity extends through position 185. This includes the K3 region that has been recognized by some researchers as a putative third -helix (YANG et al. 2003 ). The expected a and d positions of the predicted (abcdefg)_n repeats identified by YANG et al. 2003 are all very highly conserved (see online supplemental Figure 1 available at http://www.genetics.org/supplemental/). Although two of the central a sites are occupied by charged (165E) or polar (172N) residues rather than by hydrophobic amino acids, buried polar residues have been shown to play important roles in dimerization interactions between AP3 and PI in Arabidopsis (YANG et al. 2003 ), as well as other eukaryotic transcription factors (ZENG et al. 1997 ).

Following position 185, conservation decreases, with multiple indels due to the expansion of repetitive sequences, particularly in the grass homologs. At the very C-terminal end of the proteins, there are two short, highly conserved regions, which we have termed AG motif I and AG motif II (Fig 2). These motifs primarily contain hydrophobic and polar residues and have no recognizable relation to known functional motifs. They do have some similarity in makeup to the conserved C-terminal sequences of the B lineage, the PI and paleoAP3 motifs (KRAMER et al. 1998 ), but no clear positional homology is discernible. The conservation of these regions throughout seed plant AG-like sequences defines them as synapomorphies for the subfamily.

Phylogenetic analyses reveal patterns of ancient gene duplication:
A full-length nucleotide alignment of 92 AG-like sequences was analyzed using MP as implemented by PAUP 4.0b10 (SWOFFORD 2001 ) and Bayesian analysis using MRBAYES 3.0 (HUELSENBECK and RONQUIST 2002 ). Gymnosperm AG-like sequences were used to root the trees on the basis of the findings of earlier studies (HASEBE and BANKS 1997 ; WINTER et al. 1999 ; THEISSEN et al. 2000 ). The resulting MP and Bayesian phylogenies (Fig 3 and Fig 4) are largely in agreement with only minor differences (see below). Overall, the MP analysis shows lower bootstrap support for many nodes, while the Bayesian analysis has relatively high posterior probability values for a majority of nodes. However, posterior probabilities are known to be considerably less stringent than bootstrap values (SUZUKI et al. 2002 ; ALFARO et al. 2003 ; DOUADY et al. 2003 ) and should be considered upper boundaries of confidence for the relationships depicted at these nodes.

View larger version (30K): In this window In a new window Download PPT slide   Figure 3. One randomly chosen tree from 20 equally parsimonious trees of 3228 steps. The numbers next to each node give bootstrap support from 1000 replicates. Gene names shown in boldface type were identified in this study. Dashed branches collapse in the strict consensus. Branch coloring is as follows: black, gymnosperm AG-like genes; gray, monocot D lineage; dark green, magnoliid dicot, ANITA grade, and lower eudicot D lineage; light green, core eudicot D lineage; orange, monocot C lineage; red, magnoliid dicot and ANITA grade C lineage; purple, lower eudicot C lineage; dark blue, euAG core eudicot C lineage; and light blue, PLE core eudicot C lineage. The yellow triangle indicates the C/D gene duplication; the black circles, gene duplications in the grass C and D lineages; the black diamond, a gene duplication in the Ranunculales C lineage; and the yellow star, the euAG/PLE gene duplication.

View larger version (41K): In this window In a new window Download PPT slide   Figure 4. A 50% majority rule tree derived from those trees sampled after "burn-in." The numbers next to each node indicate the posterior probabilities for those branches. Branch coloration and symbols have the same significance as in Fig 3. The taxon of origin is shown in parentheses after each gene name.

Both analyses give strong support to a clade containing all of the angiosperm sequences. Consistent with this, there are many distinct amino acid apomorphies for the angiosperm and gymnosperm clades; however, the lack of an established outgroup for the AG subfamily makes it impossible to determine which character states were primitive in the ancestor of all seed plants. Within the angiosperms, the loci are divided into two major clades, which we have termed the C and D lineages. Each lineage contains representatives from throughout the angiosperms, including the basal ANITA group, indicating that they were produced by an ancient gene duplication that predated the diversification of extant angiosperms.

The designation of the "D" lineage is based on the inclusion of the so-called D class genes from Petunia, FBP7 and FBP11 (COLOMBO et al. 1995 ), and follows terminology used in previous publications (TZENG et al. 2002 ). In the D clade, the position of the Nymphaea representative NymAG3 differs between the MP and Bayesian analyses. The strongly supported basal placement of NymAG3 in the Bayesian tree (Fig 4) is more consistent with the position of the Nymphaeales in current angiosperm phylogenies (QIU et al. 1999 ; ZANIS et al. 2002 ). The monocots are represented by the Agapanthus gene ApMADS2 and a group of grass homologs that are divided into two paralogous lineages, one including Oryza P0408G07.14 and Zea ZMM25 and the other, Oryza OsMADS13 and the Zea genes ZAG2 and ZMM1. This indicates that an early gene duplication event that occurred before the common ancestor of rice and maize was followed by a later maize-specific duplication, which yielded the ZAG2/ZMM1 pair (Fig 3 and Fig 4, solid circles in D lineage). Magnoliid dicot and eudicot sequences are also present in the D clade, demonstrating that representatives of the lineage are widely conserved across the angiosperms. It is notable, however, that no D orthologs were recovered in the RT-PCR survey of the Ranunculales, a finding that is being pursued further with genomic analyses.

The D lineage has a number of distinguishing sequence characteristics, some of which are shown in Fig 5. Overall, members of the clade show higher variability in the AG motif I and II regions than do the gymnosperm AG-like genes or C-lineage homologs. Within the D lineage, the core eudicot loci are associated with a loss of conservation in the second residue of AG motif I and with the conversion of positions 6 and 7 in AG motif II to highly conserved lysine residues (Fig 2 and amino acid alignment in online supplemental data available at http://www.genetics.org/supplemental/).

View larger version (24K): In this window In a new window Download PPT slide   Figure 5. Simplified phylogeny of the AG subfamily with diagnostic character states mapped onto branches. Sequence character states refer to the amino acid alignment (see online supplemental data at http://www.genetics.org/supplemental/). Other character states were inferred on the basis of 5' RACE, comparison of genomic and cDNA sequences, and published reports of expression patterns (see text). The yellow triangle indicates the C/D duplication event while the star represents the euAG/PLE duplication.

We also investigated whether aspects of genomic structure represent a synapomorphy for the D lineage. AG homologs are unusual for MIKC-type genes in that they often possess eight introns rather than the typical six (BRUNNER et al. 2000 ; JOHANSEN et al. 2002 ). The additional two introns are positioned 5' of the MADS domain and in the last codon of AG motif II, which is commonly the last codon of the protein (YANOFSKY et al. 1990 ; BRADLEY et al. 1993 ). This organization is observed in several C-lineage members and in one gymnosperm (RUTLEDGE et al. 1998 ; BRUNNER et al. 2000 ), suggesting that the presence of eight introns is likely to be primitive in the AG subfamily. However, all of the D-lineage genes for which genomic structure is available (AGL11/STK, OsMADS13, ZAG2, and ZMM11) are missing intron 8 at the 3'-end of AG motif II (THEISSEN et al. 1995 ; ARABIDOPSIS GENOME INITIATIVE 2000; CHOISNE et al. 2002 ). Although this sampling is quite limited, it does include both core eudicot and grass species and could indicate that the loss of intron 8 is a shared character of the D lineage. To explore this possibility, we cloned and sequenced genomic fragments corresponding to the intron 8 region of C- and D-lineage representatives from Nymphaea, NymAG1 and NymAG3, respectively. Alignment of the genomic and cDNA sequences clearly shows that both NymAG1 and NymAG3 have introns at the expected position for intron 8 (see online supplemental Figures 2 and 3 available at http://www.genetics.org/supplemental/). These findings indicate that the D lineage did not lose intron 8 before the radiation of extant angiosperms. It remains possible that the D lineage lost intron 8 after the early divergence of the Nymphaeales, but it may also be that the lack of intron 8 in STK and the grass D genes arose independently. This potential clearly exists since it is also known to have occurred independently at least once in the C lineage, as evidenced by the SHP1/2 genes of Arabidopsis (MA et al. 1991 ).

The C lineage contains both of the originally described C-function genes, AG from Arabidopsis and PLE from Antirrhinum. The NymAG-1 and -2 loci do not fall at the base of the C clade in either analysis, which could indicate ancient patterns of gene duplication and extinction, but also may be an artifact due to the limited sampling from magnoliid dicots and the ANITA group. Parsimony analyses in which the Nymphaea loci are constrained to the base of the C lineage produced 30 trees only 9 steps longer than the original MP tree, a difference that is not significant by either the KH or the Templeton test. Monocot representatives include loci from the Orchidaceae, Amaryllidaceae, and Poaceae. The topology of the grass C-lineage genes suggests a pattern of gene duplication similar to what is observed in the D lineage: an early gene duplication was apparently followed by a later event in the Zea lineage. The AG homologs from the Ranunculales form a well-supported, single clade in the Bayesian analysis, but they are paraphyletic in the MP tree. In both phylogenies, the Ranunculaceae loci are separated into two paralogous lineages, indicating that they were produced by a gene duplication that at least predated the last common ancestor of the family (solid diamonds in Fig 3 and Fig 4). The position of the lower eudicot Meliosma, represented by MdAG1, differs somewhat between the MP and Bayesian analyses, with the MP position being more consistent with the most recent phylogeny of the eudicots (SOLTIS et al. 2003 ).

All of the core eudicot C-lineage loci fall into a single clade with strong support in both analyses; however, this group is deeply split into two separate lineages. PLE and other AG-like genes from Petunia, Nicotiana (tobacco), Arabidopsis, Malus (apple), Rosa, Vitis (grapevine), and Liquidambar (sweetgum) form one clade, which we refer to as the PLE lineage. Sister to this lineage is what we call the euAG lineage, which includes AG, the Antirrhinum gene FAR, and an array of AG homologs from across the core eudicots. The PLE and euAG lineages include six paralog pairs, such as FBP6 and pMADS3 from Petunia, which comprise taxa from both the Rosids and the Asterids, the two major core eudicot groups. Furthermore, loci from the Vitaceae, Caryophyllales, and Saxifragales are clearly placed in one lineage or the other. This topology indicates that the paralogous PLE and euAG lineages were produced by a gene duplication that occurred before the diversification of the core eudicots, meaning that AG and PLE are not simple genetic orthologs but relatively ancient paralogs.

To test this finding, we reanalyzed the data set using MP under a series of topological constraints. If all core eudicot loci are constrained by superorder (Rosids, Asterids, etc.), the analysis recovers 31 trees, each 35 steps longer than the MP tree, which are significantly different by both tests at P < 0.001. In these trees, the euAG and PLE lineage members still sort out into two corresponding clades within each constrained superorder group (data not shown). The use of backbone constraints that would accept the pre-core eudicot duplication but force AG and PLE to be genetic orthologs resulted in 24 trees, 20 steps longer than the original MP tree, a difference that is significant at P < 0.05. Consistent with these results, the PLE and euAG lineages each possess a number of diagnostic amino acid character states (Fig 5).

One characteristic commonly found in C-lineage members is the presence of a N-terminal extension preceding the MADS domain (JAGER et al. 2003 ), which is not typical of MIKC-type MADS-box genes (PURUGGANAN et al. 1995 ). These regions are variable in sequence and length, ranging from 13 to 52+ amino acids (see online supplemental Figure 4 at http://www.genetics.org/supplemental/; JAGER et al. 2003 ). Of the 31 complete core eudicot mRNA sequences, 29 show extensions but they are found only in three of the eight complete monocot sequences (see supplemental online Figure 4 at http://www.genetics.org/supplemental/). N-terminal extensions have not been seen in any D-lineage members or gymnosperm AG-like genes characterized to date (JAGER et al. 2003 ). Analysis of AG function in Arabidopsis indicates that the large N-terminal extension found in this protein is not essential to any major aspect of gene function (MIZUKAMI et al. 1996 ). The most likely scenario for the appearance of N-terminal extensions in C-lineage members seems to be that in-frame ATG codons have evolved several times independently within the large 5'-untranslated region that is common to the AG subfamily (JAGER et al. 2003 ). To explore the evolution of this novel domain, we performed 5' RACE on MdAG1, ThdAG1, SrhAG, and NymAG1, C-lineage loci representing the lower eudicots, magnoliid dicots, and ANITA grade. None of these cDNAs display N-terminal extensions and have the first in-frame ATG immediately preceding the MADS domain. This suggests that the frequent presence of an N-terminal extension is primarily a characteristic of the core eudicot C-lineage members, with the domain having evolved independently at least one other time in the monocots.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Implications of sequence conservation:
It is perhaps not surprising to find that members of the AG subfamily exhibit a high degree of sequence conservation, given their critical role in producing reproductive organs. Consistent with this pattern, several studies have shown that constitutive expression of heterologous AG-like genes in Arabidopsis (RUTLEDGE et al. 1998 ; TANDRE et al. 1998 ) or in Nicotiana (MANDEL et al. 1992 ; KANG et al. 1995 ) produces phenotypes similar to that of 35S:AG (MIZUKAMI and MA 1992 ). These results suggest that the sequence conservation of AG homologs reflects a similar conservation of biochemical interactions. While the M, I, and K domains have been clearly shown to be involved in DNA binding and protein dimerization (RIECHMANN et al. 1996A , RIECHMANN et al. 1996B ), the function of the C domain is poorly understood. Ectopic expression experiments have demonstrated that deletion of the entire C domain, including most of the putative K3 -helix (see online supplemental Figure 1 at http://www.genetics.org/supplemental/), produces a dominant negative form of AG (MIZUKAMI et al. 1996 ). This indicates that although the C domain is not required for DNA binding or dimerization, it is essential for full protein function. Furthermore, conserved C-terminal motifs have been identified in many lineages of MIKC-type MADS-box genes (KRAMER et al. 1998 ; JOHANSEN et al. 2002 ; LITT and IRISH 2003 ; VANDENBUSSCHE et al. 2003 ) and several lines of evidence indicate that these motifs are functionally important (KRIZEK and MEYEROWITZ 1996 ; LAMB et al. 2002 ). It remains to be determined, however, how components of the C domain, such as the K3 or C-terminal motifs, might contribute to higher-order protein interactions or other aspects of AG function.

Gene duplications in the C lineage have led to subfunctionalization and maintained redundancy:
AGAMOUS and PLENA are not simple genetic orthologs: Phylogenetic analyses of the large AG homolog data set show that PLE and AG actually represent paralogous lineages derived from a gene duplication that occurred within the lower eudicots. This confirms similar results obtained in much more limited analyses (DAVIES et al. 1999 ; KROGEN and ASHTON 2000 ; SVENSSON et al. 2000 ). Representatives of both the PLE and euAG lineages have been identified in six taxa but loss-of-function data are available only for the paralogs from Arabidopsis, Antirrhinum, and Petunia (BOWMAN et al. 1989 ; CARPENTER and COEN 1990 ; DAVIES et al. 1999 ; LILJEGREN et al. 2000 ; KAPOOR et al. 2002 ). In Arabidopsis, AG and SHP1/2 exhibit a mix of redundant and distinct functions. While AG fulfills the primary aspects of C function (BOWMAN et al. 1989 ), SHP1 and -2 play both a unique role in the differentiation of the replum margin (LILJEGREN et al. 2000 ) and a redundant one in promoting carpel and ovule identity (WESTERN and HAUGHN 1999 ; PINYOPICH et al. 2003 ). Similar to what has been found with other types of paralogs (LEE and SCHIEFELBEIN 2001 ; SKAER et al. 2002 ), SHP1/2 can substitute for aspects of AG's stamen identity function, although they do not usually perform this role (PINYOPICH et al. 2003 ). The SHP1/2 genes are thought to be genetically downstream of AG, possibly directly (SAVIDGE et al. 1995 ). In Antirrhinum, functional evolution has taken an alternate route, leaving PLE the primary C-function gene and the euAG ortholog FAR a largely redundant paralog that contributes to stamen differentiation (DAVIES et al. 1999 ). In contrast to what is observed in Arabidopsis, PLE and FAR are not functionally interchangeable and it is FAR that appears to be genetically downstream of PLE. Loss-of-function analysis of pMADS3 in Petunia suggests that pMADS3 and FBP6 are neither fully redundant nor completely separate in function, with both contributing to aspects of organ identity and meristem determinacy (KAPOOR et al. 2002 ).

Given that the combined functions of the paralog pairs in each species are roughly equivalent, the most parsimonious explanation is that most of these functions were present in the common ancestral repertoire. Following their formative gene duplication event, 100–120 million years ago (MYA; MAGALLON et al. 1999 ), it appears that subfunctionalization was the primary trend, although various degrees of maintained redundancy have also been observed. While it may be common for AG homologs to control late aspects of carpel development, the role of SHP1/2 in the replum margin could be considered a kind of neofunctionalization. It remains possible, if not likely, that alternate scenarios such as paralog loss or more dramatic neofunctionalization have occurred in other core eudicots. The phylogenetic findings do not undermine our understanding of the functional homology of PLE and AG since their highly similar functions were clearly inherited from a common ancestor. Their paralogous relationship does underscore the fluid nature of functional evolution following gene duplication and demonstrates the importance of evaluating genetic orthology and functional homology as separate entities (THEISSEN 2002 ).

Interestingly, gene duplication events have also been identified in the AP3 and AP1 gene lineages close to the base of the core eudicots (KRAMER et al. 1998 ; LITT and IRISH 2003 ). In the case of AP3, a gene duplication gave rise to the euAP3 and TM6 paralogous lineages while in AP1 an event produced the euAP1 and euFUL lineages. Further sampling in lower eudicot taxa will be necessary to determine whether the AG, AP3, and AP1 duplications were coincident. Unlike AP3 and AP1, which underwent dramatic changes in otherwise conserved motifs following their lower eudicot duplications (KRAMER et al. 1998 ; LITT and IRISH 2003 ), there are comparatively few fixed differences between the euAG and PLE lineages. It does appear, however, that the base of the core eudicots was a critical period in angiosperm evolution with many significant changes in both floral morphology (ENDRESS 1990 ) and the gene lineages that control floral organ identity.

Gene duplications have also shaped the evolution of the C lineage in the grass family: Approximately 50–70 MYA (GAUT 2002 ), a gene duplication predating the last common ancestor of Zea, Hordeum (rye), Triticum (wheat), and Oryza gave rise to the paralogous lineages defined by the Zea genes ZAG1 and ZMM2 (SCHMIDT and AMBROSE 1998 ). This was followed by a segmental allotetraploidization event in the Zea lineage (GAUT and DOEBLEY 1997 ), which produced the ZMM2/ZMM23 paralog pair (MUNSTER et al. 2002 ). The expression patterns of ZAG1 and ZMM2 indicate that the paralogs have become subfunctionalized, with ZAG1 more strongly expressed in carpels and ZMM2 in stamens (MENA et al. 1996 ). However, the phenotype of plants with insertional mutations in ZAG1 (MENA et al. 1996 ) indicates that carpel identity is redundantly controlled, possibly by other AG-like genes or novel factors similar to the DROOPING LEAF locus identified in rice (NAGASAWA et al. 2003 ). In Oryza, it remains unclear as to whether the ZMM2 ortholog OsMADS3 participates in all aspects of C function, as suggested by antisense transgenic lines (KANG et al. 1998 ), or primarily promotes stamen identity, as indicated by ectopic expression of the gene (KYOZUKA and SHIMAMOTO 2002 ). An Oryza ZAG1 ortholog has not yet been annotated in the genome, but in the closely related Hordeum, orthologs of both ZAG1 and ZMM2 have been identified. It will be interesting to learn whether these genes are subfunctionalized in a manner similar to the Zea genes or show a different pattern of functional evolution. In general, it is notable that subfunctionalization appears to be the trend for C-lineage paralogs in both the core eudicots and grasses.

The D lineage is defined by distinct aspects of protein sequence and expression pattern:
Can a distinct function be defined for the D lineage? The concept of D function was first proposed on the basis of functional studies of the FBP7 and FBP11 genes in Petunia. The elimination of FBP7/11 expression results in the transformation of ovules into pistil-like structures, while ectopic expression of FBP7 results in the production of ovules on the sepals and, occasionally, the petals (ANGENENT et al. 1995 ; COLOMBO et al. 1995 ). These results were taken to indicate that the genes could promote ovule identity in disassociation from carpel identity, thereby requiring a fourth class of gene activity (COLOMBO et al. 1995 ). In contrast, analysis of the D-lineage member from Arabidopsis, STK, has shown that ovule identity is promoted by the combined activity of both C and D orthologs (FAVARO et al. 2003 ; PINYOPICH et al. 2003 ). These contrasting results may be due, in part, to different derivations of the placenta, which initiates as free central in Petunia (ANGENENT et al. 1995 ) but is marginal in Arabidopsis (GASSER and ROBINSON-BEERS 1993 ). In any case, the Arabidopsis results indicate that an absolute separation of C- and D-lineage functions is not universally applicable. Although the concept of a D function has been embraced in the literature (THEISSEN et al. 2000 , THEISSEN et al. 2002 ; FAVARO et al. 2002 ; TZENG et al. 2002 ), which we recognize by our designation of the D lineage, the control of ovule development might also be considered a component of C function sensu lato. Consistent with this argument, several C-lineage members have independently acquired primarily ovule-specific expression patterns, including SHP1/2 in Arabidopsis (MA et al. 1991 ), CAG2 in Cucumis (PERL-TREVES et al. 1998 ), and ThdAG2 in Thalictrum (V. S. DI STILIO and E. M. KRAMER, unpublished results). At the same time, it must be acknowledged that almost all of the D-lineage members characterized to date, including core eudicot and grass orthologs, exhibit ovule-specific expression (SCHMIDT et al. 1993 ; ANGENENT et al. 1995 ; LOPEZ-DEE et al. 1999 ; BOSS et al. 2002 ; TZENG et al. 2002 ; PINYOPICH et al. 2003 ), the one exception being CAG1/CUM10 from Cucumis (KATER et al. 1998 ). Therefore, although C-lineage members have retained the potential to contribute to ovule identity, the ancient C/D duplication event does appear to have been followed by a restriction of expression in the D lineage such that the genes generally do not function in male sporogenic tissue. Further studies of the expression patterns and functions of D orthologs will be necessary to clarify the conservation of their role in ovule development and to determine whether they typically function in an exclusive manner, as in Petunia, or in a redundant one, as in Arabidopsis.

Was the C/D gene duplication significant for the evolution of the angiosperms?
Given that all gymnosperm AG-like genes examined to date are expressed in microsporophylls, megasporophylls, and ovules (RUTLEDGE et al. 1998 ; TANDRE et al. 1998 ; WINTER et al. 1999 ), the ovule-specific expression of the D lineage can be described as a subfunctionalization (JAGER et al. 2003 ). It remains unclear as to whether or not substantial redundancy in the ovule identity program has always existed between C and D orthologs. If a large degree of redundancy has always been present, the D lineage may have been retained primarily to provide genetic buffering in the crucial ovule development pathway. In either case, however, the presence of ovule-specific D-lineage genes could also have increased the degree of dissociation between the sporophyll and ovule genetic pathways. Consider the apparent gymnosperm identity programs: microsporophylls are encoded by two independent gene lineages, AP3/PI-like and AG-like, while both megasporophylls and ovules are controlled by AG-like alone. Within this context, the evolution of the D paralogs created an alternative genetic source for elaboration of ovule morphology. This remains true even if the C and D genes remained redundant to some degree, analogous to the SHP genes promoting novel aspects of Arabidopsis carpel morphology. A complete subfunctionalization could have had more profound effects on both megasporophyll and ovule evolution. It has been recognized that subfunctionalization can free paralogs to adapt specifically to narrow functional repertoires (HUGHES 1994 , HUGHES 1999 ; ZHANG 2003 ). Similarly, the process can increase the modularity of whole genetic pathways, which allows characters to evolve without pleiotropic effects (RAFF 1996 ; WAGNER and ALTENBERG 1996 ). The suggestion has been made that greater modularity, at any hierarchical level, is a kind of key innovation that may be associated with radiations in diversity (YANG 2001 ). Therefore, it is possible that subfunctionalization between the C and D lineages decoupled megasporophyll and ovule development, facilitating evolutionary modifications of both structures.

Overall, this analysis of the AG subfamily has demonstrated the dynamic nature of functional evolution following gene duplication and underscores the importance of conducting both phylogenetic and functional analyses of gene lineages. It is also quite clear that the current extent of our knowledge regarding the functions of AG-like genes is entirely restricted to the core eudicots and grasses. To achieve a more thorough understanding of the evolution of the AG subfamily, it is critical to obtain functional data for C- and D-lineage members from intervening angiosperm lineages.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank libraries under accession nos. AY464093, AY464094, AY464095, AY464096, AY464097, AY464098, AY464099, AY464100, AY464101, AY464102, AY464103, AY464104, AY464105, AY464106, AY464107, AY464108, AY464109, AY464110, AY464111, AY464112, AY464113, AY464114, AY464115, AY464116, AY464117, AY464118, AY464119, AY464120.
² Present address: Department of Biology, University of Washington, Seattle, WA 98115.

	ACKNOWLEDGMENTS

We thank Heather Watchel and Phillip Santiago for help with screening and sequencing of clones and G. Giribet and J. Wakely for the use of their computer equipment. We also thank Amy Litt, Daniel Fulop, and two anonymous reviewers for comments on the manuscript. This work was supported by a grant from the Harvard Milton Fund to E.M.K. and a Mercer Fellowship of the Arnold Arboretum to M.A.J. and V.S.D.

Manuscript received August 29, 2003; Accepted for publication November 10, 2003.

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