Rapid Molecular Evolution of CYCLOIDEA-like Genes in Antirrhinum and Its Relatives

Thomas Gübitz, Ailsa Caldwell and Andrew Hudson

Institute of Cell and Molecular Biology, University of Edinburgh

Correspondence: E-mail: andrew.hudson{at}ed.ac.uk.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
The CYCLOIDEA (CYC) and DICHOTOMA (DICH) genes encode related TCP transcription factors that control floral asymmetry in Antirrhinum majus. Analysis of sequences from relatives of Antirrhinum suggested that CYC and DICH arose from a gene duplication in an ancestor of the tribe Antirrhineae and have subsequently evolved at similar rates. Coding regions outside the conserved functional TCP and R domains differed by numerous indels, suggesting rapid evolution and low constraint on amino acid sequence. An analysis of variability within the genus Antirrhinum revealed very similar CYC alleles in 17 representative species, consistent with most of the species having diverged within the last 1 myr. Whereas substitution mutations appear to have accumulated constantly, one Antirrhinum CYC allele provided evidence for sporadic and rapid accumulation of insertion mutations.

Key Words: CYCLOIDEADICHOTOMA • TCP genes • Antirrhinum • Antirrhineae


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Antirrhinum majus (tribe Antirrhineae, family Veronicaceae sensu Olmstead et al. 2001), produces dorsiventrally asymmetric (zygomorphic) flowers. Floral asymmetry in A. majus is dependent on two closely related genes, CYCLOIDEA (CYC) and DICHOTOMA (DICH), that are expressed dorsally (adaxially) in floral meristems and developing floral organs (Carpenter and Coen 1990; Luo et al. 1996; Almeida, Rocheta, and Galego 1997; Luo et al. 1999). CYC and DICH have partially redundant roles in promoting dorsal identity in the floral meristem, corolla, and androecium, and thus zygomorphy of the flower as a whole. Almost all genera within the Antirrhineae and their more distant relatives in the Veronicaceae have zygomorphic flowers. Reduced activity of a CYC-like gene in Linaria vulgaris (a zygomorphic member of the Antirrhineae) can cause loss of floral asymmetry, indicating conserved function of CYC-like genes within this tribe (Cubas, Vincent, and Coen 1999). CYC-like genes in the distantly related eudicot, Arabidopsis thaliana, showed dorsally restricted expression in floral meristems that develop into radially symmetric (actinomorphic) flowers (Cubas et al. 2001). This suggests that an ancestral CYC-like gene was expressed asymmetrically and recruited to control asymmetric flower morphology in Veronicaceae and related families in the Lamiales with asymmetric flowers (Reeves and Olmstead 1998; Cubas, Coen, and Martínez Zapater 2001).

Other functionally characterized CYC-related genes include teosinte branched1 (tb1), which represses growth of axillary shoots and floral organs in maize, and the PCF1 and PCF2 genes of rice (Doebley, Stec, and Hubbard 1997; Kosugi and Ohashi 1997). All these belong to a larger gene family named TCP (from tb1, CYC, and PCF). This family comprises 25 members in A. thaliana, is specific to plants, and encodes noncanonical basic Helix-Loop-Helix (bHLH) transcription factors (Cubas et al. 1999; Riechmann et al. 2000). All CYC-like genes share the TCP box, encoding a 55–59 amino acid bHLH motif that is implicated in protein dimerization and DNA binding and an R box encoding a more C-terminal motif of 18 amino acids. Outside these boxes, protein coding sequences (which in CYC, DICH, and tb1 are uninterrupted by introns) show low sequence conservation, except in a region encoding a C-terminal domain of unknown function that has been termed the "end box" (Cubas et al. 1999; Möller et al. 1999; Citerne, Möller, and Cronk 2000). Because some TCP genes have known roles in floral asymmetry, and because of the potential importance of other members of the family in morphological evolution, they have been the subject of phylogenetic analysis in other genera with zygomorphic flowers (e.g., Gesneriaceae: Möller et al. 1999; Citerne, Möller, and Cronk 2000; and grasses: Lukens and Doebley 2001).

A previous study in Antirrhineae claimed to have identified at least five CYC-like loci in Antirrhinum and Misopates species, mainly on the basis of sequence phylogenies (Vieira, Vieira, and Charlesworth 1999). Paralogs and each putative ortholog appeared to be very highly conserved within the genus Antirrhinum, between genera within the Antirrhineae (Antirrhinum, Misopates, Linaria, and Cymbalaria), and with Digitalis purpurea, a more distant member of the Veronicaceae (Olmstead et al. 2001). This finding suggested that CYC-like sequences have evolved at an unusually slow rate in Veronicaceae.

To investigate further the evolutionary dynamics of CYC-like genes, we obtained CYC sequences from 17 Antirrhinum species and sequences of CYC and DICH loci from relatives of Antirrhinum. In contrast to previous studies, we found that CYC represents a single locus in Antirrhinum, that DICH is its closest paralog, and that the two loci have resulted from a gene duplication in the Veronicaceae in the lineage leading to the tribe Antirrhineae. Moreover we show that both CYC and DICH genes have evolved quickly within Veronicaceae, mainly as a result of nucleotide insertions and deletions in regions outside the functionally important TCP and R boxes. Antirrhinum species, with one exception, share similar CYC alleles, suggesting that the genus has undergone a recent radiation within the last ~5 myr.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Genomic DNA was extracted according to the method of Doyle and Doyle (1987). With the exception of Digitalis purpurea and Cymbalaria muralis, which were collected from the wild in Edinburgh, all taxa were grown from seed accessions, details of which are available on request. CYC sequences were amplified from D. purpurea, C. muralis, Chaenorhinum origanifolium, Linaria maroccana with the primers cycFS (5'-atgctaggtttcgacaagcc-3') and cycR* (5'-atgaatttgtgctgatccaaaatg-3') and from Misopates orontium, Sairocarpus nuttallianus, and all Antirrhinum species with primers cycF (5'-tcccttcactctcgcgc-3') and cycR (5'-tggcgcatagctggttcgac-3'; fig. 1). DICH sequences were amplified with primers dichF (5'-acgacgtgatttccgggtac-3') and dichR (5'-acttccccacgttctgcttca-3') or with the primer combination dichFT (5'-ttccaccttcacttaatc-3') and cycR or cycR*, neither of which is specific for CYC orthologs. No candidate DICH orthologs were obtained from Ch. origanifolium with any of these primer combinations, and further attempts to test whether DICH orthologs were present in this species were not pursued. Polymerase chain reaction (PCR) testing was carried out with Taq polymerase in buffer containing 2 mM Mg2+ at 94°C for 2 min; 30–35 cycles of 94°C for 20 s, annealing for 30 s, and extension at 72°C for 2 min, followed by a final extension at 72°C for 5 min. Different annealing temperatures were used for different primer combinations: 55°C for trnL + F primers, 60°C for cycF–cycR48, 48°C for cycFS–cycR*, and 55°C for dichF–dichR. In most cases, PCR products were cloned in pGEM-T Easy vector (Promega), and one clone from each accession was sequenced using T7 and SP6 primers. Sequences were then confirmed by direct sequencing of PCR products using the original amplification primers and the internal sequencing primers, cycF297 (5'-gccaagggacaggagagt-3') and cycR413 (5'-ctttgatagcggttttcg-3'). The sequences of CYC from M. orontium and A. charidemi, and DICH from A. siculum were obtained directly from PCR products. A. majus CYC (gi:2826867) and DICH (gi:6466187) sequences and a CYC-like sequence from L. vulgaris (gi:5566284) were extracted from GenBank and confirmed by sequencing of the PCR product from the equivalent laboratory lines. Chloroplast sequence-based phylogenies were based on DNA sequences of the trnL + F region, consisting of regions of the (UAA) and (GAA) tRNA genes and the intergenic spacer separating them, using the universal primers f and c (Taberlet et al. 1991). trnL + F sequences for Mohavea breviflora (gi:19224319) and Mimulus ringens (gi:19224308) were obtained from GenBank.



View larger version (9K):
[in this window]
[in a new window]
 
FIG. 1. Structure of the CYC gene. The position and orientation of each oligonucleotide primer used in PCR and sequence analysis is indicated by an arrowhead and positions given in relation to the start codon of the A. majus CYC reference sequence (gi:2826867), where the A of the initial ATG(Met) codon is position 1. The TCP and R boxes are situated at positions 259–435 and 599–651 of the A. majus sequence, respectively

 
Nucleotide sequences were aligned using the T-Coffee program (Notredame, Higgins, and Heringa 2000) at www.ch.embnet.org. Phylogenetic analyses on all three data sets (trnL + F, CYC, and DICH homologs from Antirrhineae and CYC from Antirrhinum) were performed using three different tree-building methods as implemented in PAUP* 4.b10 (Swofford 1998). The minimum evolution (ME) method (Kumar 1996) was used with Kimura two-parameter (K2P) distances (Kimura 1980). Maximum parsimony (MP) analysis was conducted with unordered and unweighted characters. Maximum likelihood (ML) analysis was performed with the Hasegawa, Kishino, and Yano (HKY) substitution model (Hasegawa, Kishino, and Yano 1985) with empirical base frequencies, ML, estimated transition-transversion ratio, and estimated gamma (among-site rate variation) correction. Heuristic searches were performed with starting trees obtained via random stepwise addition, tree-bisection-reconnection (TBR) branch-swapping, and the "MulTrees" option in effect. Support for nodes was assessed using the bootstrap method (Felsenstein 1985) with 1,000 replicates for ME, 500 for MP, or 250 for ML. The number of synonymous (Ks) and nonsynonymous substitution (Ka) rates was calculated with the program K-estimator (Comeron 1999) using the method of Comeron (1995).

For Southern hybridizations, 3 µg of genomic DNA was digested with Hind III, fractionated in 0.7% agarose gels, and blotted onto a nylon membrane. Sequences were detected by hybridization to 32P-labeled probes consisting of either the CYC or DICH sequences from A. majus amplified with primers cycF-cycR or dichF-dichR that had been cloned into pGEM-T. Hybridization was carried out in 2x SSC at 65°C (high stringency), 60°C (medium stringency), or 55°C (low stringency) for 18 hours. Filters were then washed in 0.2x SSC at the equivalent temperature and exposed to X-ray film for 3 days. Between hybridizations, filters were washed in 0.1% SDS at 100°C until no residual signal could be detected with a 3-day exposure to film.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
To provide a phylogenetic framework against which to assess the evolution of CYC-like genes, sequences were obtained for the trnL + F region from the chloroplast genomes of representative taxa. The resulting phylogeny (fig. 2) had essentially the same topology as one obtained using the more slowly evolving ndhF chloroplast sequence (Ghebrehiwet, Bremer, and Thulin 2000), except that it resolved Misopates as basal to a clade containing Antirrhinum and the New World genera Sairocarpus and Mohavea.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 2. Minimum evolution phylogram for chloroplast trnL + F sequences of Antirrhineae. Sequences from Mimulus ringens and Digitalis purpurea are used as outgroups. Numbers at each node give bootstrap support values for ME/MP/ML analyses respectively; values lower than 50% are not shown or are represented by dashes. The scale bar indicates Kimura two-parameter distance

 
Oligonucleotide primers complementary to opposite ends of the CYC coding sequence from A. majus (cycF and cycR; fig. 1) were then used to amplify sequences from Antirrhinum species and their relatives, S. nuttallianus, M. orontium, and Chaenorhinum origanifolium (fig. 3). DICH-like genes were amplified from A. majus, S. nuttallianus, and M. orontium using primers (dichF and dichR) complementary to regions of the A. majus DICH sequence that were not conserved in A. majus CYC. Because these primers were unable to amplify CYC and DICH loci from Linaria species, Ch. origanifolium, Cymbalaria muralis, and Digitalis purpurea, primers corresponding to the more highly conserved TCP box and end box (cycFS and cycR*) were used to obtain CYC and DICH sequences from these more distantly-related taxa. However, no DICH-like sequence was obtained from Ch. origanifolium with any of these primers. All sequences consisted of open reading frames without frameshifts. CYC sequences from the Antirrhineae (i.e., all species except D. purpurea) formed a monophyletic clade, as did the DICH sequences from the equivalent species with the Digitalis sequence as an outgroup to both (fig. 3).



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 3. Minimum evolution phylogram of CYC and DICH sequences from Antirrhineae. Bootstrap support values are shown as in figure 1. The scale bar indicates Kimura two-parameter distance

 
All sequences were similar within the TCP and R boxes, with K2P distances ranging between 0.017 and 0.17. Outside these boxes, the sequences differed by numerous insertions and deletions. Indels made sequence alignment problematic, and therefore estimates of divergence distances, and substitution rates were dependent on the alignment chosen. However, alignments based only on the TCP and R boxes produced phylogenies with the same topologies and only slightly lower resolution than more complete sequences (data not shown). The K2P distances for these regions outside the TCP and R boxes were consistently higher (between 0.07 and 0.4) than for the TCP and R boxes. Relative rate tests (Takezaki, Rzhetzky, and Nei 1995) comparing CYC and DICH sequences from A. majus, S. nuttallianus, M. orontium, L. vulgaris, L. maroccana, and Cy. muralis in relation to the CYC-like sequence from D. purpurea suggested substitution rates that were not significantly different for the two loci.

To compare the evolutionary rate of CYC with another nuclear locus, an A. majus EST collection was searched for homologs of D. purpurea nuclear loci present in GenBank. Two Antirrhinum ESTs were identified as homologs of the D. purpurea aldose reductase gene (gi:13160396). Aligning the Antirrhinum and Digitalis sequences revealed no indels and K2P distances of 0.23 and 0.24 between the Digitalis sequence and each of the Antirrhinum ESTs. In comparison, the K2P distance between Antirrhinum CYC and Digitalis CYC-like was 0.28 with 15 indels (the number of indels depending on the alignment chosen), and between Antirrhinum DICH and Digitalis CYC-like the distance was 0.30 with 22 indels. In comparison, the K2P distance between CYC and DICH loci in Antirrhinum was 0.23 with 19 indels. These results suggest that CYC and DICH loci have evolved faster than the aldose reductase locus, largely as a result of indels outside the TCP and R boxes.

The ratio of nonsynonymous to synonymous substitution rates (Ka/Ks) within the TCP and R boxes ranged from 0.1 to 0.4. Outside these regions values were higher (between 0.1 and 0.6), but comparable to those obtained for TCP genes in grasses (Lukens and Doebley 2001). These Ka/Ks values did not provide evidence for positive selection and suggested that the variation within CYC and DICH sequences reflected relaxed evolutionary constraints.

The relationships within the CYC and DICH clades (fig. 3) differed slightly from each other and from the species phylogenies based on chloroplast DNA (fig. 2). Most notably, the clade containing Antirrhinum DICH was placed closer to the Cymbalaria sequence than to Linaria, whereas the Antirrhinum CYC and trnL + F sequences appeared closer to Cymbalaria than to Linaria. Possible explanations for these differences is that they resulted from later gene duplications that gave rise to paralogs within each clade and that the sequences did not represent orthologous genes. Evidence of a later duplication was provided by the two CYC-like sequences obtained from Ch. origanifolium. These were sufficiently different from each other (K2P distance = 0.087) to represent distinct loci rather than alleles. They were, however, more similar to each other than to the sequence from any other taxon, suggesting that they had arisen from a recent duplication in the lineage leading to Chaenorhinum.

The number of CYC and DICH loci was tested further by Southern hybridization of genomic DNA. High stringency hybridization with the A. majus DICH sequence as a probe detected strongly hybridizing bands (of ~1.5 kb) in A. majus and M. orontium, but not in their more distant relatives, Cy. muralis and D. purpurea (fig. 4). This suggested that a single DICH locus was present in Antirrhinum and Misopates and that the sequences from these species were more similar to each other than to the DICH-like genes of other taxa. This relationship was consistent with the phylogeny of DICH sequences obtained from these species, except that S. nuttallianus produced a much weaker band than M. orontium in Southern hybridization, even though the DICH sequences from the two species were equally similar to the A. majus probe. One explanation for this discrepancy is that S. nuttallianus has a larger genome than M. orontium, so fewer DICH target sequences were present in equivalent amounts of DNA on Southern blots. Similarly, high stringency hybridization with the A. majus CYC probe detected strong bands in A. majus and M. orontium and weaker bands in S. nuttallianus and Ch. origanifolium. As for DICH, the relative strength of hybridization signals was consistent with the relationship between CYC sequences in these taxa, except that S. nuttallianus and L. vulgaris were weaker than expected. This discrepancy might again reflect relative differences in genome size.



View larger version (46K):
[in this window]
[in a new window]
 
FIG. 4. Detection of CYC and DICH sequences by Southern hybridization. Genomic DNA (3.5 ng) from A. majus, Sairocarpus nuttallianus, Misopates orontium, Linaria vulgaris, Chaenorhinum origanifolium, Cymbalaria muralis, or Digitalis purpurea was digested with Hind III. It was then hybridized with either the A. majus CYC or DICH sequences at high stringency (65°C), medium stringency (60°C), or low stringency (55°C). Sizes of relevant bands were estimated by interpolation from an internal size standard (BRL 1 kb ladder). All DICH hybridizations were carried out to the same filter, which was different from that used for CYC hybridizations

 
Bands hybridizing weakly with the DICH probe at high stringency were also detected in all species except D. purpurea. For example the second most strongly hybridizing band in A. majus had a size of ~4.3 kb. These bands hybridized more strongly to the DICH probe at moderate stringency (fig. 4) and strongly to the CYC probe at high stringency. This suggested that they corresponded to the CYC locus and therefore that CYC and DICH are the two most closely related TCP loci in A. majus, S. nuttallianus, and M. orontium. A single band of ~3.7 kb was detected in D. purpurea at moderate and low stringency hybridization with the DICH probe and at low stringency hybridization with CYC, as expected from the presence of a single sequence in Digitalis that is equally similar to the CYC and DICH loci of A. majus.

At low stringency both CYC and DICH probes detected multiple bands in all species, consistent with the presence of multiple members of the TCP gene family.

To investigate further the variation of CYC alleles within the genus Antirrhinum, sequences were obtained from 17 Antirrhinum species accessions representing the full morphological and geographical range of the genus. Phylogenetic analysis gave clear support for monophyly of Antirrhinum CYC in relation to the outgroups M. orontium and S. nuttallianus (fig. 5). The CYC alleles of Antirrhinum species showed low substitution divergence (average P = 0.004; maximum P = 0.017), suggesting that they had diverged relatively recently. Several groups of species were found to share the same or very closely related sequences including (1) A. graniticum and A. barellieri; (2) A. mollissimum and A. hispanicum; (3) A. cirrigherum, A. latifolium, and A. australe; and (4) A. lopesianum, A. meonanthum, and A. braun-blanquetii. However, no bootstrap support was obtained for the distinction of groups (2), (3), or (4) using ML or MP.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 5. Minimum evolution phylogram of CYC sequences from Antirrhinum species. M. orontium and S. nuttallianus are used as outgroups. Bootstrap support values are shown as in figure 1. The GenBank CYC reference sequences of A. majus is named A. majus CYC. The scale bar indicates Kimura two-parameter distance

 
As in comparisons of CYC and DICH sequences from Antirrhineae, CYC alleles from Antirrhinum differed by several insertions and deletion polymorphisms. About half the alleles shared an additional trinucleotide (ACT) in their 3' region. A second trinucleotide repeat (CAA), encoding glutamine residues N-terminal to the TCP domain, was present in five copies in most alleles, four copies in A. molle, and two copies in A. siculum. The A. molle CYC allele differed from other Antirrhinum alleles by four insertions, each of three nucleotides that maintained the reading frame.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
CYC-like and DICH-like sequences were obtained from members of the Antirrhineae and from D. purpurea. The sequences formed two well-supported clades within the Antirrhineae—one containing the A. majus CYC gene, the other A. majus DICH—with the D. purpurea sequence basal to both. The basal position of the D. purpurea sequence is consistent with the taxonomic relationship between Digitalis and the Antirrhineae (Ghebrehiwet, Bremer, and Thulin 2000; Olmstead et al. 2001). The D. purpurea sequence is also more closely related to the Antirrhineae CYC- and DICH-like genes found in this study than to paralogous TCP genes from A. majus (Zsuzsanna Schwarz-Sommer, personal communication), suggesting that it represents a CYC/DICH homolog.

Our findings differ from those of Vieira, Vieira, and Charlesworth (1999), who proposed the existence of five CYC-like loci in Antirrhineae, including A. majus. Two of their proposed loci—cyc1a and cyc1b—correspond to Antirrhinum CYC alleles in our study, and their cyc2, cyc3, and cyc4 loci correspond to Misopates CYC, Misopates DICH, and Antirrhinum DICH, respectively. The previous study is therefore likely to have classified alleles as paralogous genes. In addition, genera as distantly related to Antirrhinum as Linaria, Cymbalaria, and Digitalis were proposed to carry sequences identical, or nearly identical, to those found in Antirrhinum (Vieira, Vieira, and Charlesworth 1999). We found no evidence for sequences so similar to Antirrhinum CYC alleles in more distantly related species, and the sequences that we did obtain from these taxa were not represented in the previous study. Possible explanations for these discrepancies is that the previous PCR primers were able to amplify CYC and DICH loci only from Antirrhinum and Misopates and that samples from more distantly related taxa had been contaminated with these sequences.

We provided further support for the existence of only one CYC and one DICH locus in Antirrhineae by direct sequencing and restriction digestion of PCR products. With the exception of Chaenorhinum, each individual carried only one or two CYC sequences, as expected of alleles. Moreover, the PCR products from A. majus and A. molle, distinguished by a restriction site polymorphism, segregated as alleles of the same locus in the F2 of an A. majus x A. molle hybrid and were used to map the locus to the same position as the cyc mutation (Schwarz-Sommer et al. 2003). Southern hybridization also suggested that DICH was the locus most similar to CYC in Antirrhinum, Sairocarpus, and Misopates, and it failed to provide evidence for sequences identical to Antirrhinum CYC in the more distantly related Linaria, Cymbalaria, and Digitalis.

The sequence phylogeny is consistent with the CYC and DICH loci having arisen from a gene duplication in the Veronicaceae (after the divergence of Digitalis) in an ancestor of the Antirrhineae with asymmetric flowers (Reeves and Olmstead 1998).

Although the relationship between DICH or CYC sequences from Antirrhinum, Sairocarpus, and Misopates is not well resolved, the phylogenies of both loci place Antirrhinum basal to Sairocarpus and Misopates (fig. 3). This contrasts with the phylogeny of the chloroplast trnF + L sequences in which Antirrhinum is basal. Such discrepancies between phylogenies of organelle and nuclear sequences are not uncommon in plants at lower taxonomic levels, and they can be explained by reticulation (e.g., sorting of alleles present in a common ancestral population) or by hybridization (e.g., Schwarzbach and Rieseberg 2002).

At a higher level, the DICH sequences from Linaria were placed basal to DICH from other Antirrhineae, whereas the Linaria CYC and trnL + F sequences were nested within the Antirrhineae. This might be explained by an additional duplication of DICH followed by loss of different paralogs from Linaria and related taxa. Although the presence of two CYC sequences in Chaenorhinum suggested that a more recent duplication had occurred, it appeared likely to have been confined to the lineage leading to Chaenorhinum. Likewise, additional CYC-like or DICH-like genes in all other taxa could not be detected by PCR amplification or Southern hybridization. An alternative explanation for the anomalous position of Linaria DICH sequences is that they had been subjected to different selectional pressures (discussed below).

These comparisons suggested a slightly higher rate of nucleotide substitution in CYC-like genes compared to aldose reductase genes However, they revealed a much higher turnover of indels in CYC-like sequences. Almost all indels are present in regions outside those encoding the functionally important TCP and R boxes. Although the rapid divergence of these regions is likely to represent relaxed evolutionary constraints, adaptive changes cannot be ruled out in the absence of further functional studies.

Rapid sequence evolution can theoretically follow divergence in the expression of duplicate genes, which allows proteins to assume different specialized functions (a case of subfunctionalization; Lynch and Force 2000). Although CYC and DICH partially overlap in function in the floral meristem of A. majus, their expression domains differ, and each also has a unique role, consistent with subfunctionalization following duplication (Carpenter and Coen 1990; Luo et al. 1996, 1999). In L. vulgaris, loss of CYC activity can have the same phenotypic effect as loss of both CYC and DICH in A. majus (Cubas, Vincent, and Coen 1999). This greater dependence on CYC activity in floral asymmetry is unlikely to reflect absence of the DICH locus from Linaria, because we detected DICH sequences in both L. vulgaris and L. maroccana. It might therefore reflect a greater degree of divergence between CYC and DICH functions in Linaria, relative to Antirrhinum. Our finding that the two Linaria DICH sequences are placed basally within the DICH clade (whereas Linaria CYC sequences cluster with Chaenorhinum CYC sequences) is consistent with the Linaria DICH locus having experienced different constraints following divergence of function. An alternative explanation is that CYC and DICH have very similar functions in Linaria and that DICH activity was already absent from the L. vulgaris material in which CYC function was tested experimentally (e.g., because of a recent mutation or epimutation). Floral asymmetry in this case would appear dependent on CYC activity alone.

Rapid sequence evolution has been recorded for several duplicated transcription factor genes in plants, including CYC-like genes of Gesneriaceae and CONSTANS LIKE genes of Brassicaceae (Citerne, Möller, and Cronk 2000; Lagercrantz and Axelsson 2000). No asymmetry in the rate of molecular evolution was detected for CYC and DICH, as has been found for other duplicated transcription factors (e.g., Stauber, Jäckle, and Schmidt-Ott 2002; Stauber, Prell, and Schmidt-Ott 2002).

Only a few nucleotide substitutions were found for CYC alleles within the genus Antirrhinum. One explanation for the similarity of CYC alleles is that speciation in Antirrhinum occurred relatively recently—a view supported by the ability of Antirrhinum species to form fertile hybrids and the low degree of sequence divergence of other nuclear and chloroplast sequences within the genus (our unpublished results). Dating the divergence between the teosinte-branched (tb1) gene in maize and its closest homolog in Oryza sativa to 65 myr before present (Purugganan 1997), provides a conservative divergence estimate of 3.2 to 4.5 x 10–3 substitutions per million years for CYC-like genes. Assuming that the rates of sequence evolution in CYC and DICH are comparable to their monocot homologs, then the divergence of Antirrhinum species can be estimated to have begun less than 5.3 to 3.7 MYA, and most Antirrhinum species to have arisen within the last million years. These divergence-time estimates for Antirrhinum species are consistent with estimates from other nuclear and chloroplast sequences (our unpublished results).

Several weakly supported clades were detected within CYC alleles from different Antirrhinum species. Species groupings largely corresponded to shared morphological characters and similar geographical distributions (Sutton 1988) and to similarities of other DNA sequences (our unpublished results), suggesting that the CYC locus provides a weak phylogenetic signal at this taxonomic level. This trend, however, is disrupted by A. siculum and A. charidemi, which appear geographically and phylogenetically distinct from the other sampled species but have CYC alleles that group among them.

CYC is linked to the self-incompatibility (S) locus of Antirrhinum by less than 1 cM (Schwarz-Sommer et al. 2003). Antirrhinum species carry functionally equivalent S alleles (e.g., Sherman 1939), which are presumed to be ancient and to have been maintained by balancing selection (Vekemans and Slatkin 1994), as in Solanaceae (Ioerger et al. 1990). Where tested, Antirrhinum S allele sequences show the expected deep divergences (Xu et al. 1996) and provide little species-phylogenetic signal. Although CYC is closely linked to S, it appears not to have been subjected to the same selection, suggesting that recombination has been sufficient to uncouple the fate of the S and CYC loci. However, one exceptional CYC sequence, differing by four trinucleotide insertions, was detected in A. molle. Although this is placed basally to other Antirrhinum CYC sequences, as expected of an old allele, it differs from other Antirrhinum sequences by more trinucleotide insertions than substitutions, and it shares none of its insertions with the outgroup sequences from Misopates or Sairocarpus. The CYC allele in A. molle is therefore likely to have accumulated its insertions very recently. Because the trinucleotide insertions cause amino acid insertions, rapid evolution of the CYC allele in A. molle might have been driven by positive selection. Alternatively, it might reflect an increased rate of insertion mutations or a decreased constraint in the A. molle lineage. Further sampling of sequences within A. molle populations will be necessary to address this question.


    Note Added in Proof
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
We would like to draw the reader's attention to a study by L. C. Hileman and D. A. Baum, published recently in this journal (Molecular Biology and Evolution, 2003, 20:591–600), which provides similar DNA sequence evidence for the fast evolution of CYCLOIDEA-like genes in the Antirrhineae.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Sequence alignments are provided online. All novel sequences have been deposited in GenBank under the accession numbers AY316693 to AY316730.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Seeds were kindly provided by E. S. Coen, D. Charlesworth, I. Mateu-Andrés, C. Thébaud, and the seedbank at the Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben. We thank J. Golz for L. maroccana DNA, M. Möller for primers cycFS, cyc*R, and trnL + F, and D. Charlesworth and two anonymous reviewers for their constructive comments. This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC).


    Footnotes
 
Brandon Gant, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Supplementary Material
 Acknowledgements
 Literature Cited
 

    Almeida, J., M. Rocheta, and L. Galego. 1997. Genetic control of flower shape in Antirrhinum majus. Development 124:1387-1392.[Abstract/Free Full Text]

    Carpenter, R., and E. S. Coen. 1990. Floral homeotic mutation produced by transposon-mutagenesis in Antirrhinum majus. Genes Dev. 4:1483-1493.[Abstract]

    Citerne, H. L., M. Möller, and Q. C. B. Cronk. 2000. Diversity of cycloidea-like genes in Gesneriaceae in relation to floral symmetry. Ann. Bot. 86:167-176.[Abstract/Free Full Text]

    Comeron, J. M. 1995. A method for estimating the numbers of synonymous and nonsynonymous substitutions per site. J. Mol. Evol. 41:1152-1159.[ISI][Medline]

    Comeron, J. M. 1999. K-Estimator: calculation of the number of nucleotide substitutions per site and the confidence intervals. Bioinformatics 15:763-764.[Abstract/Free Full Text]

    Cubas, P., E. Coen, and J. M. Martínez Zapater. 2001. Ancient asymmetries in the evolution of flowers. Current Biol. 11:1050-1052.[CrossRef][ISI][Medline]

    Cubas, P., N. Lauter, J. Doebley, and E. S. Coen. 1999. The TPC domain: a motif found in proteins regulating plant growth and development. Plant J. 18:215-222.[CrossRef][ISI][Medline]

    Cubas, P., C. Vincent, and E. S. Coen. 1999. An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401:157-161.[CrossRef][ISI][Medline]

    Doebley, J., A. Stec, and L. Hubbard. 1997. The evolution of apical dominance in maize. Nature 386:485-488.[CrossRef][ISI][Medline]

    Doyle, J. J., and J. L. Doyle. 1987. A rapid DNA isolated procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-15.

    Felsenstein, J. 1985. Confidence-limits of phylogenies—an approach using the bootstrap. Evolution 39:783-791.[ISI]

    Ghebrehiwet, M., B. Bremer, and M. Thulin. 2000. Phylogeny of the tribe Antirrhineae (Scrophulariaceae) based on morphological and ndhF sequence data. Plant Syst. Evol. 220:223-239.[ISI]

    Hasegawa, M., H. Kishino, and T. A. Yano. 1985. Dating the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22:160-174.[ISI][Medline]

    Ioerger, T. R., A. E. Clarke, and T.-H. Kao. 1990. Polymorphism at the self-incompatibility locus in Solanaceae predates speciation. Proc. Natl. Acad. Sci. USA 87:9732-9735.[Abstract]

    Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.[ISI][Medline]

    Kosugi, S., and Y Ohashi. 1997. PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene. Plant Cell 9:1607-1619.[Abstract/Free Full Text]

    Kumar, S. 1996. Stepwise algorithm for finding minimum evolution trees. Mol. Biol. Evol. 13:584-593.[Abstract]

    Lagercrantz, U., and T. Axelsson. 2000. Rapid evolution of the family of CONSTANS LIKE genes in plants. Mol. Biol. Evol. 17:1499-1507.[Abstract/Free Full Text]

    Lukens, L., and J. Doebley. 2001. Molecular evolution of the teosinte branched gene among maize and related grasses. Mol. Biol. Evol. 18:627-638.[Abstract/Free Full Text]

    Luo, D., R. Carpenter, L. Copsey, C. Vincent, J. Clark, and E. S. Coen. 1999. Control of organ asymmetry in flowers of Antirrhinum. Cell 99:367-376.[ISI][Medline]

    Luo, D., R. Carpenter, C. Vincent, L. Copsey, and E. S. Coen. 1996. Origin of floral asymmetry. Nature 383:794-799.[CrossRef][ISI][Medline]

    Lynch, M., and A. Force. 2000. The probability of duplicate gene preservation by subfunctionalization. Genetics 154:459-473.[Abstract/Free Full Text]

    Möller, M., M. Clokie, P. Cubas, and Q. C. B. Cronk. 1999. Integrating molecular phylogenies and developmental genetics: a Gesneriaceae case study. Pp. 375–402 in P. M. Hollingsworth, R. M. Bateman, and R. J. Gornall, eds. Molecular systematics and plant evolution. Taylor & Francis, London.

    Notredame, C., D. G. Higgins, and J. Heringa. 2000. T-coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302:205-217.[CrossRef][ISI][Medline]

    Olmstead, R. G., C. W. de Pamphilis, A. D. Wolfe, N. D. Young, W. J. Elisens, and P. A. Reeves. 2001. Disintegration of the Scrophulariaceae. Am. J. Bot. 88:348-361.[Abstract/Free Full Text]

    Purugganan, M. D. 1997. The MADS-box floral homeotic gene lineages predate the origin of seed plants: phylogenetic and molecular clock estimates. J. Mol. Evol. 45:392-396.[ISI][Medline]

    Reeves, P.A., and R. G. Olmstead. 1998. Evolution of novel morphological and reproductive traits in a clade containing Antirrhinum majus (Scrophulariaceae). Am. J. Bot. 85:1047-1056.[Abstract]

    Riechmann, J. L., J. Heard, and G. Martin, et al. (14 co-authors). 2000. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105-2110.[Abstract/Free Full Text]

    Schwarz-Sommer, Z., E. de Andrade Silva, R. and Berndtgen, et al. (11 co-authors). 2003. A linkage map of an F2 hybrid population of Antirrhinum majus and A. molle. Genetics 163:699-710.[Abstract/Free Full Text]

    Schwarzbach, A. E., and Rieseberg L. H. 2002. Likely multiple origins of diploid hybrid sunflower species. Mol. Ecol. 11:1703-1715.[CrossRef][ISI][Medline]

    Sherman, M. 1939. The inheritance of self-sterility in various species and varieties of Antirrhinum. Z. f. indukt. Abst. Vererbungsl. 77:3-17.

    Stauber, M., H. Jäckle, and U. Schmidt-Ott. 1999. The anterior determinant bicoid of Drosophila is a derived Hox class 3 gene. Proc. Natl. Acad. Sci. USA 96:3786-3789.[Abstract/Free Full Text]

    Stauber, M., A. Prell, and U. Schmidt-Ott. 2002. A single Hox3 gene with composite bicoid and zerknüllt expression characteristics in non-Cyclorrhaphan flies. Proc. Natl. Acad. Sci. USA 99:274-279.[Abstract/Free Full Text]

    Sutton, D. A. 1988. A revision of the tribe Antirrhineae. Oxford University Press, London.

    Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, Mass.

    Taberlet, P., L. Gielly, G. Pautou, and J. Bouvet. 1991. Universal primers for amplification of three coding regions of chloroplast DNA. Plant Mol. Biol. 17:1105-1109.[ISI][Medline]

    Takezaki, N., A. Rzhetzky, and M. Nei. 1995. Phylogenetic test of the molecular clock and linearized trees. Mol. Biol. Evol. 12:823-833.[Abstract]

    Vekemans, X., and M. Slatkin. 1994. Gene and allelic genealogies at a gametophytic self-incompatibility locus. Genetics 137:1157-1165.[Abstract/Free Full Text]

    Vieira, C. P., J. Vieira, and D. Charlesworth. 1999. Evolution of the cycloidea gene family in Antirrhinum and Misopates. Mol. Biol. Evol. 16:1474-1483.[Abstract]

    Xue, Y., R. Carpenter, H. G. Dickinson, and E. S. Coen. 1996. Origin of allelic diversity in Antirrhinum S locus RNases. Plant Cell 8:805-814.[Abstract/Free Full Text]

Accepted for publication May 8, 2003.