Department of Organismic and Evolutionary Biology, Harvard University
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Abstract |
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Key Words: gene duplication nonsynonymous/synonymous rate ratio CYCLOIDEA DICHOTOMA Antirrhinum majus snapdragon
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Introduction |
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Wild-type flowers of Antirrhinum majus (snapdragon) are bilaterally symmetrical (zygomorphic) with an adaxial (dorsal) region that is distinct from the abaxial (ventral) region. In particular, the two adaxial petal lobes are enlarged relative to the lateral and abaxial lobes, and the adaxial stamen is aborted early in development (resulting in four fertile stamens at maturity). It has been shown that the genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) are required to determine adaxial flower identity in A. majus (Luo et al. 1996, 1999). In cyc-dich double mutants, fully abaxialized, radially symmetrical (peloric) flowers are formed. If one of the two genes is present in its wild-type form, a partially peloric flower develops, but the severity of this phenotype is different for each gene. Whereas dich single mutants show only slight modifications to the adaxial petals and a wild-type pattern of stamen abortion, cyc mutants produce flowers that have a high degree of abaxialization and lack stamen abortion. Cloning of CYC and DICH (Luo et al. 1996, 1999) has shown that they are closely related members of the TCP family of transcription factors, many of which appear to influence meristem and primordium growth (Cubas et al. 1999). Therefore, it is likely that the roles of CYC and DICH in flower development are derived from an ancestral function before gene duplication with subsequent changes to gene function accumulating in the CYC lineage, the DICH lineage, or both. The fact that cyc mutants show a more severe phenotype than dich mutants might lead one to suppose that there has been less change in function at the CYC locus. Consequently, one might predict that, of the two genes, CYC may show evidence of stronger purifying selection. Additionally, if DICH is in the process of acquiring novel function, one might see evidence of directional selection at this locus. Or, if DICH is evolving neutrally, having been freed from responsibility for the ancestral function, one might see evidence of neutral evolution at the DICH locus.
In this paper, we describe molecular evolution in the CYC/DICH gene family across the Antirrhineae (the tribe to which Antirrhinum belongs). All species included have zygomorphic flowers, and it is likely that this is the plesiomorphic condition inherited from their common ancestor (Coen and Nugent 1994; Donoghue, Ree, and Baum 1998; Olmstead et al. 2001). Contrary to a previous study suggesting multiple gene duplication events (Vieira, Vieira, and Charlesworth 1999), we show that CYC and DICH derive from a single duplication event before the radiation of the Antirrhineae. In addition we present evidence that both CYC and DICH loci are under purifying selection but that there may be a tendency towards relaxed selective constraint at the DICH locus. These findings are consistent with CYC and DICH both maintaining the putatively ancestral function of determining adaxial flower identity.
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Materials and Methods |
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Phylogenetic Analysis
Among the clones sequenced for each species, one was selected from each distinct sequence class to represent a given species. The sequence selected was one that lacked single-base differences from other cloned sequences because such singletons are likely to reflect nucleotide misincorporation during PCR ("PCR-errors"). The selected sequences were aligned manually with reference to both nucleotide and hypothetical amino acid information using MacClade 4.0 (Maddison and Maddison 1999). Percent divergences (uncorrected pairwise differences) among and within loci were used to assess putative allelic variation.
Phylogenetic analyses were conducted using PAUP* 4.0b1 (Swofford 2001). Fitch parsimony (MP) trees were generated with heuristic searches (100 random stepwise taxon additions and TBR branch-swapping algorithm) with gaps treated as missing data. Bootstrap support for nodes (Felsenstein 1985) was estimated with 1,000 heuristic search replicates using the same setting as the original search. Trees were rooted with Gcyc1 and Gcyc2 sequences from Haberlea ferdinandi-coburgii (Citerne, Moller, and Cronk 2000).
To compare the CYC-like sequences isolated in this analysis with those found by Vieira, Vieira, and Charlesworth (1999), maximum parsimony trees were generated (as described above) that included our CYC-like sequences, as well as Linaria cyc1B (GenBank accession number AF146862), Cymablaria cyc1B (GenBank accession number AF146861), and Digitalis cyc1A, cyc1B, cyc2, and cyc4 (GenBank accession numbers AF146847, AF146860, AF146865, and AF146876, respectively) from Vieira, Vieira, and Charlesworth (1999).
Maximum likelihood (ML) trees were generated under the general time-reversible (GTR) model of evolution with a discrete gamma model (d) allowing for four categories of rate variation among sites (Yang 1994a, 1994b). Heuristic searches under the ML optimality criterion were conducted using random stepwise taxon addition and NNI branch swapping algrorithm. ML searches excluded Gcyc1 and Gcyc2 sequences from Haberlea ferdinandi-coburgii, and were instead rooted with CYC-like sequences from Digitalis purpurea (consistent with the parsimony analysis).
We conducted Wilcoxon signed-rank (Templeton 1983; Larson 1994; Mason-Gamer and Kellogg 1996) and Kishino-Hasegawa (Kishino and Hasegawa 1989) tests based on the parsimony and ML analyses, respectively, to determine if the data could reject an ancient duplication, giving rise to the CYC and DICH paralogous lineages. Constraint trees were constructed that would support a CYC/DICH gene duplication before the divergence of Digitalis or Haberlea from the lineage leading to Antirrhinum. The optimal trees compatible with those constraints were found using heuristic searches (as above) and were evaluated relative to the optimal unconstrained trees.
Differences in the mode of molecular evolution among regions of the CYC/DICH gene were examined in a likelihood framework (Sanderson and Doyle 2001). Five data partitions corresponding to the variable and conserved regions of the TCP gene family (Cubas et al. 1999) were identified (fig. 1). The null hypothesis, that all partitions evolved according to the same model of molecular evolution (GTR + d) with one set of rate parameters, was compared with an alternative hypothesis that allowed different rate parameters in the GTR + d
model to be estimated for each of the five partitions. The ML tree was assumed, and the sum of the log-likelihood scores for the five partitions was compared with the likelihood of the data under the null hypothesis using a likelihood ratio test (Felsenstein 1981; Goldman 1993; Yang, Goldman, and Friday 1995; Huelsenbeck and Rannala 1997). If the likelihood ratio (2
), 2[-lnL1+lnL2], is significant as determined from a
2 test with the appropriate degrees of freedom, then the parameter rich model was considered to provide a significantly better explanation of the data. The degrees of freedom equal the sum of the number of free parameters in each partition of the alternative model minus the number of free parameters in the null model.
Differences in the rate of molecular evolution among these five regions were estimated by averaging over all pairwise distances between CYC and DICH sequences for each of the five regions. Pairwise distances were estimated using ML under the GTR + d model of molecular evolution using the shape parameter that was estimated in the ML search (
= 0.857).
Analyses of patterns of molecular evolution were conducted using ML trees generated under the GTR-d model as described above but with selected taxa excluded. In all four analyses, CYC paralogs from Antirrhineae taxa that lacked DICH paralogs, Cymbalaria muralis, Kickxia spuria, and Maurandya antirrhiniflora, were excluded. The outcomes are as follows: tree 1: CYC and DICH paralogs from Antirrhineae plus CYC-like sequences from D. purpurea as designated outgroups; tree 2: both CYC and DICH paralogs from Antirrhineae, rooted between the CYC and DICH clades; tree 3: CYC paralogs from the Antirrhineae, rooted with CYC from Lophospermum (consistent with the MP and ML tree [fig. 2]); tree 4: DICH paralogs from the Antirrhineae, rooted with DICH from Lophospermum (consistent with the MP and ML tree [fig. 2]).
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To test for shifts in selective constraint within the gene tree, four nested likelihood models were applied to trees 1 and 2 above. These models are based on the codon substitution model of Goldman and Yang (1994), which accounts for the genetic code structure, transition/transversion rate bias, and heterogeneity in the frequency of bases found at all three codon positions. Model A (Goldman and Yang 1994) assumes a single for all branches in the phylogeny, whereas models B, C, and D allow for variation in
among lineages (Yang 1998; Yang and Nielsen 1998; Bielawski and Yang 2001). Model B assumes two
ratios: one restricted to lineages predating a gene duplication event, the second restricted to all lineages postdating a gene duplication event. Model C assumes three
ratios: one restricted to lineages predating a gene duplication event and different ratios for each of the paralogous lineages after the gene duplication event. Model D, the most general model, assumes an independent
ratio for each branch of the phylogeny and is, therefore, useful for distinguishing shifts in
correlated with the CYC/DICH duplication from effects within the CYC and/or DICH lineages. These models do not account for among-codon variation in
. Because there was evidence for rate heterogeneity among the five different regions of the CYC/DICH gene (see Results), these were analyzed independently.
To further investigate lineage specific shifts in after gene duplication, models A and D were applied to the CYC subtree (tree 3) and the DICH subtree (tree 4). Analyses of these subtrees were needed because many insertion/deletion events are required to align CYC and DICH, effectively deleting much of the important information and introducing additional sources of uncertainty. In contrast, within the CYC and DICH clades, alignment is relatively straightforward, facilitating statistical analysis of the history of selective constraint.
The likelihood values of the pairs of nested models, A to D, were compared using a likelihood ratio test (Felsenstein 1981; Goldman 1993; Yang, Goldman, and Friday 1995; Huelsenbeck and Rannala 1997). If the likelihood ratio (2), 2[-lnL1+lnL2], is significant as determined from a
2 test with the appropriate degrees of freedom (the difference in number of
ratios estimated), then the parameter rich model was considered to provide a better explanation of the data.
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Results |
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For some individuals, multiple similar but distinct sequences were isolated from a single individual. In most cases, these extra sequences could be attributed to PCR error. However, in some cases, divergence was greater (0.53% to 1.42% nucleotide divergence [table 2]) such that PCR error is an unlikely explanation. These are interpreted as putative alleles amplified from a heterozygous individual.
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All sequences identified as putative loci coalesced within and not between species. Those clones that did not coalesce within species always showed a distinct pattern in which the 5' end of the sequence was identical to one locus (CYC or DICH) from that taxon, and the 3' end of the sequence was identical to the other locus. These clones are interpreted as recombinants generated during PCR (Bradley and Hillis 1997) and were eliminated from further analyses.
Phylogenetic Analysis
Maximum parsimony and maximum likelihood each yielded a single optimal tree. These trees differed only in the position of DICH from Chaenorhinum villosum. In the MP tree, Chaenorhinum DICH was sister to Linaria canadensis + L. vulgaris DICH, whereas in the ML tree (fig. 2), it was sister to Misopates + Antirrhinum DICH. The MP and ML gene trees (fig. 2) suggest a single gene duplication event, before the radiation of the Antirrhineae, resulting in the CYC (100% bootstrap) and the DICH (87% bootstrap) clades. In addition to this duplication, the phylogeny suggests two independent gene duplications within a monophyletic (81% bootstrap) Digitalis lineage.
Trees in which Digitalis has both CYC and DICH paralogs, implying a more ancient gene duplication event, are 20 steps longer than the most parsimonious tree. This is significant using a Templeton test (Templeton 1983), with P = 0.041, but is not significant using a Kishino-Hasegawa test (Kishino and Hasegawa 1989) with P = 0.052. Trees in which Gesneriaceae Gcyc1 and Gcyc2 sequences are divided into CYC-like and DICH-like forms can be rejected (P < 0.01 for both the Templeton and Kishino-Hasegawa tests). These results suggest that the CYC/DICH gene duplication occurred within Lamiales, and likely within the Veronicaceae (Olmstead et al. 2001), after the divergence of Digitalis from the lineage leading to Antirrhineae.
Inferred species relationships within the CYC and DICH clades are highly congruent (fig. 2). However, there is strong support for only a few relationships. Specifically, with 100% bootstrap support, the following clades are supported: Linaria canadensis + L. vulgaris (CYC and DICH), Antirrhinum + Misopates (CYC and DICH), and Lophospermum + Maurandya (CYC only).
Maximum parsimony analysis of the full data matrix plus Linaria, Cymbalaria, and Digitalis CYC-like sequences from Vieira, Vieira, and Charlesworth (1999) yielded two most parsimonious trees. In both trees, Linaria, Cymbalaria, and Digitalis CYC-like sequences from the Vieira et al. study (cyc1A, cyc1B, and cyc2) are allied to Antirrhinum and Misopates CYC rather than to sequences from Linaria, Cymbalaria, or Digitalis (fig. 3). The Digitalis DICH-like sequence from the Vieira et al. study (cyc4) is allied to Antirrhinum DICH rather than to Digitalis sequences (fig. 3). This result raises the possibility that the sequences obtained by Vieira, Vieira, and Charlesworth (1999) were contaminating Antirrhinum and Misopates sequences rather than distinct loci.
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Different Selection Among Lineages
A likelihood ratio test was used to look for evidence of variation in the rate of nonsynonymous versus synonymous substitution (dN/dS = ) on different lineages. The test was conducted independently for the five gene regions (fig. 1) on the four pruned trees (fig. 4). In most cases, the data were insufficient to reject a single underlying value of
for the entire tree. Multiple-rate models were only favored in tests involving trees 1 and 2 (and tree 4 for the TCP-domain), which included both CYC and DICH paralogs. Trees 3 and 4, which included only CYC or DICH genes, respectively, nearly always supported a single-rate model.
As summarized in table 3, estimates of with data from both CYC and DICH paralogs included (tree 1 and tree 2) were much lower for the TCP- and R-domains (0.013 to 0.022) than for the variable domains (0.189 to 0.405). This result is expected because low values of
arise when lineages have been subject to strong purifying selection. Therefore, inferred functional constraints acting on the TCP- and R-domains (Cubas et al. 1999) would be predicted to give rise to lower values of
. Nonetheless, while purifying selection is apparently stronger in the TCP- and R-domains, it may not be absent from regions A, B, and C, as indicated by values of
that are markedly less than 1.0.
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In the more variable regions, A, B, and C, most tests failed to reject the null hypothesis of a single value of (table 3). However, for regions A and C, analysis on tree 2 (and tree 1 in the case of region C) suggested a model in which there have been different rates of evolution at the CYC and DICH loci (table 3). Specifically, in each case,
was estimated to be higher at the DICH locus, suggesting relatively relaxed purifying selection. Compatible with this finding, for all three regions, the estimate of
, for tree 3 (CYC only) is lower than for tree 4 (DICH only).
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Discussion |
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Conflict with Vieira, Vieira, and Charlesworth (1999)
In a recent paper, Vieira, Vieira, and Charlesworth (1999) suggested that there are at least five CYC-like loci in Antirrhinum majus. Comparing their sequences to DICH (which appeared after Vieira, Vieira, and Charlesworth, 1999) and CYC from A. majus, it would seem that they isolated three CYC and two DICH loci. At several of these loci, they also sequenced genes from Misopates (a close relative of Antirrhinum) and other, more distantly related lineages, Linaria, Cymbalaria, and Digitalis. Surprisingly, genes amplified from these distantly related species were identical to, or differed by very few nucleotides from, sequences obtained from Antirrhinum. Their data were interpreted as suggesting a series of ancient gene duplications followed by such strong selective constraint at the nucleotide level that divergence among species all but ceased. In contrast, our results suggest that there has only been one gene duplication event, giving rise to the CYC and DICH clades, but that each gene has undergone substantial genetic divergence at the nucleotide level with obvious conservation at the amino acid level, especially in the TCP- and R-domain regions.
One possible explanation for the discrepancy between these two studies is that we missed cryptic CYC and DICH loci from Antirrhinum and also failed to amplify the CYC-like genes attributed to Linaria, Cymbalaria, and Digitalis by Vieira, Vieira, and Charlesworth (1999). Under this explanation, the CYC-like sequences that we did obtained from Linaria, Cymbalaria, and Digitalis would represent more distantly related genes descended from yet further ancient duplication events. We do not believe this explanation is correct for the following reasons. (1) We used the same primers as Vieira, Vieira, and Charlesworth (1999), making it unlikely that we would have failed to find the missing loci, despite cloning and sequencing as many as 30 clones from a single species. (2) It is improbable that we would have amplified distantly related paralogs in Linaria, Cymbalaria, and Digitalis while missing paralogs that are sequence-identical to those successfully amplified from Antirrhinum. (3) The symmetrical species relationship in the CYC and DICH subtrees would be unlikely to arise if there had been numerous hidden duplication events. (4) The level of divergence between species, for example Digitalis and Antirrhinum, that we obtained is in line with the phylogenetic distance and sequence divergence of these taxa at the rbcL, ndhF, and rps2 loci (Olmstead et al. 2001), whereas the divergences suggested by the Vieira, Vieira, and Charlesworth (1999) study are unrealistically low.
The only alternative explanation we can offer for the discrepancy between our data and Vieira, Vieira, and Charlesworth (1999) is that, despite various precautions, some of the sequences they amplified from non-Antirrhinum accessions were contaminated by Antirrhinum and Misopates sequences. In line with this explanation, the sequences they obtained closely match with sequences we obtained from Antirrhinum majus and Misopates orontium but not with other Antirrhineae or Digitalis (fig. 3). Consequently, we will assume for the remainder of this paper that our data are reliable as a basis for studying patterns of molecular evolution in the CYC/DICH gene family.
Selective Maintenance After Gene Duplication
Classical models suggest that after gene duplication, either one paralog will acquire a new function or, more often, one paralog will be lost through the fixation of null alleles (Ohno 1970; Nei and Roychoudhury 1973; Bailey, Poulter, and Stockwell 1978; Ohta 1988; Walsh 1995). Such models tend to discount long-term maintenance without functional divergence because the action of selection on duplicate loci entails a positive feedback mechanism. If one copy becomes slightly less effective than its paralog due to genetic drift, then further deleterious mutations to that locus will have less severe consequences than mutations to the gene that retains ancestral function. Thus, these models predict that, unless one gene acquires a novel function, there will be a run-away process of genetic degradation culminating in the complete loss of one paralog.
The fact that we failed to isolate DICH paralogs from three of the sampled genera (Kickxia, Maurandya, and Cymbalaria) is consistent with the idea that these genes may have been lost by genetic degradation. However, even if we assume that in the case of DICH these genes were lost (rather than simply not amplified), the classical models are hard to defend. First, gene loss, if it has occurred, has apparently proceeded more slowly than speciation with at most three, of the 11, sampled Antirrhineae lacking DICH. Second, estimated nonsynonymous/synonymous substitution rate ratios () in both the CYC and DICH clades were much less than 1.0, indicating that purifying selection has been acting at both loci. Third, there is no evidence for positive selection in the protein-coding region of either paralog, as would be expected if they had acquired a novel function, although positive selection on a small subset of sites cannot be ruled out. Finally, functional data from Antirrhinum majus suggest that CYC and DICH have similar biochemical functions despite some differences in gene expression patterns (Luo et al. 1999). These differences in patterns of gene expression may contribute to the maintenance of CYC and DICH lineages.
Given that our data, in combination with the functional data on CYC and DICH from A. majus, cannot easily be explained by classical models of molecular evolution after gene duplication, they need to be evaluated within the framework provided by newer models, which allow for the possibility of long-term selective maintenance of duplicate genes without one attaining a novel function. Specifically, two additional mechanisms have been proposed for the persistence of paralogs. First, if two duplicate genes both retain an ancestral function, purifying selection could act on both loci via dosage effects and/or error-buffering during ontogeny (Thomas 1993; Krakauer and Nowak 1999). Alternatively, if the ancestral gene fulfilled multiple biological functions, then the duplicate loci could be maintained when reduction in activity at one locus creates a selective constraint, preventing gene loss at the second locus (Stoltzfus 1999). This has been described as selection for complementary subfunctionalization (Force et al. 1999; Lynch and Force 2000). The distinction between selection maintaining dosage and selection for complementary subfunctionalization is not very clear, because the contribution of two loci to a gene-product "dose" can be thought of as subfunctionalization (the "subfunctions" being production of the first or second half of the required amount). However, they have different evolutionary consequences in that subfunctionalization causes parcellation, a reduction in pleiotropy that may facilitate subsequent independent evolution of each subfunction.
In light of these models of molecular evolution and information from genetic studies illustrating that CYC and DICH have a high degree of redundant function in A. majus, with CYC contributing more significantly to adaxial flower identity than DICH, it becomes possible to make sense of our sequence data and propose a plausible scenario (fig. 5). We suggest that immediately after the gene duplication event that gave rise to the CYC and DICH loci, there was a period of relaxed selection in which both genes accumulated mild loss-of-function mutations. The somewhat higher values of for the DICH clade, the evidence that DICH has been lost or has diverged markedly in three lineages, and the milder phenotype of dich relative to cyc mutant Antirrhinum majus suggest that the DICH locus accumulated more deleterious mutations than the CYC locus. Nonetheless, it appears that the CYC locus was not immune from such mutation because low values of
are estimated for DICH as well as CYC clades, and because Antirrhinum dich mutants do not have wild-type flowers (Luo et al. 1999). Minimally, we speculate that a reduction in CYC expression or activity in the adaxial area of the corolla resulted in a dependence on the DICH gene product to complete normal development of the adaxial petal lobes. The fact that the DICH lineage leading to A. majus shows evidence of relaxed purifying selection in the TCP-domain (table 3 and fig. 4D) raises the possibility that the role of DICH diminished recently in Antirrhinum majus. If extensive reduction in DICH function is restricted to the Antirrhinum lineage, then we expect to see a higher degree of ancestral DICH function retained across the Antirrhineae. If one were to determine both cyc and dich mutant phenotypes from other Antirrhineae taxa, we hypothesize that dich phenotypes would be quite similar to the dich mutant phenotype in A. majus. This follows because patterns of molecular evolution across the Antirrhineae do not suggest significant loss of CYC function along specific lineages. However, cyc mutant phenotypes in taxa of the Antirrhineae might be less severe than the cyc mutant phenotype in A. majus, because DICH function in these taxa would be contributing more to adaxial flower identity.
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Although slightly deleterious mutations likely fixed at both loci (Ohta 1973, 1993), it seems that the early degradation at the DICH locus was more intense than at the CYC locus. This explains the somewhat weaker purifying selection at DICH relative to CYC (table 3). Additionally, the reduced purifying selection at DICH may have increased the probability of DICH crossing the threshold beyond which purifying selection could prevent gene loss. Such independent losses of DICH (as seen in Maurandya, Cymbalaria, and Kickxia) could have been precipitated by population bottlenecks in which deleterious mutations became fixed by genetic drift or by periods of directional selection, during which mutations at the CYC locus arose that served to reduce the selective cost of loss of DICH function.
The scenario we have proposed for the molecular evolution of the CYC/DICH gene family in Antirrhineae is probably the simplest way to reconcile the molecular data and previous genetic work on Antirrhinum. However, it requires some additional assumptions to accommodate genetic data from Linaria vulgaris. Specifically, Cubas, Vincent, and Coen (1999) showed that a methylation defect at the CYC locus was sufficient to cause the formation of a completely peloric flower. Given that in Antirrhinum complete peloria requires the loss of both CYC and DICH activity, it would appear that in Linaria vulgaris, the DICH ortholog has no residual role in the determination of adaxial flower identity. Why then should the Linaria vulgaris DICHortholog be maintained and yet show no signs of relaxed purifying selection? Two explanations are possible. Either Linaria DICH function was completely lost so recently that there has been insufficient time for the gene sequence to show signs of relaxed selection or this locus has acquired a novel function but directional selection has not left a strong enough signature to be detected in our analysis. These alternatives could be distinguished by observing a Linaria vulgaris dich mutant to see whether it has floral defects, extrafloral defects, or neither. Similarly, it would be interesting to see the phenotype of a Linaria canadensis cyc mutant because if it were completely peloric (like L. vulgaris), then one could not so easily posit that L. vulgaris dich lost function very recently.
The CYC/DICH gene subfamily is a relatively simple group to study because there are just two loci in diploid Antirrhineae. Furthermore, because Antirrhinum is a model system for the study of floral symmetry, we were able to integrate genetic and functional data with analyses of sequence evolution to propose a simple molecular evolutionary scenario. Although this hypothesis is speculative, it suggests numerous follow-up studies, including measurement of the selective consequences of dich mutations in realistic field environments and characterization of mutant phenotypes in other species of Antirrhineae. Furthermore, by coupling functional studies with molecular evolutionary analyses focusing on upstream sequences of CYC and DICH, we may begin to understand whether maintenance of CYC and DICH is due in part to novel function in the cis-regulatory regions of these genes. Through such integrative studies, we believe light can be shed on the short-term and long-term evolutionary consequences of gene duplication.
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Acknowledgements |
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Footnotes |
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2 Present address: Department of Botany, University of Wisconsin, Madison, Wisconsin.
E-mail: lena.hileman{at}yale.edu.
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