Why Do Paralogs Persist? Molecular Evolution of CYCLOIDEA and
Related Floral Symmetry Genes in Antirrhineae (Veronicaceae)
Lena C. Hileman1 and David A. Baum2
Department of Organismic and Evolutionary Biology, Harvard University
CYCLOIDEA (CYC) and DICHOTOMA (DICH) are paralogous genes that determine adaxial (dorsal) flower identity in
the bilaterally symmetric flowers of Antirrhinum majus (snapdragon). We show here that the duplication leading to the
existence of both CYC and DICH in Antirrhinum occurred before the radiation of the Antirrhineae (the tribe to which
snapdragon belongs). We find no additional gene duplications within Antirrhineae. Using explicit codon-based models of
evolution in a likelihood framework, we show that patterns of molecular evolution after the duplication that gave rise to
CYC and DICH are consistent with purifying selection acting at both loci, despite their known functional redundancy in
snapdragon. However, for specific gene regions, purifying selection is significantly relaxed across DICH lineages,
relative to CYC lineages. In addition, we find evidence for relaxed purifying selection along the lineage leading to
snapdragon in one of two putative functional domains of DICH. A model of selection accounting for the persistence of
paralogous genes in the absence of diversifying selection is presented. This model takes into account differences in the
degree of purifying selection acting at the two loci and is consistent with subfunctionalization models of paralogous gene
evolution.
Introduction
1
Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut.
2
Present address: Department of Botany, University of Wisconsin,
Madison, Wisconsin.
Key words: gene duplication, nonsynonymous/synonymous rate
ratio, CYCLOIDEA, DICHOTOMA, Antirrhinum majus, snapdragon.
E-mail: [email protected].
Mol. Biol. Evol. 20(4):591–600. 2003
DOI: 10.1093/molbev/msg063
Ó 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
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 wildtype 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
591
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Gene duplication is thought to be an important evolutionary agent because duplicated genes may be co-opted
for new functions, allowing for increases in genomic
complexity (Ohno 1970). In classical models of evolution
after gene duplication (Ohno 1970; Nei and Roychoudhury 1973; Bailey, Poulter, and Stockwell 1978; Ohta
1988; Walsh 1995; Lynch and Conery 2000), two outcomes are possible. One of two paralogous loci in a population may become subject to directional selection for
some novel function, whereas the other paralog retains the
ancestral function. In this case, both genes are expected to
persist in the genome. Alternatively, and with higher
probability, one of the duplicates will accumulate deleterious loss-of-function mutations, ultimately being lost.
Thus, in classical models, long-term persistence is only
likely when paralogs acquire novel function. In contrast,
recent theoretical models allow for the possibility of selective maintenance of both paralogous genes without the
acquisition of a novel function (Thomas 1993; Nowak et al.
1997; Force et al. 1999; Krakauer and Nowak 1999;
Stoltzfus 1999; Lynch and Force 2000; Lynch et al. 2001).
In this paper, we reconstruct the history of gene
duplication in a small gene family and then use a likelihood-based analysis of sequence evolution to explore
changes in the strength and modes of selection that have
acted on duplicated genes.
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
592 Hileman and Baum
Table 1
Taxa Sampled for Analyses
GenBank Accession Numbers
Taxon
Antirrhinum majus
Asarina procumbens
Chaenorhinum villosum
Cymbalaria muralis
Kickxia spuria
Linaria canadensis
Linaria vulgaris
Lophospermum sp.
Maurandya antirrhiniflora
Misopates orontium
Digitalis purpurea
Specimen (Herbarium)
CYCLOIDEA
DICHOTOMA
##
104/99 (JBV)
P 47755 (A)
RN s.n.
RKO 8 (A)
RKO 1A (A)
RKO 23 (A)
91/97 (JBV)
H 18323 (GH)
RKO 49 (A)
##
AF512598
AF512601
AF512599
AF512595
AF512602
AF512603
AF512596
AF512597
AF512600
AF512604/AF512605/AF512606
AF512592
AF512591
AF512589
AF512590
AF512593
AF512594
NOTE.—## 5 common garden variety; H 5 Hill; P 5 Podlech; RKO 5 Ryan K. Oyama; RN 5 collected by Reto Nyffeler;
GH 5 Gray Herbarium; A 5 Herbarium of the Arnold Arboretum; JBV 5 Jardin Botanico de Valencia. Accession of
Lophospermum from JBV was identified only to the level of genus.
Materials and Methods
Study Species
In addition to the published sequences, Antirrhinum
majus CYC (GenBank accession number Y16313), A.
majus DICH (GenBank accession number AF199465),
and Linaria vulgaris CYC (GenBank accession number
AF161252) taxa were sampled from among the Antirrhineae and the closely related foxglove, Digitalis
purpurea (table 1). CYC-like sequences from Gesneriaceae, Haberlea ferdinandi-coburgii Gcyc1 (GenBank
accession number AF208322) and Gcyc2 (GenBank
accession number AF208317) were used for rooting
purposes.
Isolation and Sequencing of CYC and DICH Loci
Genomic DNA was isolated using the plant
DNAEASY kit (QIAGEN, Chatsworth, Calif.). To amplify CYC-like sequences from genomic DNA, PCR was
performed with 39 to 42 cycles each with denaturation at
948C for 30 s, annealing at 428C to 518C for 30 s and
extension at 728C for 1 minute. The forward primer, P1,
and reverse primer, P2 (Vieira, Vieira, and Charlesworth
FIG. 1.—Map of the CYC and DICH genes from A. majus. Variable
regions 5 A, B, and C. Conserved regions 5 TCP-domain and Rdomain.
1999), were used to amplify all CYC and DICH sequences
in this analysis (fig. 1). Multiple DICH-specific primers
were designed in an attempt to isolate the DICH locus
from Cymbalaria muralis, Kickxia spuria, and Maurandya
antirrhiniflora, but all attempts were unsuccessful (fig. 1,
and data not shown).
PCR products were run on 1% agarose gels, and
bands near the expected length (ca. 850 bp for CYC loci
and ca. 950 bp for DICH loci) were excised. Excised
PCR products were extracted using a gel extraction
kit (QIAGEN, Chatsworth, Calif) and cloned into the
pGEMÒ-T Easy vector (Promega, Madison, Wis). Six to
23 clones per ligation reaction were sequenced with the
vector specific primers T7 and SP6. Sequencing was performed on either an ABI PRISMÒ 377 DNA Sequencer
or an ABI PRISMÒ 3100 Genetic Analyzer, according to
manufacturer’s instructions (Applied Biosystems, Foster
City, Calif).
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).
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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.
Molecular Evolution of Floral Symmetry Genes 593
FIG. 2.—Gene tree representing relationships among CYC-like loci
of the Antirrhineae and close relatives. The maximum-likelihood tree
(2lnL 5 10668.35) is presented; the maximum parsimony tree (length
2,115 steps) differs only in the placement of Chaenorhinum DICH (sister
to Linaria DICH). Tree was rooted with Gcyc1 and Gcyc2 sequences
from Haberlea ferdinandi-coburgii; these taxa have been pruned from the
tree. Branch lengths were optimized with likelihood using the GTR 1
dÿ model of evolution. Numbers at nodes indicate parsimony bootstrap
support greater than 50%, based on 1,000 random addition, heuristic
search replicates.
evolution using the shape parameter that was estimated in
the ML search (a 5 0.857).
Analyses of patterns of molecular evolution were
conducted using ML trees generated under the GTRdÿ 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]).
Likelihood-Based Analysis of Selective Constraint
The ratio of the rate of nonsynonymous versus
synonymous substitution (dN/dS 5 x) is a measure of the
history of selection acting on a gene or gene region. Ratios
significantly less than 1 are suggestive of purifying selection, whereas ratios greater than 1 suggest directional
selection. These ratios were explored in a likelihood
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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 (dÿ) 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 1 dÿ) with one set of rate parameters, was
compared with an alternative hypothesis that allowed
different rate parameters in the GTR 1 dÿ 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 (2d), 2[2lnL11lnL2], is significant as
determined from a v2 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 1 dÿ model of molecular
594 Hileman and Baum
Table 2
Summary of Sequence Data from Taxa Sampled
Taxon
Antirrhinum majus
Asarina procumbens
Chaenorhinum villosum
Cymbalaria muralis
Kickxia spuria
Linaria canadensis
Linaria vulgaris
Lophospermum sp.
Maurandya antirrhiniflora
Misopates orontium
D. purpurea
Putative Loci
(% Divergence Between Locia)
2
2
2
1
1
2
2
2
1
2
3
(23.81)
(18.53)
(26.47)
(27.18)
(28.59)
(16.09)
(14.22)
(13.93–34.34)
Clones Sequenced/Putative Alleles
(% Divergence Between 2 Allelesa)
CYC
11/1
13/1
9/1
3b/1
11/2 (0.62)
5/1
23/2 (0.54)
4/2 (0.53)
9/1
16/1
Dp-CYC1: 16/2 (1.42)
Dp-CYC2: 8/1 Dp-CYC3: 6/1
DICH
10/1
19/1
1
0
0
5/1
7/2 (1.00)
7/1
0
4/1
a
Percent divergence based on uncorrected pairwise distance.
Three cloned PCR inserts were sequenced fully and correspond to CYC. An additional five cloned PCR inserts were identified as CYC but were only partially sequenced.
b
Friday 1995; Huelsenbeck and Rannala 1997). If the
likelihood ratio (2d), 2[2lnL11lnL2], is significant as determined from a v2 test with the appropriate degrees of
freedom (the difference in number of x ratios estimated),
then the parameter rich model was considered to provide
a better explanation of the data.
Results
Sequence Data
Between 1 and 30 of the clones sequenced from each
species correspond to genes in the TCP gene family that
could be aligned to CYC and DICH from A. majus. For
each species and putative locus, the sequence of a single
representative clone was deposited in GenBank and used
for all further analyses (table 1).
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.
Ignoring putative allelic diversity, no more than two
distinct CYC-like gene sequences were isolated from
any one species of Antirrhineae, but there were three distinct sequences in Digitalis. These were interpreted provisionally as loci. The two putative loci from a given
Antirrhineae species had a raw nucleotide divergence of
14.22% to 28.59%, whereas the three putative loci from
Digitalis purpurea differed by 13.93% to 34.34%. Two
putative loci were obtained from Asarina procumbens,
Chaenorhinum villosum, Linaria canadensis, L. vulgaris,
Lophospermum sp., and Misopates orontium, but only
a single CYC-like locus was isolated from Cymbalaria
muralis, Kickxia spuria, and Maurandya antirrhiniflora
(table 2). To increase the possibility of isolating DICH-like
sequences from the latter species, we designed two DICHspecific forward primers and one DICH-specific reverse
primer (fig. 1). In most cases, these primers did not result
in PCR products, and when products were obtained, they
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framework using the codeml program in the PAML
package (Yang 2000).
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 x for all branches in
the phylogeny, whereas models B, C, and D allow for
variation in x among lineages (Yang 1998; Yang and
Nielsen 1998; Bielawski and Yang 2001). Model B
assumes two x 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 x 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
x ratio for each branch of the phylogeny and is, therefore,
useful for distinguishing shifts in x 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 x. 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 x 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
Molecular Evolution of Floral Symmetry Genes 595
were found not to correspond to TCP genes (data not
shown).
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 59 end of the sequence was identical to one
locus (CYC or DICH) from that taxon, and the 39 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
FIG. 3.—One of two maximum parsimony gene trees showing the
placement of Linaria cyc1B, Cymbalaria cyc1B, and Digitalis cyc1A,
cyc1B, cyc2, and cyc4 (indicated with asterisks [Vieira, Vieira, and
Charlesworth 1999]) relative to CYC-like sequences isolated in this
analysis (fig. 2). Tree was rooted with Dp-CYC 1, Dp-CYC 2, and DpCYC 3. Branch lengths were optimized with likelihood using GTR 1
dÿ model of evolution.
The pruned data sets used for studies of molecular
evolution all yielded ML trees that were compatible with
the full data set (fig. 4). However, trees that included only
CYC or DICH genes generally showed higher bootstrap
support reflecting, presumably, the fact that alignmentinduced noise is minimized (fig. 4).
Variable and conserved regions of the TCP gene
family exhibit different levels of sequence divergence
(Cubas et al. 1999) and may be evolving under different
modes of molecular evolution. Likelihood ratio tests
suggest that there are significant differences in the mode
of molecular evolution among the five regions (A, B, C,
TCP-domain, and R-domain [fig. 1]) of the CYC/DICH
genes in Antirrhineae. Allowing the five data partitions to
have different rate parameters in the GTR 1 dÿ model of
molecular evolution significantly improved the likelihood
of the data relative to enforcing a single set of rate
parameters across the entire gene (P ,, 0.01, df 5 204).
Substitution rates across these five regions, as assessed by
average pairwise differences between CYC and DICH
(assuming the GTR 1 dÿ model of molecular evolution),
shows that the variable regions A, B, and C are evolving at
a faster rate (mean pairwise distance [standard deviation] 5
0.696 [0.22], 0.534 [0.18], and 0.455 [0.10] respectively)
than the conserved TCP- and R-domains (0.210 [0.04]
and 0.115 [0.07], respectively).
Different Selection Among Lineages
A likelihood ratio test was used to look for evidence
of variation in the rate of nonsynonymous versus
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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 1 L. vulgaris DICH, whereas in the ML tree
(fig. 2), it was sister to Misopates 1 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 5 0.041, but is not significant using a KishinoHasegawa test (Kishino and Hasegawa 1989) with P 5
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 1 L. vulgaris (CYC and
DICH), Antirrhinum 1 Misopates (CYC and DICH), and
Lophospermum 1 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.
596 Hileman and Baum
synonymous substitution (dN/dS 5 x) 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 x 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 x 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 x
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 x. 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 x that are markedly
less than 1.0.
In the conserved domains, there is no evidence of any
consistent difference in the strength of selection acting on
CYC and DICH. However, for tree 4, which contains only
DICH sequences, the likelihood ratio analysis for the TCPdomain rejects all models relative to model D, which
allows a different value of x for each lineage (table 3).
While specific differences in x between lineages cannot be
evaluated statistically, it is interesting to observe that x is
close to zero on eight of the lineages, nonzero but small on
two lineages (leading to Linaria canadensis and Misopates
orontium), but high on the lineage leading to Antirrhinum
majus (fig. 4D). The observation of a much higher value of
x on the lineage leading to the model species, Antirrhinum
majus, may indicate that the action of selection on the
DICH TCP-domain in A. majus may not be typical of the
entire Antirrhineae clade.
In the more variable regions, A, B, and C, most tests
failed to reject the null hypothesis of a single value of x
(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, x
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 x, for tree 3
(CYC only) is lower than for tree 4 (DICH only).
Discussion
The Timing of Gene Duplication
The optimal phylogeny of CYC-like sequences from
species in the Antirrhineae and selected outgroups (fig. 2)
suggests that there was a single duplication event before
the radiation of the Antirrhineae that gave rise to the CYC
and DICH loci. Although the optimal tree suggests that
this duplication occurred after the divergence of Antirrhineae and Digitalis, this result cannot be considered
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FIG. 4.—Four trees used for molecular evolution analyses. (A) Tree 1, including outgroup (Digitalis) and all ingroups except the CYC-like genes
from species where no DICH-like paralogs were identified. (B) Tree 2, as tree 1, but outgroup pruned. (C) Tree 3, CYC-like sequences only. (D) Tree 4,
DICH-like sequences only (numbers below branches correspond to lineage-specific estimates of x estimated for the TCP-domain). Numbers above
branches (trees 1, 3, and 4) indicate parsimony bootstrap support greater than 50%, based on 1,000 random addition, heuristic search replicates.
Molecular Evolution of Floral Symmetry Genes 597
Table 3
Variation in the Underlying Nonsynonymous/Synonymous Substitution Rate Ratio (x) Among Lineages
Region A
TCP-Domain
Optimal
Model
Estimated
x
Optimal
Model
Tree 1
A
x 5 0.245
A
Tree 2
B
A
Tree 3
Tree 4
A
A
xC
xD
x
x
5
5
5
5
0.189
0.330
0.184
0.245
A
D
Estimated
x
Region B
R-Domain
Region C
Optimal
Model
Estimated
x
Optimal
Model
Estimated
x
Optimal
Model
x 5 0.022
B
xOG 5 0.427
xC1D5 0.202
A
x 5 0.018
C
x 5 0.019
A
x 5 0.211
A
x 5 0.013
B
A
A
x 5 0.192
x 5 0.250
A
A
x 5 0.000
x 5 0.019
A
A
x
x1–8
x9
x10
x11
5
5
5
5
5
0.020
0.0001
0.0252
0.1884
0.6513
Estimated
x
xOG
xC
xD
xC
xD
x
x
5
5
5
5
5
5
5
0.251
0.197
0.405
0.204
0.352
0.189
0.394
definitive. This is because trees in which Digitalis has both
CYC and DICH orthologs could not be rejected statistically using the Kishino-Hasegawa test. Therefore, our data
suggest that gene duplication occurred soon before the
radiation of Antirrhineae but do not entirely rule out
a CYC/DICH gene duplication earlier in the radiation of
Lamiales.
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 CYClike 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 ex-
planation 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
Downloaded from http://mbe.oxfordjournals.org/ by guest on October 1, 2014
NOTE.—For each gene-region/tree combination, the model that is favored by a likelihood ratio test is given with the estimated values of x under that model. Model A
(all trees) assumes equality of x across the tree. Model B (trees 1 and 2 only) allows for a different x for ingroup taxa (Antirrhineae CYC and DICH for tree 1, DICH for
tree 2) relative to outgroups. Model C (tree 1 only) estimates independent x values for the outgroup, the Antirrhineae CYC clade, and the Antirrhineae DICH clade.
Model D (all trees) estimates a unique x for each branch. x indicates a single ratio for all branches. xOG indicates the ratio for branches predating the CYC/DICH
duplication event. xC1D indicates the ratio for all branches (CYC 1 DICH) postdating the duplication event. xC indicates the ratio for CYC lineages postdating the
duplication event. xD indicates the ratio for DICH lineages postdating the duplication event. x1–11 indicates ratios correspond to branches 1 to 11 in figure 4D.
598 Hileman and Baum
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 (x) in both the CYC and DICH clades were
much less than 1.0, indicating that purifying selection has
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FIG. 5.—A scenario for the history of selection acting on the CYC/
DICH genes. The upper panel depicts the relative activity of the CYC and
DICH genes after gene duplication (indicated with an arrow) and their
cumulative activity. The lower panel shows the relative strength of
purifying selection acting on slightly deleterious mutations before gene
duplication and after gene duplication (indicated with an arrow). It is
presumed that selection acts primarily to remove mutations that result in
cumulative gene activity that is less than some minimum value (indicated
by the shaded area in the upper panel). Immediately after gene duplication,
the activity of the two paralogs was individually equal to the ancestral gene
such that the cumulative activity greatly exceeded the minimal level
needed for functionality (upper panel). This resulted in decreased purifying
selection (lower panel) and a gradual loss of excess activity by drift.
Eventually, the cumulative gene activity returned to a level close to the
threshold for functionality (upper panel). During the period of diminished
purifying selection it is hypothesized that the DICH locus experience more
deleterious mutations. As a result, after purifying selection stabilized,
DICH activity was lower than CYC (upper panel) and DICH was subject to
lower levels of purifying selection than CYC (lower panel).
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 geneproduct ‘‘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 x 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 x are estimated for DICH as well as CYC clades,
and because Antirrhinum dich mutants do not have wildtype 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
Molecular Evolution of Floral Symmetry Genes 599
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.
Acknowledgments
The authors would like to thank Sarah Mathews,
Justin Blumenstiel, Richard Ree, Charles Bell, Deborah
Charlesworth, Elena Kramer (and members of the Kramer
lab), and two anonymous reviewers for useful discussions
about data analysis and helpful comments on early
versions of this manuscript. This research was funded by
an NSF grant (DEB-9972647) to L.H. and D.B., and a
student research grant to L.H. from the Department of
Organismic and Evolutionary Biology, Harvard University.
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