Download The evolutionary history of Antirrhinum suggests that ancestral

The Plant Journal (2011) 66, 1032–1043
doi: 10.1111/j.1365-313X.2011.04563.x
The evolutionary history of Antirrhinum suggests that
ancestral phenotype combinations survived repeated
hybridizations
Yvette Wilson and Andrew Hudson*,†
Institute of Molecular Plant Sciences, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JH, UK
Received 29 November 2010; revised 25 February 2011; accepted 1 March 2011; published online 6 April 2011.
*
For correspondence (fax +44 131 650 5392; e-mail [email protected]).
†
Present address: Division of Plant Science, University of Dundee at SCRI, Invergowrie, Dundee, DD2 5DA, UK.
SUMMARY
The model species Antirrhinum majus (the garden snapdragon) has over 20 close wild relatives that are
morphologically diverse and adapted to different Mediterranean environments. Hybrids between Antirrhinum
species have been used successfully to identify genes underlying their phenotypic differences, and to infer
how selection acts on them. To better understand the genetic basis for this diversity, we have examined the
evolutionary relationships between Antirrhinum species and how these relate to geography and patterns of
phenotypic variation in the genus as a whole. Large population samples and both plastid and multilocus
nuclear genotypes resolved the relationships between many species and provided some support for the
traditional taxonomic division of the genus into morphological subsections. Morphometric analysis of plants
grown in controlled conditions supported the phenotypic distinction of the two largest subsections, and the
involvement of multiple underlying genes. Incongruence between nuclear and plastid genotypes further
suggested that several species have arisen after hybridization between subsections, and that some species
continue to hybridize. However, all potential hybrids appear to have retained a phenotype similar to one of
their ancestors, suggesting that ancestral combinations of characters are maintained by selection at many
different loci.
Keywords: Antirrhinum, snapdragon, phylogeny, morphological evolution.
INTRODUCTION
Much of our understanding of the genes involved in morphological evolution and speciation has come from taxa that
are sufficiently different to be regarded as separate species,
but that retain the ability to form fertile hybrids. Both natural
and artificial hybrids have been used to detect loci underlying differences between the parental species. In some cases,
the genes and mutations have themselves been identified.
This has been particularly successful when the research
infrastructure developed in a closely related model species
is available, for example in Drosophila (McGregor et al.,
2007; Jeong et al., 2008).
The garden snapdragon, Antirrhinum majus (Plantaginaceae) has been used as a model to study inheritance, and the
genetic control of development and flower colour (SchwarzSommer et al., 2003). Its close relatives are native to the
western Mediterranean region, mostly the Iberian peninsular, and comprise a monophyletic group – the traditional
1032
genus Antirrhinum – in which between 17 and 27 distinct
species and subspecies have been recognized in different
taxonomic accounts (Rothmaler, 1956; Webb, 1971; Sutton,
1988). Although morphologically diverse and adapted to
different, often extreme, environments, all Antirrhinum
species can form fertile hybrids with each other and with
A. majus when artificially cross-pollinated. Such hybrids
have identified genes underlying differences in morphology
and flower colour between their parents (Hackbarth et al.,
1942; Langlade et al., 2005; Schwinn et al., 2006; Feng et al.,
2009). Natural Antirrhinum hybrids have also identified
genes involved in flower colour variation, and have suggested how selection acts on them (Whibley et al., 2006).
The genus Antirrhinum can therefore provide a model for
understanding the genetic basis for patterns of phenotypic
diversity and adaptation around the species level, which
may be typical of many recently evolved Mediterranean
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
Constrained evolution in the genus Antirrhinum 1033
taxa (Thompson, 2005). However, it has been difficult to
relate the genetic differences between pairs of parental
species to variation in the genus as a whole because the
relationships between Antirrhinum species have not been
resolved. For instance, it is currently not possible to infer the
ancestral state of a character and whether similar phenotypes might have evolved multiple times within the genus.
One obstacle to resolving evolutionary relationships
within Antirrhinum is reflected in the unclear taxonomy of
its species, many of which have not been reported to have
a unique, fixed character (a synapomorphy; Webb, 1971).
Therefore, species might not represent discrete genetic
entities because they have been delimited artificially.
Nevertheless, support for the genetic distinction of some
recognized species has been provided by allozymes and
DNA sequence variation (Mateu-Andres and SegarraMoragues, 2000, 2003; Jimenez et al., 2005a; Mateu-Andres
and de Paco, 2005). Relationships above the species level
are also unclear. The genus has been divided into three
morphological subsections – Antirrhinum, Streptosepalum
and Kickxiella – but no subsection has been defined by a
synapomorphy, and all have been suggested to overlap in
phenotype (Rothmaler, 1956; Webb, 1971). The subsections
also correlate with ecology: most members of subsection
Kickxiella are small prostrate alpines or xerophytes that
grow on rock faces, whereas subsections Antirrhinum and
Streptosepalum comprise larger, more upright plants that
are able to grow in competition (Figure 1). This raises the
possibility that each subsection represents an ecotype, and
that its species have evolved similar characters independently as adaptations to a particular environment, rather
than sharing characters through common descent.
Attempts to resolve a species phylogeny for Antirrhinum
from DNA sequences have been unsuccessful. Relatively
little sequence variation has been found in the genus,
consistent with its recent origin, and the variation is not
distributed consistently between taxa, so that different
genes support different relationships between species
(Jimenez et al., 2005b; Vargas et al., 2009). Sparse sampling
of the taxa used for DNA sequence analysis might also have
contributed to a lack of phylogenetic resolution, given that
young species are likely to share many unfixed alleles that
might not be represented in small taxon samples.
Here, we examine evolutionary relationships within the
genus Antirrhinum by comparing populations sampled
from across the geographic range of each species. Plastid
and multilocus nuclear genotypes resolve the relationships
between many species, and further suggest that the traditional morphological subsections largely correspond to
separate evolutionary lineages. They also suggest that
hybridization has occurred repeatedly between the two
major ancestral lineages where they overlap in range.
Morphometric analysis of plants grown in common garden
conditions supported the phenotypic distinction of the two
Figure 1. The three morphological subsections of Antirrhinum.
A representative of each subsection is shown in situ and in cultivation.
Subsection Antirrhinum is represented by A. pseudomajus, subsection
Streptosepalum is represented by A. braun-blanquetii and subsection Kickxiella is represented by A. pulverulentum. Scale bars: 150 mm for cultivated
plants, and the ruler shown with plants in the field is 35 mm wide.
major subsections, and the involvement of many underlying
genes. However, all putative hybrid species appeared to
resemble one of their parents, suggesting that ancestral
suites of phenotypes have survived hybridization as a result
of selection at multiple loci.
RESULTS
Antirrhinum taxonomy reflects discontinuous phenotypic
variation
Antirrhinum populations were sampled from across the
geographic range of each recognized species and subspecies, so that the depth of sampling in each taxon broadly
corresponded to its abundance (Figure 2; Tables 1 and S1).
The only recognized species that remained un-sampled was
Antirrhinum martenii, which could not be found at its original collection sites in the Moroccan Rif. For convenience,
we treated all taxa at the rank of species, including those that
are often regarded as subspecies of A. majus.
To assess phenotypic variation within Antirrhinum we
grew plants from a representative subset of 98 populations
together in a glasshouse, and recorded phenotypes for an
average of 5.8 plants from each population (Table 2). We
chose phenotypes that differed between populations, without regard to their differences between species or subsections, to avoid any bias towards characters that might
automatically support traditional taxonomic divisions. Characters were then selected for further analysis on two genetic
criteria. The first attempted to reduce the use of characters
that were strongly influenced by environment. It assumed
that members of the same species are genetically most
similar to each other. Therefore, a comparison of the
variation within species and between species gives an
estimate of the extent to which a phenotype is genetically
determined (approximating its broad-sense heritability).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
1034 Yvette Wilson and Andrew Hudson
Figure 2. The distribution of recognized Antirrhinum species.
Species ranges were estimated from collection sites of this and previous studies. The range of A. tortuosum extends further eastwards than is shown here. The
names of members of subsection Antirrhinum are shown in red, members of Kickxiella are shown in blue and members of Streptosepalum are shown in orange.
Each species range is coloured to represent its geographic location, so that species with similar locations are shown in colours with similar hues. For A. tortuosum
and A. linkianum, subpopulations that occur in distinct geographic regions are shown with different colours.
Phenotypes for which more than 35% of the total variance
occurred within species (stomatal indices in leaves, stems or
petals, and leaf epidermal cell size) were excluded from
further analysis.
The second criterion for screening phenotypes attempted
to minimize repeated sampling of the same genetic differences. Differences in leaf and flower size and shape (allometry), for example, which have been used as taxonomic
characters in Antirrhinum, do not vary independently of
each other because they are affected by the same genes: i.e.
the characters are developmentally constrained (Langlade
et al., 2005; Feng et al., 2009). Instead of treating them
separately, we therefore quantified variation in the allometries of leaves and flowers together using a computer shape
model of flattened node-4 leaves, flattened dorsal petals
and side views of intact flowers, in which other aspects of
variation are apparent (e.g. the angles at which petal lobes
are presented). The shape model captures co-variation in
the positions of points placed around the organ outlines as
orthogonal principal components (PCs; Figure 3). The first
four PCs accounted for 93% of the variance within the genus,
allowing the leaf and flower allometry of each plant to be
described accurately with these four PC values. The types of
variation captured by the PCs are shown in Figure 3(b). PC1
describes mainly size variation, showing that organ size is
the major difference between plants, although this is correlated with other aspects of shape variation. For instance, an
increase along PC1, which involves an increase in leaf and
flower size, is accompanied by a narrower leaf shape and
more reflexed dorsal petals. PC2 and PC3 capture other
aspects of shape and size variation in both leaves and
flowers, whereas PC4 mainly describes the extent to which
flowers vary independently of leaves (Figure 3b). Because
the PCs are uncorrelated to each other, each was treated as a
separate phenotypic character.
We also avoided using other phenotypes that high
correlations suggested might be developmentally constrained (e.g. the lengths of the style and stamens), and
chose flower colour phenotypes to represent the effects of
genes known to be involved in interspecies differences
(Schwinn et al., 2006; Martin et al., 1987; see Experimental
procedures for details).
For the 22 remaining characters, average values were
calculated for each of the 98 populations, and the range of
the mean values were adjusted to a common linear scale of
0–22. These rescaled values were used in de-trended correspondence analysis (DCA) to position each population in
phenotypic space, defined by the two major axes of
co-variation in the data set (Parnell and Waldren, 1996).
A notable feature of the distribution of populations in
phenotypic space was that members of subsection Kickxiella
formed a cluster distinct from subsections Antirrhinum and
Streptosepalum, with Kickxiella populations mainly sharing
higher values along DCA axis 1 (Figure 4a). Kickxiella
therefore appears to be phenotypically distinct from
the other two subsections. The only exception involved
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
Constrained evolution in the genus Antirrhinum 1035
Table 1 Antirrhinum species names and abbreviations
Species
Subsection Antirrhinum
A. australe Rothm.
A. barrelieri Boreau
A. boissieri Rothm.
A. cirrhigerum Filcahob
A. graniticum Rothm.
A. grosii Font Quer
A. latifolium Miller
A. linkianum Boiss. & Reuterb
A. litigiosum Paub
A. majus L.b
A. pseudomajus
Fernández-Casasb
A. siculum Millar
A. striatum Fernández-Casasb
A. tortuosum Vent.b
Subsection Kickxiella
A. charidemi Lange
A. hispanicum Chav.
A. hispanicum
(Moroccan accessions)
A. lopesianum Rothm.
A. microphyllum Rothm.
A. molle Lange
A. mollisimum Rothm.
A. pertegasii Rothm.
A. pulverulentum Lazaro
A. rupestre Rothm.
A. sempervirens Lapeyr.
A. subbaeticum Guemes
A. valentinum Font Quer
Subsection Streptosepalum
A. braun-blanquetii Rothm.
A. meonanthum
Hoffmans. & Link
Abbreviation
Numbers
used in
morphmetricsa
au
ba
bo
ci
gr
go
la
li
lt
ma
ps
8/48
3/30
1/12
4/33
7/33
si
st
to
7/16
6/17
9/99
ch
hi
Mh
1/4
3/16
3/16
lo
mi
mo
ms
pe
pu
ru
se
su
va
1/3
1/3
6/47
3/30
1/6
2/24
5/20
2/11
1/3
1/3
bb
me
1/9
1/6
5/10
6/25
7/29
3/15
a
The number of populations of each species used in morphometric
analysis is shown, followed by the total number of individuals
sampled.
b
These taxa have also been considered subspecies of A. majus, e.g.
A. majus ssp. striatum or A. majus ssp. majus.
populations from Morocco that had previously been
included in Antirrhinum hispanicum (subsection Kickxiella;
Rothmaler, 1956). These mapped in the same phenotypic
space as subsections Antirrhinum and Streptosepalum,
whereas Spanish populations of A. hispanicum, from which
the species was originally described, fell within the Kickxiella cluster. Because Moroccan populations could not be
classified as A. hispanicum on grounds of morphology,
they were subsequently treated as a separate taxon within
subsection Antirrhinum.
Although DCA supported the phenotypic distinctiveness
of Kickxiella, subsections Streptosepalum and Antirrhinum
were not separated from each other. At the species level,
populations of the same species tended to cluster together,
although many overlapped. The overlap between species
in a space involving 22 phenotypes does not rule out the
possibility that individual species might be defined by
fewer phenotypes or by characters that were not considered here.
The DCA allows co-variation between characters to be
represented in the same phenotype space as the populations
(Figure 4b), illustrating their effects on the separation of
populations. The spread of characters along axis 1 suggested that the phenotypic distinctiveness of subsection
Kickxiella, which has higher axis-1 values, does not depend
solely on one type of character, for example size or flower
colour. This was further supported by the finding that
subsection Kickxiella differed significantly from the other
two subsections for all phenotypes, except PC2, PC4 and Ve
(P £ 0.05 in Student’s t-tests). Therefore, subsection Kickxiella can be defined by a combination of phenotypes that are
likely to reflect variation in a number of different genes.
In contrast, all subsections overlapped along DCA axis 2,
which also reflects variation in a number of different
characters, although members of the same species tended
to cluster with each other along this axis.
Plastid haplotypes support distinct evolutionary lineages
One explanation for the partial overlap of Antirrhinum species in phenotypic space is that the genus behaves as a
single interbreeding population, in which the genetic
differences between individuals reflect their geographic
separation (isolation by distance). In this case, the similar
phenotypes of Kickxiella species should either reflect their
closer genetic relatedness and geographic proximity to each
other, or may be independent of both relatedness and
geography if similar phenotypes have evolved independently. We took this scenario of a single population as the
null hypothesis against which to test alternatives, including
the possibility that members of subsection Kickxiella share
similar phenotypes because they descend from a separate
evolutionary lineage.
We first examined the relatedness of individuals from
their plastid haplotypes. Because previous studies had
detected relatively little sequence variation in Antirrhinum
plastids (Jimenez et al., 2005b), we compared 19 non-coding
loci across 10 Antirrhinum species, and identified the three
most variable (trnD-trnT, trn-S-trnR and trnS-trnFM; Shaw
et al., 2005, 2007). Their sequences were then obtained from
90 populations that represented the geographic range of
each species, and were found to contain 54 polymorphisms
arranged into 34 different haplotypes. The relationships
between haplotypes were inferred by parsimony. To allow
for the resolution of a purely branching haplotype network
(Figure 5), 12 single-nucleotide polymorphisms that were
present in rare haplotypes and not fixed in any species were
assumed to be homoplasious to mutations supporting
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
1036 Yvette Wilson and Andrew Hudson
Table 2 Phenotypes used in de-trended correspondence analysis (DCA)
Character
H2a
Description
Scaleb
PC1
PC2
PC3
PC4
Anth
Branch
CarpL
El
FlNode
Ht
InfNode
IntHairs
LfHair
PeCell
PedL
StemW
StRatio
Sulf
Ve
YelFace
YelHair
YelTube
81
83
75
73
91
67
88
73
67
73
84
88
90
65
75
60
73
87
68
78
75
68
First PC from the allometry model of leaves and flowers
Second PC from the allometry model of leaves and flowers
Third PC from the allometry model of leaves and flowers
Fourth PC from the allometry model of leaves and flowers
Intensity of red anthocyanin pigmentation in the corolla
Ratio of the length of the longest axillary branch to Ht (below)
Carpel length (from ovary base to stigma tip)
Presence of anthocyanin outside the corolla face and dorsal petal sinus
Number of the node at which flowers were first produced
Length of the stem from cotyledons to first flower
Maximum number of nodes bearing open flowers at any one time
Density of stem trichomes between nodes 1 and 2
Density of adaxial epidermal trichomes in node-4 leaves
Density of adaxial epidermal cells in dorsal petals
Pedicel length
Diameter of the stem midway between nodes 1 and 2
Ratio of ventral to dorsal stamen lengths
Presence of widespread yellow aurone pigment in the corolla
Presence of darker anthocyanin pigmentation over corolla veins
Extent of yellow aurone pigmentation in the corolla face
Extent of yellow trichomes within the ventral corolla tube
Extent of yellow pigmentation within the ventral corolla tube
C
C
C
C
D, 5
C
C
D, 2
D, 29
C
D
C
C
C
C
C
C
D, 2
D, 2
D, 4
D, 5
D, 4
a
Percentage of the total variance in each character that is contributed by differences between species, which approximates to the heritability (H 2) of
the character in the broad-sense.
b
Characters were either measured on a continuous scale (C) or scored in discrete categories (D), in which case the number of character states is
given (e.g. D, 2 refers to a binary character). Units are not given for continuous characters because they were subject to linear rescaling, which
made them dimensionless.
deeper branches. Sequences from Misopates and Chaenorrhinum, each of which has been suggested to be sister
to Antirrhinum (Ghebrehiwet et al., 2000; Oyama and
Baum, 2004; Vargas et al., 2004), both rooted the Antirrhinum
haplotype network in the same position.
The network contained four main clades (Figure 5).
Clade I was restricted to Antirrhinum siculum. Clade II
occurred mainly in subsections Antirrhinum and Streptosepalum, and Clades III and IV occurred mainly in
subsection Kickxiella. This distribution of haplotypes
cannot be explained solely by geographic separation,
and therefore is inconsistent with the null hypothesis that
the genus behaves as a single, unstructured population.
For instance, members of subsection Antirrhinum carrying
a clade-II haplotype occur throughout the geographic
range of the genus, and overlap with Kickxiella species
carrying clade-IV haplotypes. A more plausible explanation is that the distribution of plastid haplotypes reflects
the evolutionary relationships between species: i.e. cladeIII and -IV haplotypes were present in the Kickxiella
ancestor; clade-II haplotypes were present in the separate
lineage leading to subsections Antirrhinum and Streptosepalum; and clade-I haplotypes were present in the
lineage represented by A. siculum only. However, several
taxa have haplotypes that are incongruent with their
phenotypes, in one of two respects. First, A. latifolium
(subsection Antirrhinum) has only clade-IV Kickxiella
haplotypes, and A. sempervirens, A. lopesianum,
A. pertegasii and A. molle in subsection Kickxiella appear
fixed for clade-II haplotypes usually found in subsections
Antirrhinum and Streptosepalum. Secondly, A. tortuosum,
A. barrelieri and A. australe from subsection Antirrhinum,
and A. pulverulentum from subsection Kickxiella, carry
both clade-II and clade-IV haplotypes.
The depth of taxon sampling was increased by genotyping another 273 individuals from an additional 156 populations (Table S1). Clade-IV haplotypes were identified by the
presence of an MseI site in trnD–trnT, and clade-II haplotypes were identified by a TfiI site in trnS–trnR. Individuals
lacking both these sites were assumed to carry clade-III
haplotypes, unless they were accessions of A. siculum
(clade I). The additional samples confirmed the distribution
of haplotypes identified by DNA sequencing, and also
revealed that populations of A. litigiosum (subsection
Antirrhinum) from the Valencia region carried clade-IV
(Kickxiella) haplotypes, whereas those from central Spain
carried clade-II haplotypes only.
One explanation for the cases of incongruence between
plastid haplotype and morphology is that ancestral polymorphisms have persisted in the lineages, leading to
different subsections (lineage sorting). However, this cannot easily account for the presence of highly diverged
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
Constrained evolution in the genus Antirrhinum 1037
(a)
(b)
Figure 3. Models describing organ shape and size variation in the genus
Antirrhinum.
(a) Points were placed around node-4 leaves, flattened dorsal petals and
images of intact corollas. Green points were placed manually and red points
were spaced automatically between them. Variation in the positions of all the
points within the genus were described by principal components (PCs). The
effects of variation of 2SDs in the first four PCs are shown relative to
the mean organ outlines in black. Var shows the percentage of the variation in
the genus that is captured by each PC.
haplotypes within the same species. For example, the
clade-II and clade-IV haplotypes found in A. tortuosum
(subsection Antirrhinum) differ by up to 21 mutations, and
those in Spanish A. hispanicum (subsection Kickxiella) differ
by up to 17 mutations, although only 24 mutations distinguish the most dissimilar haplotypes within the genus as
a whole (Figure 5). Such cases of incongruence are more
consistent with transfer of haplotypes by hybridization
between lineages. Further evidence for hybridization is
provided by the location of populations with incongruent
haplotypes. They occur where Kickxiella species overlap
with members of the other subsections: for example, with
subsection Antirrhinum in the Sierra Nevada of south-east
Spain (Figure 6). In contrast, haplotypes are congruent with
morphology where subsections grow in isolation (e.g.
subsection Antirrhinum in Morocco, western Portugal and
northern Spain, and Kickxiella in central Spain). The only
notable exception is that of A. latifolium, which carries a
clade-IV haplotype, is restricted to the Alps and does not
currently co-occur with a Kickxiella species, although this
does not rule out the possibility of hybridization with a
previously sympatric Kickxiella.
Nuclear genotypes support species delimitations and
evolutionary lineages
Because maternally inherited plastid genomes provide only
limited information about the relationships between individuals, particularly for hybrids, we also examined variation
in nuclear genes. Previous studies had found relatively
little nuclear sequence variation and inconsistent distribution between Antirrhinum species (Gübitz et al., 2003;
Vargas et al., 2009). We therefore sampled multiple nuclear
loci as amplified fragment length polymorphisms (AFLPs)
from the set of 293 accessions that had been used for plastid
haplotype analysis. Removal of potentially homoplasious
markers left 381 informative AFLPs.
The AFLPs were used to infer the population structure
within Antirrhinum using a Bayesian model, STRUCTURE,
which assigns individuals to a specified number of populations by minimizing genetic disequilibria within each population (Falush et al., 2003). This method is not dependent
on prior taxonomic assumptions, and is able to represent
hybrids as genetic admixtures of other populations.
When STRUCTURE was used to assign each accession to
one of two populations (K = 2 in Figure 7), one population
comprised subsection Streptosepalum and most of subsection Kickxiella, and the second consisted of subsection
Antirrhinum and the Kickxiella species from south-east Spain
(A. hispanicum, A. mollissimum, A. rupestre and A. charidemi). This division therefore supported the genetic distinction of Streptosepalum and northern Kickxiella species from
subsection Antirrhinum and southern Kickxiella. A. latifolium and A. molle, which showed incongruence of plastid
haplotypes and morphology consistent with hybrid origins,
were suggested to have admixed nuclear genomes, as was
A. siculum.
A maximum of 14 different populations could be identified by STRUCTURE (Figures 7 and S3). Only two of these –
A. graniticum and A. latifolium – consisted almost entirely of
unique haplotypes, suggesting a high level of coalescence
within these species. The two Streptosepalums remained
within the same population as A. grosii and A. lopesianum
from subsection Kickxiella. The central Spanish Kickxiella
species, which are all local endemics and do not overlap
in range, were assigned to another population, which
we subsequently refer to as core Kickxiella. Many of the
remaining species fell into geographic groups with similar
genetic compositions. In south-east Spain, for example,
A. mollissimum was designated an admixture of genomes
from neighbouring A. charidemi and A. rupestre, and the
A. rupestre genome was also found admixed in A. hispanicum and A. barrelieri nearby.
Cultivars of A. majus were assigned to the same population as A. pseudomajus and A. striatum from around the
eastern Pyrenees, supporting the domestication of A. majus
from this population.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
1038 Yvette Wilson and Andrew Hudson
Figure 4. Distribution of Antirrhinum species in phenotype space.
The two major axes of co-variation in 22 different phenotypic characters were identified by de-trended correspondence analysis. The mean values for 98 populations
were plotted in the space define by these axes (a).Members of subsection Antirrhinum are shown as triangles, members of subsection Streptosepalum are shown as
squares and members of subsection Kickxiella are shown as circles. Populations are coloured as in Figure 2, to reflect their geographic origins. The label mh denotes
Moroccan accessions previously assigned to A. hispanicum. The contribution of each character to the phenotypic space is shown in (b).
Characters and their abbreviations are detailed in Table 2, and accession numbers for populations are presented in Figure S2.
Figure 5. Relationships between plastid haplotypes.
Sampled haplotypes are shown in boxes containing the species names and accession numbers of the plants carrying them.
The most parsimonious relationships between haplotypes are shown by lines connecting boxes with cross lines that represent the number of mutations by which
haplotypes differ. Diagonal cross lines show mutations that were assumed to be homoplasious with earlier mutations. Branches in which mutations introduced an
MseI or TfiI restriction site are also shown. The tree is rooted with sequences from Misopates orontium, which differs from the in-group by 19 mutations.
The ability to progressively subdivide the genus into
populations with boundaries that corresponded to classical
species delimitations provided support for traditional tax-
onomy, and suggested that AFLP genotypes might resolve
relationships between species, even though extensive
admixture was inferred for all but A. graniticum and
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
Constrained evolution in the genus Antirrhinum 1039
Figure 6. Geographic distribution of plastid haplotypes.
Members of subsection Antirrhinum are shown as circles, members of subsection Kickxiella are shown as triangles and members of subsection Streptosepalum are
shown as squares. Filled shapes represent haplotypes that are congruent with morphology; unfilled shapes show incongruent haplotypes.
A. latifolium. Therefore, genetic distances were calculated
between all pairwise combinations of individuals, and were
then used in neighbour-joining analysis with Misopates
orontium as the out-group. When all individuals were
included in the analysis, members of the same species
usually clustered with each other, as they did in the STRUCTURE analysis, but the relationships between species were
poorly resolved (Figure S4). One explanation for poor resolution is that some of the taxa have hybrid origins, and so
carry alleles from multiple lineages. These shared alleles
could reduce resolution both within and between the
parental lineages. We therefore excluded potential hybrids
– i.e. plants with incongruent nuclear and plastid genotypes,
or apparently admixed nuclear genomes – if they reduced
the overall support. The remaining accessions were
resolved into three major clades (Figure 8). One comprised
the core Kickxiella, within which A. pulverulentum formed
a supported group with its central Spanish neighbour
A. microphyllum, and A. pertegasii grouped with the geographically distant A. subbaeticum. The second major clade
consisted of species from subsection Antirrhinum, with
A. siculum at its base and A. graniticum sister to the
remaining taxa, which included two geographic groupings:
A. pseudomajus, A. striatum and A. litigiosum from around
the eastern Pyrenees, and A. linkianum and A. cirrhigerum
from the west coast of Iberia. All accessions of Moroccan
‘A. hispanicum’ clustered together within subsection
Antirrhinum, supporting the phenotypic evidence for their
membership of this subsection. The third major clade
consisted of A. meonanthum and A. braun-blanquetii
(subsection Streptosepalum) from north-west Iberia,
together with two of their neighbouring Kickxiella species,
A. grosii and A. lopesianum.
DISCUSSION
By comparing the genotypes of Antirrhinum populations at
multiple nuclear loci we have identified three well-supported
clades of species. The two largest clades do not correspond
to individuals from the same geographic locations, although
they contain geographic subgroups within them, and so
cannot be explained by a genetic structure that relates solely
to geographic isolation. The clades are therefore likely to
represent different evolutionary lineages.
One clade consists of species from subsection Kickxiella,
which was originally defined by the shared phenotypes of
its members. Morphometric analysis under common garden
conditions confirmed the phenotypic distinctiveness of
this subsection, with common characters that include small
stature, small, ovate and hairy leaves, and small pale flowers
with reflexed petals. The ancestral Kickxiella is therefore
likely to have shared these characters, and, like its descendants, to have lived on rock faces. The second major clade
comprised mainly members of subsection Antirrhinum.
These are dark-pink- or yellow-flowered species that form
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
1040 Yvette Wilson and Andrew Hudson
Figure 7. Assignment of individuals to populations.
Plants were assigned to different ancestral populations, each shown in a different colour, by their nuclear genotypes. Representative assignments are shown for
models in which the maximum number of populations (K) was set to either two or 17. Black bars denote members of subsection Antirrhinum, dark-grey bars denote
members of subsection Streptosepalum and light-grey bars denote members of subsection Kickxiella.
Figure 8. A neighbour-joining tree of Antirrhinum species.
The tree was made from pairwise nuclear
genetic distances between inferred non-hybrid
accessions. Support values are shown for nodes
that were recovered in more than 50% of 1000
bootstrap replicates.
large, upright plants, with larger, more elongated leaves,
and are able to grow in competition. The third major clade
contains both of the species from subsection Streptosepalum and two species with Kickxiella phenotypes. The
classical division of Antirrhinum into three morphological
subsections therefore appears partly natural.
The overlap of the three subsections along DCA axis 2
implies that all subsections vary to a similar extent for the
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
Constrained evolution in the genus Antirrhinum 1041
different morphological and flower-colour characters that
contribute to this axis. This is consistent with ancestral
variation that has not been fixed during divergence of
subsections, although fixation may have occurred during
speciation because members of the same species tend to
cluster along axis 2.
Distinct plastid lineages were also identified, and each
was found predominantly in subsections Antirrhinum and
Streptosepalum, or in Kickxiella. Many species carry congruent nuclear and plastid genomes, for instance A. microphyllum, A. subbaeticum and A. valentinum have genotypes
expected of direct descendants of the core Kickxiella lineage, whereas A. siculum, A. graniticum, A. pseudomajus,
A. striatum, A. cirrhigerum and A. linkianum have both
plastid and nuclear genomes representative of subsection
Antirrhinum.
However, several species appear to have nuclear and
plastid genomes from different lineages. These include
A. sempervirens, A. pertegasii and A. pulverulentum, which
have subsection Antirrhinum plastids but core Kickxiella
nuclei. Such cytonuclear incongruence can be explained
if hybridization between subsections was followed by introgression involving Kickxiella, leading to the capture of an
Antirrhinum plastid by a pre-dominantly Kickxiella nuclear
genome. Similarly, A. tortuosum could also have captured
its diverse incongruent plastids from multiple Kickxiella
donors.
The remaining species share their nuclear genotypes with
both Antirrhinum and Kickxiella lineages, and consequently
cannot be assigned to one of the three major clades.
However, they share plastids and nuclear genes with their
geographic neighbours, suggesting that they are the result
of hybridizations between subsections Antirrhinum and
Kickxiella without significant introgression. This is particularly apparent in the species from south-east Spain, which
have similar nuclear genotypes and carry both Kickxiella
and Antirrhinum plastids. Hybridization could also have
given rise to the nuclear clade consisting of the two
Streptosepalum species together with A. grosii and
A. lopesianum from subsection Kickxiella, because these
species are found together in north-west Iberia and carry
diverged plastids. Alternatively, the clade might represent a
third ancestral lineage in which some members have
captured incongruent plastids from subsection Kickxiella.
It is not possible to resolve the order in which the three
major lineages diverged from each other. However, the
distribution of the core Kickxiella species between the
mountains of central Spain suggests that this lineage was
once more widespread, and became fragmented on contraction to its current mountain refugia. This fragmentation
could have contributed to the formation of distinct species.
A similar range contraction has been proposed to account
for the genetic relationships in several alpine species that
are currently restricted to mountains around the western
Mediterranean (Kropf et al., 2003; Dixon et al., 2007).
Contraction to alpine refugia can also explain why all high
mountain ranges in Iberia and southern France either have
their own endemic core Kickxiella species or, in the case
of the Alps (A. latifolium), the Pyrenees (A. molle and
A. sempervirens) and the Baetic Cordillera (the south-east
Spanish species), retain vestiges of the Kickxiella lineage in
their plastid and nuclear genotypes. Populations from the Rif
Mountains of Morocco have been recognized as a species
within subsection Kickxiella (A. martenii; Rothmaler, 1956),
suggesting that Kickxiella might once have extended into
North Africa. As Kickxiella species are found on dry rock
faces, mostly at higher elevations, contraction in their
ranges could have occurred during periods of increasing
rainfall and warming. Several major events of this kind have
occurred in the Mediterranean region since the end of the
last ice age (Fletcher et al., 2010). The same environmental
changes that reduced the range of Kickxiella could have
allowed subsection Antirrhinum to spread through lowland
regions, perhaps from coastal refugia, and therefore to
hybridize with Kickxiella in regions of contact.
Character evolution
The genetic relationships between Antirrhinum species
suggest that several hybridization events occurred between
subsections Antirrhinum and Kickxiella, yet the phenotypes
of the two subsections remain distinct from each other. This
is most apparent in two species that are sympatric in the
Sierra Nevada of south-east Spain: A. rupestre (Kickxiella
phenotype) and A. barrelieri (Antirrhinum phenotype),
which have indistinguishable AFLP and plastid genotypes,
consistent with their continuing hybridization. Therefore,
although hybridization has the potential to create new
adaptive combinations of phenotypes (e.g., Rieseberg et al.,
2003), evolution in Antirrhinum appears to be constrained.
Two factors suggest that the evolutionary constraints
within Antirrhinum are not developmental. Firstly, the
phenotypic characters that distinguish subsections Kickxiella and Antirrhinum were chosen to represent variation in
multiple genes with independent effects. Secondly, hybrids
between A. majus and A. charidemi have shown that multiple genes underlie the differences between their Antirrhinum and Kickxiella phenotypes (Langlade et al., 2005; Feng
et al., 2009). For instance, 10 genes with additive effects
were found to affect leaf size, so that F1 hybrids and almost
all F2 progeny had leaf phenotypes that were intermediate
between the two parents (i.e. they occupied the gap in
phenotypic space between subsections Antirrhinum and
Kickxiella).
A more likely explanation is therefore that Antirrhinum
phenotypes are constrained by selection. For some characters this might reflect contrasting adaptations to life on bare
rock faces or in competition: for instance, small organs and
dense hairs might be advantageous in limiting water loss on
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
1042 Yvette Wilson and Andrew Hudson
dry rock faces, but a disadvantage in competing with other
species (Parkhurst and Loucks, 1972; Ehleringer et al., 1976).
Hybrids with intermediate phenotypes could therefore be
maladapted in both parental habitats. Because the differences between Antirrhinum and Kickxiella phenotypes
appear to involve multiple unlinked genes, selection would
have to act co-ordinately on many loci.
The proposed evolutionary history of Antirrhinum, which
involves hybridization between emerging lineages, influenced by environmentally induced range changes, might be
typical of the early stages of species formation in many taxa.
It suggests that adaptation can exert a major constraint on
diversity, even when environments change rapidly and
novel genotypes are produced frequently in hybrids.
One testable prediction of our hypothesis that ancestral
combinations of genes are reselected following hybridization in Antirrhinum is that similar phenotypes will have a
similar genetic basis, even in species that share little of their
nuclear genomes. The genus Antirrhinum, and particularly
its recent hybrid species, might therefore allow adaptive
genes to be identified through genotype–phenotype association.
EXPERIMENTAL PROCEDURES
Taxon sampling
The populations used in this study are detailed in Table S1. Populations of most species were sampled in 2006–2007. Sampling sites
included locations used in previous taxonomic accounts to aid
identification. Seeds of these accessions are available on request.
Other accessions were kindly provided by Isabel Mateu-Andrés,
Christophe Thébaud, Thomas Gübitz, Enrico Coen and their
colleagues.
Phenotype analysis
Plants for phenotype analysis were grown from field-collected
seeds or from seeds produced by intercrossing members of the
same population. Germination was synchronized in March 2007 by
imbibing seeds in 10 lM gibberellin (GA3), and plants were grown in
natural light in a glasshouse. Phenotypes were recorded as the fifth
flower opened. Phenotypes and their abbreviations are listed in
Table 2. Cell and hair densities were measured from impressions
made in cyanoacrylate glue on microscope slides by counting the
number of cells or hairs within a 2 mm2 area. Other phenotypes
were measured directly from plants or from digital images. Flower
images were used to score the presence of yellow aurone pigments
outside the corolla face, which reflects variation in the sulfurea gene
(sulf; Whibley et al., 2006), darker anthocyanin in petal cells overlying major veins (Ve, conditioned by the Venosa locus; Schwinn
et al., 2006), and restriction of anthocyanin pigmentation to the
corolla face and between dorsal petal lobes, which is characteristic
of mutations in the Eluta gene (El; Martin et al., 1987). Although
these three genes show epistatic interactions, they have independent effects that allow all combinations of genotypes to be inferred
(Martin et al., 1987).
Shape models were initially made for individual organs (node-4
leaves, flattened dorsal petals or intact flowers) using the method of
Langlade et al. (2005), in which points were positioned around the
outlines of each organ. Matrices of point co-ordinates were then
translated and rotated to set their centroids at the origin, and to
minimize variance in the positions of points (a Procrustes alignment
without scaling). Principal component analysis (PCA) was used to
partition the variance between plants into orthogonal PCs. For each
type of organ, PC1 identified size as the major source of variation
between plants (Figure S1). To minimize re-sampling this size
variation (which could have a common genetic basis in the different
organs), we therefore made a single shape model for all three
organs together. The leaf matrices were first rescaled so that the
total variance between plants in the leaf data set was the same as for
petals and flowers together. The three point matrices (leaf, petal and
flower) representing each individual were combined and subject to
PCA.
Plastid haplotype analysis
To compare plastid haplotypes, 19 non-coding loci (Shaw et al.,
2005, 2007) were amplified from 10 Antirrhinum species, and
sequences of the three loci with the highest proportion of nucleotide
substitutions relative to indels were obtained from all populations.
Further accessions were genotyped by incubating the product from
trnD–trnT with MseI and from trnS–trnR with TfiI. Where lack of
cleavage suggested a novel haploptype for the species, PCR products were sequenced to confirm the absence of the restriction site.
Amplified fragment length polymorphisms (AFLPs) were produced from DNA digested with PstI and MseI, and were amplified
with four primer combinations (P11–M49, P11–M41, P12–M37 and
P14–M35, in Keygene nomenclature). AFLPs that did not represent
genotypes consistently were identified by carrying out 10 AFLP
reactions on each of four independent DNA extractions from the
same set of genetically diverse plants. Fragments that were not
detected in all replicates of the same plant were removed from
the larger data set. Significant negative correlations between AFLP
band size and frequency were detected for two primer combinations, suggesting size homoplasy among smaller bands. Fragments
smaller than 100 nucleotides were therefore excluded from the
analysis.
Individuals were clustered into populations using STRUCTURE
(Falush et al., 2003, 2007). Priors and parameters relating to
correlated allele frequencies and the extent of population subdivision were found to have only minimal effects on population
assignments and likelihoods of models fitting the data (Jakobsson
and Rosenberg, 2007). At least eight simulations were carried out
for each value of K (number of populations), without varying other
parameters. Each simulation comprised 20 000 burn-in and 120 000
experimental replications. Results were visualized with DISTRUCT
(Rosenberg, 2004).
Pairwise genetic distances (Jaccard) between individuals were
calculated from AFLP data in PAST (Hammer et al., 2004), and
neighbour-joining analyses of distance matrices in PHYLIP (Felsenstein, 1989).
ACKNOWLEDGEMENTS
We are grateful to Maureen Erasmus, Kim Coulson, Mary Coulson,
Niall Wilson, Monique Burrus and Christophe Thébaud for their
considerable help with fieldwork. This work was supported by
BBSRC, through grant BB/D552089/1, and a postgraduate studentship to YW, and by a Small Project Grant from the University of
Edinburgh Fund.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Allometric variation in Antirrhinum leaves and flowers.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1032–1043
Constrained evolution in the genus Antirrhinum 1043
Figure S2. Distribution of numbered Antirrhinum populations in
phenotype space.
Figure S3. Assignment of Antirrhinum accessions to between two
and 14 populations.
Figure S4. Neighbour-joining analysis of all Antirrhinum accessions.
Table S1. Antirrhinum populations used in this study.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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ª 2011 The Authors
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