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Post-pollination barriers do not explain the persistence of two distinct

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Plant Syst Evol (2010) 286:223–234
DOI 10.1007/s00606-010-0303-4
ORIGINAL ARTICLE
Post-pollination barriers do not explain the persistence of two
distinct Antirrhinum subspecies with parapatric distribution
C. Andalo • M. B. Cruzan • C. Cazettes • B. Pujol •
M. Burrus • C. Thébaud
Received: 8 December 2009 / Accepted: 21 April 2010 / Published online: 25 May 2010
Ó Springer-Verlag 2010
Abstract Empirical studies of post-pollination barriers to
gene flow between recently diverged plant species are
important to understand ecological processes underlying
speciation. Using greenhouse and common garden experi-
ments, we investigated the strength of post-pollination
barriers that restrict or prevent gene flow between two
subspecies of Antirrhinum: Antirrhinum majus pseudoma-
jus and A. m. striatum. The two are distributed parapatri-
cally but share the same major pollinators (bumblebees),
and form narrow hybrid zone in many areas of southern
France and northern Spain where they come into close
contact. We assessed the strength and symmetry of mating
barriers and their homogeneity among populations by
comparing fruit set, seed set and offspring performances
between intra- and inter-subspecific crosses performed in
parental and hybrid populations. Although all populations
showed high levels of self-incompatibility, we found very
little evidence for barriers to gene flow once pollen had
been transferred to the stigma. We suggest that reproduc-
tive isolation in this system mostly involves barriers related
to processes occurring before pollination, with little or no
role of post-pollination barriers.
C. Andalo (&)  C. Cazettes  B. Pujol  M. Burrus 
C. Thébaud
Laboratoire Evolution et Diversité Biologique,
UMR 5174 CNRS-Université Paul Sabatier,
118 route de Narbonne, 31062 Toulouse Cedex 9, France
e-mail: andalo@cict.fr
M. B. Cruzan
Department of Biology, Portland State University,
PO Box 751, Portland, OR 97207, USA
Keywords Antirrhinum  Hybridization 
Pollinator sharing  Post-pollination barriers 
Reproductive isolation
Introduction
Beginning with Dobzhansky (1937), evolutionary biolo-
gists have tried to explain the origin and evolution of
reproductive isolation, i.e. mechanisms that prevent gene
flow between closely related species (Coyne and Orr
2004). Reproductive isolation is typically viewed as aris-
ing from two broad classes of mechanisms: pre-zygotic
and post-zygotic barriers. Pre-zygotic barriers are further
divided into pre-mating and post-mating barriers (of
course, all post-zygotic barriers are also post-mating)
depending on whether or not inter-specific matings occur
when two species meet. In plants, pre-pollination (pre-
mating) barriers can prevent hybridization by limiting
pollen transfer between hetero-specific individuals through
habitat or pollinator partitioning, temporal isolation, or
floral divergence between species. In contrast, post-polli-
nation (post-mating) barriers act against gene flow once
pollen has been transferred to the stigma. Post-pollination
barriers involve pre-zygotic processes such as pollen
incompatibility and pollen competition or post-zygotic
processes such as hybrid breakdown (Arnold 1997). This
last process may involve environment-dependent (exoge-
nous) or environment-independent (endogenous) selection
against hybrids, or both.
Various post-pollination mechanisms (both pre- and
post-zygotic) can operate, and this can lead to asymmetries
in post-pollination barriers, with important consequences
for hybridization dynamics (Tiffin et al. 2001). For exam-
ple, differences in style length, self-compatibility or ploidy
123
224 C. Andalo et al.

level (reviewed in Levin 1971) may all produce asymme-

tries in reproductive isolation at early stages such as seed
and fruit sets. Later in the life cycle, nuclear-cytoplasmic
incompatibilities between the two parental genomes can
produce asymmetries in post-zygotic isolation, with the
severity of hybrid breakdown dependent on the maternal
species used. By contrast, nuclear incompatibilities usually
produce symmetric post-zygotic barriers with a decrease of
relative fitness of hybrids independent of the direction of
the cross. The quantification of asymmetry is thus crucial
for understanding the evolution of isolating mechanisms
when hetero-specific pollen transfer is possible.
In this paper, we examine the relative role of post-
pollination barriers in Antirrhinum (snapdragon—Scroph-
ulariaceae). This plant group is characterized by floral
divergence and the potential for substantial pollinator
sharing when two or more species occur in close proximity.
The Antirrhinum clade is both species rich and ecologically
diverse, with most species showing extensive divergence in
floral traits, such as shape, size and colour (Mather 1947;
Rothmaler 1956; Langlade et al. 2005). However, there
has been no previous quantitative assessment of potential
reproductive barriers between the different species or
subspecies.
We used a series of field and greenhouse experiments to
quantify post-pollination barriers between two Pyrenean
subspecies of the Antirrhinum majus complex (Antirrhinum
subsection Antirrhinum sensu Rothmaler 1956):
A. m. pseudomajus and A. m. striatum. These subspecies
differ strikingly in flower colour (Fig. 1a, b) and are related
as sister clades. They occur parapatrically in the eastern Fig. 1 Flowers of Antirrhinum majus pseudomajus (a) and Antirrhi-
num majus striatum (b)
part of the Pyrenees, where they come into contact in a
zone stretching 150 km along the Sierra del Cadi in
Catalunya, Spain (Whibley et al. 2006). A number of odour will usually translate into pre-pollination barriers if
herbarium specimens available in collections show that this floral divergence is causally linked to pollinator special-
contact zone has existed for a large number of generations ization (e.g. Grant 1992; Hodges et al. 1996; Campbell
(C. Thébaud, pers. obs.), an observation consistent with the et al. 2002; Ramsey et al. 2003; Ippolito et al. 2004). It is
hypothesis that it originated through ancient secondary hard to imagine such a mechanism at play in our study
contact between populations that had diverged in allopatry system, since the two subspecies are pollinated exclusively
(Rothmaler 1956). In the Pyrenees, A. m. pseudomajus and by large bees (mostly Bombus lucorum, B. hortorum,
A. m. striatum reach 1,900 m altitude and form a hybrid B. lapidarius and Xylocopa violacea), and flowers are
zone wherever they meet closely along the contact zone, visited by the same pollinators wherever they occur in
regardless of environmental features such as altitude, close proximity (C. Andalo and C. Thébaud, unpublished
rainfall, soil type and human disturbance (C. Andalo and data). When partially sympatric sister species share their
C. Thébaud, unpublished data). The two subspecies pollinators, pre-pollination barriers depend exclusively on
hybridize readily where they meet, but when samples are pollinator fidelity to a particular morphotype (i.e. behav-
taken along transects through the hybrid zones, flower ioural constancy) (Waser et al. 1996; Waser 2006) and are
colour changes abruptly from one pure state to the other thus likely to be variable in time and space (e.g. Aldridge
over just a few kilometres. This implies that some form of and Campbell 2007). In addition, flower constancy has
reproductive isolation exists between the two A. majus been shown to be weaker when species differ in few floral
subspecies (Whibley et al. 2006). traits (Gegear and Laverty 2005). Thus, it seems possible
In plants that are pollinated by animals, differentiation that species or subspecies integrity is at least partially
between species in floral traits such as shape, colour or maintained by post-pollination barriers in such situations.

123
Post-pollination barriers in Antirrhinum
Here we address the following questions as a first step to
understand the evolution of isolating mechanisms in
Antirrhinum: (1) To what extent does reproductive isola-
tion between A. m. pseudomajus and A. m. striatum
depend on post-pollination barriers? (2) Are these barriers
asymmetric, i.e. does their strength depend on the direction
of the cross? And, if so, (3) does asymmetry differ among
components of reproductive isolation?
Materials and methods
Population and individual sampling
Populations were chosen to span the geographic and
elevational ranges of the subspecies’ distributions and to
obtain samples from both parapatric and distantly allopatric
populations (Fig. 2). The allopatric populations sampled
were from La Preste (PRES; 42°240 N, 2°240 E, elevation
1,214 m, France) and from Pomas (POM; 43°70 N, 2°160 E,
elevation 262 m, France) for A. m. pseudomajus and
Fig. 2 Location of the five
Antirrhinum majus populations
studied (black points). Grey
area represents the distribution
of A. m. striatum. Grey lines
represent elevation isolines
above 1,800 m
225
A. m. striatum, respectively. The parapatric populations
were sampled from either side of a contact zone between
A. m. pseudomajus in Pardines (PAR; 42°190 N, 2°120 E,
elevation 1,118 m, Spain) and A. m. striatum near Collades
de Tosas (TOS; 42°220 N, 1°560 E, elevation 1,492 m,
Spain). A fifth population, in which individuals displayed
hybrid phenotypes with intermediate flower colour, was
sampled from this contact zone (nearly midway between
the PAR and TOS populations) near Planoles (PLA;
42°190 N, 2°50 E, elevation 1,285 m, Spain).
Mother plants were randomly selected in the different
localities (5, 5, 9, 9 and 20 in PRES, POM, TOS, PAR and
PLA, respectively). Mature seeds were harvested ran-
domly from these plants; all seeds from the same mother
plant constituted a maternal family. A larger number of
maternal families was sampled in TOS, PAR and PLA, to
better take into account the higher genetic diversity
observed in these populations (A. Khimoun, unpublished
data).
Prior to crossing, plants were grown to maturity from
seed. Conditions in the greenhouse were maintained as
123
226 C. Andalo et al.

Table 1 Number of crosses performed in each population

Paternal parent
PRES (pseud.) PAR (pseud.) POM (stria.) TOS (stria.) PLA (hybrids)
Auto. Btw. Self Auto. Btw. Self Auto. Btw. Self Auto. Btw. Self Auto. Btw. Self

Maternal parent
PRES (pseud.) 16 16 (5) 14 13 (5) 12 (5) 13 (5)
PAR (pseud.) 22 (5) 24 24 (5) 23 23 (5) 24 (5)
POM (stria.) 13 (5) 13 (5) 15 14 (5) 15 13 (5)
TOS (stria.) 23 (5) 23 (5) 23 (5) 27 25 (5) 25
PLA (hybrids) 31 (5) 30 (5) 37 31 (5) 37
Sample sizes for inter-subspecific and intra-subspecific crosses are presented in bold and italics, respectively. The numbers of fruits sampled for
the common garden experiment are presented in brackets
Auto unaided autogamy, Btw crosses between two different individuals, Self self-crosses, Pseud Antirrhinum majus pseudomajus, Stria
A. m. striatum

constant as possible with a 16 h/8 h day/night light regime
and an evaporative cooling system that regulated temper-
ature at around 20°C.
Crossing design
Crosses were carried out to assess the level and symmetry
of post-mating isolation within and between taxa. Since
Antirrhinum flowers are hermaphroditic and protandrous,
flower buds were emasculated by removal of the four sta-
mens and individual plants were enclosed in pollinator
exclusion bags that were made with bridal veil. Five days
after emasculation, the receptive stigmas were hand-polli-
nated with pollen from male-phase flowers (Table 1; 619
flowers were treated in total). To test for potential pollen
contamination, a control treatment was performed in which
flower buds were emasculated and enclosed in pollination
bags, but not pollinated. Of the 87 control flowers treated in
this way, only one produced a fruit. Thus, we can safely
assume that pollen contamination was minimal. For crosses
involving a pollen donor different from the pollen receiver
we avoided inbreeding and incompatible pollination as
much as possible by pooling pollen from two or three
donors from different maternal families whenever possible
(more than 76% of crosses). One to seven flowers per plant
were randomly allocated to a particular pollination treat-
ment with no replication of treatments within an individual.
We first tested for self-incompatibility because it influ-
ences hybridization ability (Lewis and Crowe 1958; de
Nettancourt 1977; Levin 1978; Harder et al. 1993). We
assessed the level of self-compatibility and autogamy in
each population (Table 1). We compared reproductive
performances for the following three treatments:
– Unaided autogamy: Flower buds were not emasculated
before enclosure in pollination bags, and left without
any pollination treatment.
– Self-cross: Flowers were hand-pollinated with pollen
from a male-phase flower of the same plant.
– Intra-population cross: Flowers were hand-pollinated
with pollen from other individuals of the same
population.
Then, we tested for cross-compatibility between sub-
species by comparing: (1) crosses within and between
populations of the same subspecies and (2) crosses between
populations of different subspecies (Table 1). We expected
the latter crosses to be less successful in terms of progeny
number and/or fitness if post-pollination barriers exist.
Hybrid breakdown was estimated by comparing intra-
population pollination (pollen from hybrid plants) and
backcross pollination (pollen from parental plants) for
hybrid plants from PLA. The intra-population treatment
produced hybrid progenies, whereas backcross treatments
produced parental-like progenies. Again, we expected
crosses to be less successful in terms of progeny number
and/or fitness when using hybrid pollen if post-pollination
barriers exist. Hence, backcross pollinations with both
A. m. pseudomajus and A. m. striatum, from PAR and TOS
(parental populations sampled from either side of the
contact zone), respectively, were also performed on hybrid
individuals (Table 1).
In the crossing designs described above, intra-popula-
tion crosses were performed within each of the five pop-
ulations (Table 1). These crosses produced parental
progenies in the four parental populations and hybrid
progenies in the hybrid population. A lower number of
progenies, lower fitness in the hybrid population, or both,

123
Post-pollination barriers in Antirrhinum
would thus suggest that effective post-pollination barriers
contribute to reproductive isolation of the two subspecies.
Traits measured
Fruit and seed set
A few weeks after crossing, when fruits were almost ready
to dehisce, each treated flower was scored for fruit set. We
measured the length, width and thickness of every fruit
produced and multiplied these three dimensions to estimate
fruit size. As seed number per fruit is very large in Antir-
rhinum, and because fruits dehisce quite rapidly, it was not
possible to count the number of seeds for each successful
cross. Hence, we estimated the correlation between fruit
size (length 9 width 9 thickness) and seed number per
fruit for a sample of 32 mature but undehisced fruits from
all five populations studied and from all cross types. This
correlation was positive and significant (r = 0.54; n = 32
fruits; P = 0.0012). Thus, for all our crosses, we used fruit
size as an estimator of the number of seeds per fruit.
Analyses of progenies: seed quality
From each fruit we weighed approximately 30 seeds and
estimated the mean seed weight. Within subspecies, the
latter trait is expected to be correlated with seed quality
parameters such as seed longevity (Fenner 1985; Banovetz
and Scheiner 1994). Furthermore, seed weight reflects the
quantity of food reserves available and could be related to
the success of individual offspring (Westoby et al. 1992).
Germination was monitored for seeds from 209 fruits
harvested in the greenhouse in December 2003. Seeds were
sown in 55 9 15 mm Petri dishes containing filter paper
moistened with distilled water (n = 6,198 total seeds). To
cultivate progenies from these crosses, seeds from 176
fruits (n = 5,202 total seeds) were sown in March 2006 in
pots filled with compost and set out in a greenhouse at
15°C. In both germination rounds the final number of
germinated seeds was recorded; no more seeds germinated
after 25 days, and average germination rates were 0.96 and
0.90 in 2003 and 2006, respectively.
Analyses of progenies: reproductive and vegetative
performances
In April 2006, the fitness of the progenies from our crossing
experiment was estimated in a large, common garden
experiment. In each of the five experimental populations,
five recipient plants that originated from five different
maternal families were randomly selected. For each
recipient plant, seeds from fruits from all the different allo-
pollination treatments were randomly sampled (Table 1;
227
95 fruits in total). Ten seedlings per fruit (950 seedlings in
total) were transplanted into peat pellets (jiffy-7) and then
planted in a common garden established at the CNRS field
station of Moulis in the Pyrenees at the end of April. We
used a simple randomized block design without replication
within a block. Ten plots of 33 m2 each were used as block
and separated by 2 m intervals. In each plot, the 95 trans-
plants each from a different fruit were randomly assigned in
a 32 9 3 grid with plants 50 cm apart. During transplant-
ing, two half plots were accidentally destroyed, giving a
total of 854 plants that were cultivated and monitored
throughout the experiment.
On each individual plant we measured fitness compo-
nents relating to both survival and fecundity. At the end of
the growing season in September, we recorded the number
of fruits per plant and measured the height of the two
longest axes. Vegetative size was estimated by summing
the height of the two longest axes. This composite variable
was a good proxy of vigour because it is strongly positively
correlated with total (shoot plus root) dry plant biomass
(r = 0.460, n = 151, P \ 0.001). In addition, large root-
to-shoot ratio is known to enhance plant survival in
drought-prone and nutrient-poor environment (Lloret et al.
1999) such as those encountered by wild Antirrhinum
populations. Above-ground and below-ground biomass of a
random sub-sample of 155 plants were harvested sepa-
rately, in September 2006. Roots and shoots, including
leaves, were separated and placed in paper envelopes, dried
at 80°C for 1 week and weighed to the nearest 0.1 g.
Statistical analysis
Fruit set and seed germination rate were either very low or
very high, with no intermediate values whatever the pol-
lination treatment and population considered. Thus, we
used Fisher’s exact test, suitable for low-frequency events,
to analyse these two life-history traits (Table 2). Fruit size,
mean seed weight, number of fruits, vegetative size and
root-to-shoot ratio were all analysed using parametric
mixed linear model with the MIXED procedure of SAS
(SAS Institute 2000; Table 2). Normality of distributions
was always tested for every dependent variable and, if
necessary, adapted Box–Cox transformation was applied
(Sokal and Rohlf 1995). Power transformation was esti-
mated using the Companion to Applied Regression pack-
age in R software (Venables and Ripley 1999; Fox 2002).
We assessed the factor affecting whether plants ever
reproduced (probability of survival to reproduction) using
mixed models with binomial errors with the GENMOD
procedure of SAS (SAS Institute 2000).
For studying cross compatibility and F1 hybrid perfor-
mances, we analysed crosses performed within and between
the four parental populations (Table 2). Two populations
123
228 C. Andalo et al.

Table 2 Summary of the analyses performed

Question Pop. Crosses compared Trait measured Statistical
used analysis

Self- All Unaided autogamy and self-crosses versus Fruit set Fisher’s exact test
incompatibility intra-population crosses
Cross Parental Intra-subspecific crosses (crosses within Fruit set Fisher’s exact test
compatibility population and between populations of the Fruit sizea Linear mixed model
same subspecies) versus inter-subspecific
crosses (crosses with both the parapatric
and allopatric populations of the other
subspecies)

F1 performances Parental Intra-subspecific crosses (crosses within Germination rate Fisher’s exact test
population and between populations of the Mean seed weight, vegetative size, Linear mixed model
same subspecies) versus inter-subspecific number of fruits, probability of
crosses (crosses with both the parapatric survival to reproduction,
and allopatric populations of the other root-to-shoot ratio
subspecies)

Hybrid Hybrid Backcrosses with A. m. pseudomajus and Fruit set, germination rate Fisher’s exact test
performances A. m. striatum versus intra-population Fruit sizea, mean seed weight, Linear mixed model
crosses vegetative size, number of fruits,
probability of survival to
reproduction, root-to-shoot ratio

All Intra-population crosses within the parental
populations versus intra-population
crosses within the hybrid population
Fruit set, germination rate Fisher’s exact test
a
Fruit size , mean seed weight, Linear mixed model
vegetative size, number of fruits,
probability of survival to
reproduction, root-to-shoot ratio

a
Fruit size surrogates for seed set

per taxon and several maternal families per population
were used which allow to test for a fixed population effect
(within taxon) and for a random maternal family effect
(within taxon and population). Because several different
crosses were done on each cultivated plant, a nested plant
recipient random effect (within taxon, population and
family) was included in the models. The geographic dis-
tance between the two populations crossed in each case
and its corresponding quadratic term were also included
as covariates to account for potential inbreeding or out-
breeding depression effects (Lynch 1991). We also tested
for the fixed effects of taxon and pollination treatment
with two levels: intra-subspecific crosses (crosses within
population and between populations of the same subspe-
cies) versus inter-subspecific crosses (crosses with both
the parapatric and the allopatric populations of the
other subspecies). The interaction between taxon and
pollination treatment effects was used to test the symmetry
of the crossing barriers. This test allows asymmetry to
be detected if the difference between intra- and inter-
subspecific crosses depends on the taxon used as maternal
plant. Such approach is more conservative than a compar-
ison limited to the two reciprocal inter-subspecific
crosses (e.g. Tiffin et al. 2001) because the latter would
detect asymmetry every time taxa differ in their mean trait
values.

123
Post-pollination barriers in Antirrhinum
To study hybrid performances, we first analysed crosses
performed on hybrid plants from the population PLA
(Table 2). We tested for a fixed effect of pollination
treatment with two levels: backcross pollinations with
A. m. pseudomajus or A. m. striatum and intra-population
crosses. Secondly, we analysed intra-population crosses
performed within each of the five populations (Table 2).
We tested for a fixed population effect with two levels:
parental populations versus hybrid population. In both
analyses, we controlled for family (within population) and
plant recipient (within population and family) random
effects.
In the common garden experiment, plants were grouped
by plot position. For traits measured on progenies, a fixed
block effect was thus added in the statistical models to
remove spatial effect. The only exception was for the study
of the probability of survival to reproduction for hybrid
plants. Because of the lack of information in the data,
parameters of the full model were not estimable. A sim-
plified mixed model with binomial error but without plot
effect was thus used.
Results
Self-incompatibility
All populations, including hybrids, were shown to be
strongly self-incompatible. There was no significant dif-
ference between manual and unaided self-pollination
treatments for fruit set (Fisher’s exact tests: P [ 0.4 in all
five populations). Pooling both self-pollination treatments,
we observed extremely low selfing rates in A. m. pseudo-
majus, A. m. striatum and hybrids (0–4%): only 7 fruits
were produced from a total of 233 self-pollinated flowers.
Across all five populations, fruit set and fruit size were
much higher in intra-population pollination than in crosses
resulting from manual selfing.
Cross compatibility between taxa
Overall, cross compatibility tests did not detect incompat-
ibility between the two Antirrhinum subspecies whatever
the direction of the cross and the population used. Fruit set
was consistently very high, and no statistically significant
difference was found between inter- and intra-subspecific
crosses (Fisher’s exact test: P [ 0.21 in all four parental
populations; Fig. 3a). In general, hand pollinations
between different individuals were very efficient in pro-
ducing fruits, since more than 97% of allo-pollinated
flowers produced fruits. Fruit size was lower in inter-
compared with intra-subspecific crosses (Fig. 3b), but
again this effect never reached statistical significance
229
(Table 3). None of the interactions between pollination
treatment and subspecies or between pollination treatment
and population within subspecies were significant
(Table 3). Fruit size was also not affected by the geo-
graphic distance between the two populations that were
crossed (Table 3).
F1 performances
The germination rate of F1 seeds was significantly lower
than for parental seeds in one population (PRES; Fisher’s
exact test P = 0.017). However, germination rates were
consistently very high (Fig. 3d). There was no significant
difference between intra- and inter-subspecific crosses in
mean seed weight (Table 3; Fig. 3c). Altogether, seed trait
analyses indicated that F1 seeds from inter-subspecific
crosses were of similar quality to those obtained from intra-
subspecific crosses. Similar results were found for vege-
tative size, number of fruits, probability of survival to
reproduction and root-to-shoot ratio of progenies (Table 3;
Fig. 4). In summary, no decrease in performance was
detected in F1 hybrids whatever the traits and the life cycle
stage considered.
Hybrid performances
Focussing on the hybrid population, we found no effect of
pollination treatment on fruit set (Fisher’s exact test:
P = 1; Fig. 5a). Fruit size was not significantly different
between backcross and intra-population pollinations
(Fig. 5b; F1,10 = 0.10, P = 0.7551). These results suggest
that hybrid pollen is as efficient in fertilizing ovules as are
pollen grains from parental subspecies.
Furthermore, no significant differences between backcross
and intra-population pollination treatments were observed
for mean seed weight (Fig. 5c; F1,10 = 0.19, P = 0.6757),
vegetative size (Fig. 6a; F1,4 = 0.48, P = 0.5284), number
of fruits (Fig. 6b; F1,4 = 0.44, P = 0.5444), probability of
survival to reproduction (Fig. 6c; v2 = 1.23, df = 1,
P = 0.2675) and root-to-shoot ratio (Fig. 6d; F1,2 = 0.12,
P = 0.7618). For germination rates, we found a slightly
significant difference between backcross and intra-
population pollination treatments (Fisher’s exact test:
P = 0.049; Fig. 5d). Nevertheless, the germination rate
was always above 93%. Therefore, the proportion of
parental genome contributed by either A. m. pseudomajus
or A. m. striatum did not appear to influence variation in
growth and reproductive traits in hybrids.
Using only intra-population crosses within each of the
five populations, fruit set and seed germination rates were
not significantly different between hybrid and parental
populations (Fisher’s exact test: P = 0.49 and P = 0.51
for fruit set and germination rate, respectively). Fruit size,
123
230 C. Andalo et al.

Fig. 3 Fruit set (a), fruit size as a proxy of seed set (b), mean seed weight (c) and seed germination rate (d) for intra- and inter-subspecific
crosses. Values are means ± standard error (SE). Numbers in brackets indicate sample sizes, for abbreviations, see Table 1

Table 3 Analysis of variance of fruit and seed traits for crosses performed in the four parental populations and of fitness components of the
derived progenies
Source of variation Parents (greenhouse experiment) Progenies (common garden experiment)
(fixed effects only) a
Fruit size Mean seed Vegetative Number Prob. of survival Root-to-shoot
weight size of fruits to reproduction ratio
df F df F df F df F df v2 df F

Plot 9 1.16 NS 9 2.74** 9 10.83 NS 8 0.83 NS

Distance between populations 1 3.35 NS 1 2.09 NS 1 1.61 NS 1 1.19 NS 1 2.69 NS 1 0.00 NS
Squared distance btw. pop. 1 2.49 NS 1 1.48 NS 1 1.40 NS 1 0.25 NS 1 2.73 NS 1 0.07 NS
Pollination treatment 1 0.01 NS 1 0.46 NS 1 1.04 NS 1 0.51 NS 1 0.05 NS 1 0.20 NS
Taxon 1 17.78*** 1 0.32 NS 1 2.21 NS 1 4.76* 1 0.90 NS 1 0.08 NS
Population (taxon) 2 1.49 NS 2 4.40* 2 1.27 NS 2 1.21 NS 2 2.87 NS 2 1.24 NS
Poll. treat. 9 taxon 1 3.70 NS 1 1.50 NS 1 1.64 NS 1 0.47 NS 1 1.31 NS 1 0.15 NS
Poll. treat. 9 pop. (taxon) 2 0.31 NS 2 0.26 NS 2 0.42 NS 2 0.07 NS 2 4.54 NS 2 0.04 NS
Residuals 129 126 599 537 703 82
NS not significant
a
Fruit size surrogates for seed set
*** P \ 0.001, ** P \ 0.01, *P \ 0.05

mean seed weight, vegetative size, number of fruits per most closely approached significance was observed for
individual, probability of survival to reproduction and root- seed weight F1,44 = 3.1, P = 0.0854). Thus, the hybrid
to-shoot ratio did not show any significant difference population seemed to produce as many progenies with
between hybrids and parental populations (the P value that similar fitness as did parental populations.

123
Post-pollination barriers in Antirrhinum 231

Fig. 4 Vegetative size (a), number of fruits (b), probability of garden experiment. Values are means ± standard error (SE). Num-
survival to reproduction (c) and root-to-shoot ratio (d) for progenies bers in brackets indicate sample sizes, for abbreviations, see Table 1
from intra- and inter-subspecific crosses cultivated in a common

Fig. 5 Fruit set (a), fruit size as

a proxy of seed set (b), mean
seed weight (c) and seed
germination rate (d) for intra-
population crosses and
backcrosses performed on
hybrid plants from the PLA
population. Values are
means ± standard error (SE).
Numbers in brackets indicate
sample sizes

123
232
Fig. 6 Vegetative size (a),
number of fruits (b), probability
of survival to reproduction (c)
and root-to-shoot ratio (d) for
progenies from intra-population
crosses and backcrosses
performed on hybrid plants
from the PLA population. These
progenies were cultivated in a
common garden experiment.
Values are means ± standard
error (SE). Numbers in brackets
indicate sample sizes
Discussion
The aim of our study is to investigate the strength, the
symmetry and the homogeneity of post-pollination barriers
and to clarify their potential role in explaining the evolu-
tion of reproductive isolation between two closely related
Antirrhinum subspecies. Through different pollination
experiments, we found no difference between intra-
subspecific and inter-subspecific crosses. Furthermore, the
sample size used in our experiments (619 crosses and 854
cultivated plants) should have been sufficient to detect
differences between pollination treatments, if they exist.
Our results reveal the lack of post-pollination barriers to
gene flow. Barriers could have been expected if there were
differences between parental species in style length, self-
compatibility or ploidy, all of which can lead to asym-
metrical post-pollination barriers (Levin 1978; Harder
et al. 1993; Arnold 1997). However, all Old World Anti-
rrhinum species are diploid (Thompson 1988), and com-
parisons of floral measurements in 35 natural populations
(for a total of 543 plants) showed no differences in style
length among taxa (C. Andalo, unpublished data). Fur-
thermore, A. m. pseudomajus and A. m. striatum did not
differ in their degree of self-incompatibility, which was
always very high, in line with those documented in other
123
C. Andalo et al.
Antirrhinum species (East 1940; Torres et al. 2002;
Zwettler et al. 2002; Mateu-Andrés and Segarra-Moragues
2004) and in other diploid species (Mable 2004). Genetic
divergence between A. m. pseudomajus and A. m. striatum
appears not to be large enough to produce strong, asym-
metric, post-pollination barriers to gene flow that are
observed in many divergent plant taxa (Tiffin et al. 2001).
In addition to high seed set in inter-subspecific crosses,
neither hybrid inviability nor infertility was detected. We
also found little or no reduction in seed germination, or
vegetative and reproductive performance of F1 hybrids and
progenies from hybrid parents grown in the common gar-
den experiment. Likewise, hybrids did not exhibit reduced
pollen efficiency in fertilizing ovules, and they produced as
many fruits and seeds as did parental plants. The absence
of strong post-zygotic barriers, with hybrids being rela-
tively fit, is commonly found in both animals and plants
(Arnold 1997). However, as shown by Campbell and
Waser (2001) in Ipomopsis and by Johnston et al. (2001) in
Iris, complex genotype–environment interactions can
generate environment-dependent selection in the wild.
Hence, it is likely that selection against hybrids almost
entirely results from interactions with biotic or abiotic
environmental factors. Furthermore, hybrid fertility (both
male and female) was estimated from plants with unknown
Post-pollination barriers in Antirrhinum
hybrid index. It is therefore possible that selection had
already taken place in our hybrid study population and
swamped initial genetic variation for some fitness-related
traits. In other words, only relatively fit hybrids reached
maturity and were sampled. Finally, since fitness may be
increased in F1 individuals because of heterosis, hybrid
breakdown could occur in the F2 and backcross genera-
tions, a situation that is very common in plants (Clausen
1951; Rhode and Cruzan 2005). These potential factors
were not quantified in this study, and more work will be
needed before we can fully evaluate their potential con-
tribution to reproductive isolation in this system. Further-
more, because of the parapatric distributions of
A. m. pseudomajus and A. m. striatum (Fig. 2), ecogeo-
graphic barriers may also help to keep the two subspecies
distinct. In all hybrid zones, however, reproductive isola-
tion in Antirrhinum must involve some other barriers
related to processes occurring before pollination.
While pollinators can transfer pollen from one plant
species to another when they share the same pollinators,
this is not necessarily the case, and pollinators may play a
major role as selection agents to maintain geographic dif-
ferentiation. Pollinators such as bumblebees often show
colour preferences that can be influenced by floral traits at a
local scale (Briscoe and Chittka 2001). Unlike other poll-
inators, they often exhibit flower constancy, the degree of
which is largely determined by how distinctive individual
flower colours are in a local plant community (Chittka
et al. 1997). Colour preferences and flower constancy are
likely to be decisive in determining mating opportunities
between individuals from different plant species, especially
when the colour of their flowers is perceived as very
different by bumblebees. Recent results suggest that
flower colour differences between A. m. pseudomajus and
A. m striatum are well above the discrimination threshold
in bumblebee colour space (Tastard et al. 2008). Also, it is
possible that, when hybridization occurs, it results in novel
flower colour phenotypes that are either not preferred or
even difficult to see by bumblebees. Jones and Reithel
(2001) found evidence that pollinator behaviour influenced
mating and reproduction in an experiment using genetic
lines of A. majus. Thus, flower colour divergence between
A. m. pseudomajus and A. m. striatum could be a key
factor in their reproductive isolation through its influence
on pollinator behaviour, causing assortative mating.
Whereas post-pollination isolation is expected to be
strong between populations or species that experience
hetero-specific pollen transfer through pollinator sharing,
both the lack of post-mating barriers demonstrated by our
study, and the observation of abrupt changes in flower
colour between differentiated groups of populations sug-
gest that pollinators play a critical role in maintaining
phenotypic divergence between these subspecies. Because
233
of their relative ease of study and the occurrence of narrow
transition zones between species or subspecies, Antirrhi-
num may prove to be an excellent system to further explore
the role of ecological barriers in the origin of species.
Acknowledgments We thank L. Mantione, E. Lance, E. Beaudoin,
V. Manchon, F. Andalo, E. Tastard, N. Norden, F. Jabot and D. Guery
for assistance with crosses, measurements and plant culture; and
J. Clobert for permission to work at the CNRS field station in Moulis.
We are also grateful to D. Rosenthal, H. de Glanville, P. Sochacki,
J. Picotte, L. Copsey, M. Dufaÿ and D. Mckey for constructive
comments on a previous draft of the manuscript.
References
Aldridge G, Campbell DR (2007) Variation in pollinator preference
between two Ipomopsis contact sites that differ in hybridization
rate. Evolution 61:99–110
Arnold ML (1997) Natural hybridization and evolution. Oxford
University Press, New York
Banovetz SJ, Scheiner SM (1994) The effects of seed mass and the
seed ecology of Coreopsis lanceolata. Am Midl Nat 131:65–74
Briscoe AD, Chittka L (2001) The evolution of color vision in insects.
Annu Rev Entomol 46:471–510
Campbell DR, Waser NM (2001) Genotype-by-environment interac-
tion and the fitness of plant hybrids in the wild. Evolution
55:669–676
Campbell DR, Waser NM, Pederson GT (2002) Predicting patterns of
mating and potential hybridization from pollinator behavior. Am
Nat 159:438–450
Chittka L, Gumbert A, Kunze J (1997) Foraging dynamics of bumble
bees: correlates of movements within and between plant species.
Behav Ecol 8:239–249
Clausen J (1951) Stages in the evolution of plant species. Cornell
University Press, New York
Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Sunderland
De Nettancourt D (1977) Incompatibility in angiosperms. Springer-
Verlag, Berlin
Dobzhansky T (1937) Genetics and the origin of species. Columbia
University Press, New York
East EM (1940) The distribution of self-fertility in the flowering
plants. Proc Am Philos Soc 82:449–517
Fenner M (1985) Seed ecology. Chapman and Hall, New York
Fox J (2002) An R and S-plus Companion to applied regression. Sage,
London
Gegear RJ, Laverty TM (2005) Flower constancy in bumblebees: a
test of the trait variability hypothesis. Anim Behav 69:939–949
Grant V (1992) Floral isolation between ornithophilous and sphing-
ophilous species of Ipomopsis and Aquilegia. Proc Natl Acad Sci
USA 89:11828–11831
Harder LD, Cruzan MB, Thompson JD (1993) Unilateral incompat-
ibility and the effects of interspeccific pollination for Erythro-
nium americanum and Erythronium albidum (Liliaceae). Can
J Bot 71:353–358
Hodges SA, Burke JM, Arnold ML (1996) Natural formation of Iris
hybrids: experimental evidence on the establishment of hybrid
zones. Evolution 50:2504–2509
Ippolito AG, Fernandes W, Holtsford TP (2004) Pollinator prefer-
ences for Nicotiana alata, N. forgetiana, and their F1 hybrids.
Evolution 58:2634–2644
Johnston JA, Grise DJ, Donovan LA, Arnold ML (2001) Environ-
ment-dependent performance and fitness of Iris brevicaulis,
I. Fulva (Iridaceae), and hybrids. Am J Bot 88:933–938
123
234
Jones KN, Reithel J (2001) Pollinator-mediated selection on flower
color polymorphism in experimental populations of Antirrhinum
(Scrophulariaceae). Am J Bot 88:447–454
Langlade NB, Feng X, Dransfield T, Copsey L, Hanna AI, Thébaud
C, Bangham A, Hudson A, Coen E (2005) Evolution through
genetically controlled allometry space. Proc Natl Acad Sci USA
102:10221–10226
Levin DA (1971) The origin of reproductive isolating mechanisms in
flowering plants. Taxon 20:91–113
Levin DA (1978) The origin of isolating mechanisms in flowering
plants. In: Hecht MK, Steere WC, Wallace B (eds) Evolutionary
Biology, vol II. Appleton Century Crofts, New York, pp 185–
317
Lewis D, Crowe LK (1958) Unilateral interspecific incompatibility in
flowering plants. Heredity 12:233–256
Lloret F, Casanovas C, Penuelas J (1999) Seedling survival of
Mediterranean shrubland species in relation to root:shoot ratio,
seed size and water and nitrogen use. Funct Ecol 13:210–216
Lynch M (1991) The genetic interpretation of inbreeding depression
and outbreeding depression. Evolution 45:622–629
Mable BK (2004) Polyploidy and self-compatibility: is there an
association? New Phytol 162:803–811
Mateu-Andrés I, Segarra-Moragues JG (2004) Reproductive system
in the Iberian endangered endemic Antirrhinum valentinum F.Q.
(Antirrhineae, Scrophulariaceae): consequences for species con-
servation. Int J Plant Sci 165:773–778
Mather K (1947) Species crosses in Antirrhinum. I. Genetic isolation
of the species majus, glutinosum and orontium. Heredity 1:175–
186
Ramsey J, Bradshaw HD, Schemske DW (2003) Components of
reproductive isolation between the monkeyflowers Mimulus
lewisii and Mimulus cardinalis (Phrymaceae). Evolution
57:1520–1534
Rhode JM, Cruzan MB (2005) Contribution of heterosis and epistasis
to hybrid fitness. Am Nat 166:E124–E139
123
View publication stats
C. Andalo et al.
Rothmaler W (1956) Taxonomische Monographie der Gattung
Antirrhinum. Akademie-Verlag, Berlin
SAS (2000) SAS OnlineDocÒ (Version 8). SAS institute, Cary
Sokal RR, Rohlf JF (1995) Biometry, 3rd edn. WH Freeman, New
York
Tastard E, Andalo C, Giurfa M, Burrus M, Thébaud C (2008) Flower
colour variation across a hybrid zone in Antirrhinum as
perceived by bumblebee pollinators. Arthropod Plant Interact
2:237–246
Thompson DM (1988) Systematics of Antirrhinum (Scrophulariaceae)
in the New World. Syst Bot Monogr 22:1–142
Tiffin P, Olson MS, Moyle LC (2001) Asymmetrical crossing barriers
in angiosperms. Proc R Soc Lond 268:861–867
Torres E, Iriondo JM, Pérez C (2002) Vulnerability and determinant
of reproductive success in the narrow endemic Antirrhinum
microphyllum (Scrophulariaceae). Am J Bot 89:1171–1179
Venables WN, Ripley BD (1999) Modern Applied Statistics with
S-Plus, 3rd edn. Springer, New York
Waser NM (2006) Specialization and generalization in plant-pollina-
tor interactions: a historical perspective. In: Waser NM, Ollerton
J (eds) Plant-pollinator interactions: from specialization to
generalization. The University of Chicago Press, Chicago,
pp 3–17
Waser NM, Chittka L, Price MV, Williams N, Ollerton J (1996)
Generalization in pollination systems, and why it matters.
Ecology 77:279–296
Westoby M, Jurado E, Leishman M (1992) Comparative evolutionary
ecology of seed size. Trends Ecol Evol 7:368–372
Whibley AC, Langlade NB, Andalo C, Hanna AI, Bangham A,
Thébaud C, Coen E (2006) Evolutionary paths underlying flower
color variation in Antirrhinum. Science 313:963–966
Zwettler D, Vieira CP, Schlötterer C (2002) Polymorphic microsat-
ellites in Antirrhinum (Scrophulariaceae), a genus with low
levels of nuclear sequence variability. J Hered 93:217–221