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Geneticandenvironmentalfactorsaffecting
reproductivevariationinAlliumvineale
ArticleinJournalofEvolutionaryBiology·September2001
ImpactFactor:3.23·DOI:10.1046/j.1420-9101.2001.00330.x
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Genetic and environmental factors affecting reproductive
variation in Allium vineale
A. CEPLITIS
Department of Genetics, Lund University, Lund, Sweden
Keywords:
Allium vineale;
asexual reproduction;
genetic variation;
RAPD;
reproductive characters;
reproductive variation.
Abstract
Traits related to allocation of resources to sexual and asexual reproduction,
together with seed production, were scored on Allium vineale plants sampled
from ®ve sites in southern Sweden during a period of 4 years. In addition,
1 random ampli®ed polymorphic DNA (RAPD) ®ngerprinting of the sampled
plants allowed the identi®cation of genets. Integration of genetic and
phenotypic data from ®eld and greenhouse provided for the analysis of
among-year, among-site, and among-genet variance components. These
variance components were taken to represent the in¯uences of short-term
environmental changes, persistent site divergence, and within-site genet
differences, respectively.
It was shown that differences among sites and among genets explained a
large part of the phenotypic variation of allocation traits, whereas among-year
differences had a larger in¯uence on the variation in seed production.
Together, the results support the conclusions of a recent model on the
evolution of mixed reproductive systems, that predicts a stable balance
between sexual and asexual reproduction because of annual ¯uctuations in
fecundity through the two modes.
Introduction
Evolutionary theory predicts a disadvantage ± a `cost' ± to
sexual reproduction as a result of a higher growth rate of
asexual females in dioecious species or, analogously, to a
transmission advantage of genes for asexuality in hermaphrodite species (Williams, 1975; Maynard Smith,
1978). Consequently, whenever genetic variation for
reproductive mode is present in a population ± and all
else is equal ± asexual reproduction is expected to replace
sexual reproduction (e.g. Charlesworth, 1980; Marshall
& Brown, 1981; Uyenoyama, 1984). For this reason,
organisms able to combine sexual and asexual reproduction are of particular interest for the study of the
evolutionary maintenance of sex (Williams, 1975).
Correspondence: Alf Ceplitis, Department of Conservation Biology and
Genetics, Evolutionary Biology Centre, Uppsala University, NorbyvaÈgen
18D, SE-752 63 Uppsala, Sweden.
Tel.: + 46 18 4716408; fax: + 46 18 4716424;
e-mail: [email protected]
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
There are many examples of organisms capable of
producing offspring both sexually and asexually. Plants,
in particular, possess a number of specialized structures
for asexual or vegetative reproduction such as apomictic
seeds, bulbils, runners and rhizomes (Richards, 1986).
Also in animals mixed sexual and asexual reproduction
occurs, as in certain marine invertebrates (Hughes &
Cancino, 1985), and in organisms reproducing by cyclical
parthenogenesis, such as aphids (Moran, 1992) and
Daphnia species (Lynch et al., 1989). In the plant Allium
vineale (wild garlic), and in a number of other Allium
species, ¯owers are often partly or entirely replaced by
bulbils. Thus, offspring are derived from a mixture of
sexual seeds and asexual bulbils. The two propagule
types are both adapted to dispersal and show similar
ecological characteristics (Ronsheim, 1994). Furthermore, the numbers of ¯owers and bulbils in individual
in¯orescences show a striking variation in natural
A. vineale populations (Richens, 1947). It has, however,
remained unclear to what extent this variation is
genetically determined. Early work by Levan (1937),
721
722
A. CEPLITIS
including experimental crosses between two related
species, A. carinatum and A. pulchellum, suggested that
in these species, the presence of bulbils in the in¯orescence is governed by a dominant allele at a single locus.
Salisbury (1942) claimed that the numbers of ¯owers and
bulbils in A. vineale are determined entirely by environmental in¯uences, whereas Ronsheim & Bever (2000)
reported high broad-sense heritability estimates.
Whereas a great multitude of models have been
constructed to explain the persistence of sexual reproduction, only very few of these have considered the
possibility of a mixed reproductive system (Hurst & Peck,
1996). Maynard Smith (1978) argued that in partly
asexual species ± like A. vineale ± the balance between the
reproductive modes may be maintained as a result of
different ecological adaptations of sexually and asexually
produced propagules. This assumption has, however,
only recently begun to receive formal analytical treatment in evolutionary models. Rispe & Pierre (1998)
speci®cally modelled a situation where a cyclically
parthenogenetic lineage is confronted with an obligately
asexual lineage producing a small number of sexual
offspring. Similarly, Bengtsson & Ceplitis (2000) analysed
the conditions for evolutionary stability of a reproductive
system where individuals of a hermaphrodite organism
(e.g. a plant species) produce variable and genetically
determined proportions of sexual and asexual offspring.
In both cases, the maintenance of a mixed reproductive
system depends critically on suf®cient temporal variation
in the relative success of the sexual and asexual reproductive modes, as a result of, for example, fecundity or
viability differences ¯uctuating between years or seasons.
Thus, under such circumstances, genetic variation for the
proportion of sexually produced offspring can be maintained.
Several studies have been carried out on the ecology
and genetics of various life-history characters in partly
asexual organisms (e.g. Douglas, 1981; Law et al., 1983;
Bauert, 1993; Lindgaard Hansen & Molau, 1994; see also
Richards, 1986 and references therein). The results have,
however, seldom been discussed in the context of the
evolutionary stability of a mixed reproductive system.
The present study examines genetic and environmental
variation for sexual and asexual reproduction in the
partly asexual plant species A. vineale. In particular, two
questions are addressed: (i) is there genetic variation for
traits directly in¯uencing allocation to sexual and asexual
reproduction under ®eld conditions, and (ii) do sexual
and asexual fecundity vary as a result of ¯uctuations in
the environment over time. In order to answer these
questions, RAPD ®ngerprinting was used as a means to
discriminate among genets sampled in the ®eld over
several years. It is shown that differences among genets
make a considerable contribution to the phenotypic
variability in reproductive allocation patterns, whereas
environmental factors have a larger in¯uence on the
variability in sexual fecundity. The results are consistent
with recent models predicting an evolutionarily stable
balance between sexual and asexual reproduction as a
result of temporal ¯uctuations in the success of the
different reproductive modes.
Materials and methods
Study species
Allium vineale L. is a weedy, bulbous perennial plant
distributed over most of west and central Europe,
extending northwards to central Scandinavia. It also
occurs in, but is not native to, North America, Australia
and New Zealand. In continental Europe, A. vineale
prefers dry, open habitats such as old ®elds and roadsides.
In Sweden it is mostly found along the coastline and in
È land
the steppe-like grasslands of the Baltic islands of O
and Gotland.
The ¯owering season starts in May or June, when the
scape, or ¯owering stalk, emerges and the spathe bursts
open to expose the in¯orescence. Each ¯owering plant
carries only a single in¯orescence. Flowers are strongly
protandrous and are visited by insects, mainly bumblebees. The fruit is trilocular with two seeds in each
loculus, but it is rare that all seeds develop (Richens,
1947). Allium vineale is self-compatible; however, the
strong protandry and the observation that insect pollinators are required for seed-set (personal observation)
suggest that outcrossing is the norm (see also Ronsheim,
1996). This conclusion is supported by allozyme data
(A. Ceplitis, unpublished data).
Bulbils mature in August to September, and seeds
usually mature 1±2 months later. Both bulbils and seeds
are dispersed by gravity. After ¯owering the scape dies
back and underground offset bulbs replace the parent
bulb. As a means of vegetative propagation, the offset
bulbs are presumably of minor importance compared
with the numerically superior bulbils, in particular as the
underground bulbs are known to have a high mortality
rate as a result of fungal infections (Richens, 1947;
HaÊkansson, 1963).
Sampling procedure
During a period of 4 years (1995±98) A. vineale plants
were sampled from ®ve geographical locations (sites) in
southern Sweden (Fig. 1). Each year, sampling took
place at two separate occasions. In the ®rst sampling,
carried out in July or August, in¯orescences were
collected from individual plants. For each in¯orescence,
three traits were scored: the number of ¯owers, the
number of bulbils, and the proportion of ¯owers (the
number of ¯owers divided by the sum of the numbers of
¯owers and bulbils). The number of ¯owers and the
number of bulbils were chosen to represent the individual plant's allocation of resources to sexual and aboveground asexual reproduction, respectively. The
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
Reproductive variation in Allium vineale
723
Fig. 1 Map of southern Sweden showing the
®ve sites from which samples of Allium
vineale were taken each year between 1995
and 1998. The insert map depicts the area
using a larger scale.
proportion of ¯owers was taken to represent the relative
importance of sexual reproduction. These three traits are
henceforth collectively referred to as allocation traits.
In late September to October, the second sampling was
carried out. At this point, in¯orescences from ¯owering
individuals were collected to obtain data on seed
production. For each in¯orescence, the average number
of seeds per ¯ower was determined. This quantity was
de®ned as the total number of seeds produced by the
in¯orescence divided by the total number of ¯owers, and
it is taken to represent the relative ef®ciency of sexual
reproduction.
The reason for sampling at two different occasions each
year is that many plants have lost some or most of their
bulbils when seeds mature, thereby making it impossible
to correctly determine the number of bulbils in the
in¯orescences. The average sample size per site and per
year was 78 and 97, respectively, for the ®rst sampling,
and 84 and 106, respectively, for the second sampling. At
each sampling occasion, plants were sampled at intervals
of at least 2 m, while trying to cover the entire area taken
up by the plant population. Final sample sizes were
unequal, as plants that had lost bulbils (®rst sampling) or
seeds (second sampling) were discarded.
In addition to collecting data directly from the ®eld,
asexually derived (bulbil) offspring of individuals sampled in 1995 (®rst sample) were grown under uniform
conditions in a greenhouse. Bulbils from the mother
plants were germinated in Petri dishes. The `seedlings',
one from each mother, were transferred to individual
pots approximately one week after germination. The
offspring plants were randomized in the greenhouse and
subjected to cold treatment in order to induce ¯owering.
The plants ¯owered in the following year when the
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
number of ¯owers, the number of bulbils, and the
proportion of ¯owers in the individual in¯orescences
were determined. As all offspring plants produced an
in¯orescence, sample sizes were equal to those for the
1995 sample (Table 1).
RAPD analysis
Multilocus genotypes were inferred from RAPD ®ngerprints for all individuals that were sampled on the ®rst
occasion every year, i.e. individuals that were scored for
allocation traits (sample sizes are given in Table 1). As
the RAPD analysis were performed on DNA extracted
from bulbils which are asexually produced propagules,
the genotype of the plant that produced the bulbils, and
on which all allocation traits were scored, was assumed
to be identical to that of the bulbil.
Five to ten bulbils from each individual chosen for
RAPD-analysis were germinated in Petri dishes. One
germinated bulbil per mother plant was used for DNA
extraction. Total DNA was extracted from fresh leaf
material using the method of Edwards et al. (1991) as
modi®ed by Rasmussen & Rasmussen (1995).
One hundred and fourty random decamer primers (kit
2 A-G, Operon Technologies, Inc., Alameda, CA, USA)
were used in an initial screening procedure. Five primers
(B05, C13, D08, E11, and F04) gave stable and reproducible band patterns, and these were chosen for the
analysis of genet identity.
Polymerase chain reactions (PCR) contained 10 mM
Tris±HCl, 2.0 mM MgCl2, 50 mM KCl, 100 lM each of the
four dNTPs, 0.4 lM primer, and 1 unit of Taq polymerase
(Boehringer, Mannheim, Germany) in a total of 25 lL
reaction mixture. Polymerase chain reactions were
724
A. CEPLITIS
Table 1 The number of genets found in samples from ®ve A. vineale sites in southern Sweden during 4 years; sample sizes are given in
parentheses. The sites are numbered SE1 to SE5 and each location name is given below the corresponding site number. `Single-site genets' and
`single-year genets' is the proportion of genets restricted to a single site and a single year, respectively, relative to all genets found at that site.
Site
SE1
KaÊseberga
Year
1995
1996
1997
1998
5
4
8
2
Site total
(23)
(20)
(20)
(20)
SE2
Habo Ljung
6
5
5
3
(20)
(20)
(20)
(20)
SE3
KaÈmpinge
4
4
8
3
SE4
Enerum I
SE5
Enerum II
Year total
(14)
(20)
(20)
(20)
7 (12)
7 (20)
12 (20)
11 (19)
13 (21)
10 (20)
9 (20)
13 (20)
34
29
41
31
96 (389)
(90)
(100)
(100)
(99)
12 (83)
15 (80)
16 (74)
26 (71)
31 (81)
Single-site genets
1.00
1.00
1.00
0.85
0.87
0.96
Single-year genets
0.67
0.73
0.94
0.69
0.68
0.74
carried out in a Perkin±Elmer Cetus Thermocycler with
the following programme: 3 min at 94 °C, followed by 45
cycles of 1 min at 94 °C, 1 min at 36 °C and 2 min at
72 °C, ending with 5 min at 72 °C.
Two replicate PCR reactions were performed for each
individual. Ampli®ed fragments were visualized on ethidium bromide-stained 1.5-% agarose gels. Fragment
patterns from replicate PCR reactions were identical
and only clear and easily detected fragments were used.
In a few cases, when the presence/absence of some
fragments was uncertain, additional PCR reactions were
performed. Individuals with identical fragment patterns
were considered to belong to the same genet, whereas
individuals with different fragment patterns were treated
as belonging to different genets.
Analysis of variation in allocation traits
Using the information from the RAPD analysis, variation
for each of the allocation traits was analysed using a
partially nested model:
Pijkl ˆ l ‡ yi ‡ sj ‡ g‰sŠjk ‡ yg‰sŠijk ‡ eijkl ;
where Pijkl is the phenotypic value of the lth individual
plant belonging to the kth genet at the jth site in the ith
year, l is the overall mean, yi is the contribution from the
ith year, sj is the contribution from the jth site, g[s]jk is the
contribution from the kth genet nested within the jth
site, yág[s]ijk is the contribution from the interaction
between the ith year and the kth genet nested within the
jth site, and eijkl is the error term. From this model the
total phenotypic variation (r2p) of each allocation trait
was partitioned into variance components due to year
(r2y), site (r2s), genet within site (r2g[s]), and the interaction
between year and genet within site (r2yág[s]).
The rationale of this design was the analysis of the
relative contributions from different sources of variation,
i.e. to estimate the variance components. Thus, all effects
were treated as random in the analyses. Treating the
main effects (year and site) as random can be justi®ed by
regarding them as samples from an underlying distribution of factors that cause variation in the mean pheno3 type (see Bennington & Thayne, 1994). The among-year
variation component was interpreted as re¯ecting shortterm environmental ¯uctuations, whereas the amongsite variation component was supposed to result from
long-term environmental and/or genetic differences. The
magnitude of the among-genet variation component is
directly related to the amount of genetic variation found,
on average, at a site. Finally, the interaction term
indicates the extent to which differences between genets
within sites vary over years, thus giving an estimate of
the importance of genotype±environment interactions to
the phenotypic variation (cf. Scheiner & Goodnight,
1984).
Among-genet variance components for the allocation
traits were also estimated separately for each population
and year. These one-way analyses were performed to
obtain a more detailed picture of the amount of withinsite genetic variation that is expressed under ®eld
conditions and to see whether genet differences remain
stable over years. The signi®cance levels in these analyses
were adjusted by a sequential Bonferroni technique
(Rice, 1989) to account for the multiple simultaneous
test performed on each trait.
Data from the greenhouse-grown bulbil-derived offspring plants were subjected to one-way analysis of
variance (A N O V A S ) to test to what extent site differences
and/or genet differences for allocation traits found under
natural conditions could also be detected in a uniform
environment. To investigate whether the ranks of the site
means remained similar in the greenhouse environment,
Kendall's coef®cient of rank correlation, s, was used to
compare the site ranks of the bulbil-derived plants with
the site ranks of their ®eld mothers (from the 1995
sample).
Because of the unbalanced nature of the data, general
linear model A N O V A was used to evaluate the statistical
signi®cance of different factors, whereas a maximumlikelihood method was used to estimate the variance
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
Reproductive variation in Allium vineale
components. The latter method has been shown to
provide more accurate estimates of variance components
from unbalanced data than other methods (Swallow &
Monahan, 1984; Huber et al., 1994). To meet the
assumptions of normality required in the analyses, the
following transformations were used: number of ¯owers,
X' ˆ log(Ö(X + 1)); number of bulbils, Y¢ ˆ ÖY; ¯ower
proportion, Z¢ ˆ log(ÖZ + Ö(Z + 2)).
As the genealogical relationships within the samples
were unknown and non-identical genets may show
various degrees of relatedness, no attempt was made to
partition the variation among genets into additive and
non-additive components (Mitchell-Olds & Rutledge,
1986; Falconer, 1989). However, if the allocation traits
under study are to some extent genetically determined,
closely related genets are expected to show a higher
phenotypic resemblance than more distantly related ones
(Falconer, 1989). To explore this assumption, an analysis
of the correlation between phenotypic similarity (estimated as the distance between clonal means, see,
e.g. Dunn & Everitt, 1982) and genetic similarity
(estimated as the proportion of shared RAPD bands;
Lynch, 1990) was performed using a Mantel test (Sokal &
Rohlf, 1995). This procedure thus constitutes an additional test for phenotypic trait differences among genets.
Analysis of variation in seed production
The analyses of the average number of seeds per ¯ower
were similar to the analyses of allocation traits. Total
variation was partitioned into components because of
variation among years, among sites, and to the interaction between years and sites. As information on genet
identity in this data set was available only for one of the
4 years (1996), a full partitioning into the variance
components postulated for the allocation traits was not
performed. A separate A N O V A was, however, carried out
on the 1996 sample to examine whether there were any
differences in seed-set among genets.
Statistical signi®cance testing and variance component
estimation procedures were the same as those for the
analyses of the allocation traits. No normality transfor-
725
mation was required for the seed production data. All
statistical analyses were carried out using the GLM,
VARCOMP and FREQ procedures of the SAS statistical
package (SAS Institute, 1982).
Results
The structure of genetic variation
The ®ve RAPD-primers generated scorable bands from 27
loci, of which 26 were variable among the investigated
plants. Among a total of 389 analysed individuals,
96 multilocus genotypes (genets) were identi®ed. The
majority of genets occurred in low frequencies; as many
as 50% of all genets were represented by a single
individual and 78% of the genets had a frequency of
1% or less, i.e. were represented by four or fewer
individuals. A few genets were found in higher frequencies; thus, seven genets made up more than half (52%) of
all sampled plants.
The vast majority of the genets had a limited geographical distribution; only four of the 96 genets were
found in samples from more than one site (Table 2).
These four genets were found at sites SE4 and SE5, which
are separated by a distance of <1 km.
Summing over years, between 12 and 31 genets were
found at the various sites (Table 2). Among these genets,
one or a few dominated the samples from each of the ®ve
sites. Typically, the dominant genets were found in high
frequencies in all of the 4 years. An exception to this
pattern was found at site SE2 where different genets
dominated each year. Among the low-frequency genets,
there was little overlap in occurrence between years. At
the investigated sites between 67 and 94% of all the
recorded genets were found in only one of the 4 years
(Table 2).
Patterns of variation in allocation traits
Flower number, bulbil number and ¯ower proportion in
the individual in¯orescences were all highly variable. For
¯ower number and ¯ower proportion the coef®cient of
Table 2 Variance components of allocation traits and of seed production in A. vineale. Variance components involving differences among
genets were not estimated for seed production as information on genet identity was only available from 1 year in that data set (see Materials
and methods).
Allocation traits
Variance component
d.f.
Flower number
Bulbil number
Flower proportion
Seeds per ¯ower
Year …r2y †
Site …r2s †
4
3
12
118
251
0.0
13.3***
17.0*
5.3
64.4
4.7*
29.7***
15.2*
1.0
49.3
1.2
6.0*
21.4**
4.9
66.5
36.6***
15.1***
±
±
48.2
Genet within site …r2g…s† †
Year ´ genet within site …r2yg…s† †
Error
The numbers are percentages of total phenotypic variation; d.f. denotes the degrees of freedom in the A N O V A . The symbols *, **, and ***
indicate that the component contributes signi®cantly to the total variation at P < 0.05, 0.01 and 0.001, respectively.
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
726
A. CEPLITIS
variation (CV) averaged 178 and 154%, respectively,
over all sites and years, whereas for bulbil number the CV
was lower, averaging 68%.
When the total phenotypic variation was partitioned
into different components (Table 2), the variation attributable to differences among years was found to be small
(<5%) for all three allocation traits. In fact, for ¯ower
number and ¯ower proportion, the among-year variance
component was statistically non-signi®cant. The amongsite component accounted for a larger proportion of
variation, ranging from 6% for ¯ower proportion to 30%
for bulbil number. For all three allocation traits, a
signi®cant among-genet component was detected,
accounting for 15±21% of the total variation (Table 2).
In all cases, the component of variation as a result of the
interaction between years and genets within sites was
small (5% or less) and non-signi®cant.
Statistically signi®cant among-site differences for
all three allocation traits were also detected in the
bulbil-derived greenhouse-grown plants (P < 0.001 from
a one-way A N O V A for each of the traits). When the
greenhouse-grown offspring plants were compared with
their ®eld mothers, a signi®cant correlation of site ranks
was found for all three traits (¯ower number, s ˆ 1.00;
bulbil number, s ˆ 0.60; ¯ower proportion, s ˆ 0.80;
P < 0.05 in all cases).
Genetic variation within sites
To investigate the effects of variation among genets
within sites and years in more detail, variance components for the allocation traits were estimated separately
for each site and year.
At all sites signi®cant among-genet differences were
detected in at least one year for at least one of the three
allocation traits under study, also after Bonferroni correction of signi®cance levels (Table 3). However, the
magnitude of genet differences varied both among sites
and among years. At site SE3, for example, genets
differed signi®cantly for all three traits in 1996, whereas
no differences for any trait were found in 1997. Differences among genets sometimes resulted in very large
variance components, as was seen at site SE4 in 1995,
where variation among genets accounted for 97.5 and
87% of the within-site variation for ¯ower number and
¯ower proportion, respectively.
When analysing the greenhouse-grown offspring
plants, signi®cant differences were detected for bulbil
number among genets from sites SE2, SE4 and SE5, and
for ¯ower number and ¯ower proportion among genets
from site SE4. Signi®cant among-genet differences for
these traits, except for bulbil number at site SE4, were
also found in the 1995 ®eld sample from which the
offspring plants were derived.
When analysing the correlation between phenotypic
and genetic similarity among the ®eld-sampled plants, a
signi®cant correlation was found for ¯ower number and
Table 3 The among-genet variance component of allocation traits
in A. vineale for each year and site. The numbers are percentages of
total within-site phenotypic variation.
Allocation trait
Year
Site
Flower number Bulbil number
Flower proportion
1995
SE1
SE2
SE3
SE4
SE5
0.0
0.0
0.0
97.5**
11.0
0.0
70.1*
0.0
4.8
78.8*
0.4
0.0
0.0
86.6**
21.9
1996
SE1
SE2
SE3
SE4
SE5
43.7(*)
0.0
25.2(*)
15.2
23.8(*)
0.0
4.3
60.7*
0.0
0.0
30.3(*)
0.0
51.8*
17.5
53.4(*)
1997
SE1
SE2
SE3
SE4
SE5
71.8**
0.0
0.0
30.0
0.0
23.6
45.5(*)
0.0
0.0
0.0
46.9(*)
0.0
0.0
16.0
0.0
1998
SE1
SE2
SE3
SE4
SE5
0.0
0.0
0.0
0.0
20.2
0.0
19.8
66.5(*)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
The symbols * and ** indicate a signi®cant among-genet variance
component at P < 0.05 and 0.01, respectively, after sequential
Bonferroni correction for multiple test; (*)-symbols within parentheses indicate variance components signi®cant before Bonferroni
correction.
for bulbil number (r ˆ 0.16 and 0.19, signi®cant at
P ˆ 0.019 and 0.006, respectively, according to a permutation test). For ¯ower proportion (r ˆ 0.14), the statistical signi®cance of the correlation fell just short of
reaching the 5% limit (P ˆ 0.069).
Patterns of variation in seed production
In the sample as a whole, there was a strong and highly
signi®cant correlation between the number of ¯owers in
a plant's in¯orescence and the total number of seeds that
the plant produced (r ˆ 0.77, P < 0.001). The average
number of seeds produced per in¯orescence (plant)
ranged from 3.2 at site SE2 in 1998 to 70.4 at site SE1
in 1997, and averaged 27.2 over all sites and years. The
average number of seeds per ¯ower varied between 0.27
at site SE2 in 1997 and 1.63 at site SE3 in 1995
(Supplementary material). With an overall CV of 67%,
the average number of seeds per ¯ower was certainly
variable, but not to the same extent as, for example, the
proportion of ¯owers.
In contrast to the pattern of variation in allocation
traits, the variation in the number of seeds per ¯ower
showed a large and signi®cant among-year component
(Table 2). Of the total variation in the number of seeds
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
Reproductive variation in Allium vineale
per ¯ower, 37% could be accounted for by variation
among years, whereas a smaller proportion (15%) was
explained by variation among sites. A complete partitioning of the variance in seed number, analogous to the
analysis of the allocation traits, was not possible as
information on genet identity was only available for the
1996 sample. However, a separate analysis of the 1996
sample revealed no difference in the number of seeds per
¯ower among genets, either within or among sites (data
not shown).
Discussion
A large number of plant and animal species are able to
reproduce both sexually and asexually. However, relatively few studies have attempted to reveal if any
underlying genetic polymorphism for reproductive mode
exists in natural populations of such species, and by what
mechanism it may be maintained; issues that are of vital
importance for our understanding of the evolutionary
signi®cance of sexual reproduction.
Genetic variation for traits related to sexual and
asexual reproductive output has been demonstrated
under controlled conditions in some organisms. For
example, in the cyclical parthenogen Daphnia magna,
4 Yampolsky (1992) found differences among clones from
a single population in the production of sexual females
and males. Similar results were obtained by Deng (1996),
who observed signi®cant broad-sense heritabilities for
the number of sexual eggs produced by clones of
D. pulicaria. In plants, variation among clones has been
found for the allocation to sexual reproduction and
clonal growth (e.g. Platenkamp & Shaw, 1992; Cheplick,
1995), while Bauert (1993) concluded that the ratio of
¯owers to bulbils in Polygonum viviparum was partly
genetically controlled. Ronsheim & Bever (2000) reported high broad-sense heritabilities for ¯ower number
(0.79) and bulbil number (0.26) in Allium vinelae. These
estimates were derived from among-genotype variance
components in greenhouse-grown material; the genotypes were, however, all taken from a single population
and potential clonal relationships were not checked by
genetic markers.
The importance of these ®nding notwithstanding,
direct evidence for the existence of genetic variation in
the ®eld for any trait can only be provided through the
identi®cation of genotypes. Nevertheless, molecular
markers have only rarely been employed to examine
genetic variation under ®eld conditions for reproductive
traits in clonal plant species (SkaÂlova et al., 1997). In the
present study, genotypic ®ngerprinting by RAPD markers
was used as a tool to identify genets. With 26 variable
loci, it was possible to discriminate among a very large
number of genotypes. Because of the dominant nature of
RAPD markers, the maximum number of detectable
genotypes is 226, as there are 26 variable loci each with
two alleles. Thus, although marker frequencies varied
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
727
among loci, it must be considered highly unlikely that,
among the 389 investigated plants, identical RAPD
pro®les would be found in two or more individuals
unless these were asexually derived from the same
parent, i.e. belong to the same genet. Moreover, in the
case of rare instances of identical RAPD pro®les being
found among otherwise genetically non-identical individuals, genet differences would be underestimated in
the A N O V A . The present approach thus provides a
conservative method of analysing phenotypic differences
among genets in the ®eld.
The results of the present study show that reproductive
characters determining the amount of sexually and
asexually produced propagules are extremely variable
in natural populations of the partly asexual plant Allium
vineale. All of the examined allocation traits were characterized by very large coef®cients of variation, often
exceeding 100%. Moreover, phenotypic variance components attributable to genetic differences played a more
in¯uential role than those more likely to be caused by
environmental factors. Most importantly, genetic variation within sites made a substantial contribution to the
overall variation in the allocation traits in A. vineale: the
component of variation as a result of differences among
genets within sites was often the largest single variance
component (Table 2).
Differences among sites also contributed markedly to
the phenotypic variation for all allocation traits (Table 2),
a situation that may be caused either by consistent
environmental differences among sites or by genetic
divergence (or by a combination of both factors). Site
differences as well as site ranks were preserved when
bulbil offspring from the various sites were grown under
uniform conditions in the greenhouse. Although this
observation is compatible with a genetic basis for site
divergence, it may also be caused by non-genetic maternal effects, a phenomenon which may be particularly
5 important in clonal plants (Schwaegerle et al., 2000).
However, Ronsheim & Bever (2000) maintained that
maternal effects do not appear to in¯uence clonal parentoffspring similarities in reproductive characters in A. vineale. As for the cause of site differentiation, selection has
been shown to have promoted population divergence in
life-history and reproductive characters in some plant
species (e.g. Schemske, 1984; Kelly, 1992). The data in
this study do not, however, allow for any such conclusions to be drawn regarding the mechanism behind the
genetic divergence among sites.
Although differences among genets were important in
shaping the overall pattern of variation in reproductive
allocation traits, there was considerable heterogeneity
among sites in any given year, as well as among years at
any given site, in the amount of genetic variation detected.
For example, at site SE3 in 1997, 61% of the variation in
bulbil number was explained by among-genet variation,
whereas at site SE4 in the same year, there was no
detectable among-genet variation for this trait (Table 3).
728
A. CEPLITIS
Likewise, in 1997 at site SE1, 72% of the variation in
¯ower number could be accounted for by variation among
genets, whereas in 1998 this trait did not show any
variation among genets at the same site (Table 3).
Genotype±environment (G±E) interactions may cause
differences among environments in the amount of genetic
variation that is found for a trait (Mazer & Schick, 1991).
Consequently, G±E interactions could explain the ®nding
that the magnitude of the among-genet variance components of the allocation traits varied from one year to
another (Table 3). However, the lack of a signi®cant yearby-genet-within-site effect in the overall variance component analysis (Table 2) suggests that G±E interactions
are of little importance for the variation in allocation traits
in A. vineale. This is true at least on the scale on which
observations were made; it cannot be ruled out that
within-site spatial heterogeneity contributes to some
extent to the variation in these traits. At the same time,
it must be remembered that every year a somewhat
different set of genets was detected in the samples from
each of the sites, and that these genets may not all be
equally different. Therefore, it is possible that in some
years, genets with very similar phenotypes were found,
whereas in other years, a different collection of genets,
more phenotypically diverse, was present in the sample.
In fact, the correlation between phenotypic and genetic
similarity indicates that closely related genets show a
higher phenotypic resemblance than more distantly
related ones. These results suggest that the changes in
genet composition that were apparent at all sites also
affected the magnitude of genet differences in allocation
traits. It deserves to be mentioned here that the apparent
rapid turn-over of low-frequency genets that was
observed at all sites probably results from the procedure
used to collect the material. Each year, only reproducing
plants (i.e. plants bearing an in¯orescence) were collected. Richens (1947) reported that, typically, in any given
year only 30% of the plants in a population produce an
in¯orescence. Presumably, the commoner genets have a
wider age distribution where at least some of the
individuals ¯ower each year, whereas most or all individuals of the rare genets may ¯ower in the same year.
In contrast to genetic effects, seasonal environmental
¯uctuations do not seem to in¯uence the variability of
allocation traits to any appreciable extent. This is indicated by the small and non-signi®cant variance component attributable to differences among years (Table 2).
On the other hand, seed production exhibited a large and
highly signi®cant among-year variance component
accounting for a third of the total variation (Table 2).
This is not surprising as seed-set is known to be strongly
in¯uenced by environmental conditions such as pollen
and resource limitation (Lee, 1988). For example, the
summer of 1998 was exceptionally wet and cold in
Sweden, circumstances that probably had a negative
effect on the abundance of insect pollinators resulting in
the low levels of seed-set that were observed at the
A. vineale sites that year (Supplementary material).
Hence, the large among-year variance component for
seed-set presumably is a product of ¯uctuations in
environmental conditions over years. Information on
genet identity was available for one of the years (1996)
only. This precluded a complete partitioning of variation,
analogous to that of the allocation traits. However, no
difference in seed production among genets in the 1996
6 sample was found. Thus, whereas environmental factors
contribute signi®cantly to the ef®ciency of sexual reproduction ± in terms of the number of seeds per ¯ower ±
genetic factors play an important role in determining the
amount of sexually produced offspring as a result of the
strong correlation between ¯ower number and total seed
number. In conclusion, the results of the present study
demonstrate that, in A. vineale, the exact proportions of
sexually and asexually produced offspring appear to be
the outcome of a complex interplay between genes and
environment, and that genetic variation for traits directly
in¯uencing this proportion exists under ®eld conditions.
Given that sexual reproduction should be strongly
disfavoured because of its inherent `cost', how can the
observed genetic variation for the balance between sex
and asex be maintained in A. vineale?
The expected evolutionary fate for a species or population polymorphic for reproductive mode is that, when
®tness values are constant, either mode of reproduction
will go to ®xation (Joshi & Moody, 1995), depending on
the relative ®tness of sexually and asexually produced
offspring. It was pointed out by Maynard Smith (1978)
that, in such species, sexually and asexually produced
offspring are often adapted to different ecological situations, so that a mixed mode of reproduction might be
maintained irrespective of any costs or bene®ts of sex
itself. Among plants, for example, many species reproduce sexually by seed and asexually by some mode of
vegetative propagation, e.g. stolons, rhizomes, or ± as in
A. vineale ± bulbils. Seeds often differ from such structures
in their dispersal abilities, dormancy patterns, and
potential for transmitting viruses to offspring
(e.g. Silander, 1985; Richards, 1986; Clay & van der
Putten, 1999), and these differences may account for the
maintenance of a balance between reproductive modes.
Similar mechanisms may favour cyclical parthenogenesis
in organisms such as aphids and Daphnia, where sexually
produced eggs are a necessary means of survival during
adverse environmental circumstances (Hebert, 1987;
Moran, 1992). Recently, Burt (2000) argued that different ecological adaptations of sexual and asexual reproduction are indeed `necessary for the stable persistence of
both in a single life-cycle'. More formal analyses of the
evolution of reproductive mode in partly asexual organisms have con®rmed this presumption, provided that
selection alternatively favours sexually or asexually
produced offspring, or that there are large enough
temporal variation in sexual vs. asexual fecundity (Rispe
& Pierre, 1998; Bengtsson & Ceplitis, 2000).
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
Reproductive variation in Allium vineale
Signi®cant ecological differences between seed and
bulbils could thus explain the persistence of a mixed
reproductive system in A. vineale. However, Ronsheim
(1994) found differences in ecological characteristics,
such as dispersal pattern and viability, between seeds and
bulbils of A. vineale to be small and concluded that the
crucial difference between seeds and bulbils necessary
for the maintenance of both modes of reproduction
would probably be genetic rather than ecological.
Although other unknown factors may in¯uence the
®tness of seed- and bulbil-produced offspring in
A. vineale, the large between-year variation in seed
production detected in the present study may bring
about ¯uctuations in sexual and asexual fecundity of a
suf®cient magnitude to keep the two reproductive modes
in a balance. This is predicted by the model of Bengtsson
& Ceplitis (2000): Given that a bulbil, on average, is as
expensive to produce as a ¯ower and that pollen
production increases linearly with ¯ower proportion,
then the inverse of the average number of seeds per
¯ower can be viewed as the relative `®tness' of bulbils (in
this way an average of 0.5 seeds per ¯ower would give
bulbils a relative ®tness of 2, etc.). Under these assumptions, Bengtsson & Ceplitis (2000; p. 419) derive the
following condition for the maintenance of a mixed
reproductive system:
H…v† < 1=2 < E…v† ÿ 1=2;
where v denotes the relative ®tness of bulbils and H and E
are the harmonic and arithmetic means, respectively. In
the present study, H(v) ˆ 0.28 and E(v) ± 1/2 ˆ 0.86
(Supplementary material), thus satisfying the condition
above. While, for the present situation, this scenario
makes assumptions of unknown validity and fails to take
several other potentially important factors into account,
it nevertheless suggests that even in the absence of any
®tness differences between seeds and bulbils in A. vineale,
a mixed reproductive system may still be favoured
because of among-year ¯uctuations in sexual and asexual
fecundity. In other words, the considerable between-year
variation in seed production will cause sexual reproduction (by seed) to be more favourable when the number of
seeds per ¯ower is high, and asexual reproduction (by
bulbils) to be more ef®cient when only low numbers of
seeds are produced. Thus, A. vineale populations may
constantly be tracking a shifting optimal balance, leading
to a situation where genetic variation for a mixed mode
of reproduction is maintained over evolutionary time.
Acknowledgments
I wish to thank B. O. Bengtsson, S. Andersson, and
H. Andersson, for critical readings of the manuscripts and
many helpful comments. The research was ®nancially
supported by the JoÈrgen LindstroÈm fund, and by a grant
from the Swedish Natural Science Research Council to
B. O. Bengtsson.
J. EVOL. BIOL. 14 (2001) 721±730 ã 2001 BLACKWELL SCIENCE LTD
729
Supplementary material
The following material is available from http://blackwellscience.com/products/journals/suppmat/JEB/JEB330/JEB
330sm.htm
Table S1 Means and standard deviations (within
parentheses) for the number of ¯owers, the number of
bulbils, the proportion of ¯owers and the average
number of seeds per ¯ower in individual in¯orescences
of A. vineale plants from ®ve sites in southern Sweden
during four years.
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7 Received 23 July 2001; accepted 3 August 2001
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