Pollen source affects female reproductive success and

Plant Species Biology (2011) 26, 244–253
doi: 10.1111/j.1442-1984.2011.00326.x
Pollen source affects female reproductive success and
early offspring traits in the rare endemic plant
Polemonium vanbruntiae (Polemoniaceae)
psbi_326
244..253
LAURA HILL BERMINGHAM* and ALISON K. BRODY†
Departments of *Plant Biology and †Biology, University of Vermont, 63 Carrigan Dr., 111 Jeffords Hall, Burlington, VT 05405
USA
Abstract
Understanding the relative magnitudes of inbreeding and outbreeding depression in rare
plant populations is increasingly important for effective management strategies. There
may be positive and negative effects of crossing individuals in fragmented populations.
Conservation strategies may include introducing new genetic material into rare plant
populations, which may be beneficial or detrimental based on whether hybrid offspring
are of increased or decreased quality. Thus, it is important to determine the effects of
pollen source on offspring fitness in rare plants. We established pollen crosses (i.e.
geitonogamous-self, autonomous-self, intrasite-outcross, intersite-outcross and openpollinated controls) to determine the effects of pollen source on fitness (seeds/fruit and
seed mass) and early offspring traits (probability of germination, number of leaves, leaf
area and seedling height) in the rare plant Polemonium vanbruntiae. Open-pollinated,
intrasite-outcross and geitonogamous-self treatments did not differ in fitness. However,
plants receiving autonomous-self pollen had the lowest fitness and the lowest probability
of seed germination. Intersite-outcross plants contained fewer seeds/fruit, but seeds germinated at higher frequencies and seedlings were more vigorous. We also detected
heterosis at the seed germination stage. These data may imply that natural populations of
P. vanbruntiae exhibit low genetic variation and little gene flow. Evidence suggests that
deleterious alleles were not responsible for reduced germination; rather environmental
factors, dichogamy, herkogamy and/or lack of competition among pollen grains may have
caused low germinability in selfed offspring. Although self-pollination may provide
some reproductive assurance in P. vanbruntiae, the result is a reduction in germination
and size-related early traits for selfed offspring.
Keywords: heterosis, inbreeding depression, outbreeding depression, rare plant, relative
performance.
Received 23 May 2010; revision received 7 September 2010; accepted 30 December 2010
Introduction
Rare plants existing within fragmented populations may
be exposed to higher rates of self-fertilization or mating
with close relatives, which may result in a reduction in
plant fitness because of inbreeding depression (Buza et al.
2000; Kéry et al. 2000). Inbreeding depression is maintained in natural populations as generations of inbreeding
or mating with close relatives reveal deleterious recessive
alleles (Charlesworth & Charlesworth 1987). Inbreeding
Correspondence: Laura Hill Bermingham
Email: [email protected]
depression may decrease if deleterious alleles are purged
over generations of inbreeding (Ellstrand & Elam 1993).
However, plant populations that have been recently isolated or reduced in size may not have sufficient time to
reduce the frequency of deleterious alleles by genetic
purging. If inbreeding depression leads to a reduction in
plant fitness, populations may experience an increased
risk of extinction, which may be particularly detrimental
for threatened and endangered species (Barrett & Kohn
1991; Ellstrand & Elam 1993; Frankham 2005).
The relative intensity of inbreeding depression depends
on the level of gene flow among populations, which is
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
POLLEN SOURCE AFFECTS RARE PLANTS
influenced by both the plant’s mating system and dispersal ability (Hamrick et al. 1979). If the genetic distance
among fragmented populations is large and dispersal
among populations is low, a decrease in fitness may occur
if distant populations are crossed (i.e. outbreeding
depression; Price & Waser 1979; Waser 1993). However,
if heterozygous individuals exhibit a fitness advantage
over homozygotes (‘overdominance’; Lande & Schemske
1985), then hybridization between fragmented populations may rescue a population from inbreeding depression and genetic drift by introducing new genetic material
(Lynch 1991; Hufford & Mazer 2003). Highly selfcompatible species with low interpopulation gene flow
are expected to exhibit higher frequencies of outbreeding
depression when compared with self-incompatible, welldispersed species (Dudash & Fenster 2000). However,
most species and populations fall somewhere in between
these two stable evolutionary endpoints depending on the
relative performance of selfed versus outcrossed progeny
(Eckert & Barrett 1994).
In the present study, we examined whether seed production and offspring performance differed between
selfed and outcrossed progeny in the globally threatened
perennial plant Polemonium vanbruntiae Britton (Eastern
Jacob’s ladder, Polemoniaceae). Populations of P. vanbruntiae may suffer from both inbreeding and outbreeding
depression because of its low dispersal ability and mixedmating system, including the capacity for self-fertilization
and clonal reproduction (Hill et al. 2008). Clonal reproduction occurs in P. vanbruntiae with vegetative shoots developing from a thick underground rhizome. In natural
populations, P. vanbruntiae clonal fragments contain an
average of two ramets/clonal fragments, although variation is high (range: 1–22 ramets/clonal fragments; Hill
Bermingham 2010).
Self-fertilization in P. vanbruntiae can result from both
within-flower (autogamous) and between-flower (geitonogamous) pollen transfer (Herlihy & Eckert 2002).
Within-flower selfing may be either spontaneous
(‘autonomy’) or pollinator-facilitated (‘facilitated autogamy’), but geitonogamous pollination always requires a
pollen vector (Richards 1986; Schoen & Lloyd 1992).
When pollen is not limiting, autonomous selfing can
result in pollen discounting (i.e. the reduction of pollen
available for outcrossing, sensu Harder & Wilson 1998).
However, if populations are limited by pollen, only
autonomous selfing provides reproductive assurance,
thus allowing offspring to be produced even with little to
no pollinator service (Baker 1955; Stebbins 1957; Charnov
1982; Schoen & Brown 1991; Lloyd 1992). The capacity for
autonomous selfing may be adaptive for rare plant populations, such as P. vanbruntiae, which exist in small, fragmented populations that may receive stochastic pollinator
service (Kennedy & Elle 2008).
Plant Species Biology 26, 244–253
245
In contrast, geitonogamous selfing may cause pollen
discounting and offers no reproductive assurance for
populations experiencing pollinator scarcity (Goodwillie
et al. 2005). However, geitonogamous selfing may be an
inevitable consequence of plants producing large floral
displays to attract pollinators (de Jong et al. 1993). Geitonogamy may be particularly widespread in clonal plants,
such as P. vanbruntiae, which produce multiple flowering
ramets of the same genet (Harder & Barrett 1995). Sex
allocation theory posits that increased plant size and large
floral displays should increase male reproductive success
(i.e. pollen deposition) and that these fitness gains will
decrease with plant size (Charnov 1982). However, female
reproductive success (i.e. seed production) may decrease
with plant size if geitonogamously produced offspring
are of lesser quality (de Jong et al. 1993). It is important to
discern the effects of geitonogamous selfing on offspring
fitness to predict patterns of selection on plant reproductive and life-history traits, as well as determine whether
the relative performance of offspring differs between
selfed and outcrossed progeny.
To determine whether the pollen source affects the relative performance of P. vanbruntiae offspring, we experimentally applied five pollen crosses (autonomous-self,
geitonogamous-self, intrasite-outcross, intersite-outcross
and open-pollinated controls) to treatment plants in
natural populations and to plants propagated in growth
chambers. A series of plants were hand-pollinated in the
laboratory in growth chambers because of concerns that
intersite crosses would result in outbreeding depression
and negatively impact natural populations. We compared
female reproductive fitness (seed production, seed
mass and germination) among all pollen crosses. In addition, we compared offspring vigor (leaf number, leaf size
and seedling height) between intersite-outcross and
geitonogamous-self progeny.
We asked three main questions: (i) is there evidence for
inbreeding depression in natural populations of the rare
plant P. vanbruntiae (if inbreeding depression occurs, is it
evident in terms of a reduction in female reproductive
fitness, offspring vigor or both); (ii) does delivering outcross pollen from a distant population increase female
reproductive success and/or offspring performance compared with plants receiving outcross pollen from the same
population and plants receiving self-pollen; and (iii) do
intersite-outcross offspring exhibit heterosis for early lifehistory traits?
Materials and methods
Study species
Polemonium vanbruntiae Britton (Eastern Jacob’s ladder) is
a globally threatened clonal perennial plant in the Phlox
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
246 L . H . B E R M I N G H A M A N D A . K . B R O D Y
family (Polemoniaceae). This species has a global conservation rank of G3 (‘vulnerable to extirpation or extinction’;
NatureServe 2010), and fewer than 100 populations occur
in eastern North America (Vermont Non-game and
Natural Heritage Program, Vermont Department of Fish
and Wildlife 2001). Polemonium vanbruntiae is narrowly
endemic to cool, mountainous, wetland regions containing calcareous basal till soils with acidic to circumneutral
pH in eastern North America (Deller 2002; Hill Bermingham 2010).
Autonomous selfing in P. vanbruntiae may be limited
because of both spatial separation of anthers and stigma
(herkogamy) and temporal separation of male and female
function (dichogamy). Because P. vanbruntiae reproduces
clonally, geitonogamy may occur among ramets of a
genet. Refer to Hill et al. (2008) for more detailed information on the reproductive biology of P. vanbruntiae.
Pollination experiments
We collected seeds from 70 plants in two Vermont populations, Forest Road 233 (FR) and Abbey Pond (AP),
located in the Green Mountain National Forest in central
Vermont, USA, in September 2004. We collected ripened
fruits from 30 plants at site FR (1424 seeds) and from 40
plants at site AP (6019 seeds). We kept 10% of the seeds for
the pollination experiments and returned the remaining
seeds to each respective population and passively dispersed the seeds around the maternal plant. The seeds for
the pollination experiments were randomly mixed and
germinated in the laboratory in December 2004. Eleven
plants produced flowers in March 2006 and these were
used for hand-pollination treatments in the laboratory. In
addition, in June 2006, we conducted hand-pollinations in
a natural population located in Lordsland Preserve (LP) in
Otsego County, New York. Precise GPS coordinates for
the site locations are not provided for conservation
reasons.
To examine the effects of pollen source on seed production and early offspring traits, we haphazardly selected
118 treatment plants throughout the entire LP population
in 2006. We randomly assigned individual flowers of a
single plant to one of four pollination treatments: (i)
geitonogamous-self-bagged, emasculated, self-pollinated
by hand with a mixture of within-plant self-pollen; (ii)
autonomous-self-bagged, no emasculation, no handpollination; (iii) intrasite-outcross-bagged, emasculated,
hand-pollinated with pollen from another flower on a
flowering stem at least 8 m away; and (iv) open-pollinated
controls. All four pollination treatments were applied to
flowers within a plant. We carried out the pollination
treatments twice per week on all open female-phase
flowers throughout the blooming period. If stigmas of the
treatment flowers were still receptive on the next visit,
pollen was again applied. This ensured that the majority
of flowers received a pollen treatment, as P. vanbruntiae
flowers remain receptive for an average of 3–5 days (Hill
et al. 2008).
For plants grown in the laboratory in growth chambers,
we carried out all of the aforementioned pollen crosses,
with the exception of open-pollinated, but also included
intersite-outcross hand-pollinations. Intersite crosses
were only carried out in the laboratory because of stipulations outlined in the Vermont Threatened and Endangered Species permit. We carried out intersite crosses by
collecting pollen on a paintbrush from all flowers dehiscing pollen on a single donor plant from either site AP or
FR. We delivered the outcross pollen to the receptive
stigmas of a plant originating from the other site, resulting
in an AP ¥ FR cross. In both the laboratory and the field
crosses, our design was such that all pollination treatments were applied to each plant. We later collected fruits,
scored them as expanded or not, counted all seeds per
fruit and weighed all seeds in the laboratory.
Effect of pollen source on early offspring traits
We planted seeds from each of the five pollination treatments in germination mix in 4.5 cm pots with six cells per
pot. Each pot contained seeds from a single pollen treatment; the pots were randomly arranged in plastic trays in
the growth chambers. We germinated all seeds from both
laboratory and field treatment plants after a 12-week
stratification period at 4°C in a growth chamber (Model
I-37LXX; Percival Scientific, Perry, IA USA) to examine if
pollen source affected offspring quality. After the 12-week
stratification, we activated the 40 W fluorescent bulbs in
the chambers, which were programmed to run on an
18h/6h light/dark cycle. We humidified the chambers
and kept the air temperature constant at 23°C. We checked
the trays daily and watered the seedlings as needed so that
the soil remained saturated. We randomized the position
of the trays in the chamber weekly throughout the plant
trials. Seedling germination began to occur in March 2007
after 4 days under the lights. We assessed successful germination by emergence of the hypocotyl and calculated
the probability of germination (0/6–6/6). The seedlings
began to open their first true leaves at 16 days, and all
seedlings had true leaves 40 days after germination. We
measured the following variables of all surviving seedlings at 100 days after seedling germination: number of
true leaves, leaf area (mm2) and seedling height (cm).
Data analysis
Differences in female reproductive fitness and offspring traits
We analyzed the data separately for treatment plants that
were pollinated in natural populations (‘field’) from those
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
Plant Species Biology 26, 244–253
POLLEN SOURCE AFFECTS RARE PLANTS
that were pollinated in the growth chambers (‘lab’). The
pollination treatments carried out in the field included
autonomous-self, geitonogamous-self, intrasite-outcross
and open-pollinated controls, but lacked intersiteoutcross. The pollination treatments carried out in the
growth
chamber
included
autonomous-self,
geitonogamous-self, intrasite-outcross and intersiteoutcross, but lacked open-pollinated controls.
To determine whether seeds/fruit and seed mass differed significantly among pollination crosses, we used
separate linear mixed models. We designated ‘flower’
nested within ‘plant’, both as random factors, and parent
plant height (cm) as the covariate. We used the parent
height of the plants that were pollen receptors as the
covariate to adjust for maternal effects (Becker et al. 2006).
Pollination treatment was the independent variable and a
fixed factor. We used a Bonferroni adjustment for pairwise
comparisons among all pollen crosses. Because we could
only assess seed production on a per-flower basis, our
analysis does not account for the potential of some flowers
to produce variable numbers of seeds. In addition, the
results must be carefully interpreted as plants may shunt
resources to flowers receiving higher quality pollen, and
those flowers may produce fruits containing higherquality seeds (Mooney & McGraw 2007). To determine the
relationship between female reproductive success (i.e.
seed production) and maternal plant size, we used a
general linear model with plant height as the independent
variable and seeds/fruit as the dependent variable.
We used a Poisson loglinear model to determine
whether the probability of germination (0/6–6/6 germinants) differed significantly among pollination crosses. A
Poisson distribution was chosen because it best fits the
count data. We designated the number of germinants
as the dependent variable and pollination treatment as
the predictor variable. We analyzed the data separately
for differences in germination in laboratory and field
crosses.
A growth-chamber malfunction after seedling germination reduced the sample sizes of seedlings. Thus, we were
only able to compare offspring performance between
intersite-outcross and geitonogamous-self progeny.
Although our offspring sample sizes were reduced, we
were still able to compare later-offspring traits (number of
leaves, leaf area and height) of the seedlings that survived.
All offspring examined for later-offspring performance
traits were the progeny of laboratory-raised plants.
Because offspring performance estimates may be correlated, we initially ran a manova. We assigned treatment as
a fixed factor and offspring height (cm), number of leaves
and leaf area (mm2) as response variables. We also carried
out univariate tests for all response variables following a
significant manova. We carried out the following transformations for offspring performance data to meet anova
Plant Species Biology 26, 244–253
247
assumptions of homoscedascity and normality: squareroot number of leaves and natural log of leaf area and
offspring height.
Heterosis and relative performance of offspring The ‘relative
performance of crosstypes’ (RP) estimate of intersiteoutcross and geitonogamous-self offspring was modified
from the methods of Ågren and Schemske (1993) as Equation 1:
RPi =
[ zo − zs ]
zmax
(1)
where zo is the mean phenotype of intersite-outcross
progeny, zs is the mean performance of geitonogamousself progeny, and zmax = zo when zo > zs and zmax = zs when
zs > zo (Ågren & Schemske 1993; Johnston & Schoen 1994).
ws
equation of
We chose to not use the traditional ∂ = 1 −
wo
inbreeding depression because the relative performance
equation gives equal weight to inbreeding and outbreeding depression, whereas the traditional equation to
estimate ∂ does not (Ågren & Schemske 1993). We also did
not use the traditional estimate of W for the relative performance indices because we did not follow offspring to
flowering, and we therefore lack a complete estimate of
offspring fitness (W) of different crosstypes. The RP index
ranges from -1 to +1, and positive values indicate inbreeding depression.
Similarly, we used the relative performance index of
intrasite and intersite outcross laboratory pollen crosses
as an estimate of outbreeding depression as Equation 2:
RPo =
[ zintra − zinter ]
zmax
(2)
where zintra is the mean performance of intrasite progeny,
zinter is the mean performance of intersite progeny, and
zmax = zintra when zintra > zinter and zmax = zinter when zinter > zintra.
Positive values indicate outbreeding depression.
We used the following equation to examine whether
offspring resulting from crosses between isolated populations exhibited heterosis (i.e. hybrid vigor):
H=
[ zinter − zintra ]
zintra
(3)
The value zintra represents the mean performance of
intrasite progeny and zinter represents the mean performance of intersite progeny. Positive values indicate heterosis (Busch 2006).
We estimated RPi, RPo and H for all early offspring
traits, including seeds/fruit, seed mass, probability of germination and the cumulative-offspring measure (seed
mass ¥ probability of germination). Because all intersiteoutcross pollinations were carried out in the laboratory,
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
248 L . H . B E R M I N G H A M A N D A . K . B R O D Y
we only used geitonogamous-self and intrasite-outcross
treatments carried out in the laboratory for the relative
performance indices. We generated 95% confidence intervals to test whether the RPi, RPo and H indices deviated
significantly from zero.
Our statistical analyses were carried out using the JMP
version 7.0 (SAS 2000) and SPSS version 16.0 statistical
software programs (SPSS 2001).
(a)
Results
Differences in early offspring traits
Pollen source had a significant effect on early offspring
traits for all treatment plants, including those that were
hand-pollinated in the field and in the growth chambers.
The number of seeds per fruit was significantly different
among pollination treatments for plants pollinated in
the field (F4,156 = 4.99, P = 0.0008) and in the growth
chambers (F4,52 = 3.24, P = 0.02). In the field, flowers from
the geitonogamous-self, intrasite-outcross and openpollinated controls set equivalent seeds/fruit, and
autonomous-self flowers set the fewest seeds/fruit
(Fig. 1a). The same trend was observed for the pollen
crosses carried out in the laboratory as autonomouslyselfed flowers set the fewest seeds. However, there was no
statistical difference between autonomous-self, intersiteoutcross or intrasite-outcross flowers in the laboratory
crosses; the only significant difference was between
geitonogamous-self and autonomous-self flowers
(Fig. 1b). There were no significant differences between the
number of seeds/fruit set by plants in natural populations
compared with those raised in a growth chamber
(F1,299 = 0.02, P = 0.88). Parent height in the ancova models
was not significant in any of the analyses and therefore the
results are presented without the covariate. Seed mass was
not different among hand-pollination treatments
(F4,161 = 0.64, P = 0.64). Larger plants, based on plant height,
produced significantly more seeds/fruit than their smaller
counterparts (F1,92 = 11.19, P = 0.001; Fig. 2).
Only the offspring resulting from field-raised parents
exhibited significant differences in probability of germination among the pollination treatments (field: c2 = 8.1,
P = 0.04, d.f. = 3; Fig. 3a; laboratory: c2 = 4.8, P = 0.19,
d.f. = 3; Fig. 3b). Seeds from intersite pollen crosses tended
to have the highest probability of germination, but intersite germination was statistically equivalent to openpollinated, intrasite and geitonogamous seedlings.
Autonomously selfed seeds had the lowest probability of
germination.
Owing to the growth chamber malfunction reducing the
sample sizes of seedlings, we were only able to compare
offspring performance between the laboratory pollinated
intersite-outcross and geitonogamous-self seedlings.
(b)
Fig. 1 Differences in female reproductive success of Polemonium
vanbruntiae among pollination treatments in (a) field-pollinated
plants and (b) laboratory-pollinated plants. The numbers under
the x-axis pollination treatments refer to the sample size (number
of flowers) for each pollination treatment. Error bars represent
the standard error of the mean. Pollination treatments with the
same lowercase letter do not differ significantly.
Nonetheless, we did detect significant differences in later
offspring traits between progeny resulting from outcross
and self pollination. Intersite-outcross seedlings performed significantly better than the geitonogamous-self
seedlings in the number of leaves produced (F1,7 = 17.03,
P = 0.006; Fig. 4a), leaf area (F1,7 = 18.83, P = 0.005; Fig. 4b)
and seedling height (F1,7 = 29.82, P = 0.002; Fig. 4c).
Heterosis and relative performance of offspring
Overall, intersite-outcross offspring were of higher
quality than geitonogamous-self offspring, indicated by
positive RPi values for seed mass, probability of germina-
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
Plant Species Biology 26, 244–253
POLLEN SOURCE AFFECTS RARE PLANTS
249
(a)
Fig. 2 Female reproductive success as a function of plant size
(r2 = 0.11).
(b)
(c)
Fig. 3 Probability of germination of Polemonium vanbruntiae
among pollination treatments in (a) field-pollinated plants and
(b) laboratory-pollinated plants. Error bars represent the standard error of the mean. Pollination treatments with the same
lowercase letter do not differ significantly.
tion and the cumulative measure of offspring quality
(Fig. 5a). Yet, RPi was significantly greater than zero for
germination (RPi = 0.11) and cumulative offspring quality
(RPi = 0.11), but was not significant for seed mass (i.e. 95%
confidence intervals overlapped zero).
Plant Species Biology 26, 244–253
Fig. 4 Comparison of early offspring traits of Polemonium vanbruntiae seedlings resulting from intersite-outcross and
geitonogamous-self pollination treatments. (a) Number of true
leaves, (b) leaf area and (c) seedling height. Error bars represent
the standard error of the mean.
Intrasite-outcross plants produced more and larger offspring than intersite-outcross treatment plants, as indicated by the significant RPo index for seeds/fruit and seed
mass (seeds/fruit: RPo = 0.21; seed mass: RPo = 0.13;
Fig. 5b). The RPo index was significantly less than zero for
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
250 L . H . B E R M I N G H A M A N D A . K . B R O D Y
germination (RPo = –0.16), implying that intersite offspring germinated at a higher rate than intrasite-outcross
offspring. This trend is again observed as significant heterosis for intersite-outcross offspring germination
(Fig. 5c). The cumulative measure of offspring quality for
heterosis was not significantly different from zero
(H = 0.07); thus, heterosis is only significant at the seedling germination stage (H = 0.20).
Discussion
Fig. 5 Relative performance (RP) and heterosis (H) estimates of
early offspring traits in the rare plant Polemonium vanbruntiae.
Following the tradition of Ågren and Schemske (1993), RPi is an
estimate for inbreeding depression (a), RPo is an estimate for
outbreeding depression (b) and H is an estimate of heterosis (c).
The cumulative function is the product of seed mass and probability of germination. The RP and H estimates are significant if
the 95% confidence intervals do not overlap zero and if the RP
and H values are greater than zero. Significant values are denoted
by an asterisk.
We found that pollen source has a significant effect on
reproductive success and early offspring traits in P.
vanbruntiae. Open-pollinated, intrasite-outcross and
geitonogamous-self treatments did not differ in reproductive success, which may indicate that natural populations
of P. vanbruntiae exhibit low genetic variation because of
either a lack of gene flow, past population bottlenecks or
both (Charlesworth & Charlesworth 1987). The combination of simultaneously open flowers on a single plant and
clonal reproduction in P. vanbruntiae increases the
probability of geitonogamy as pollinators regularly visit
multiple flowers on the same genetic individual, thus
increasing the selfing rate (de Jong et al. 1992; Eckert
2000). Plants receiving only autonomous-self pollen consistently performed most poorly in terms of the number of
seeds/fruit and germination. Intersite-outcross seedlings
germinated at higher frequencies, had more, larger leaves,
and were taller than offspring produced from
geitonogamous-self pollen. Although self-pollination may
provide some reproductive assurance of offspring production in P. vanbruntiae, the result is a reduction in germination and size-related early offspring traits for
geitonogamously selfed offspring.
Self-fertilization is expected to expose and purge deleterious alleles from populations, therefore reducing
levels of inbreeding depression, particularly at loci affecting early life-history traits (Lande & Schemske 1985; Charlesworth & Charlesworth 1987; Husband & Schemske
1996; Byers & Waller 1999). Yet, in selfing populations, the
mutation rate may be high enough to overcome the effects
of purging and maintain inbreeding depression (Ågren &
Schemske 1993). In fact, deleterious alleles having a small
effect may also become fixed as a result of genetic drift in
small populations. Based on our detection of heterosis at
the germination stage as a result of outcrossing from a
distant site, small, isolated populations of P. vanbruntiae
may contain a substantial mutational load caused by drift
(Crow 1948; Levin 1984; Ouborg & van Treuren 1994;
Paland & Schmid 2003; Busch 2006). It is also plausible
that the clonal nature of P. vanbruntiae has limited the
purging of deleterious alleles that act on early seed traits
(Hill et al. 2007).
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
Plant Species Biology 26, 244–253
POLLEN SOURCE AFFECTS RARE PLANTS
Sex allocation theory (Charnov 1982) speculates that
increased plant size and large floral displays should
increase male reproductive success (i.e. pollen deposition), but female reproductive success (i.e. seed production) may decrease with plant size if geitonogamous-self
offspring are of lesser quality (de Jong et al. 1993). We did
not find significant differences in female reproductive
success between geitonogamous-self and outcross
progeny, but did find that geitonogamously produced offspring are of lesser quality when compared with intersiteoutcross offspring. In addition, smaller plants produce
fewer seeds/fruit; thus, size affects female reproductive
success in P. vanbruntiae. Taken together, these results
may indicate selection against increased plant size and
large floral displays in P. vanbruntiae in order to avoid
inbreeding depression and the production of inferior geitonogamously selfed offspring.
We expect self-fertilization to be maintained in populations of P. vanbruntiae because natural selection will favor
decreases in selfing if the inbreeding depression index is
>0.5 (Byers & Waller 1999). Our estimates of inbreeding
depression fall far below this threshold. Husband and
Schemske (1996) found average inbreeding depression
levels of 0.23 for self-fertilizing species and 0.53 in predominantly outcrossing angiosperms. Polemonium vanbruntiae is fully self-compatible (Hill et al. 2008), so low
levels of inbreeding depression as a result of the purging
of deleterious alleles is to be expected in this rare plant
(Charlesworth & Charlesworth 1987) because the genetic
load has been exposed to selective pressures (Eckert &
Barrett 1994). Inbreeding depression was most prevalent
at the germination stage, which is an important stage in
the life cycle for establishment in natural populations. In
addition, previous studies of P. vanbruntiae and other
clonal species have found that seedling survival and
growth contribute significantly to future population
growth, highlighting the importance of offspring recruitment, establishment and survival in order for rare plant
populations to persist over time (de Kroon et al. 1987;
Kéry et al. 2000; Hill Bermingham 2010).
Although we detected inbreeding depression in the
relative performance index RPi for germination, we must
be cautious in our interpretation of these results. First, we
assessed offspring quality and performance under greenhouse conditions, whereas effects of inbreeding depression are often magnified under field conditions
(Goodwillie et al. 2005). However, field experiments are
inherently risky because of stochastic events that may
obscure differences in offspring performance and underestimate inbreeding depression (Byers & Waller 1999).
Ultimately, though, sowing seeds in a natural environment simulates the actual environment in which the
plants will mature (Schemske, 1983). Second, seeds from
the two self-pollen crosses showed significant differences
Plant Species Biology 26, 244–253
251
in germination; autonomous-self seeds germinated at a
significantly lower rate than geitonogamous-self seeds. If
deleterious alleles were responsible for decreased germinability, then both types of selfing should have low
germination. Instead, the low germination rate for the
autonomously self cross may result from older pollen
because P. vanbruntiae exhibits temporal separation of
male and female function (dichogamy) where the anthers
dehisce pollen prior to the stigma becoming receptive.
When the stigma becomes receptive and can receive
pollen, within-flower pollen is no longer freshly dehisced.
In addition, P. vanbruntiae has spatial separation of
anthers and stigma (herkogamy) where the stigma is
exserted beyond the anthers and corolla (Hill et al. 2008).
For this reason, few pollen grains may be reaching the
receptive stigma without pollinator assistance. Finally,
autonomous pollen was from a single donor, whereas in
the four other crosses pollen was collected from multiple
flowers or donor plants. Young and Stanton (1990)
showed that when pollen is received from multiple
donors and there is competition among pollen grains, the
pollen of superior quality fertilizes the ovules. In general,
the quality of offspring from multiple-donor pollinations
has been shown to be higher than that resulting from
single-donor crosses (Schemske & Paulter 1984; Young &
Stanton 1990), as was shown here with the single-donor
autonomous-self pollen cross.
We detected outbreeding depression for female reproductive success in plants receiving intersite-outcross
pollen. However, these effects may be negligible as plants
receiving outcross pollen from a distant site had a higher
probability of germinating, thus exhibiting heterosis for
the germination stage. Offspring that have a higher germination rate and grow significantly faster are more vigorous
and may outcompete neighboring plants for resources
under natural conditions, thus giving them a selective
advantage. Previous studies in other plant species have
also found increased performance of first generation
hybrids relative to parents; however, hybrid fitness can
decrease in later generations owing to the breakup of
co-adapted gene complexes (Hufford & Mazer 2003).
Our findings corroborate studies examining the offspring quality of long-lived perennial plants with mixed
mating systems existing within small populations. For
example, Mooney and McGraw (2007) examined offspring quality in self and cross-pollinated populations of
Panax quinquefolius and found a significant reduction in
leaf area and height in selfed offspring when compared
with outcross offspring. Michaels et al. (2008) detected
substantial inbreeding depression in populations of the
perennial plant Lupinus perennis because self-pollination
reduced seed production, seedling emergence and seedling growth. As in these studies, our experiment did not
follow the offspring to reproduction because of the mul-
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
252 L . H . B E R M I N G H A M A N D A . K . B R O D Y
tiple years it takes for long-lived perennial plants to
flower. This limits our ability to measure the cumulative
effects of inbreeding depression on offspring survival and
fitness to determine whether inbreeding depression poses
significant fitness consequences for P. vanbruntiae.
However, if selfed offspring suffer from a reduction in
survival to reproductive maturity, there could be selection
against self-fertilization in P. vanbruntiae populations
(Eckert & Allen 1997; Morgan et al. 1997).
For plants like P. vanbruntiae with a mixed mating
system, self-pollination can provide reproductive assurance when pollen is scarce. However, even self-fertile
species can have inbreeding depression at various lifehistory stages, as we observed in P. vanbruntiae, which can
result in reduced offspring quality of selfed progeny.
Genetically mixing plant populations has been shown to
lead to substantial levels of heterosis in the survival and
reproduction of interpopulation hybrid offspring (Levin
1984; van Treuren et al. 1993; Heschel & Paige 1995; Paland
& Schmid 2003) and may be an effective conservation
management strategy for P. vanbruntiae populations if
hybrid fitness does not decrease in later generations. Even
so, evidence of inbreeding depression may not warrant
the introduction of genotypes from distant populations,
given the possibility of outbreeding depression in subsequent generations. Overall, our results indicate that
female reproductive success and offspring vigor depend
on the pollen source, and inbreeding depression may pose
a significant threat to the population persistence of P.
vanbruntiae, a globally threatened plant species.
Acknowledgments
We thank the Vermont Experimental Program to Stimulate Competitive Research (EPSCoR) (EPS0236976) for
research funding. Thank you to Connie L. Tedesco for
providing P. vanbruntiae seed for the offspring quality
experiments, and to Sarah A. Friend for assistance in data
collection. We are grateful to the US Forest Service and
Vermont Non-game and Natural Heritage Program for
research permits.
References
Ågren J. & Schemske D. W. (1993) Outcrossing rate and inbreeding depression in two annual monoecious herbs, Begonia
hirsuta and B. semiovata. Evolution 47: 125–135.
Baker H. G. (1955) Self-compatibility and establishment after long
distance dispersal. Evolution 9: 347–349.
Barrett S. C. H. & Kohn J. R. (1991) Genetic and evolutionary
consequences of small population size in plants: implications
for conservation. In: Falk D. A. & Holsinger K. E. (eds). Genetics and Conservation of Rare Plants. Oxford University Press,
New York, NY, pp. 3–30.
Becker U., Reinhold T. & Matthies D. (2006) Effects of pollination
distance on reproduction and offspring performance in
Hypochoeris radicata: experiments with plants from three European regions. Biological Conservation 132: 109–118.
Busch J. W. (2006) Heterosis in an isolated, effectively small, and
self-fertilizing population of the flowering plant Leavenworthia alabamica. Evolution 60: 184–191.
Buza L., Young A. & Thrall P. (2000) Genetic erosion, inbreeding
and reduced fitness in fragmented populations of the endangered tetraploid pea Swainsona recta. Biological Conservation
93: 177–186.
Byers D. L. & Waller D. M. (1999) Do plant population purge their
genetic load? Effects of population size and mating history on
inbreeding depression. Annual Review of Ecology and Systematics 30: 479–513.
Charlesworth D. & Charlesworth B. (1987) Inbreeding depression and its evolutionary consequences. Annual Review of
Ecology and Systematics 18: 237–268.
Charnov E. L. (1982) The Theory of Sex Allocation. Princeton University Press, Princeton, NJ.
Crow J. F. (1948) Alternative hypotheses of hybrid vigor. Genetics
33: 477–487.
Deller M. B. (2002) Polemonium vanbruntiae Britton (Appalachian
Jacob’s ladder) New England Plant Conservation Program
Conservation and Research Plan for U.S. Forest Service
Region 9. New England Wild Flower Society, Framingham,
Massachusetts.
Dudash M. R. & Fenster C. B. (2000) Inbreeding and outbreeding
depression in fragmented populations. In: Young A. & Clarke
G. (eds). Genetics, Demography, and Viability of Fragmented
Populations. Cambridge University Press, Cambridge,
pp. 35–53.
Eckert C. G. (2000) Contributions of autogamy and geitonogamy
to self-fertilization in a mass-flowering clonal plant. Ecology
81: 532–542.
Eckert C. G. & Allen M. (1997) Cryptic self-incompatibility in
tristylous Decodon verticillatus (Lythraceae). American Journal
of Botany 84: 1391–1397.
Eckert C. G. & Barrett S. C. H. (1994) Inbreeding depression in
partially self-fertilizing Decodon verticillatus (Lythraceae):
population-genetic and experimental analyses. Evolution 48:
952–964.
Ellstrand N. C. & Elam D. R. (1993) Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24: 217–242.
Frankham R. (2005) Genetics and extinction. Biological Conservation 126: 131–140.
Goodwillie C., Kalisz S. & Eckert C. G. (2005) The evolutionary
enigma of mixed mating systems in plants: occurrence, theoretical explanations, and empirical evidence. Annual Review of
Ecology and Systematics 36: 47–79.
Hamrick J. L., Linhart Y. B. & Mitton J. B. (1979) Relationships
between life history characteristics and electrophoretically
detectable genetic variation in plants. Annual Review of Ecology
and Systematics 10: 173–200.
Harder L. D. & Barrett S. C. H. (1995) Mating cost of large floral
displays in hermaphrodite plants. Nature 373: 512–515.
Harder L. D. & Wilson W. G. (1998) A clarification of pollen
discounting and its joint effects with inbreeding depression
on mating system evolution. The American Naturalist 152: 684–
695.
Herlihy C. R. & Eckert C. G. (2002) Genetic cost of reproductive
assurance in a self-fertilizing plant. Nature 416: 320–323.
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology
Plant Species Biology 26, 244–253
POLLEN SOURCE AFFECTS RARE PLANTS
Heschel M. S. & Paige K. N. (1995) Inbreeding depression, environmental stress, and population size variation in scarlet gilia
(Ipomopsis aggregata). Conservation Biology 9: 126–133.
Hill Bermingham L. (2010) Deer herbivory and habitat type influence long-term population dynamics of a rare wetland plant.
Plant Ecology 210: 359–378.
Hill L. M., Brody A. K. & Tedesco C. L. (2008) Mating strategies
and pollen limitation in a globally threatened perennial Polemonium vanbruntiae. Acta Oecologica 33: 314–323.
Hill N. M., Myra M. T. D. & Johnston M. O. (2007) Breeding
system and early stage inbreeding depression in a Nova
Scotian population of the global rarity, Sabatia kennedyana
(Gentianaceae). Rhodora 108: 307–328.
Hufford K. M. & Mazer S. J. (2003) Plant ecotypes: genetic differentiation in the age of ecological restoration. Trends in Ecology
& Evolution 18: 147–155.
Husband B. C. & Schemske D. W. (1996) Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50: 54–70.
Johnston M. O. & Schoen D. J. (1994) On the measurement of
inbreeding depression. Evolution 48: 1735–1741.
de Jong T. J., Waser N. M. & Klinkhamer P. G. L. (1993) Geitonogamy: the neglected side of selfing. Trends in Ecology &
Evolution 8: 321–325.
de Jong T. J., Waser N. M., Price M. V. & Ring R. M. (1992) Plant
size, geitonogamy and seed set in Ipomopsis aggregata. Oecologia 89: 310–315.
Kennedy B. F. & Elle E. (2008) The reproductive assurance benefit
of selfing: importance of flower size and population size.
Oecologia 155: 469–477.
Kéry M., Matthies D. & Spillmann H. (2000) Reduced fecundity
and offspring performance in small populations of the declining grassland plants Primula veris and Gentiana lutea. Journal of
Ecology 88: 17–30.
de Kroon H., Plaisier A. & van Groenendael J. (1987) Densitydependent simulation of the population dynamics of a perennial grassland species, Hypochaeris radicata. Oikos 50: 3–12.
Lande R. & Schemske D. W. (1985) The evolution of selffertilization and inbreeding depression in plants. I. Genetic
models. Evolution 39: 24–40.
Levin D. A. (1984) Inbreeding depression and proximitydependent crossing success in Phlox drummondii. Evolution 38:
116–127.
Lloyd D. G. (1992) Self-fertilization and cross-fertilization in
plants II. The selection of self-fertilization. International Journal
of Plant Sciences 153: 370–380.
Lynch M. (1991) The genetic interpretation of inbreeding depression and outbreeding depression. Evolution 45: 622–629.
Michaels H. J., Shi X. J. & Mitchell R. J. (2008) Effects of population size on performance and inbreeding depression in
Lupinus perennis. Oecologia 154: 651–661.
Mooney E. H. & McGraw J. B. (2007) Effects of self-pollination
and outcrossing with cultivated plants in small natural populations of American ginseng, Panax quinquefolius (Araliaceae).
American Journal of Botany 94: 1677–1687.
Plant Species Biology 26, 244–253
253
Morgan M. T., Schoen D. J. & Bataillon T. M. (1997) The evolution
of self-fertilization in perennials. The American Naturalist 150:
618–638.
NatureServe (2010) NatureServe: an Online Encyclopedia of Life,
version 4.4. Association for Biodiversity Information, Arlington, VA. [cited 23 May 2010.] Available from URL: http://
www.natureserve.org/
Ouborg N. J. & van Treuren B. (1994) The significance of genetic
erosion in the process of extinction. IV. Inbreeding load and
heterosis in relation to population size in the mint Salvia pratensis. Evolution 48: 996–1008.
Paland S. & Schmid B. (2003) Population size and the nature of
genetic load in Gentianella germanica. Evolution 57: 2242–2251.
Price M. V. & Waser N. M. (1979) Pollen dispersal and optimal
outcrossing in Delphinium nelsoni. Nature 277: 294–297.
Richards A. J. (1986) Plant Breeding Systems. Allen & Unwin,
London.
SAS (2000) SAS Institute. JMP Statistical Discovery Software,
version 7.0. SAS Institute, Cary.
Schemske D. W. (1983) Relative fitness of selfed and outcrossed
progeny in Gilia achilleifolia (Polemoniaceae). Evolution 37:
292–301.
Schemske D. W. & Paulter L. P. (1984) The effects of pollen composition on fitness components in a neotropical herb. Oecologia 62: 31–36.
Schoen D. J. & Brown A. H. D. (1991) Whole-flower and partflower self-pollination in Glycine clandestina and G. argyrea
and the evolution of autogamy. Evolution 45: 1651–1664.
Schoen D. J. & Lloyd D. G. (1992) Self- and cross-fertilization in
plants. III. Methods for studying modes and functional
aspects of self-fertilization. International Journal of Plant Sciences 153: 381–393.
SPSS (2001) SPSS for Windows, release 11.0.1. SPSS, Chicago.
Stebbins G. L. (1957) Self fertilization and population variability
in the higher plants. The American Naturalist 91: 337–354.
van Treuren R., Bijlsma R., Ouborg N. J. & van Welden W. (1993)
The significance of genetic erosion in the process of extinction.
IV. Inbreeding depression and heterosis effects caused by
selfing and outcrossing in Scabiosa columbaria. Evolution 47:
1669–1680.
Vermont Non-game and Natural Heritage Program, Vermont
Department of Fish and Wildlife (2001) The Biological and Conservation Data System, Rare Species and Significant Natural Community Digital Data Set. Vermont Department of Fish and
Wildlife in Waterbury, VT USA, Waterbury.
Waser N. M. (1993) Population structure, optimal outbreeding,
and assortative mating in angiosperms. In: Thornhill N. W.
(ed.). The Natural History of Inbreeding and Outbreeding, Theoretical and Empirical Perspectives. University of Chicago Press,
Chicago, pp. 173–199.
Young H. J. & Stanton M. L. (1990) Influence of environmental
quality on pollen competitive ability in wild radish. Science
248: 1631–1633.
© 2011 The Authors
Journal compilation © 2011 The Society for the Study of Species Biology