Available online at www.sciencedirect.

com

South African Journal of Botany 77 (2011) 280 – 291

www.elsevier.com/locate/sajb

Relationship between reproductive assurance and mixed mating in perennial

Kosteletzkya virginica
C.-J. Ruan a,⁎, P. Qin b , J.A. Teixeira da Silva c
a
Key Laboratory of Biotechnology & Bio-Resources Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University,
Dalian City, Liaoning 116600, China
b
Halophyte Research Lab, School of Life Sciences, Nanjing University, Nanjing 210093, Jiangsu, China
c
Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
Received 9 March 2010; received in revised form 29 June 2010; accepted 11 August 2010

Abstract

Recent works have shown that mixed mating systems often evolve despite strong inbreeding depression and reproductive assurance, which is
one of the widely accepted explanations for the evolution of selfing. However, there have been few empirical studies on the relationship between
mixed mating and reproductive assurance in perennial plants. In the herbaceous perennial, Kosteletzkya virginica, delayed selfing induced from
context-dependent style curvature offers reproductive assurance, and adverse weather conditions significantly reduce pollinator visitation rates. In
this study, our goals were (i) to experimentally evaluate pollinator failure rate, reproductive assurance, selfing rate and the relationships between
them, and (ii) to measure inbreeding depression across multiple growth seasons. Results indicate that both population selfing rates and
reproductive assurance are significantly and positively correlated with field estimates of pollinator failure rates, and there is a strong relationship
between selfing rates and reproductive assurance. Inbreeding depression across multiple growth seasons ranged from 0.621 to 0.665, and there
were no significant differences among different seasons. Our data demonstrates that a mixed mating system is beneficial because frequent
pollinator failure has allowed reproductive assurance to evolve through delayed selfing which minimizes the risk of seed discounting and is still
advantageous despite high inbreeding depression.
© 2010 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Adult inbreeding depression; Kosteletzkya virginica; Pollinator failure rate; Reproductive assurance; Selfing rate

1. Introduction selfing increases ovule success when seed production is limited

by pollen transfer (reproductive assurance hypothesis) (Darwin,
Mixed mating, in which hermaphrodite plant species repro- 1876; Jain, 1976). Reproductive assurance, in which self-
duce by both self- and cross-fertilization, is frequent in seed plants fertilization ensures seed production when pollinators and/or
(Barrett et al., 1996; Vogler and Kalisz, 2001) and often evolves potential mates are scarce, is one of the most longstanding and
despite strong inbreeding depression (the reduced fitness of self- widely accepted explanations for the evolution of selfing (Baker,
fertilized relative to outcrossed progeny) (Goodwillie et al., 1955; Barrett, 1998; Herlihy and Eckert, 2002; Holsinger, 1996;
2005). The selective advantage of selfing can arise in one of two Jain, 1976; Lloyd, 1992). A substantial proportion of species with
ways: 1) selfing exhibits an intrinsic advantage in pollen intermediate outcrossing (t) (0.2 b t ≤ 0.8 for 42% of 345 species
transmission (automatic selection or cost of outcrossing) in 78 families, Goodwillie et al., 2005) are at least partially
(Fisher, 1941). Genes favoring selfing (mating system modifiers) capable of autonomous selfing, indicating a possible role for
are automatically selected because they benefit from a 50% reproductive assurance in their mixed mating systems. However,
transmission advantage compared to “outcrossing” genes; and 2) a model of the evolution of self-fertilization under a stochastic
pollination environment revealed that the criterion of seed
⁎ Corresponding author. Tel.: +86 411 87656015. maximization under a stochastic environment is not accurate
E-mail address: ruancj@yahoo.com.cn (C.-J. Ruan). from an evolutionary point of view, and the evolutionary stable
0254-6299/$ - see front matter © 2010 SAAB. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.sajb.2010.08.012
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 281

strategies (mixed mating) do not maximize seed production (Chang and Rausher, 1999) and Hibiscus trionum var. trionum
(Cheptou and Schoen, 2007). This model includes pollen (Ramsey et al., 2003). Variation in the level of inbreeding
limitation (reproductive assurance) inspired from Lloyd's depression among maternal families may reflect differences in
(1979) work, extending it to stochastic environments — a variant the history of selfing and opportunities for purging among
of Morgan and Wilson's (2005) model, and considering a families that may be related to variation in floral traits (Ramsey
hermaphroditic annual plant population experiencing inbreeding et al., 2003). Inbreeding depression in adults diminishes the
depression and pollinator limitation. genetic transmission advantage associated with selfing, espe-
Delayed selfing, in which selfing occurs after the opportunity cially in long-lived perennials that experience inbreeding
for receiving outcross pollen has passed (Lloyd, 1979), permits depression over many seasons; however, perennials may evolve
mixed mating and allows plants to reproduce when pollinators to minimize the between-year seed discount, which reduces the
are temporally or spatially variable (Kalisz and Vogler, 2003; cost of reproductive assurance (Morgan et al., 1997). However,
Kalisz et al., 2004; Lloyd and Schoen, 1992; Lloyd, 1992). to date, there have been few empirical studies on the link
Although delayed self-fertilization has been reported in some between evolutionary stable mixed mating strategies and
annuals (e.g., Hibiscus laevis, Klips and Snow, 1997; Collinsia maximum seed production (reproductive assurance), especially
verna, Kalisz et al., 1999; Hibiscus trionum, Ramsey et al., for perennial plants.
2003; and Incarvillea sinensis, Qu et al., 2007) or perennials In the herbaceous perennial, Kosteletzkya virginica, styles
(e.g., Kalmia latifolia, Rathcke and Real, 1993; Crotalaria remain erect if they become pollinated, but if no pollination
micans, Etcheverry et al., 2003; Caulokaempferia coenobialis, occurs, styles gradually curve downward, to establish a contact
Wang et al., 2004; and Holcoglossum amesianum, Liu et al., between the anthers and stigmas, resulting in delayed selfing
2006), it is much more common in annual than in perennial (Ruan et al., 2004). Adverse weather conditions significantly
plants (Barrett et al., 1997; Stebbins, 1950). This association has reduce pollinator visitation rates, and increase the incidence of
been commonly interpreted in terms of an effect of life history style curvature in K. virginica (Ruan et al., 2009a). Delayed
on the mating system (Zhang, 2000). Annual plants have a selfing induced from this context-dependent style curvature
higher reproductive allocation than perennial plants (Bazzaz et offers reproductive assurance under unpredictable pollinator
al., 1987; Silvertown and Dodd, 1997). Natural history environments (Ruan et al., 2008; Ruan et al., 2009a). In
observations suggest associations between the annual habit addition, K. virginica displays mixed mating in response to the
and selfing and between the woody growth form and out- pollinator environment despite high inbreeding depression
crossing. For instance, Barrett et al.'s survey (1997) finds (Ruan et al., 2009b).
significant differences in average selfing rate (s) between 74 To understand the relationship between mating system and
annual species (s = 0.64), 47 herbaceous perennials (s = 0.41), reproductive assurance, in the same two naturalized populations
and 96 woody perennials (s = 0.18). of K. virginica during 2005–2008, we measured adult
Inbreeding depression (δ) is a key force opposing the inbreeding depression by quantifying the performance of selfed
evolution of selfing (Charlesworth and Charlesworth, 1987; and outcrossed progeny across multiple life-cycle stages,
Charlesworth and Charlesworth, 1990; Lande and Schemske, pollination failure rates, reproductive assurances and selfing
1985; Lloyd, 1979). The magnitude of inbreeding depression in rates estimated by AFLP (amplified fragment length polymor-
species with mixed mating will influence the evolution of phism) markers. Based on their variation between populations
mating strategies. The selfing rate of annual Collinsia verna and among years and the relationships between them, we tested
(δ b 0.15) increases when the pollination environment in wild (i) if reproductive assurance will be important in perennials, (ii)
populations necessitates reproductive assurance, mechanistical- if reproductive assurance and delayed selfing will be beneficial
ly linking reproductive assurance to intermediate selfing rates when inbreeding depression is high, and (iii) if plants will rely
through mixed mating (Kalisz et al., 2004). For perennial more on reproductive assurance when pollinator failure is high.
Aquilegia canadensis, the extent of selfing at the population
level (~75%) is unrelated to the magnitude of pollinator failure
(the flowers cannot be pollinated by pollinators), with strong 2. Materials and methods
inbreeding depression (δ = 0.98) (Herlihy and Eckert, 2002).
Holsinger (1991) predicts that selfing can evolve and mixed 2.1. Study species and study area
mating can be maintained regardless of the relative fitness of
selfed and outcrossed progeny. In the 64 species investigated by Kosteletzkya virginica (L.) Presl. (Malvaceae) is native to
Goodwillie et al. (2005), 88% of them exhibited broadly defined eastern salt marshes in North America (Gallagher, 1985). It
mixed mating systems (0.2 ≤ s b 0.8); inbreeding depression of reproduces sexually (not clonally) via fruit capsules that contain
72% of species was ≥ 0.5, with a mean δ of 0.81. However, five ventricles, each with a single ovule. Roots remain alive
there have been few experimental tests of this observation on throughout winter and the perennial root crown can produce
the evolution of mixed mating systems despite strong new stems each year. K. virginica flowers are hermaphroditic,
inbreeding depression. entomophilous, and non-agamospermous (Ruan et al., 2004). In
The difference in the magnitude of inbreeding depression K. virginica, the filaments are united to form a column, termed
among different families has been reported in Hibiscus monadelphous stamens. This column encircles the styles and
moscheutos (Snow and Spira, 1993), Ipomoea purpurea forms a special form of herkogamy, approach herkogamy. In the
282 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291

flowering period, each plant produces 1–25 open flowers daily, selfing (within flowers). In addition, it does not reduce the
and each flower lasts only one day (Ruan et al., 2005a). attractiveness of flowers to pollinators (Ruan et al., 2008). This
The seeds, collected from the Halophyte Biotechnology experiment was repeated three times across the peak flowering
Center of the University of Delaware in the USA in 1992, were period each year (N = 900 flowers per treatment per population
planted in a field plantation at Dafeng (DF) County of per year). At 13:00–15:00, intact flowers were hand outcrossed
Yancheng City of Jiangsu Province, China in 1994. Dafeng with pollen from five to 10 pollen donors located at least 3 m
lies at 119°27′–120°54′E, 32°34′–34°28′N. The annual mean away whereas emasculated flowers received pollen only from
temperature is 14.7 °C, and the annual mean rainfall is 900– natural pollinator visits (open-pollinated). Fruit set was the
1060 mm. It has a 230-day non-frost period with an annual total number of flowers that produced fruit per 100 flowers. The
radiation of 2241–2496 h. The main soil type is sullage-puddle number of seeds per capsule was calculated. Pollinator failure
soil, with 0.2–1.0% organic matter, and 0.5–1.2% salinity and rate was calculated by the following formula: 1 − (% fruit set of
varies with season (higher in spring and in autumn, and lower in emasculated flowers/% fruit set of hand outcrossed intact
summer). There were approximately 100,000 individuals in the flowers) (Kalisz et al., 2004). The annual population pollinator
naturalized DF K. virginica population in 2005 and the mean failure rate was calculated by averaging nine values [three
plant density was 18 ± 1.2 plants/m2 (n = 25). Selfing rates of values from 300 paired flowers (one value per 100 paired
populations of annual C. verna directly respond to the pollinator flowers) for each of three time periods] during the peak
environment (Kalisz et al., 2004). For K. virginica, adverse flowering time each year (July–September for 2005 and 2006,
weather conditions significantly reduced pollinator visitation June–August for 2007 and 2008). We estimated variation in
rates, and increased the incidence of style curvature, which pollinator failure rate between two populations and among years
resulted in delayed selfing (Ruan et al., 2009a). Hence, to using one-way ANOVA followed by the contrast test, and tested
effectively test the influence of pollination conditions on the the interaction of population and year using two-way ANOVA.
mating system, we collected approximately 20,000 seeds from We estimated the reproductive assurance of delayed selfing
the DF site in 2004 and sowed them in the Dalian (DL) site in in the DF and DL naturalized populations of K. virginica in
spring 2005 where the weather conditions are obviously 2005, 2006, 2007 and 2008. We randomly selected 80 pairs of
different from those of the DF site. In autumn 2005, we gained plants in each population each year. Members of each pair were
over 16,000 plants in the DL site, and these constituted its located 3 m apart, with different pairs at least 10 apart. In each
population. The DL site is located in the coastal wetlands of flowering season, we partially emasculated (only the apical 1–3
Dalian City (120°58′–123°31′E, 38°43′–40°10′N), Liaoning cycled stamens) all flowers on one plant per pair to prevent
Province, China. It has a maritime climate with an annual mean delayed selfing. All flowers on the second plant in the pair were
temperature of 10.2 °C, and an annual mean rainfall of left intact. Each flower in the two treatments was monitored. If
658.7 mm. It has 191 frost-free days and a total annual radiation fruit set occurred, the mature fruits were harvested about
of 2764 h. Soil salinity was 0.6–1.7% and varies with season. 5 weeks later, and the number of seeds per capsule was counted.
In our two studied populations, K. virginica was pollinated For every 10 pairs of plants, reproductive assurance (the gain in
by several insects (e.g. bees, wasps, butterflies, moths, etc.), in seed production via delayed selfing) was calculated as the
which common (Apis cerana) and little (A. florae) bees are main difference between the mean seed production of intact flowers
pollinators, and big bees (Bombus speciosus), white butterfly and that of emasculated flowers (Herlihy and Eckert, 2002;
(Pieris rapae) and one species of ants are fluctuating vectors Lloyd, 1992; Schoen and Lloyd, 1992). Reproductive assurance
(Ruan et al., 2005a). for each population per year was calculated by averaging eight
reproductive assurance values. We estimated the variation in
2.2. Pollinator failure rate, reproductive assurance and selfing reproductive assurance between two populations and among
rate years using one-way ANOVA followed by the contrast test, and
tested the interaction of population and year using two-way
In the DF and DL naturalized populations of K. virginica in ANOVA.
2005, 2006, 2007 and 2008, groups of 300 flowers from 300 To determine the selfing rate, all seeds from 60 randomly
different plants (one flower per plant) were partially emascu- chosen plants per population per year were germinated in
lated before anthers dehisced (04:00–04:30) by cutting only the growth chambers with 45 μmol s− 1 m− 2 light, a 12-h photope-
apical 1–3 cycled stamens on the monadelphous column (only riod, 70–80% relative humidity and at 28 °C. As Thompson and
some anthers within a flower were removed), using sharp, Ritland (2006) used AFLP markers to estimate outcrossing rate
sterile scissors. These were paired with groups of 300 intact of herbaceous perennial Townsendia hookeri, in this study we
flowers in 300 selected plants (one flower per plant). This also used AFLP markers to determine annual selfing rate per
emasculation eliminated the possibility of delayed selfing, as population per year, selecting 25–40 maternal plants per
indicated by the lack of seed set in partially emasculated flowers population per year with at least 15 seedlings. Young leaves
that were bagged before one day opening, then, bags were from the apical end of selected maternal plants and their selected
removed after two days flowering (Ruan et al., 2009a). It also seedlings per population were collected. Total genomic DNA
minimized the reproductive impact of complete emasculation was extracted from fresh leaf tissue using the protocol of Ruan
(all anthers within a flower were removed) at the level of et al. (2003): Samples were placed in a mortar with liquid
geitonogamous self-pollination (among flowers) and facilitated nitrogen and ground to a fine power, and then transferred to a
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 283

1.5 ml microcentrifuge tube. To the tube, 800 μl extraction selfed progeny relative to outcrossed progeny (Keller and
buffer (100 mM Tris–HCl (pH = 8.0), 1% PVP–40,000, 2% (w/v) Waller, 2002), using 20 plants that were selfed or outcrossed; 15
CTAB, 25 mM EDTA (pH = 8.0), and 10 mM diethyldithio- progeny from each treatment were grown in a greenhouse
carbamic acid) was added. The tube was then incubated at 65 °C (N = 20 × 15 × 2 = 600 plants).
for a total of 30 min, with one inversion of the tube at 15 min. The At the end of the 2003 growing season, we randomly selected
tube was centrifuged at 10,000 ×g for 10 min at 4 °C. To remove 30 individuals at least 10 m apart at the DF population, and
enzymes and any other contaminating protein, the pellet was randomly collected 10 fruits per plant. In spring of 2004, 10 days
rinsed twice with hydroxybenzene/chloroform/isopentanol after germination, one seedling was randomly chosen from each
(25:24:41, v/v/v). The resulting DNA pellet was washed with of the original 30 individuals until a sample size of 20 parents
75% ethanol, and then the isolated DNA was resuspended in TE was achieved; then seedlings were transplanted into individual
buffer. Typically, 1 μl of leaf DNA was used in a PCR 500-cm3 polystyrene pots containing sand, loam and peat
amplification reaction. The purity and concentration of DNA (1:1:1). Pots were placed in an insect-free greenhouse, watered
samples were assessed by 1% agarose gel electrophoresis regularly, and fertilized every 3 weeks with Hoagland's nutrient
compared to known quantities of calf thymus DNA and by UV- solution (Gamborg and Wetter, 1975). Daily in the flowering
spectrophotometry. period, two flowers on each individual were randomly selected
As suggested by Ruan et al. (2005b), AFLP analysis was and emasculated and then randomly allocated to one of two
performed using Analysis System II (GIBCO-BRL Life pollination treatments: hand-crossing or hand-selfing. Cross-
Technologies, Shanghai, China) according to the manufac- pollinated flowers received mixed pollen from one to four other
turer's protocol. After inactivation (15 min at 72 °C), EcoRI and plants growing in an insect-free greenhouse, but which were
MseI adaptors were ligated to the ends of genomic DNA different from all individuals of 20 selected parents. Self-
restriction fragments. The digested and ligated template DNA pollinated flowers received pollen from the same plant. The
was pre-amplified using EcoRI + A (5′-GACTGCGTAC- average number of flowers per individual was more than 64
CAATTCA-3′) and MseI + C (5′-GATGATGCCTGAG- (ranging from 53 to 86). There were more than 1280 crossed and
TAAC-3′) primers in a total volume of 50 μl containing 5 μl 1280 selfed flowers. Mature fruits were harvested about 5 weeks
ligation mixture (diluted 10-times in TE). Selective amplifica- after pollination and seeds were counted.
tion was performed in a final volume of 50 μl containing 5 μl In spring of 2005, the selfed and crossed seeds were
pre-amplification products (diluted 50-times in TE). The germinated as described above, with three replications for each
selective amplification primer combinations had three addition- family grown. The number of seeds germinated was counted by
al nucleotides at their 3′ end. In all reactions, only the EcoRI the method of Ramsey et al. (2003). Ten days after germination,
primers were 5′-labelled with 33P. Based on the number and 15 selfed and 15 outcrossed seedlings from each family were
quality of polymorphic fragments, 10 primer combinations were randomly chosen and planted into pots as described above. To
selected and selective amplification was carried out on all avoid the effects of micro-environment variation on seedling
samples, including in E + AAC/M + CTC, E + AAG/M + CTG, growth, pots were randomly placed into three separate family
E + AAC/M + CTT, E + ACA/M + CAT, E + AAC/M + CAA, E + trays, each containing five selfed and five crossed progeny;
AAG/M + CAG, E + ACT/M + CAT, E + ACC/M + CAT, E + trays were then assigned to random positions in the glasshouse
ACG/M + CTA, E + AGG/M + CAC, E + AGG/M + CAG, E + and reassigned every 2–3 days. Individuals were managed as
AGG/M + CTA and E + AGG/M + CTG. PCR-amplified products described above. Across a range of K. virginica life-cycle stages
were separated in 6% (w/v) polyacrylamide gels, and visualised from the seedling growth to the fruit mature in 2005, we
by autoradiography. measured the following three traits: percentage survival to
Scoring of AFLP bands was done considering only two flowering, flower number, and seed number per capsule. The
possible alleles: presence (1) or absence (0) of bands. Multilocus number of plants surviving to flowering was recorded. We
(tm) outcrossing rates were estimated using Ritland's MLTR harvested their fruits and counted the number of seeds in each.
(multilocus t and r) software for Windows (version 3.4, written When plants were 200 days old that was time of the end of the
by Kermit Ritland, revised Sept. 2009) (Ritland, 2002). Selfing flowering, we counted the total number of open flowers per
rate (s) was calculated using the following formula: s = 1 − tm. plant. In spring of each year from 2006 to 2008, we investigated
We estimate variation in selfing rate between two populations the number of living roots and whether the root crown could
using one-way ANOVA. produce new stems; then the above three traits were measured.
Linear regression was used to test for relationships between To estimate inbreeding depression for individual maternal
the pollinator failure rate and the selfing rate, between the families we used a subset of the above five fitness traits,
pollinator failure rate and reproductive assurance, and between including seeds per capsule of maternal plant, germination or
the selfing rate and reproductive assurance, using SPSS v. 11.0 survival of perennial root, survival to flowering, number of
software. flowers, and seeds per capsule of F1 progeny. These traits were
chosen because they are related to overall fitness and are
2.3. Adult inbreeding depression probably independent from one another (Ramsey et al., 2003).
Relative self-performance (RP) for each maternal family was
During 2005–2008, we measure inbreeding depression of K. calculated by RP = Ws/WC, where Ws and WC were the mean
virginica across multiple growth seasons, based on the fitness of performance of selfed and crossed progeny, respectively
284 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291

Fig. 1. Selfing rates (SR) (A), pollinator failure rates (PFR) (B) and reproductive assurance (RA) (C) at Dalian (DL) (black bar) and Dafeng (DF) (white bar)
naturalized Kosteletzkya virginica populations during 2005–2008.
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 285

Table 1 the cumulative relative self-performance (Charlesworth and

Variations in pollinator failure rates (PFR) between populations (DL and DF) Charlesworth, 1987), which was calculated for each maternal
and among years (2005, 2006, 2007 and 2008) tested using one-way ANOVA,
and an interaction of years and populations tested using two-way ANOVA.
family as the product of the RP of the above five traits. For the
inbreeding depression of different families in different growth
Variation of PFR between populations using one-way ANOVA analysis
seasons and of the same family across the growth seasons that
Sum of df Mean F P lasted from 2005 to 2008, we conducted descriptive statistics of
squares square
frequencies and descriptives using SPSS v. 11.0 software.
Between groups 0.005 1 0.005 6.413 0.014 As suggested by Ramsey et al. (2003), we estimated the RP
Within groups 0.060 70 0.001
of selfed progeny for all traits by using the means of 20 maternal
Total 0.065 71
individuals and calculated the population level RP. Cumulative
Variation of PFR among years at DL population using one-way ANOVA followed performance (CP) per growth season was calculated as the
by the contrast test product of the RP of the above five fitness traits. Inbreeding
One-way ANOVA depression (δ) per growth season was calculated as 1 − CP. We
Sum of df Mean F P
followed Johnston and Schoen (1994) and transformed the
squares square original data to a logarithmic scale to assess the differences in
Between groups 0.010 3 0.003 15.307 0.000
levels of inbreeding depression among different growth seasons
Within groups 0.007 32 0.000 from 2005 to 2008, using one-way ANOVA.
Total 0.016 35
3. Results
Contrast tests
Contrast Value of Std. error t df P 3.1. Reproductive assurance and selfing rate
contrast
2005 vs. 2006 0.008044 0.0067867 1.185 32 0.245 Pollinator failure rates varied from 0.091 ± 0.002 to 0.174 ±
2005 vs. 2007 − 0.015633 0.0067867 −2.304 32 0.028 0.008 in the DF and DL populations of K. virginica from 2005
2005 vs. 2008 − 0.034522 0.0067867 −5.087 32 0.000
to 2008 (Fig. 1A), with an average of 0.118 ± 0.010. There were
2006 vs. 2007 − 0.023678 0.0067867 −3.489 32 0.001
2006 vs. 2008 − 0.042567 0.0067867 −6.272 32 0.000 significant differences in pollinator failure rate between the two
2007 vs. 2008 − 0.018889 0.0067867 −2.783 32 0.009 populations (P b 0.05); these differences were also found among
years at the DL and DF populations (both P b 0.001), and their
Variation of PFR among years at DF population interaction on pollination failure rate was significant (P b 0.001)
One-way ANOVA (Table 1). In the DL population, a difference in pollinator failure
Sum of df Mean F P rate between the two years was found between 2005 and 2007
squares square or 2008, between 2006 and 2007 or 2008, and between 2007
Between Groups 0.031 3 0.010 25.275 0.000 and 2008 (all P b 0.05), but not between 2005 and 2006
Within Groups 0.013 32 0.000 (P = 0.245) (Table 1). For the DF population, a difference in
Total 0.044 35 pollinator failure rate was not found between 2005 and 2007
(P = 0.378), but was found between 2005 and 2006 or 2008,
Contrast tests
between 2006 and 2007 or 2008, and between 2007 and 2008
Contrast Value of Std. error t df P (all P b 0.05) (Table 1).
contrast
Estimates of the multilocus population outcrossing rate (tm),
2005 vs. 2006 − 0.049622 0.0094836 −5.232 32 0.000 the single-locus population outcrossing rate (ts) and the single-
2005 vs. 2007 0.008478 0.0094836 0.894 32 0.378 locus inbreeding coefficient of maternal parents (F) obtained
2005 vs. 2008 0.030100 0.0094836 3.174 32 0.003
2006 vs. 2007 0.058100 0.0094836 6.126 32 0.000
from MLTR-based AFLP data clearly indicate a mixed mating
2006 vs. 2008 0.079722 0.0094836 8.406 32 0.000 system in the K. virginica open-pollinated breeding populations
2007 vs. 2008 0.021622 0.0094836 2.280 32 0.029 (Table 2). Multilocus estimates did not significantly differ from

An interaction among “years” and “population”

Table 2
Source Type III sum df Mean F P Estimates of the multilocus population outcrossing rate (tm), the single-locus
of squares square population outcrossing rate (ts) and the single-locus inbreeding coefficient of
Corrected model 0.04568 7 0.006526 21.328 0.000 maternal parents (F) based on AFLP data, at the Dalian and Dafeng populations
Intercept 1.001 1 1.001 3272.877 0.000 of Kosteletzkya virginica during 2005–2008, using the MLTR program
Population 0.005478 1 0.005478 17.901 0.000 software.
Year 0.00.919 3 0.001640 5.358 0.002 Item Dalian population Dafeng population
Population * year 0.03529 3 0.01.176 38.439 0.000
Total 1.067 72 2005 2006 2007 2008 2005 2006 2007 2008
tm 0.708 0.753 0.614 0.611 0.647 0.532 0.638 0.731
(Ramsey et al., 2003). The magnitude of inbreeding depression ts 0.693 0.748 0.602 0.596 0.639 0.518 0.627 0.724
(δ values) for each maternal family was calculated as suggested by tm − ts 0.015 0.005 0.012 0.015 0.008 0.014 0.011 0.007
F 0.071 0.053 0.102 0.109 0.090 0.135 0.080 0.049
Ramsey et al. (2003): δ = 1 - Ws/WC, where Ws/WC represented
286 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291

the single-locus estimates (Table 2). Selfing rates varied from Table 4
0.247 to 0.468 in the DL and DF populations of K. virginica Variations in reproductive assurance (RA) between populations (DL and DF)
and among years (2005, 2006, 2007 and 2008) tested using one-way ANOVA,
from 2005 to 2008 (Fig. 1B), with an average of 0.346 ± 0.026. and an interaction of years and populations tested using two-way ANOVA.
There were no significant differences in selfing rate among
Variation of RA between two populations
different seasons following one-way ANOVA (F1, 7 = 0.409,
P = 0.546) (Table 3). Sum of df Mean F P
squares square
Reproductive assurance varied from 0.803 ± 0.002 to 2.108 ±
0.008 in the DF and DL populations of K. virginica from 2005 Between groups 0.302 1 0.302 1.204 0.277
Within groups 15.571 62 0.251
to 2008 (Fig. 1B), with an average of 1.560 ± 0.063. Significant
Total 15.873 63
differences in reproductive assurance were found among
different years at the DF and DL populations (both P b 0.001), Variation of RA among years at DL population using one-way ANOVA followed
but there were none between the two populations (P = 0.277) by the contrast test
(Table 4). There is an interaction between populations and years One-way ANOVA
on reproductive assurance (P = 0.000 b 0.001) (Table 4). In the Sum of df Mean F P
DL population, a difference in reproductive assurance was not squares square
found between 2007 and 2008; but there were significant Between groups 6.451 3 2.150 30.580 0.000
differences in reproductive assurance between 2005 and 2006, Within groups 1.969 28 .070
2007 or 2008, and between 2006 and 2007 or 2008 (all P b 0.05) Total 8.420 31
(Table 4). For the DF population, a difference in reproductive
assurance was not found between 2005 and 2007 (P = 0.621), Contrast tests
but was found between 2005 and 2006 or 2008, between 2006 Contrast Value of Std. error t df P
and 2007 or 2008, and between 2007 and 2008 (all P b 0.05) Contrast
(Table 4). 2005 vs. 2006 0.588875 0.1325884 4.441 28 0.000
Population selfing rates were significantly and positively 2005 vs. 2007 − 0.585875 0.1372421 − 4.269 28 0.000
correlated with field estimates of pollinator failure rates 2005 vs. 2008 − 0.420097 0.1288528 − 3.260 28 0.003
2006 vs. 2007 − 1.174750 0.1372421 − 8.560 28 0.000
(regression, R = 0.949; F1, 7 = 54.658, P = 0.000 b 0.001; linear 2006 vs. 2008 − 1.008972 0.1288528 − 7.830 28 0.000
term: y = 0.04401 + 2.548 x, t = 7.393, P = 0.000; Fig. 2A). A 2007 vs. 2008 0.165778 0.1336366 1.241 28 0.225
positive correlation was also found between reproductive
assurance and pollinator failure rates (regression, R = 0.836; Variation of RA among years at DF population using one-way ANOVA followed
F1, 7 = 13.948, P = 0.01 b 0.05; linear term: y = 0.204 + 13.4 x, by the contrast test
t = 3.735, P = 0.01 b 0.05; Fig. 2B). For the DF naturalized K. Sum of df Mean F P
virginica population, a 22% increase in pollinator failure rate squares square
(0.094 in 2008 vs 0.115 in 2007) led to a 34% increase in the Between groups 3.600 3 1.200 9.461 0.000
selfing rate (0.269 in 2008 vs 0.362 in 2007) and a 38% increase Within groups 3.551 28 .127
Total 7.151 31
in reproductive assurance (1.145 in 2008 vs 1.582 in 2007).
There was a significantly positive correlation between Contrast tests
selfing rates and reproductive assurance (regression,
Contrast Value of Std. error t df P
R = 0.946; F1, 7 = 54.322, P = 0.000 b 0.001; linear term: contrast
y = 0.09655 + 0.159 x, t = 7.371, P = 0.000 b 0.001; Fig. 2C).
2005 vs. 2006 − 0.4287 0.17806 − 2.407 28 0.023
For the DF naturalized K. virginica population, when the selfing 2005 vs. 2007 0.0921 0.18431 0.500 28 0.621
rate increased 33% (0.353 in 2005 vs 0.468 in 2006), the 2005 vs. 2008 0.4890 0.17304 2.826 28 0.009
reproductive assurance increased 26% (1.679 in 2005 vs 2.108 2006 vs. 2007 0.5207 0.18431 2.825 28 0.009
in 2008). 2006 vs. 2008 0.9177 0.17304 5.303 28 0.000
2007 vs. 2008 0.3969 0.17947 2.212 28 0.035
3.2. Adult inbreeding depression An interaction among “years” and “populations”
Source Type III sum df Mean F P
Inbreeding depression values for different families across of squares square
four growth seasons from 2005 to 2008 at the DF naturalized
Corrected model 10.353 7 1.479 15.005 0.000
Intercept 156.264 1 156.264 1585.302 0.000
Population 0.332 1 0.332 3.373 0.072
Table 3 year 0.940 3 0.313 3.178 0.031
The variance in selfing rate between the two populations tested using one-way Population * year 9.111 3 3.037 30.811 0.000
ANOVA. Total 171.616 64
Sum of Squares df Mean square F P
Between Groups 0.002 1 0.002 0.409 0.546 populations of K. virginica ranged from 0.15 to 0.91 (Fig. 3),
Within Groups 0.035 6 0.006 with a normal distribution (one-sample Kolmogorov–Smirnov
Total 0.037 7
Test: Z = 0.803, P = 0.540, Fig. 4). Inbreeding depression of 20
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 287

Fig. 2. Relationships between pollinator failure rates (PFR) and selfing rates (SR) (A), between PFR and reproductive assurance (RA) (B) and between PFR and
RA (C).
288 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291

Fig. 3. Inbreeding depression (ID) for individual families at Dafeng naturalized Kosteletzkya virginica populations across multiple growth seasons from 2005 to 2008.

families varied from 0.32 to 0.91, with a 25.86% coefficient of reproductive assurance to evolve through delayed selfing
variation in 2005, from 0.38 to 0.84, with a 20.94% coefficient which minimizes the risk of seed discounting under a stochastic
of variation in 2006, from 0.15 to 0.86, with a 27.74% pollination environments; (2) delayed selfing in K. virginica is
coefficient of variation in 2007, and from 0.39 to 0.82, with a still advantageous despite high inbreeding depression; and
17.36% coefficient of variation in 2008. The inbreeding (3) there are strong relationships between selfing rate and
depression of individual families did not increase as growth pollinator failure rate, pollinator failure rate and reproductive
age increased from 2005 to 2008 (Fig. 3) and its coefficient of assurance as well as reproductive assurance and selfing rate.
variation across four growth seasons from 2005 to 2008 varied The evolution of a mixed mating may be because it promotes
from 6.89 to 49.83%, with an average of 20.47 ± 2.40%. outcrossing but provides reproductive assurance when pollina-
For the DF naturalized population of K. virginica, mean tors or mates are scarce, combining the advantages of both
inbreeding depression values at the population level varied from reproductive strategies (Becerra and Lloyd, 1992; Cruden and
0.621 ± 0.036 to 0.665 ± 0.026 during 2005 to 2008 (Table 5), Lyon, 1989); but so far, a clear theoretical understanding of the
with an average of 0.637 ± 0.016. There were no significant role of reproductive assurance in the evolution of mixed mating
differences in inbreeding depression among different seasons has not yet emerged, and empirical work lags behind theory
following one-way ANOVA (F3, 79 = 0.352, P = 0.788) (Table 6). (Goodwillie et al., 2005). Delayed autonomous selfing in K.
virginica provides reproductive assurance under unpredictable
4. Discussions pollinator environments. This is because (1) a reduction in
pollinator visitation increased selfing rate, which mainly
Our data demonstrates that (1) a mixed mating system is consists of delayed autonomous selfing, (2) the mean number
beneficial because frequent pollinator failure has allowed of seeds per capsule from intact open-pollinated flowers was
higher than that of partially emasculated open-pollinated
flowers, and (3) delayed selfing in K. virginica occurs just
before corolla closure (Ruan et al., 2004), after which pollen is
unavailable, and un-fertilized ovules cannot be outcross
pollinated; so style curvature-mediated delayed selfing in this
species incurred minimal pollen and seed discounting (Lloyd,
1992; Schoen and Brown, 1991). This evidence satisfy the
criteria to test if autonomous selfing provides reproductive
assurance when pollination is unpredictable (Barrett, 2002,
2003; Herlihy and Eckert, 2002; Kalisz et al., 2004), which
include that plants must fail to receive outcross pollen, but this
failure need not occur every season. During periods of low or no
outcross pollen receipt, autonomous selfing must boost seed
production. Finally, the combined costs (seed discounting,
pollen discounting and inbreeding depression) must not
completely negate the fitness gain of selfing.
A population adapted to a stochastic pollination environment
Fig. 4. Frequency of histogram of inbreeding depression (ID) in Kosteletzkya is one that maximizes seed production over years (Ashman
virginica. et al., 2004); i.e. the best strategy is a mix of outcrossing for
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 289

Table 5
Inbreeding depression (δ) at population level across different growth seasons from 2005 to 2008 in DF naturalized population of Kosteletzkya virginica. Relative self-
performances = RP, cumulative performance = CP.
Growth season (year) 1 (2005) 2 (2006) 3 (2007) 4 (2008)
RP (mean ± SE)
Maternal plant: seeds per capsule 0.996 ± 0.011 0.996 ± 0.011 0.996 ± 0.011 0.996 ± 0.011
Germination (g)/survival of perennial root (sp) 0.734 ± 0.041g 0.833 ± 0.009 sp 0.812 ± 0.035 sp 0.824 ± 0.016 sp
Survival to flowering 0.992 ± 0.003 0.961 ± 0.028 0.943 ± 0.010 0.936 ± 0.008
No. of flowers 0.538 ± 0.037 0.512 ± 0.031 0.548 ± 0.018 0.523 ± 0.026
F1 progeny: seeds per capsule 0.971 ± 0.024 0.915 ± 0.027 0.868 ± 0.033 0.832 ± 0.015
CP (mean ± SE) 0.379 ± 0.032 0.373 ± 0.019 0.363 ± 0.031 0.335 ± 0.024
δ (mean ± SE) 0.621 ± 0.036 0.627 ± 0.029 0.637 ± 0.040 0.665 ± 0.026

good pollination years and selfing for bad ones. For perennial K. inferred that self-fertilization has evolved in many species
virginica, when pollinator visits decrease, intermediate selfing despite strong inbreeding depression. Based on a model to
rates significantly increase through an increase in the proportion predict optimal selfing rates that includes a range of possible
of delayed selfing, which maximizes seed reproduction. In relationships among the three components of reproductive
North America, since K. virginica is perennial and produces fitness, as well as the effects of evolving inbreeding depression
more stems per plant during the first few years (Gallagher, caused by deleterious mutations and of selection on total seed
1985), the population density of this species may be higher; number, Johnston et al. (2009) demonstrated that intermediate
although this species has general pollinators (e.g. bees, selfing is optimal for a wide variety of relationships among
butterflies, hawkmoth, etc.), it displays high frequency style fitness components and that inbreeding depression is not a good
curvature resulting in delayed selfing (Dr. John L. Gallagher predictor of selfing-rate evolution. In A. canadensis, reproduc-
personal communication), which may be because variable tive assurance through self-fertilization increases seed produc-
weather conditions in the salt marshes often bring out an tion (1.77 seeds per carpel), but this benefit is greatly
unpredictable pollinator environment. In our two studied outweighed by severe seed discounting (− 3.13 seeds per
populations, adverse weather conditions significantly reduced carpel) and inbreeding depression (δ = 0.98) (Herlihy and
pollinator visitation rates, and increased the incidence of style Eckert, 2002); furthermore, high autogamy in A. canadensis,
curvature, which resulted in delayed selfing (Ruan et al., which seems disadvantageous, is independent of pollinator
2009a). These indicate, for perennial K. virginica, that failure (Herlihy and Eckert, 2002, 2004). In our study, K.
unpredictable pollinator environments may promote the evolu- virginica presents a mixed mating system (selfing rate ranging
tion of delayed selfing to achieve reproductive assurance when from 0.247 to 0.468) in response to pollinator environments
pollinators are scarce or absent. This is consistent with the despite high inbreeding depression over multiple seasons (δ
results of Kalisz and Vogler (2003) in which autonomous ranged from 0.621 to 0.665), which is significantly greater than
selfing in C. verna, which provides reproductive assurance, is 0.5 (one-sample t-test: t = 13.993, P b 0.01), the theoretical value
adaptive under variable pollinator conditions. Under the poor of inbreeding depression above which the automatic gene
pollination conditions, autonomous selfing has been selected in transmission advantage conveyed by self-fertilization is negated
Roscoea schneideriana (Zingiberaceae) because it provides (Charlesworth and Charlesworth, 1987; Lande and Schemske,
substantial reproductive assurance with very low costs (δ = 0.23) 1985). For Hibiscus trionum var. vesicarius, delayed selfing
(Zhang and Li, 2008). provides considerable mating flexibility, with mean inbreeding
Theory suggests that inbreeding depression, the main depression = 0.64 (Seed et al., 2006).
selective factor opposing the evolution of selfing, can be There was considerable variation in the magnitude of
purged with self-fertilization, a process that is expected to yield inbreeding depression among maternal families, which may
pure strategies of either outcrossing or selfing (Lande and be from the variation in the extent or speed of stylar movement
Schemske, 1985; Schemske and Lande, 1985). In contrast, that influences the occurrence of outcrossing or delayed selfing
based on a review of self-fertilization rates and inbreeding (Ruan et al., 2009a). This difference of among-family
coefficient of 150 plant species, Goodwillie et al. (2005) inbreeding depression was detected in Hibiscus moscheutos,
family Malvaceae (Snow and Spira, 1993), Ipomoea purpurea,
family Convolvulaceae (Chang and Rausher, 1999) and
Table 6 Hibiscus trionum var. trionum, family Malvaceae (Ramsey
The variance of inbreeding depression among growth seasons from 2005 to
2008, using one-way ANOVA.
et al., 2003). On the other hand, inbreeding depression of K.
virginica remains similar across multiple growth seasons.
One-way ANOVA
Although inbreeding depression gradually increased with an
Sum of squares df Mean square F P increase in growth age from 2005 to 2008, this increase is low
Between groups 0.023 3 0.008 0.352 0.788 with a 0.044 increase in range.
Within groups 1.663 76 0.022 However, the theory model of Cheptou and Schoen (2007)
Total 1.686 79
showed that mere observation of a population that appears to be
290 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291

employing selfing for optimizing seed production in the face of Darwin, C., 1876. The Effects of Cross and Self-fertilization in Vegetable
uncertain pollination does not constitute proof of adaptation to Kingdom. John Murray, London.
Eckert, C.G., Herlihy, C.R., 2004. Using a cost-benefit approach to understand
its environment. To better understand the evolution of a mating the evolution of self-fertilization in plants: the perplexing case of Aquilegia
system and reproductive assurance in K. virginica, it will be canadensis (Ranunculaceae). Plant Species Biology 19, 159–173.
necessary to (i) dissect patterns of selfing into individual Etcheverry, A.V., Protomastro, J.J., Westerkamp, C., 2003. Delayed autono-
components (Goodwillie et al., 2005), (ii) integrate demograph- mous self-pollination in the colonizer Crotalaria micans (Fabaceae:
Papilionoideae): structural and functional aspects. Plant Systematics and
ic and genetic approaches to investigate the evolution of mating
Evolution 239, 15–28.
systems (Cheptou and Schoen, 2007), and (iii) manipulate Fisher, R.A., 1941. Average excess and average effect of a gene substitution.
plants in the field to test the effects of reproductive assurance on Annals of Eugenics 11, 53–63.
future survivorship and reproduction (Morgan et al., 1997), Gallagher, J.L., 1985. Halophytic crops for cultivation at seawater salinity. Plant
combining a cost-benefit approach (Eckert and Herlihy, 2004). and Soil 89, 323–336.
Gamborg, O.L., Wetter, L.R., 1975. Plant Tissue Culture Methods. National
Research Council, Saskatoon, Canada.
Acknowledgements Goodwillie, C., Kalisz, S., Eckert, C.G., 2005. The evolutionary enigma of
mixed mating systems in plants: occurrence, theoretical explanations, and
The authors wish to thank Ms. H Li, Y Zhang, Q Wang and empirical evidence. Annual Reviews of Ecology and Systematics 36,
Mr. M W Wang for their field assistance. This work was funded 47–79.
by the National Natural Science Foundation of China (Grant Herlihy, C.R., Eckert, C.G., 2002. Genetic cost of reproductive assurance in a
self-fertilizing plant. Nature 416, 320–323.
no. 30500071 to C.-J. Ruan). Herlihy, C.R., Eckert, C.G., 2004. Experimental dissection of inbreeding and its
adaptive significance in a flowering plant, Aquilegia canadensis (Ranuncu-
laceae). Evolution 58, 2693–2703.
References Holsinger, K.E., 1991. Mass action models of plant mating systems: the
evolutionary stability of mixed mating systems. The American Naturalist
Ashman, T.L., Knight, T.M., Steets, J.A., Amarasekare, P., Burd, M., Campbell, 138, 606–622.
D.R., Dudash, M.R., Johnston, M.O., Mazer, S.J., Mitchell, R.J., Morgan, Holsinger, K.E., 1996. Pollination biology and the evolution of mating systems
M.T., Wilson, W.G., 2004. Pollen limitation of plant reproduction: in flowering plants. Evolutionary Biology 29, 107–149.
ecological and evolutionary causes and consequences. Ecology 85, Jain, S.K., 1976. The evolution of inbreeding in plants. Annual Reviews of
2408–2421. Ecology and Systematics 7, 469–495.
Baker, H.G., 1955. Self-compatibility and establishment after ‘long-distance’ Johnston, M.O., Schoen, D.J., 1994. On the measurement of inbreeding
dispersal. Evolution 9, 347–348. depression. Evolution 48, 1735–1741.
Barrett, S.C.H., 1998. The evolution of mating strategies in flowering plants. Johnston, M.O., Porcher, E., Cheptou, P.-O., Eckert, C.G., Elle, E., Geber, M.A.,
Trends in Plant Science 3, 335–341. Kalisz, S., Kelly, J.K., Moeller, D.A., Vallejo-Marin, M., Winn, A.A., 2009.
Barrett, S.C.H., 2002. The evolution of plant sexual diversity. Nature Reviews. Correlations among fertility components can maintain mixed mating in
Genetics 3, 274–284. plants. The American Naturalist 173, 1–11.
Barrett, S.C.H., 2003. Mating strategies in flowering plants: the outcrossing- Kalisz, S., Vogler, D.W., 2003. Benefits of autonomous selfing under
selfing paradigm and beyond. Philosophical Transactions of the Royal unpredictable pollinator environments. Ecology 84, 2928–2942.
Society of London. Series B: Biological Sciences 358, 991–1004. Kalisz, S., Vogler, D., Fails, B., Finer, M., Shepard, E., Herman, T., Gonzales,
Barrett, S.C.H., Harder, L.D., Worley, A.C., 1996. The comparative biology of R., 1999. The mechanism of delayed selfing in Collinsia verna
pollination and mating in flowering plants. Philosophical Transactions of (Scrophulariaceae). American Journal of Botany 86, 1239–1247.
the Royal Society of London. Series B: Biological Sciences 351, Kalisz, S., Vogler, D.W., Hanley, K.M., 2004. Context-dependent autonomous
1271–1280. self-fertilization yields reproductive assurance and mixed mating. Nature
Barrett, S.C.H., Harder, L.D., Worley, A.C., 1997. The comparative biology of 430, 884–887.
pollination and mating in flowering plants. In: Silvertown, J., Franco, M., Keller, L.F., Waller, D.M., 2002. Inbreeding effects in wild populations. Trends
Harper, J.L. (Eds.), Plant Life Histories: Ecology, Phylogeny and Evolution. in Ecology & Evolution 17, 230–241.
Cambridge University, Cambridge, pp. 57–76. Klips, R.A., Snow, A.A., 1997. Delayed autonomous self-pollination in
Bazzaz, F.A., Chiariello, N.R., Coley, P.D., Pitelka, L.E., 1987. Allocating Hibiscus laevis (Malvaceae). American Journal of Botany 84, 48–53.
resources to reproduction and defense. Bioscience 37, 58–67. Lande, R., Schemske, D.W., 1985. The evolution of self-fertilization and
Becerra, J.X., Lloyd, D.G., 1992. Competition-dependent abscission of self- inbreeding depression in plants. I. Genetic models. Evolution 39, 22–40.
pollinated flowers of Phormium tenax (Agavaceae): a second action of self- Liu, K.W., Liu, Z.J., Huang, L.Q., Li, L.Q., Chen, L.J., Tang, G.D., 2006. Self-
incompatibility at the whole flower level. Evolution 46, 458–469. fertilization strategy in an orchid. Nature 441, 945–946.
Chang, S.M., Rausher, M.D., 1999. The role of inbreeding depression in Lloyd, D.G., 1979. Some reproductive factors affecting the selection of self-
maintaining the mixed mating system of the common morning glory, fertilization in plants. The American Naturalist 113, 67–79.
Ipomoea purpurea. Evolution 53, 1366–1376. Lloyd, D.G., 1992. Self- and cross-fertilization in plants. II. The selection of
Charlesworth, D., Charlesworth, B., 1987. Inbreeding depression and its self-fertilization. International Journal of Plant Sciences 153, 370–380.
evolutionary consequences. Annual Review of Ecology and Systematics 18, Lloyd, D.G., Schoen, D.J., 1992. Self- and cross-fertilization in plants. I.
237–268. Functional dimensions. International Journal of Plant Sciences 153,
Charlesworth, D., Charlesworth, B., 1990. Inbreeding depression with 358–369.
heterozygote advantage and its effect on selection for modifiers changing Morgan, M.T., Wilson, W.G., 2005. Self-fertilization and the escape from
the outcrossing rate. Evolution 44, 870–888. pollen limitation in variable pollination environments. Evolution 59,
Cheptou, P.-O., Schoen, D.J., 2007. Combining population genetics and 1143–1148.
demographical approaches in evolutionary studies of plant mating systems. Morgan, M.T., Schoen, D.J., Bataillon, T.M., 1997. The evolution of self-
Oikos 116, 271–279. fertilization in perennials. The American Naturalist 150, 618–638.
Cruden, R.W., Lyon, D.L., 1989. Facultative xenogamy: examination of a Qu, R., Li, X., Luo, Y., Dong, M., Xu, H., Chen, X., Dafni, A., 2007. Wind-
mixed mating system. In: Bock, J.H., Linhart, Y.B. (Eds.), The evolutionary dragged corolla enhances self-pollination: a new mechanism of delayed self-
Ecology of Plants. Boulder, Co, Westview, pp. 171–207. pollination. Annals of Botany 100, 1155–1164.
 C.-J. Ruan et al. / South African Journal of Botany 77 (2011) 280–291 291

Ramsey, M., Seed, L., Vaughton, G., 2003. Delayed selfing and low levels of Schoen, D.J., Brown, A.H.D., 1991. Whole- and part-flower self pollination in
inbreeding depression in Hibiscus trionum (Malvaceae). Australian Journal Glycine clandestine and G. argyrea and the evolution of autogamy.
of Botany 51, 275–281. Evolution 45, 1665–1674.
Rathcke, B., Real, L., 1993. Autogamy and inbreeding depression in Mountain Schoen, D.J., Lloyd, D.G., 1992. Self- and cross fertilization in plants. III.
Laurel, Kalmia latifolia (Ericaceae). American Journal of Botany 80, Methods for studying modes and functional aspects of self fertilization.
143–146. International Journal of Plant Sciences 153, 381–393.
Ritland, K., 2002. Extensions of models for the estimation of mating systems Seed, L., Vaughton, G., Ramsey, M., 2006. Delayed autonomous selfing and
using n independent loci. Heredity 88, 221–228. inbreeding depression in the Australian annual Hibiscus trionum var.
Ruan, C.J., Qin, P., Han, R.M., 2003. Identification of hybrids of Kosteletzkya vesicarius (Malvaceae). Australian Journal of Botany 54, 27–34.
virginica using RAPD markers. Collection of Thesis of High-tech Forum of Silvertown, J., Dodd, M., 1997. Comparing plants and connecting traits. In:
Marine Organisms, Beijing. Silvertown, J., Franco, M., Harper, J.L. (Eds.), Plant Life Histories: Ecology,
Ruan, C.J., Qin, P., He, Z.X., 2004. Delayed autonomous selfing in Kosteletzkya Phylogeny and Evolution. Cambridge University, Cambridge, pp. 3–16.
virginica (Malvaceae). South African Journal of Botany 70, 640–645. Snow, A.A., Spira, T.P., 1993. Individual variation in the vigor of self pollen
Ruan, C.J., Qin, P., Han, R.M., He, Z.X., 2005a. Identification of hybrids of and selfed progeny in Hibiscus moscheutos (Malvaceae). American Journal
Kosteletzkya virginica using AFLP markers. Journal of Nanjing Forestry of Botany 80, 160–164.
University (Natural Sciences Edition) 29, 20–24. Stebbins, G.L., 1950. Variation and Evolution in Plants. Columbia University,
Ruan, C.J., Qin, P., Xi, Y.G., 2005b. Floral traits and pollination modes in New York.
Kosteletzkya virginica (Malvaceae). Belgian Journal of Botany 138, 39–46. Thompson, S.L., Ritland, K., 2006. A novel mating system analysis for modes
Ruan, C.J., Zhou, L.J., Zeng, F.Y., Han, R.M., Qin, P., Lutts, S., Saad, L., Mahy, of self-oriented mating applied to diploid and polyploidy arctic Easter
G., 2008. Contribution of delayed autonomous selfing to reproductive daisies (Townsendia hookeri). Heredity 97, 119–126.
success in Kosteletzkya virginica. Belgian Journal of Botany 141, 3–10. Vogler, D.W., Kalisz, S., 2001. Sex among the flowers: the distribution of plant
Ruan, C.J., Li, H., Mopper, S., 2009a. Kosteletzkya virginica displays mixed mating systems. Evolution 55, 202–204.
mating system responding to pollinator environment despite strong Wang, Y., Zhang, D., Renner, S.S., Chen, Z., 2004. A new self-pollination
inbreeding depression. Plant Ecology 203, 183–193. mechanism. Nature 431, 39–40.
Ruan, C.J., Mopper, S., Teixeira da Silva, J.A., Qin, P., Shan, Y., 2009b. Zhang, D.Y., 2000. Resource allocation and the evolution of self-fertilization in
Context-dependent style curvature within flowers offers reproductive plants. The American Naturalist 155, 187–199.
assurance under unpredictable pollinator environments. Plant Systematics Zhang, Z.Q., Li, Q.J., 2008. Autonomous selfing provides reproductive
and Evolution 277, 207–215. assurance in an alpine ginger Roscoea schneideriana (Zingiberaceae).
Schemske, D.W., Lande, R., 1985. The evolution of self-fertilization and Annals of Botany 102, 531–538.
inbreeding depression in plants. II. Empirical observations. Evolution 39,
41–52.

Edited by A Pauw