ORIGINAL RESEARCH

published: 24 June 2016

doi: 10.3389/fpls.2016.00874

Invasion Fosters Change:

Independent Evolutionary Shifts in
Reproductive Traits after Oxalis
pes-caprae L. Introduction
Sílvia Castro 1*† , Mariana Castro 1† , Victoria Ferrero 2,3 , Joana Costa 1 , Daniela Tavares 1 ,
Luis Navarro 2 and João Loureiro 1
1
Centre for Functional Ecology and Department of Life Sciences, University of Coimbra, Coimbra, Portugal, 2 Department of
Plant Biology, Faculty of Science, University of Vigo, Vigo, Spain, 3 Department of Ecology and Evolutionary Biology,
University of Toronto, Toronto, ON, Canada

Edited by:
Peter Alpert,
Biological invasions offer optimal scenarios to study evolutionary changes under
University of Massachusetts Amherst, contemporary timescales. After long-distance dispersal, exotic species have to cope
USA with strong mate limitation, and shifts toward uniparental reproduction have been
Reviewed by: hypothesized to be selectively advantageous. Oxalis pes-caprae is a clonal tristylous
Gerhard Karrer,
University of Natural Resources species native to South Africa, and invasive in Mediterranean regions worldwide. It
and Life Sciences Vienna, Austria reproduces sexually and asexually but the importance of each strategy differs between
Mukhtar Ahmed,
Washington State University, USA
ranges. Native populations reproduce mostly sexually while in invasive ones asexual
*Correspondence:
reproduction is the prevailing strategy due to the dominance of pentaploid monomorphic
Sílvia Castro populations. Nevertheless, two contrasting scenarios have been observed after
[email protected] introduction: transition toward clonality, and re-acquisition of sexuality fueled by multiple
† These authors have contributed introductions of compatible mates. Here, we aimed to assess evolutionary changes of
equally to this work.
reproductive traits in O. pes-caprae invasive populations and evaluate whether these
Specialty section: traits could be related with invasion success and prevalence of certain forms in the
This article was submitted to
western Mediterranean basin. Sexual and asexual reproduction traits were quantified
Agroecology and Land Use Systems,
a section of the journal under optimal conditions in a common garden experiment including native and invasive
Frontiers in Plant Science sexual, predominately asexual, and obligated asexual individuals. Different reproductive,
Received: 13 January 2016 ecological, and genetic constraints created by long-distance dispersal seem to have
Accepted: 03 June 2016
Published: 24 June 2016
generated different selective pressures in sexual and asexual traits, with our results
Citation:
supporting evolutionary changes in invasive populations of O. pes-caprae. Native plants
Castro S, Castro M, Ferrero V, Costa had higher sexual fitness, while a transition toward clonality was clear for invasive forms,
J, Tavares D, Navarro L and Loureiro
supporting clonal reproduction as a major trait driving invasion. Differences were also
J (2016) Invasion Fosters Change:
Independent Evolutionary Shifts in observed among invasive plants, with sexual forms having increased dispersal potential;
Reproductive Traits after Oxalis thus, they are expected to be in advantage in comparison with predominantly asexual
pes-caprae L. Introduction.
Front. Plant Sci. 7:874.
and obligated asexual plants, and may become widespread in the future. Historical
doi: 10.3389/fpls.2016.00874 processes, like the initial introduction of predominantly asexual forms followed by sexual

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Castro et al. Shifts in Reproductive Traits after Introduction

forms more recently, could be in the origin of current distribution patterns of O. pes-
caprae in the western Mediterranean. This study shows that invasion processes are
very dynamic and that ecological and genetic constraints determined by the invasion
process may originate different reproductive strategies that are likely to determine
invasion success.
Keywords: clonality, evolution of reproduction, Mediterranean regions, pentaploid, polyploidy, reproductive
strategy, sexual and asexual reproduction, tristyly

INTRODUCTION recombination, thus contributing not only to ameliorate loss

of genetic diversity due to founder events, but also to fuel the
Biological invasions are a serious threat to biodiversity and have opportunities for local adaptation and the ability of colonizers
long been recognized to comprise significant ecological and to respond to unpredictable environmental fluctuations in the
evolutionary consequences, not only for the communities being new range(s) (Eckert, 2002; Novak and Mack, 2005; reviewed in
invaded, but also for the invasive species themselves (Brown and Barrett, 2011). Despite the clear advantages of sexuality, asexual
Eckert, 2005; Barrett et al., 2008; Pyšek et al., 2012; Oduor, 2013). reproduction might be favored under unreliable circumstances,
For these reasons, since the seminal works of Elton (1958) and as it provides reproductive assurance and enables the persistence
Baker and Stebbins (1965), biological invasions have attracted of individuals in unfavorable habitats for sexual reproduction or
much attention of researchers in an attempt to identify traits avoids the costs associated with sexual reproduction, allowing
that might confer an advantage during colonization of new small populations and adaptive genotypes to rapidly establish and
habitats (Pyšek and Richardson, 2007; Hayes and Barry, 2008; van spread (Eckert, 2002; Barrett, 2015; reviewed in Vallejo-Marín
Kleunen et al., 2010) and to understand the biotic and abiotic et al., 2010).
factors that determine invasion success (e.g., Souza et al., 2011; Long-distance dispersal is frequently associated with strong
Wisz et al., 2013). It also became clear that the introduction of founder effects and loss of genetic diversity, thus exposing
sub-populations in new ecological scenarios generates valuable founder individual(s) to strong mate limitation both at the
oportunities to study evolutionary transitions over contemporary establishment of the first viable population(s) and during
time scales (e.g., Sakai et al., 2001; Brown and Eckert, 2005; range expansion (Baker, 1955, 1965; Stebbins, 1957). This is
Barrett et al., 2008; Prentis et al., 2008). These studies contribute particularly relevant in obligated outcrossing species, such as
to a better understanding of the factors triggering a successful self-incompatible or heterostylous plants, in which compatible
invasion, and provide new insights on the evolutionary history mates might be lost during long-distance dispersal (e.g., Ornduff,
of specific traits, such as those related to the reproductive system 1987; Hollingsworth and Bailey, 2000; Barrett et al., 2008;
(Barrett et al., 2008; Barrett, 2011). Zhang et al., 2010). Under this scenario, a switch to uniparental
Reproduction is one of the key factors involved in the reproduction, either through self-fertilization or increased
successful establishment and spread of a given organism asexual reproduction, might be selectively advantageous and
after long-distance dispersal (e.g., Sakai et al., 2001; Barrett foster invasion (reviewed in Pannell et al., 2015). Transitions
et al., 2008; Hayes and Barry, 2008; Castro-Díez et al., 2014; to asexual reproduction or selfing have been documented
Moravcová et al., 2015). Reproductive modes determine the for several introduced species, such as the clonals Fallopia
production, dispersal and genetic composition of propagules, japonica in the UK (Hollingsworth and Bailey, 2000), Eichhornia
thus influencing the genetic and demographic structure of crassipes in China (Zhang et al., 2010), Oxalis pes-caprae in
populations, as well as the dispersal ability and evolutionary Mediterranean regions (Baker, 1965; Ornduff, 1987), and Arundo
potential of introduced individuals or sub-populations, that, by donax in Australia (Haddadchi et al., 2013), and the self-
its turn, will also determine the reproductive strategy (Sakai compatibles Echinochloa microstachya in Australia (Barrett and
et al., 2001; Novak and Mack, 2005; Barrett et al., 2008; Husband, 1990), Echium plantagineum in Australia and Canary
Ness et al., 2010). Flowering plants exhibit an outstanding Islands (Petanidou et al., 2012), and Gomphocarpus physocarpus
diversity of reproductive strategies, from sexual to asexual modes in Australia (Ward et al., 2012). Additionally, higher rates
and from self-compatible to obligated outcrossers (reviewed of uniparental reproduction in introduced and in invasive
in Barrett, 2002), that frequently occur in combination and species compared with natives or with species that failed to
reveal liability under certain ecological and genetic stressful establish, have been reported by several studies (Mulligan and
conditions (Dorken and Eckert, 2001; Eckert, 2002; Goodwillie Findlay, 1970; Rambuda and Johnson, 2004; Silvertown, 2008;
et al., 2005; Herben et al., 2015). Thus, the relative contribution van Kleunen et al., 2008; Marco et al., 2010). Nevertheless,
of each strategy to the fitness of a population/individual is comparative studies of plant reproductive strategies in native
expected to vary under the novel conditions and will play a and invaded ranges are scarce (but see Brown and Eckert, 2005;
major role in the establishment and spread of the introduced Lavergne and Molofsky, 2007; Petanidou et al., 2012).
individual(s) (e.g., Brown and Eckert, 2005; Lui et al., 2005; Sexual and asexual reproductive strategies frequently co-occur
Barrett et al., 2008; Silvertown, 2008). Sexual reproduction in flowering plants and, although this dual strategy was proven to
provides the possibility for increasing genetic diversity through be advantageous (Silander, 1985; Bengtsson and Ceplitis, 2000;

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Castro et al. Shifts in Reproductive Traits after Introduction

Van Drunen et al., 2015), it can also lead to allocation trade- sterile double-flowered form was also reported to be successfully
offs and antagonist interactions between reproductive modes, spreading in south western Iberian Peninsula (Castro et al.,
such as the interference generated by clonal growth in the 2007). However, we have recently detected the re-acquisition of
opportunities for mating (Handel, 1985; Vallejo-Marín et al., sexual reproduction likely fueled by multiple introductions of
2010; Barrett, 2015; Van Drunen et al., 2015). By reducing the compatible mating partners [tetraploid (4x) L-, M-, and S-morph
number of mating partners and by increasing the opportunities individuals; Castro et al., 2013; Ferrero et al., 2015]. This is the
for geitonogamous pollen dispersal, clonal growth interferes with first study exploring the role of reproductive traits in the invasion
sexual reproduction in reducing not only the offspring sired, success of O. pes-caprae.
but also its quality and fitness (e.g., Handel, 1985; Charpentier, The objective of this study was to quantify changes in
2002; Somme et al., 2014; but see Van Drunen et al., 2015). reproductive traits in invasive populations of O. pes-caprae and
Allocation trade-offs occur when the production of sexual and evaluate whether these differences could explain the prevalence
asexual structures compete for the resources available from the of some floral forms in the invaded range of the western
total resource pool (van Kleunen et al., 2002; Thompson and Mediterranean basin and be involved in the invasion success
Eckert, 2004; Liu et al., 2009), or through the replacement of of this species. We compared the investment in sexual and
sexual structures by asexual ones or vice-versa (e.g., production asexual reproduction between native and invasive individuals,
of inflorescences instead of vegetative shoots, Geber et al., 1992; and among sexual (4x L-, M-, and S-morph), predominately
production of bulbils in the inflorescences instead of flowers, asexual (5x S-morph) and obligated asexual individuals (4x
Ronsheim and Bever, 2000; or production of flowers from sterile double-flowered form) found in the invaded range.
meristems that in previous years resulted in vegetative tissue, Based on the invasion history of O. pes-caprae and on a
Savinykh, 2003). Therefore, it is expected that differential fitness trade-off hypothesis between allocation to sexual and asexual
of the two strategies will affect the balance between sexual and reproduction, we expected that, in the invaded range, selection
asexual reproduction in the population over time (Silvertown, has promoted individuals with an increased capacity for
2008; Vallejo-Marín et al., 2010; Van Drunen et al., 2015). investment in asexual reproduction in detriment of sexual
If strong trade-offs between investment in sexual and asexual reproduction, especially among the mostly clonal forms; in
reproduction occur, rapid clonal expansion may limit allocation sexual forms the trade-off between the two strategies might be
to flowering and seed production (Vallejo-Marín et al., 2010). more dependent of the environmental context. Still, the low
However, evidence for fitness trade-offs between sexual and sexual success of sexual forms in the invaded area (due to
asexual reproduction is ambiguous (Van Drunen et al., 2015). low mate availability and/or genetic depauperated populations;
Although several studies support a trade-off between the two Castro et al., 2013; Ferrero et al., 2015) may generate a context
strategies (e.g., van Kleunen et al., 2002; Thompson and Eckert, promoting asexuality in comparison with sexual forms from
2004; Liu et al., 2009; Van Drunen and Dorken, 2012), studies at native populations. Thus, we hypothesized that asexual forms
the genet level are scarce and many of them failed to detect such would have significantly higher asexual potential than sexual
trade-offs between reproductive strategies (Vallejo-Marín et al., forms in order to become dominant in the invaded range, and
2010; Van Drunen and Dorken, 2012). that both would have significantly higher asexual potential than
Oxalis pes-caprae is a clonal tristylous species native to South sexual forms from the native area where sexual reproduction
Africa and invasive throughout all Mediterranean regions of the prevails. Our findings are discussed in the light of biological
world. This species reproduces by two contrasting strategies: invasions and of the role of reproductive traits in successful
asexually through the profuse production of bulbs (Pütz, 1994; invasion.
Vilà et al., 2006) and sexually trough a highly specialized
mechanism, tristyly and heteromorphic self-incompatibility
system (Ornduff, 1987) that promote cross-fertilization and MATERIALS AND METHODS
increased genetic diversity (Barrett, 2002). Sexual and asexual
reproduction occurs in both native and invaded areas, but the Plant Species
contribution of each reproductive mode differs between ranges Oxalis pes-caprae L. (Oxalidaceae), Bermuda buttercup, is a
(Castro et al., 2007, 2013; Ferrero et al., 2015). In the native range, geophyte with a deeply buried annual bulb that produces
isoplethic populations occur (Ornduff, 1987; Turketti, 2010; subterranean stems bearing a rosette of leaves and several
Ferrero et al., 2015), i.e., populations with similar proportions of inflorescences of yellow flowers arranged in umbellate cymes
the three reciprocal style morphs (long-, mid-, and short-styled (Vilà et al., 2006; Sánchez-Pedraja, 2015). It is a tristylous species
morphs, hereafter L-, M-, and S-morph, respectively), indicating with a heteromorphic self-incompatibility system (Ornduff,
that populations are in equilibrium and that sexual reproduction 1987). Thus, the production of viable offspring only occurs
is expected to be the main reproductive mode. In the invaded after legitimate pollination between individuals with reciprocal
range, two different scenarios appear to be occurring. Until very style morphs. Double-flowered sterile individuals have also been
recently, the main scenario was a transition toward clonality in frequently observed in the western Mediterranean basin (Castro
which the pentaploid (5x) S-morph was the dominant form, and et al., 2007) and sporadically in South Africa (Salter, 1944;
thus asexual reproduction through bulbs has been pointed as Suda and Oberlander, personal communication). The Bermuda
the prevailing mechanism of reproduction and spread (Baker, buttercup has a high capacity for asexual reproduction through
1965; Ornduff, 1987; Castro et al., 2007). Additionally, a complete a profuse production of bulbs. The main bulb produces a

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Castro et al. Shifts in Reproductive Traits after Introduction

fasciculate root with contractile properties that grows deeper Common Garden Experiment
in the soil some centimeters each year (Pütz, 1994), and To investigate if there were differences in sexual and asexual
later in the season or under stressful conditions (e.g., soil reproductive traits between native and invasive plants, we
perturbation), the subterranean stems produce a high number conducted a common garden experiment at the Botanical Garden
of small bulbs (Young, 1968; Verdaguer et al., 2010; authors of the University of Coimbra, where individual plants from both
personal observations). Furthermore, O. pes-caprae is a polyploid areas were grown outdoors under similar optimal conditions. To
species, with diploid (2n = 2x = 14 chromosomes), tetraploid remove potential maternal effects, bulbs from SA and the MB
(2n = 4x = 28 chromosomes), and pentaploid (2n = 5x = 35 were grown for one and two generations, respectively, before
chromosomes) individuals. In South Africa, all cytotypes have sexual and asexual investment traits were measured. In June
been reported, although the 5x cytotype appears to be extremely 2012, all bulbs were harvested and stored in paper bags. During
rare (Ornduff, 1987; te Beest et al., 2012; Ferrero et al., 2015). September 2012, they were weighed and weight values were
Contrarily, the 5x is the dominant cytotype in the invaded ranges recorded as initial bulb weight. The analysis of the dispersion
worldwide, although in Australia and recently in the western of the initial bulb weight allowed us to select one bulb per
Mediterranean region, the 4x has also been reported (Symon, individual matching similar overall mean weights (mean ± SD,
1961; Michael, 1964; Castro et al., 2007, 2013). 0.463 ± 0.086 g). In total, 338 bulbs were selected, representing
The Bermuda buttercup was introduced into the 29 populations and 137 individuals from the native area, and
Mediterranean basin in the end of the 18th century, most 13 populations and 201 individuals from the invaded range
probably multiple times (Vignoli, 1937; Galil, 1968; Signorini (Supplementary Table 1). This selection reflected the different
et al., 2011), and spread widely afterward. The species was reproductive strategies found in SA and MB: sexual (4x L-,
soon recognized as a weed in several Mediterranean areas M-, and S-morphs), predominately asexual (5x S-morph), and
(e.g., Sicily, Hildebrand, 1887; Canary Islands, Morris, 1895; obligated asexual (4x sterile double-flowered form).
Algeria, Ducellier, 1914; Balearic Islands, Knoche, 1922; Bulbs were individually planted ∼2.0 cm below the soil
Malta and neighbor islands, Borg, 1927; Tunisia, Chabrolin, surface in 2 L plastic pots (9.6 cm × 9.6 cm × 21.5 cm)
1934), including Portugal where it was described as abundant filled with commercial substrate, and pots were randomized at
in orchards (Henriques, 1920; Vasconcelos and Moreira, the beginning of the experiment. Before flowering, plants were
1976). Early introductions of the plant occurred due to covered with a mosquito net to avoid undesirable pollination. To
its ornamental value, and later through soil movement in characterize sexual and asexual reproduction performance, we
agriculture, horticulture, and gardening (Michael, 1964; measured the following traits: (a) bulb viability, (b) occurrence
Signorini et al., 2011). Still, the routes of (repeated) introduction of flowering; (c) floral display; (d) biomass invested in sexual
to the Mediterranean basin and other invaded regions are not and asexual structures; and (e) production of diaspores through
clear. sexual and asexual means (fruit, seed, and bulb production).
During the flowering peak, we classified each individual as
Field Sampling either vegetative or reproductive, and we collected one flower
Extensive field sampling for bulb harvesting was conducted per inflorescence, when produced, into individual paper bags
during February and March 2010 in the invaded range of the for later estimation of: (a) mean flower weight and (b) total
western Mediterranean basin (MB), and during August 2011 in weight investment in flowers per plant. Inflorescences were
the native area, South Africa (SA). All the necessary permits periodically monitored, and were collected when senescent,
for plant collection were obtained. In the invaded range, an allowing us to simultaneously assess: (a) the total number of
additional effort was made to sample throughout the regions flowers produced per plant, i.e., floral display, and (b) the total
where trimorphic populations and the sterile double-flowered investment in inflorescences measured as dry weight. Total and
form are more common (Castro et al., 2007, 2013). All floral average investments in the production of sexual structures were
forms and cytotypes found in this invaded range were included estimated for each plant. Fruit and seed production were obtained
in our study, i.e., the 4x L-morph, 4x M-morph, 4x S-morph, by cross pollinating three flowers per plant using reciprocal style
5x S-morph, and the 4x sterile double-flowered individuals morphs of the same area of origin. Fruit set was calculated as
(Supplementary Table 1). In the native range, the field sampling the proportion of flowers that developed into fruits and seed
was conducted across most of the latitudinal and longitudinal production as the mean number of seeds per fruit. We calculated
distribution of the species (Salter, 1944; Supplementary Table 1). a measure of sexual potential for each plant by multiplying the
Our extensive sampling in the native range confirmed previous total number of flowers produced by the mean fruit and seed
results showing that the 5x S-morph and the double-flowered production. Bulbs were harvested by the end of the season. The
individuals are extremely rare in South Africa (Ferrero et al., investment in asexual structures was quantified by assessing the
2015). For this reason, only 4x L-morph, 4x M-morph, and number of bulbs produced per plant, and total and mean bulb
4x S-morph native plants were included in this comparative weight per plant.
study. In each population, we sampled bulbs from 10 individuals
per floral form, separated at least 5-m apart to avoid re-
sampling clones of the same individual. Sampled populations Statistical Analysis
were characterized for style morph frequency and cytotype Data was grouped according to the following criteria: (a) area
composition as described in Castro et al. (2013). of origin (South Africa native range, SA, and invaded range of

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Castro et al. Shifts in Reproductive Traits after Introduction

the Mediterranean Basin, MB) and (b) reproductive strategy. We sexual individuals (SA4xSex), with no statistically significant
defined reproductive strategy as: sexual, including 4x plants with differences being observed among groups (χ2 3,338 = 3.49,
morphologically functional flowers (Sex); predominantly asexual, P = 0.322; Supplementary Figure 1A).
5x S-morph plants that reproduce mostly asexually despite some The probability of producing floral structures differed among
sporadic ability to produce viable offspring (Asex; Castro et al., groups (χ2 3,305 = 11.23, P = 0.011), with the obligated asexual
2013; Costa et al., 2014, in press); and, obligated asexual, 4x individuals (MB4xSt) having a significantly higher probability to
double-flowered sterile mutants (St). Accordingly, all individuals remain vegetative than the other invasive groups (i.e., MB4xSex
were assigned to one of the following groups: South African 4x and MB5xAsex; P < 0.05), while native sexual plants had
sexual forms (SA4xSex), Mediterranean basin 4x sexual forms intermediate values between the two extremes (Supplementary
(MB4xSex), Mediterranean basin 5x predominantly asexual form Figure 1B).
(MB5xAsex) and Mediterranean basin 4x obligated asexual form
(MB4xSt, double-flowered sterile mutant). Sexual Traits: Floral Display
The above groups were defined as fixed factor in generalized Floral display differed among groups in terms of the number
linear mixed models (GLMM) to assess differences in sexual of flowers per inflorescence (χ2 3,276 = 19.07, P < 0.001),
and asexual traits. GLMMs enabled us to model variables that number of inflorescences per plant (χ2 3,276 = 47.89, P < 0.001)
did not completely fulfill the assumptions of a standard linear and total number of flowers per plant (χ2 3,276 = 31.68,
model and had the advantage to allow the incorporation of P < 0.001; Figures 1A–C). The native sexual plants and the
random factors in the models (Bolker et al., 2009). Although invasive predominantly asexual plants (MB5xAsex) produced
the initial bulb weight was fairly homogenous, this variable inflorescences with more flowers than the invasive sexual
was included as covariate to account for possible differences individuals (MB4xSex; P < 0.05), while the obligated asexual
caused by bulb weight. Population and individual were defined form (MB4xSt) had a lower and highly variable mean value not
as random factors and these were removed from the models differing from the other three groups (Figure 1A). The groups
whenever their variance was lower than the variance of the with ability for sexual reproduction (SA4xSex and MB4xSex),
residuals (Bolker et al., 2009). When both random factors were even if only sporadically (MB5xAsex), produced significantly
removed, a generalized linear model (GLM) was used instead more inflorescences than the obligated asexual individuals,
(Supplementary Table 2). A binomial distribution with a logit resulting in larger total floral display per plant (P < 0.05;
link function was used to model bulb viability and probability Figures 1B,C).
of flowering; a Poisson distribution with a log link function Significant differences among groups were also observed in
was used to model the number of flowers, inflorescences and the biomass invested for the production of sexual structures
bulbs per plant; and a Gaussian distribution with an identity (Figures 1D–F, Supplementary Figure 1), namely in the mean
link function was used to model the mean number of flowers flower and inflorescence weight (χ2 3,276 = 820.80, P < 0.001
per inflorescence, mean flower and inflorescence weight, total and χ2 3,276 = 51.78, P < 0.001, respectively; Figures 1D–E),
flower and inflorescence weight per plant, total weight of total flower and inflorescence weight per plant (χ2 3,276 = 28.08,
sexual structures per plant, fruit set (arcsine transformed), P < 0.001 and χ2 3,276 = 44.39, P < 0.001, respectively;
mean seed production, sexual potential, mean and total bulb Supplementary Figures 1C,D), and total weight of sexual
weight. In all cases, differences between least-square means were structures per plant (χ2 3,276 = 32.30, P < 0.001; Figure 1F).
tested pairwise through multiple comparisons. To evaluate the Obligated asexual individuals (MB4xSt) had significantly heavier
existence of trade-offs between sexual and asexual investments, flowers, followed by the MB5xAsex, and the native and
correlations between the amount of biomass invested in invasive sexual individuals had lower flower weights (P < 0.05;
sexual and asexual structures were calculated for the entire Figure 1D). The same trend was observed for total flower
dataset and for each group separately. All statistical analyses weight per plant, except for the obligated asexual individuals
were performed in R version 3.1.1 (R Core Team, 2014) (MB4xSt) which produced less inflorescences (Figure 1B) and
using the packages “car” for GLMs and Type-III analysis of consequently less flowers (Figure 1C), lower total flower weight
variance (Fox and Weisberg, 2015), “nlme” for linear and non- (Figure 1F) and reduced investment in total flower biomass
linear mixed models (Pinheiro et al., 2015), and “multcomp” (Supplementary Figure 1C). A different scenario was found for
for multiple comparisons after Type-III analysis of variance mean inflorescence weight, which was significantly lower for the
(Hothorn et al., 2008), and “stats” for GLMs (R Core Team, obligated asexual individuals and sexual native plants. Sexual
2014). invasive individuals presented intermediate inflorescence weight,
and invasive predominantly asexual plants had significantly
heavier inflorescences (P < 0.05; Figure 1E; a similar pattern
RESULTS is observed for the total inflorescence weight per plant;
Supplementary Figure 1D). Despite the differences in the
Bulb Viability and Probability of number and biomass of reproductive structures among all
Flowering groups, the total investments in the production of sexual
Results from all statistical analyses are summarized in structures per plant did not differ among groups except for
Supplementary Table 2. Bulb viability was high, varying between the MB5xAsex, which presented significantly higher weights
88% in invasive sexual individuals (MB4xSex) and 94% in native (P < 0.05; Figure 1F).

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Castro et al. Shifts in Reproductive Traits after Introduction

FIGURE 1 | Floral display and biomass invested in sexual reproductive structures among different forms of Oxalis pes-caprae from its native and
invaded areas and with distinct reproductive strategies: South African 4x sexual forms (SA4xSex), Mediterranean basin 4x sexual forms (MB4xSex),
Mediterranean basin 5x predominantly asexual form (MB5xAsex), and Mediterranean basin 4x obligated asexual form (MB4xSt, sterile
double-flowered form). (A) Mean number of flowers per inflorescence; (B) Mean number of inflorescences per plant; (C) Total number of flowers per plant;
(D) Mean flower weight (mg); (E) Mean inflorescence weight (mg); (F) Total weight of sexual structures per plant (mg). Values are given as model-adjusted
back-transformed least-square means and 95% confident intervals. Significant differences among factors are indicated with different letters (P < 0.05).

Production of Dispersal Units: Sexual highly heterogeneous bulbs that did not differ significantly
and Asexual Strategies from the other invasive groups (Figure 2E). There was a clear
and significantly higher investment in total bulb weight by
Sexual fitness differed significantly among groups (fruit set:
the invasive sexual and predominantly asexual plants than the
χ2 2,251 = 47.38, P < 0.001; seed production: χ2 2,246 = 89.44,
other groups, as well as in the obligated asexual individuals in
P < 0.001; and sexual potential: χ2 2,251 = 15.77, P < 0.001).
comparison with native sexual plants (P < 0.05; Figure 2F).
Fruit and seed production were significantly lower in invasive
plants than in native ones and, among invasive groups, it was
significantly lower in the predominantly asexual individuals Trade-off between Sexual and Asexual
(P < 0.05; Figures 2A,B). The calculation of a measure of Investment
sexual potential revealed that native sexual individuals had a No trade-off was observed in the biomass invested in sexual and
significantly higher success than invasive plants (P < 0.05); asexual structures. On the contrary, the production of sexual
however, no significant differences were detected in sexual structures was positively correlated with the biomass invested in
potential between sexual invasive and predominantly asexual the production of bulbs, except for invasive sexuals and obligated
invasive individuals (Figure 2C). asexuals (total: r = 0.214, P < 0.001; analyses by group: SA4xSex:
Asexual traits also differed significantly among groups, either r = 0.286, P = 0.0182; MB4xSex: r = −0.0223, P = 0.800;
measured as number of bulbs (χ2 3,305 = 350.13, P < 0.001), MB5xAsex: r = 0.449, P < 0.001; MB4xSt: r = 0.183, P = 0.381;
mean bulb weight (χ2 3,305 = 27.36, P < 0.001) or total bulb Figure 3).
weight per plant (χ2 3,305 = 211.13, P < 0.001). Invasive sexual
plants produced more bulbs per plant than predominantly
asexual individuals, which also produced more bulbs than native DISCUSSION
sexual and invasive obligated asexual individuals (P < 0.05;
Figure 2D). However, predominantly asexual individuals had Our results indicate the occurrence of evolutionary changes in
significantly heavier bulbs than native and invasive sexual plants the reproductive traits of invasive populations of O. pes-caprae.
(P < 0.05), while obligated asexuals had fairly heavy, but Indeed, most of the traits evaluated, differed between native

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Castro et al. Shifts in Reproductive Traits after Introduction

FIGURE 2 | Production of sexual and asexual diaspores among different forms of Oxalis pes-caprae from its native and invaded areas and with
distinct reproductive strategies: South African 4x sexual forms (SA4xSex), Mediterranean basin 4x sexual forms (MB4xSex), Mediterranean basin 5x
predominantly asexual form (MB5xAsex) and Mediterranean basin 4x obligated asexual form (MB4xSt, sterile double-flowered form). (A) Fruit set,
given as arcsine of the proportion of flowers developing into fruit; (B) Seed production, given as the mean number of seeds per fruit; (C) Sexual potential, given as a
measure of sexual ability of the plant obtained multiplying the number of flower produced in a plant by the fruit and seed production; (D) Mean number of bulbs per
plant; (E) Mean bulb weight (mg); (F) Total weight of the bulbs produced per plant (mg). Values are given as model-adjusted back-transformed least-square means
and 95% confident intervals. Significant differences among factors are indicated with different letters (P < 0.05).

and invasive populations and among individuals with different and draw hypotheses on how reproductive traits could have
reproductive strategies when grown in the same environment. been involved in the invasion success and in the prevalence of
In particular, we found that: (1) overall, plants with sexual some forms in the invaded range of the western Mediterranean
ability (including the 5x S-morph) had higher probability of region.
flowering and larger floral displays than the sterile forms; (2)
the total investment in the production of floral structures was Reproduction: Traits and Strategies
significantly higher in the 5x form than in the remaining forms; Reproduction determines the number and genetic composition
this suggests an effect of the ploidy level in the overall size of the of dispersal units, being vital for the establishment and spread of
structures and, in the case of the sterile double-flowered form, plant populations after long-distance dispersal (e.g., Sakai et al.,
a trade-off between the number of flowering structures and the 2001; Ness et al., 2010). The relative contribution of different
resources needed to produce them (i.e., heavier sterile flowers reproductive modes varies depending on the ecological and
resulting in lower number of inflorescences); (3) differences in genetic factors under which colonizers are subjected (e.g., Dorken
the production of bulbs and seeds revealed that native plants had and Eckert, 2001; Eckert, 2002; Herben et al., 2015). Our results
higher sexual fitness, while a transition toward clonality was clear showed remarkable differences in several reproductive traits
for the invasive forms; (4) differences were also observed among between ranges and among forms with different reproductive
invasive individuals, with the sexual forms producing more strategies. Overall, a transition to clonality was observed among
dispersal units (seeds and small bulbs), the predominantly asexual invasive plants: native individuals had a higher sexual fitness than
form producing an inter-medium number of large bulbs, and invasive ones, which in contrast had higher asexual fitness than
the sterile form being apparently less aggressive and producing natives. These observations matched our expectations mainly by
less, yet large, bulbs; (5) finally, no trade-off between sexual two reasons, described below.
and asexual investments was observed. Below, we discuss our Firstly, genetic diversity of native populations is higher
results in light of the complex invasion history of O. pes-caprae than that of invasive ones (Ferrero et al., 2015), and this is

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Castro et al. Shifts in Reproductive Traits after Introduction

FIGURE 3 | Correlation between biomass invested in the production of sexual and asexual structures among different forms of Oxalis pes-caprae
from its native and invaded areas and with distinct reproductive strategies: South African 4x sexual forms (SA4xSex), Mediterranean basin 4x sexual
forms (MB4xSex), Mediterranean basin 5x predominantly asexual form (MB5xAsex) and Mediterranean basin 4x obligated asexual form (MB4xSt,
sterile double-flowered form).

expected to affect the sexual fitness. Sexual reproduction is the we expected that selection would benefit individuals with an
main mechanism of reproduction in the native range, where increased capacity for investment in asexual reproduction.
floral polymorphism promotes outcrossing and thus genetic Indeed, uniparental reproduction has been proposed to be
diversity and frequency dependent selection governs isoplethic selectively advantageous under scenarios of strong mate
populations (Ornduff, 1987; Turketti, 2010; Ferrero et al., 2015). limitation, such as invasions, rapid range expansion, island
Contrarily, invasive populations are highly constrained by the colonization and meta-population dynamics (Baker’s Law;
scarcity of compatible mates (Castro et al., 2007, 2013), being Baker, 1965; Pannell et al., 2015). Our results corroborate
dominated by 5x S-morphs with residual sexual reproduction this prediction: regardless of the reproductive strategy,
(Costa et al., 2014, in press), and thus with low recombination invasive O. pes-caprae forms invested significantly more in
probabilities. Additionally, strong founder effects led to a asexual reproduction than natives. An enhancement in clonal
decrease in genetic diversity of invasive populations (Ferrero reproduction in invasive populations in comparison with natives
et al., 2015). Altogether, these factors significantly impacted has also been observed in several other species. For example,
genetic composition of invasive populations and, consequently, invasive Butomus umbellatus individuals were more likely to
plant fitness under outcross pollinations. Genetic depauperation produce bulbils than native individuals (Brown and Eckert,
after long distance dispersal has been described in several 2005); rapid selection of genotypes with stronger vegetative
other invasive species (e.g., Dlugosch and Parker, 2008; Zhang growth was observed in Phalaris arundinacea (Lavergne
et al., 2010), and although multiple introductions can ameliorate and Molofsky, 2007), and greater vegetative reproduction in
their consequences (Novak and Mack, 2005; Dlugosch and the invasive ranges of Achillea millefolium and Hypericum
Parker, 2008; Simberloff, 2009), negative impacts of low genetic perforatum than on their native ranges (Beckmann et al., 2009).
diversity for plant reproduction have been shown (Barrett, 2002; Interestingly, differences in bulb production have also been
Crawford and Whitney, 2010). However, genetic bottlenecks do observed among invasive populations of O. pes-caprae in the
not necessarily hinder the adaptive potential of invasive species Mediterranean basin, with insular populations having higher
(Barrett et al., 2008; Dlugosch and Parker, 2008; Rollins et al., dispersal potential than continental ones (Vilà and Gimeno,
2013). 2006). Clonal reproduction was one of the traits identified by
Secondly, under unfavorable conditions for sexual Baker (1965) for the “ideal weed” and there are several studies
reproduction in invasive populations, i.e., strong mate addressing invasive species traits that support this reproductive
limitation (either due to monomorphic populations or due strategy as one of the features involved with successful invasions
to the predominance of asexual 5x individuals; Baker, 1965; (e.g., Pyšek and Richardson, 2007; Silvertown, 2008; Marco et al.,
Ornduff, 1987; Castro et al., 2007, 2013; Ferrero et al., 2015), 2010).

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Castro et al. Shifts in Reproductive Traits after Introduction

As expected, individuals with the ability to reproduce sexually, been pointed as an important feature of the invasion process,
including the predominantly asexual plants, invested more in the especially under stressful conditions (Lane, 1984; Sala et al.,
production of floral structures than the sterile form. This is in 2007), with parental bulb weight significantly impacting plant
accordance with a strategy to promote sexual reproduction either biomass in shaded environments, as evident by the production
by investing in attractive floral display (i.e., larger inflorescences, of significantly more leaves in plants originated from bigger
inflorescences with larger flower displays) or by producing more bulbs than from smaller ones (Verdaguer et al., 2010). However,
sexual potential units (Barrett, 2002). A trade-off between the although fitting nicely the results, there are several lines of
number of inflorescences produced and the considerable amount evidence that do not completely support this hypothesis and
of energy necessary to produce double-flowers might explain the make our findings difficult to interpret. First, it is difficult to
reduced floral display of the sterile double-flowered form (see disentangle the effects of ploidy level from those related with
below). Interestingly, the 5x form produced larger flowers and evolutionary changes. Although, bulb size of the offspring of 4x
larger inflorescences that resulted in a higher biomass investment sterile double-flowered form and the 5x S-morph were similar,
without a detrimental impact in the floral display. The production the larger bulbs in the 5x individuals might be driven by ploidy,
of larger reproductive structures is likely related with the ploidy similarly to the pattern observed in the flowering structures
level, since polyploidy is hypothesized to drive significant changes (results herein) and to the patterns observed in other polyploid
in cell size and, consequently, in overall organ size (Levin, 2002). complexes (Levin, 2002). Second, 5x S-morph individuals showed
Apparently, this higher investment did not lead to allocation that parent bulb weight has a small overall effect on O. pes-
trade-offs since the 5x cytotype produced similar floral display to caprae plant biomass (Sala et al., 2007; Verdaguer et al., 2010). In
other sexual forms. general, bulbs emerged successfully and vigorously regardless of
Besides the differences in the production of sexual and their sizes (Vilà et al., 2006; Verdaguer et al., 2010), still, parent
asexual diaspores detected between ranges, different strategies bulb size might be particularly important for plant emergence
were also observed among invasive forms. As expected, sexual and initial development, depending on the conditions where the
fitness was higher in the 4x sexual forms than in the 5x form, plant is growing (Vilà et al., 2006; Sala et al., 2007; Verdaguer
and null in the sterile double-flowered form where the sexual et al., 2010). Regardless of the effects in early stages, bulb size
organs were replaced by petals due to a mutation in the genes was not determinant for the development of adult plants and
responsible for the floral development (Weigel and Meyerowitz, subsequent offspring production, possibly because further plant
1994). The lower sexual fitness of the 5x individuals after growth might become independent of this storage organ once the
outcrossing is mainly due to its odd ploidy level; although these plant starts to photosynthesize. Finally, the production of bulbs
5x individuals are able to produce some viable gametes, they in O. pes-caprae was shown to be plastic and highly dependent
also produce unviable gametes with variable ploidies (Vignoli, of nutrient availability (Sala et al., 2007). If bulb weight has
1937; Signorini et al., 2013; Costa et al., 2014), diminishing no fitness advantage, then producing many small bulbs would
significantly the production of offspring through seed. However, be advantageous, especially when mate limitation is strong and
and as described above, the 5x individuals produced slightly no allocation trade-off between sexual and asexual investment
larger floral displays and bigger floral structures; these features is observed (see below). In this context, sexual invasive forms
increase the number of gametes and the attractiveness of the have a higher dispersal potential, through both sexual and
plants for pollinators, which might contribute to ameliorate asexual means than the other invasive forms and might become
the low sexual potential of the 5x individuals. Additionally, widespread in the future.
differences in the asexual traits were also detected among invasive
forms. Under optimal resource conditions, the obligated asexuals
invested significantly less in bulb production than the other Bermuda Buttercup Invasion History:
invasive forms, producing larger bulbs but in smaller amounts What Have We Learned So Far?
(like native plants) than the other invasive forms. Interestingly, The Bermuda buttercup is a classic example in biological
the sexual and predominately asexual forms allocated a similar invasions, known as a strictly asexual form that successfully
amount of energy to the production of bulbs; however, while spread in Mediterranean climate regions of the world (5x
the former invested resources in producing many small bulbs, S-morph; Baker, 1965). However, the origin of this invasive
the later invested in less but larger bulbs. These patterns agree form is still unclear and the colonization history revealed to be
with trade-off models for propagule number and size which more complex and dynamic than previously envisaged. Native
predict that in optimal environments it is preferable to maximize populations are composed of the 4x cytotype, with the 5x
offspring quantity, whereas in stressful conditions (such as the S-morph being extremely rare (Michael, 1964; te Beest et al.,
limitation of sexual partners) it is preferable to invest in offspring 2012; Signorini et al., 2013; Ferrero et al., 2015). Contrarily, the
quality (Smith and Fretwell, 1974; Sadras, 2007). Based on this 5x S-morph dominates all Mediterranean climate regions, except
model, we could hypothesize that selective pressures during in Australia where both asexual (monomorphic 5x S-morph
the invasion of the predominantly asexual form might have populations) and sexual populations (4x trimorphic populations)
benefited larger bulbs, while selective pressures over asexual have been reported (Symon, 1961; Michael, 1964; Ferrero,
propagule production are not expected to be so strong for the personal observations). The most accepted hypothesis is that
sexual forms that have an additional reproductive mode (Costa the 5x S-morph has been originated from 4x individuals in the
et al., unpublished results). Oxalis pes-caprae bulb weight has introduced range and subsequently introduced in several areas of

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Castro et al. Shifts in Reproductive Traits after Introduction

the world (Krejčíková et al., 2013; Signorini et al., 2013; Ferrero and therefore it is likely under a competitive disadvantage with
et al., 2015; most probably multiple times in the Mediterranean other floral forms. Recent molecular studies have shown a
basin, Signorini et al., 2011), including South Africa, where it close relationship between these individuals and native plants,
was recently reported in a new semi-natural location for the first supporting the occurrence of several multiple introductions.
time (Ferrero et al., 2015). The combination of several factors, These multiple introductions might have provided a sufficiently
including strong heteromorphic incompatibility system, lack of high number of propagules to mediate a successful invasion
compatible mates and odd ploidy, constrained the production process. Although species traits are extremely important, several
of dispersal units mostly to asexual means, and consequently studies have shown that propagule pressure is also a determinant
the successful spread of this form in introduced ranges became factor for successful invasion (Novak and Mack, 2005; Colautti
dependent on bulb production (Baker, 1965; Ornduff, 1987). Our et al., 2006; Dlugosch and Parker, 2008; Simberloff, 2009).
results strongly support this hypothesis showing a clear selection Interestingly, propagule pressure was also shown to be important
toward clonality through a significantly increase in the number of in colonization by O. pes-caprae along altitudinal gradients
bulbs as well as in their size (the latter driven or not exclusively within invaded areas (Ross et al., 2008). Additionally, besides
by the ploidy level) in comparison with natives. Producing more ecological and life history traits, human mediated dispersal (e.g.,
bulbs would be selectively advantageous since it increases the in earlier stages as ornamental plant, and currently through
number of propagules, while larger bulbs may confer significant soil movements in agriculture, horticulture and gardening, or
advantages under stressful environments, allowing faster plant through land translocations during road constructions; Michael,
emergence and providing more reserves, which will translate into 1964; Signorini et al., 2011; Castro et al., 2013) might have also
larger plants (Vilà and Gimeno, 2006; Sala et al., 2007; Verdaguer promoted the dispersal of this invasive form, as well as the others
et al., 2010; Tavares, 2014). (Pyšek and Richardson, 2007).
However, the story does not end here. In the western
Mediterranean region, invasive populations seem to be changing Trade-offs between Sexual and Asexual
very rapidly (Castro et al., 2007, 2013; Costa et al., 2014, in Strategies
press). Molecular studies have shown an invasion punctuated by No trade-off between the production of sexual and asexual
multiple introductions of other floral morphs comprised of the structures has been detected in O. pes-caprae. These observations
tetraploid ploidy level (Ferrero et al., 2015), and field surveys agree with studies in other species (Vallejo-Marín et al., 2010;
detected a reacquisition of sexual reproduction in this region Van Drunen and Dorken, 2012) and with previous experiments
(Castro et al., 2013; Costa et al., 2014, in press). Although with O. pes-caprae (Vilà and Gimeno, 2006; Verdaguer et al.,
the introduction or incipient occurrence of M- and L-morphs 2010). This lack of a trade-off might be explained by the
would constitute a source of compatible mates, these individuals particular developmental processes of the plant as the production
are still under a scenario of strong mate limitation due to the of flowering structures and bulbs are asynchronous in O. pes-
dominance of the predominantly asexual 5x S-morph. Thus, they caprae likely reducing the competition for resources between
will be subjected to similar strong selective pressures toward both reproductive processes. In the first half of the plants’ life
uniparental reproduction. Our results clearly support that these cycle, most of the energy is redirected to growth and flowering,
new individuals have also diverged from native populations and only afterward, when the aboveground part of the plant starts
and present an inversion toward uniparental reproduction via to senesce, energy is directed to the production of underground
asexual reproduction (results herein), but also via changes in the structures, namely to the production of bulbs (Pütz, 1994;
strength of the incompatibility system (Costa et al., unpublished Verdaguer et al., 2010). Indeed, we observed the opposed pattern,
results). Additionally, our results show that these sexual forms with a positive correlation between bulb and flower biomass. This
have superior reproductive fitness in comparison with the 5x could simply be a reflection of plant size rather than resource
S-morph and the 4x sterile double-flowered form. So, how can management strategies.
we explain the distribution patterns in the western Mediterranean
basin? Based on the reproductive traits, the current distribution
patterns can only be explained under a scenario of different CONCLUSION
introduction timings, first with the introduction and spread of
the 5x individuals and more recently with the introduction of 4x Different sexual and asexual reproductive traits were quantified
sexual plants that are starting to become more dominant than between native and invasive populations, as well as among
previously documented. Given the superior reproductive fitness different forms within invasive populations. Different
of the 4x sexual individuals, they are expected to become more reproductive strategies and ecological and genetic contexts
dominant in the future. Still, other life-history transition stages created by long-distance dispersal seem to generate divergent
and ecological responses, including bulb viability and emergence, selective pressures in several sexual and asexual reproductive
competitive ability, resistance to herbivory and response to soil traits. The introduction process seems to have promoted
disturbance, need to be addressed in future studies in order to clonal reproduction and this is most probably the major trait
fully characterize the fitness of each form. driving the invasion success of O. pes-caprae; however, invasive
The successful spread of the sterile double-flowered form sexual forms have increased dispersal potential and additional
in south–west Iberian Peninsula is particularly intriguing. This means to produce dispersal units and promote heterozygosity.
form had the lowest dispersal potential among invasive forms Consequently, invasive sexual forms are expected to be in

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Castro et al. Shifts in Reproductive Traits after Introduction

competitive advantage in relation to the predominately asexual and Technology (FCT) funds in the ambit of the project
and obligated asexual plants, and thus could become widespread PTDC/BIA-BIC/110824/2009, CRUP Acções Integradas Luso-
in the invaded range in the future. Historical processes, with the Espanholas 2010 with the project E10/10, MCI-Programa de
introduction of the predominantly asexual 5x S-morph first and Internacionalización de la I+D (PT2009-0068) of the Spanish
more recently of the 4x sexual morphs, were probably important DGICYT (CGL2009-10466 and CGL2013-45941) and the
in establishing the current distributional patterns of the different Xunta de Galicia (INCITE09-3103009PR and R2014/036). FCT
forms in the western Mediterranean basin. This study shows also supported the work of SC (BPD/41200/2007, Starting
that invasion processes can be incredibly complex and dynamic, Grant IF/01267/2013), MC (PTDC/BIA-BIC/110824/2009;
while the interaction between ecological and genetic constraints SFRH/BD/89617/2012) and JC (SFRH/BD/89910/2012), and
determined by the invasion process might result in different Xunta de Galicia supported the work of VF (I2C Program).
reproductive strategies which in turn determine the success of
invasive populations.
ACKNOWLEDGMENTS
AUTHOR CONTRIBUTIONS
Authors are thankful to Western Cape Nature Conservation
SC, VF, LN, and JL designed the experiment; SC, VF, JC, and JL Board and Department of Environment and Nature
conducted field collections; MC, with the collaboration of all the Conservation, Northern Cape, for providing authorizations to
authors, conducted the common garden experiment; SC analyzed collect and undertake scientific research (no. AAA005-00218-
the data with the other authors participating in the discussion of 0028). The authors are also thankful to Ruben Heleno for critical
the results; SC and MC, with contribution of all the authors, wrote reading and English review.
the manuscript.

FUNDING SUPPLEMENTARY MATERIAL

This research was supported by FEDER funds through the The Supplementary Material for this article can be found online
COMPETE Program and by Portuguese Foundation for Science at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00874

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Stellenbosch University, Stellenbosch. This is an open-access article distributed under the terms of the Creative Commons
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evolutionary consequences of clonality for plant mating. Ann. Rev. Ecol. Evol. is permitted, provided the original author(s) or licensor are credited and that the
Syst. 41, 193–213. doi: 10.1073/pnas.1501712112 original publication in this journal is cited, in accordance with accepted academic
Van Drunen, W. E., and Dorken, M. E. (2012). Trade-offs between clonal and practice. No use, distribution or reproduction is permitted which does not comply
sexual reproduction in Sagittaria latifolia (Alismataceae) scale up to affect with these terms.

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