Modes and rates of selfing and associated inbreeding depression in the self-incompatible plant Senecio squalidus (Asteraceae): a successful colonizing species in the British Isles

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Blackwell Publishing, Ltd.

Modes and rates of selfing and associated inbreeding depression in the self-incompatible plant

Senecio squalidus

(Asteraceae): a successful colonizing species

in the British Isles

Adrian C. Brennan

1

, Stephen A. Harris

2

and Simon J. Hiscock

1

1

School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 IUG, UK;

2

Department of Plant Sciences, University of Oxford, South Parks

Road, Oxford OX1 3RB, UK

Summary

• The strength of the self-incompatibility (SI) response in

Senecio squalidus

wasmeasured across its British range. Geographic variation in SI was investigated andthe extent and inheritance of pseudo-self-compatibility (PSC) and inbreedingdepression were determined.• Mean self-fruit-set per capitulum was calculated for individuals and samplepopulations. The heritability of PSC and the magnitude of inbreeding depressionwere assessed by comparing selfing rates and fitness trait values between SI and PSCparent–progeny lines.• SI was found to be strongly expressed in

S. squalidus

throughout its British range,with only 3.1% of the individuals sampled showing PSC. This PSC had relativelylow heritability with stronger expression of SI in selfed progeny relative to PSC parents.Inbreeding depression was shown to be great in

S. squalidus,

with mean life historystage values ranging from 0.18 to 0.25.• The strength of SI in

S. squalidus

appears not to have weakened in response toits rapid colonization of Britain. The avoidance of inbreeding depression is likely tobe the primary factor maintaining strong SI in this successful colonizing species.

Key words

: Baker’s rule, colonization, inbreeding depression, pseudo-self-compatibility,selfing rate,

Senecio squalidus

, sporophytic self-incompatibility.

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doi

: 10.1111/j.1469-8137.2005.01517.x

Author for correspondence:

Simon Hiscock Tel: +44 (0)117 9546835 Fax: +44 (0)117 9257374 Email: [email protected]

Received:

22 April 2005

Accepted:

16 June 2005

Introduction

In recent years, there has been a shift from the traditionalview of plant mating systems as either entirely outcrossingor entirely selfing to a view that there is typically much moremixed-mating in nature (Stephenson

et al

., 2000; Vogler &Kalisz, 2001; Barrett, 2003). Mating systems based upon self-incompatibility (SI) come close to the ideal of a stable matingsystem with complete outcrossing, because self-fertilizationis prevented by genetic factors that identify and reject self-pollen (Hiscock & Mcinnis, 2003). In SI taxa, outcrossingmaximizes the advantages associated with sexual reproduction

by limiting the reduced fitness effects of deleterious mutationsand maintaining the genetic diversity and potential adaptabilityof offspring (Lloyd, 1992). However, observations of matingbehaviour show that, even for species typically viewed asoutcrossers, low to intermediate levels of selfing (0–20%) area common feature (Schemske & Lande, 1985; Barrett &Eckert, 1990; Vogler & Kalisz, 2001). Studies of SI in naturalpopulations have begun to highlight the importance of‘flexibility’ provided by a latent element of selfing in otherwiseSI species (Levin, 1996; Hiscock, 2000a; Stephenson

et al

.,2000). Indeed, mating system shifts from outcrossing toselfing are a frequent response to population disturbance

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(Barrett

et al

., 1989; Goodwillie, 2001; Busch, 2005). Theprinciple that selfing ability provides reproductive assuranceduring colonization (i.e. ‘Baker’s rule’; Baker, 1967) explainswhy many weedy species that undergo frequent local extinctionand recolonization events are selfers (Pannell & Barrett, 1998).

Senecio squalidus

(Oxford ragwort) has been used as amodel to investigate mating system and selfing rate in a highlysuccessful colonizing species known to be SI (Abbott &Forbes, 1993; Hiscock, 2000a,b).

S. squalidus

is a good modelfor such investigations because its recent colonizing historyin Britain has been well documented (Harris, 2002; Preston

et al

., 2002; R. J. Eastwood & S. A. Harris, University ofOxford, Oxford, UK, unpublished).

S. squalidus

was intro-duced to Britain via Oxford’s Botanic Garden

c

. 300 yr agoand has colonized most urban habitats in Britain as far northas central Scotland during the last 150 yr.

S. squalidus

hasalso attracted attention because of hybridization with

Seneciovulgaris

to create three new

Senecio

taxa within the last 70 yr(Abbott, 1992; Ingram & Noltie, 1995; Lowe & Abbott,2003). Self-incompatibility within the Asteraceae is generallybased upon a sporophytic system of self-incompatibility (SSI),whereby a single polyallelic locus,

S

, expressed in the diploidtapetal tissue of the anther, determines the incompatibilityphenotype of the pollen (Hiscock & Mcinnis, 2003). A wholerange of mating system responses have been observed indifferent SI Asteraceae taxa as a result of recent populationdisturbance (e.g. Abbott & Forbes, 1993; DeMauro, 1993;Reinartz & Les, 1994).

Here, we describe an investigation to determine the extentto which the SSI mating system of

S. squalidus

has beeninfluenced by the recent population history of this species inBritain. Important questions are: how did

S. squalidus

dealwith the initial population bottleneck associated with a singleintroduction event and small-scale cultivation in Oxford’sBotanic Garden over a period of

c

. 150 yr, and what featuresof the mating system may have helped facilitate the rapidcolonization of Britain of

S. squalidus

following its escapefrom the Oxford Botanic Garden?

According to Baker’s rule, it might be predicted that thestrength of expression of SSI in

S. squalidus

may have weakenedor broken down to self-compatibility (SC) as a means of enhancingreproductive assurance during colonization (Baker, 1967).Such a shift from SSI to SC has happened in

Scalesia affinis

during its colonization of the Galapagos Islands (Nielson

et al

., 2003) and in

Aster furcatus

as a consequence of populationfragmentation and decline in central North America (Reinartz& Les, 1994). However, previous studies indicate that

S. squalidus

has retained a strong and fully functional SSI system, becauseestimates of selfing rates are very low, typically 0.2–4.6% (Gibbs

et al

., 1975; Abbot & Forbes, 1993; Brennan

et al

., 2002).Thus,

S. squalidus

, better represents one of the rare exceptionsto Baker’s rule, where maintenance of a strong outcrossingmating system has not prevented successful range expansion.Similarly, another Asteraceae species,

Centaurea solstitialis

,

appears to have maintained a highly outcrossing matingsystem through its colonization of western North America fromnative Europe and Asia (Sun & Ritland, 1998). However,population bottleneck founder events in

C. solstitialis

wouldhave been ameliorated by repeated introductions resultingfrom agricultural and industrial practices in the last century.

Rather than simply considering either strong SI or SC asthe only alternative mating strategies available to

S. squalidus

as it colonized Britain, there is growing evidence that matingsystem ‘flexibility’ provides resilience to populations facingdisturbance (Levin, 1996; Stephenson

et al

., 2000). There-fore, it is important from an ecological and evolutionaryperspective to investigate the potential adaptive importanceof qualitative variation in SI mating systems. For example,failing sexual reproduction leading to further populationdecline has been observed in two Asteraceae species,

Hymenoxisacaulis

and

Centaurea corymbosa

, that have maintained stronglyexpressed SSI through population decline and fragmentation(DeMauro, 1993; Colas

et al

., 1997). In contrast to thesetaxa, temporary partial breakdown of SSI with recovery tofull SSI has been documented for another Asteraceae species,

Crepis sancta

, during the colonization and establishmentof new populations in the French Mediterranean region(Cheptou

et al

., 2002). We hypothesize that a process similarto that found in

C. sancta

may have occurred in

S. squalidus

during its colonization of Britain. Under this scenario, selfingwould be favoured during the founding stages of new popu-lations when reproductive assurance through outcrossing islimited. Later, populations would experience a recoveryto full SSI as immigration introduces extra

S

allele diversity,which increases mate availability, and as selection againstinbreeding depression, which promotes outcrossing, predom-inates again. To explore this possibility, the strength of SSIand the prevalence of selfing were compared in

S. squalidus

populations from both long-established central UK popula-tions and new peripheral populations at the edges of its Britishrange.

Low rates of selfing in individuals from otherwise SI speciesappear to be widespread in nature, a phenomenon knownas pseudo-self-compatibility (PSC; Levin, 1996). The geneticfactors responsible for PSC include self-fertility or null allelesat the

S

locus and

S

modifier loci not linked to

S

. PSC hasbeen observed in many species and is typically a quantitativetrait, the phenotype of which is the result of multiple genes ofsmall effect and environmental factors such as temperature,flower aging and prior fertilization success (Bixby & Levin,1996; Levin, 1996; Vogler

et al

., 1998; Stephenson

et al

., 2000).This supports theoretical studies concluding that low selfingrates, as part of SI systems, are most stably maintainedwhen controlled by additive or dominant alleles at multipleloci rather than recessive alleles at single or few loci (Latta &Ritland, 1993; Latta, 1994).

The widespread occurrence of selfing and biparentalinbreeding in taxa usually considered as being strongly SI

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raises the important issue of inbreeding depression and itsimplications for the evolution of SI mating systems (Lande& Schemske, 1985; Charlesworth & Charlesworth, 1987;Husband & Schemske, 1996). Certainly, the avoidance ofinbreeding depression is the primary selective force maintain-ing outcrossing systems such as SI (Lande & Schemske, 1985;Charlesworth & Charlesworth, 1987; Glemin

et al

., 2001;Castric & Vekemans, 2004). However, simple mating systemmodels suggest that outcrossing is vulnerable to invasion andreplacement by selfing because of the 2-fold transmissionadvantage of selfing genotypes over outcrossing genotypes(Fisher, 1941). Thus, inbreeding depression must reduce thefitness of selfed progeny by more than half for outcrossingmating systems to persist. Because of the enforced outcrossingbehaviour of SI plant species, deleterious alleles responsiblefor inbreeding depression are sheltered from purging selectionin the heterozygous state and the accumulated genetic loadcan be great (Charlesworth & Charlesworth, 1987; Husband& Schemske, 1996; Glemin

et al

., 2001). This sheltering ofgenetic load is accentuated even further when deleteriousalleles accumulate in the zone of reduced recombinationsurrounding the

S

locus (Glemin

et al

., 2001; Castric &Vekemans, 2004; Stone, 2004). It is vital to better understandthe magnitude of inbreeding depression and the ability ofselection to purge deleterious alleles, as these factors haveconsiderable consequences for the maintenance and recoveryof SI mating systems after population disturbance (Colas

et al

., 1997; Cheptou

et al

., 2002; Busch, 2005). Inbreedingdepression has been observed in two other typically outcross-ing

Senecio

species,

Senecio integrifolius

(Widén, 1993) and

Senecio vernalis

(Comes, 1994), and it is important to exam-ine the magnitude of inbreeding depression in

S. squalidus

aspart of studies of its mating behaviour.

Here we report an extensive survey of selfing rates andinbreeding in

S. squalidus

across its entire British range. Theprimary aim of this study was to gain a comprehensiveunderstanding of how the SSI mating system of

S. squalidus

hasbeen maintained during colonization. Patterns of selfing rates inpopulations of

S. squalidus

throughout Britain were investigated,with an emphasis on comparing selfing rates in established andactively colonizing populations. The environmental and geneticcomponents of selfing in

S. squalidus

and their mode of expressionwere investigated to determine how they might evolve in wildpopulations. Finally, the extent of inbreeding depression in

S. squalidus

was measured to examine the extent to which it isresponsible for maintaining SSI in the species in Britain.

Methods

Plant material

Achenes were sampled from five to 25

Senecio squalidus

L. maternal plants per population (191 plants in total)from 11 wild populations (Bristol, Carlisle, Crewe, Glasgow,

Holyhead, Inverness, Kirriemuir, Oxford, St. Blazey, Stoke-on-Trent and Tamworth, labelled Bl, Cl, Cw, Gw, Hd, Is, Kr,Ox, SB, ST and Tt, respectively) during the flowering seasonsbetween 1999 and 2001 (Fig. 1), except for the Bristolpopulation which was sampled in 1990 and the Oxfordpopulation from which young plants, rather than achenes,were sampled. These populations can be grouped into‘central’ populations established pre-1950 (Bl, Cw, Hd, Ox,ST and Tt) and ‘peripheral’ populations established post-1950 (Cl, Gw, Is, Kr and SB). Achenes were stored dry at 4

°

Cuntil needed. Three achenes per maternal plant were sownin moist soil-based compost in 10-cm-diameter pots in aglasshouse and one plant was then potted on. Sample plantswere grown to flowering size in the glasshouse with aminimum night temperature of 10

°

C and 16 h day-length.Subsequently, sample plants were propagated through vegetativepropagation (Hiscock, 2000b).

Measurement of selfing rates

Previous controlled pollination experiments in

S. squalidus

demonstrated that self-fruit-set was not enhanced byphysically transferring self-pollen onto stigmas using sablebrushes or by gently brushing flowering capitula together(Abbott & Forbes, 1993; authors’ unpublished data). Thus,each sample plant was tested for the strength of its SIby placing branches with immature capitula in clear plasticpollination bags and occasionally agitating them to dispersepollen until the capitula had flowered and dehisced. Intactcapitula were then dissected and the numbers of viable fruitsrecorded as those that were filled and pigmented comparedto unfilled, pale, unfertilized ovules. Mean self-fruit-setper capitulum was calculated as a measure of selfing rate forindividuals and sample populations. Because

S. squalidus

capitula typically consist of

c

. 100 uniovulate florets (authors’pers. obs.), mean self-fruit-set per capitulum is approximatelyequivalent to percentage self-fruit-set values measured inother SI studies. Plants were classified as self-incompatible(SI) when mean self-fruit-set values were

≤

2 and as pseudo-self-compatible (PSC) when mean self-fruit-set values were>2 but < the typical outcross self-fruit-set of

c

. 25 (Hiscock,2000b). The significance of differences between meansample population selfing rates was calculated by comparingit with 1000 bootstrap samples of equivalent sample size fromthe entire dataset.

Inheritance of selfing rates

Twelve

S. squalidus

individuals were chosen as representativeof the full range of selfing rates observed. These plants couldbe grouped into three classes (1, 2 and 3).

Class 1. PSC plants generated by forced-selfing wild-sampled Oxford plants; labelled A34.4.21, Bill1, Bill21,Bill35 and OS9 (Hiscock, 2000a,b).

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Class 2. Naturally occurring PSC plants among wild-sampled

S. squalidus

individuals; labelled Ox10 andTt13.

Class 3. Outcrossed SI plants derived from compatiblecrosses between wild-sampled SI individuals from Oxford andGlasgow; labelled Ox5*Gw3, Ox17*Gw7, Ox20*Gw12,Ox25*Gw2 and Ox26*Gw18 (Brennan

et al

., 2003).Selfed fruits from classes 1 and 2 and crossed fruits from

class 3 were used to create 12 progeny arrays (9–22 individualseach) labelled according to their parent plant(s). Mean self-fruitvalues per capitulum were measured for each individual fromeach progeny array and the three different classes of progenyarrays were compared. The correlation of selfing rates betweenparent and progeny arrays was evaluated by regressingparental self-fruit per capitulum values against their equiva-lent mean progeny values. Additionally, standard measures ofinheritance of quantitative traits, narrow sense heritability(

h

2

) and the percentage additive genetic coefficient of varia-tion or evolvability (

CV

a

), were calculated according toEquations 1 and 2.

Eqn 1

(Falconer, 1989;

x

, parental trait values;

y

, progeny traitvalues)

Eqn 2

(Houlé, 1992;

x

, mean parental trait value).

Mating system and inbreeding depression

In addition to measuring selfing rates, nine traits weremeasured on the 12 progeny arrays derived from differentmating regimes and four additional progeny arrays consistingof three progeny arrays derived by forced-selfing (A34.3,A34.9 and OS.3; 56, 68 and 41 plants, respectively) andone array derived by outcrossing (A34.3

×

OS.3; 58 plants).The traits were:(A) percentage seed germination on moist filter-paper in thelaboratory;(B) days to germinate on moist filter-paper in the laboratory;(C) mid-cauline leaf length measured from pedical base to leaf tip;(D) number of lobes of the mid-cauline leaf;(E) height from soil level to first capitulum;

Fig. 1 Locations of populations from which Senecio squalidus samples were obtained. Fruits were sampled from all populations in 1999 and/or 2000 by A.C.B., except for Bristol (1990; Richard Abbott), Oxford (as young plants; 1999; S.A.H. and S.J.H.) and St. Blazey (2001; S.J.H.).

hx yx

2 2

cov( , )var( )

=

CVx y

a cov( , )

=100 2

x

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(F) days until ray florets opened on the first capitulum;(G) number of capitula on the primary flowering branch;(H) maximum number of simultaneously flowering capitula;(I) percentage pollen viability measured using fluorescenediacetate (FDA; Hiscock, 2000a).

Each of these traits influences fitness at one of three life historystages, defined as: (i) germination and seed survival (A, B);(ii) vegetative growth and vigour (C–F), and (iii) reproductionand fecundity (G–I). Data were grouped into those progenyarrays that had been derived through outcrossing and throughselfing, respectively, and two-sample t-tests and nonparametricMann–Whitney tests were performed. Inbreeding depres-sion (δ) was calculated for each fitness trait according toEquation 3.

Eqn 3

(ws, fitness of selfed progeny; wx, fitness of crossed progeny).The fitness traits days to germinate and days to flower were

inverse-transformed before calculating inbreeding depressionvalues to allow comparisons with inbreeding depression

values calculated for other traits, as shorter germination anddevelopment times correspond to fitter phenotypes. Meaninbreeding depression values were calculated for the traitsbelonging to each of the three life history stages.

Results

Selfing rates in S. squalidus populations

A frequency histogram of self-fruit-set values for the 191wild-sampled S. squalidus plants revealed a highly right-skewed distribution of selfing rates (Fig. 2a). The majorityof individuals were strongly SI, with mean self-fruit-set percapitulum values of ≤ 2, whilst only six individuals withmean self-fruit-set values per capitulum > 2 but < 25 could beclassified as PSC (Fig. 2a). When these PSC individuals wereomitted from the frequency histogram of self-fruit-set, thedistribution remained highly right-skewed, with the majorityof individuals (61.8%) exhibiting zero self-fruit-set (Fig. 2b).Whole population samples were all highly SI, with mean self-fruit-set per capitulum values all < 2. The greater populationmean self-fruit-set per capitulum values for Cl, Cw, Kr, Ox

Fig. 2 (a) Self-fruit-set per capitulum values for the entire sample of 191 Senecio squalidus individuals from 11 British sample populations. Pseudo-self-compatible individuals with mean self-fruit-set per capitulum values between 2 and 25 are labelled individually. (b) Self-fruit-set per capitulum values for a subset of 185 confirmed self-incompatible S. squalidus individuals from 11 British sample populations.

δ = −1w

ws

x

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and Tt, relative to the other populations, were primarily aresult of the presence of PSC individuals in these populationsamples (Fig. 3). No sample population exhibited significantlyhigher selfing rates compared with the others after Bonferronicorrection for multiple tests of significance (range of P-valuesfrom 0.05 to 0.95; α = 0.005; Kr and Bl, respectively; Fig. 3).


The strength of SI in the three classes of parental plants(forced-self PSC plants, naturally occurring PSC plants andoutcrossed SI plants) followed the same patterns in theirprogeny. However, self-fruit-set values were considerablyreduced in the progeny (Fig. 4). There was a nonsignificantpositive correlation between the selfing rates of parental plantsand of those their progeny (P = 0.09, Fig. 5). Narrow-senseheritability was low (h2 = 0.06; Fig. 5) compared with thecoefficient of additive genetic variance (CVa = 30.67; Fig. 5).

Mating system and inbreeding depression

A comparison of nine fitness trait values measured for selfedand outcrossed progeny arrays demonstrated that all exceptone (days to flower; Table 1) were of the expected relative

magnitude if the assumption that outcrossed progeny werefitter than selfed progeny holds. A statistical comparisonbetween selfed and outcrossed progeny confirmed that thesedifferences were significant at the 5% confidence level forsix of the eight traits (Table 1). These differences in meanmeasured fitness traits between selfed and outcrossed progenycorrespond to inbreeding depression values ranging from−0.03 to 0.38 (days to flower and leaf length, respectively;Table 1). Mean inbreeding depression at each life historystage ranged from 0.18 to 0.25 (reproduction and germina-tion, respectively; Table 2). A variety of characteristic ‘unfit’phenotypes were identified within the selfed progeny arraysstudied, including ‘dwarfed’, ‘leafy’, ‘twisted’ and ‘floppy’phenotypes (Fig. 6).

Discussion

Selfing rates in S. squalidus populations

Senecio squalidus is strongly SI throughout its British range.Low selfing rates were observed across its entire range, withan overall mean self-fruit-set per capitulum value of just 0.4(Fig. 3). These values confirm previous findings of low selfingrates in individual sample populations of S. squalidus that

Fig. 3 Mean self-fruit per capitulum values including and excluding self-compatible individuals for 11 Senecio squalidus sample populations. Diamonds, all plants; squares, all SI plants. Bl, Bristol; Cl, Carlisle; Cw, Crewe; Gw, Glasgow; Hd, Holyhead; Is, Inverness; Kr, Kirriemuir; Ox, Oxford; SB, St. Blazey; ST, Stoke-on-Trent; Tt, Tamworth.

Fig. 4 Mean self-fruit-set per capitulum values for Senecio squalidus plants and their progeny derived from (1) pseudo-self-compatible (PSC) plants generated through forced selfing, (2) naturally occurring PSC plants and (3) outcrossing self-incompatible (SI) plants. Numbers in brackets after class descriptions are sample sizes for parents and progeny, respectively. Standard error bars refer to interindividual variation in self-fruit-set.


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ranged from 0.2 to 4.6% self-fruit-set (Gibbs et al., 1975;Abbott & Forbes, 1993; Hiscock, 2000a,b; Brennan et al.,2002). Furthermore, there was a clear division between the96.9% of SI individuals with 1.4 or fewer self-fruits percapitulum and six naturally occurring PSC individuals withhigher mean self-fruit-set per capitulum values (3.6–15.5:Fig. 2a,b). This confirms that the semiarbitrary limit of twofruits set per capitulum chosen to distinguish compatible and

incompatible cross interactions in previous studies of SIin S. squalidus is a suitable cut-off point for assessing SI(Hiscock, 2000a,b; Brennan et al., 2002). Additionally, thisrelatively large difference in self-fruit-set per capitulum valuesbetween SI and PSC individuals suggests that the S-modifierloci that influence SI expression in S. squalidus may be few innumber and consist of alleles with large phenotypic effect.

A low frequency of 3.1% PSC individuals that had inter-mediate selfing rates was observed in the overall sample set(Figs 2a, 3). Furthermore, these PSC individuals are a minor-ity within the populations in which they were identified(3.8% to 8.0%) and do not affect the conclusion that theoverall sample populations are strongly self-incompatible(Fig. 3). The presence of PSC at low to intermediate frequen-cies ranging from 0.0 to 17.8% is commonly observed inpopulations of wild SI species (Levin, 1996). The observedfrequency of PSC in S. squalidus is at the lower end of therange of these observations and indicates that SI is not break-ing down to SC as has been observed for other Asteraceae

Fig. 5 Inheritance of selfing rates for seven pseudo-self-compatible (PSC) and five self-incompatible (SI) parental Senecio squalidus plants and their F1 progeny. Mean parental values are presented where the cross involves two different parents. Standard error bars indicate variation between progeny. h2, narrow sense heritability calculated according to Equation 1. CVa, percentage additive genetic coefficient of variation calculated according to Equation 2.

Table 1 Comparison of nine mean fitness trait values and inbreeding depression values for 10 inbred progeny arrays and six outcrossed Senecio squalidus progeny arrays

Fitness characterMean inbred value (SE, sample size)

Mean outcrossed value (SE, sample size)

Two-sample t-test (df)

Mann–Whitney U-test

Inbreeding depressionvalue (δ)

% seed germinationa 96.8 (0.01, 5) 87.6 (0.07, 7) n/ad 0.3845d 0.09Days to germinatea 6.45 (0.26, 281) 3.84 (0.12, 213) < 0.001 (385) < 0.0001 0.40Leaf length (cm)b 10.98 (0.22, 154) 17.57 (0.39, 57) < 0.001 (92) < 0.0001 0.38Lobes/leafb 7.78 (0.14, 154) 10.97 (0.45, 57) < 0.001 (66) < 0.0001 0.29Height (cm)b 44.03 (1.50, 154) 51.56 (2.95, 57) 0.025 (86) 0.0357 0.15Days to flowerb 104.15 (1.86, 62) 107.22 (3.21, 46) 0.410 (74) 0.6343 −0.03Capitula/primary inflorescencec 7.71 (0.29, 65) 8.92 (0.35, 48) 0.009 (98) 0.0295 0.14Maximum flowering capitula/dayc 22.01 (1.31, 119) 28.00 (3.64, 52) 0.126 (64) 0.9318 0.21% viable pollenc 38.18 (1.95, 119) 47.82 (3.23, 38) 0.013 (66) 0.0099 0.20

Superscripts after fitness traits refer to agermination, bvegetative growth and creproduction life history stages.dRefers to the significance of differences in germination counts tested using the χ2 test (1 degree of freedom) only, because sample size was limited by number of progeny arrays.SE, standard error; df, degrees of freedom.

Table 2 Mean inbreeding depression values over three life history stages for 10 inbred progeny arrays and six outcrossed Senecio squalidus progeny arrays

Life history stageMean inbreeding depression value (δ)

Germination 0.25Vegetative growth 0.20Reproduction 0.18


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species under population stress. For example, 23.8% of Asterfurcatus individuals were observed to be selfers and theirpresence was hypothesized to be attributable to selection forreproductive assurance in response to population disturbanceas habitat destruction reduces and fragments its range incentral USA (Reinartz & Les, 1994).

SI was consistently strongly expressed in S. squaliduspopulation samples from throughout its British range (0.1–1.1 mean self-fruit-set per capitulum per population; Fig. 3).Furthermore, the six PSC individuals that were identifiedwere distributed in three long-established populations (Cw,Ox and Tt; pre-1950) and two more recently colonizedpopulations (Cl and Kr; post-1950). If Baker’s rule applies toS. squalidus, then we might predict that recently colonizedpopulations would show greater selfing ability than longerestablished populations in response to increased selectionfor reproductive assurance in these new populations. How-ever, as all study populations exhibited consistently stronglyexpressed SI regardless of time since colonization, it appearsthat the strength of selection maintaining outcrossing andSI in S. squalidus is greater than the opposing selective forcefor reproductive assurance, even in small and colonizingpopulations.

Alternatively, perhaps the PSC observed in S. squaliduspopulations represents an element of mating system‘flexibility’ that permits successful colonization (Levin,1996; Stephenson et al., 2000). Selection pressures on matingbehaviour are likely to change from the time populations arefirst colonized until they become established. Such a phenom-enon has already been observed in C. sancta, where selectionfor reproductive assurance in newly colonizing populationsfavours an initial increase in the frequency of PSC, followedby selection to re-establish SI as mate availability increaseswith the immigration of extra S allele diversity into popula-tions (Cheptou et al., 2002). In this study, no relationshipwas observed between S allele number and the prevalence ofPSC in S. squalidus populations (data not shown). Transientincreases in population selfing rates during colonization couldbe particularly difficult to detect if there was a large environ-mental component to the expression of PSC. For example, ifselfing ability increases with flower age, as it does in Campan-ula rapunculoides (Stephenson et al., 2000), then selfingability is also sensitive to population demographics because lowerpollination rates associated with small population size wouldresult in older but more self-fertile flowers receiving pollen

more often (Good-Avila et al., 2001). Under conditions oftransient and highly responsive selfing rates in S. squalidus, itcould be that the sample populations represented in thisstudy have not adequately captured the colonizing front of thecurrent British range of S. squalidus. Testing the differencebetween the hypotheses (i) that SI is maintained throughoutthe colonization process in S. squalidus and (ii) that it is tran-siently compromised during active colonization wouldrequire detailed investigations of the genetic and environ-mental factors that both influence the expression of PSC andmaintain the selective advantage of an outcrossing SSI system inS. squalidus.


The investigation of the inheritance of selfing factors inS. squalidus was limited because so few suitable individualswere available for study. Sample size was limited because ofthe rarity of PSC in wild-sampled plant material and the fewPSC plants generated in previous selfing experiments ofS. squalidus. However, the genetic control of PSC is likely tobe the same for both naturally occurring PSC and force-selfedglasshouse PSC plants because of their very similar selfing ratecharacteristics and the results for both sets of individuals areconsidered jointly (Fig. 4).

A considerable environmental component appeared toinfluence the expression of PSC, according to the small measuredheritability (h2) value of just 0.06 and the nonsignificantcorrelation of selfing rates between parental plants and theirprogeny (Fig. 5). The particular environmental factors en-couraging plasticity in SI expression were not investigated andremain obscure. However, a range of environmental factors,including temperature, humidity, ambient carbon dioxideconcentration, sodium chloride concentration at pistil surface,flower age and prior fertilization success, have been shown toinfluence the strength of SI in other species (Levin, 1996;Stephenson et al., 2000). In S. squalidus, SI can be overcomeby treating stigmas with dilute sodium chloride solutions,which interferes with the inhibition response of stigma tissue(Hiscock, 2000a). The occurrence of sodium chloride in someS. squalidus habitats such as roadsides and railway lines hasbeen hypothesized to influence selfing rates in the wild(Abbott & Forbes, 1993; Hiscock, 2000a,b).

Nevertheless, environmental factors alone are unlikely toexplain variable selfing rates between parents and progeny, as

Fig. 6 A selected range of representative phenotype classes observed in forced-selfed progeny arrays of Senecio squalidus. Plants were photographed at approx. 100 d old when wild-type phenotype progeny were beginning to flower. (a) All crossed progeny and most force-selfed progeny were indistinguishable from the wild-type phenotype presented here. (b) ‘Leafy’ phenotypes tended to developed leaves in place of captula (19% force-selfed A34 progeny). (c) ‘Twisted’ phenotypes had twisted stems and leaf petioles (16% force-selfed Bill progeny). (d) ‘Dwarf’ phenotypes were much smaller than wild-type phenotypes and were often chlorotic and failed to flower (17% force-selfed A34 progeny). (e) ‘Floppy’ phenotypes were an extreme form of the ‘twisted’ phenotypes where the stems could no longer support the leaves in an upright position (26% force-selfed Bill progeny). (f ) Range of morphologies observed for mid-cauline leaves among the force-selfed and cross progeny arrays. The wild-type is sixth counting from the left.


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the magnitude of the measured coefficient of additive geneticvariance (CVa = 30.67; Fig. 5) clearly indicates that there isa heritable component to selfing rates. Thus, observedself-fruit-set per capitulum values remained consistently higherfor plants classified as PSC relative to plants classified asSI across one generation, despite a large reduction in themagnitude of observed self-fruit-set per capitulum values(Fig. 4). This large reduction in selfing rates observed acrossa single generation of selfed PSC individuals is perhaps themost conspicuous and unexpected finding of this study(Fig. 4). If it is a general characteristic of PSC in S. squalidus,then it suggests an automatic tendency for SI to recover inwild populations even if founded by selfing PSC individuals.It is only possible to speculate on the possible causes of thephenomenon at this stage. The reduction in selfing ratesacross a single generation could be a result of selection againstlethal and deleterious loci linked to genetic factors responsiblefor selfing or simply general inbreeding depression expressedin selfed individuals (Glemin et al., 2001; Castric & Vekemans,2004; Stone, 2005). However, there is little evidence for purg-ing of selfing genotypes in the form of reduced germinationsuccess in selfed progeny arrays relative to outcrossed progenyarrays (0.87 vs 0.97 mean seed germination for selfed vs outcrossedprogeny arrays, respectively; Table 1). Alternatively, it maybethat crucial environmental factors, responsible for influencingthe strength of expression of SI, varied between the growingseason during which the parental PSC plants were identifiedand the later season during which the selfing rates of the prog-eny arrays were measured. Finally, the mode of inheritance ofPSC factors themselves may explain the observed pattern ofreduced PSC. If dominant alleles or epistatic interactionsbetween alleles at different loci controlled the expression ofPSC, then segregation of these loci would tend to restore SIin a proportion of the progeny (Latta & Ritland, 1993; Latta,1994). However, this hypothesis conflicts with the idea thatthe exposure of recessive PSC alleles in homozygous geno-types was responsible for the origin of the five glasshouse PSClines (A34.4.21, Bill1, Bill21, Bill35 and OS9) that werederived through a round of forced selfing carried out onwild-sampled SI S. squalidus plants (Hiscock, 2000a,b). Clearly,further studies with additional plant material are required tobetter understand the mode of inheritance of PSC inS. squalidus and the particular environmental factors import-ant in influencing its expression.

Inbreeding depression

Significant inbreeding depression was observed for six of the ninemeasured fitness traits, confirming that outcrossed S. squalidusindividuals tend to be fitter than selfed individuals (Table 1).The degree of inbreeding depression detected varied considerablydependent on the measured fitness trait, but mean inbreedingdepression values indicated that all life history stages, includingseedling, vegetative and reproductive developmental stages,

were detrimentally affected to a similar extent by selfing.Inbreeding depression at the seedling stage in S. squalidus(0.25; Table 2) is of similar magnitude to that measuredin full-sib inbred arrays of another Senecio species with SSI,S. integrifolius, based on the same fitness traits of percentagegermination and days to germinate (0.10; Widén, 1993). Thesemeasures were made under benign laboratory and glasshouseconditions that underestimate actual fitness reductions in thewild (Dudash, 1990; Cheptou et al., 2000; Hayes et al., 2005).Thus, under natural conditions, it is likely that cumulativeinbreeding depression could exceed the 0.50 fitness reductionthreshold at which outcrossing is favoured over selfing(Lloyd, 1992; Husband & Schemske, 1996). Furthermore, thepresence of distinct and recognizable ‘unfit’ phenotype classeswithin the selfed progeny arrays is probably attributable to theexpression of recessive alleles of large deleterious effect com-prising part of the genetic load in S. squalidus (Fig. 6). Similarly,unfit progeny expressing alleles of large deleterious effectswere also observed upon selfing in another SI Senecio species,S. vernalis (Comes, 1994). It is clear that inbreeding depres-sion and genetic load are severe and the balance of selectionfavours outcrossing and SI in British S. squalidus. Even if thereis a temporary weakening of SSI during the initial coloniza-tion phases, PSC and other selfing phenotypes cannot invadeor persist in S. squalidus populations because of severeinbreeding depression. This study of inbreeding depressionfocused primarily on plants from Oxford, so it would beinformative to investigate inbreeding depression in plantsfrom peripheral populations to check if any purging of geneticload has occurred in response to colonization. Certainly, itappears that little purging of genetic load occurred inS. squalidus during its initial population bottleneck duringintroduction via Oxford. Neither has this occurred for otheroutcrossing Senecio species, S. integrifolius and S. vernalis, alsosubject to population disturbance, adding to the evidence forthe association between inbreeding depression and themaintenance of SSI in Senecio and more generally in theAsteraceae (Widén, 1993; Comes, 1994).

Finally, it is worth noting that the intermediate within-population levels of inbreeding inferred by population geneticsurveys of genetic and isozyme diversity in S. squalidus( f = 0.00–0.47 at various loci; Abbott et al., 2000; Brennanet al., 2003; authors’ unpublished data) could not be gener-ated by the low selfing rates observed in this study (Fig. 2a,b).Rather than selfing, these levels of inbreeding in S. squalidusmay be caused indirectly by biparental inbreeding. The exten-sive dominance interactions observed between the S allelesthat make up the SSI system in S. squalidus would certainlypermit regular matings between closely related plants (Abbott& Forbes, 1993; Hiscock, 2000b; Brennan et al., 2002,2003). Perhaps S. squalidus can tolerate these more moderatelevels of inbreeding induced by family matings rather thanselfing as a necessary trade-off between minimizing inbreedingdepression and maximizing reproductive assurance.


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Acknowledgements

This work was supported by a BBSRC studentship to A.C.B.and a BBSRC David Phillips research fellowship to S.J.H.Extra maintenance funding for A.C.B. was provided by theDepartment of Plant Sciences, University of Oxford, MarkQuested Award; Fishmonger’s Company, London, MargaretPollock Award; Somerville College, University of Oxford; andThe Lawlor Foundation, Belfast. We thank two anonymousreviewers for improving the paper, Phil Smith for maintainingthe plants, Melanie Edrich for measuring fitness traits andProf. Richard Abbott, Dr Paul Ashton, Dr David Tabah, DrSuzanne O’Shea, Dr Catherine Jones and Mr Ian Howarth,who all contributed to field sampling.

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Modes and rates of selfing and associated inbreeding depression in the self-incompatible plant Senecio squalidus (Asteraceae): a successful colonizing species in the British Isles