Self-incompatibility in Senecio squalidus L. (Asteraceae)

Annals of Botany 85 (Supplement A): 181-190, 2000doi:10.1006/anbo.1999.1058, available online at http://www.idealibrary.com on IDEIa

Self-incompatibility in Senecio squalidus L. (Asteraceae)

SIMON J. HISCOCK*

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX] 3RB, UK

Received: 21 July 1999 Returned for revision: 1 September 1999 Accepted: 11 November 1999

The Asteraceae, one of the largest families of flowering plants, shows an extreme diversity of breeding systems withmany species having a pronounced flexibility in breeding strategy a factor thought to be crucial to the success of thisfamily in colonizing most major biogeographical regions. Despite self-incompatibility (SI) being the most commonmode of outcrossing in the Asteraceae, there have been few detailed studies of SI within this family. This paperdiscusses the SI system operating in Senecio squalidus (Oxford ragwort), an invasive alien species that has colonizedmost parts of the UK within the last 150 years. In S. squalidus, SI is sporophytic and controlled by a single multiallelicS locus. Preliminary findings suggest that UK populations contain few S alleles. Pseudo self-compatibility (PSC) hasbeen found to operate in S. squalidus and leads to total or partial loss of S-specific discrimination as females, thoughpollen S function is unaffected. Exceptional compatible and semi-compatible crosses in normally incompatible groupssuggest that a cryptic gametophytic element (the G gene) can influence the outcome of certain crosses. It is suggestedthat PSC, mediated by modifier loci unlinked to the S locus, together with the apparent activity of a G gene indetermining SI, may have provided flexibility in the SI system of S. squalidus that was crucial to its success as acolonist. Basic aspects of the cell biology of the pollen stigma interaction during compatible and incompatiblepollinations in S. squalidus are discussed in the light of previous studies of species in the Asteraceae and Brassicaceae.

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Key words: Senecio squalidus, Asteraceae, self-incompatibility, S-alleles, pollen-stigma interactions.

INTRODUCTION

One of the most fundamental facts to emerge from studiesof self-incompatibility (SI) in flowering plants is theextraordinary diversity of systems through which SI canoperate (Hiscock et al., 1996; Hiscock and Kies, 1999).Even within the tight genetic constraints of gametophyticand sporophytic determination there is great variation in:(a) the number of loci involved; (b) the number of allelesinvolved; (c) the sites of pollen tube recognition and'inhibition'; and now, very importantly (d) in the moleculesand cell signalling pathways that mediate SI responses. Themolecular diversity of SI systems in flowering plants is instark contrast to the relative conservation of the molecularmachinery that determines SI through mating type in fungi.Mating type systems in fungi probably have a commonancestral 'type' from which other systems have beenelaborated, but in flowering plants diversity among SIsystems most certainly reflects multiple origins of differentSI systems during angiosperm diversification (Hiscock andKiles, 1999). However, the relative abundance of 'late-acting' SI systems (Sage et al., 1994) and gametophytic SI(GSI) systems together with the apparent lack of sporo-phytic SI (SSI) systems in basal angiosperm lineagessuggests that certain late-acting and gametophytic SIsystems (particularly those with multiple loci) are likelyto be more ancient than the sporophytic systems whichappear to be exclusive to more 'advanced' angiospermfamilies (Hiscock and Kies, 1999). The recent report of

* Fax +44-1865 275074, e-mail simon.hiscock(dplant-sciences.ox.ac.uk

0305-7364/00/0A0181 + 10 $35.00/00

both gametophytic and sporophytic SI in a single family,the Polemoniaceae (Goodwillie, 1996), poses some inter-esting evolutionary questions about the relative ages of thetwo SI systems within this family-could it be thatselection is favouring the superimposition of the moreefficient sporophytic systems ('efficient' in terms of itsgreater restriction on biparental inbreeding) upon anancient gametophytic system? Evidence from the Brassica-ceae (Lewis et al., 1988; Zuberi and Lewis, 1988) certainlysuggests that in Brassica campestris and Raphanus sativusthe sporophytic SI system is superimposed upon anunderlying gametophytic SI system that is detectable onlyin certain exceptional crosses between plants containing Salleles high in the dominance series. Whether this crypticgametophytic system is the relic of an ancient system ofGSI inherited by brassicaceous species from an earlyancestor that has subsequently been succeeded by acqui-sition of SSI is a matter for speculation (see Lewis, 1994).Alternatively the current sporophytic system may haveevolved from this GSI system by a switch in the time ofgene expression. Speculations such as this, relating to thephylogenetic and evolutionary relationships between SIsystems can only be addressed if more detailed genetic andmolecular genetic studies are carried out on SI systems inspecies from families that have hitherto received little or noattention. It was with this in mind that the current study ofSI in the Asteraceae was initiated using Senecio squalidus(Oxford ragwort) as a model species.

Genetic studies carried out in the 1950s on SI in theAsteraceae demonstrated that in Crepisfoetida (Hughes andBabcock, 1950), Parthenium argentium (Gerstel, 1950) and

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Hiscock et al.-Self-incompatibility in Senecio squalidus L.

Cosmos bipinnatus (Crowe, 1954) SI is under the sporo-phytic control of a single multiallelic S locus. Indeed thefirst demonstration and explanation of the sporophyticsystem was that of Hughes and Babcock (1950) for Crepisfoetida, a work previously consigned to the PhD thesis ofHughes (1943) at Berkeley (cited by Lewis, 1994). Sincethese pioneering studies of sporophytic SI in the Asteraceaefew genetic, and no molecular genetic studies have followed(but see Verma and Singla, 1990; Lewis, 1994) so we do notknow whether SSI in species of the Asteraceae operatesthrough the same receptor kinase/secreted glycoproteinsystem (SRK/SLG) that mediates SSI in the Brassicaceae(Nasrallah et al., 1994) or whether an alternative molecularsystem is involved. Structural and cytological studies ofpollen-stigma interactions following compatible andincompatible pollinations in Cosmos and Helianthus havehighlighted similarities between the incompatibility reactionin the Asteraceae and the Brassicaceae; such similaritiesinclude the release of wall-held pollenkit (rich in lipids andproteins) upon contact between pollen and stigma (Knox,1973; Howlett et al., 1975), the arrest of incompatible pollenon the stigma surface before penetration of the cuticle(Vithanage and Knox, 1977) and deposition of the 31-3glucan callose within pollen tubes and adjacent stigmaticpapillar cells during or after pollen tube arrest (Knox, 1973;Vithanage and Knox, 1977). These structural studies alsosuggested that the stigma surface of Cosmos and Helianthusis, like Brassica, of the 'dry' type (Heslop-Harrison andShivanna, 1977) with a continuous cuticle overlaid by a thinproteinaceous pellicle and with no obvious surface secretion(Vithanage and Knox, 1977). A later TEM study of pollen-stigma interactions in Cosmos and Helianthus usinganhydrous fixation conditions, however, indicated that thestigma surfaces of these species may not be entirely 'dry'and that stigmatic secretions were evidently induced bypollen-stigma contact (Elleman et al., 1992). In this paperstudies on the genetics of SI in S. squalidus are reportedtogether with an account of the main cellular events thatoccur during compatible and incompatible pollen-stigmainteractions. Before discussing the details of the SI system inS. squalidus it is worth reflecting upon the uniquepopulation biology of this species within the British Islesbecause the nature and behaviour of the SI system must beviewed and interpreted in the light of this populationhistory.

THE UNIQUE POPULATION BIOLOGY OFS. SQUALIDUS

S. squalidus was introduced into Britain in the late 17thCentury from Sicily via the Botanic Garden in Oxford(hence 'Oxford ragwort'). Here it remained for nearly150 years (Kent, 1956) until during the late 18th Centurywhen it 'escaped' from the gardens and became noticeablegrowing in old college walls where it spread to most parts ofthe city. The rise of the railways during the 19th Centuryand the expansion of the railway network from Oxfordprovided further suitable, nutrient-poor arid environmentsfor S. squalidus to colonize (the clinker beds) and graduallyit spread to most parts of Britain, so that today there are

only a few parts of Scotland and Wales where Oxfordragwort cannot be found (Kent, 1960). Indeed it is widelybelieved that the entire UK population of S. squalidus isderived from this single introduction event (Kent, 1960;Crisp, 1972; Abbott, 1992; Abbott and Forbes, 1993). Suchan astonishing fact raises many very important questionsabout the SI system of S. squalidus; for instance, how andwhy has such an obligate outcrosser been so successful asan invasive colonizer when colonizing species are usuallyself-compatible (Stebbins, 1957; Baker, 1967; Abbott andForbes, 1993)? Additionally, how has the SI system inS. squalidus been maintained, apparently intact, despite aserious initial population bottleneck within the founderpopulation? Previous studies have shown that underpopulation bottlenecks, where mating potential is reducedas a consequence of diminished numbers of shared S alleles,SI systems may break down, leading to self-compatibility(Reinartz and Les, 1994). Thus, as well as being an idealspecies in which to study the molecular genetics of SSI inthe Asteraceae, S. squalidus also offers unique opportunitiesto study the maintenance and dynamics of an SI system in a'natural' population that has experienced extreme popu-lation changes within a well documented time period.

THE SI RESPONSE IN S. SQUALIDUS

As with other members of the Asteraceae, the SI response inS. squalidus takes place at the stigma surface (Fig. IC. D).In an incompatible pollen-stigma interaction most pollengrains fail to develop beyond the hydration stage (Fig. C)and if pollen tubes are produced they are arrested on thestigmatic surface after very little subsequent growth or veryrarely after penetration of the stigma (Fig. D). When thestrength of the incompatibility system was tested by seed setfollowing controlled self-pollinations most plants tested setno seed, while the few individuals that did set seed set onlyone five seeds per capitulum compared to the 20-60 seedscharacteristically set following cross pollination (S. Hiscockunpubl. res.). Not surprisingly the incompatibility responseappears the same on stigmas of both disc florets (hermaph-rodite) and ray florets (female), although the receptivity ofdisc florets towards compatible pollen appeared to begreater than that of ray florets. Indeed, consistently morecompatible pollen grains were found to germinate andpenetrate the stigmas of disc florets than developed on thestigmas of ray florets (Fig. 2). The significance of thisobservation is unclear, but if the primary function of rayflorets is to attract pollinators (hoverflies) (Abbott andIrwin, 1988), and not therefore exclusively to motheroffspring, one could speculate that female fertility resourcesmight be better directed towards the central disc floretswhere pollinators alight and where most pollen is generallydeposited. Because of the fairly low percentage penetrationrates for pollen tubes under compatible pollinations, a testfor pollen viability was carried out using fluoresceindiacetate (Heslop-Harrison and Heslop-Harrison, 1970).This test revealed pollen viabilities ranging from 16.5 to53% with a mean value of 29.6%, compared to the controlestimate for Brassica oleracea pollen of 88% viable pollen.These low pollen viability estimates for S. squalidus could

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FIG. 1. Fluorescence micrographs of compatible and incompatible pollen-stigma interactions in S. squalidus. Pollen tubes were visualized usingdecolourized aniline blue (Linskens and Esser, 1957; see Fig. 2) and photographed using a Zeiss Axiophot microscope under ultravioletillumination. A and B, Compatible pollinations-pollen tubes can be seen clearly penetrating the stigma surface. Note in B no callose deposits canbe seen in the pollen tubes or adjacent papillar cells (arrows) even though patches of callose are visible in some parts of the stigma (triangles). Cand D, Incompatible pollinations-in C pollen grains have hydrated but failed to germinate and callose can be seen within nearby papillar cells(arrow). D shows an incompatible pollen grain that has germinated whose pollen tube has penetrated the stigma surface. Arrest is characterized bya swelling of the tube tip and deposition of callose within the tube and within adjacent papillar cells (arrows). The second incompatible pollen tube

was arrested before penetration, again with callose deposition in and around the tube.

certainly help to explain the low penetration countsobserved in compatible pollinations and are similar toestimates of 26% viability obtained by Abbott and Forbes(1993). Nevertheless, only a single successful penetration isrequired to fertilize the single ovule of each flower.

GENETIC DETERMINATION OF SI

Genetic analysis of SI in S. squalidus has demonstrated that,as in other previously investigated species in the Asteraceae,SI is determined sporophytically and is under the control ofa single polyallelic S locus. These data are presentedelsewhere (Hiscock, 2000), so only a brief summary of theresults will be discussed here. The genetic analysis was

carried out in order to (a) confirm the assumed sporophyticnature of SI in S. squalidus and (b) to generate lines ofplants with defined S alleles, particularly S homozygotes,for use in the molecular analysis of SI. Plants from twoOxford populations were collected and tested for thestrength of their SI system by pollen tube growth andseed set. Four individuals with strong SI, two from eachpopulation, were subjected to 'forced' selfing by treatingtheir stigmas with dilute salt solution prior to pollination(see below). Assuming that SI is controlled by a singlesporophytic locus and that parental plants selected wereheterozygous at the S locus, their two S alleles wouldbe expected to segregate in a 1:2:1 ratio of homozygoteA:heterozygotes AB:homozygote B. Intercrossing such a

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FIG. 2. The strength of the SI system operating in disc and ray florets.Individual florets were removed from capitula and maintained in watersuspended in a perspex capillary block. Florets were pollinated byapplying appropriate pollen with a fine sable paint brush whilst viewingthe stigma under a binocular microscope. Six- 18 h later pistils wereremoved from the pollinated florets and fixed in chloroform:absolutealcohol:acetic acid (6:3: 1) for I h, softened in IM NaOH for I h andthen squashed between a microscope slide and a cover slip andmounted in 0.1% aniline blue (Linskens and Esser, 1957). Pollinatedstigmas were then observed under UV illumination (see Fig. ) and thenumber of pollen tubes penetrating the stigma counted. Twenty five-30pollinations were carried out for each type of cross or self. Vertical lines

represent standard errors.

progeny array would than make it possible to identifythe homozygotes and heterozygotes by their compatibilityand incompatibility relationships (see Lewis, 1979 fordetails).

Progenies derived from parental plants from the firstpopulation (OS, Oxford Station) showed an identicalpattern of incompatibility, with two incompatibility groupsbeing clearly definable (Table 1). This two-group pattern iseasily explained by single locus sporophytic control byassuming complete dominance of one allele in both pollenand stigma (Table 1). One incompatibility group thusconsists of plants homozygous for the recessive allelewhile the second incompatibility group consists of plantshomozygous for the dominant allele and also of hetero-zygotes-the two being indistinguishable by SI phenotype.

TABLE 1. Summary of results rom full diallel crosses of twoselfed progeny arrays of parent plants from the OS population

SwSw SwSx SxSx

SwvSwSS_- SxSx +

Both diallels showed the same two incompatibility group patternwhich is consistent with sporophytic determination of SI and one Sallele being completely dominant in both pollen and stigma (Sx). Notethat because of the dominance effect, individuals SxSx and SwSx showidentical incompatibility phenotypes.

Interestingly, the two progeny arrays derived from parentalplants from the second population (A34, a major trunk-road) also shared an identical inter-compatibility pattern.However, this time three incompatibility groups could beidentified, with two groups showing clear reciprocaldifferences in incompatibility-a feature exclusively associ-ated with SSI and reflecting differential dominance effectsof an allele in either pollen or stigma. This pattern is easilyexplained by assuming dominance of one allele in thestigma and co-dominance in the pollen (Table 2). All allelicdesignations were subsequently tested and confirmed by abest-fit analysis using a computer program written by YanWong (Department of Plant Sciences, University ofOxford, UK) which tested all possible dominance scenarioswithin the sporophytic system.

TABLE 2. Summary of results from full diallel crosses oftwo selfed progeny arrays of parent plants from the A34

population

SaSa SbSb SaSh

SaSa - +ShSh + -SaSh +

Both diallels showed identical three incompatibility group patternswith a clear reciprocal difference in incompatibility between two of thegroups. This pattern is consistent with sporophytic SI and one S allele(Sb) being dominant in the stigma; S alleles show codominance inpollen.

HOW MANY S ALLELES?

Plants with defined S genotypes were intercrossed to deter-mine how many S alleles were shared between the originalfour parental plants (Hiscock, 2000). Surprisingly fourhomozygotes were incompatible with each other andtherefore shared the same S allele (designated S, ) whichwas the allele shown to be recessive in the stigma in allprogeny arrays and recessive in the pollen in the two OSprogeny arrays. The remaining two A34 homozygotes wereinter-compatible and also compatible with the SIS t homo-zygotes and so were designated S2S2 and S3S 3. When plantsof the OS progeny arrays, consisting of homozygotes andheterozygotes indistinguishable by phenotype were inter-crossed they were found to be incompatible, indicating thatthey shared a second S allele (designated S4) in addition tothe Sl allele; hence both original OS parental plants wereSS 4 . Thus the four original parental plants contained justfour S alleles suggesting that the number of S alleles withinand between populations of S. squalidus is low. Such afinding is consistent with the extant UK population beingdescended from just a few original plants. Work is currentlyin progress to obtain more objective counts of the numbersof S alleles within UK populations of S. squalidus so thatan estimate can be made of the total number of S allelespresent in British S. squalidus.

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IS A SECOND GAMETOPHYTIC (G) LOCUSINVOLVED IN DETERMINING SI IN

S. SQUALIDUS?

A regular finding during the genetic analysis of SI inS. squalidus was the occasional, but regular occurrence of'anomalous' crossing results, particularly the occurrence ofexceptional compatible or semi-compatible crosses ingroups that were shown to be predominantly incompatible(Hiscock, 2000). Repeated crossing and analyses of pollentube growth and seed set confirmed the reality of theseanomalies such that they could not be attributed to 'noise'in the system (Table 3). The scattered and unpredictablenature of these anomalous crosses defies explanation by asecond sporophytic locus. Exceptional crossing results,especially compatible ones, in predominantly incompatiblegroups appear to be a common feature of sporophytic SIsystems in the Brassicaceae and Asteraceae (Lewis et al.,1988; Zuberi and Lewis, 1988; Lewis, 1994; Table 4). Aftera series of meticulous genetic analyses of bud-self-derivedprogeny arrays from such exceptional ('anomalous') crossesin Brassica campestris and Raphanus sativus (Brassicaceae),Lewis and his co-workers 'in desperation invoked anheretical second gametophytic gene' which showed nearnormal gametophytic segregation patterns in selfed progenyarrays (reviewed by Lewis, 1994). This gene, G, whichappeared to have only two alleles in the accessionsanalysed, was proposed to exist usually in the homozygousstate, especially in cultivated varieties, and so was normallyundetectable. Only in heteroallelic stocks and wild popu-lations could the presence of the G gene be detected as aconsequence of exceptional compatible crossing results.Lewis (1994) then went on to suggest that this underlyinggametophytic SI system may be common to all sporophyticSI systems. Certainly within all of the selfed progeny arraysanalysed in S. squalidus anomalous compatible crosses weredetected at a mean frequency of 19-2% (Table 3), suggest-ing that if such anomalies are indeed due to alleles of the Glocus, then S. squalidus may well possess the sporophytic-gametophytic SI system proposed by Lewis. Preliminarygenetic analyses of progenies derived from anomalouscompatible crosses do suggest that a 'gametophyticelement' could account for the production of the observedpatterns of compatibility and incompatibility (data notshown). However, further genetic analyses are needed,particularly of incompatibility/compatibility segregationpatterns in forced self progeny arrays, before the presenceof such a G gene can be demonstrated conclusively forSenecio. If, indeed, the G gene is a feature of all sporophyticsystems (as predicted by Lewis, 1994) we have to ask whatthe G gene does in relation to the gene products of the Slocus and if the G gene is being maintained in thesporophytic system or if it is being lost. In short, is the Ggene just a curious relict of an ancient gametophyticSI system, or is the G gene an essential part of the extant SIsystem? If G is selectively maintained as part of the SIsystem perhaps its role might be to add flexibility to themore stringent sporophytic SI systems by increasing matingpotential when S allele numbers are low and when co-dominance of alleles drastically reduces mating potential

TABLE 3. Percentage of 'anomalous' crossing resultsobtained in the Jour full diallel crosses of selfed progeny

arrays (adapted from Hiscock, 2000)

Family Percentage anomalous +/- Percentage anomalous -and + compatibilities in incompatibilities inincompatible (-) groups compatible (+) groups

OS-Bill self 22-1 4-5OS-2 self 13.5 1.6A34-3 self 22.0 -A34-4 self 19.3 4.1

Total 19.2 2.6

Note that most anomalies represent exceptional compatible andsemi-compatible outcomes in generally incompatible groups. OS-Bill,OS2, A34-3 and A34-4 are the designations given to parental plantsfrom which selfed progeny arrays were derived.

TABLE 4. Percentage of anomalous compatible and semi-compatible crosses in S. squalidus compared with percentagesof anomalous compatible and semi-compatible crosses inother species with sporophytic SI from the Asteraceae and

Brassicaceae (adapted from Hiscock, 2000)

Species Percentage anomalous +/- and +compatibilities in incompatible groups

Senecio squalidus 19-2Crepis foetida 14.3Hypochoeris radicata 34.5

Brassica campestris 12-5Raphanus raphanistrum 9.2Raphanus sativus 7.9Brassica oleracea 7.8

Adapted from Lewis et al. (1988) and Lewis (1994).

between siblings? Evidence indicating gametophytic activityof S alleles in the pollen of Cerastium arvensis (Lundqvist,1990), which has SSI and high levels of codominancebetween S alleles, supports such a prediction, but whetherthe apparent gametophytic expression of a normallysporophytically-expressed S gene in Cerastium actuallyreflects the activity of G is uncertain (Lewis, 1994).

PSEUDO SELF-COMPATIBILITY INS. SQUALIDUS

Flexibility within the SI system of S. squalidus also appearsto arise from the well-known phenomenon of pseudo self-compatibility (PSC) (reviewed in de Nettancourt, 1977).Pseudo self-compatibility leads to the production of selfseed by, normally, SI individuals, but seed set after cross-pollinations is always considerably greater than seed setfollowing selfing (Pandey, 1959; de Nettancourt, 1977;Levin, 1996). Pandey (1959) defined three classes of pseudoself-compatibility: (a) environmental, a consequence of, forinstance, temperature, day length, age etc; (b) artificial,brought about by mechanical or chemical treatment ofplants or flowers, for instance 'bud' pollination or CO2treatment; and (c) genetic, caused by the action of modifier

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Hiscock et al.-Self-incompatibility in

loci unlinked to the S-locus. All three types of pseudoself-compatibility have been observed in S. squalidus(S. Hiscock, unpubl. res.) but only two (artificial andgenetic) will be discussed here.

Artificial PSC

In an attempt to find ways of weakening SI in S. squalidusto produce inbred progenies, various treatments known toovercome SI in other species were tested. Bud pollinationwas impossible because receptive stigmatic surfaces onlybecome exposed when flowers are sufficiently mature for theSI system to be functional. Treating stigmas with dilute saltsolutions prior to pollination did, however, prove to besuccessful in weakening the SI response in most plantstested such that elevated levels of pollen tube penetrationwere observed (Fig. 3) and self seed was produced.Interestingly, salt treatment of stigmas from disc floretsreduced pollen tube penetration in compatible pollinationsrelative to the water control but either had no effect orincreased compatible pollen tube penetrations on stigmasfrom ray florets (Fig. 3). Despite the widespread use of salt-treatment by plant breeders to produce inbred lines ofbrassicas (Fu et al., 1992) the mechanism by which saltweakens the SI system is not known, nor is it known howmany different SI systems are sensitive to salt treatment. Itis interesting that salt weakens the SI system of S. squalidus,because large populations of this plant grow along majorroadsides where salinity levels are usually artificiallyelevated as a consequence of road salting during winter.Our results suggest that in these 'salty' environments the SIsystem could be weakened allowing an increase in matingpotential that could facilitate the continued spread of thespecies in situations where there are high levels of S allelesharing.

To determine whether other treatments known to over-come SSI in Brassica also overcome SSI in S. squalidus, theeffect of cycloheximide (a protein synthesis inhibitor) on SIwas tested. Cycloheximide overcomes the stigmatic barrierto pollen tube penetration in Brassica by a mechanism asyet unknown (Sarker et al., 1988) but pollen tubes thencease growing in the stigma or style, presumably becausethe tubes lose their ability to synthesize protein. Similareffects were found in S. squalidus florets treated withcycloheximide (Fig. 4)-stigmatic incompatibility was over-come but pollen tubes later ceased growing within the style.Interestingly, this treatment, like salt treatment, slightlyreduced the number of compatible pollen tubes penetratingstigmas of disc florets while at the same time slightlyincreasing the number of compatible pollen tubes penetrat-ing stigmas of ray florets. These findings suggest thatwhatever the mechanism of SSI in Brassica and Senecioboth species share a common physiological requirement forcontinuous protein synthesis during the stigmatic SSIresponse.

Genetic PSC

When individuals from forced self progeny arrays weretested for self-incompatibility, approximately 11% of the

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FIG. 3. Effect of salt treatment on the SI response in disc and rayflorets. Florets were maintained as in Fig. 2. Prior to pollinationstigmas were gently dabbed with a sable paint brush soaked in 1% or5% aqueous NaCI containing 0.1% Triton X100 as a wetting agent.Stigmas were allowed to dry for 2-3 h and then pollinated as in Fig. 2.Pistils were then fixed, stained and observed as in Fig. 2. Vertical lines

indicate standard errors.

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FIG. 4. Effect of cycloheximide treatment on the SI response in disc andray florets. Florets were maintained in perspex capillary blocks as inFig. 2 except that the water contains 10 mM cycloheximide. Pollinatedstigmas were prepared and observed as in Fig. 2. A total of 20 replicateswere carried out for each treatment. Vertical lines indicate standard

errors.

progeny were found to be pseudo self-compatible (Hiscock,2000). PSC individuals set seed to the extent of approx.three-five seeds per capitulum after selfing compared toapprox. 20-60 seeds per capitulum after cross pollination.These data suggest that individuals are highly heterozygousat S gene modifier loci. Interestingly, the pseudo self-compatible phenotype was also associated with a total orpartial loss of S-specific discrimination by the stigma suchthat all PSC individuals were generally compatible asfemale parent with all other individuals, irrespective of Sgenotype (Table 5; Hiscock, 2000). However, when pollencompatibilities were observed the PSC plants clearlyretained an S imprint in their pollen which allowed their

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TABLE 5. Compatibility behaviour of pseudo self-compatible(PSC) individuals towards other SI individuals within a

selfed progeny array diallel

SIS, SIs4/s4s4 PSC

SIS] - + s,s4s4 + -PSC + + +/- + +/-

Note that PSC individuals show total or partial loss of S-specificdiscrimination as female but retain their S imprint in the pollen.

S genotype to be determined by compatibility/incompat-ibility relationships with other individuals. It was concludedtherefore that the modifier genes responsible for producingthe PSC phenotypes had their effects exclusively on S genesexpressed in stigmatic tissues.

CELLULAR EVENTS AT THE STIGMASURFACE DURING COMPATIBLE AND

INCOMPATIBLE POLLINATIONIN S. SQUALIDUS

Stigmatic surfaces of members of the Asteraceae haveusually been described as 'dry' (Heslop-Harrison andShivanna, 1977). Initial TEM observations of the papillatestigma of S. squalidus revealed, however, a distinct secretedmatrix on the surface of the stigma between the papillarcells (Fig. 5A). This secretion, containing lipids, proteinsand carbohydrates (Hiscock and Hoedemaekers, unpubl.res.), appears not to extend over the entire surface of thepapillae which is 'dry' and cuticularized, suggesting thatthis type of stigma is intermediate between the 'dry' typeand the 'wet' type (Heslop-Harrison and Shivanna, 1977).It is proposed, therefore, that the Senecio type of stigma bereferred to as 'dry-wet'. During both compatible andincompatible pollen-stigma interactions, stigmatic secre-tion is enhanced in papillar cells subjacent to the adheringpollen grains apparently in response to the dramaticoutpouring of pollenkit from the exine region of the pollenwall (Fig. 5B-D). At this time (approx. 30-60 min afterpollination) distinct 'secretory bodies' can be seen withinthe cell wall (Fig. 5C) that appear to empty their contentsinto the extracellular spaces between papillar cells, wheretheir contents mix with the pollenkit (Fig. 5D). It isprobable that this interaction between pollen-derived andstigma-derived material which together form a character-istic attachment 'foot' between pollen grain and papillar cellrepresents an important stage in the signalling processesthat lead to acceptance or rejection.

Compatible interactions

During compatible pollen-stigma interactions, 'foot'formation is accompanied by full hydration of the pollengrain and emergence of a pollen tube through one of threecolpi. The pollen tube then grows between papillar cellswithout direct penetration of the cuticle. Penetration of thestigmatic cuticle occurs at the base of the papillae where the

cells are closely packed and where the cuticle disappears(Fig. IA,B). Pollen tubes then grow intercellularly betweenstigmatic cells and after a short distance of downwardgrowth tubes turn through 90° and follow the angle of thestigmatic branch until they reach the style where theycontinue to grow intercellularly downwards until they reachand fertilize the single ovule.

Incompatible interactions

In most incompatible pollen-stigma interactions, pollendevelopment ceases before germination of the grain withmany incompatible grains failing to adhere to or hydratefully on the stigma (Fig. C,D). Arrest of these pollen grainsis frequently accompanied by the accumulation of materialwithin the cell walls of papillar cells directly beneath thegrains (Fig. 5E,F). This material which must be derivedfrom the stigma has yet to be fully characterized, althoughit appears not to be callose, appearing more 'lipid-like'under TEM. When this wall material was observed intissue subjected to anhydrous fixation in osmium vapour itappeared electron dense, a feature typical of osmiophilliclipids (Fig. 5E). When conventionally fixed tissue wasobserved, however, the region of wall swelling appearedboth electron dense and electron lucent (Fig. 5F),suggesting that some of the osmiophillic wall material hadbeen washed out in the initial stages of aqueous fixation, asmight be expected for a lipid-based substance. In someinstances, incompatible pollen grains germinate and pro-duce a short tube which is arrested as it grows betweenpapillar cells with a concomitant deposition of callose bothwithin the tube and within adjacent papillar cells (Fig. D).In exceptional cases incompatible tubes can be seenpenetrating the cuticle and growing into the conductivetissue of the stigma (Fig. 1 D) only to be arrested before theybegin the 900 turn-arrest, in this case, being characterizedby a swelling of the tube tip and deposition of callose withinthe tube and surrounding stigmatic cells. This latterincompatible phenotype is particularly associated withpollen tubes that fail to reach the stylar tissue in exceptional('anomalous') semi-compatible crosses. If this correlation isshown to be correct, it may be that this unusual incom-patibility phenotype reflects the action of G alleles (in a halfcompatible gametophytic scenario) which produce anincompatibility reaction more reminiscent of GSI than SSI.

CONCLUSIONS

Data presented in this paper indicate an intrinsic flexibilitywithin the sporophytic SI system of S. squalidus. If thenumber of S alleles within British populations is indeedvery low, as suggested by our preliminary data and impliedby the population history of S. squalidus in the UK,then such flexibility would be a significant advantage toS. squalidus as an outcrossing colonizer likely to experiencepopulation bottlenecks. Outcrossers are generally thoughtnot to make successful colonists (Stebbins, 1957; Baker,1967) although a recent reassessment of 'Baker's Law'(which basically states that self-compatible individuals arelikely to be more successful as colonists than SI individuals)

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FIG. 5. Transmission electron micrographs of pollen-stigma interactions in S. squalidus. A-E, Fixed in osmium vapour and post-fixed inglutaraldehyde according to Elleman and Dickinson (1986). F, Fixed using a modified Karnovsky's formulation (Elleman et al., 1992). Thinsections (60-100 nm) were stained with uranyl acetate and lead citrate according to standard protocols. A, Transverse section of stigmatic papillarcells. Note the extracellular stigmatic secretion present between the stigmatic papillae (M). B, Longitudinal section through pollen-stigmainterface showing the attachment 'foot' consisting of pollenkit, derived from the exine (E) of the pollen and material derived from enhancedsecretion by the stigmatic papillar cells (arrows). Note that the cell wall of the papillar cells directly below the pollen grain has expanded andcontains 'secretory bodies'. C, Longitudinal section through papillar cell wall directly below pollen grain. Note the 'secretory bodies' present in thecell wall (arrows) apparently emptying their contents into the extracellular space containing pollenkit. D, Detail of 'secretory bodies' in cell wall(w) of papillar cells. Material with electron opacity similar to the secretory bodies is clearly present in the extracellular space in contact withpollenkit (p). E, Longitudinal section through pollen-stigma interface of incompatible pollination showing the pronounced swelling of thepapillar cell wall (w). The material within the swelling has similar electron opacity to the material present in secretory bodies. E. Exine wall ofpollen grain. F, Detail of papillar wall swelling seen in incompatible pollinations after conventional aqueous fixation. Note wall swelling containselectron-dense material only in the upper region; the presence of an electron luscent region below suggests that a proportion of the electron-dense

material has been washed out during fixation, implying that this material could be lipidic.

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indicates that under certain conditions SI individuals can besuccessful colonists, particularly if they are perennial orhave good seed longevity (Pannell and Barrett, 1998).S. squalidus is a short-lived perennial and individuals canpersist for 3-5 years (Hiscock, unpubl. res.). Thus, acombination of perenniality and a flexible breeding systemcould be responsible for the success of S. squalidus as acolonizing outcrosser and account for its rapid spread tomost parts of Britain during the last 150 years.

Two sources of flexibility in the SI system of S. squalidushave been highlighted. First, the relatively high frequency(19-2%) of exceptional compatible or semi-compatiblematings in predominantly incompatible groups suggeststhe action of a cryptic gametophytic element (the G gene?)in controlling the outcome of certain crosses where S allelesare shared but G alleles probably are different. Thisgametophytic/sporophytic type of SI determination waspreviously demonstrated by Lewis et al. (1988) and Zuberiand Lewis (1988) for the Brassicaceae and by Lundqvist(1990, 1995) for the Caryophyllaceae (although with slightdifferences in interpretation). One might predict that such agametophytic/sporophytic system would have a selectiveadvantage in situations where population numbers fluctuateand where population bottlenecks occur because the Gsystem would increase the mating potential of individualswith shared S alleles. The fact that anomalous compatiblecrosses are more frequent among plants derived from wildpopulations than they are in plants residing in plantbreeders' inbred stock lines (Lewis, 1994, and this study)may reflect the selective maintenance of larger numbers ofdifferent G alleles in wild populations.

The second flexibility element in the breeding system ofS. squalidus is pseudo self-compatibility. Forced inbreedinggenerated PSC individuals at a frequency of approximately11% suggesting that S. squalidus is highly heterozygous atloci that modify S gene action. Even though selfing is rarein natural populations of S. squalidus, biparental inbreedingappears to be common (Abbott and Forbes, 1993, and thisstudy) and could account for the generation of PSCindividuals. The existence of PSC individuals in naturalpopulations, albeit at low frequency (Hiscock, unpubl. res.),suggests that PSC is a natural source of flexibility within thebreeding system. PSC individuals were also found to becompatible or semi-compatible as female parent with allother plants tested even though their pollen S allelic imprintremained unaffected. This lack of female S discriminationfurther increases the mating potential of PSC individualswithin a population which would give these individuals adistinct advantage in bottleneck conditions. Extremebottlenecks can cause the total breakdown of SI systemsto yield self-compatible individuals (Reinartz and Les,1994) so flexibility within an SI system must be crucial to itsmaintenance in strong functional form through bottlenecks.Indeed, Levin (1996) suggested that genetic PSC is a keyreproductive strategy enabling SI species to negotiatepopulation bottlenecks without dissolution of the out-breeding system. During population bottlenecks whenmating potential is restricted by reduced numbers of Salleles, selection will favour the spread of pseudo self-compatible individuals and allow population numbers to

increase. The offspring of pseudo self-compatible parentsmay suffer the effects of inbreeding depression, but this maybe outweighed by reproductive assurance. As the popu-lation size increases, selection will once again favouroutcrossing as a consequence of the elevated levels ofinbreeding depression and the SI system will once againassume its former strength, especially if, during thispopulation expansion, new S alleles have entered thepopulation by migration or perhaps through the generationof new S specificities. In this way the SI system can assumea flexibility during population bottlenecks and then returnto full strength when the bottleneck is alleviated. Thepresent studies on Senecio tend to support this hypothesis.Furthermore, Levin's predicted requirement for inbreedingdepression as the selective force needed to reinstate fullyfunctional SI when population numbers increase, alsoappears to be met by the apparently high genetic loadcarried by individuals of S. squalidus because progeniesfrom forced selfings showed clear signs of the effects ofinbreeding depression-reduced germination rates, reducedgrowth rates, reduced inflorescence production, reducedpollen viability, abnormal morphologies and increasedsusceptibility to infection (Hiscock, unpubl. res.).

In conclusion, current evidence suggests that within thestrong SSI system of S. squalidus there is enough intrinsicflexibility, in the form of PSC and probably also in theform of a cryptic gametophytic SI system, to make asignificant contribution to its ability to negotiate popula-tion bottlenecks and be a successful colonizer. Futurestudies will aim to measure the level and extent of thisbreeding system flexibility in selected stable and activelycolonizing populations throughout Britain.

ACKNOWLEDGEMENTS

Thanks are due especially to Hugh Dickinson for manyhelpful discussions and invaluable advice and guidance.Thanks are also due to Yan Wong for writing a computerprogram which proved invaluable for testing sporophyticallelic designations, to Sarah Widdowson, Karin Hoede-maekers and Adrian Brennan for help through under-graduate project work and to Cledwyn Merriman andCarole Elleman for help with EM. Finally, special thanksare due to Jack and Yolande Heslop-Harrison for invalu-able help and advice during the inception of this project.S.J.H. is funded by a BBSRC David Phillips ResearchFellowship.

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Self-incompatibility in Senecio squalidus L. (Asteraceae)

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