Florl morphology nd reproductive success in herkogmous Nrcissus cyclmineus (Amryllidcee) Asier R. Lrring Æ Plo Guitiá n Æ Jose Luis Grrido Æ Jvier Guitiá n Astrct We studied the vrition on florl trits of Nrcissus cyclmineus, species endemic to the northwestern Ierin Peninsul. We nlyzed the effect of different reeding systems nd the degree of herkogmy nd stigmtic exertion on femle reproductive success. Results showed tht, while vrition on florl structures is reltively low (i.e. less thn 25% for ll sterile structures), CVs for herkogmy nd stigmtic exertion re much higher. We lso found considerle vrition mong popultions in the reltive position of stigms nd nthers nd significnt effect of reeding system (fcilitted utogmy, spontneous utogmy or xenogmy) on fruit set nd seed production. The numer of seeds per fruit ers no reltion with coron size, degree of stigmtic exertion or herkogmy. However, herkogmy showed mrginlly significnt effect on men seed weight per fruit. The results highlight the need to nlyze the influence of continuous vritions in the position of florl orgns on the reproductive success of the monomorphic species of the genus Nrcissus. A. R. Lrring (&) Instituto Mediterráneo de Estudios Avnzdos, Miquel Mrqués, 21, 07190 Esporles, Bleric Islnds, Spin e-mil: sier.rodriguez@ui.es P. Guitián J. Guitián Deprtmento de Botánic, Universidde of Sntigo de Compostel, Fcultde de Frmci, Cmpus Sur s/n, 15782 Sntigo de Compostel, A Coruñ, Spin J. L. Grrido Estció n Bioló gic de Doñ n, Avd. Mrí Luis, s/n, Pelló n del Perú, 41013 Seville, Spin Keywords Reproductive success Herkogmy Stigmtic exertion Florl vrition Breeding system Nrcissus cyclmineus Introduction The grouping of mle nd femle gmetes into specilised reproductive structures, together with the possiility of selffertilistion, represent two essentil chrcteristics of hermphrodite plnts which hve undoutedly contriuted to the extrordinry diversity of sexul systems (Brrett 2003). Trditionlly, mny of the vritions in reproductive systems hve een interpreted s mechnisms tht promote outcrossing (Brrett nd Hrder 1996). For exmple, the sptil nd temporl segregtion of sex orgns, within nd etween flowers, my ct s n nti-selfing mechnism, preventing the dverse effects of selfing nd inreeding depression (Lloyd nd We 1986). However, mny species exhiiting this trit re self-incomptile nd therefore, lredy protected from these negtive effects. In such cses, the segregtion of sex orgns my ct y reducing interference etween mle nd femle functions nd enhncing pollen export efficiency (Lloyd nd We 1986; We nd Lloyd 1986; Hrder nd Brrett 1996; Brrett 2002, 2003; Medrno et l. 2005). One of these mechnisms, shred y niml-pollinted plnts, is the sptil seprtion of pollen presenttion nd pollen receipt, lso known s herkogmy. Severl different forms of herkogmy re known to exist, ut the most common, y fr, is pproch herkogmy, strtegy y which stigms re locted ove the level of the nthers, cusing pollintors to first contct the stigm s they enter the flowers (We nd Lloyd 1986; Brrett 2003). Avoidnce of selfing hs lso een cited s driving force underlying the
evolution of herkogmy. In fct, while fr from eing generl pttern (Medrno et l. 2005), the distnce seprting stigms nd nthers ws found to influence mternl outcrossing rtes in severl self-comptile species (see Beloussoff nd Shore 1995; Krron et l. 1997; Brunet nd Eckert 1998). The degree of herkogmy my lso e considered trde-off etween the reduction of sexul interference nd the increse in pollen trnsfer precision (Brrett 2002). By decresing sexul interference, the flower my reduce the mount of pollen loss on its own stigm (pollen discount), resulting in reduction of the men investment per dispersed pollen grin (Brrett 2002 nd references therein). If herkogmous flowers show incresed outcrossing rtes, we would expect higher femle reproductive success relted to herkogmy. On the contrry, n increse in the reproductive success of the mle function would occur if the reduction of sexul interference is wht drives the evolution of herkogmy. The genus Nrcissus encompsses 65 species of perennil geophytes distriuted throughout the western Mediterrnen region (Arroyo 2002), feturing species tht re monomorphic, dimorphic nd trimorphic for style length. Furthermore, the genus includes monomorphic tx, oth self-comptile (e.g. N. longispthus) nd self-incomptile (e.g. N. ulocodium), together with others with no evidence of ny linkge etween style polymorphism nd the incomptiility rection (N. ssonus) (Herrer 1995; Brrett et l. 1996; Brrett et l. 2004; Grhm nd Brrett 2004; Brrett nd Hodgins 2006). This wide diversity of sexul systems hs mde the genus Nrcissus n excellent model for understnding the evolution of florl polymorphisms, despite the fct tht informtion on the nturl history of some tx is still lcking. Nrcissus cyclmineus is species endemic to the northwestern prt of the Ierin Peninsul nd is included in the Pseudonrcissus section, one of the ten recognized for this genus. Although the species hs een descried s self-incomptile (Btemn 1954), preliminry dt hve reveled high levels of fruit set cross the popultions, despite very low rtes of pollintor visits (Lrring et l. unpulished dt), suggesting tht this species might show certin degree of self-comptiility. As this species exhiits limited florl disply (one flower per plnt), its self-comptiility mkes it suitle model for the nlysis of the functionl role of herkogmy. In this work, we nlyze the morphologicl vrition in the florl trits of N. cyclmineus, focusing prticulrly on the femle function. The following specific questions hve een ddressed: (1) Wht is the effect of the different reeding systems (nmely, fcilitted utogmy, spontneous utogmy, nd xenogmy) on fruit set nd seed production? nd (2) Wht is the effect of the degree of herkogmy nd stigmtic exertion on femle reproductive success? Methods Study species nd re Nrcissus cyclmineus DC. (Amryllidcee) is 20 30 cm ulous geophyte tht ppers long the nks of wtercourses nd dmp medows in the Atlntic ctchment res of the northern Ierin Peninsul. Its scpe develops single, pendulous yellow flower with 15 25 mm spthe. The coron is cylindriclly tuulr enclosing six equl sized stmens nd discoidl stigm with vrile degree of herkogmy. Flowering tkes plce from Ferury to Mrch, nd fruits ripen from June to July. Fruit set levels re high in ll popultions (rnge 70 90%; Lrring et l., unpulished dt). The reproductive nd vegettive structures of the plnt re suject to hevy predtion y slugs nd rodents. A recent prospecting study of potentil distriution res enled us to locte 46 popultions, rnging in numer from 5 to 10,000 flowering individuls (Lrring et l. unpulished dt). The study ws crried out on four popultions in the province of A Coruñ (Spin): Grixo (Sntigo de Compostel, numer of reproductive individuls within the popultion = 1,646), Snt Com (n = 562), Cstro (Zs, n = 9,880) nd Trsufre (Muxi, n = 450). All the popultions re locted in seprte, geogrphiclly isolted river sins. Florl morphology For ech popultion 41 45 flowers were collected nd immeditely plced in plstic gs nd tken to the lortory to prevent wilting. Once there, y mens of digitl clliper, the following mesurements were recorded (Fig. 1): coron length (L cor ), coron dimeter (D cor ), coron perture (A cor ), stmen length (L st ), nther length (L nt ), filment length (L fil ), nd style length (L sty ) (Fig. 1). The degree of stigmtic exertion ws clculted for ech individul flower s E = L sty L cor, nd herkogmy ws computed s H = L sty L st. Therefore, positive vlues of stigmtic exertion indicte exerted stigms, nd positive herkogmy vlues point to the existence of pproch herkogmy. We did not find ny flowers with reverse herkogmy, s the negtive vlues of herkogmy in our smples corresponded to flowers whose stigms re t the sme level s the nthers, ut never lower thn their proximl end. Breeding systems To nlyze the effects of different reeding systems on reproductive success 90 plnts distriuted throughout the popultions of Cstro nd Trsufre (45 ech) were gged with tulle. Once nthesis took plce, we pollinted 15 plnts 123
Style Anther Stmen Coron weighed. To check for the presence of ontogenic chnges in herkogmy, we collected 25 plnts presenting flower uds. Once in the l, they were plnted in individul pots nd their herkogmy ws mesured every dy from the strt of the nthesis to the end of the flowering period. Dt nlysis Coron Dimeter Coron Aperture Fig. 1 Schemtic drwing of Nrcissus cyclmineus flower nd the mesures collected for the present study. Derived mesures were clculted s follows: filment clculted s stmen length nther length, exertion s style length coron length, nd herkogmy s style length stmen length (see text for detils) with their own pollen (fcilitted utogmy) in ech popultion, nd 15 with pollen from other locl individuls (fter removing the stmens with pincers; xenogmy), leving nother 15 individuls unmnipulted to e used s experimentl controls (spontneous utogmy). Ech flower sujected to the xenogmy tretment received mixture of pollen from ten different donors which were hphzrdly selected from the whole popultion, voiding the immedite vicinity of the focl flower. When fruits ripened, gs were removed nd seeds were individully counted nd weighed (with 10-1 mg precision lnce). Fruit ripening ws ssessed y their yellowish color, which occurs one or two dys efore fruit opening. N. cyclmineus often produces non-vile seeds, which despite eing lckish in color like helthy mture seeds, re empty nd esy to recognize. Although these orted seeds were counted nd weighed, they were omitted from the nlyses, except in cses where orted seed numer ws included s covrite. Otherwise, the numer of seeds lwys refers to mture seeds. Stigmtic exertion nd pproch herkogmy In order to study the effect of pproch herkogmy on femle reproductive success, we selected 30 individuls in the Cstro popultion, spnning sustntil grdient of seprtion etween stmens nd stigm. To prevent involuntry pollen trnsfer, smll, disposle pper rulers were used to mesure the distnce etween the stigm nd the coron rim, s well s the distnce etween the ltter nd the closest end of the nthers with precision of 1 mm. The plnts were left in nturl pollintion conditions nd once fruit setting took plce, individuls were gged to void predtion. Mture fruits were then collected nd the seeds were counted nd Vrition in florl morphology trits mong the four popultions ws ssessed y mens of univrite one wy ANOVAs. Bivrite correltion nylses etween the flower trits mesured were lso performed for the whole smple. As the numer of ANOVAs nd correltion nlyses performed ws high, Bonferroni s sequentil correction ws pplied (Rice 1989). To prevent the reeding system experiment from filing, owing to ny dverse circumstnces tht could ffect prticulr smpling site, the tril ws conducted on two popultions, lthough this study did not im to evlute ny Popultion effect. Thus, the Popultion effect ws included s fixed fctor in ll the nlyses in order to control the differences tht might rise etween these two popultions. The effect of pollintion tretment on fruit set nd its vrition mong popultions ws nlyzed y mens of Generlized Liner Model, with dt djusted to inomil distriution nd logit s the link function, including pollintion tretment nd popultion s fixed fctors. We reduced the model y eliminting the effects tht presented P [ 0.25, following the recommendtions of Winner et l. (1991) nd Underwood (1997). Moreover, in ll cses the model selected in this wy presented the lowest AIC. All P vlues were estimted y mens of the type III likelihood rtio test. The effect of pollintion tretment on seed production nd its vrition mong popultions ws nlyzed y mens of n ANOVA. The response vrile (numer of seeds) ws squre root trnsformed. Popultion nd pollintion tretment were considered fixed fctors. Plnts whose fruit ws lredy open when collected were discrded, since the smll seed size nd the gging system could not gurntee the collection of every seed. Flowers tht did not set fruit were not considered in this nlysis. Two priori contrsts were lso performed, one testing fcilitted versus spontneous utogmy tretment, nd the other testing fcilitted utogmy versus xenogmy tretment. The effect of the pollintion tretment on seed weight ws nlyzed y Generl Liner Model with slope heterogeneity. Popultion nd pollintion tretment fctors were considered fixed nd the squre root of the numer of ripe seeds ws included s covrite. Fruits lredy open when collected nd those hving no ripe seeds (due to the inclusion of ripe seed numer s squre root trnsformed covrite) were discrded. As in previous nlyses, the full
model ws simplified y progressively eliminting the effects presenting the highest P vlues, provided tht they 25 were greter thn 0.25 (Winner et l. 1991; Underwood 1997). The finl model showed the lowest AIC vlue of ll the models tested. The fct tht there is n interction 20 etween the covrite nd the fctors detrcts from the ltter s significnce (Huitem 1980). For this reson we resorted to Johnson Neymn nlysis (Huitem 1980) to 15 clculte the regions of significnce. Finlly, we nlyzed the influence of pproch herkogmy nd the degree of stigmtic exertion on the numer 10 nd weight of the seeds of the unmnipulted flowers in our herkogmy exertion experiment (see ove), through multiple regression where oth seed numer vriles 5 (mture nd orted) were squre root trnsformed. The effect of coron length ws included to monitor the possile influence of individul plnt size. Vrile selection 0 ws performed s in previous nlyses (see ove). The dily evolution of herkogmy ws nlyzed y mens of Generlized Liner Mixed Model, with repeted mesures design, where time of mesurement ws the within-suject fctor. All nlyses were performed using Sttistic 6.0 nd 20 SPSS 11.5, nd 14 softwre. For the Johnson Neymn nlyses, we modified the routines descried y O Connor (1998) to tke into ccount the numer of comprisons crried out fter Huitem (1980). 15 Results 10 # of flowers # of flowers -4,00-2,00 exertion 0,00 2,00 Florl morphology Nrcissus cyclmineus is monomorphic species which presents considerle vrition in the reltive position of stigms nd nthers (Fig. 2). Descriptive sttistics for ll flowers show tht, overll, most vriles exhiited CV of round 10 20%, except stigmtic exertion nd herkogmy level whose CV vlues were very high. Vrition mong popultions ws significnt in coron dimeter nd perture, s well s in sex orgn-relted trits (stmen nd style length, nd the degree of herkogmy; Tle 1, Fig. 3). The vrition pttern is consistent mong popultions: flowers elonging to the Cstro popultion exhiited the lrgest sexul orgns, wheres those in Grixo showed the smllest. Not surprisingly, ll size-relted vriles showed medium to strong correltions nd exertion nd herkogmy were correlted s expected with style nd coron size. We lso found positive reltionship for herkogmy nd exertion, reflecting the fct tht oth vriles incresed with style length (Tle 2). 5 0-1,00 0,00 1,00 2,00 3,00 4,00 herkogmy Fig. 2 Histogrms of herkogmy nd exertion (see text for detils), where oth vriles cn e seen to e highly vrile nd show unimodl distriution Breeding systems Men fruit set did not differ significntly etween popultions nd since the Popultion effect presented P vlue higher thn 0.25, it ws omitted from the finl model. As cn e seen in Fig. 4(, ), pollintion tretment exerted significnt effect on the men fruit set (df = 2, v 2 = 15.58, P \ 0.0001), lthough mrginlly significnt interction effect (df = 2, v 2 = 5.21, P = 0.074) suggests tht these 123
Tle 1 Min descriptive sttistics nd popultion vrition of florl trits (N = 173, mesure units: mm) Popultion effect ws estimted through univrite ANOVAs. Significnt vlues, fter pplying Bonferroni s sequentil correction, re old typed. The high vlue of the coefficient of vrition presented y oth herkogmy s well s stigmtic exertion is noteworthy Florl trit Men Minimum Mximum SD CV (%) F (3,169) P Coron length 16.79 11.95 23.13 2.03 12.12 2.08 0.1050 Coron dimeter 6.67 4.76 9.86 0.91 13.66 3.37 0.0199 Coron perture 5.80 3.22 9.57 1.26 21.73 6.45 0.0004 Stmen length 13.21 8.78 18.21 1.88 14.27 23.24 0.0000 Filment length 6.37 3.22 10.11 1.44 22.57 24.52 0.0000 Anther length 6.84 4.41 10.12 1.13 16.51 5.78 0.0009 Florl trit Men Minimum Mximum SD CV (%) F (3,168) P Style length 14.95 11.03 Stigmtic exertion -1.82-5.92 Herkogmy 1.77-1.98 22.26 1.94 12.95 4.87 0.0028 2.40 1.51 82.78 1.64 0.1824 4.96 1.31 74.44 8.58 0.0000 differences in pollintion tretments my vry depending on the popultion considered. Seed production per fruit rnged from 1 to 39 (men = 12.46, SD = 10.89), showing pttern of vrition similr to tht of fructifiction rtes (Fig. 5). After eliminting the Popultion effect from the model (P [ 0.25), the only significnt effect ws Pollintion Tretment (df PolTret = 2, df error = 32, F = 8.05, P = 0.001; Popultion 9 Pollintion Tretment interction: df Pop*PolTret = 2, df error = 32, F = 2.56, P = 0.093). As shown in Fig. 5, the two utogmous tretments did not revel significnt differences ( priori contrst, t = -0.545, P = 0.453), while the numer of seeds produced y fruits pollinted with xenogmous pollen ws greter thn the numer resulting from fcilitted utogmy ( priori contrst, t = -1.715, P = 0.002). Averge seed weight nd the reltionship etween the numer of seeds per fruit nd their weight vried etween popultions nd etween pollintion tretments (Tle 3). In the ltter cse, the differences depended on the vlue of the covrite, numer of seeds per fruit, with spontneous utogmy showing shrper trde-off etween seed numer nd men weight (Fig. 6). Stigmtic exertion nd pproch herkogmy None of the individuls exmined in the l showed ny vrition in herkogmy over the flowering period (df mes = 6, df error = 54.249, F = 0.774, P = 0.593). All the flowers mrked in the field did set fruit. The seed production per fruit ws not relted to the degree of stigmtic exertion, coron size, or level of herkogmy, s indicted y the poor fit of the model (df model = 3, df residul = 17, F = 0.656, P = 0.590) nd the lck of significnce for ny of the effects (herkogmy, slope = -0.114, t = 0.247, P = 0.808; exertion, slope = 0.190, t = 1.131, P = 0.274 nd coron slope = 0.222, t = 1.274, P = 0.220). The degree of herkogmy hd mrginlly significnt nd positive effect on the men seed weight per fruit (slope = 0.966, t = 1.781, P = 0.091; model R 2 = 0.143, df model = 1, df residul = 19, F = 3.173, P = 0.091). Seed weight under nturl pollintion conditions ws not significntly ffected y exertion, coron length, or numer of seeds (either orted or ripe) (ll P [ 0.25). Discussion The results show tht N. cyclmineus is self-comptile, monomorphic species, oth of which re ncestrl chrcteristics within the genus Nrcissus (Grhm nd Brrett 2004; Brrett nd Hrder 2005; Brrett nd Hodgins 2006). Furthermore, it exhiits considerle vrition in the reltive position of the style with respect to the stmens (pproch herkogmy). While the coefficients of vrition of the florl structures re reltively low (i.e. sizes of sterile structures less thn 25% in ll cses), those relted to herkogmic nd stigmtic exertion re much higher. Stylr size nd herkogmic vlues re similr to those found for the monomorphic Nrcissi of the Apodnthi section (Pérez et l. 2004). Although prt of this vrition cn e ttriuted to ontogenic chnges in flower trits, the effect on fitness surrogtes found here would suggest tht t lest prt of the vrition mesured is due to inter-individul differences in herkogmy nd stigmtic exertion. Likewise, in N. longispthus, nother monomorphic species with vrition in herkogmy levels, temporl chnge in the seprtion of sexul orgns throughout the flower s life is not significnt (Medrno et l. 2005). In N. cyclmineus, the previous monitoring of 600 flowers throughout their whole life cycle pointed to lck of ontogenic chnges in exertion (personl oservtion). In the present work, we hve confirmed lck of ontogenic chnges in the degree of herkogmy for this species, y monitoring the flowers individully in the l.
Fig. 3 Vrition etween popultions in vriles studied. Remrkly, the four popultions distriute long flower size nd herkogmy grdient. Different letters indicte significnt difference (Tukey post-hoc test) 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 Filment 7.8 Anther 7.6 7.4 7.2 7.0 6.8 c 6.6 6.4 6.2 Cstro St. Com Trsufre Grixo 6.0 Cstro St. Com Trsufre Grixo 16.0 Stmen 17.0 Style 15.5 15.0 16.5 14.5 16.0 14.0 15.5, 13.5 13.0 15.0 12.5 12.0 11.5 c 14.5 14.0 11.0 13,5 Cstro St. Com Trsufre Grixo Cstro St. Com Trsufre Grixo -0.8-1 -1.2-1.4-1.6-1.8-2 -2.2-2.4-2.6-2.8 Stigmtic Exertion 3.5 Herkogmy 3.0 2.5 2.0 1.5 1.0 0.5 Cstro St. Com Trsufre Grixo Cstro St. Com Trsufre Grixo Nerly ll the monomorphic chrcteristics studied, with the exception of flower size, exhiited vrition etween popultions. In the cse of herkogmy, this vrition is common phenomenon in mny different tx (see for exmple Holtsford nd Ellstrnd 1992; Crr nd Fenster 1994; Crr et l. 1997). As Fig. 2 shows, pproch herkogmy is more mrked in popultions with smller sexul orgns, lthough the limited numer of popultions studied would preclude generliztion. This reltionship is not present t the plnt level; moreover, the lrger the flower, the higher the herkogmy nd stigmtic exertion (results not shown). Breeding systems, herkogmy, nd stigmtic exertion In contrst to Btemn s indictions (1954), our results show tht N. cyclmineus is self-comptile species le to produce seeds in oth fcilitted nd spontneous selfpollintion conditions. Anlyses crried out on fruit set nd seed production point to possile inreeding effect on this species. Both the numer of ripe fruits nd the numer of seeds per fruit re higher for outcrossed flowers (xenogmy tretment) thn for self-pollinted ones (fcilitted utogmy), despite the fct tht hnd pollintion is supposed to void pollen limittion effects. Under inreeding depression, outcrossing mting systems would e selectively fvored, lthough models incorporting the costs nd enefits of selfing predict stle mixed mting systems in nturl popultions (Lloyd nd Schoen 1992; Goodwillie et l. 2005). We found no reltion etween herkogmy nd the numer of seeds produced per fruit. Although our dt on herkogmy do not llow us to ssess the rte of self/ 123
Tle 2 Person correltion coefficients etween florl trits of ll plnts tken together (N = 173) Coron Significnt vlues fter pplying Bonferroni s sequentil correction re old typed Coron Dimeter Coron Aperture Stmen Filment Anther Style Exertion Herkogmy,609,368,628,416,519,711 -,430,170 Coron Dimeter,617,459,256,441,487 -,185,106 Coron Aperture,310,184,283,261 -,139 -,012 Stmen,801,649,761,142 -,293 Filment,065,572,189 -,283 Anther,532 -,006 -,125 Style,328,398 Exertion,283 Herkogmy outcross pollen rriving to experimentl flowers, these results suggest tht herkogmy does not increse the mount of outcross pollen. Fruit Set 0.8 0.7 0.6 0.5 0.4 0.3 A Our nlyses suggest tht pollen origin hs n importnt effect on the reltionship etween seed weight nd numer. While spontneous utogmy resulted in igger seeds only for low seed numers (\9 seeds), fcilitted utogmy resulted in igger seeds for lrger seed numers, which re usully found in the field (from 20.25 to 34.44). Although fruits from spontneous utogmy lwys yielded low seed numers, this chnge in the trde-off etween seed numer nd weight deserves further ttention. Plnts with greter seprtion etween stigms nd style seem to produce hevier seeds thn those presenting Fruit Set 0.2 0.1 0.0 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 B Xenogmy Fcilitted utogmy Spontneous utogmy Trsufre Cstro Numer of mture seeds (sqrt trnsformed) -0. 5 Xenogmy Fcilitted Spontneous Xenogmy Fcilitted Spontneous utogmy utogmy utogmy utogmy 5. 0 4. 5 4. 0 3. 5 3. 0 2. 5 2. 0 1. 5 1. 0 0. 5 0. 0 Fig. 4 Estimted grnd mens of the proility of fruit set for the three pollintion tretments () nd estimted mens for ech tretment t ech of the sites () Fig. 5 Estimted grnd mens of the numer of mture seeds for the three pollintion tretments. Outcrossing flowers produced more seeds thn self-pollinted ones
Tle 3 Anlysis of the effects of popultion, pollintion tretment nd numer of seeds on men seed weight Weigth (mg) recently reported tht positive correltion etween herkogmy nd outcrossing rte is not generl pttern in SS df MS F P the genus Nrcissus. An lterntive hypothesis postulted tht different qulity of pollen would rrive t the stigm Popultion (P) 16.065 1 16.07 6.484 0.017 s function of herkogmy rte, medited y pollintor Tretment (T) 24.945 2 12.47 5.034 0.014 ehviour. Although our results do not llow us to evlute Seeds (S) 32.984 1 32.98 13.313 0.001 this hypothesis, we did find negtive reltionship etween P 9 S 11.658 1 11.66 4.705 0.039 herkogmy rtes nd inter-individul vrition of seed T 9 S 21.144 2 10.57 4.267 0.025 weight (dt not shown), suggesting tht pollen qulity P 9 T 9 S 13.447 2 6.72 2.714 0.085 differences could e relted to the effect of herkogmy Error 64.417 26 2.47 oserved on seed weight. The popultion 9 tretment effect ws eliminted from the model due to P vlue higher thn 0.25 Seeds, numer of mture seeds, squre root trnsformed 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 # of seeds (sqrt-trnsformed) Spontneous utogmy Fcilitted utogmy Xenogmy Fig. 6 Simple slopes of the reltion etween the numer of mture seeds per fruit (squre root trnsformed) nd the verge seed weight per fruit. Dotted line: xenogmy, dshed line: induced utogmy, solid line: spontneous utogmy. Verticl dshed lines indicte the region of significnce for the comprison etween the two types of utogmy; when the squre root of the numer of seeds flls out of this region, the difference etween the two tretments is sttisticlly significnt (Johnson Neymn nlysis; no region of significnce for the remining vrile pirs) Contrry to our findings in the reeding system experiment, seed numer ws not relted to seed weight for our nturlly pollinted flowers, when nlyzing the role of herkogmy. However, we cnnot rule out the possiility tht gging the whole scpe efore florl nthesis might hve strongly ffected the level of ville resources for fruit production nd ripening. In fct, our experimentl gged flowers showed lower fruit set nd seed numer thn unmnipulted flowers monitored over the course of two yers in three different popultions (unpulished dt). In conclusion, our results revel considerle vrition in florl morphology s well s in the chrcteristics ssocited with sexul orgns (herkogmy nd stigmtic exertion) etween popultions of N. cyclmineus. The reltive position of the stigms nd nthers hs significnt effect on femle reproductive success, which highlights the need to nlyze the reproductive consequences of continuous vritions in the monomorphic species of the genus Nrcissus. Acknowledgments This work ws finnced y the Rmó n Areces Foundtion under the Reserch Project Aid progrm. Jime Fgú ndez nd Borj Frpó n collorted on the field work nd Crlos Teir crried out florl mesurements. We thnk Ainho Mgrch for English correction nd editing. The comments y two nonymous referees hve significntly improved the first drft of the mnuscript. more exerted stigms, lthough this reltionship ws only mrginlly significnt. One possile explntion could e tht higher degree of herkogmy diminishes rtes of pollintion with the plnt s own pollen nd produces etter qulity seeds. Herkogmy hs een deemed mechnism for reducing self-fecundtion (see Brrett 2002 nd references in this work), strong positive link tht hs often een found etween the degree of herkogmy nd outcrossing rtes (Holtsford nd Ellstrnd 1992; Beloussoff nd Shore 1995; Krron et l. 1997; Herlihy nd Eckert 2004). However, our results on the reeding system of N. cyclmineus do not support this hypothesis, s there re no significnt differences in seed weight etween fcilitted utogmy nd xenogmy. Medrno et l. (2005) References Arroyo J (2002) Nrcissus (Amryllidcee), l evolució n de los polimorfismos florles y l conservció n más llá de ls lists rojs (The evolution of florl polymorphism nd conservtion eyond the red lists ). Rev Chil Hist Nt 75:39 55 Brrett SCH (2002) The evolution of sexul plnt diversity. Nt Rev Genet 3:274 284 Brrett SCH (2003) Mting strtegies in flowering plnts: the outcrossing-selfing prdigm nd eyond. Phil Trns R Soc Lond B 358:991 1004 Brrett SCH, Hrder LD (1996) Ecology nd evolution of plnt mting. Trends Ecol Evol 11:73 79 Brrett SCH, Hrder LD (2005) The evolution of polymorphic sexul systems in dffodils (Nrcissus). New Phytol 165:45 53 Brrett SCH, Hodgins A (2006) Florl design nd the evolution of symmetricl mting systems. In: Hrder LD, Brrett SCH (eds) Ecology nd evolution of flowers. Oxford University Press, UK 123
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