Incompatibility and pollen competition in Alnus glutinosa: Evidence from pollination experiments

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  • Genetica 105: 259271, 1999. 2000 Kluwer Academic Publishers. Printed in the Netherlands. 259

    Incompatibility and pollen competition in Alnus glutinosa: Evidence frompollination experiments

    Wilfried Steiner & Hans-Rolf GregoriusInstitut fr Forstgenetik und Forstpflanzenzchtung, Universitt Gttingen, Bsgenweg 2, D-37077 Gttingen,Fed. Rep. Germany; Author for correspondence: (Fax: +49 / 551 / 398367;E-mail: gregorius@ufogen.uni-forst.gwdg.de)Received 1 March 1998 Accepted 6 August 1998

    Key words: Alnus glutinosa, incompatibility, pollen competition, pollination experiments

    Abstract

    Different types of incompatibility systems were found to operate simultaneously in Alnus glutinosa in the courseof numerous pollination experiments, including self-pollination and pollination with controlled pollen mixtures.Isozyme genetic markers were used to identify the pollen parent of each offspring from the mixed pollinationexperiments, thus allowing specification of the fertilization success of each pollen parent. In a first step, theseresults were compared with observations on in vitro pollen germination experiments. This comparison allowsfor exploration of the explanatory value of different germination media as models of germination conditions onstigmas. In most cases, the data suggest that the in vitro germination conditions resemble the fertilization conditionsin vivo, at least in the sense that they favor the same pollen parents. By providing a generic and operable definitionof the two basic types of incompatibility, eliminating (inability to fertilize ovules) and cryptic (resulting in loweredfertilization success of a pollen parent under competition), evidence was detected for the existence of both typesof incompatibility in Alnus glutinosa, where eliminating incompatibility occurred as self-incompatibility only.However, since this incompatibility seems to act primarily via pollen elimination, seed production is not likely tobe negatively affected in natural populations, even for comparatively large amounts of self-pollination.

    Glossary

    Cryptic incompatibility of a pollen parent; a pollenparent being opposed (see entry opposition) by somebut not by all ovule parents.Cryptic pollen deficiency of a pollen parent; a pollenparent being opposed (see entry opposition) by allovule parents.Cryptic self-fertility of a plant; self-pollen applied tostigmas under exclusion of cross-pollen inhibits fer-tilization, whereas a mixture of self- and cross-pollenresults in the formation of some progeny from self-fertilization.Eliminating incompatibility of a pollen parent; infer-tility with some but not with other ovule parents.Infertility of a pollen parent with the ovules of anovule parent; the proportion of unfertilized ovules in-creases with the share of the pollen parent among allpollen pollinating the ovule parent.

    Interference of two pollen types; the performance(germination and growth) of one pollen type is af-fected by the presence of the other; interference cantake place uni- and bilaterally.Opposition of an ovule parent to a pollen parent;the relative success of a pollen parent in fertilizing theovules of a plant is below average in competition withpollen from other plants.Pollen deficiency of a pollen parent; infertility of thepollen parent with all ovule parents.

    Introduction

    Generative reproduction in plants allows for variableforms of selection during the phase from the formationof gametes to their fusion in zygotes and the initialstages of seed development. Although this reproduc-tive phase normally covers only a comparatively small

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    portion of an organisms total life span, its overallselective potential may be considerable. Especially inlong-lived, highly iteroparous organisms like trees, itmay even exceed the potential for selection during theextensive vegetative phase.

    Effects of selection on mating processes (selectivemating) have long been discussed in the populationgenetics literature. They include aspects such as dif-ferential intensity and asynchrony of flowering as asource of differential mating participation and mat-ing preferences, segregation distortion as implied byselection during gamete formation, gametophytic se-lection, and early postzygotic incompatibility or se-lective embryo abortion. In fact, even the specificallycombinational effects of mating that appear, for ex-ample, in various prezygotic incompatibility relations,present themselves as a result of selection processesamong the potential mating partners of an individualor group of individuals. Differential mating successamong the potential mating partners defines the mat-ing preferences of the individual or group (Gregorius,1989). By combining a multitude of different formsand intensities of selection, the generative phase ofreproduction thus establishes a complex system ofselection mechanisms.

    The results on mating system characteristics of Al-nus glutinosa reported in the following highlight malegametophytic selection caused by pollen competitionfor fertilization. Pollen competition is understood hereas all aspects involved in the striving of pollen to fer-tilize the same ovule. Different factors may contributeto this kind of selection. (i) Pollen vigor basically de-termines the capacity of an individual pollen grain togerminate, grow, and fertilize; physiological consti-tution and genetic characteristics of the pollen or itsproducer may affect this capacity (see e.g., Mulcahy,Muleahy & Ottaviano, 1986, chapter on Gene ex-pression in pollen). Here, the conditions for selectionare shaped over the whole reproductive process fromthe beginning of gametophyte formation to the finalfertilization event, and these conditions strongly influ-ence the mating (fertilization) success of the differentpollen types.

    After pollination of the female flowers, (ii) femalemate choice, sexual selection, and prezygotic incom-patibility mechanisms become relevant. Interactionbetween female flower and pollen can lead to selectionamong the pollen present on a stigma. Especially incombination with incompatibility phenomena, femalerecognition and rejection processes are well known(for reviews, see de Nettancourt, 1977; McCubbin &

    Kao, 1996). Further selection conditions arise from(iii) malemale interactions in the sense of pollen in-terference (for example, via allelopathic substances).Several investigations have demonstrated that neigh-boring pollen on a stigma or pollen tubes in a style canmore or less directly influence each others germina-tion or pollen tube growth, thus resulting in selection(Marshall et al., 1996; Kanchan & Jayachandra, 1980;Murphy & Aarssen, 1989). Since these three factorsdo not act in isolation from each other, they are verydifficult to distinguish experimentally.

    Alnus glutinosa is monoecious and predominantlywind-pollinated. The female and male inflorescencesare well separated. Only in very rare cases couldinflorescences with male flowers at the bottom andfemale flowers at the top of the same inflorescencesbe observed. As the vast majority of inflorescenceson every tree do not comprise flowers of both sexualtypes, such combinations of sexes in one inflorescencecan be considered as malformations. No such inflores-cences were used in our experiments. The existence ofa self-incompatibility system in this species seems tobe likely despite some conflicting reports in the liter-ature (Johnsson, 1951; Weiss, 1964; Hagman, 1970,1975 report self-incompatibility in contrast to Heit-mller, 1957; Rohmeder & Schnbach, 1959 [p. 71];Holzer, 1961; Weisgerber, 1974). The repeated occur-rence of self-incompatibility hints at the existence ofa genetic mechanism that enables the species to se-lectively discriminate between self- and cross-pollen.Such mechanisms, however, may (probably to a lesserextent) also imply the possibility of selective fertiliza-tion among cross-pollen that differs in specific charac-teristics, thus defining more complex incompatibilityrelations.

    The present experimental approach is intended tocontribute to the clarification of these conflicting re-ports and to enable a more detailed analysis of theproblem of incompatibility in Alnus glutinosa. Takingthe possibility of (cryptic) incompatibility relationsshowing under pollen competition only into account,the majority of the pollination experiments are com-posed of pollen mixtures consisting of defined propor-tions of two pollen parents. The two pollen parents andthe ovule parent are chosen on the basis of isozymegenotypes that allow identification of the pollen par-ent of each offspring from the pollination experiment(the principle of this method goes back to Bateman,1956). Particular attention is given to an unambigu-ous and operational formulation of the basic types ofincompatibility relations in order to allow detection

  • 261

    of variants of incompatibility and of fertilization re-lations that do not fit into the common concepts ofincompatibility (for convenience, the relevant termsare compiled in a brief glossary at the end of thepaper).

    Materials and methods

    Tree material

    All pollination experiments were carried out on treesgrowing in two seed orchards (Jungviehweide andGahrenberg) of the Hessian Forest Research Institu-tion. All pollen parents except one were also chosenamong the seed orchard trees. The only exception isa planted roadside tree (ATW2) of unknown originand age. All seed orchard trees are named with theirofficial numbers consisting of clone name and rametnumber. As literature reports, the general possibilityof misclassification in seed orchards, different rametsof the same clone were treated as separate trees inour experiments. Neither pollen samples nor seed lotsfrom different ramets of the same clone were mixed,although electrophoretic data from isozymes were inaccordance with the official classification of the treesexamined in the course of our investigations. The pos-sibility that two different clones may show identicalpatterns at all isozyme systems under investigation hadto be taken into account. Seed lots resulting from re-peats of the same pollination experiment on the sameclone were also kept separate.

    Pollen sampling and mixing

    Pollen sampling was done as described by Steiner andGregorius (1998). The proportions of single pollenparents in pollen mixtures was defined by their weightproportions. All mixtures consisted of pollen from twoparents. Pollen mixtures were produced after exposingthe pollen samples to constant climatic conditions inorder to enable exchange of humidity with the sur-rounding atmosphere until equilibrium was reached.As Alnus pollen turned out to be very hygroscopic, thispretreatment ensures that the weight proportions of thepollen mixtures reflect biomass proportions and notdifferences in water content. To test for weight con-stancy as an indicator of equilibrium, very small pollensamples were weighed on a highly sensitive scale(107 g) and observed for weight constancy for severalminutes. Only newly collected pollen was used in allexperiments, and in vitro germination tests preceded

    the in vivo use of this pollen (Steiner & Gregorius,1998). Pollen parents were chosen for use in pol-len mixtures according to their genotypes at isozymegene markers. In order to identify the male parent ofeach progeny plant, the pollen parents carried differ-ent alleles at a minimum of one gene locus, while theovule parent was homozygous, or carried no allele ofpaternal type at this locus.

    Flower isolation and pollination

    Removal of male catkins and isolation of female in-florescences was done at the end of winter about twoweeks before the expected flowering date. Becauseof bad weather conditions (strong winds, snowstorms,rain) and their unpredictability, many isolation bagswere destroyed and could not be repaired or replaced.The additional weight of snow or wetness, togetherwith an increased susceptibility to wind, caused thebreaking of many branches. For this reason, not allplanned experiments could be successfully performed.

    As soon as the first tips of the stigmas could beobserved between the opening bud scales of the femaleinflorescences, pollination was performed by usinga small brush to apply pollen directly to the femaleflowers (as described by Linares, 1984). The isolationbags were opened at one side and closed immediatelyafter pollination. Several days after the first pollina-tion, when flowering intensity was at its maximum, asecond pollination was carried out in order to pollin-ate flowers that probably had not been receptive at thetime of the first pollination. As the trees in the seedorchard differed in phenology, all pollinations couldnot be performed on the same day but had to be spreadover three to four days. Consequently, the second pol-lination of the early flowering trees nearly coincidedwith the first pollination of the late flowering trees.Isolation bags were kept closed until the end of theflowering period in the seed orchard, when nearly allmale catkins had fallen to the ground. At this time,the bags were removed and the branches were labeled.In autumn (September or October), the ripened seedswere collected.

    The following types of pollination variants wereperformed, if possible on the same ovule parent: (i)Pollinations using a pollen mixture. (ii) Single pollencrosses: Whenever possible, a pollen mixture experi-ment was accompanied by its constituent two singlepollen crosses on the same ovule parent. This enablesinvestigation of single pollen fertilization in the ab-sence of pollen competition. If a single pollen cross

  • 262

    fails to produce viable progeny, the failure of thesame pollen type in a pollen mixture experiment can-not be attributed to (malemale) pollen competitioneffects. This failure can likely be explained as theresult of male deficiency or malefemale incompat-ibility interactions. On the other hand, if a pollentype is successful in a single pollen cross but notin a pollen mixture cross, this must be treated as asign of effective pollen competition. (iii) Control pol-linations: To test the possibility of contamination byairborne pollen, some control bags were also opened,and pollination was simulated with a brush as usual butwithout applying pollen. All progeny resulting fromthis kind of treatment must be the result of contam-ination during pollination or by undetected damageof the isolation bags. (iv) Consecutive pollinations:This variant consisted of applying one pollen typeat the first pollination and another pollen type at thesecond pollination. This kind of experiment enablesthe investigation of first pollination primacy.

    Electrophoretic analysis of progenyHorizontal starch gel electrophoresis of isozymes wasused to genetically analyze the progeny resulting frompollination experiments. The enzyme systems, theirinheritance, and the procedures used will be publishedelsewhere. All enzyme systems showing variationamong the seed orchard trees were examined, althoughonly one specific system was necessary for paternityanalysis. The investigation of the other enzyme sys-tems served as a screening for pollen contaminationduring artificial pollination.

    In order to detect progeny resulting from pollencontamination during artificial pollination, not onlythe specific enzyme system used to identify patern-ity was analyzed, but in addition, all enzyme systemsthat had shown variation among the seed orchard treeswere examined. As the small seeds of Alnus glutinosado not contain enough embryo material to analyze sev-eral enzyme systems simultaneously, the seeds weregerminated so that the resulting plants could be usedfor electrophoretic analysis.

    Data analysis

    The analysis addresses two objectives, one relatingthe observations from the pollination experiments tothe results obtained from earlier in vitro pollen ger-mination experiments using the same pollen samples(Steiner & Gregorius, 1998). The other objectiveconcerns opportunities for extracting information on

    pollen deficiency (reduced vigor, sterility, infertility)and on (cryptic) incompatibility from the differen-tial fertilization relations observed in the pollinationexperiments.

    For the first objective, germination is viewed asa step during the fertilization process and analyzedwith respect to its effect on the fertilization successof the two pollen parents involved in each pollinationexperiment. For this purpose, the pollen germina-tion percentages obtained separately for each pollenparent on two germination media are assumed to com-pletely determine fertilization success. In this model,the germination medium takes the place of the pol-len germination conditions realized on the stigmas ofan ovule parent, and pollen types are assumed notto interfere with each other during germination. Agiven ovule parent (which may correspond to eitherof the two germination media) shall be pollinated bya mixture of pollen from two pollen parents 1 and2 in proportions r1 and r2 (fertilization references,r1 C r2 D 1). The pollen of these two parents germin-ates in proportions c1 and c2. The model assumptionsimply that the proportion f 0i of the ith pollen parent(i D 1; 2) among the fertilized ovules of the ovuleparent (the prime indicates the modeled proportion)equals

    f 0i Dri ci

    r1 c1 C r2 c2 :

    The hypothesis to be tested here for each pollina-tion experiment is that the actual fertilization success(fi=ri , with fi s = actually observed fertilization pro-portions) of pollen parent i equals its modeled coun-terpart (f 0i =ri ). In this case, one would accept that theactual fertilization mechanisms are explainable by themodel assumptions. In all statistical tests, the confid-ences (or p-values, i.e. the probability of not obtaininga result more probable than the observed under thehypothesis to be tested) will be given to allow directjudgement of the goodness of fit of the modeled (hy-pothesized) to the actual observation. Only pollinationexperiments which yielded a progeny of at least 10(analyzed) plants are included in the analysis.

    Concerning the second objective, pollen deficiencyor incompatibility are usually considered to be con-nected with the failure to fertilize, as becomes mani-fest in unfertilized ovules. The underlying idea ofinfertility can be made more precise by stating infertil-ity of a pollen parent with the ovules of an ovule parent(see Glossary), if the proportion of unfertilized ovulesincreases with the share of the pollen parent among

  • 263

    all pollen pollinating the ovule parent. This includesthe possibility that a strict increase is limited to verysmall proportions only, such as may be the case forpollen or ovule elimination (see e.g., Finney, 1952).Consequently, pollen deficiency of a tree follows frominfertility of this pollen parent with all ovule parents.In contrast, a pollen parent shows incompatibility if itis infertile with some but not with other ovule parents.To prevent confusion of this definition with other con-cepts of incompatibility it will be termed eliminatingincompatibility.

    Cryptic incompatibility of a pollen parent, in turn,is distinguished from eliminating incompatibility bythe fact that the share of this parent among all pollina-tions of an ovule parent has no effect on the proportionof unfertilized ovules; special pollen or ovule elim-ination effects are thus excluded. Hence, under thiscondition, incompatibility relations can be recognizedonly if there are cases in which the (relative) successof a pollen parent in fertilizing the ovules (fertiliz-ation success as defined above) of a given tree isbelow the average in competition with pollen fromother trees. This situation can be conceived of asan ovule parent being in opposition to a pollen par-ent. Therefore, a pollen parent can be stated to showcryptic incompatibility if it is opposed by some butnot by all ovule parents. The analogue to pollen de-ficiency is now realized if a pollen parent is opposedby all ovule parents, in which case it is meaningful tospeak of cryptic pollen deficiency. While ovule elim-ination cannot lead to cryptic incompatibility, purepollen elimination can effectively produce cryptic ef-fects if pollination always provides sufficient numbersof compatible pollen. The latter can be assumed to bethe rule in natural populations.

    These principles form the basis of the followinganalysis, taking into special consideration the presentsituation of incomplete crossing designs.

    Results

    Self-pollination

    Several self-pollination experiments were performedeither by isolating female and male catkins togetherin the same isolation bag (pollination was ensured bywind movements and additional shaking of the bagby hand) or by a pollination experiment as describedabove, where the self-pollen was previously collectedand then applied by a brush to the female flowers.

    All experiments using defined pollen mixtures of self-and cross-pollen failed because of broken branches ordestroyed isolation bags. The female flowers showednormal development after self-pollination. But thenumber of inflorescences developing to ripe coneswas smaller for self-pollinated ones than for cross-pollinated ones. Due to the reduced number of selfingexperiments, this difference did not reveal statist-ical significance. Cones resulting from self-pollinationyielded many seeds, but the germination percentageamong all seeds produced after selfing was very poor(2%) compared to the average germination percent-age of seeds resulting from cross-pollinations (16%in 1993 and 26% in 1994). As could be seen afteropening the ungerminated seeds, most of these seedswere empty. As the proportion of embryo-containingbut non-germinating seeds among all seeds was notsignificantly different for seeds from self- and cross-pollination experiments, the poor germination of seedsfrom selfing must be explained by the highly in-creased proportion of empty seeds among the seedsfrom selfing. Since the pollen parents used in the self-ing experiments produced viable seeds in outcrosses,a system of eliminating self-incompatibility exists,acting either prezygotically or postzygotically duringearly embryo development. None of the seedlings ori-ginating from selfing survived for more than someweeks under greenhouse conditions. Depending onthe actual time of failure, this might be consideredas indicating late acting postzygotic incompatibility orinbreeding depression.

    Control experiments, pollen contamination

    The control pollination experiments with placebopollen yielded only small cones and the loss during de-velopment from flower buds to cones was higher thanthe loss of self- or cross-pollinated flowers. The conescontained seeds, but nearly all were empty. The ger-mination percentage was even lower than that of selfedseed (due to the extremely high proportion of emptyseeds), but the few seedlings arising did not show thispronounced inbreeding depression. Most control ex-periments did not yield any germinating seeds. Mostof the few plants that survived until electrophoreticinvestigation must be the result of cross-fertilization,because they showed alleles that could not have beentransmitted by the ovule parent. For the remainingplants, neither self nor cross origin could be excluded,but as self-pollen possesses only very little fertilizationcapacity, it seems most probable that these plants also

  • 264

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  • 266

    originated from cross-fertilization with a paternal con-tribution that was indistinguishable from the maternalcontribution. It is, therefore, extremely unlikely thatapomixis phenomena play a role in the reproductionof Alnus glutinosa.

    The progeny obtained from cross-fertilization inthese control experiments show that pollen contamin-ation cannot be ruled out for this method. However,it has to be taken into account that in the control ex-periments each single contaminating pollen grain hada good chance of fertilizing, as there were no othercompeting pollen grains. Under normal experimentalpollination conditions, however, contaminating pollenrepresents only a very small proportion of the totalpollen available for fertilization. Among the familieslisted in Table 1 only 33 cases of pollen contaminationcould be detected and excluded from further analysiswhile the other 1865 plants could be attributed to oneof the two potential pollen parents. This correspondsto a total contamination percentage of 1.7%. Therestill exists an unknown proportion of undetectablecontamination, especially in the cases of pollen mix-tures where several different paternal contributions areclassified as legitimate. The rate of detectable contam-ination among the single pollen crosses is, however,only negligibly higher (2.0% for a total of 407 plantsexamined). The method thus can be considered to bevery reliable and suitable for this kind of investigation.

    Pollinations with pollen mixtures

    The results of the mixed pollination experiments aresummarized in Table 1. Each progeny set originatingfrom a single isolation bag is designated by a numbercomposed of the year of the experiment (93 or 94) andby an individual number given to each isolation bagin the respective year. Each progeny set results frompollination of a tree with a mixture of pollen from twopollen parents, and therefore is composed of half-sibsand two types of full-sibs. Progenies identified as res-ulting from pollen contamination are not consideredin the data presented in Table 1. In the first columnof Table 1, the designation of each progeny set (alsotermed family in the following) is given together withthe number of the respective ovule parent (in bracketsbelow). The second column specifies the two pollenparents used for the respective pollen mixtures. Thethird and fourth columns contain the proportions ofthe two pollen parents in siring the progeny as ob-served by electrophoretic analysis of the progeny andthe proportions according to which of the two pollen

    parents are represented in the pollen mixture. Theseobservations are then used in testing three hypothesesin the remaining columns. These hypotheses are:

    (1) equal fertilization success of the two pollenparents involved in a pollination experiment: The ob-served fertilization proportions and the proportions inthe pollen mixture are compared in Table 1 under theheading hypothesis 1. The central column gives theconfidence (p-value) of the statistical test (with theunderlying sample size N given in parentheses be-low), the right column gives the the distance d0 (afterGregorius, 1974) between these proportions.

    (2) fertilization success is determined by the invitro performance on medium A.

    (3) fertilization success is determined by the invitro performance on medium B.

    The in vitro germination experiments on two dif-ferent media (a solid agar medium A and a liquidsaccharose medium B) are described in detail inSteiner and Gregorius (1998). Their results were usedto model fertilization proportions (for the pertainingmodel formulation see the Data analysis section).These modeled proportions are then tested for accord-ance with the observed fertilization proportions. Thein vitro germination percentages, the modeled fertil-ization proportions, the confidences of the test, andthe distances between actually observed and modeledfertilization proportions are given in Table 1 under theheadings hypothesis 2 (germination on medium A)and hypothesis 3 (germination on medium B).

    As the results in Table 1 show, hypothesis 1 is re-jected in most of the cases. Only in three out of theseven families originating from the 1993 experiments,the observation does not contradict the hypothesis ofequal fertilization success at the 5%-level of signi-ficance. Yet, in all 17 families of 1994 there aresignificant deviations from this hypothesis, in 14 evenat the 0.1% level. In most of the cases, low confidencevalues go along with large distances between ob-served and hypothesized fertilization proportions. Onthe whole, these distances are distinctly larger in 1994than in 1993. In eight families, fertilization was evencompletely inhibited for one of the pollen parents.According to the above explanations, cryptic pollendeficiency can, however, be excluded as a potentialcause of this observation. Cryptic incompatibility asan alternative cause will be discussed later.

    Hypothesis 2 shows no significant deviations fromthe observations in four out of 17 cases, while thisholds for two out of 12 cases for hypothesis 3. Amongthe other families the deviations are mostly highly

  • 267

    significant (very low confidences) and show larger dis-tances between observed and hypothesized (modeled)fertilization proportions. It is remarkable that in theclear majority of cases the confidences of hypotheses 2and 3 exceed those of hypothesis 1, and this tend-ency is mirrored in the distances between observed andhypothesized fertilization proportions. This suggeststhat in these cases the in vitro germination conditionsresemble to some degree the fertilization conditionsin vivo. For those families not following this pic-ture, pollen parent ATW2 marks the strongest con-trast by practically inverting the in vitro observationsfor medium A (hypothesis 2) under in vivo condi-tions. The consistently superior fertilization successof pollen parent ATW2 in all families in associationwith its consistent inferiority in pollen germination onmedium A and average germination performance onmedium B (hypothesis 3) combines aspects of crypticpollen deficiency under certain conditions with aspectsof consistent competitive superiority under the studiedin vivo conditions. This situation requires further ana-lysis in terms of incompatibility phenomena and willbe picked up in the discussion section.

    Fertilization success of pollen types undercompetition

    Fertilization success, fi=ri as defined above, is ameasure of relative fitness of the ith pollen type infertilizing the ovules of a given individual under com-petition with other pollen types. Hence, the fi s canbe considered to result from selection acting on theris, and the amount of selection involved in this pro-cess can be consistently measured by the distance d0between pollination (ri ) and fertilization (fi) propor-tions (see Gregorius & Degen, 1994). The determinedamount of selection is combined in Figure 1 withthe information about the selectively superior pollentype in each pollination experiment to enable detectionof patterns of competitive superiority among pollentypes.

    Each arrow in the figure represents one pollen mix-ture, and the arrow points towards the pollen typethat is inferior in fertilization success; double-headedarrows signify that no significant difference in fertiliz-ation success occurs between observed and modeledproportions. The intensity of shading of the arrow-heads indicates the degree of inferiority in terms ofthe amount of selection involved in fertilization, andit decreases from complete inferiority (black) to theabsence of differences in fertilization success (white).

    Figure 1. Consistent superiority relations in fertilization successamong pairs of competing pollen parents. Arrows point at theinferior pollen type; double-headed arrows indicate absence of in-feriority; number of lines equals number of experiments; shading ofarrowheads corresponds to degree of inferiority.

    The number of lines associated with one arrow (from1 to 3) equals the number of pollination experimentsperformed with the same pollen mixture and withthe same inferiority relations among the involved pol-len parents. No cases of reversed inferiority relationswithin a pollen mixture occurred in our investigation.

    Whenever arrows point both to and away froma pollen parent, this signals that the parent showsno cryptic pollen deficiency and may therefore bea candidate for cryptic incompatibility according tothe previous explanations in the data analysis section.Among the nine pollen parents studied in 1994, sixare such candidates. Inferiority relations are presen-ted separately for the two years, because in vitrogermination experiments revealed significantly differ-ent germination patterns in different years (Steiner &Gregorius, 1998). Moreover, as only one pollen parent(Th4) was available in both years, no additional rela-tions would have been revealed by combination of thetwo graphics.

    The pollen mixture experiments (7 in 1993 and17 in 1994) include four identical repeats (93-001 =93-004 in 1993; 94-083 = 94-091, 94-101 = 94-102and 94-167 = 94-168 in 1994), in the sense that thesame pollen mixture was applied a second time onthe same ovule parent in an independent experimentin a separate isolation bag. No significant differencesamong repeats were found on the basis of Pearsons2-tests for homogeneity. There is thus no indication

  • 268

    of variation in relative fertilization conditions withinan ovule parent.

    Frequency dependence of fertilization successIf pollen competition takes place through direct inter-ference (such as allelopathic interaction), it is conceiv-able that a pollen types fertilization success increaseswith its relative pollination frequency. Since frequencygradients are not realized within the pollen mixturesin the data presented here, only a rough idea of thisform of positive frequency dependence can be sought.Such a dependence could be suspected if the morefrequent type among the pollinations also is the moresuccessful type in fertilization. If all pollen mixtureswith one pollen type exceeding frequency 0.55 areconsidered in order to avoid situations of indecision,there are nine cases of superior fertilization successof the more frequently pollinating type (fi=ri > 1 toa statistically significant degree for ri > 0:55), fourcases with inferior success, and one case without stat-istically significant differences in fertilization success.A generally best pollination primacy therefore doesnot seem to exist, but the possibility of frequency de-pendent ranking of fertilization successes cannot beruled out.

    Consistency of fertilization relationsSome of the pollen mixtures have been applied re-peatedly in different pollination experiments, as in-dicated by the number of lines per arrow in Figure 1.The four cases of identical repeats have already beenpointed out to be consistent in fertilization success,but some cases where the same pollen mixture wasapplied to different ovule parents still remain. The pol-len mixtures Sf1CS1 and S1CWb16 were appliedto two ovule parents (resulting in progeny sizes of 93and 80 for the first mixture, and 60 and 35 for thesecond), and the pollen mixture Th5CMe8 was ap-plied to three ovule parents (resulting in progeny sizesof 62, 63, and 31). In all cases, the ranking in fertil-ization success is consistent over the respective ovuleparents, and even the degrees of superiority are sim-ilar. The mixtures Sf1C S1 and Th5CMe8 resultedin superiority of Sf1 and Th5, respectively, with stillconsiderable fertilization success of the inferior pollenparent. In the pollen mixture S1CWb16, however,S1 is completely superior, leaving no offspring to besired by Wb16 on any ovule parent. A test of homo-geneity of the different progenies resulting from thesame pollen mixture yielded a minimum confidencevalue of 23.7%. In summary, these results indicate

    the existence of cryptic incompatibility, but it must bestated that the requirements of the above mentioneddefinition (see glossary) are not fullfilled: The limitednumber of female parents does not allow statements ofwhether a given pollen parent is opposed by all ovuleparents (cryptic pollen deficiency) or by some but notall ovule parents (cryptic incompatibility). This aspectwill also be treated in the discussion section.

    Complementary pollination experiments

    Each pollen mixture experiment was planned to beaccompanied by the two corresponding pure crossesinvolving only one of the pollen parents. Not all com-binations could be realized because of the enormousexperimental problems under field conditions, andamong the realized pure crosses, only a small partcould be analyzed because of limited financial re-sources. In particular, no data were obtained for ac-companying pure crosses for those cases in which pol-len mixtures produced complete failure of one pollenparent to fertilize the ovule parent. As a consequence,even in these extreme cases no clear distinction canbe made between eliminating incompatibility (purecross would also fail) and cryptic incompatibility (purecross would be successful) as possible causes for theobservations.

    Consecutive pollinationsTo get an idea about effects of temporal synchroniz-ation of male and female flowering, additional pol-lination experiments were carried out in which twopollen types were applied separately and consecutively(after a period of some days) to the same ovules. Thematerial and the results are summarized in Table 2.The observed fertilization proportions are given to-gether with the fertilization and pollination propor-tions of the corresponding (simultaneous) mixture ex-periments (transferred from Table 1). The results fromovule parent Me5-700 support the hypothesis of firstpollination primacy because only one plant out of 50was sired by the second pollen type, even though thissecond type had shown superiority or at least absenceof inferiority in the simultaneous mixtures. Contra-dictory to the first pollination primacy hypothesis arethe results obtained for ovule parent Me13-622: In allthree cases, a second pollination primacy can be ob-served independently of the order of the pollen types.Obviously, despite the observation of female flower-ing, the state of receptivity (at least for the pollen used)was not reached at the time of first pollination.

  • 269

    Table 2. Overview of consecutive pollination experiments accompanying mixed pollination experiments

    Ovule parent: Me5-700 Ovule parent: Me5-700 Ovule parent: Me13-622Pollen parents: Me1, Th4 Pollen parents: Me7, Th4 Pollen parents: Sf1, S1

    Pollination Pollen Progeny proport. Pollen Progeny proport. Pollen Progeny proport.type types Observ. Hypoth. types Observ. Hypoth. types Observ. Hypoth.

    Simultan Fam. 93-090 .N D 42/ Fam. 93-095 .N D 53/ Fam. 94-115 .N D 93/mixture Me1 0.26 0.44 Me7 0.32 0.39 Sf1 0.63 0.40

    Th4 0.74 0.56 Th4 0.68 0.61 S1 0.37 0.60

    Consecutive Fam. 93-097 .N D 14/ Fam. 93-096 .N D 20/ Fam. 94-117 .N D 23/mixture 1. Me1 1.00 1. Me7 1.00 1. Sf1 0.04

    2. Th4 0.00 2. Th4 0.00 2. S1 0.96

    Consecutive Fam. 93-106 .N D 16/ Fam. 94-125 .N D 109/mixture 1. Me1 0.94 1. Sf1 0.09

    2. Th4 0.06 2. S1 0.91

    Consecutive Fam. 94-118 .N D 76/mixture 1. S1 0.09

    2. Sf1 0.91

    For the consecutive pollen mixtures, the first and second pollination is indicated by 1. and 2., respectively, before the nameof the pollen type.

    Discussion

    In order to enable evaluation of the potential relevanceof our observations for the detection of incompatiblityrelations, it is neccessary to briefly recall some resultsthat refer to the distinction between eliminating andcryptic forces. So far, only self-incompatibility couldbe clearly demonstrated to exist and to be of the elim-inating form. It thus remains to address the possibilityof cross-incompatibility.

    According to the earlier explanations at the endof the data analysis section, cryptic incompatibilitywould be a meaningful candidate for analysis of ourresults if there were indications of the existence ofpure pollen elimination. In fact, during an initial self-pollination test, a tiny fraction of pollen from a knownsecond pollen parent entered the pollination bag, andin the resulting small progeny all plants, except one,were sired by the second father; the one exceptionalso could not be excluded as an offspring of thisfather. In view of the overwhelming abundance ofself-pollinations, the occurrence of cross-fertilizationswould, however, be extremely improbable if self-pollen would have had the capacity to fertilize theovules. This, in turn, would have resulted in inviablezygotes. Hence, a considerable portion of the self-pollen must have been eliminated before fertilizationin order to reserve sufficient opportunities for fertil-ization by the few cross-pollen. Pollen elimination

    is, therefore, indeed the more likely form of elim-ination, which, in turn, suggests effectively crypticforms of cross-incompatibility for the analysis of ourobservations.

    The evolutionary advantage of this cryptic formis obvious. It maximizes the conversion rate of vi-able ovules into zygotes while allowing for purging ofdisadvantageous genes through self-pollination. Un-der natural conditions, cross-pollination will alwaystake place at a sufficient rate to guarantee fertilizationof all ovules, so that genetic purging, taking placeamong self-pollen, is limited to the prezygotic phase.Postzygotic genetic purging as a consequence of self-pollination is expected to considerable degrees only ifcross-pollination is negligible. Since no mechanismsfor prevention of self-pollination are known in Alnusglutinosa, genetic purging in the species is likely totake place according to these principles.

    Mechanisms of pollen competitionBasically, the forces acting during the pollinationfertilization phase can be classified into those due tothe interplay among pollen parents (usually referred toas pollen competition) and those due to the interplaybetween ovule and pollen parents. The latter class in-cludes mechanisms of modifying pollen competitionrelations through ovule parent characteristics, whilethe first class includes pollen competition in the form

  • 270

    of interference. Herewith, two pollen types are said toshow interference if the performance (germination andgrowth) of one type is affected by the presence of theother; interference can take place uni- and bilaterally.

    If ovule parents exert no modifying effects on pol-len competition relations, incompatibility is not anissue. If, in addition, pollen interference is absent, theranking in relative fertilization success of the pollenparents can be expected to be consistent and transitive.Hence, pollen deficiency will be realized for a num-ber of pollen parents. It follows that, in the absenceof modifying effects by ovule parents, pollen inter-ference is required to overcome the danger of pollendeficiency. Non-interfering conditions can easily beimagined to be realized for variable speed of pollentube growth or germination.

    Even in the case of overlapping distributions inspeed of growth for different pollen parents, distincteffects on fertilization success may be very common.Therefore, only some simple preconditions have to bemet: in the group of fastest growing pollen, one parentis represented more frequently than any other; and thepollination density is sufficiently high.

    However, in our investigations at least one obser-vation suggests the existence of considerable polleninterference, possibly connected with the stage ofpollen germination. The suggestion follows from acomparison of the in vivo fertilization performanceof several pollen types with their in vivo germinationbehavior as reported by Steiner and Gregorius (1998)and as included in Table 1. There, pollen parent ATW2is seen to have shown very poor in vitro germination,while it reached extremely high fertilization successesin all combinations. If germination were to play a de-cisive role in the overall fertilization process (which itis likely to do), a plausible explanation of this behaviorwould result from pollen interference acting unilater-ally, for example, through allelopathic forces (see e.g.,Kanchan & Jayachandra, 1980, or Murphy & Aarssen,1989) on the pollen of all competitors of ATW2.

    Modification of pollen competition relations byovule parents is necessary for incompatibility to exist,and it is also an important source for avoiding pollendeficiency. In our experiments, pollen deficiency oc-curred in three cases, two in 1993 and one in 1994 (atthe bottoms of the respective representations in Fig-ure 1). Since this deficiency was observed only forone pollen mixture in each case, this observation isprobably not very significant. Among the three pol-len mixtures, each of which was applied to differentovule parents, no modification of pollen competition

    relations was observed. But if in all other cases modi-fication were also absent, this would imply that onlytwo of the nine pollen parents in 1994 would outcom-pete all the rest. The high evolutionary improbabilityof such a situation, however, suggests that in at leastsome cases modification of competition by ovule par-ents takes place. Pollen parent Wb16 would then bea likely candidate for showing cryptic incompatibil-ity, since it is opposed in three competition situationsby all involved ovule parents, while it is superiorin fertilization success in competition with Th4 on adifferent ovule parent (see Figure 1). This indirect ex-perimental evidence justifies expectations that crypticincompatibility be realized for a number of pollenparents.

    In any case, it should be emphasized that the state-ment of cryptic incompatibility relations is restrictedto the present set of data. For example, if a pollenparent that is consistently opposed by an ovule par-ent under various competition conditions with otherpollen parents attains above-average fertilization suc-cess under a new competition condition (e.g., ATW2in competition with Me8), the claim of cryptic incom-patibility cannot be maintained for this pollen parentif it previously was opposed by this ovule parent only.

    Beyond the likelihood of pollen elimination,the outcome of the self-pollination test experimentprovides another interesting indication referring to amechanism of pollen competition known as crypticself-fertility. This term was introduced by Bertinand Sullivan (1988) to describe the phenomenonthat self-pollen applied to stigmas under exclusionof cross-pollen results in effective inhibition of self-fertilization, whereas a mixture of self- and cross-pollen results in the formation of some selfed progenyas a possible consequence of some kind of mentor ef-fect provided by the compatible cross-pollen for theotherwise incompatible self-pollen. The high rate ofloss of offspring in the test experiment thus suggeststhat cross-pollen did not exert a significant mentor ef-fect on self-pollen, so that cryptic self-fertility may notplay an important role in our investigations.

    Environmental stability of competition relations

    The good replicability of the results with pollen mix-tures (four experiments have been repeated identically,resulting in a total progeny of 660 individuals) sug-gests that the competition relations may not be verysensitive to the scale of environmental modificationsrealized under the experimental field conditions. This

  • 271

    contrasts with the results of the accompanying invitro pollination experiments (Steiner & Gregorius,1998), where large germination differences amongpollen samples from genetically identical parents ap-peared to be the result of small-scale environmentalmodifications during pollen development. The in vivoreproduction process as a whole, therefore, seemsstable to a degree which suggests that competition dif-ferences are affected by a strong genetic component inthe mating partners involved.

    Acknowledgements

    This work was supported by grant Gr. 435/14 from theDeutsche Forschungsgemeinschaft (DFG). We thankI. Grote-Talartschik for technical assistance, H. Weis-gerber for making seed orchards available for ex-periments, and one anonymous reviewer for helpfulcomments and suggestions.

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