949

American Journal of Botany 90(6): 949–953. 2003.

BRIEF COMMUNICATION

MALES OUTCOMPETE HERMAPHRODITES FOR SEED

SIRING SUCCESS IN CONTROLLED CROSSES IN THE

POLYGAMOUS FRAXINUS EXCELSIOR (OLEACEAE)1

MARIE-ELISE MORAND-PRIEUR,2 CHRISTIAN RAQUIN,JACQUI A. SHYKOFF, AND NATHALIE FRASCARIA-LACOSTE

Laboratoire Ecologie, Systematique et Evolution (ESE), UMR CNRS-ENGREF 8079, Batiment 360, Universite Paris-Sud,91 405 Orsay Cedex, France

Polygamy (including trioecy and subdioecy), the co-occurrence of males, hermaphrodites, and females in natural populations, is arare and poorly studied breeding system expressed in Fraxinus excelsior L. (Oleaceae), a wind-pollinated tree. Here we investigatesiring ability of pollen from male vs. hermaphrodite individuals to better understand this sex polymorphism. We conducted single-donor and two-donor pollination experiments and compared both fruit set and seed siring success, assessed with polymorphic micro-satellite markers, of male and hermaphrodite individuals. Single pollen donor crosses allowed us to verify the male function ofhermaphrodites. However, pollen from hermaphrodites was much less proficient than male pollen, with males siring 10 times as manyfruits in single donor pollination treatments. This result was strengthened by the surprisingly low reproductive success of hermaphroditesin pollen competition conditions: of the 110 seedlings analyzed three were selfed and only one was sired by the hermaphrodite donor.The remaining 106 were sired by the male pollen donor. These results raise the question of the maintenance of male fertility inhermaphrodites in Fraxinus excelsior. Male function of hermaphrodites in this species now needs to be assessed under field conditions.

Key words: androdioecy; Fraxinus excelsior; male reproductive success; microsatellites; Oleaceae; paternity assignment; pollencompetition; polygamous mating system; trioecy.

Breeding systems show great diversity in angiosperms butthe co-occurrence of males, hermaphrodites, and females with-in the same population, identified by the general term ‘‘polyg-amy’’ (including trioecy and subdioecy), appears to be rare,representing 3.6% of species (Yampolsky and Yampolsky,1922; Richards, 1997), and is poorly described.

Fraxinus excelsior L. (Oleaceae), a temperate tree species,has been variously described as polygamous, trioecious, orsubdioecious (Binggeli and Power, 1999). The maintenance ofdiverse sexual forms in this species has not yet been studied.In particular, the relative male fertility of single sexed puremales vs. hermaphrodite individuals, though intensively in-vestigated in other androdioecious species, remains unexploredin this one. Androdioecy, the rarest breeding system in angio-sperms (Yampolsky and Yampolsky, 1922; Charlesworth,1984), is defined by the co-occurrence of male and hermaph-rodite individuals in natural populations. Only six plant spe-cies, three belonging to the Oleaceae family, are apparentlyfunctionally androdioecious, i.e., with male-fertile hermaph-rodites coexisting with pure males (Liston et al., 1990; Lepartand Dommee, 1992; Pannell, 1997a; Ishida and Hiura, 1998;Akimoto et al., 1999; Dommee et al., 1999; Vassiliadis et al.,2002). Moreover, maintenance of androdioecy requires a malefertility advantage of males over hermaphrodites. This maleadvantage was found for pollen production (Philbrick and Rie-seberg, 1994; Pannell, 1997b) and for fertilization ability in

1 Manuscript received 10 October 2002; revision accepted 10 January 2003.The authors thank P. Bertolino, O. Cudelou and O. Jonot for laboratory

work. The first author acknowledges an FCPR grant from the French Ministryof Agriculture (FMA). The ENGREF institution from the FMA provided fi-nancial support.

2 Author for reprint requests (E-mail: marie-elise.morand@ese.u-psud.fr).

controlled crosses using single donor pollinations (Ishida andHiura, 1998; Dommee et al., 1999; Vassiliadis et al., 2000).In natural populations however, male and hermaphrodite in-dividuals may compete for seed siring (Pannell and Ojeda,2000), and paternity analysis is needed to determine the ef-fective male reproductive success of male vs. hermaphroditeindividuals. Indeed in the androdioecious Phillyrea angusti-folia pollen from hermaphrodites and males is equivalent inseed siring efficiency estimated by paternity analyses (Vassi-liadis et al., 2002) although a male advantage is found undersingle donor conditions (Vassiliadis et al., 2000).

An important aspect of male success of pollen from malevs. hermaphrodite individuals could be seed siring ability incompetition. To date no paternity analyses of different (i.e.,male vs. hermaphrodite) pollen donors in controlled crosseshave been reported in androdioecious species. More generally,although seed paternity is nonrandom when pollen from twoor more pollen donors is applied to stigmas (see Marshall andOliveras [2001] for a review of these studies), this has usuallybeen related to pollen tube growth rate differences rather thanphenotypic differences between the pollen donors (see for ex-ample; Bjorkman et al., 1995; Snow and Spira, 1996; Pasonenet al., 1999). On the other hand, in Caenorhabditis elegans, awell-studied androdioecious nematode, male sperm is superiorto sperm of selfing hermaphrodites for fertilizing ova (Wardand Carrel, 1979; Hodgkin and Barnes, 1991; Singson et al.,1999), with a male advantage from two- to fourfold in off-spring production (Hodgkin and Barnes, 1991).

The aim of the present study was to give some insights intothe polygamous breeding system of Fraxinus excelsior undercontrolled conditions. Several single-donor and two-donorcrosses were performed. We analyzed fruit set and also as-

950 [Vol. 90AMERICAN JOURNAL OF BOTANY

signed paternity using microsatellite markers to (1) estimatepotential male fertility of hermaphrodites, (2) compare malereproductive success of males and hermaphrodites in single-donor pollinations, and (3) assess seed siring success of malesand hermaphrodites in pollen competition conditions.

MATERIALS AND METHODS

Study species—Common ash (Fraxinus excelsior L., Oleaceae) is a decid-uous forest tree with wind-pollinated flowers and wind-dispersed fruits, dis-tributed throughout Europe and Asia Minor (Wardle, 1961). Sexual types pre-sent a continuum from pure male to pure female individuals, with a range ofhermaphrodites in between (Wardle, 1961; Picard, 1982; Binggeli and Power,1999; Wallander, 2001). A single individual can have varying proportions ofmore than one sort of flowers (staminate plus perfect or pistillate plus perfect)(Binggeli and Power, 1999; Wallander, 2001). Perfect flowers consist of onepistil and two stamens attached to the base of the ovary (Binggeli and Power,1999; Wallander, 2001). Flowers have neither calyx nor corolla and are tightlyclumped in inflorescences borne on lateral buds. Flowering occurs in the northof France in March–April and usually lasts 3–4 wk within a given population.Perfect flowers are protogynous, although anther dehiscence occurs while thestigma is still receptive. The ovary contains four ovules, and fruits are gen-erally one-seeded winged samaras.

Controlled crosses—Six Fraxinus excelsior individuals (four hermaphro-dite trees and two males) located on the Orsay University campus were usedas parents in the controlled crosses. No pure female trees were present, sothe hermaphrodites H1, H2, H3, and H4 were used as maternal trees. Two ofthese (H1 and H2) were pure hermaphrodites, bearing only perfect flowers,and these were also chosen as hermaphrodite pollen donors. Two pure males(M1 and M2), bearing only staminate flowers, were chosen as the male pollendonors. Hermaphrodite trees were tested for self-compatibility in several yearsby bagging inflorescences before bud opening. Though early development offruits was normal, immature samaras all abscissed and fell, suggesting self-incompatibility (see Morand et al., 2002).

In March 1999, flowering branches of pollen donors were cut for pollencollection. These branches were put into water in the laboratory and storedat room temperature until flowering. Pollen was harvested either on sheets ofaluminum foil (pollen donors M1 and H1) or in maize pollen-collecting bags(donors M2 and H2). Pollen germination rate was determined for all fourindividuals by counting 2 3 100 pollen grains incubated for 6 h at 248C onpollen growth medium containing 1.27 mmol/L Ca(NO3)2, 1.62 mmol/LH3BO3, 0.2 mmol/L KH2PO4, 0.05 mmol/L K2HPO4, and 351 mmol/L sucrose(Mulcahy and Mulcahy, 1983) and solidified with 5 g/L Phytagel (SigmaAldrich, Saint Quentin Fallavier, France) before autoclaving. Pollen germi-nation was similar between individuals with the same pollen collection meth-od but differed between the following collection methods: aluminum foil invitro pollen germination rate of 0.43 6 0.02 (mean 6 1 SD; N 5 4); maizepollen bags in vitro pollen germination rate of 0.16 6 0.05 (N 5 4). Thenumber of inflorescences from which we collected pollen was equivalent fromone tree to another.

Three pollination treatments were performed by bagging inflorescences be-fore bud opening and brushing pollen over the open stigmas as soon as theyappeared and 1 wk later: (1) pollination with pollen from one hermaphroditetree (H1 or H2), (2) pollination with pollen from one male tree (M1 or M2),(3) pollination with a mass for mass mixture of pollen from one male treeand one hermaphrodite tree (pollen competition treatment). For this last treat-ment, we mixed pollen with equivalent in vitro germination rates, giving twodifferent pollen mixtures: (M2 1 H2) and (M1 1 H1). A total of 25 controlledcrosses on one pair of inflorescences each were carried out, using each of thefour mother trees for all three treatments (Table 1). Because the flowers aresmall, the number of flowers per inflorescence could not be counted for eachpollination; however, within a pollen recipient tree, the number of flowers perinflorescence is comparable. One to three controlled crosses was performedper pollination combination (donor[s]) and recipient) (Table 1). Finally, as thefour mother trees were hermaphrodite, autonomous selfing was possible since

the numerous tiny hermaphrodite flowers could not be emasculated. However,no self-pollen was manually added. Bags were removed 3 wk after the firstpollination treatment and fruits were collected and counted in mid-August.Seedlings for molecular analysis were obtained as described in Raquin et al.(2002), with a slight modification of the H10 culture medium (containing 28mmol/L sucrose instead of 14 mmol/L sucrose and 14 mmol/L maltose).

Microsatellite analysis—A total of 146 seedlings were analyzed with mi-crosatellite markers to check paternity. Table 1 summarizes the distributionof seedlings over the various pollination treatments. All seven viable seedlingsresulting from pollination with a hermaphrodite pollen donor were analyzedto verify whether they were sired by the putative donor or resulted from pollencontamination. A sample of seedlings from the male pollen donor treatmentwas analyzed and for the pollen competition treatment, a sample of offspringfrom each pollinated branch was genotyped.

Leaves of parent trees and young seedlings were collected and frozen at2808C in the laboratory before DNA extraction. Total DNA was extractedeither from 100 mg of material using the DNeasy Plant Mini Kit (Qiagen,Courtaboeuf, France) or from 50 mg of material using the DNeasy 96 PlantKit (Qiagen). Among the microsatellite markers isolated on Fraxinus excelsiorby Brachet et al. (1999) and Lefort et al. (1999), six loci were used: M2–30,FEMSATL 4, 11, 12, 16, and 19. The 20 mL of polymerase chain reaction(PCR) mixture contained 15 ng of template DNA for the parents and 30 ngfor the seedlings, 90 mmol/L of dNTP, 0.375 mmol/L of each primer, 1.5 mLof 103 buffer, and 0.375 units of Taq polymerase (Q-Biogene, Illkirch,France). For the locus FEMSATL 12, 0.375 mmol/L of MgCl2 was also added.Amplification reactions were carried out as described in Brachet et al. (1999)and Lefort et al. (1999). The PCR products were separated by electrophoresisin 6% polyacrylamide gels and visualized by silver staining according toStreiff and Lefort (1997).

Statistical analyses—A mixed partially nested ANOVA model using JMP5.0 software (SAS, 1995) was used to test the effect of pollen donor identity(nested within pollination treatment), recipient identity (nested within polli-nation treatment), and pollination treatment (three levels: male, hermaphro-dite, pollen mixture) on fruit set.

RESULTS

Male fertilities of hermaphrodites and males—The molec-ular analysis of 143 of the 146 seedlings revealed no paternityinconsistent with the genotype of the experimental pollen do-nor, and the other three seedlings had genotypes consistentwith selfing, giving no evidence for pollen contamination. Her-maphrodites were thus capable of siring seed, although theirmale fertility was lower than that of males (see below) andwe found the first evidence to our knowledge for partial self-compatibility of Fraxinus excelsior hermaphrodites.

No significant differences were observed between pollendonors (nested within pollination treatment; F3,10 5 0.24, P 50.87) or between hermaphrodite recipients (nested within pol-lination treatment; F9,10 5 0.82, P 5 0.61), so we carried outa simplified ANOVA that detected a significant effect of pol-lination treatment on fruit set per pollinated branch (Welchunequal variance; F2,11 5 14.44, P 5 0.0008). Multiple com-parisons separated hermaphrodite donors from the other twopollination treatments, with male pollen donors siring 10 timesmore fruits than hermaphrodites (Fig. 1).

Male and hermaphrodite seed siring success in pollencompetition—Of the 107 outcrossed seedlings analyzed, 106were sired by the male and only one by the hermaphroditepollen donor (Fig. 2). In our controlled conditions of pollencompetition, the two Fraxinus excelsior males generally ex-cluded the two hermaphrodite pollen donors from siring seed.

June 2003] 951MORAND-PRIEUR ET AL.—MALE FERTILITIES IN FRAXINUS EXCELSIOR

TABLE 1. Numbers of Fraxinus excelsior fruits (Fruit) and seeds (Seed) obtained for each pollination. Number of viable embryos (Embryos)obtained after rehydration following Raquin et al. (2002) is also given. N is the number of seedlings analyzed with the microsatellite markers.Paternity M, Paternity H, and Selfed are, respectively, the number of seedlings assigned to the male pollen donor, the number of seedlingsassigned to the hermaphrodite pollen donor, and the number of selfed seedlings.

Recipients

Pollen donors

Hermaphrodites

H1 H2

Males

M1 M2

Pollen mixture

M1 1 H1 M2 1 H2

H1FruitSeedEmbryosNPaternity MPaternity HSelfed

———————

———————

1110

86060

1100000

———————

———————

———————

2523—b

—b

—b

—b

—b

7776—b

—b

—b

—b

—b

———————

———————

686617a

171511

979617a

111100

H2FruitSeedEmbryosNPaternity MPaternity HSelfed

3311010

1100000

———————

———————

1110—b

—b

—b

—b

—b

5346

—b

—b

—b

—b

—b

1210

87700

———————

———————

16876501

564119a

1312

01

———————

———————

H3FruitSeedEmbryosNPaternity MPaternity HSelfed

0000000

———————

1100000

———————

1717141111

00

130121

14a

1111

00

———————

474729—c

—c

—c

—c

———————

5853472222

00

———————

2019199900

545237151500

H4FruitSeedEmbryosNPaternity MPaternity HSelfed

———————

———————

0000000

———————

———————

———————

———————

171410—c

—c

—c

—c

———————

———————

———————

2320149900

212088800

Note: A dash indicates that no cross was made.a Only a sample of seeds was germinated (cross H3 3 M1, 22 seeds; crosses H1 3 [M2 1 H2], 20 seeds per cross; cross H2 3 [M1 1 H1], 32

seeds per cross).b No seeds were germinated.c No seedlings were analyzed with the microsatellite markers.

DISCUSSION

We found that (1) hermaphrodites are male-fertile and par-tially self-compatible, (2) in single donor pollinations, malepollen donors have a greater seed siring success than her-maphrodites, and (3) in pollen competition conditions, maledonors almost always exclude hermaphrodites from ovule fer-tilization.

The two pure hermaphrodites used in our controlled crossesboth successfully sired seed. However, the very low fruit setand the few viable embryos obtained from these single-donorpollinations suggest far lower pollen fertility of hermaphro-dites than males. Indeed, we found a 10-fold male fertilityadvantage for male pollen donors over hermaphrodites thatwas further accentuated under pollen competition conditions,where males sired almost all analyzed seeds. Male advantagein fertilization success, but not of this magnitude, is known inother androdioecious species (Ishida and Hiura, 1998; Dom-mee et al., 1999; Vassiliadis et al., 2000).

Low fertilization success of hermaphrodites could result ei-

ther from a poor male function of this sex phenotype or fromgenetic incompatibility between the few individuals tested,since it is possible that the few hermaphrodite donors and re-cipients shared common incompatibility alleles. These twopoints remain to be elucidated, for example by examining pol-len tube growth in two-donor pollinations to assess pollenquality of both sexual forms and by performing additionalcrosses between genetically distant individuals.

The three selfed offspring found in the pollen competitiontreatment suggest that Fraxinus excelsior is partially self-com-patible despite general failure of fruit production followingselfing (Morand et al., 2002). Although we can now assert thatcommon ash seems partially self-compatible under certain pol-lination regimes, selfing rates in natural populations remain tobe assessed.

In natural conditions where several pollen donors competefor ovule fertilization, male reproductive success of hermaph-rodites may be very low in Fraxinus excelsior. In Phillyreaangustifolia, however, where the relative male fertilities of

952 [Vol. 90AMERICAN JOURNAL OF BOTANY

Fig. 1. Mean number of fruit per cross (11 SD) after different pollinationtreatments on four hermaphrodite recipients (see Table 1). 3 H, pollinationusing pollen from hermaphrodite donors; 3 M, pollination using pollen frommale donors; 3 (M 1 H), pollination using a pollen mixture from one her-maphrodite donor and one male donor. Means associated with the same low-ercase letter were not significantly different (nonparametric multiple compar-isons, P , 0.05).

Fig. 2. An example of a 6% polyacrylamide gel after silver staining at the microsatellite locus M2-30. Mo, the hermaphrodite recipient tree H3; M, themale pollen donor M1; H, the hermaphrodite pollen donor H1; lanes 1–21, the progeny obtained with this mixture pollen treatment (M1 1 H1). Allelic sizesare given in base pairs. As no allele is shared between the three heterozygote parents at locus M2-30, paternity could be unambiguously attributed to the malepollen donor for each seedling.

males and hermaphrodites were assessed in single-donor pol-linations and in a natural population, single donor fertilizationsuccess did not predict relative siring success of males vs.hermaphrodites (Vassiliadis et al., 2000, 2002). These resultsemphasize the importance of assessing relative male reproduc-tive success of hermaphrodites under natural conditions. Thus,the next step in our study of the functional mating system ofFraxinus excelsior will be to perform a paternity analysis ina natural population using the available microsatellite markers.

If the very high advantage in fertility of males over her-maphrodites is verified in natural populations of common ash,the maintenance of a male function in hermaphrodite individ-uals becomes a puzzle with two types of explanation. First,hermaphrodites may be functionally male fertile, but able toreproduce only in the absence of competing males, for ex-ample in populations composed of only hermaphrodites andfemales. This might be the case in newly colonized forest gapsor open habitats, if the numerous seeds of females comprisemost of the seed rain. Although to date the genetic basis ofsex determination in this species is unknown, there appears tobe a strong genetic component as individuals of the same clonehave similar sex phenotype (M.-E. Morand-Prieur and C. Ra-quin, personal observation). If female or hermaphrodite prog-enies are more likely to segregate female and hermaphroditeoffspring than male ones, newly founded populations maycontain few or no males. Secondly, pollen production and therelics of male fertility may represent a stage in the transitionfrom hermaphroditism towards dioecy in this species. In fact,the low male fertility of hermaphrodites can be considered asa female-biased functional gender, as defined in Lloyd (1980).

June 2003] 953MORAND-PRIEUR ET AL.—MALE FERTILITIES IN FRAXINUS EXCELSIOR

In conclusion, in Fraxinus excelsior, male individuals havehigher fertilization success than do hermaphrodites both aloneand in competition. The question of the maintenance of malefunction in hermaphrodites needs further investigation and re-quires paternity analysis under field conditions. The resolutionof this question will help to define more precisely the matingsystem of this species (polygamy in the strict sense, trioecy,or subdioecy) and allow us to better understand it from anevolutionary point of view.

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