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Reproductive Biology |
Laboratoire d'Ecologie Alpine, UMR CNRS 5553, Université J. Fourier, BP 53, F-38041 Grenoble Cedex 09, France
Received for publication August 6, 2002. Accepted for publication November 21, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Apiaceae • conservation • Eryngium alpinum • inbreeding depression • microsatellites • pollination • protandry • self-incompatibility • selfing rate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), which are both influenced by the breeding system. First, reproductive success partly determines the growth rate of a population. Second, mating patterns control genetic structure and play a major role in evolutionary diversification within species. Moreover, a general relation between heterozygosity and fitness is suspected (Ledig, 1986
; Mitton, 1993
). Thus, depending on the breeding system, the consequences of, for example, demographic stochasticity and/or inbreeding depression can be more or less dramatic, and the breeding system is a characteristic of the species that can either increase or buffer extinction probabilities.
Plants exhibit an extremely wide variation in reproductive strategy. Inbreeding levels, which can span from very low to very high (Husband and Schemske, 1996
), are influenced by a diversity of mechanisms, either extrinsic or intrinsic (Barrett and Harder, 1996
; Barrett, 1998
). First, because they are sessile, plants rely on pollen vectors for their reproduction. By establishing the dispersion of pollen grains among flowers, these biotic (insects, birds) and/or abiotic (wind, water) agents determine mating opportunities and inbreeding may result from both selfing and biparental inbreeding. Second, floral design (i.e., characteristics of individual flowers) and floral display (i.e., the spatial arrangement of flowers) influence the probability of selfing. For example, this probability increases when plants display a high number of large inflorescences (geitonogamy; de Jong et al., 1993
; Harder and Barrett, 1995
; Vrieling et al., 1999
; Eckert, 2000
). Third, floral architecture can be further complicated by temporal (dichogamy) and spatial (herkogamy) separation of male and female functions. Fourth, various types of genetic self-incompatibility can act after pollination, on the stigma, or in the style. Other types of pollen–pistil interaction can be observed, in particular the prepotency of cross vs. self-pollen (Eckert and Allen, 1997
). Most of these systems tend to be facultative, thus providing flexibility under ecological conditions in which outcrossing cannot always be guaranteed. Finally, inbreeding depression may influence inbreeding levels in adult plant populations by decreasing survival and reproduction of inbred zygotes (Darwin, 1876
; Charlesworth and Charlesworth, 1987
; Husband and Schemske, 1996
).
Numerous experimental and theoretical studies have considered selfing and its evolution. Most studies have concluded that only two stable outcomes were possible (Lande and Schemske, 1985
; Charlesworth and Charlesworth, 1987
): predominant outcrossing associated with strong inbreeding depression or predominant self-fertilization with weak inbreeding. However, it is now accepted that mixed-mating systems may also be stable, depending on the balance between positive and negative consequences of selfing (Schemske and Lande, 1985
; Holsinger, 1991
). Selfing can be considered adaptive in many cases. First, selfing has a genetic advantage, given that selfers are both mother and father to their own seeds (Fischer, 1941
; Jain, 1976
). Second, selfing is advantageous when it occurs because outcross pollen is scarce (reproductive assurance hypothesis; Lloyd, 1992
). Third, if the exact mode of self-pollination is strict autogamy (as opposed to geitonogamy), the quantity of pollen lost during its transfer to stigma is lowered compared to that of outcrossing (Barrett and Harder, 1996
). Fourth, the ability to self is advantageous for the successful colonization by a single propagule following long-distance dispersal (Baker, 1955
). Last, selfing facilitates and accelerates local adaptation and ensures reproductive isolation between sympatric species (Jain, 1976
). On the other hand, selfing can also have negative consequences. Even if more empirical work is needed, there is some evidence for reduced within-population genetic variance in highly inbred populations compared with outbreeders (Charlesworth and Charlesworth, 1995
). Thus, in the long term, the adaptive potential in selfing populations could be lower than in outcrossing ones. In the shorter term, in species that outcross, selfing may lead to a decrease in survival and/or fecundity of inbred individuals ("inbreeding depression"; Darwin, 1876
; Charlesworth and Charlesworth, 1987
; Husband and Schemske, 1996
). These possible negative outcomes are accompanied by the reduced opportunities for pollen export when selfing occurs ("pollen discounting"; Nagylaki, 1976
; Holsinger et al., 1984
; Harder and Wilson, 1998
).
Thus, inbreeding in itself is not necessarily negative, and historically highly selfing species are thought to have purged deleterious alleles (Lande and Schemske, 1985
; Schemske and Lande, 1985
; Barrett and Charlesworth, 1991
; Latta and Ritland, 1994
; Husband and Schemske, 1996
; also controversial, see Eckert and Barrett, 1994
; Affre and Thompson, 1999
; Byers and Waller, 1999
). Nevertheless, a rapid increase in selfing in primarily outcrossing populations can be responsible for their extinction because of inbreeding depression and increased genetic drift (Frankham, 1998
; Saccheri et al., 1998
). As a result, it appears necessary, from a conservation perspective, to be aware of the precise situation of a species. Is it selfing without any evidence of inbreeding depression, suggesting an old habit of selfing? Is it completely (or almost completely) outcrossing? Or is it selfing and suffering high inbreeding depression, in which case conservation measures are urgently needed?
In the present study, we aim at determining the reproductive status of E. alpinum, an endangered sub-alpine species (Gillot and Garraud, 1995
): Is selfing possible? What is the selfing rate? What is the impact of major extrinsic and intrinsic factors, such as pollinators' foraging behavior, self-incompatibility, and inbreeding depression on selfing rate? To answer these questions, we pursued in the field and in the laboratory various avenues, such as observing pollinators, determining the selfing rate on seed progenies (using microsatellite markers), making controlled crosses, testing for inbreeding depression at the germination stage, and studying stigma/stamen maturity and pollen viability over time. The characterization of these mechanisms and their interaction should lead to a global understanding of the reproductive biology of the species and enable us, in the short term, to understand the current status of the populations and to propose an efficient plan for conservation management. In the long term, interpreting such complementary results could lead to a global understanding of dynamic population processes and evolution (Ouborg et al., 1999
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The aerial parts of the plant (leaves and stems) dry out in the fall and are initiated from the taproot in the spring. Each flowering individual generally produces 1–5 stems, each of them bearing 1–5 inflorescences: one terminal inflorescence and 0–4 axillary inflorescences. Each inflorescence produces 200–300 flowers. At maturity, the upper leaves and bracts turn blue (giving an intense and showy blue color to the whole population). Flowering usually starts a few days later. On each inflorescence, flowers are arranged on spirals and open in sequence from bottom to top. The spirals spanning from the bottom to the top of a given inflorescence are all composed of the same number of flowers (hereafter referred to as the "number of ranks of flowers of the inflorescence"). Each flower produces two ovules and five stamens, which are folded within the petals and are not visible until maturity. They are exserted a few hours before dehiscence. After dehiscence, stamens and petals fall. Fruits are schizocarpous diachenes. The species is patchily distributed in France, Italy, Switzerland, Austria, and Croatia (Cherel and Lavagne, 1982
). The plant, emblematic of alpine flora, grows in open habitats (avalanche corridors or hayfields), at altitudes between 1300 and 2500 m a.s.l. The species is threatened by human activities, mainly by cutting for commercial use (flowering stems were cut extensively to be sold as dried bouquets in towns) and by changes in land use leading to habitat destruction and fragmentation. Indeed, former hayfields are now either used as pastures, which limits flowering, or are abandoned, leading to habitat closure (unfavorable to E. alpinum). Although locally abundant, some populations are known to decrease in size while others have disappeared. Eryngium alpinum is now protected all over Europe (European Habitat Directive; Wyse and Akeroyd, 1994
) and is considered vulnerable by the International Union for the Conservation of Nature (IUCN; Gillot and Garraud, 1995
). A genetic survey, performed in 12 widespread French populations, showed that all populations were at Hardy-Weinberg equilibrium (Fis = 0; M. Gaudeul, unpublished data).
Study sites
In situ experiments were performed at two sites in the Fournel Valley (44°79' N, 6°53' E), located 10 km south of the city of Briançon, France. Eryngium alpinum is patchily distributed throughout the 12-km-long, east/west-oriented valley. The first site, called les Bernards, is at the eastern entrance of the valley, 1550 m a.s.l.; it is an abandoned 1-ha hayfield, which is currently undergoing extensive shrub development. The second one, les Deslioures, is 8 km deeper into the valley, 1600 m a.s.l., at the bottom of an avalanche corridor. Both sites are in the periphery of the Ecrins National Park. Les Deslioures has the densest and largest population of E. alpinum in Europe (
12 ha) with several thousand plants. Sheep graze this site yearly in the fall. The valley has a complex geology with limestone bedrock at the entrance, while the sides of the valley are mainly composed of schist. The climate is continental, with great differences in temperature through the year (maximum in July) and low precipitation (minimum in June). The snow cover usually lasts from November to April. Bee hives are implanted in the vicinity of both sites, potentially leading to an overestimation of the honey bee (Apis mellifera) frequency in E. alpinum natural sites.
Malaval has a very small population (10 individuals) located in the Ecrins National Park, near the Lautaret pass, 1300 m a.s.l. (45°04' N, 6°31' E). Plants grow on a very steep and very stony slope. Champagny has an intermediate-size population (around 100 individuals) situated in the Vanoise National Park, near Champagny-la-Vanoise, 1850 m a.s.l. (45°45' N, 6°70' E).
Observation of pollinators
In summer 2000 at the Deslioures site, between 1200 and 1400 (solar time), we observed arbitrarily chosen pollinators on inflorescences and noted whether or not the next inflorescence visited belonged to the same plant or not. We calculated the percentage of flights within a single plant (potentially leading to geitonogamy) as opposed to the flights between distinct plants (potentially leading to outcrossing).
Controlled crosses
In summer 1998 and 2000, 6–25 individuals were randomly chosen at Bernards and Deslioures before the beginning of flowering. Within each individual, we performed different pollination treatments on different inflorescences: natural pollination (also referred to as control), autonomous selfing, natural outcrossing, manual selfing, manual outcrossing, and negative control (Table 1).
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The same protocol was adopted in 2000, except for the following: as we suspected bagging may affect seed set, whole plants (except one control stem) were surrounded with a nylon mesh tent. Moreover, because flowering was very early and because of lack of time, we did not perform all of the 1998 treatments (Table 1).
At the end of summer 1998 and 2000, all treated and control inflorescences were bagged, enabling us to collect seeds for counting before they fell. Seed set was calculated for each inflorescence by dividing the number of viable seeds by the total number of ovules (comprising viable, aborted, and unfertilized fruits). It was desirable to perform all treatments on each plant such that maternal effects do not obscure differences in reproductive success. Paired t tests were used to determine the effect of pollination treatment on seed set. This analysis focused on comparisons among inflorescences receiving different treatments on the same plant. Within each site and for each year, t tests were performed for each pair of treatments with SAS software (SAS Institute, 1990
). Significance levels were adjusted for multiple comparisons (Bonferroni correction).
Selfing rate
At the end of summer 2001, we sampled five to six random maternal plants and 15 open-pollinated seeds per plant in one large population at Deslioures and in two restricted sites, Malaval and Champagny, which had about 10 (only five were flowering) and 100 individuals, respectively. For maternal plants, leaf material was conserved in silica gel. DNA was extracted from leaf and seeds with the DNeasy 96 Plant Kit (QIAGEN, Courtaboeuf, France), following the manufacturer's protocol. Each DNA sample was individually genotyped at three microsatellite loci (Ealp040, EalpD268, and Ealp035; Gaudeul et al., 2002
). To ensure high reproducibility in spite of low DNA quantity (around 1 ng/µL for seeds), a two-step amplification strategy was adopted. Pre-amplification was performed in a 20-µL volume containing about 8 µL of template DNA, 0.1 mmol/L of each dNTP (Perkin-Elmer, Applied Biosystems, Courtaboeuf, France), 0.05 µmol/L of each non-fluorescent primer (Genset Oligo, Paris, France), 2 mmol/L MgCl2 (Perkin-Elmer), 1 unit (U) Taq polymerase (Perkin-Elmer), and 1x Taq buffer (Perkin-Elmer). The cycling conditions were 10 min at 95°C, 25 cycles composed of 30 s denaturing at 95°C, 30 s annealing at 45°C, and 30 s extension at 72°C, and 7 min at 72°C to complete extension. Then, for each locus, a second amplification reaction was prepared in a 12-µL volume containing 2 µL of pre-amplified DNA, 0.1 mmol/L of each dNTP, 0.5 µmol/L of both primers (one of them labeled with fluorescence), 2 mmol/L MgCl2 (1.5 mmol/L for Ealp035), 1 U Taq polymerase and 1x Taq buffer. Reactions were run for 35 cycles with hybridization temperatures of 45°C for Ealp040 and 50°C for Ealp035 and EalpD268. Finally, amplified fragments were loaded on a 6% Long Ranger polyacrylamide gel, and electrophoresis was run for 3 h on an automated sequencer ABI 377 (Perkin-Elmer). Microsatellite patterns were visualized with Genotyper 2.0 (Perkin-Elmer). Reproducibility was confirmed by performing three independent trials on eight DNA samples obtained from seeds.
Each population was analyzed separately under the mixed-mating model of Ritland and Jain (1981) and Ritland (1989)
, implemented with the software MLTR (Multilocus t and r, version 0.9; Ritland, 1990
). We estimated the single locus (ts) and multilocus (tm) outcrossing rates, the average single-locus inbreeding coefficients of maternal plants (f), and the extent of biparental inbreeding (ts-tm). The Newton-Raphson method (Chabert, 1999
) was used to fit the observed proportions of genotypes descended from a known maternal genotype to the proportions expected under the mixed-mating model. For each parameter, 1000 bootstraps were performed and 95% confidence intervals (CI) were calculated.
Stigma/stamen maturity and pollen viability
Seven phenological stages were defined: no exserted stamens on the inflorescence, dehiscent stamens on the first five ranks of flowers, on the first 10 ranks, on the first 15 ranks, on 20 ranks (roughly equivalent to dehiscent stamens on the complete inflorescence), stamens fallen on the first five ranks, and stamens and petals fallen on the first five ranks. On 5 August 2001 at the Deslioures site, we randomly chose five inflorescences per phenological stage and collected 20 styles per inflorescence among the first three ranks of flowers. We immediately fixed styles in a 1 : 2 acetic acid/absolute ethanol solution, in which they were conserved for a few days. After washing with running tap water, they were softened for 20 min at 65°C in 2 mol/L sodium hydroxide, rinsed with tap water, stained with a 1 : 1 mix of glycerol and fresh Martin solution (5% aniline blue, 0.1 mol/L potassium phosphate) and squashed under a cover slip (adapted from Dafni, 1992
). Finally, we observed styles with a fluorescence microscope and UV filter (excitation wave length: 450–490 nm). Pollen-tube walls and callose plugs fluoresce a bright yellow-green. We noted the presence and number of pollen grains and pollen tubes for each style.
To estimate the viability of pollen grains, five plants were isolated in individual nylon mesh tents before the stamens were exserted (Deslioures, 31 July 2001). As soon as stamens dehisced, we hand-pollinated one terminal inflorescence per tent with outcross pollen. We then daily collected eight styles per inflorescence (from the bottom rows of flowers) for 4 d following pollination and at 9 d. Following the same protocol as described before, we subsequently looked for pollen tubes with a microscope.
Inbreeding depression
At the end of summer 2000, we germinated seeds from the controlled crosses at Deslioures (N = 19 plants). For the control, manual outcrossing + selfing, and manual selfing treatments, groups of 20 seeds per plant were weighed and placed in petri dishes. Dishes were randomly arranged in a refrigerator (5°C, darkness) and watered daily with a solution of gibberellic acid (0.5 mg GA/mL water). Germination was monitored twice a week for 4 mo. To investigate the effect of pollination treatments, we performed t tests between all pairs of treatments on the mass and number of germinated seeds.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Controlled crosses
In 1998, seed sets of control inflorescences did not differ significantly from seed sets of the cut, control inflorescences, neither at Deslioures nor at Bernards (0.60 vs. 0.54 for Deslioures and 0.68 vs. 0.74 for Bernards). Thus, the number of flowers in an inflorescence did not change its seed set. The negative controls had extremely low seed sets (0.02, Deslioures; 0.01, Bernards). The seeds that did form were probably due to accidental cross pollen transfer on stigmas. We do not interpret this result as evidence for agamospermous seed production.
In 1998 and 2000, seed sets of control inflorescences were about 0.70 and all treated inflorescences, either manually or naturally pollinated, had significantly lower seed sets (except naturally outcrossed inflorescences in 1998, Bernards; Fig. 1). This reduction suggested that the experimental design was somehow limiting seed production and that we were less effective at pollinating than are natural pollinators. Nevertheless, in both years, outcrossed inflorescences appeared to have an intermediate seed set (0.18 to 0.61) between the selfed (0.08 to 0.38) and controls (0.60 to 0.73) (Fig. 1).
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On the plants that we isolated before anther dehiscence, we observed pollen tubes on two out of 40 styles collected 4 d after hand pollination. Nine days after pollination, a high number of pollen tubes were visible in all the styles. Thus, pollen grains can remain for at least 4–5 d on a stigma before germinating.
Inbreeding depression
Paired t tests evidenced statistically significant differences in seed mass and number of germinated seeds among pollination treatments. However, these effects differed for the two variables: outcrossed + selfed seeds were lighter than selfed and naturally pollinated ones, but their germination rates equaled those of naturally pollinated ones and surpassed those of selfed seeds (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
The almost complete outcrossing habit of E. alpinum was demonstrated by the microsatellite genotyping of seed progenies. This habit was in agreement with a previous genetic survey of 12 populations, which revealed null within-population fixation indices (M. Gaudeul, unpublished data). Moreover, naturally pollinated inflorescences had high seed sets compared to selfed inflorescences (0.60 to 0.73 vs. 0.08 to 0.30), and we showed inbreeding depression at the germination stage. Both results are congruent with a primarily outcrossing breeding system. Indeed, although still controversial, inbreeding depression should decrease with continued selfing (Eckert and Barrett, 1994
; Affre and Thompson, 1999
; Byers and Waller, 1999
) because recessive deleterious alleles are expressed in homozygote individuals and purged through selection (Barrett and Charlesworth, 1991
; Latta and Ritland, 1994
; Husband and Schemske, 1996
). In addition, numerous experimental studies on species with a wide range of selfing rates suggest that selfers typically express late inbreeding depression (if any), whereas outcrossers usually suffer early-acting depression (e.g., on germination, as is the case here for E. alpinum; Husband and Schemske, 1996
). Our high outcrossing rate was rather unexpected given the pollination ecology of E. alpinum, which tends to suggest a high potential for geitonogamy (70% of insect flights occur between inflorescences within a plant). The controlled crosses and the timing of stigma/stamen maturation provided information on potential mechanisms limiting selfing.
The temporal study of pollen germination on stigmas showed an important time lag between stamen dehiscence and pollen germination. This is not likely to be due to the lack of pollen deposition on stigmas. Indeed, pollinators' visitation rates are high as soon as a few stamens of a given inflorescence are dehiscent (data not shown). Moreover, hand pollinations on isolated inflorescences also lead to delayed germination of pollen grains. Thus, increased pollination success with increased flower number cannot explain the observed pattern that is consequently interpreted as the evidence of protandry. Within a flower, stigmas did not become mature until about 4–5 d after stamens had deshisced. Such temporal dioecy is common among members of the Apiaceae family and has been found in several Eryngium species in Mexico (Cruden and Hermann-Parker, 1977
; Cruden, 1988
; Molano-Flores, 2001
).
In E. alpinum, given the observed protandry and the sequential flowering within an inflorescence (Myrium Gaudel, personal observation), insects usually move from male-phase flowers to immature flowers or from female- to male-phase flowers. At first sight, because of the lack of synchronization between these male and female phases, geitonogamous selfing between flowers within an inflorescence appears unlikely compared to outcrossing. However, we also showed that pollen grains remain viable for at least 4–5 d, which is the estimated time lag between stamen and stigma maturity. Thus, within-inflorescence fertilization may be possible if pollen does not fall from immature stigmas. We only observed pollen grains on stigmas of flowers with fallen stamens and with visible pollen tubes in the style, suggesting that pollen can only adhere to mature stigmas, a mechanism that can at least partly prevent selfing. In agreement, Molano-Flores (2001)
precisely showed that stigmas of E. yuccifolium initially have a dome shape and that they spread and become sticky only when receptive, explaining the time elapsed between pollen deposition and tube elongation. However, flowers that were manually fertilized when anthers were dehiscent did produce seeds (controlled crosses). Thus, protandry is not likely to be a complete barrier to pollen germination and to ovule fertilization among flowers of the same inflorescence.
Moreover, E. alpinum is characterized by strong mass flowering, and several studies found a significant positive correlation between the number of simultaneously opened flowers and the selfing rate (Harder and Barrett, 1995
; de Jong et al., 1999
). Given that 70% of the flights occur between inflorescences of a single plant and that each inflorescence is composed of at least 200 flowers, the temporal separation of the maturity of stamens and stigmas at the single-flower level is obviously not sufficient to eliminate geitonogamous selfing.
The controlled crosses confirmed the long-term viability of pollen. Indeed, autonomously selfed inflorescences produced seeds, which must arise from within-flower or near-flower fertilization (seed sets from 0.08 to 0.26). Thus, the plants can reproduce even in the absence of pollinators, and new sites can be colonized successfully following the dispersal of single propagules.
However, outcrossed seed set was higher than selfed seed set. This result was particularly clear at Bernards in 1998 and at Deslioures in 2000. Thus, selfing seems to be limited by some genetic mechanism, either from maternal control before fertilization (i.e., partial self-incompatibility) or from inbreeding depression very shortly after fertilization. The exact timing and mechanism of this phenomenon cannot be inferred from the experiments we performed. In addition, the interpretation of these results is complicated by the fact that in 1998, naturally pollinated inflorescences sometimes set more seeds than manually pollinated ones. This reduced seed set was probably partly explained by the negative effect of bagging: indeed, we observed that nylon mesh bags tend to retain more insect parasites and to increase humidity around the inflorescence, thus facilitating the fungal growth. This effect had been previously observed in such studies (Gugerli, 1997
) and was confirmed by experiments that we performed during summer 1999 (data not shown). Moreover, the lower seed set after manual pollination than after natural pollination could be linked to the unsuspected protandry, to the traumatizing effect of emasculation, and/or to an inadequate protocol for pollen deposition (e.g., decreased survival and vigor of pollen produced on bagged inflorescences, too much or not enough pollen deposited on stigmas; Young and Young, 1992
). However, these experimental caveats do not affect the overall observation of higher seed sets through outcrossing compared to selfing.
Which mechanism—either partial self-incompatibility or early acting inbreeding depression—is most likely to explain such a result? Even if inbreeding depression affected germination rates, it seems rather unlikely between ovule fertilization and seed maturation. Indeed, although probably receiving both self and outcross pollen, naturally pollinated inflorescences have high seed sets (0.60 to 0.73) compared to selfed inflorescences (0.08 to 0.30). The negative effect of bagging alone is probably not responsible for this large difference. But if there were significant early-acting inbreeding depression, the differential viability of selfed vs. outcrossed zygotes would probably have decreased the reproductive success of control inflorescences. As a consequence, higher seed set through outcrossing is more likely to result from the better germination of cross vs. self-pollen (even if inbreeding depression cannot be ruled out). Such self-incompatibility phenomenon may evolve in species with large floral display, when geitonogamous pollination has a detrimental effect on fitness. This effect was found in E. alpinum, as germination rates of selfed seeds were lower than those of outcrossed ones. Because geitonogamy often presents the disadvantages of selfing without offering its advantages (e.g., reproductive assurance), it is usually not considered as adaptive, but rather as a by-product of large plant size and mass flowering (Brunet and Eckert, 1998
; Eckert, 2000
). That is why in plant species, high potential for geitonogamy is often associated with self-incompatibility (Arroyo, 1976
).
In spite of protandry and partial self-incompatibility, selfing is possible and could provide reproductive assurance: when stamens dehisce, self-pollen is probably transported to stigmas before outcross pollen. However, since the stigmas are not mature, pollen grains probably do not germinate easily or rapidly. During this female-inactive phase, cross pollen can be transferred onto the stigmas. In this case, it would subsequently compete against self-pollen and probably manage to germinate in most cases. On the contrary, if no cross pollen is available because of low density of plants or reduced pollinator activity, early-deposited pollen could germinate and selfing would occur. This temporal mechanism is in agreement with microsatellite estimates of breeding system parameters: although the number of maternal families was rather low in each population (five or six), leading to large 95% confidence intervals, higher selfing rate and higher parental inbreeding coefficient were observed in the Malaval population, which is composed of only 10 plants (and only five flowering) and which is therefore more threatened by reduced pollinator activity than in the larger populations at Deslioures and Champagny. Indeed, in this study, pollinator exclusion rather strongly reduced seed set, indicating that pollinator limitation could potentially reduce plant fitness. In this case, autonomous self-fertilization can provide reproductive assurance, as has been demonstrated in many, mostly annual plant species. Whether such a phenomenon should be considered as adaptive in long-lived perennials is controversial. Indeed, Morgan and Schoen (1997)
emphasized the fact that, unlike annuals, such species would experience inbreeding depression over many seasons and that they may pay a greater cost when selfing increases total seed set at the expense of future survival and reproduction. The importance of perennial strategy (see also Calvo, 1993
) could explain that in E. alpinum, not all the flowers are fertilized and transformed into fruits. Indeed, we observed that the apical flowers of terminal inflorescences, as well as most flowers of axillary inflorescences, only rarely set seeds and sometimes do not even open (M. Gaudeul, personal observation).
From a conservation perspective, this study suggests that the risks associated with the reproductive ecology of E. alpinum are rather low in large populations. Indeed, selfing is strongly limited by protandry and partial self-incompatibility, and preponderant outcrossing is likely to maximize the adaptive potential and minimize inbreeding depression. Meanwhile, the species can self-fertilize autonomously, and consequently, does not rely completely on pollen vectors to reproduce. However, we found that the smallest population, Malaval, displayed a higher selfing rate than the other two (tm = 0.65; 95% CI = 0.39–0.90), probably due to the reduced attractiveness of the plants (from their small number) and to the subsequently reduced pollinator activity. As we considered only one small population, this trend needs to be confirmed but it appears similar to the Allee effect (Allee, 1951
) that has been demonstrated for a variety of plant species (see Groom, 1998
; Hackney and McGraw, 2000
; Moody-Weiss and Heywood, 2001
). This could lead small populations of E. alpinum to have lower seed sets and/or higher inbreeding depression and thus to enter a vicious circle leading to extinction (the extinction vortex; Gilpin and Soulé, 1986
).
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FOOTNOTES |
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2 Author for correspondence and reprint requests. Current address: Department of Plant Ecology, Evolutionary Biology Centre, Uppsala University, Villavägen 14, SE-752 36 Uppsala, Sweden. myriam.guadel{at}ebc.uu.se
.
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