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Density-dependent mating and reproductive assurance in the temperate forest herb Paris quadrifolia (Trilliaceae)¹

² Division Plant Ecology and Systematics, University of Leuven, Arenbergpark 31, B-3001 Heverlee, Belgium ³ Research Institute for Nature and Forest, Kliniekstraat 25, B-1070 Brussels, Belgium

Received for publication 13 August 2007. Accepted for publication 8 January 2008.

ABSTRACT

In animal-pollinated plants, autonomous selfing provides reproductive assurance under conditions of infrequent pollinator visits or a lack of mates, but few data are available for wind-pollinated species or species with combined insect and wind-pollination, for which it is often assumed that pollen availability does not limit reproduction. In this study, the capacity of autonomous selfing was investigated in the temperate forest herb Paris quadrifolia, and an emasculation experiment was performed under natural field conditions to investigate the contribution of autonomous selfing to total seed set across a continuous gradient of densities of flowering conspecifics. In the absence of wind or pollinators, autonomous selfing was observed through anthers approaching stigmas at the end of flowering and the capacity for autonomous pollination was about 0.34. Under natural conditions, considerable outcross pollination was observed, but the proportion of ovules successfully fertilized significantly decreased with decreasing density of conspecifics when flowers were emasculated, but not when flowers were left intact. These results indicate that autonomous selfing resulted in reproductive assurance (RA = 0.16) and thus support the hypothesis that autonomous selfing can also provide reproductive assurance in wind-pollinated species.

Key Words: autogamy • delayed selfing • mixed mating • Paris quadrifolia

In animal-pollinated plants, autonomous selfing provides reproductive assurance under conditions of infrequent pollinator visits or a lack of mates (e.g., Kalisz et al., 2004; Herlihy and Eckert, 2002), but few data are available for wind-pollinated species or species with combined insect and wind pollination (ambophilous pollination) for which it is often assumed that pollen availability does not limit reproduction. Nonetheless, in wind-pollinated species the spatial distribution of pollen around paternal plants is typically leptokurtic (e.g., Gleaves, 1973; Levin and Kerster, 1974; Rognli et al., 2000) and the proportion of fertilized ovules and seed set in recipient plants has been found to decrease rapidly with increasing distance from the pollen donor (Allison, 1990; Davis et al., 2004). However, there are few experimental tests of whether the capacity of self-fertilization increases seed set and thus provides reproductive assurance in wind-pollinated species. Indeed, Eckert et al. (2006) recently reviewed the literature available and found experimental results for 29 taxa, none of which were wind pollinated. As in animal-pollinated species, the most obvious way to test that self-pollination provides reproductive assurance, is to remove anthers from flowers before dehiscence and to compare seed set between control and emasculated plants (Cruden and Lyon, 1989; Lloyd and Schoen, 1992). However, since the contribution of self-pollination to seed set in wind-pollinated species may depend on the local density of conspecifics (Farris and Mitton, 1984; Wolff et al., 1988; Robledo-Arnuncio et al., 2004; Buggs and Pannell, 2006), experiments ideally also should consider the natural variation in density of flowering individuals within a population (Eckert and Schaefer, 1998).

Here, we experimentally study the capacity for autonomous self-fertilization in the long-lived clonal forest herb Paris quadrifolia L. (Trilliaceae) and investigate whether it provides reproductive assurance. This species is a clonal, rhizomatous herb that occurs in the understorey of moist deciduous forests of the temperate regions of Europe and Asia (Hegi, 1975). Although the black-purple, fleshy ovary may attract small flies (Hegi, 1975), the species has many traits associated with wind rather than biotic pollination (Culley et al., 2002). The flowers consist of four petals and four sepals (Fig. 1), are inconspicuously colored (yellowish-green), produce no nectar (Müller, 1873; Knuth, 1898) and are scentless (Daumann, 1959). Pollen to ovule ratios are relatively high (Götzenberger et al., 2006), whereas pollen diameters (50 µm) are at the high end of the range typically observed for wind-pollinated species (10–50 µm) (Culley et al., 2002). Flowers are also strongly protogynous: at anthesis stigmas are already developed and anthers open a few days later at a time that stigmas are still fresh and receptive (Daumann, 1959). Protogyny combined with the fact that anthers produce large amounts of pollen provides opportunities for plants to first capture outcross pollen. Data on pollen vectors for this species are very scarce, and the few available data also suggest that the species is either wind pollinated or for the largest part selfing. Daumann (1959) has recorded only very few visits of the flies Pollenia vagabunda and Phaonia morio. Müller (1873) recorded several meat-flies visiting P. quadrifolia flowers (among others Scatophaga merdaria), but there is no evidence that these species actually served as pollinators. Selfing, on the other hand, could occur when anthers progressively approach the stigma at the end of flowering (i.e., delayed autonomous selfing) (Fig. 1) or on very rare occasions as the result of pollinator activity (i.e., facilitated selfing). Jacquemyn et al. (2005), for example, mentioned that the pollen-robbing Agriotes pilosellus may facilitate selfing when it searches for pollen.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Delayed selfing in Paris quadrifolia: position of stigmas and anthers (A) at start of anthesis, (B) two weeks after anthesis and (C) at the end of flowering, when anthers make physical contact with stigmas.

MATERIALS AND METHODS

Capacity for autonomous pollination
To investigate the capacity for autonomous pollination in P. quadrifolia, we transplanted 80 shoots into the greenhouse to exclude them from pollinators and possible wind pollination. Twenty shoots were randomly selected and assigned to one of four pollination treatments: (1) stigmas were hand pollinated with self pollen; (2) stigmas were hand pollinated with outcross pollen; (3) plants were left unpollinated, but had their anthers intact, and (4) shoots were left unpollinated, but their anthers were removed. The latter treatment was added to confirm that autonomous seed set did not occur through apomixis or accidental pollination by insects or wind. Because flowering shoots of P. quadrifolia bear only one flower, any potential for geitonogamous pollination influencing the results of the pollination experiment was excluded a priori. For pollination treatments 1 and 2, stigmas were pollinated 3 d after opening of flowers. Outcross pollen was taken from several different shoots within the same population but at considerable distances (> 10 m) from the shoots taken for pollination.

Three months after the pollinations, fruits were harvested, and for each fruit, we counted the number of seeds and the number of ovules that were not successfully pollinated. For each fruit, we calculated the percentage of successfully pollinated ovules. The capacity for autonomous pollination, or autofertility (AF), was calculated as F_C ÷ F_I, where F_C is the proportion of ovules successfully fertilized in flowers that were excluded from pollinators and wind and F_I is the proportion of ovules successfully fertilized in naturally pollinated flowers (Lloyd and Schoen, 1992; Eckert et al., 2006).

Effects of emasculation and plant density on seed set
To study the contribution of autonomous selfing to seed set, we followed an experimental set-up similar to that of Eckert and Schaefer (1998). In a large population of P. quadrifolia (>10000 flowering individuals), a 14 x 5 m plot was laid out in May 2007 (Fig. 3A). In this plot, each flowering shoot was mapped to the nearest centimeter. We haphazardly selected and marked 100 individuals, 50 of which were left unmanipulated, whereas in the remaining 50 plants, the capacity for autogamy was removed by emasculating flowers before anther dehiscence. For each marked individual, we calculated the average distance to the nearest 10 shoots and determined local plant density as the number of flowering shoots within radii of 50, 100, and 150 cm. Fruits were harvested in mid July, and the number of seeds and the number of ovules that were not fertilized were counted. Seed set was determined as the proportion of ovules successfully fertilized to the total number of ovules. A measure of reproductive assurance (RA) was calculated as (F_I – F_E) ÷ F_I, where F_I and F_E are the proportion of ovules successfully pollinated in intact open and emasculated flowers, respectively (Eckert et al., 2006, p. 187). All experiments were conducted between May and July 2007 in Voeren (Belgium) (see Jacquemyn et al. [2005, 2006] for more details on the study area).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Experimental set-up of the pollination experiment to assess whether seed set is related to the density of flowering conspecifics and to test the hypothesis that autonomous selfing provides reproductive assurance in Paris quadrifolia (see Materials and Methods for details). (A) Detailed map of all flowering individuals within the investigated plot. Dark red circles represent plants that were emasculated; light red circles were plants that were left unmanipulated and were sampled for seed set: open circles represent the remaining plants that acted as potential pollen donors. Seed set (i.e., the proportion of ovules successfully fertilized) in relation to (B) the number of flowering conspecifics within a radius of 0.5 m and (C) the distance to the 10 nearest flowering conspecifics.

To test the hypothesis that emasculation affected seed set in P. quadrifolia and whether this was affected by the density of conspecifics, we used an analysis of covariance (ANCOVA). First, the hypothesis that the effect of density on the proportion of successfully fertilized ovules was not related to emasculation treatment (emasculated vs. intact flowers) was tested by fitting a model containing the main effects of emasculation treatment and local density, as well as the emasculation treatment x local density interaction. In this model, the interaction term provides the test of the null hypothesis of equal slopes. Because a few plants were lost due to predation by snails and the number of plants differed between the two treatments, significance was tested using a type III (partial) sum of squares. In case of clear evidence of inequality of regression slopes, separate slopes estimates were assessed by fitting a model containing a main effect of emasculation treatment and an interaction effect of emasculation treatment x local density. The emasculation treatment x local density parameter estimates then give the simple slope estimates within each level of emasculation treatment. Because the numbers of flowering individuals within radii of 50, 100, and 150 cm (P < 0.0001) were strongly correlated, only two different measures were used to assess local density of conspecifics: (1) the number of flowering individuals within a radius of 50 cm and (2) the mean distance to the 10 nearest flowering individuals.

RESULTS

Capacity for autonomous selfing
The proportion of ovules successfully fertilized was not significantly different between flowers that were hand-pollinated with self or cross pollen (93.6 and 94.0%, respectively) (Fig. 2). In contrast, the percentage of ovules successfully fertilized was significantly lower in unmanipulated plants in the greenhouse, with about 32% of all ovules being successfully fertilized (Fig. 2). The level of autonomous selfing in unmanipulated plants (AF) was 0.34. Finally, emasculated flowers in the greenhouse did not set any seed at all indicating that apomictic fertilization does not take place in P. quadrifolia and that our results were not disturbed by occasional wind or insect pollination in the greenhouse (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2. The proportion of ovules successfully fertilized under different pollinator treatments in the greenhouse. Treatments applied were: hand selfing (stigmas were hand pollinated with self pollen); hand outcrossing (stigmas were hand pollinated with outcross pollen); unmanipulated (plants were left unpollinated, but had their anthers intact); emasculated (plants were left unpollinated, but their anthers were removed).

Self-fertilization in Paris quadrifolia
Lloyd and Schoen (1992) distinguished three modes of selfing, depending on the period of potential outcross pollination: prior selfing, competing selfing, and delayed selfing. With outcross pollen limitation, all modes of autonomous selfing, and especially delayed selfing can confer reproductive assurance with little or no pollen and seed discounting (Lloyd, 1979). In P. quadrifolia, we have shown that autonomous selfing significantly contributed to seed set. Plants in the greenhouse, where wind or insects were completely excluded, indeed showed successfully pollinated ovules, although pollination success was lower than when experimentally pollinated with self or cross pollen or when plants were pollinated in natural conditions. Plants that were emasculated in the greenhouse, on the other hand, did not set any fruit, indicating that apomictic selfing does not occur.

It was further shown that autonomous selfing occurred in the developmental course of the flowers through anthers approaching to the stigmas and finally making physical contact with the stigmas (H. Jacquemyn and R. Brys, personal observation; see also Fig. 1) (i.e., delayed selfing). Delayed selfing has been demonstrated in several plant species, but mechanisms may vary from one species to the next. In Collinsia verna, for example, delayed selfing is characterized by both diminished herkogamy and dichogamy (Kalisz et al., 1999). In Aquilegia canadensis, the stamens progressively elongate toward the exserted stigma (Eckert and Schaefer, 1998). Similar to the mechanism reported here, anthers collapse on the stigma on the final day of floral development in Kalmia latifolia (Rathcke and Real, 1993) and Sanguinaria canadensis (Lyon, 1992).

Self-fertilization and reproductive assurance
Direct tests of reproductive assurance that compare seed set by emasculated flowers vs. intact control flowers subject to open pollination are still scarce (reviewed in Goodwillie et al., 2005 and Eckert et al., 2006), and results differ largely among species. Eckert et al. (2006) recently compiled all available data on experimental tests that investigated the capacity for autonomous selfing and reproductive assurance. They showed a wide variation in both RA and AF, but found no covariation between them. Furthermore, a considerable number of species did not seem to benefit from autofertility, because autogamy hardly contributed to seed production (Eckert et al., 2006).

In P. quadrifolia, reproductive assurance was rather low (0.16). However, given that the species is wind-pollinated, considerable outcross pollination can be expected within populations, reducing the contribution of autonomous pollination to total seed set. Indeed, considerable seed set was observed in emasculated flowers (71% of all ovules was significantly fertilized when flowers were emasculated vs. 84% when flowers were left intact), suggesting substantial outcross pollination within populations. Nevertheless, when plants were further away from flowering conspecifics or when the density of flowering conspecifics decreased, emasculation clearly resulted in decreased seed to ovule ratios. These results are consistent with density-dependent or distance-dependent outcross pollination, with decreasing probabilities of successful pollination when plants become isolated and confirm earlier findings of Allison (1990) and Davis et al. (2004) that seed set in wind-pollinated plants may to a certain extent be subject to Allee effects. Similarly, Eppley and Pannell (2007) showed that outcrossing rates decreased with decreasing density in the annual Mercurialis annua.

In intact flowers, on the other hand, successful seed set was completely independent of local density. These results are in sharp contrast with findings of Eckert and Schaefer (1998), who could not detect any significant effects of emasculation or density on seed set in the animal-pollinated Aquilegia canadensis. Thus, our results indicate that autonomous selfing significantly contributed to seed set and may support the hypothesis that autonomous selfing can provide reproductive assurance in wind-pollinated species or species with combined insect and wind pollination. Indeed, when only those plants more than 0.5 m away from the 10 nearest conspecifics are included in the analysis, the measure of reproductive assurance increases from 0.16 to 0.23.

Mixed mating and wind-pollination
A recent review showed that ca. 42% of investigated species have a mixed mating system, with animal-pollinated species having a much higher frequency of mixed mating than species pollinated by wind or water, where either complete selfing or complete outcrossing were the dominant modes of reproduction (Goodwillie et al., 2005). The authors suggested that the factors that lend evolutionary stability to mixed mating systems are more prevalent in animal- than in wind-pollinated species, possibly because pollinator services tend to have higher spatio-temporal variation in animal-pollinated species than in wind-pollinated species (Goodwillie et al., 2005). In wind-pollinated species, considerable spatial variation in reproductive success exists (e.g., Koenig and Ashley, 2003; Davis et al., 2004; Eppley and Pannell, 2007), which may create selective forces similar to those imposed by variable pollinator environments in animal-pollinated species (Koenig and Ashley, 2003). Results from previous analyses of the spatial genetic structure both within (Jacquemyn et al., 2005) and among populations (Jacquemyn et al., 2006) suggested that P. quadrifolia is predominantly selfing. These results are somewhat surprising given that emasculated flowers had ca. 71% of their ovules successfully fertilized and thus suggest high outcrossing rates. Because P. quadrifolia shows pronounced clonal growth (Jacquemyn et al., 2006) and because clones are often clustered within a population (Jacquemyn et al., 2005), geitonogamous pollination (i.e., pollen transfer from one flower to another within the same genet) probably plays an important role in determining selfing rates in this species. Clearly, more research is needed that specifically addresses the relative importance of self- vs. cross-pollination in this species. Because the maintenance of mixed mating requires a cost of selfing (e.g., inbreeding depression), future experiments should also determine fitness of crossed and selfed seeds to assess potential costs of selfing. Preliminary results, however, suggest that there is very little inbreeding depression with regards to seed number, seed weight, and germination (H. Jacquemyn, unpublished results).

FOOTNOTES

¹ The authors are grateful to O. Honnay, S. Barrett, J. Friedman, M. Vallejo-Marín, and two anonymous reviewers for constructive comments on an earlier version of this manuscript. This research was supported by the Flemish Fund for Scientific Research (FWO) to H.J. and R.B.

⁴ Author for correspondence (e-mail: hans.jacquemyn{at}bio.kuleuven.be)

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This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jacquemyn, H. Articles by Brys, R. Search for Related Content PubMed Articles by Jacquemyn, H. Articles by Brys, R. Agricola Articles by Jacquemyn, H. Articles by Brys, R.