• Knowledge of pollination services provided by flower visitors is a prerequisite for understanding (co)evolutionary processes between plants and their pollinators, for evaluating the degree of specialization in the pollination system, and for assessing threats from a potential pollination crisis.
• This study examined pollination efficiency and visitation frequency of pollinators – key traits of pollinator-mediated fecundity – in a natural population of the wild carnation Dianthus carthusianorum.
• The five lepidopteran pollinator species observed differed in pollination efficiency and visitation frequency. Pollinator importance, the product of pollination efficiency and visitation frequency, was determined by the pollinator's visitation frequency. Pollination of D. carthusianorum depended essentially on only two of the five recorded pollinator species. Seed set was pollen-limited and followed a saturating dose–response function with a threshold of c. 50 deposited pollen grains for fruit development.
• Our results confirm that D. carthusianorum is specialized to lepidopteran pollinators, but is not particularly adapted to the two main pollinator species identified. The local persistence of D. carthusianorum is likely to be at risk as its reproduction depends essentially on only two of the locally abundant, but generally vulnerable, butterfly species.
Flowering plants rely primarily on insect vectors for efficient pollen transfer (Bond, 1995; Kearns & Inouye, 1997; Waser & Campbell, 2004). Since Darwin, the tremendous diversity of flower shapes, colours and odours has been interpreted as an adaptation to pollinators (Darwin, 1862). Flowers and pollinators were implicitly assumed to be coadapted because of their obvious associations. Eventually, the descriptive concept of pollination syndromes established a seemingly sound explanation for the functionally related diversity of flowers and pollinators (Vogel, 1954; Faegri & van der Pijl, 1979). Altogether, this led to the biased perception that plant–pollinator relationships are rather specialized. In recent years this paradigm has come under scrutiny (Ollerton, 1996; Waser et al., 1996; Ollerton, 1998; Johnson & Steiner, 2000); for example, analyses of plant–pollinator webs revealed that most plant species within a community were visited by numerous pollinators stemming from a broad taxonomic spectrum (Memmott, 1999). However, visitation does not imply successful pollination (Olsen, 1997). Rather, the relevance of a pollinator species is its effective contribution to the plant's fecundity. Therefore only detailed investigations will uncover whether a plant's flowers are specialized or generalized with respect to the pollination system. The degree of specialization vs generalization in plant–pollinator relationships has implications for conservation biology. Recent speculation about the effect of a decreasing pollinator fauna on plant fitness (pollination crisis: Bond, 1995; Allen-Wardell et al., 1998; Kearns et al., 1998; Karrenberg & Jensen, 2000) makes it even more important to identify the essential factors of the interaction between plants and their pollinators.
The carnation Dianthus carthusianorum (Caryophyllaceae) is a prime example of a plant species that appears to have a specialized pollination. The most obvious characteristic feature of D. carthusianorum are its conspicuous, tubular shaped flowers which provide a mechanical barrier to nectar consumption for flower visitors with too short proboscides. By quantitatively analysing the dependence on its pollinator species, the risk of extinction through potentially decreasing pollinator species can be assessed.
A pollinator's importance to a plant equals its relative contribution to the plant's reproduction, and consists of its abundance, visitation rate and pollination effectiveness (Waser et al., 1996; see also Sugden, 1986; Herrera, 1987; Armbruster, 1988; Young, 1988; Herrera, 1989; Olsen, 1997). These separate components of pollinator importance provide a detailed qualitative and quantitative characterization of the plant's dependence on its flower visitors. For instance, a plant species is considered to be highly specialized if only one out of several visiting species effectively pollinates the plant's flowers. In contrast, a plant is considered generalized if a diverse guild of flower visitors are similar in their pollination effectiveness. Therefore the components of pollinator importance tell us how and to what a degree a plant species depends on its different pollinator species, and eventually indicates whether a plant species is generalized or specialized with respect to its pollinators. Furthermore, the relative contributions of the different components of pollinator importance provide insight into the current selection regime, and hence the evolutionary processes of the plant–pollinator relationship (Waser et al., 1996). For instance, only frequently visiting pollinator species can exert ample selection pressure to cause an evolutionary response toward specialization (an increase in pollination effectiveness).
In this study we recorded the flower visitors, estimated their components of pollinator importance, and evaluated their relative contribution to fecundity of the butterfly-pollinated carnation D. carthusianorum. The aims were: (1) to identify the relevant pollinator species in terms of pollinator importance; (2) to estimate for each pollinator species the relative influence of the separate components (visitation frequency and pollination efficiency) on its pollinator importance; (3) to judge the degree of specialization in the pollination system; and (4) to assess whether the loss of pollinator species might present a risk to the local persistence of D. carthusianorum. We further investigated the relationship between different pollen quantities deposited on the stigma and the resulting reproductive success in D. carthusianorum. This provides the link between pollination efficiency, the raw pollination effect and pollination effectiveness, the pollinator's contribution to plant fecundity.
Materials and Methods
Study plant and study site
Dianthus carthusianorum L. (Caryophyllaceae) is a gynodioecious perennial herb, forming a rosette of grass-like leaves and one to several, mostly unramified shoots 30–45 cm high. As far as is known, D. carthusianorum is not clonal and reproduces only sexually (Hegi, 1979). During the flowering period (June–October) these shoots produce an inflorescence with numerous protandrous flowers. The crimson-coloured petals are enclosed in a narrow calyx tube and end in a flat rim, which serves as a landing platform for pollinators. The flowers have two stigma lobes and 10 stamens, and are mainly visited and pollinated by butterflies that feed on the nectar secreted from nectaries at the base of the filaments at the very bottom of the calyx (Knuth, 1898). The calyx tubes at our study site had a mean length of 15.3 ± 2.6 mm (1 SD) with a diameter of 3–5 mm. Anthesis of a single flower lasted for 2–5 d (D. Bloch, A. Erhardt, pers. obs.). Selfing is generally prevented by protandry. However, selfing is possible through a back-curling movement of the stigma lobes at the end of anthesis.
Field work was conducted in a large natural population of D. carthusianorum (> 1000 individuals) growing in a rocky steppe (Festucion valesiacae, according to Ellenberg, 1996), on a south-facing slope near Leuk (Valais) in the Rhone valley in the Swiss Alps. Dianthus carthusianorum was the dominant flowering plant at the study site during the observation period, which lasted from the end of June to the end of July 2002.
Pollination efficiency of different insect visitors of D. carthusianorum was assessed by covering 40 randomly chosen plants with cages to exclude pollinators from the end of June until the end of July 2002. A cage consisted of a thin, cubic wooden frame (20 × 20 × 50 cm) covered with nylon mesh (pore diameter 0.25 mm). These caged plants provided the virgin flowers for measuring the pollination efficiency of the different pollinators. Plants with virgin flowers in the female stage of anthesis were exposed to foraging insects and were observed continuously under good weather conditions. Observed visitors were identified, and the stigma lobes were harvested immediately after the departure of each insect. The harvested stigma lobes were fixed on a slide with a jelly fixative containing glycerol, gelatine, distilled water and Safranin stain (C20H19ClN4) (Beattie, 1971). Pollen grains adhering to the stigma lobes were counted under the microscope. As experimental flowers were not emasculated, the measurements may also include self-pollen transferred to the stigma lobes by movements of the foraging insect. Stigma lobes of unvisited caged flowers were harvested regularly at the end of anthesis to assess the number of pollen grains deposited by selfing under insect exclusion.
The visitation frequency of floral visitors was estimated by counting visits of foraging insects on D. carthusianorum within three equally sized observation areas (8 × 8 m). Each visit was recorded and the corresponding species identified. The observed visitation frequencies of pollinators represent their activity on D. carthusianorum, but do not necessarily reflect their general abundances. Observation periods were distributed randomly throughout the day (from 09 : 00 to 16 : 00 h, which covers the most important foraging period; D. Bloch, A. Erhardt, pers. obs.), and the three observation plots were distributed randomly within the D. carthusianorum population. Each observation period lasted 30 min, each observation area contained the same flower density (20 inflorescences), and observations were conducted during optimal weather conditions for foraging butterflies (sunny, at most a slight breeze). This standard setting is referred to as an ‘observation unit’. During July 2002, a total of 16 observation units were conducted. Butterflies foraged primarily during the morning because of strong thermic winds in the afternoon.
Pollinator importance was calculated as the product of pollination efficiency and visitation frequency of a given pollinator species (for details see Statistical analysis). Thus pollinator importance indicates the significance of each pollinator species to the reproduction of D. carthusianorum at our study site.
Relation between pollen quantity deposited on stigma and seed set
The relationship between the quantity of pollen deposited on the stigma and seed set was investigated by measuring seed set after deposition of varying pollen loads. This allowed us to verify whether, and how, pollination efficiency (the number of pollen grains deposited by a single visit of a pollinator) is related to realized fecundity, and thus whether pollination efficiency is a proper estimate for pollination effectiveness, the contribution to fecundity by each pollinator visit. Twenty-four randomly chosen plants were covered with cages to exclude pollinators. Emasculated virgin flowers in female phase were hand-pollinated with three categories of pollen load (small, intermediate, large). The pollen was deposited on the stigma lobes using a small brush. Each of the three categories was deposited on three different flowers of each study plant. If a plant produced three more flowers, the procedure was repeated, allowing resource-rich plants to express their resource availability through production of further flowers and fruits, rather than measuring the development of an experimentally limited, low number of fruits. Stigma lobes were harvested 3 d after hand pollination (ensuring fertilization) and were fixed in Safranin jelly, and the number of pollen grains on the stigma lobes was counted under the microscope. An intermediate distance of 15 m between pollen donor plant and pollen acceptor plant was chosen to reduce the probability of inbreeding or outbreeding effects (Schemske, 1983; Waser & Price, 1983; Waser, 1993). Two to three unpollinated flowers per plant were cut off and used for counting the mean ovule number per plant. By mid-August, when the focal fruits were ripe, each of the 24 plants in the experiment had produced five to 12 flowers, and the plants soon ceased producing further flowers. Matured fruits were harvested, and seed set was determined dividing seed number by mean ovule number per plant. The mean per plant was used, as counting ovules is a destructive method and it is therefore impossible to count first ovules and then seeds of the same fruit. However, analysis of ovule distribution showed that ovule number is fairly stable within the flowers of a plant (mean of coefficients of variation = 13.9 ± 5.0% (1 SD), n = 6).
Pollen loads deposited on stigmas of flowers with unlimited exposure to pollinators were assessed using stigma lobes from uncaged plants that were harvested at the end of anthesis and fixed in Safranin jelly. These flowers were not emasculated, hence the number of pollen grains represents the sum of insect-deposited pollen including cross-pollination and selfing. This is regarded as the number of pollen potentially determining reproductive success in the lifetime of a flower under natural conditions.
Statistical analyses were calculated with R Statistical Software (R Development Core Team, 2003). Conclusions about pollination efficiency were based on a linear mixed-effects model (Pinheiro & Bates, 2000) with the pollinator species as fixed and plant individuals as random factors. Violation of homoscedasticity required the omission of three pollinator species with low sample sizes (Papilio machaon L., n = 4; Macroglossum stellatarum L., n = 3; Thymelicus sylvestris (Poda), n = 2) and the square-root transformation of the dependent variable ‘number of pollen grains’. Pollen deposition by selfing was included for comparison with the numbers of pollen deposited by the pollinator species. P values were corrected for multiple testing according to sequential Bonferroni technique (Holm, 1979).
Visitation frequencies of pollinator species were analysed using a generalized linear model (McCullagh & Nelder, 1989). As visitation frequency, the response variable, consisted of counts, we employed a model based on a Poisson distribution and controlled for overdispersion (var = mean, link = log). Two of the five observed pollinator species, M. stellatarum and T. sylvestris, were excluded from the analysis because they were not recorded at any observation unit.
Pollinator importance was estimated as the product of pollination efficiency and visitation frequency. To prevent pseudoreplicated samples of pollinator importance, we multiplied each measure of visitation frequency with the mean of a random subsample of pollination efficiency (subsample for Satyrus ferula (Fabricius), n = 5; Melanargia galathea L., n = 2). Statistical analysis was conducted as a linear model for the two pollinator species S. ferula and M. galathea. Sample sizes of the other three pollinator species were too small for this analysis. The pollinator importance of S. ferula and M. galathea was square-root transformed to fulfil the assumptions of error distribution. For comparison, pollinator importance of the three omitted species was calculated from the means of their pollination efficiencies and visitation frequencies.
The relation between the number of pollen grains deposited on a stigma and seed set was analysed with a nonlinear mixed-effects model (Pinheiro & Bates, 2000). Prior examination of data distribution and calculations of the relationship between pollen quantity and seed set required log-transformation of pollen quantity, and indicated a threshold value for seed set. Therefore we fitted a model of the form y = b × (x −a) by constraining y to 0 for x < a, where y = seed set; x = number of pollen grains; b = slope; a = threshold. As different pollen quantities were applied to different flowers of the same plant, we defined plants as random factors. Thus the parameters of the dose–response-function were estimated for each plant individual. In some plants, herbivory critically reduced the number of flowers to sample sizes too small to provide reliable estimates. Model fitting was therefore based on 13 out of 24 plants. However, this did not cause a biased estimate, as we could not detect a systematic pattern of herbivory.
During the 4 wk of the study, 136 insect visits to the target flowers were recorded. All visits were made by individuals of five different lepidopteran taxa (Fig. 1a), most frequently by S. ferula (Satyridae, 63% of visits) and M. galathea (Satyridae, 30%), and rarely by P. machaon (Papilionidae, 3%), M. stellatarum (Sphingidae, 2.5%), and T. sylvestris (Hesperiidae, 1.5%). The number of pollen grains deposited per visit on stigma lobes (F2,130 = 814.6, P = 0.0005) differed between S. ferula (9.92 ± 1.73; mean ± 95% CI), M. galathea (13.13 ± 2.67; mean ± 95% CI), and the number of pollen found on self-pollinated flowers (7.41 ± 1.90; mean ± 95% CI). Contrasts corrected by sequential Bonferroni technique revealed significant differences in pollen deposition between S. ferula and M. galathea (P = 0.0292) and between self-pollinated flowers and the two pollinator species (M. galathea, P = 0.0003; S. ferula, P = 0.0210).
In the observation plots, S. ferula, M. galathea and P. machaon differed in their visitation frequencies (F2,45 = 23.557, P = 0.0009, dispersion = 2.18). In the 16 observation units conducted, S. ferula was missing only once, while M. galathea was absent five times and P. machaon as many as 15 times. Satyrus ferula was the most frequently observed visitor with a mean of 1.57 (± 0.11, 1 SE) individuals per observation unit, followed by M. galathea (0.36 ± 0.31, 1 SE) and the rare visits of P. machaon (−2.77 ± 0.10, 1 SE). Contrasts corrected by sequential Bonferroni technique revealed different visitation rates for S. ferula and M. galathea (P = 0.0025); for S. ferula and P. machaon (P = 0.0005); and for M. galathea and P. machaon (P = 0.0435). Visits by M. stellatarum and T. sylvestris were never recorded during the observation units (Fig. 1b). To calculate their pollinator importance, we assumed their visitation frequency to be 0.06 individuals per observation unit (mean visitation frequency of P. machaon).
Pollinator importance tended to be higher for S. ferula compared with M. galathea (F1,30 = 2.93, P = 0.095; 23.3 ± 2.93 and 16.16 ± 2.93, respectively; mean ± 1 SE). The pollinator importance of S. ferula was by far higher than that of P. machaon, M. stellatarum and T. sylvestris (6.63%, 1.81% and 0.66% relative to S. ferula, respectively; Fig. 1c).
Relationship between pollen quantity deposited on stigma and seed set
Seed set of the study plants was positively related to pollen quantity deposited on the stigma lobes (F1,71 = 38.10, n = 85, P < 0.0001). However, this relationship was observed only after a threshold of 3.92 ± 0.16 (mean ± 1 SE; F1,71 = 669.45, n = 85, P < 0.0001; Fig. 2). Note that in a nonlogarithmic plot, the function would asymptotically approach an upper limit of approx. 40% seed set (Fig. 3). However, the semilogarithmic plot shows the threshold level as well as the linear relationship more clearly (Fig. 2). During the flowering period, 16.13% of freely accessible D. carthusianorum flowers accumulated < 50 pollen grains during their anthesis (Fig. 3), and 71% of the flowers accumulated < 200 pollen grains (187 ± 34; mean ± 1 SE; n = 31). Thus seed set of these flowers was clearly pollen-limited under natural conditions.
Pollinator importance, visitation frequency and pollination efficiency
In our research population, the bulk of the reproductive success of D. carthusianorum was mediated by the visitation frequency of two pollinator species. We also confirmed that D. carthusianorum is mainly, if not exclusively, pollinated by diurnal Lepidoptera (Knuth, 1898; Hegi, 1979). Furthermore, we found considerable differences in pollination efficiency, visitation frequency and hence pollinator importance among the five observed pollinator species. Pollinator importance was determined by visitation frequency rather than by the pollination efficiency of pollinator species. The most important pollinator in this study was S. ferula. Although this species was only third in the hierarchy of pollination efficiency (Fig. 1a), it was the most important pollinator of D. carthusianorum because of its high visitation frequency (Fig. 1b). Earlier studies (Schemske, 1983; Spears, 1983; Waser & Price, 1983; Olsen, 1997) have also demonstrated that a pollinator's visitation frequency was the dominating factor for assessing its final level of pollinator importance. Thus the visitation frequency of pollinators was the principal determinant of plant fecundity regarding pollination, as seed set under natural conditions was strongly limited by pollen supply, be it from selfing and/or provided by pollinators. Our results also suggest that D. carthusianorum, although adapted to butterfly pollinators, is not particularly specialized to its main two butterfly pollinators at the study site, and thus appears to be rather a generalist with respect to its pollinating butterfly species. However, seasonal stability in visitation frequencies of the two main pollinator species (D. Bloch, A. Erhardt, pers. obs. at the study site, 2000–05) would probably promote specialization through pollinator-mediated selection, that is, adaptation for increased pollination effectiveness of the main pollinator species (Waser et al., 1996).
Pollinator-limited fecundity and pollination effectiveness
Seed set of the study population was positively related to the number of pollen applied, a pattern found in many plant taxa (Jaquemart, 1997; Johnson & Bond, 1997; Bosch & Waser, 1999, 2001; Pflugshaupt et al., 2002). This result confirmed our assumption that pollen deposition on the stigma by a single pollinator visit is a fair estimate of pollination effectiveness, the contribution to fecundity of a single pollinator visit. Seed set of flowers with unlimited access to pollinators was strongly pollen-limited (Fig. 3): 70% of the flowers received pollen quantities, which correspond to only 20% seed set. Sixteen per cent of the flowers accumulated even fewer pollen grains than necessary for the development of any seeds. However, resource availability is also relevant for realized fecundity, as indicated by the presence of a threshold value of pollen quantity deposited on the stigma lobes for fruit formation. A plant's investment policy in fruits is obviously to abort fruits of flowers that accumulate only a low pollen quantity, to the advantage of better-pollinated flowers in which limited resources are invested. As differences in microhabitat quality and/or genotype also affect seed set (Waser, 1993), resource availability is expected to influence seed set (Campbell & Halama, 1993; Baker et al., 2000). A similar threshold between pollen quantity and seed number, as found in the present study, was described in a Hibiscus species almost 250 yr ago by Koelreuter (1761). He also noted that the observed threshold varied considerably according to environmental conditions. Thus differences in microhabitat and/or genotypic differences may affect responses in seed formation to pollen availability on an individual scale. The threshold itself, a refusal or incapacity to develop small numbers of seeds with the high costs of fruit formation, can also be viewed as a response to resource limitation on the habitat scale – an adaptation to the overall nutrient poor soil conditions of dry grasslands. The evolution of such a threshold in D. carthusianorum could correspond to the life strategy of stress-tolerant plants according to the CSR model described by Grime (1977).
Recently, several authors have claimed that the sexual reproduction of many plant species is threatened by declining pollinator frequencies because of human impact (Allen-Wardell et al., 1998; Kearns et al., 1998; Karrenberg & Jensen, 2000). Although various butterfly species visit D. carthusianorum in the investigated population, a decline in abundance, or a loss of one or even both of the two main pollinator species, could cause a serious threat to the local persistence of D. carthusianorum. Such a threat is realistic as both species, although locally abundant, are generally rare, weak dispersers, and are restricted to the ecological conditions of the local environment of rocky steppes (Benz et al., 1994), itself a rare, species-rich habitat drastically reduced by viticulture, housing development and fertilization (Delarze et al., 1999). In the Rhone valley of south-eastern Switzerland both butterfly species, M. galathea and S. ferula, have vanished from 29 of 67 and 31 of 62 sites, respectively, since 1970 (Gonseth, 1987). Thus the population density of both pollinator species shows a tendency to collapse or to go locally extinct. Furthermore, the observed pollinator limitation does not suggest that pollinator species compete significantly for nectar in D. carthusianorum. It therefore seems unlikely that the remaining pollinator species could compensate for a decrease in the primary pollinator species. However, as D. carthusianorum is self-compatible (no apomixis), a minimal sexual reproduction could be assured by selfing, although pollen quantity deposited by selfing is precariously close to the threshold for fruit formation. Thus the risk of local extinction will eventually depend on the demographic consequences of inbreeding (Schemske, 1983; Waser, 1983; Waser & Price, 1983; Bond, 1995). Furthermore, flight distances of pollinators between visited flowers affect the progeny's fitness (Price & Waser, 1979; Levin, 1981). Butterflies cover greater flight distances than other insects, and therefore are able to sustain a higher effective population size (Beattie & Culver, 1979; Schmitt, 1980). Hence a stronger inbreeding depression is likely if a plant population becomes dependent on selfing because of a lack of pollinators. Consequently, D. carthusianorum may face serious inbreeding depression in the case of decreasing pollinator abundances. Other impacts of human activities, whether habitat deterioration or destruction and fragmentation (Jennersten, 1988), further threaten this plant–pollinator system.
This study shows that realized fecundity in the D. carthusianorum population investigated depends strongly on pollinator service. Two of the five lepidopteran pollinator species are by far the most important pollinators. Visitation frequency rather than pollination efficiency of pollinators was the main determinant of pollinator importance. Although pollination efficiencies do not indicate a particular specialization to the two main pollinator species, D. carthusianorum is specialized to pollination by diurnal Lepidoptera. Seed set under natural field conditions was strongly limited by pollen supply, whether by selfing or provided by pollinators. The local persistence of D. carthusianorum is at risk, as its reproduction depends essentially on two locally abundant, but generally vulnerable, pollinator species.
We thank Sabine Berger, Deborah Renz, Dominique Haller and Thomas Hofer for assistance in the field, Andreas Schoetzau for statistical support, Peter Keusch of the Dienststelle fuer Wald und Landschaft (DWL) of the Canton Valais and the Burgergemeinde Leuk (VS) for access to the study site, Lars Wenger for cage construction and finally the Speck Crew for kindly lending their van. Jovanne Mevi-Schütz and Georg F. Armbruster provided many helpful comments on the manuscript. This study was funded by the Swiss National Science Foundation (31-63562.00 to A. Erhardt).