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(American Journal of Botany. 2008;95:925-930.) doi: 10.3732/ajb.0800036 © 2008 Botanical Society of America, Inc. |
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Ecology |
2 Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake, Sakyo, Kyoto 606-8502, Japan 3 Graduate School of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan 4 Tohoku Research Center, Forestry and Forest Products Research Institute, Morioka, Iwate 020-0123, Japan
Received for publication 28 January 2008. Accepted for publication 23 May 2008.
ABSTRACT
The amount and genetic composition of pollen grains that are transported to flowers influence the reproduction and fitness of plants. Despite the importance of insect-pollination systems, an understanding of those systems is still lacking due to the absence of a genetic analysis of pollen grains that are transported to flowers. We evaluated the pollination efficiencies of bumblebees (Apidae, Bombus spp.), flower beetles (Scarabaeidae, subfamily Cetoniinae, Protaetia and Eucetonia sp.), and small beetles (Lagriidae, Arthromacra sp.) that visited the flowers of Magnolia obovata (Magnoliaceae) using quantitative (flower visitation frequency, amount of adherent pollen per insect) and qualitative (origin and genetic diversity of adherent pollen per insect) criteria. Most of the pollen adhering to bumblebees and small beetles was self-pollen. This result suggests that visitation by these insects may cause geitonogamous pollen flow and negatively affect the reproduction of M. obovata, causing inbreeding depression. In contrast, flower beetles transported large amounts of genetically diverse outcross pollen. Our results suggest that certain beetle species contribute quantitatively and qualitatively to the pollination of M. obovata. Direct genetic analysis of pollen grains will advance our understanding of plant mating systems and may shed light on the mutualism and coevolution of plants and flower visitors.
Key Words: beetle • bumblebee • insect pollination • Japan • Magnoliaceae • microsatellite • pollen genotyping • pollination efficiency
Gene flow is crucial to plant reproduction because it influences the reproductive success and fitness of individuals and the genetic structure of the population. Genetic analysis has revealed that pollen grains tend to be transported over longer distances than seeds (Chase et al., 1996
; Dick et al., 2003; Isagi et al., 2007). Many angiosperms rely on animals for pollination, with insects being the major animal vectors (Goulson, 1999). Flowers are often visited by a variety of insect species, which vary widely in morphological and behavioral features (Herrera, 1987; Mayfield et al., 2001; Adler and Irwin, 2006). Thus, pollinators may have different effects on the reproductive success, fitness, and population structures of the plants they pollinate. To understand plant mating systems, plant–insect interactions, and coevolution, it is essential to study the effects of flower-visiting insects on pollination.
Pollination efficiency, the contribution of flower visitors to plant pollination, has been evaluated in various ways. The basic approach used to estimate a given pollinator’s contribution to pollination involves observing flower visits and evaluating variables such as visitation frequency and visitation duration (Primack and Silander, 1975; Galen and Newport, 1987; Herrera, 1989). To evaluate a pollinator’s ability to transfer pollen, researchers have examined the number of pollen grains transported (Primack and Silander, 1975; Mayfield et al., 2001; Ivey et al., 2003), the degree of pollen removal (Conner et al., 1995), the amount of pollen deposited (Herrera, 1987; Muchhala and Potts, 2007), and the distances and patterns of movement (Olesen and Warncke, 1989; Widén and Widén, 1990; Schulke and Waser, 2001). Fluorescent dye has also been used as a pollen analog to estimate pollinator movement (Murawski and Gilbert, 1986; Adler and Irwin, 2006). However, none of these measurement techniques provide information on the genetic composition or diversity of the pollen grains that are transported to flowers.
The genetic composition and diversity of transported pollen strongly influences fertilization success and offspring fitness via self-incompatibility and inbreeding depression. For example, pollen grains that are transported to another part of the same plant of a self-incompatible species produce no seeds, and even in self-compatible species, self-pollination may yield seeds of lower fitness (Husband and Schemske, 1996; Keller and Waller, 2002). Therefore, to assess the contribution of pollinators to effective pollination, it is necessary to determine quantitative and genetic qualitative characteristics of transported pollen grains. Several methods of distinguishing different pollen donors have been used. Color polymorphism of pollen grains (Jorgensen et al., 2006) can be readily assessed, but the discrimination is relatively poor and adaptation is restricted to specific species. Although gene transformation (Havens and Delph, 1996; Hudson et al., 2001) and differences in ploidy (Williams et al., 1999) can also distinguish pollen genotypes in vivo, these methods are unsuitable for processing the large numbers of samples required for analysis of natural populations.
We previously developed a method for direct genotyping of single pollen grainsMatsuki et al., 2007) that allows pollen from different pollen donors to be distinguished with high resolution using multiple microsatellite markers. Genetic tagging of individual pollen grains is useful for the detailed analysis of plant mating systems and drawing correlations between plants and flower visitors. In this study, the pollen adhering to insects was analyzed using quantitative and qualitative criteria to evaluate the pollination efficiencies of different insect taxa visiting Magnolia obovata.
MATERIALS AND METHODS
Plant material
Magnolia obovata Thunb. (Magnoliaceae) is a large (up to 20–30 m tall), common deciduous tree species that is native to temperate forests in Japan. The standing density of adult trees is relatively low, only a few trees per hectare (Isagi et al., 2000). The flowers are hermaphroditic and protogynous. On the first day of flowering of M. obovata, the female-phase of flowering lasts only an hour to a half-day of daytime, after which the petals close and the stigmas are pressed against the gynoecium in the afternoon or evening (Kikuzawa and Mizui, 1990). The next day, the petals open again, the stamens release, and pollen is available. Although the flowering period of each flower is 3–4 d, flowering persists for individual trees for up to 40 d (Kikuzawa and Mizui, 1990; Ishida et al., 2003). Thus, geitonogamy occurs frequently because of the simultaneous presence of female- and male-phase flowers on an individual tree (Ishida et al., 2003). The rate of self-pollination is high, approximately 80% at the embryo and seed stages (Ishida, 2006) and 70% at the seedling stage (Isagi et al., 2004). The early life stages of M. obovata undergo substantial inbreeding depression: in one study, the cumulative survival rate from the embryo to the seedling stage for selfed progeny was found to be less than 10% of the corresponding value for outcrossed progeny (Ishida, 2006). No selfed saplings were found in a natural population (Isagi et al., 2000). The main pollinators of M. obovata are thought to be beetles (Kikuzawa and Mizui, 1990), which spend much time crawling within the flowers (Thien, 1974). Like other beetle-pollinated plants (Dieringer et al., 1999; Gottsberger, 1999; Sakai and Inoue, 1999), M. obovata flowers emit a strong odor to attract pollinators. In addition to beetles, bees and hover flies have also been observed to visit the flowers of this species (Tanaka and Yahara, 1988).
Study site and field sampling
A field survey was carried out at Ogawa Forest Reserve, Ibaraki Prefecture, central Japan (36°56'N, 140°35'E; 610–660 m a.s.l). The annual mean air temperature and precipitation over 10 years (1986–1995), measured at a meteorological station in Ogawa (36°54'N, 140°35'E), were 10.7°C and 1910 mm, respectively (Mizoguchi et al., 2002). The area is covered by deciduous broad-leaved forest, and the dominant woody species in the canopy are Quercus serrata, Fagus japonica, and F. crenata.
From late May to mid-June in 2005, flower-visiting insects were observed and collected from the canopies of six adult Magnolia obovata trees. Because the stigmas of M. obovata female-phase flowers generally become receptive to pollen in the daytime (Kikuzawa and Mizui, 1990), insect observations and collections were conducted between 0900 and 1600 hours. Two to three trees were each observed for 2–3 h per day, except on days that were rainy, windy, or below 15°C, for a total of 51.7 h for all six trees combined. Simultaneous with the observation period, insects were collected with insect nets, placed into separate plastic vials, and stored at –30°C prior to DNA analysis. The collected insects were classified into the following groups on the basis of their taxonomic and behavioral features: bumblebees (Apidae, Bombus spp.), other Hymenoptera (Andrenidae and Halictidae), flower beetles (Scarabaeidae, Protaetia sp., Eucetonia sp.), small beetles (Lagriidae, Arthromacra spp., Elateridae), and Diptera (Syrphidae).
Number of pollen grains adhering to insects
The number of adhering pollen grains was counted for all insects collected. To remove the adherent pollen, we washed each insect was washed in 2 mL of 1% sodium dodecyl sulfate (SDS) by vortexing for 1 min. The pollen present in 10 µL of the resulting solution was counted under a stereomicroscope. The total number of pollen grains adhering to each insect was estimated from the average of three replicate counts. For bumblebees, the pollen loads packed onto the bumblebee’s hind-leg pollen-transport structure were not included in the counting because the packed pollen was no longer available for pollination as a result of the addition of nectar and oil by the bumblebees (Thorp, 1979, 2000).
Genotype determination of pollen grains adhering to insects
The microsatellite genotypes of pollen grains adhering to flower-visiting insects were analyzed according to Matsuki et al. (2007). "Other Hymenoptera" and "Diptera" were not included because their visitation frequencies were low and the number of pollen grains adhering to these insects was not sufficient for statistical analysis. The microsatellite genotypes of each pollen grain were scored using nine pairs of microsatellite primers developed for M. obovata by Isagi et al. (1999). Pollen samples for which genotypes were determined for more than five microsatellite loci were used for further analysis. Pollen samples that had the same alleles at all analyzed loci as the tree from which the insects were collected were considered self-pollen (pollen that was transported to a different region on one tree). Pollen samples that had alleles different from those of the collection tree were considered outcross pollen. The probabilities of a haploid "self" genotype being formed by chance by other adult trees in the study site were calculated for all pollen grains that were considered self-pollen in the present analysis using the allele frequencies of adult trees in the study site (Isagi et al., 2000; Y. Matsuki, unpublished data). The probabilities were quite low (5.25 x 10–11–9.80 x 10–4; average 1.34 x10–5), and therefore the determination of self-pollen was sufficiently reliable. The proportion of self-pollen in the total pollen load was calculated for each insect. The genetic diversity of the pollen adhering to insects was expressed in terms of gene diversity (the probability that two alleles chosen randomly are different; Nei, 1973). The gene diversity of the pollen load that adhered to each insect and its standard deviation were calculated using the program Microsatellite Toolkit (Park, 2001). Statistical analyses were conducted using SPSS for Windows 10.1.3J (SPSS, Chicago, USA). Proportional data were arcsine-transformed prior to the statistical analysis. Differences in the mean values of the analyzed criteria among insect groups were analyzed using a one-way ANOVA followed by a Scheffé’s multiple comparison test to detect differences among the insect groups. For all statistical tests, the significance was measured at the P 
0.05 level.
RESULTS
Flower-visiting insects
Flowers of Magnolia obovata were mainly visited by Hymenoptera and Coleoptera. In total, 349 insects were observed, and 225 insects were collected for identification and analysis of the adherent pollen grains (Table 1).
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0.001). The amount of self-pollen on insects (number of adherent pollen grains on each insect x average proportion of self-pollen for each insect group) differed among insect groups (F = 89.6, df = 2, P < 0.001; Fig. 2A). The amount of self-pollen on small beetles (815 ± 1868; 0–14 388) was less than that on bumblebees (15 092 ± 11 533; 0–48 283; P < 0.001) and flower beetles (11 104 ± 8308; 507–31 760; P < 0.001). The amount of outcross pollen on insects (number of adherent pollen grains on each insect x average proportion of outcross pollen for each insect group) differed among insect groups (F = 132.8, df = 2, P < 0.001; Fig. 2A). Flower beetles had more outcross pollen (16 657 ± 12 462; 760–47 640) than did bumblebees (2058 ± 1573; 0–6584; P < 0.001) and small beetles (101 ± 231; 0–1778; P < 0.001).
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DISCUSSION
Pollination characteristics of each insect group
Bumblebees
For some plant species, especially many herbaceous plants, bumblebees are effective pollinators that contribute to the pollination and reproduction of pollinated plants (Mayfield et al., 2001; Schulke and Waser, 2001). However, the foraging movement patterns of bees are strongly affected by resource distribution patterns (Levin and Kerster, 1969; Schmitt, 1980; Mitchell et al., 2004); bees tend to travel shorter distances in places where the standing density of flowering individuals and the flowering intensity are high (Widén and Widén, 1990; Larson and Barrett, 1999; Utelli and Roy, 2000). In this study, although bumblebees transported large amounts of pollen, most of the pollen load was self-pollen. This phenomenon can be attributed to the effective foraging behavior of bumblebees: bumblebees might collect pollen intensively from one M. obovata individual on which many flowers opened simultaneously.
The genetic diversity of pollen grains on bumblebees was lower than that of pollen on flower beetles. This phenomenon might result from a combination of bumblebee’s grooming behavior and the high content of self-pollen found on their bodies. Bumblebees often groom their body during foraging, packing the collected pollen with nectar and oil onto transport structures on their hind legs effectively making it unavailable for pollination (Rademaker et al., 1997; Thorp, 2000). Although these pollen grains were not included in our analysis, reduction of pollen carryover as a result of grooming behavior (Thomson, 1986) could explain the low genetic diversity of a bumblebee’s pollen load. Due to grooming, most of the available pollen on the bumblebee’s body would have been recently collected and, coupled with a bumblebee’s intensive foraging within the same tree, may result in the dominance of self-pollen and overall low genetic diversity.
The genetic analysis of pollen grains suggested geitonogamous pollen movement by bumblebees. More self-pollen grains were transported by bumblebees than by flower beetles or small beetles. Visitation by bumblebees can increase the opportunity for self-pollination, which causes inbreeding depression in M. obovata seedlings and saplings. However, in the current study, because the sexual phases of the flowers that were visited were not identified, there is an undeniable possibility that bumblebees did not visit unrewarding female-phase flowers of M. obovata. Thus, bumblebees may not serve as pollinators of M. obovata (however, in the 2006 and 2007 surveys, visitation rates to male- and female-phase flowers did not differ [Y. Matsuki, unpublished data]). Further observations and genetic analysis that take into account the sexual phase of pollinated flowers are needed.
Flower beetles
Although beetle-pollination syndrome had been thought to be a primitive and relatively inefficient pollination system (e.g., Faegri and van der Pijl, 1971), in the past few decades, it has been revealed that beetle-pollination systems are highly specialized and arise through long-term coevolution (Bernhardt, 2000). For example, Scarabaeidae beetles play an important role in the pollination of the Araceae and Dipterocarpaceae species in tropical regions (Young, 1988; Gibernau et al., 1999; Sakai et al., 1999). Also, the pollination systems of the Magnoliaceae are considered highly specialized systems that operate in cooperation with beetles (Thien, 1974). In this study, flower beetles (Scarabaeidae, Cetoniinae; Protaetia cataphracta and Eucetonia pilifera) that visited M. obovata flowers had proportionally less self-pollen and higher genetic diversity of pollen compared with bumblebees and small beetles. Moreover, flower beetles transported the greatest amount of genetically diverse outcross pollen, reflecting the active interplant movement of flower beetles. Englund (1993) observed frequent interplant flights of flower beetles (Scarabaeidae, Cetoniinae; Cetonia sp.) in European temperate zones. Most beetles of the subfamily Cetoniinae can fly more rapidly than other beetles because they are able to spread their wings without raising the elytra (Iijima and Tamura, 2001). The high genetic diversity of the pollen transported by flower beetles in M. obovata is in accordance with previous assessments of the contribution to pollination of the Cetoniinae (Englund, 1993; Iijima and Tamura, 2001). Flower beetles are likely to contribute significantly to the reproduction of M. obovata by transporting a large number of outcross pollen grains with high genetic diversity.
Small beetles
Anthophilous beetles visit flowers not only to obtain edible resources but also to carry out mating activities, to hide from predators, and to produce heat (Bernhardt, 2000; Young, 1988; Gottsberger, 1999). In the current study, small beetles were often observed in M. obovata flowers throughout the female and male phases. The large proportion of self-pollen and the low genetic diversity found among pollen grains adhering to small beetles suggests that these insects move mostly within trees, with infrequent movement between trees. Their behavioral patterns and high visitation frequencies indicate that small beetles promote the self-pollination of M. obovata.
Pollination of M. obovata
The flowers of the Magnoliaceae are thought to have been adapted to beetle pollination (Thien, 1974; Bernhardt, 2000). The large protogynous and odoriferous flowers, the timing of flowering, and the large quantities of pollen produced are typical features of beetle-pollinated species. Our results also suggest that M. obovata flowers are pollinated effectively by certain beetles. The female phase of M. obovata is short (lasting from 1 h to a maximum of half a day) and does not provide any reward to foraging insects (Kikuzawa and Mizui, 1990). Kikuzawa and Mizui (1990) concluded that the female-phase M. obovata flowers imitate the male-phase flowers, which offer abundant pollen, thus misleading flower-visiting insects (automimicry). Despite this, opportunities for successful pollination might still be restricted because of a shortage of pollinators (Kikuzawa and Mizui, 1990) and pollen (Ishida et al., 2003). Substantial selfing rates were reported in M. obovata (50–68% in endosperm, Ishida et al., 2003; 71% in seedlings, Isagi et al., 2004), and the selfing rates varied greatly among fruits (Isagi et al., 2004). The present results suggest that selfing is caused primarily by bumblebees and small beetles, whereas outcrossing is enhanced by flower beetles. The flowers of M. obovata were visited by various insects that carried genetically varied loads of adherent pollen. The genetic composition of seed set in M. obovata may be determined by only a few insects that happened to visit the flower. Magnolia obovata has obvious features of beetle-pollination syndrome, and some beetles that visit flowers tend to transport outcross pollen effectively. Therefore, despite the evidence for a high degree of selfing, the beetle pollination system of M. obovata has been selected through long-term coevolution. The high levels of inbreeding depression (Ishida, 2006; Isagi et al., 2000) also suggest the importance of outcrossing.
Conclusions and future directions
Various criteria have been used to estimate the pollination efficiencies of pollinators in many pollination systems (e.g., Primack and Silander, 1975; Mayfield et al., 2001). We measured the amount of self- and outcross pollen transported by different insect groups and taxa and found large quantitative and qualitative differences in the pollen loads of these insects. Our results emphasize the need for genetic analysis of pollen grains when evaluating the effect of pollinators on plant mating systems.
Genetic tagging of single pollen grains is a valuable technique for the study of pollination ecology. This approach will facilitate the study of pollination, plant reproduction and interactions, and mechanisms of coevolution between plants and flower visitors. Differences in climatic conditions, standing density, flowering intensity, and years might also result in differences in the quantity and quality of pollen grains that are transported to flowers. Further detailed analyses that involve multiple sites and years are needed. Moreover, it is important to analyze not only pollen adhering to pollinator bodies, but also pollen deposited on the stigma for a more detailed assessment of the contribution of pollinators to plant reproduction. Further analysis will clarify the influence of the genetic origins of pollen grains on pollen-tube competition, self-incompatibility, and inbreeding depression. In addition, our approach will contribute to the study of the evolution of mutualism. Although pollination is essential for the maintenance and regeneration of plant communities, knowledge of pollination systems is still limited. This limitation can be partly attributed to the difficulties associated with observing pollinators, especially their interplant movements. Genotyping of pollen grains can be used to directly determine the composition of pollen donors from pollen grains transported to flowers and will facilitate the study of pollination and plant mating systems, plant–pollinator relationships, and the influence of habitat degradation on pollination systems.
FOOTNOTES
1 The authors thank the Forestry and Forest Product Research Institute for permission to work in the Ogawa Forest Reserve. This research was partly supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Research Institute for Humanity and Nature, and the 21st Century Center of Excellence Program at Kyoto University. 
5 Author for correspondence (e-mail: matkyu{at}kais.kyoto-u.ac.jp)
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