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Small populations suffer increased risk of extinction as a result of both demographic and genetic factors (Ellstrand, 1992; Clarke & Young, 2000; Holsinger, 2000; Richards, 2000a). Most species are not distributed continually over space and time but instead occur as sometimes very small subpopulations with unique genetic and demographic characteristics. These subpopulations are connected via gene flow. In angiosperms, gene flow is accomplished during two different life stages: the pollen and seed stages. Gene flow via these two mechanisms may operate at different spatial scales such that one mode may be more important at small and the other at larger scales (McCauley, 1997). Pollen-mediated gene flow is important for outcrossing plants, so much so that many plant species have become specialized for particular animal pollinators (Wyatt, 1983; Ellstrand, 1992). Characteristic suites of floral traits, known as ‘pollination syndromes’ (Baker & Hurd, 1968; Wyatt, 1983), are associated with specific groups of pollinators.
When plants have evolved to depend on animals to move their pollen, the degree of gene flow will be limited in part by the behavior of the animal disperser. Some pollinators behave differently in small plant populations than in large ones. For example, in three species of pollen-limited orchid (Orchis spitzelii Sauter, O. palustris Jacq., Anacamptis pyramidatis (L.) Rich.) studied in Sweden, pollinator visits increased with increasing plant population size (Fritz & Nilsson, 1994). A similar result was found for bumblebees visiting Lychnis viscaria; bees preferred large over small populations but probed more flowers within plants when in small populations (Mustajärvi et al., 2001). In particularly small populations of animal-pollinated plants, mate numbers decrease with decreasing population size and may drop to a point at which pollinator service deteriorates, resulting in population extinction (Bronstein et al., 1990; Fritz & Nilsson, 1994). Further, some pollinators (e.g. bees) forage primarily within rather than between patches (Waddington, 1983) and may change behavior in response to habitat fragmentation. For example, bumblebees pollinating Betonica officinalis visited plants in fragments less often and for less time than they visited plants in unfragmented patches (Goverde et al., 2002). The degree of population isolation can also influence pollinator behavior. For example, pollinators were less abundant but visited more plants and flowers within isolated populations of Delphinium nuttallianum compared with populations that were not isolated (Schulke & Waser, 2001).
Many of these studies designed to examine the response of pollinators to small plant populations have focused on a single type of pollinator (e.g. bumblebees; Mustajärvi et al., 2001; Goverde et al., 2002). In spite of the conceptual cleanliness of the pollination syndrome concept, most insect-pollinated plants are generalists rather than specialists in terms of their attractiveness to pollinators and are visited by many species (Pellmyr & Thompson, 1996; Waser et al., 1996; Johnson & Steiner, 2000; Fenster et al., 2004). Different types of pollinators exhibit different foraging behaviors. In contrast to bumblebees, some pollinators such as hummingbirds, butterflies and large moths (e.g. noctuids and sphingids) spend more time moving between patches and travel longer distances in search of pollen or nectar (Schmitt, 1980; Courtney et al., 1981; Miyake & Yahara, 1998). For plants that are generalists, pollinated by a diversity of animals, population genetic structure may be influenced by the proportion of pollen transferred by different pollinators (Schmitt, 1980). To fully understand the role of pollen movement in gene flow in small plant populations therefore requires decomposition of the effects on gene flow of different types of pollinators with different behaviors. This kind of study is particularly important in light of the current ‘pollination crisis’ (Allen-Wardell et al., 1998; Kearns et al., 1998; but also see Ghazoul, 2005) so that we can understand the likely impact of declines of certain pollinators on plants.
In this study, we examined the relative contributions of nocturnal vs diurnal pollinators in facilitating gene flow between small Silene alba Poiret populations experiencing different degrees of spatial isolation. This species has been the subject of extensive long-term research in which natural populations in a 25 × 25 km study area in Giles County, VA, USA have been followed through space and time (Antonovics et al., 1994). Subpopulations of S. alba are typically spaced within 160 m of one another (Richards, 2000a). These subpopulations vary widely in size, from as few as a single plant to > 255 plants, and are ephemeral, showing substantial turnover, although the population as a whole persists. Small subpopulations experience much higher rates of extinction than do large subpopulations (Antonovics et al., 1994). These dynamics are characteristic of a metapopulation (Levins, 1970) in which the long-term persistence of the species is dependent upon the pattern of colonization and extinction of subpopulations. Newly colonized subpopulations of S. alba can be very small, consisting of as few as five individuals (Antonovics et al., 1994), and experience severe inbreeding depression (Richards, 2000a).
In S. alba, fruit set and gene flow are both influenced by the distance between subpopulations. In a manipulative experiment, Richards et al. (1999) found that mean fruit set, a measure of pollination success, dropped from 0.25 fruits per female flower in populations separated by 20 m to 0.05 fruits per flower in populations separated by 80 m. Similarly, the immigration rate of pollen into populations was 47% for populations separated by 20 m but dropped to 6% in populations separated by 80 m. Comparison of population genetic structure at this spatial scale using both nuclear and cytoplasmically inherited markers revealed that in S. alba pollen movement has a much larger role than seed movement in bringing about gene flow (McCauley, 1994, 1997).
Silene alba flowers have a long corolla tube, white petals, and a sweet odor, traits typical of plants pollinated by nocturnal moths (Baker & Hurd, 1968; Young, 2002). However, flowers are visited by a wide variety of both nocturnal and diurnal pollinators including noctuid, sphingid and geometrid moths as well as syrphid flies, a variety of bees, and butterflies (Shykoff & Bucheli, 1995; Altizer et al., 1998; Young, 2002). Some studies report higher flower visitation rates for nocturnal pollinators (Shykoff & Bucheli, 1995) and other studies for diurnal pollinators (Altizer et al., 1998). Nocturnal pollinators such as large moths are long-distance fliers (Linhart & Mendenhall, 1977) that have been observed feeding in widely spaced populations of S. alba (Altizer et al., 1998), whereas many diurnal pollinators such as bees tend to forage primarily within rather than between flower patches (Altizer et al., 1998). Further, many nocturnal pollinators rely on olfaction to discover even small patches of flowers (reviewed in Kelber et al., 2003). In contrast, many diurnal pollinators rely on vision to detect flowers and are generally more attracted to large floral displays (Handel, 1983). For these reasons, we predicted that, for small populations of S. alba, nocturnal pollinators would play a larger role in pollen-mediated gene flow than would diurnal pollinators, especially for populations isolated by greater distances. Specifically, we set out to determine (1) the relative role of nocturnal vs diurnal pollinators in pollen-mediated gene flow between subpopulations of S. alba, and (2) whether larger, longer flying nocturnal pollinators are better at moving pollen longer distances than their smaller, shorter flying diurnal counterparts.
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Our major finding is that nocturnal and diurnal pollinator guilds did not have equal effects on pollen-mediated gene flow between small S. alba populations. To be effective in moving pollen between populations, a pollinator must not only visit flowers and collect pollen in one population but must also be effective in depositing that pollen onto the receptive stigmas of flowers in another population. Although plants subjected to the diurnal pollinator treatment produced significantly more flowers than did plants in either the nocturnal or the control treatments, the same plants produced significantly fewer capsules, the first indication that diurnal pollinators were less effective in pollinating flowers. Further, plants exposed only to diurnal pollinators had reduced per plant fruit sets compared with other plants. Finally, nocturnal pollinators were responsible for substantially higher rates of gene flow than were diurnal pollinators. Our findings have important implications in (1) clarifying the role of nocturnal vs diurnal pollinators on gene flow in a well-studied model experimental system, (2) understanding the role of pollen-mediated gene flow in small populations, and (3) broadening our understanding of the concept of pollination syndromes.
The results of our study, the first to use genetic markers to explicitly measure pollen-mediated gene flow by nocturnal and diurnal pollinators, are well corroborated by other research. We observed more flowers but fewer capsules produced by plants exposed only to diurnal pollinators. This result is probably related to differential resource allocation by plants to flowers rather than capsules when pollination is limited. Wright & Meagher (2003) found that flower production in S. alba is related to pollination; in their study, average daily flower production was almost three times higher for nonpollinated than for 100% pollinated plants. In the related species Silene vulgaris, the number of flowers declined significantly as capsule production increased (Colosi & Cavers, 1984).
We measured pollination success as fruit set, the per plant number of capsules produced per female flower, and found that fruit set was considerably lower in plants exposed to diurnal rather than nocturnal pollinators. In a study of S. alba populations in Colorado that used seed set as a measure of pollination success, seed set in flowers visited by only nocturnal pollinators was more than twice that of flowers visited by only diurnal pollinators (Young, 2002). Capsules resulting from visitation by diurnal bees produced fewer seeds than did capsules resulting from visitation by nocturnal moth pollinators. Further, moths moved fluorescent dye powder (an experimental analog for pollen) more than five times the distance that bees did (Young, 2002). Two studies examined patterns of pollinator visitation in S. alba in the context of transmission of anther-smut fungus, a venereal disease in plants (Shykoff & Bucheli, 1995; Altizer et al., 1998). Again using fluorescent dye powder as a pollen analog, Shykoff & Bucheli (1995) found that only 8% of plants with open flowers received dye during the day, whereas 39% received dye during the night. Our observed pattern, in which plants in the nocturnal pollinator treatment group showed 15.5% more outcrossing than plants in the diurnal treatment group, suggests that use of dye as a pollen analog accurately portrays the trend in the direction and magnitude of the difference between pollination by nocturnal and diurnal pollinators but that it may overestimate actual rates of gene flow.
Several factors could explain the increased effectiveness of nocturnal pollinators in our system. First, S. alba plants show a typical moth pollination syndrome (Baker & Hurd, 1968) and are perhaps simply more attractive to nocturnal than to diurnal pollinators. This supposition would be supported by higher flower visitation rates by nocturnal than by diurnal pollinators. Although we did not measure pollinator visitation rates in our study, Shykoff & Bucheli (1995) found that nocturnal pollinators had significantly higher flower visitation rates than did diurnal pollinators. However, Altizer et al. (1998) found the opposite. The degree to which a plant attracts pollinators is likely to change depending on the total pollination milieu, the combination of flowering plant species and pollinator species present in the same space and time, which may explain different visitation rates between nocturnal and diurnal pollinators in different studies.
Even if nocturnal pollinators visit S. alba flowers less frequently than do their diurnal counterparts, they could be more effective at transmitting genes if they have a greater tendency to transfer pollen between plants. There are several examples of plant species for which nocturnal pollinators have a greater per-visit effectiveness than do diurnal pollinators (reviewed in Young, 2002). For example, in the Japanese honeysuckle Lonicera japonica, diurnal bees removed but also consumed more pollen than did nocturnal moth pollinators, resulting in an overall lower pollen transfer efficiency for bees (Miyake & Yahara, 1998). Because we were only measuring instances of gene flow between rather than within populations, the increased gene flow by nocturnal pollinators likely results from a greater tendency of nocturnal pollinators in our system to forage between rather than within patches. Although nocturnal pollinator behavior has been the subject of fewer studies than the behavior of diurnal pollinators and is thus much less well understood, Young (2002) showed that, in a S. alba system, nocturnal pollinators carry fluorescent dye further than do diurnal pollinators, though she was only able to measure dye movement on a scale of centimeters rather than the tens of meters scale used in this study. A large body of evidence exists to indicate that, in other plant systems, diurnal pollinators such as bumblebees and honey bees have a tendency to spend more time foraging within than between patches (Waddington, 1983). Further work is called for on the role of different types of pollinators in moving pollen between rather than within populations.
We measured gene flow receipt as the proportion of outcrossed capsules produced in a population. Using this measure, we found relatively low rates of outcrossing compared with the work of Richards et al. (1999). The highest rate of gene flow receipt we measured was 8.74%, whereas Richards et al. found that gene flow receipt reached as high as 100% in populations spaced 20 m apart near large source populations. The reduced amounts in our study may be related to fewer pollinators in the area or to the small size of adjacent populations serving as pollen sources. Nocturnal moth pollinators are known to fluctuate widely in abundance within and between seasons (Pettersson, 1991).
The distance by which populations are separated from one another also has an important role in determining rates of pollen-mediated gene flow. Our finding that plant populations separated by only 20 m have much higher rates of gene flow than do populations separated by 80 m is consistent with the findings of Richards et al. (1999) who looked at the effects of population size and degree of population isolation on pollen-mediated gene flow in S. alba. Here we hypothesized that nocturnal pollinators, by virtue of their long-distance flight behavior, would be better at facilitating gene flow in populations separated by longer distances than would diurnal pollinators. The trend we observed, in which nocturnal pollinators were responsible for three times as much gene flow as diurnal pollinators for plants in the 80-m distance treatments, is consistent with our hypothesis but not statistically significant. Basically, gene flow rates were so low for the 80-m populations that it would be very difficult to statistically confirm a difference in effect between nocturnal and diurnal pollinators. Nevertheless, we did find a significant pollinator type by distance effect on the rate of gene flow. This finding is particularly important when placed in the context of the interplay among dispersal, extinction and colonization in plant metapopulations. Changes in the frequency of different types of pollinators could have enormous implications for metapopulation structure if key pollen vectors decline in number. The numerical decline in pollinators could result from direct anthropogenic disturbance of the pollinators (Kearns et al., 1998) or from habitat fragmentation if plant populations occurring in fragments are less able to attract (because of isolation) or support (because of population size) an adequate number of pollinators (Moody-Weis & Heywood, 2001; Donaldson et al., 2002).
Finally, although we clearly determined that for our S. alba system nocturnal pollinators played a much greater role in between-population gene flow than did diurnal pollinators, diurnal pollinators nevertheless contributed to pollen-mediated gene flow. This finding supports the notion advanced by Waser et al. (1996) that plant pollination systems are actually more generalized than the traditional ‘pollination syndrome’ view might suggest. Thus, although the apparent pollination syndrome of a plant may well be suited to a suite of particular pollinators, the concept of pollination syndrome should be used not as a strict rule but as a guide for the design of experiments that empirically test the role of different pollinators in mediating gene flow. Remarkably little is known about the foraging behavior, both within and between patches, of the large moths that function as nocturnal pollinators in this system.
‘Genetic rescue’ (Richards, 2000b) via pollen-mediated gene flow may reduce the negative consequences of inbreeding depression and thus reduce the likelihood of population extinction in small, isolated plant populations. Although we showed that nocturnal pollinators are considerably more effective than diurnal pollinators at moving genes in our system, their effectiveness declined in populations separated by 80 m. At fine spatial scales, nocturnal pollinators are likely the key agents of pollen movement in S. alba metapopulations. As the frequency of pollen-mediated gene flow decreases with increasing subpopulation isolation, plants must rely on other mechanisms (e.g. demographic rescue via immigration of new individual plants) in order to persist through time.