AOBPreview originally published online on March 22, 2004
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Annals of Botany 93: 603-607, 2004
© 2004 Annals of Botany Company
Does the ‘Old Bag’ Make a Good ‘Wind Bag’?: Comparison of Four Fabrics Commonly Used as Exclusion Bags in Studies of Pollination and Reproductive Biology
1 Department of Ecology and Evolutionary Biology, Torrey Life Sciences Building, 75 North Eagleville Road, U-43, University of Connecticut, Storrs, CT 06269-3043, USA
* For correspondence. E-mail Paul.Neal{at}UConn.edu
Received: 13 August 2003; Returned for revision: 2 October 2003; Accepted: 22 December 2003 Published electronically: 22 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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• Background and Aims Fabrics used in pollination bags may exclude pollen carried by biotic vectors, but have varying degrees of permeability to wind-borne pollen. The permeability of bags to wind-borne pollen may have important consequences in studies of pollination and reproductive biology. The permeability of four fabrics commonly used in the construction of pollination bags was examined.
• Methods Deposition of wind-borne pollen on horizontally and vertically oriented microscope slides was assessed on slides enclosed in pollination bags, as well as on control slides.
• Key Results It was found that the permeability of fabrics to wind-borne pollen, as measured by deposition on both horizontally and vertically oriented slides, decreased with pore size. However, deposition on horizontal slides was always greater than on vertical slides for a given fabric; this could manifest itself as differential success of pollination of flowers in bags—dependent on flower orientation.
• Conclusions Obviously, bags with mesh size smaller than most pollen grains are impermeable to pollen. However, material for such bags is very expensive. In addition, it was also observed that bags with even moderately small pore size, such as pores (approx. 200 µm) in twisted fibre cotton muslin, offered highly significant barriers to passage of wind-borne pollen. Such bags are sufficiently effective in most large-sample-size reproductive biology studies.
Key words: Amphiboly, anemophily, pollen exclusion bag, pollination, pollination bag, pollinator exclusion bag, technique, wind pollination.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Bags that exclude various kinds of pollinators or pollen delivery systems are fundamental to studies of pollination and plant reproductive biology (Dafni, 1992; Kearns and Inouye, 1993). The pore size of bags may exclude some pollen vectors but not others. Selective exclusion due to pore size may be an intentional part of the study (Vanstone and Patton, 1988; Pellmyr, 1989), or a problem to be overcome (Barrett and Helenurm, 1987; Cruden et al., 1990). The effectiveness of exclusion through pore size is relatively easy to demonstrate for biotic pollinators by direct observation. However, the relative effectiveness of various bags in excluding wind-borne pollen is more difficult to ascertain, given that the pollen vector cannot be directly observed.
In studies concerned with wind-borne pollen, fabric pollination bags have been used to exclude all sources of pollen (e.g. Arroyo and Squeo, 1987), or to allow wind-borne pollen but exclude biotic pollen vectors (e.g. Pellmyr, 1989). Rarely are fabric pollination bags quantitatively tested for their ability to accomplish these tasks. However, there are exceptions. For example, Sacchi and Price (1988) found no difference in the amount of pollen deposited on glass slides coated with silicone grease and placed in mesh bags (0·9 x 0·9 mm mesh) compared with slides left uncovered. Anderson (1976) used Fraxinus americana L. (a dioecious, stereotypically wind-pollinated species upon which he observed no insect visitation) to test the permeability of nylon monofilament screen bags (pore size = 1·21 mm) to wind-borne pollen (pollen diameter = 33·2 µm). He found fruit set in inflorescences covered by screen bags to be 70 % of uncovered inflorescences, while in inflorescences covered by glassine envelopes fruit set was entirely eliminated. Goodwillie (1999) tested for bag effects by comparing counts of wind-borne pollen deposited on petroleum jelly-coated microscope slides enclosed in bags with slides that were not enclosed. She found that slides in bags had fewer pollen grains than those in the open, but that the difference was not significant.
Pollen exclusion bags can be constructed from fabrics with widely varying characteristics (e.g. material, pore size, thread type), depending on the level of exclusion desired. Fabric that excludes all but the smallest biotic pollen vectors or that admits at least some wind-borne pollen can be easily and cheaply obtained from retail fabric shops. Exclusion of all pollen may require a material with a uniform pore size that is smaller than the wind-carried pollen. However, such material can only be obtained from specialty manufacturers, and it is often very expensive. Knowledge of the permeability of various fabrics to wind-borne pollen will be crucial when interpreting the results obtained when using pollen exclusion bags. Herein, we systematically examine four fabrics typically or potentially used in constructing pollen exclusion bags for field studies of pollination and plant reproductive biology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The permeability of the pollination bags to wind-borne pollen was tested using five replicates of each of five treatments. In the control treatment, no exclusion bag was used (i.e. the natural deposition of pollen was measured). The deposition of pollen was also measured in pollination bags constructed from four different fabrics: (1) large mesh (Fig. 1A)—a fabric often used to exclude larger biotic pollinators, but that is also assumed to allow the passage of wind-borne pollen; (2) small mesh (Fig. 1B)—a fabric that would exclude all but the smallest biotic pollinators (e.g. thrips); (3) cotton muslin (Fig. 1C)—a fabric that might be expected to prevent entry of wind-blown pollen; and (4) filter fabric (Fig. 1D)—a specialty screening fabric with extremely tiny, uniform pores designed to be used in filters. Characteristics of the fabrics are given in Table 1. All bags were newly constructed for the study (i.e. had not been previously exposed to field conditions). Fabric for the bags was obtained from a local fabric store, except the screening fabric, which was obtained from the manufacturer (Table 1).
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Pollen deposition was assessed by placing two glass slides inside each pollination bag. A light smear of silicone grease (Dow Corning High Vacuum Grease; Dow Corning Corp., Midland, MI, USA) was applied across the middle (approx. 1·5 cm wide) of each slide. The grease is a silicon lubricant for glass stopcocks, joints and glass–rubber connections. It is heat stable, so the grease did not run while the slides were in direct sunlight. The two slides were placed on 5 cm square wood blocks (Fig. 2). One slide was placed horizontally in the middle of the block (‘H’ in Fig. 2). The second slide was held in a vertical position (‘V’ in Fig. 2) by inserting it in a 1·5 mm wide, 2 mm deep groove in the block. Modelling clay was used on the backsides of horizontal slides and in the groove for vertical slides to prevent the slides from shifting. A wire hoop was placed inside each bag to prevent the fabric bags from coming into contact with the surface of the slides (Fig. 2). To avoid spurious deposition of pollen on the slides, the bags were installed immediately after the slides were placed on the blocks. Staples were used to tightly bind the base of the bags to the wooden blocks.
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The randomized groups of one each of the five treatments were arranged in two rows with the vertical slides facing out on a table 1 m off the ground in a lawn. The lawn was adjacent to extensive native hardwood forests, and an open New England hay field. Among the anemophilous native species: (a) hickory trees (Carya spp.) (to the west and south within 25 m of the test site) were just completing flowering; (b) white pine (Pinus strobus) (two mature trees 30 m to the north of the test site) cones were open with visible quantities of yellow pine pollen on vegetation, and even in the air during some wind gusts; and (c) there were abundant grasses in flower in the adjacent uncut hay field (<50 m to the north of the test site) (Digitaria sp. and various other species).
Tests were conduced over 2.5 days in mid-June (15–17 June 2003) in Storrs, CT, USA. The weather during this time consisted of moderate, temperate summer days, with daytime highs of about 23 °C, and night-time lows of about 7 °C. The wind was light during the days, and it was calm in the nights. The bags were left in place for about 54 h, and removed when rain was predicted; thus, the tests were conducted only during dry, mild weather.
When the experiment was terminated, the slides were immediately placed on slide trays and stored in closed boxes. The pollen on the slides was counted by scoring all pollen in ten randomly chosen, non-overlapping fields (3·6 mm diameter) with a x4 objective on a Nikon compound microscope. A combination of bright field and dark field microscopy was used to be sure that all pollen grains, and only pollen grains (not clay micelles, or bits of plant cells, airborne algae, etc.) were counted. No distinction was made among the pollen grains, but the vast majority was Pinus strobus pollen. The pine pollen varied in size, with a minimum distance across the grain ranging from 40 to 50 µm. The maximum distance across the grain ranged from 50 to 70 µm, and depended on the expansion of the pollen bladders. Approximately 5 % of the pollen was from Carya spp., which was 20–30 µm in diameter.
An unplanned, nonparametric multiple comparisons test by STP (Sokal and Rohlf, 1995) was used to compare pollen deposition on slides within and between pollination bag treatments.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The rank order of pollen quantity deposited in each pollination bag treatment was the same for horizontal and vertical slides (Fig. 3): control > large mesh bags > small mesh bags > cotton muslin bags > filter fabric bags. Considering the controls as having unlimited or infinite pore size, this rank order is consistent with that of pore size for the five treatments (Table 1). The decline in pollen deposited on slides with decreasing pore size may be the result of pollen being blocked by the fabric itself or of reduced penetration of the wind due to aerodynamic disturbances. The pool of pollen was not filtered by pollen size. Casual observation of pollen size on slides of different fabrics shows that the larger pine pollen was found on all slides where pollen was deposited. In addition, the pore size of large mesh and small mesh bags is much larger than the largest pollen deposited on slides, suggesting that filtration by size would not be important. Although the rank of pollen deposition matches pore size of the fabrics, the differences in pollen quantity did not differ significantly between all fabric types. For both horizontal and vertical slides, the difference in deposition between control and large mesh bags was not significant (Fig. 3).
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0·05). The value below treatment name is the ratio of the number of grains deposited on vertical vs. horizontal slides.Within each pollination bag treatment, more pollen was deposited on horizontal slides compared with vertical slides (Fig. 3). This difference was significant for all treatments except filter fabric. However, the very few pollen grains deposited on slides in filter fabric bags were probably the result of experimental error, given the pore size of the fabric is much smaller than the diameter of all pollen we observed. During the experimental sampling period, wind conditions varied from calm to a light breeze. In the bag treatments other than the filter fabric, additional pollen may have been deposited on horizontal slides during relatively calm periods. In these three bag treatments (i.e. other than the filter fabric bags), the relative proportion of pollen on vertical vs. horizontal slides is not constant (Fig. 3), but decreases more rapidly with pore size. This may be the result of aerodynamics within the bag, where wind velocity is presumably reduced and pollen settles more quickly, and thus does not have sufficient velocity to reach or adhere to the vertical slides.
The variation in pollen deposition associated with bags of various fabrics, and on horizontal vs. vertical slides suggests that the bags may affect the environmental conditions inside the bag relative to those outside the bag. Although no specific test of these conditions for the fabrics was made, several studies have shown that pollination exclusion bags can have a significant affect on temperature and humidity in the bags (Corbet and Wilmer, 1981; Pleasants and Chaplin, 1983; Wyatt et al., 1992). Synthetic fibres have the advantage of drying quickly; the fabric of cotton muslin bags may retain moisture causing a change in humidity (and temperature) within the bag. Similarly, the small pore size of filter fabric bags may also cause moisture to be retained within the bag. Depending on the species under investigation, floral characteristics such as pollen dehiscence, stigmatic receptivity, nectar production, or development time may be affected. Furthermore, personal experience indicates that the probability of fungal infection of the plants may be increased. This may be a problem, especially in humid, rainy or dewy environments.
The bags used in this study were newly constructed, and as such, the pore size was fairly consistent. Inspection of our supply of previously used bags found many bags with scattered larger pores due to damage from snagging vegetation and shifting of the weave. Only filter fabric bags appeared to be exempt from this problem, probably due to the extremely tight weave. However, we also found that cotton muslin bags generally have a reduced pore size as bags weather and the fibres untwist and/or unravel. Even in new muslin fabric, some pores are open (at arrow a in Fig. 1C), while others are occluded with fibre ends (as in arrow b in Fig. 1C). Unraveling is likely to continue to increase with continued use of the bags.
Our results indicate that the material used for pollination bags should be chosen with care when wind-borne pollen is or could be an important factor. Bags with large mesh would be effective at excluding larger biotic pollinators, while still allowing free flow of wind-borne pollen. This might be especially important in studies with ambophilous pollination (Free, 1964; Sacchi and Price, 1988; Testolin et al., 1991; Tamura and Kudo, 2000; Culley et al., 2002) where the investigator is considering the importance of this mode of pollen transport. These bags, with large pores, and nylon or polyester fabric, would offer the additional advantages of not creating an artificial environment within the bags, by retaining humidity or heat, and they would dry out more quickly after rain or heavy dew.
The small mesh nylon bags would obviously keep out virtually all biotic pollinators (except, perhaps, thrips), but they also significantly reduce the quantity of wind-delivered pollen, and would thus not provide good estimates of effective anemophily. The polyester small mesh bags we used have the additional advantage of the quick drying of the synthetic fibre due to relatively good porosity. The cotton muslin bags would exclude all relevant biotic pollinators. Additionally, they were nearly as effective as the filter fabric bags at excluding wind-borne pollen, and their cost is negligible. A drawback to these bags, however, is their slower drying rate.
The filter fabric bags allowed essentially no pollen in, and, obviously, no biotic pollinators. However, compared with other fabrics, they may produce an environment within bags most different from ambient conditions. Additionally, they are very expensive (cost for our material was about $20 per bag). They do keep out all wind-borne pollen, so that is obviously an advantage. However, given the relative effectiveness of the cotton muslin bags and the relative biological insignificance of the wind-borne pollen that does get through the cotton bags, researchers may wish to consider the diminishing returns of the extra expense.
We recommend that researchers consider the potential in each study for being misled by pollination bags that are semi-permeable to wind-borne (i.e. not excluding or allowing all wind-borne pollen), and that they carefully select the fabric used in the construction of pollen exclusion bags. It may be wise to include preliminary experiments or controls to quantify bag effects on pollen deposition. In addition, depending on their choice of fabric, researchers may also need to quantify the effects of bags on the floral environment within the bag (e.g. temperature, humidity).
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ACKNOWLEDGEMENTS |
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We thank Mona Anderson for help with the project, C. Martine for comments on an early draft of the manuscript, J. D. Ackerman and C. Goodwillie for critical reviews, and the Department of Ecology and Evolutionary Biology of the University of Connecticut support. P.R.N. thanks the Saloma Foundation for continuing support.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Anderson GJ. 1976. The pollination biology of Tilia. American Journal of Botany 63: 1203–1212.[CrossRef]
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Barrett SCH, Helenurm K. 1987. The reproductive biology of boreal forest herbs. I. Breeding systems and pollination. Canadian Journal of Botany 65: 2036–2046.
Corbet SA, Wilmer PG. 1981. The nectar of Justicia and Columnea: composition and concentration in a humid tropical climate. Oecologia 51: 412–418.[CrossRef]
Cruden RW, Baker KK, Cullinan TE, Disbrow KA, Douglas KL et al. 1990. The mating systems and pollination biology of three species of Verbena (Verbenaceae). Journal of the Iowa Academy of Science 97: 178–183.
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Sacchi CF, Price PW. 1988. Pollination of the arroyo willow, Salix lasiolepis: role of insects and wind. American Journal of Botany 75: 1387–1393.
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Vanstone VA, Patton DC. 1988. Extrafloral nectaries and pollination of Acacia pycnantha Benth. by birds. Australian Journal of Botany 36: 519–531.
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