Name: field experiments of pollination ecology: the case of lycoris sanguinea var. sanguinea
Uploaded: 2016-11-25T15:00:00
Description: Watch this Scientific Journal Video about Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea at JoVE.com

The authors thank the three anonymous reviewers for their helpful comments on the manuscript. This work was partly supported by Grant-in-Aid for JSPS Fellows (26.11613).

NOTE: This article is based on our previous work30. Some parts are reprinted with permission from The Botanical Society of Japan and Springer Japan.
1 . Observation of Floral Visitors
<ol>
	<li>Selection of the Observation Fields
	<ol>
		<li>Search the areas where the plant materials are distributed, and select the candidate study sites using reliable resources, such as picture books, academic journals, etc. Adjust the number of study sites to fit the research objectives (<em...

Flower observations and bagging experiments were employed in this study to reveal the visitation frequencies and the female reproductive success of plants, respectively. In Dafni (1992)38, the videotape method was effective because it could record the timing and duration of visitors for analysis and prevent observer bias. However, at the time, this method required expensive equipment, and the observation times were limited by battery life. Recently, the cost of equipment for producing video records has decline...

Plant-pollinator interactions are prime examples for the study of evolutionary biology and ecology. The mutualistic relationships between flowers and pollinators are thought to have promoted the diversification of angiosperm1,2 as a result of natural selection, although other biotic and abiotic factors have also exerted influence3,4,5. It is also thought that floral traits have changed to adapt to the most effective pollinators and to produce more fruit and seeds6. These beliefs have been constructed though large studies based on different indices, such as pollination effectiveness, that involve various interpretations7.
Flowering plants that have generalized pollination systems are visited by various types of pollinators8. Herein, a flower visitor was defined as an animal species that visited to get a floral reward, and pollinators were defined as floral visitors that pollinate. Some of these visitors carry conspecific pollen grains to the stigmata of the flowers visited and can be classified as pollinators. Other visitors may also have some intraspecific pollen; they might conduct less pollination due to behavioral or morphological mismatches between the pollinators and the flowers. These comparable differences in the contribution to plant reproduction could produce varying degrees of selective pressure on floral traits9 and could cause the adaptive divergence of flowering plants. Therefore, although the composition of the pollinator community and the relative species abundance are important10, the accurate evaluation of each visitor&#39;s effectiveness is also critical to determine the adaptive and/or evolutionary processes of the plants.
In this study, quantitative estimations of pollinator efficiencies, defined as the fruit and seed production per visitation frequency, were determined11. The species and frequency of each floral visitor were observed, and reproductive effects on the visited flowers were estimated. The recording of floral visits through human observations is a classical method in pollination ecology. However, this method imposes a large burden on observers, who are required to remain in front of the plants and to take careful, long-term measurements. Recently, the technologies of filming and recording have rapidly developed, and low-cost digital video cameras have enabled the introduction of video recording to pollinator observations12,13. These methods can facilitate the gathering of basic information on floral visitors and could help to develop an understanding of the pollination ecology of a target plant species.
However, the visitation frequencies of the pollinators are not necessarily correlated to their pollination effectiveness7,14, and it is important to evaluate the qualitative effects of each pollinator on flower fitness. Pollination effectiveness has been estimated through the number of pollen grains on the stigmata15,16, pollen tube growth17,18and fruit and/or seed production19,20. Bagging experiments, conducted using visitor-exclusive bags, are the typical methods for testing self-compatibility, autogamy21,22, and the presence of apomixes23. Additionally, the evaluation of pollination effectiveness for a certain pollinator in the visitor assemblage has been frequently conducted in environments where other floral visitors have been restricted (i.e., a wire cage, net, or bag with a mesh small enough to exclude larger pollinators that is set on flowering plants). For example, bagging experiments with small mesh bags were conducted to reveal the pollination ability of ants or thrips24,25. Moreover, bird exclusion experiments using a wire cage or net have shown the effective pollinators of the Aloe taxa26-28.
The objectives of this study were: 1) to introduce the methods used in a previous paper and 2) to improve these methods for general use in other studies on floral visitors, their visiting frequencies, and their effects on plant fitness. Lycoris sanguinea var. sanguinea is one of the species included in the genus Lycoris, which is distributed broadly throughout Japan and narrowly in Korea29and has funnel-shaped reddish-orange flowers (Figure 1a). A previous study revealed that L. sanguinea var. sanguinea was visited by multiple insect species, including an unidentified small bee species and the larger species Amegilla florea29. However, the visitation frequencies and pollination effectiveness of these visitors were not identified. Pollinator observations for the identification of floral visitors were performed first. Visitation by small bees was observed on flowers that had not completely opened yet (breaking buds; Figures 1b, c). Small bees moved hurriedly around the undehisced anthers in the breaking buds and collected pollen using their mandibles. The hypothesis was that the small bees could be pollinators at the breaking-bud stage because the gaps between the anthers and the stigmata in the flowers were smaller than the body length of the bees. Therefore, bagging experiments were conducted to test the pollination ability of small bees at the breaking-bud stage, and additionally to examine the reproductive strategies of L. sanguinea var. sanguinea. These buds were bagged after the small bees visited, which allowed an estimation of the pollination ability of the bees. The individuals were caged with unopened buds. A small-mesh cage was used, through which only small bees could pass, allowing an estimation of the pollination efficiencies of small bees throughout the entire flowering stage.

<table border="0" cellpadding="0" cellspacing="0" width="535">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:200px;">recording sheet</td>
			<td style="width:159px;">any</td>
			<td style="width:112px;">NA</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">insect net</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pooter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ethyl acetate</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Ethanol</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic tube</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic case</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soft bag</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">digital video camera(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tripod(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bags</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">wire or plastic mesh boards</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iron wires</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">labeling tape</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stick supporters</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soft strings or wire</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pincette(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

field experiments of pollination ecology: the case of lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Five populations were selected for pollinator observations. In the pre-observation phase, visitations of various insect species to opening flowers and small bees to breaking buds were confirmed. Floral visitor observations revealed that most of the visitors to all five study sites were individuals of the small bee species Lasioglossum japonicum. The total visitation record showed that the visitation ratios of this species were more than 90% at three sites (Figure 2</stron...

Watch this Scientific Journal Video about Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea at JoVE.com

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

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