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">
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		<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 ...

The overall goal of this experiment is to investigate the pollinators of Lycoris sanguinea, variety sanguinea, and their pollination abilities. This method can help researchers in the pollination ecology field. For example, in the decreasing of observers around the time of the will focus on specific pollinators'effect per plant.The main advantage of this technique is that it facilitates the investigation of floral visitor species and frequencies for analysis of specific visitors'flower pollination efficiency. For floral visitor pre-observation first decide which target individuals in each area will be observed during the same time interval. Next, select 5 to 10 freshly opened flowers who are breaking buds as target flowers, and begin the visual observation for the period of an entire day, as the flowers can be visited by various pollinators in different time ranges.On the recording sheets, record the species name of each floral visitor, and the time of each visitation per hour, per flower. Closely observe whether floral visitors touch the anthers and/or stigmata. If they do, record the visitors as pollinators.To capture the floral visitors for their identification and preservation, use a handmade aspirator. Then kill the trapped visitors quickly with ethyl acetate and preserve them in a plastic tube with 100%ethanol. At the end of the observation period, use the morphological features of the visitors to identify the specific names of the specimens.Select the target flowers as just demonstrated. Then fix the video cameras to tripods and set the tripods approximately 50 centimeters away from the individual targets. Confirm the appropriate focus and light quantity on the screen displays of each target.Then begin the visual observation and video filming for the appropriate time zones as estimated by the pre-observations. Identify the species names of the visitors and pollinators. At the end of the observation period, check the video clips and note the species'names and their visit times in the same way as for the pre-observation.For a bagging experiment, select 30 individuals per treatment that have no damage from herbivores or from a severe environment. Label the target flowers in the field. Then, taking care not to touch the anthers or stigmata, bag the flowers with a bag with 0.5 to 1 millimeter mesh holes.Use supports to keep the individuals upright as necessary, and use a wire to carefully fix the bags to the flowers. Then, adjust the positions of the flowers to prevent contact and pollen depositions between the different stigmata to reject the influences from before the cage establishment. Afterwards, cover the target plant individuals with prepared cages.Using iron poles, attach the cages firmly to the ground to prevent entry by visitors between the cage bases and the ground. At the end of the flowering season, collect all of the caged and labeled flowers by cutting and separating them from the maternal individuals. Preserve each labeled sample separately to prevent contamination.Then check for the presence or absence of a fruit set on each labeled flower, recording the seed numbers of each fruit in the case of setting fruit. In this representative experiment, floral visitor observations revealed that most of the visitors to all five study sites were individuals of the small bee species Lasioglossum Japonicum, with a visitation ratio of more than 90%at three sites. In contrast, the ratios of the second most frequent visitor, Amegilla Florea, were lower than 10%in these fields.Both bee species also touched the stigmata of visited flowers and were recorded as pollinators. At one site, the visitation frequencies of small bees recorded by visual observations were significantly higher than those recorded by video. The breaking bud treatment of bagged or caged flowers demonstrates the pollination ability of small bees visiting breaking buds with a higher fruit set than the auto self-pollination observed in this experiment, indicating the presence of pollen deposition by small bees to stigmata at the breaking bud stage.Comparing the fruit and seed sets between the treatments reveals the reproductive traits of Lycoris sanguinea, variety sanguinea, which is predominantly animal-pollinated, with partially pollen-limited conditions. Further, these comparisons also suggest that the plant in this experiment has a self-compatibility consistent with previous reports. Moreover, comparisons of the other treatments confirm the pollination abilities of breaking bud pollination in Lycoris sanguinea, variety sanguinea.Once mastered, the plant cage technique can be performed on most growing plants for a variety of visitors. For the pollinator with patience, pre-research of the floral visitors, applying suitable recording techniques and selection of optimal are essential for accurate data collection. The introduction of recording equipment reduces incidence of possible human recording errors, such as over-observation of floral visitors.Following these procedures as a method, like molecular analysis using polymorphic micro saccharide markers can be performed to answer additional questions about pollinator-mediated gene flow between individuals. After watching this video, you should have a good understanding of how to record the interactions of target plants and pollinators, through observation of floral visitors and estimations of their pollination efficiencies.

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Modifying the Bank Erosion Hazard Index BEHI Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio

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Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream&#8217;s function and habitat it provides. ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

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VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

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Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

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Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

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With the advent of small, low-cost environmental sensors, it is now possible to deploy high-density networks of sensors to measure hyper localized temperature variation. Here, we provide a detailed methodology for constructing a compact version of a previously described custom-fabricated radiation shield for use with inexpensive thermochrons.

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Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides in attempts to control its populations eliminates natural enemies and competitor flower thrips species, thereby increasing its populations. An unsustainable situation develops with concomitant resistant pest populations, secondary pest outbreaks, and environmental degradation. Integrated pest management utilizes knowledge of pest and natural enemy relationships to implement tactics that are environmentally friendly and sustainable. Minute pirate bugs are the most important worldwide predators of thrips. They can suppress and ultimately control Frankliniella species flower thrips. Flower samples taken at least weekly are needed to understand predator-prey dynamics. Shown here is the sampling of the flowers of fruiting vegetables and companion plants to estimate the densities of individual thrips and minute pirate bug species. Representative data illustrates how the protocol is used to determine the efficacy of management tactics over time and how to evaluate the benefits of predation by minute pirate bugs. The sampling protocol is similarly adaptable to sampling thrips and minute pirate bugs in other plant species hosts.

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides ...