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).

注:本文是根据我们以前的工作30。一些部分转载来自日本和斯普林格日本植物学会许可。 1。访花观察<ol><li> 观测场的选择 <ol><li>搜索中，植物材料分布的地区，采用可靠的资源，如图画书，学术期刊等调整研究地点的数量，以适应研究目标（选择候选研究地点比如，一个广泛的位置花的游客在日本的比较）。 </li><li>检查所选择的候选群体的来自相关的出发点，如科研院所和住宿，位置和距离，如果需要长期的研究。 </li><li>通过在一个单位面积计数植物个体的数目大致估计每个种群规模。选择实验操纵大量人群S使用许多植物的个体。 </li><li>访花的预观察<ol><li>决定靶向个体在每个区域将在同一时间间隔期间观察到的，这取决于人口密度。选择5-10刚刚开的花或花蕾突破作为目标的花朵，并拒绝已经开通退化的。 </li><li>启动一整天的时间肉眼观察，因为花可以通过在不同的时间不同的传粉范围31,32参观。 </li><li>在记录片，记录访花的种类名称，每人花小时每个探视时间。 </li><li>密切观察访花是否触及花药和/或红斑;如果他们这样做，记录下参观者授粉。如果访花只能访问花草不要触摸生殖器官，并录制成游客。 </li><li>使用防虫网或手工制作的吸引，捕捉花香游客识别和保留它们作为标本（ 图1F）。用乙酸乙酯或手指压向胸部快速杀灭被困访客，并在软袋或在塑料管或壳体用100％乙醇保留它们。 </li><li>观察后，确定使用形态特征样本的具体名称，或在鉴定中的困难时，请专家为各分类群的帮助。 </li></ol></li></ol></li><li> 观察领域 <ol><li>选择根据自己的可及性，人口规模和花卉观众数量研究场所。选择的时间段，其中包括在开花季节花卉游客的代表性样本，并基于研究目的观察期（ 例如，通过访花探视频率波动的研究，长期的时间段应设置）。 </li><li>准备相应的录音设备，如数码摄像机（例如，对于夜间记录，应使用摄像机用红外线功能）。 </li><li>以同样的方式作为1.1.4选择目标的花朵。 </li><li>固定摄像机铝三脚架。设置目标的个人与前面的三脚架相机，约50厘米远。 </li><li>检查并拍摄前修改光对摄像机的屏幕显示的数量和材料的重点。 </li><li>开始在同一时间通过预观测资料估算适当的时区的目视观测和拍摄的视频（在本案中，观察期分别为平均六个多小时，大约从上午05时至下午13:00 ）。 </li><li>识别访客和传粉昆虫的种类名称，并记录他们的名字和他们的记录片材访问的次数。 </li><li>捕获并确定以同样的方式访客和授粉为1.1.4.5。 </li><li>重复步骤1.2.1至1.2.7各观测中ñ时期。设置基础上，研究目标和观测数据前的观察期。 </li></ol></li><li> 数据分析后观察 <ol><li>以同样的方式传粉观测如1.1.4.6后确定捕获试样的种类名称。 </li><li>检查视频剪辑，并注意以同样的方式作为用于目视物种名称和它们的访问时间。 </li><li>从视觉观察和录像数据计算每人每次花的游客小时花探视频率。使用基于基本统计并使用合适的软件，如R，SPSS和/或SAS 33-35（ 例如，方差双向分析（ANOVA适当的统计方法统计比较每个访问者的站点和岁之间的这些频率）与R中的软件杜克的诚实差异显著（HSD）的测试）。 </li></ol></li></ol> 2。套袋和保持架实验<ol><li> 筹备和套袋实验笼 <ol><li>对于套袋实验，编制袋有小网目尺寸（〜0.5-1毫米），并用它们来完全防止花探视（ 例如，分别在2011年和2012年用于装袋实验无纺布袋）。 </li><li>为保持架实验中，准备金属丝或塑料丝网板的直径，允许目标越小访问者通过，但不包括较大的，并连接这些网眼板，以形成一个笼子。确保有不低于网直径大的差距。调整基于所述目标植物物种和个体数笼的尺寸和形状。 </li></ol></li><li> 套袋试验 <ol><li>选择每处理30个人，没有从食草动物或严重破坏环境。对于治疗，从各个工厂一花独放，或单株使用单独的花朵。 </li><li>袋ŧ他用胶带贴标签后，他们在目标领域的花（编号和字母; 图1G）。要小心，不要触摸塑料袋内的花药或魂飞魄散，以避免自花授粉的可能性。 </li><li>精心修复袋用软绳或线（ 图1d）花。 </li><li>小心地用软绳或线支持在必要时将袋或风，重压之下对倾斜或塌陷的个人支持设置单独的花朵直立。扭曲或缠绕细绳或金属丝轻轻围绕目标个体的茎，以免建立损坏。 </li><li>在套袋试验治疗<ol><li>对于"控制"的待遇，附加标签的目标鲜花和进行任何治疗。允许花卉游客自由参观。 </li><li>对于"异交"的待遇，涵盖了芽，直到他们的花，然后取出的袋装花花药。把一些polle从上留下烙印多个个体ñ粒。 </li><li>对于"自交"的待遇，涵盖了芽，直到花开放，并从同花的花粉把自己的上魂飞魄散。再次覆盖这些治疗花。 </li><li>对于"自动自我"治疗，盖上袋子，直到开花赛季结束的味蕾。 </li><li>对于"断芽"的待遇，确定已访问了破芽，观察小蜜蜂的出入口（ 图1b，C）。取出破芽的花药，以防止在同一花重复花粉沉积由小蜜蜂探视后，和袋这些芽迅速，以防止进一步的探访。 </li><li>对于"开花"的待遇，包芽，直到开场阶段，以避免在磨合萌芽阶段，不胜其烦。在此之后，取出包，并让参观者收集花蜜和花粉。 </li><li>对于"芽"的待遇，移除未开封的花蕾花药和人为异交他们。这些袋子迅速芽，防止花，不胜其烦。 </li></ol></li></ol></li><li> 凯奇实验 <ol><li>盖上笼准备目标植物的个体。手工调节的笼花朵的位置，以防止不同痕（ 图1E）之间的接触和花粉沉积。 </li><li>对于"笼子"的治疗，选择未开封的花蕾的个人和扣帽子对他们的选择鲜花的鉴定。凯奇与标记的芽个人从建立笼（ 即，花卉虫不能访问未开封的花蕾，可估计只网箱放置后参观昆虫的影响）之前拒绝的影响。 </li><li>牢固安装笼使用铁极，以防止通过笼子基极和地之间的访客条目地面。 </li></ol></li><li> 数据分析房颤之三套袋和保持架实验<ol><li>收集所有的标记花朵在开花季节（ 图1G）通过切割并从母体的个体中分离它们的端部。单独保留每个标记的样品，以防止其污染。 </li><li>检查存在或不存在的水果上的每个标记设置的flower.Record在设定果的情况下，每个果实的种子数目。 </li><li>计算（坐果比率，其定义为水果由花的数量除以数）和每成熟果实（种子集比，定义为通过数除以种子数种子数的每花果实数的比胚珠）使用所有记录的数字。 </li><li>统计学比较使用适当的方法和软件，如那些在1.3.3中列出的治疗之间的加工水果，种子组的比率（ 例如，单向ANOVA和Tukey的HSD或Fisher中的R软件33精确检验）。 </li><li>使用装袋实验的结果的材料的调查<ol><li>为了测试动物传粉的需要，在统计学上的"控制"和"自动自"治疗之间的比较结果。 </li><li>为了估计花粉限制的程度，比较"控制"和"异型杂交"治疗。 </li><li>为了测试自交亲和性，比较"异交"和"自交"治疗。 </li></ol></li><li>破芽授粉的影响评估<ol><li>比较"控制"和"芽"治疗，以确定破芽痕是否繁殖成熟，是否可以使用在破芽期进行控制的"控制"治疗的价值。 </li><li>比较"自动自"和"打破芽"处理，以确定是否花的繁殖成功的断裂与小蜜蜂授粉-bud阶段是统计学上比进行自花授粉（ 即，测试断裂芽授粉的存在）植物更高。在此之后，这两种治疗方法比较"控制"估计破芽授粉的授粉效率。 </li><li>比较"断芽"，"笼"和"花"处理，以估计破芽授粉繁殖的影响。 </li></ol></li></ol></li></ol>

The authors declare that they have no competing financial interests.

花观测和装袋实验在该研究中采用的以显示探视频率和植物的雌性繁殖成功，分别。在Dafni酒店（1992）38，因为它可以记录用于分析观众的定时和持续时间，并防止观察者偏压录像带方法是有效的。但是，在当时，该方法需要昂贵的设备，和观察时间是由电池寿命的限制。近来，装备用于产生视频记录的成本下降，并且可以在其他授粉研究可以采用这一技术方法。在这项研究中，探视频率分别在站点3（ 表2）的视觉和视频观测之间显著不同。这可能是造成花的游客过观察，人为错误，如这可以被拒绝。 Dafni酒店（1992）也提到装袋或净实验研究繁育系统38。非WOVEN用于箱包面料，这些都没有pollen-或防水。L.荪变种荪不是风媒物种，但雨水可能影响袋装花的繁殖成功率。铁股份被用于支持个人与湿书包的重量袋装花，但这些因素可能影响花的生殖能力。覆盖昆虫独家网络全厂与支持者可能会消除这些方法论问题的最佳选择。此外，被用于蜂种的分离，这是一个植物笼研究的第一个例子笼中。这项研究证明了这一方法的有效性，我们可以将其应用到其他的研究，其目标需要不同的官能团，如小型和大型蜜蜂授粉效果的判断。 这些授粉观察发现，大多数血红石蒜的花卉游客</EM&gt;变种荪整个花期是Lasioglossum血吸虫 。为了使成功的观测，在一些候选研究地点靶材料的预观测应，这是在协议部分步骤1.1中描述。例如，一些访花的标识是根据在现场观察或相机拍摄的照片制成。一些花卉访问者属于该人难以确定在第一分类群组由于它们无法区分形态性状或快速参观活动，如halictid蜜蜂或夜间天蛾，分别。因此，对花卉走访初步的研究可以帮助确认并记录每次访问。要充分理解环境条件的影响，以选择合适的观测研究点是很重要的。例如，雨天条件不适合用于传粉的观察，因为授粉的外观和图案可以改变。如果所选网站已在环境条件下的波动，也可能没有收集，分析研究目的足够的观测数据。交替，结果可能被误解由于气候条件的影响授粉社区39的组合物差异。 在这项研究中，用肉眼观察和录像（ 表1）进行了检查访花。这两种方法各有优缺点。在视觉观察中，对象可以从多个角度观看，并且可以更具体地观察到。然而，在目标获得的信息是有限的，因为记录仍然只能作为田野笔记或数码照片。相比之下，花卉游客可反复使用录制的视频进行检查。不幸的是，这种方法往往会产生用于分析不适合记录，如外的焦点和不充分明亮的图像。除了这些方法，索姆E专用记录技术已经开发了在最近几年。例如，在罕见的授粉者，如一些兰科植物开花植物，用数码相机拍摄的间隔是授粉识别40的有效途径。数码摄像机与视频移动侦测传感器，可在花授粉记录的运动的清晰图像，在夜间41甚至快速移动的传粉者。此外，一高速摄像机也已用于观察缓慢授粉的动作，例如每个授粉的接触到柱头42。视频录制和数码摄影是实地观察常用的方法，并了解每种方法的特点，并选择最合适的是很重要的。 装袋实验表明度数L的授粉的依赖血红变种血红 （ 表2）。在这些方法中，关键的步骤s为的袋子和笼子的准备。在这种情况下，所用的袋分别为花（ 图1d）的适当大小;然而，可能有必要以制备较大尺寸或昆虫排除网，它可以覆盖整个植物的个体。 "自动自"处理过的水果不多，但较大的种子设定的比例，这可能已被羞辱和花粉附着袋之间的接触引起的。可以用每个实验的目标的适当方法可以防止这样的错误。套袋为打破芽呈小蜜蜂授粉在磨合萌芽阶段（ 表2）。小蜜蜂往往处理破芽收集花粉长于开放参观花其他昆虫。这些行为差异可能表明，磨合蕾期授粉不具有比其他授粉过程更高授粉效率。为了揭示这些行为上的差异，每授粉成功每个传粉者单次访问或授粉效率应评估43,44。通过制备未开封，袋装芽，有可能估计对破芽授粉的生殖方面的单访问的效果。另外，在保持架实验中，未开封的芽保持。此方法不能被用来评价授粉的由小蜜蜂仅在开花期的效果。一种替代方法是覆盖具有比所有的访花，直到笼花全开的体积缩小差距的布笼。 虽然目前的实验提供了良好的效果，现场实验揭示植物授粉相互作用的信息有限。例如，有人推测由小蜜蜂破蕾花授粉可以促进自交或geitonogamous授粉。当小蜜蜂在打破芽左右移动，会出现此授粉过程。一些花粉粒能容易地从同一花到柱头携带。此外，通过他们的身体尺寸估计小蜜蜂觅食范围分别为短，促进花粉传播45,46的短距离。这些预测是困难仅使用现场实验，调查，虽然个人之间的花粉运动可以使用标记有荧光染料47,48花粉进行跟踪。例如，扩增片段长度多态性或微卫星标记可被用来估计是否每个种子的花粉供体从相同或不同的个体49,50而得。近年来，同种植物之间花粉运动都使用单一花粉基因分型技术35得到执行。这种分子的方法已被用于显示授粉效率36的评价的意义。在目前的情况下，这种分子技术可以揭示破芽的花粉粒，其心肌梗死的到来位置GHT表明破芽和完全开放的花朵之间的度的基因流。因此，全面研究计划的基础上，这两个领域的工作和分子分析，是必要的揭示传粉的效果。

植物授粉的相互作用是进化生物学和生态学研究最好的例子。花和授粉之间的互惠关系被认为促进了被子植物1,2-作为自然选择的结果的多样化，尽管其他生物和非生物因素也产生影响3,4,5。它也被认为花性状已经改变，以适应最有效的传粉者，并产生更多的果实和种子6。这些信念已建成但基于不同的指数，如授粉效果的大型研究，涉及各种不同的解释7。 已经广义传粉系统的开花植物是由各种类型的授粉8参观。在这里，花贵宾在定义为访问得到花香奖励，并传粉被定义为授粉花卉游客的动物物种。一些游客携带同种的花粉来参观花的柱头，可以划分为传粉者。其他游客还可能有一些种内的花粉;他们可能由于授粉和花之间的行为或形态不匹配进行授粉少。在植物繁殖的贡献相媲美，这些差异可能产生对花性状9不同程度的选择性压力，并可能导致开花植物的适应性分歧。因此，虽然授粉群落的组成和相对物种丰度是重要的10中，每个访问者的效果的准确评估也很关键，以确定所述自适应和/或植物的进化过程。 在这项研究中，传粉的效率，定义为每探视频率果实和种子生产，定量估计被确定11。该规范观察IES每个花卉游客的频率，估计在参观花繁殖产生影响。通过人类观察花卉访问的记录是传粉生态学一个经典方法。但是，这种方法强加观察员，谁被要求保持在植物的前面，并采取谨慎，长期测量一个很大的负担。近日，拍摄和记录的技术已经发展迅速，低成本的数码摄像机已经启用了引进录像来观察授粉12,13。这些方法可以方便的对花卉的游客基本信息的收集，并有助于培养对象植物的传粉生态学的理解。 然而，传粉的探视频率不一定相关，其授粉有效性7,14，并评价各授粉的定性影响是很重要的花健身。授粉有效性已经通过花粉在柱头15,16的数目，花粉管生长17,18和果实和/或种子产量19,20估算。装袋的实验中，进行了使用访客排他性袋，是用于测试的自相容性，自花授粉21,22，和无融合生殖23的存在下，典型的方法。此外，授粉效果在访问者组合一定授粉评价已经频繁的环境中进行的，而其他花的游客已被限制（即一个铁丝笼，净，或用网眼足够小袋子，以排除大授粉即在开花植物集）。例如，进行了小网袋套袋实验揭示蚂蚁或蓟马24,25的授粉能力。此外，使用一个金属丝笼或网状鸟排除实验已经示出的芦荟类群的有效传粉26-28。 这项研究的目的是:1）引入在以前的文章中使用的方法和2），以改善这些方法在花卉的游客，他们的访问频率等的研究一般使用，以及它们对植物健身效果石蒜荪变种。 荪是包含在石蒜属，这是广泛分布在日本和狭义韩国29和具有漏斗形红橙色的花朵（ 图1a）的品种之一。先前的研究表明，L.荪变种是血红多个昆虫物种，其中包括一名身份不明的小蜂种和较大的品种Amegilla弗洛雷亚 29访问。然而，这些游客探视频率和授粉效果尚未发现。首先进行的访花的授粉鉴定意见。通过探访小蜜蜂是表外实体RVED上还没有完全没有开通花（花蕾破; 图1b，C）。小蜜蜂赶紧搬来搬去在打破芽undehisced花药和使用他们的下颚采集花粉。该假设是小蜜蜂可能是在磨合蕾期授粉，因为花药和花的柱头之间的缝隙比蜜蜂的体长较小。因此，进行了套袋试验在磨合萌芽阶段，以测试小蜜蜂授粉能力，还考察L.的繁殖策略荪变种荪 。这些芽参观了小蜜蜂，这让蜜蜂的授粉能力的估计之后袋装。个人用未开封的芽笼。小网罩使用，通过它只有小蜜蜂可以传递，让整个flowe小蜜蜂授粉效率的估计环阶段。

<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.

被选定为授粉观察五个群体。在观测前阶段，在打开的鲜花和小蜜蜂破芽各种昆虫物种的探视得到了证实。花先生观察发现，大部分游客到所有五个研究点小蜂种Lasioglossum血吸虫个人。总探视记录表明该物种的探视比率在三个位点（ 图2）分别为90％以上。与此相反，第二最常客，Amegilla弗洛雷亚 ，的比率分别在这些领域中低于10％。这些蜂种也触及参观花痕，它们被记录为授粉。其他常见的花的游客，如黑带食蚜蝇 ，收集花粉不与柱头接触，他们被认定为游客。在一个站点中，数据分析表明，探视通过肉眼观察记录小蜜蜂的频率较那些视频（ 表1中记录显著高:单因素方差分析:网站2:DF = 1，F = 0.471，P = 0.50;网站3:DF = 1，F = 12.12 ，P &lt;0.001;网站4:DF = 1，F = 1.019，P = 0.33;站点5:DF = 1，F = 1.605，P = 0.22，分别）。 装袋和笼实验是在站点1在2011年进行的，2012年无纺布袋，用较小的网孔尺寸比花香的游客身体尺寸和丝网笼，具有比小蜜蜂，但小的大网眼比其他游客，制备。观察小蜜蜂对花笼频繁的探访，并采用笼。 "分断芽"的治疗结果显示小蜜蜂来访打破芽的授粉能力。水果的比较在2.4.5中描述的处理之间种子套透露血红石蒜 血红变种，这主要是动物授粉（"控制"与"自动自我"的繁殖性状:费舍尔对坐果精确检验，P &lt;0.001 ）与部分花粉限制条件（"控制"与"异交":费舍尔对坐果精确检验，P = 0.16;费舍尔的种子集精确检验:P = 0.37）。攀比还建议，这种植物具有自交亲和性（"异交"与"自交":费舍尔对坐果精确检验，P = 0.48;费舍尔的种子集精确检验:P = 0.32），这与先前的报道一致36,37。此外，其他治疗方法比较建议打破芽授粉的授粉能力的L.荪变种荪 。这种植物的耻辱成熟在萌芽阶段（＃34;控制"与"芽":费舍尔对坐果精确检验，P = 0.80;费舍尔的种子集精确检验:P = 0.41），和值"控制"是用于在breaking-攀比，蕾期"分断芽"的治疗表现出较高的坐果率比"自动自我"（费希尔对坐果精确检验，P = 0.02），说明花粉沉积在破芽存在小蜜蜂魂飞魄散阶段。然而，这是不可能的演示此授粉过程的进展。在三种处理中2.4.6.2详述（"控制"，"自动自"和"断芽"）显示显著差异（单向ANOVA对于坐果; DF = 2，F = 18.46，P &lt;0.001;单向ANOVA用于种子组:DF = 2，F = 3.6593，P = 0.03），但Tukey的HSD表明破芽授粉不是很有效（"断芽";与"自动自我":水果盘:P = 0.10;结实:P = 0.03）。另外，在2.4.6.3三种治疗之间的比较表明无显著差异（双因素方差分析:DF = 2，F = 0.6881，P = 0.50;双向ANOVA用于种子组:DF = 2，F = 1.2376， P = 0.30）。 <img alt="图1" src="/files/ftp_upload/54728/54728fig1.jpg" /> 图1:植物材料和实验设备 （ 一 ）目标植物的花， 石蒜变种血红 血红 。 （b）一个破萌芽状态。 （ 三 ）小蜜蜂逛破芽收集花粉。 （d）与装袋实验的一个例子。 （e）在丝网笼覆盖一些未开封的味蕾。 （六）被主要用于小蜜蜂的采集吸气。 （G </st荣&gt;）标签系统的一个例子。 "C"是指用于装袋实验打破芽这种花。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54728/54728fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54728/54728fig2.jpg" /> 图2:Lasioglossum血吸虫探视率全开花朵和断裂芽和Amegilla弗洛雷亚来开花该图的比例是基于以前的工作25。最常见的访问者是小蜜蜂L.血吸虫 ，接下来是更大的蜜蜂A.弗洛雷亚 。该研究站点的信息如下:网站1 =泉自然公园，千叶县;网站2 = Sonnou之森，千叶县;网站3 =菅原神社，神奈川县;网站4 = Mannyou自然公园，枥木县;和网站5 = Kogushi Katakuri之乡，群马县。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54728/54728fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54728/54728table1.jpg" /> 表1:花卉的游客在2013年该表显示了观测时间， 视觉和录像观测值之间的比较观察到每个站点的花数，大大小小的蜜蜂访问次数。 "视觉"和"视频"从视觉观察和录像的值，分别为。探视频率与标准偏差在括号中。相比通过单向ANOVA其他观察方法有星号的值是显著不同（ 即，小蜜蜂在目视观察探视比值比视频重新显著更高在站点3线）。 <img alt="表2" src="/files/ftp_upload/54728/54728table2.jpg" /> 表2:套袋在2011年和2012年的缩写结果和实验笼如下:在每个治疗中使用N =花数;没有。水果=坐果数;没有。种子=种子集数;坐果=坐果率;和结实=种子设定比例。结实率都给出标准偏差。破折号表示没有数据。

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

以显示一个给定植物物种的授粉有效性的田间试验的多种方法已被开发出来。这项研究表明了传粉生态学使用血红石蒜变种的血红的案例研究和小说授粉机制，打破芽授粉现场实验的基本方法。

传粉生态学的田间试验:为例<em&gt;血红石蒜</em&gt;变种。<em&gt;荪</em

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

11.4K 次观看.  Chiba University. 以显示一个给定植物物种的授粉有效性的田间试验的多种方法已被开发出来。这项研究表明了传粉生态学使用血红石蒜变种的血红的案例研究和小说授粉机制，打破芽授粉现场实验的基本方法。 

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

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

Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream&#8217;s function and habitat it provides. Sediments from highly eroding streambanks are a major source of pollution in a stream system and have the potential to jeopardize habitat, infrastructure, and stream function. Watershed management practices throughout the Cleveland Metroparks attempt to locate and inventory the source and rate the risk of potential streambank erosion to assist in formulating effect stream, riparian, and habitat management recommendations. The Bank Erosion Hazard Index (BEHI), developed by David Rosgen of Wildland Hydrology is a fluvial geomorphic assessment procedure used to evaluate the susceptibility of potential streambank erosion based on a combination of several variables that are sensitive to various processes of erosion. This protocol can be time consuming, difficult for non-professionals, and confined to specific geomorphic regions. To address these constraints and assist in maintaining consistency and reducing user bias, modifications to this protocol include a &#8220;Pre-Screening Questionnaire&#8221;, elimination of the Study Bank-Height Ratio metric including the bankfull determination, and an adjusted scoring system. This modified protocol was used to assess several high priority streams within the Cleveland Metroparks. The original BEHI protocol was also used to confirm the results of the modified BEHI protocol. After using the modified assessment in the field, and comparing it to the original BEHI method, the two were found to produce comparable BEHI ratings of the streambanks, while significantly reducing the amount of time and resources needed to complete the modified protocol.

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

Streambank erosion potential can be evaluated and ranked using David Rosgen&#8217;s Bank Erosion Hazard Index (BEHI), however this protocol has significant limitations. Here we present protocol modifications to address time constraints, allow nonprofessionals to complete accurate assessments, and account for non-alluvial stream conditions in Northeast Ohio.

修改银行侵蚀危险指数（BEHI）协议的河岸侵蚀美国俄亥俄州东北部的快速评估

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

科研

环境科学

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

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

校准被动采样 - NH多情节场测量<sub&gt; 3</sub&gt;与动态管法和被动采样的组合排放

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

通过他们的足迹识别个体:合模猎豹

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

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

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

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

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

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.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

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

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Construction of a Compact Low-Cost Radiation Shield for Air-Temperature Sensors in Ecological Field Studies

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although cost-effective, if not deployed with proper solar radiation shielding, the observations recorded from these sensors will be biased and inaccurate. Manufactured radiation shields are effective at minimizing this bias, but are expensive compared to the cost of these sensors. Here, we provide a detailed methodology for constructing a compact version of a previously described custom fabricated radiation shield, which is more accurate than other published shielding methods that attempt to minimize shield size or construction costs. The method requires very little material: corrugated plastic sheets, aluminum foil duct tape, and cable ties. One 15 cm and two 10 cm squares of corrugated plastic are used for each shield. After cutting, scoring, taping and stapling of the sheets, the 10 cm squares form the bottom two layers of the solar radiation shield, while the 15 cm square forms the top layer. The three sheets are held together with cable ties. This compact solar radiation shield can be suspended, or placed against any flat surface. Care must be taken to ensure that the shield is completely parallel to the ground to prevent direct solar radiation from reaching the sensor, possibly causing increased warm biases in sun-exposed sites in the morning and afternoon relative to the original, larger design. Even so, differences in recorded temperatures between the smaller, compact shield design and the original design were small (mean daytime bias = 0.06 &#176;C). Construction costs are less than half of the original shield design, and the new design results in a less conspicuous instrument that may be advantageous in many field ecology settings.

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.

一种用于空气温度传感器的紧凑型低成本辐射屏蔽的生态场研究

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

农药在田间对病媒蚊子的影响

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

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.

生态动物的长期视频跟踪: 挪威龙虾 (诺菲氏肾病) 日生物活动的案例研究

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.

台湾蝙蝠种群感染病毒监测的标准操作程序

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

传粉生态学的田间试验:为例

摘要

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