广布小蝶兰及两种根茎兰属植物的传粉生物学研究

植物与传粉者的关系并非一门独立的学科，它包含很多进化生物学的内容。传粉生物学研究为我们理解植物进化的一些关键问题，如生殖隔离、物种形成、适应进化等提供重要证据。兰科植物由于其精巧的花部结构及其独特的传粉机制，备受传粉生物学家的关注。从达尔文时代以来，有大量的文献记载了欧洲兰族植物的传粉生物学的各个方面，其中，共涉及4种欺骗性传粉方式，它们几乎代表了兰科植物中近1/3没有报偿兰花所有的欺骗类型。然而，到目前为止，尽管在中国有丰富的兰族植物资源，中国兰科植物的传粉生物学研究仍然很缺乏。 在本研究中，作者以黄龙自然保护区内兰族的2种根茎兰属植物和1种小蝶兰属植物为材料，研究它们的传粉生物学，并探讨他们之间的生殖隔离机制。 1． 根茎兰属Galearis 及小蝶兰属Ponerorchis植物的交配系统 所研究的2种根茎兰属植物和1种小蝶兰属植物人工自交授粉均可以成功结实。在自然条件下，广布小蝶兰和二叶根茎兰都必须依赖于昆虫才能结实成功，而黄龙根茎兰则是以自花授粉为主。 2． 广布小蝶兰Ponerorchis chusua的传粉系统 在黄龙自然保护区进行了连续2 年的野外观测和实验，结果表明，广布小蝶兰是一种自交亲和，但需要昆虫传粉的欺骗性植物。广布小蝶兰的主要传粉者为雌性Bombus rufofasciatus，雌性B. consobrinus wittenburg，B. cucorun的蜂王和雄性B. pyrosona。熊蜂对广布小蝶兰具有典型的欺骗性兰花传粉的特征，即访问十分迅速，并且一般一个植株只访问一朵花，避免了同株异花授粉。 3． 二叶根茎兰Galearis diantha的传粉系统 野外观察表明，二叶根茎兰只有一种有效的传粉者，其访问频率不高，访 问时间短，并且会一次访问一个植株的两朵花，不可避免的产生同株异花授粉。自然结实率为23.0－26.8％，人工授粉实验证明传粉者限制是结实率较低的主要原因。 4. 黄龙根茎兰Galearis huanglongensis的传粉系统 研究结果表明，黄龙根茎兰为自动自花授粉的植物，黄龙根茎兰的花粉约在花开放4天后，花粉块从花粉囊中掉出，由于重力作用，花粉块柄弯曲使花粉接触到柱头，完成传粉。 5. 根茎兰属Galearis及小蝶兰属Ponerorchis植物的生殖隔离机制 本文的研究表明，根茎兰属内的杂交及与小蝶兰属间植物之间人工杂交授粉均可以成功结实，它们物种之间的生殖隔离主要是通过受精前隔离机制。具有相同传粉者（B. rufofasciatus）的广布小蝶兰与二叶根茎兰通过机械隔离机制保持物种界限。

四川黄龙沟兰科植物多样性及影响繁殖成功的生态因子

植物生态学特性诸如植物的分布特征、与生境的关系、生物学特性、繁殖成功特点及其影响因素等方面的详细资料是研究珍稀植物种群结构和动态以及开展保育生物学研究的基础。 黄龙沟草本植物群落具有较为丰富的植物种类。在所调查样方中(N=662)共有维管植物124种，分属于37科91属，有54个中国特有种。其中兰科植物多达21属33种，中国特有种有12个，新种一个。如此众多的地生兰科植物聚集在面积不到1平方千米的沟内，而且部分兰科植物种类在沟内形成优势草本群落，这在中国地生兰的分布区域中是不多见的。大多数物种，包括兰科植物在内，出现的频率都较小，并且分布不均匀。种间关联分析和相关分析表明黄龙沟优势兰科植物可以分为两组。一组包括无苞杓兰、黄花杓兰、二叶根茎兰、广布小碟兰、少花鹤顶兰和西藏杓兰，主要分布在光线充足但又具有一定遮阴条件的环境中;另一组包括筒距兰、沼兰、珊瑚兰、小斑叶兰、布袋兰、小花舌唇兰和小叶对叶兰，它们主要分布于荫蔽的环境中。这些兰科植物在组内大多呈现显著的正相关关系，组间大多呈现显著的负相关关系，说明黄龙沟兰科植物在资源利用方式上可能产生了分化。 黄龙沟兰科植物主要分布在两种生境中，即钙化滩流地和一小片森林中。在这两种生境中微环境条件以及兰科植物的种类组成、数量特征和分布格局的差异都很大。钙化滩流地的兰科植物种类数目和每个样方中包含的兰科植物种类数目都比森林生境中的高。钙化滩流地中发现有30种兰科植物，六种最为常见的是无苞杓兰、黄花杓兰、西藏杓兰、广布小碟兰、二叶根茎兰和少花鹤顶兰，它们的密度与树木盖度呈负相关关系。森林生境中有21种兰科植物分布，其中筒距兰和斑叶兰出现最多，其密度与树木盖度呈正相关关系。光照强度可能对兰科植物的分布起到重要的决定作用。兰科植物彼此之间对繁殖成功以及植株间建立的相互促进作用以及土壤中高的钙含量很可能是形成黄龙沟兰科植物多样性的重要原因，而钙化滩流地中的溪流对建立和维持兰科植物生存所必须的稳定环境条件起到至关重要的作用，如果随意改变水流的方向或减少水流的流量，将给某些兰科植物带来灾难性的后果。 黄龙沟钙化滩流地中斑块的大小对物种数（含兰科植物）具有强烈的影响，即斑块越大，所包含的物种数量越多。物种-面积关系符合幂函数方程S=cAZ的规律。中等尺度的斑块(1-10 m2)包含的物种数（含兰科植物）的增长速度最快，而在斑块面积大于10 m2时，物种数增长速度最小。 少花鹤顶兰是多年生、多次结实的具有克隆能力的地生兰科植物，是中国特有种。黄龙沟少花鹤顶兰每个花序的平均花朵数为3(1-7)朵。叶和花的形态指标在年季间无显著变异。2005-2007年3年间的开花物候没有明显的差异，花期始于6月中旬，于7月底至8月初结束，持续约6-7周，80-90%的花在7月初的开花高峰期开放，属花集中开放模式。花寿命与是否授粉密切相关，成功授粉的花寿命比没有授粉的花寿命短。少花鹤顶兰是自交亲和的，但其结实完全依赖于两种熊蜂的传粉，不存在自动自花授粉和无融合生殖现象。柱头可授性和花粉活力可维持15天左右。繁殖成功率年季间的变化很大，并表现出逐年下降的趋势，花粉移走率为18-51%，自然结实率为10-36%。繁殖成功与开花物候之间的关系不显著，但与某一特定时刻的开花数量（密度）有关。生境对少花鹤顶兰的形态和繁殖成功率有显著的影响。在荫蔽生境中的个体比开阔地中的个体大，但繁殖成功率却低于开阔地的。花序密度（丛的大小）对少花鹤顶兰繁殖成功的影响比较复杂，不同大小的丛，对花粉移走率的影响格局和方式不同，而对结实率几乎没有影响。花序大小对繁殖成功率没有显著的影响。花位置对繁殖成功的影响在不同大小的丛中具有不同的格局，在最小的丛中，花位置对雄性和雌性繁殖成功的影响都很显著，但在最大丛中都不显著。边缘效应对少花鹤顶兰和广布小碟兰的繁殖成功有显著的影响，花粉移走率和结实率均随离栈道的距离增加而增加。在离栈道10米以内的植株受到的影响最为严重。这种影响可能是由边缘效应和在栈道上的游客共同作用下，对传粉者产生了干扰，导致传粉者的行为改变所引起的。如果这种边缘效应是一个普遍现象的话，那么在进行自然保护区设计时，应该对这种效应加以重视。

鄂尔多斯高原景观要素空间格局及植物与环境关系的定量解析

鄂尔多斯高原是一个多层次、复杂的生态过渡带，具有复杂多样的环境条件、生态特点，因此也就具有复杂多样的植物与环境关系。本文从群落和景观两个尺度水平上研究鄂尔多斯高原植物或植被与环境关系及景观空间格局。利用鄂尔多斯高原野外植物群落样方调坦数据、微生境环境数据、气候数据，以典范对应分析（Canonical Correspondence Analysis, CCA）的方法分析了鄂尔多斯高原植物分布空间格局与环境要素的关系并对秋类环境要素对鄂尔多斯高原植物空间分布格局的贡献进行了定量分解;利用1:500 000 鄂尔多斯高原植被、土壤、土地利用、土地沙漠化类型等专题地图在GIS支持下分析了鄂尔多斯高原景观空间格局、并利用上述专题图数据加上鄂尔多斯高原气候数据库分析了土壤、土地利用、土地利用、土地沙漠化和气候等对鄂尔多斯高原植被空间分布格局的作用。通过分析，得到了以下主要结论： 1 在分析方法上、利用典范对应分析的方法，把植物分布的空间因素与环境因素分离的方法发展为植物分布空间格局不同类型影响因素作用的定量分离，提出了相应的概念模型和实现方法。 2 影响鄂尔多斯高原植物分布空间格局的主要微生境环境要素是基质类型、地下水位、覆沙厚度等，而影响鄂尔多斯高原分布空间格局的主要气候要素中，降水和干湿指标的作用大于温度和热量指标的作用。 3 通过对鄂尔多斯高原植物分布空间格局与环境关系的研究，以植物对微生境环境要素的反应为根据，把鄂尔多斯高原主要植物划分为4个大类群：梁地植物、沙地植物、草甸植物和耐盐植物。根据它们对气候要素的反应，把鄂尔多斯高原主要植物划分为典型草原植物、荒漠草原植物和草原化荒漠植物3大类。进一步，根据鄂尔多劳动保护高原植物与环境关系的研究，进行了鄂尔多斯高原植被功能型划分的尝试，得到了鄂尔多斯高原的12种主要植被功能型。 4 对鄂尔多斯高原植物分布的空间格局的影响环境因素的贡献作了定量地分解。分析结果显示：鄂尔多斯高原植物分布空间格局中有27.02％可由已知环境变量得到解释，其中21.56％与微生境环境要素相关，7.51％与气候要素的作用有关，而气候与微生境环境要素的耦合作用的份额为2.05％。根据植物生长是否直接受到地下水的影响，鄂尔多斯高原存在两大类型生态特点差异明显的生境类型：中性立地和隐域生境，对两大类型生境上影响植物空间分布格局的环境要素的作用也进行了定量分解。分析结果表明：对于植物生长不直接受地下水影响的中性立地，已知环境要素的作用可以解释植物空间分布格局总信息的29.36％，稍大于对总体上鄂尔多斯高原植物分布空间格局的解释，其中9.23％与气候要素相关，22.08％与微生境环境要素相关，而两种类型环境要素的耦合作用则占1.95％。对于植物生长直接受到地下水影响的隐域生境，所有已知环境要素对植物分布空间格局的贡献率为72.28％，其中气候要素的作用为30.31％，微生境环境要素的作用为49.08％，两类环境要素的耦合作用为7.11％。 5 描述景观空间格局的指数多种多样，这些能数在描述特定区域的景观空间格局时是有信息冗余的。本文对利用FRAGSTATS所获得的鄂尔多斯高原植被、土壤、土地利用、土地沙漠化等景观分量的20个景观指数实施了因子分析。通过因子分析，我们可以把描述鄂尔多斯高原景观空间格局的景观指数归并为以下8类：多样性指数、斑块多度指数、斑块类型丰富度指数、斑块面积指数、斑块形状指数、分形维数、空间配置指数和斑块面积变异指数。通过因子分析，还得到了这些景观指数对描述鄂尔多斯景观格局的共性特征：在描述鄂尔多斯高原景观空间格局时，作用最大的是多样性指数、斑块多度指数、面积加权平均斑块形状指数和面积加权平均分形维数，其次是斑块类型丰富度指数、平均分维指数、平均形状指数和斑块面积指数，而空间配置指数（扩散与毗连指数）和斑块面积变异指数的作用则比较微弱。 6 对鄂尔多斯高原景观指数的因子分析是非常有效和成功的。因子分析对鄂尔多斯高原植被、土壤、土地利用、土地沙漠化景观指数的分析分别得到了5-6个主要因子，可以表达原有20个景观指数所表达信息的91.1-96.0％，即可以反应鄂尔多斯高原景观空间格局的大部分信息。本文所进行的因子分析对因子进行了方差最大化(Varimax)正交旋转的处理，因子分析得到的每一个主要因子都有一个或几个与之相关性非常高的景观指数与之对应，因此，就可以用与因子分析所得主要因子相关性最高的景观指数代替该主要因子来表达鄂尔多斯高原的景观空间格局。另外还因为有些景观指数之间具有极高的相关系数，所以对因子分析所得到的景观指数可以进一步精减，最后利用因子分析成功地把原有20个景观指数减少到了11个。最后被选来描述鄂尔多斯高原景观格局的景观指数有下列11个：MSIEI（修正的Simpson均匀度指数）、AWMPFD（面积加权平均斑块分形维数）、AWMSI（面积加权平均形状指数）、NP（斑块数目）、PR（斑块类型丰富度）、MSI（平均形状指数）、MPFD（平均斑块分形维数）、MPS（平均斑块面积）、PSCV（斑块面积变异系数）、DLFD（双对数分形维数）和IJI（扩散与毗连指数）。 7 鄂尔多斯高原植被、土壤、土地利用在景观组成结构上具有一个共同特点，就是各种类型的面积差异极大，少数类型占有极大比重，而其余面积则很小。产生这一情形的原因主要与人为活动的强烈影响有关，表现在地带性的植被与土壤面积所占的比重不高，沙地、沙生植被与风沙土则占有很大比重。 8 以地带性植被和滩地隐域性植被表示的鄂尔多斯高原的原生植被仅占高原面积的不足30％，而以地带性土壤和滩地隐域性土壤表示的原生性土壤占鄂尔多斯高原总面积的近40％，说明土壤退化不如植被退化严重，或滞后于植被退化。 9 鄂尔多斯高原各景观指数的空间变化曲线，植被与土壤很相近，具有非常相似的格局;土地利用景观格局空间变化特征与植被、土壤等明显不同;土地沙漠化的景观格局空间变化曲线介植被曲线、土壤曲线与土地利用曲线之间，说明土地沙漠化不仅是一个受人为活动影响的过程，而且与自然过程密切相关。 10 鄂尔多斯高原景观格局的空间梯度变化表现出了东西向和南北向的梯度，但总体上以东西向的变化比较明显。 11 通过鄂尔多斯高原土壤类型、土地利用、土地沙漠化等景观要素、气候、空间要素与鄂尔多斯高原植被空间分布格局的CCA分析，探讨了它们之间的相互关系。以对鄂尔多斯高原植被组成数据的总方差解释的百分率为标准，土壤对鄂尔多斯植被分布的空间格局的作用最大，其方差贡献率可达44.28％，其次是土地利用与鄂尔多斯高原植被的关系也很密切，土地利用对鄂尔多斯高原植被空间分布格局的方差贡献率为22.45％，空间因素对鄂尔多斯高原植被空间分布格局的贡献率为17.51％，土地沙漠化对鄂尔多斯高原植被空间分布格局的贡献为15.65％，排在第四位;气候因素对鄂尔多斯高原植被空间格局的贡献率为11.95％，居第五位。 12 在气候要素对鄂尔多斯高原植被空间分布格局的作用中，降水与干湿指标的作用大于温度与热量指标的作用。这一点与利用野外调查样方的群落数据植物与气候关系的分析是完全一致的。CCA分析还表明鄂尔多斯高原植被空间格局的东西向变化大于南北向分异。 13 在群落和景观水平上，鄂尔多斯高原植物空间分布或植被格局的影响因素的作用具有相似的格局，即气候因子的作用明显地小于地质、土壤、水文等微生境环境要素（群落水平）或土壤（景观水平）的作用，并在这两个尺度上气候要素对植物空间分布或植被格局的定量解释份额上也是非常相近的，都仅有10％左右。气候因子对鄂尔多斯高原植物空间分布格局的这种弱的解释能力，从侧面说明了人为活动等非自然因素对鄂尔多斯高原植物空间分布格局的强烈作用。 14 在鄂尔多斯高原生态系统管理上，应协调人与自然的关系;加强鄂尔多斯高原的生物多样性保育，对于本区生态和经济对非常重要的滩地，应协调好对其开发利用与保护的关系;在鄂尔多斯高原土地沙漠化防治方面，应把调整人地关系与自然生态背景与条件相结合，如使用“三圈”模式等生态系统管理模式等。

CO2浓度升高对三种树木叶片化学和舞毒蛾幼虫取食及生长的影响

探讨全球气候变化的生物学和生态学效应是当今生态学中的热点，研究大气CO2浓度升高对植物-昆虫相互作用关系的影响具有重要的理论和实践意义。本文使用开顶式气室（Open-top chamber，OTC）在野外条件下研究了CO2浓度升高对三种树木（小青杨、白桦和蒙古栎）叶片化学成分含量的影响，以及树木叶片品质变化对一种广食性森林昆虫（舞毒蛾）幼虫取食、生长发育和取食偏嗜性的影响。得出如下结果：（1）CO2浓度升高对3个受试树种叶片中的营养成分及次生代谢物含量均有显著影响，总体表现为氮含量降低，而碳氮比、非结构性碳水化合物、总酚和缩合丹宁含量增加。叶片中的化学成分含量可随时间发生显著变化，不同树种、甚至同一树种不同冠层高度的叶片对CO2浓度升高的响应强度也是不同的。叶片的干物质含量和比叶重对CO2浓度升高的响应不显著。（2）室内非选择性取食实验、室内选择性取食实验以及上树取食饲养方式下的多龄期取食实验，均发现高浓度CO2处理组内舞毒蛾幼虫的生长发育受到显著抑制。但对四龄舞毒蛾幼虫所进行的短期生物测定并未发现不同CO2浓度处理下幼虫的生长发育速率、对食物的取食率和转化率等昆虫营养指标存在显著差异。（3）叶片品质的降低是导致舞毒蛾幼虫生长发育受抑制的主要原因。但是总体上，CO2浓度升高导致的叶片品质变化并未显著影响幼虫的取食率和取食量。（4）舞毒蛾幼虫对不同叶片种类表现出清晰的取食选择性，这种选择性在其幼龄期就可表现出来。幼虫对小青杨上层叶片有最显著的偏嗜性，对蒙古栎下层叶片有最明显的拒食性。但是CO2浓度升高导致的叶片品质变化对舞毒蛾幼虫的取食选择性和寄主偏嗜行为并未产生显著影响。（5）检测出高浓度CO2处理组内舞毒蛾幼虫虫粪中含有浓度更高的植物次生代谢物质（总酚和缩合单宁），这很可能是昆虫整体生长发育受抑制的重要原因之一。

Do leaves of plants on phosphorus-impoverished soils contain high concentrations of phenolic defence compounds?

Prominent theories of plant defence have predicted that plants growing on nutrient-poor soils produce more phenolic defence compounds than those on richer soils. Only recently has the Protein Competition Model (PCM) of phenolic allocation suggested that N and P limitation could have different effects because the nutrients are involved in different cellular metabolic processes. 2. We extend the prediction of the PCM and hypothesize that N will have a greater influence on the production of phenolic defensive compounds than P availability, because N limitation reduces protein production and thus competition for phenylalanine, a precursor of many phenolic compounds. In contrast, P acts as a recyclable cofactor in these reactions, allowing protein and hence phenolic production to continue under low P conditions. 3. We test this hypothesis by comparing the foliar concentrations of phenolic compounds in (i) phenotypes of 21 species growing on P-rich alluvial terraces and P-depleted marine terraces in southern New Zealand, and (ii) 87 species growing under similar climates on comparatively P-rich soils in New Zealand vs. P-depleted soils in Tasmania. 4. Foliar P concentrations of plants from the marine terraces were about half those of plants from alluvial soils, and much lower in Tasmania than in New Zealand. However, foliar concentrations of N and phenolic compounds were similar across sites in both comparisons, supporting the hypothesis that N availability is a more important determinant of plant investment in phenolic defensive compounds than P availability. We found no indication that reduced soil P levels influenced plant concentrations of phenolic compounds. There was wide variation in the foliar N and P concentrations among species, and those with low foliar nutrient concentrations produced more phenolics (including condensed tannins). 5. Our study is the first trait comparison extending beyond standard leaf economics to include secondary metabolites related to defence in forest plants, and emphasizes that N and P have different influences on the production of phenolic defence compounds. © 2009 British Ecological Society.

Impacts of culling and exclusion of browsers on vegetation recovery across New Zealand forests

Introduced browsing animals negatively impact New Zealand's indigenous ecosystems. Eradicating introduced browsers is currently unfeasible at large scales, but culling since the 1960s has successfully reduced populations to a fraction of their earlier sizes. Here we ask whether culling of ungulates has allowed populations of woody plant species to recover across New Zealand forests. Using 73 pairs of permanent fenced exclosure and unfenced control plots, we found rapid increases in sapling densities within exclosures located in disturbed forests, particularly if a seedling bank was already present. Recovery was slower in thinning stands and hampered by dense fern cover. We inferred ungulate diet preference from species recovery rates inside exclosures to test whether culling increased abundance of preferred species across a national network of 574 unfenced permanent forest plots. Across this network, saplings were observed irrespective of their preference to ungulates in the 1970s, but preferred species were rarer within disturbed sites in the 1990s after long-term culling and despite nationwide increases in sapling densities. This indicates that preferred species are relatively heavily affected by browsing after culling, presumably because remaining animals will increase consumption of preferred species as competition is reduced. Our results clearly suggest that culling will not return preferred plants to the landscape immediately, even given suitable conditions for regeneration. Complete removal of ungulates rather than simply reducing their densities may be required for recovery in heavily browsed temperate forests, but since this is only feasible at small spatial scales, management efforts must target sites of high conservation value. © 2012 Elsevier Ltd.

Microbe-mediated recovery of salt marshes contaminated with oil hydrocarbons

Salt marshes are highly productive intertidal habitats that serve as nursery grounds for many commercially and economically important species. Because of their location and physical and biological characteristics, salt marshes are considered to be particularly vulnerable to anthropogenic inputs of oil hydrocarbons. Sediment contamination with oil is especially dangerous for salt marsh vegetation, since low molecular weight aromatic hydrocarbons can affect plants at all stages of development. However, the use of vegetation for bioremediation (phytoremediation), by removal or sequestration of contaminants, has been intensively studied. Phytoremediation is an efficient, inexpensive and environmental friendly approach for the removal of aromatic hydrocarbons, through direct incorporation by the plant and by the intervention of degrading microbial populations in the rhizosphere (microbe-assisted phytoremediation). Rhizosphere microbial communities are enriched in important catabolic genotypes for degradation of oil hydrocarbons (OH) which may have a potential for detoxification of the sediment surrounding the roots. In addition, since rhizosphere bacterial populations may also internalize into plant tissues (endophytes), rhizocompetent AH degrading populations may be important for in planta AH degradation and detoxification. The present study involved field work and microcosms experiments aiming the characterization of relevant plant-microbe interactions in oilimpacted salt marshes and the understanding of the effect of rhizosphere and endosphere bacteria in the role of salt marsh plants as potential phytoremediation agents. In the field approach, molecular tools were used to assess how plant species- and OH pollution affect sediment bacterial composition [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis subsp. perennis] in a temperate estuary (Ria de Aveiro, Portugal) chronically exposed to OH pollution. In addition, the 16S rRNA gene sequences retrieved in this study were used to generate in silico metagenomes and to evaluate the distribution of potential bacterial traits in different microhabitats. Moreover, a combination of culture-dependent and -independent approaches was used to investigate the effect of oil hydrocarbons contamination on the structure and function of endophytic bacterial communities of salt marsh plants.Root systems of H. portulacoides and S. perennis subsp. perennis appear to be able to exert a strong influence on bacterial composition and in silico metagenome analysis showed enrichment of genes involved in the process of polycyclic aromatic hydrocarbon (PAH) degradation in the rhizosphere of halophyte plants. The culturable fraction of endophytic degraders was essentially closely related to known OH-degrading Pseudomonas species and endophytic communities revealed sitespecific effects related to the level of OH contamination in the sediment. In order to determine the effects of oil contamination on plant condition and on the responses in terms of structure and function of the bacterial community associated with plant roots (rhizosphere, endosphere), a microcosms approach was set up. The salt marsh plant Halimione portulacoides was inoculated with a previous isolated Pseudomonas sp. endophytic degrader and the 2-methylnaphthalene was used as model PAH contaminant. The results showed that H. portulacoides health and growth were not affected by the contamination with the tested concentration. Moreover, the decrease of 2-methylnaphthalene at the end of experiment, can suggest that H. portulacoides can be considered as a potential plant for future uses in phytoremedition approaches of contaminated salt marsh. The acceleration of hydrocarbon degradation by inoculation of the plants with the hydrocarbon-degrading Pseudomonas sp. could not, however, be demonstrated, although the effects of inoculation on the structure of the endophytic community observed at the end of the experiment indicate that the strain may be an efficient colonizer of H. portulacoides roots. The results obtained in this work suggest that H. portulacoides tolerates moderate concentrations of 2-methylnaphthalene and can be regarded as a promising agent for phytoremedition approaches in salt marshes contaminated with oil hydrocarbons. Plant/microbe interactions may have an important role in the degradation process, as plants support a diverse endophytic bacterial community, enriched in genetic factors (genes and plasmids) for hydrocarbon degradation.

Micromorphology of the labellum and structure of the osmophore in a group of closely related species of the sexually deceptive Orchid genus Ophrys (orchidaceae)

Tese de doutoramento, Biologia (Biologia Celular), Universidade de Lisboa, Faculdade de Ciências, 2016

The fate and toxicity of the flavonoids naringenin and formononetin in soil

The flavonoid class of plant secondary metabolites play a multifunctional role in below-ground plant-microbe interactions with their best known function as signals in the nitrogen fixing legume-rhizobia symbiosis. Flavonoids enter rhizosphere soil as a result of root exudation and senescence but little is known about their subsequent fate or impacts on microbial activity. Therefore, the present study examined the sorptive behaviour, biodegradation and impact on dehydrogenase activity (as determined by iodonitrotetrazolium chloride reduction) of the flavonoids naringenin and formononetin in soil. Organic carbon normalised partition coefficients, log K-oc, of 3.12 (formononetin) and 3.19 (naringenin) were estimated from sorption isotherms and, after comparison with literature log K-oc values for compounds whose soil behaviour is better characterised, the test flavonoids were deemed to be moderately sorbed. Naringenin (spiked at 50 mu g g(-1)) was biodegraded without a detectable lag phase with concentrations reduced to 0.13 +/- 0.01 mu g g(-1) at the end of the 96 h time course. Biodegradation of formononetin proceeded after a lag phase of similar to 24 with concentrations reduced to 4.5 +/- 1% of the sterile control after 72 h. Most probable number (MPN) analysis revealed that prior to the addition of flavonoids, the soil contained 5.4 x 10(6) MPNg(-1) (naringenin) and 7.9 x 10(5) MPNg(-1) (formononetin) catabolic microbes. Formononetin concentration had no significant (p > 0.05) effect on soil dehydrogenase activity, whereas naringenin concentration had an overall but non-systematic impact (p = 0.045). These results are discussed with reference to likely total and bioavailable concentrations of flavonoids experienced by microbes in the rhizosphere. (c) 2007 Elsevier Ltd. All rights reserved.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Conservation ecology of bees: populations, species and communities

Recent concerns regarding the decline of plant and pollinator species, and the impact on ecosystem functioning, has focused attention on the local and global threats to bee diversity. As evidence for bee declines is now accumulating from over broad taxonomic and geographic scales, we review the role of ecology in bee conservation at the levels of species, populations and communities. Bee populations and communities are typified by considerable spatiotemporal variation; whereby autecological traits, population size and growth rate, and plant-pollinator network architecture all play a role in their vulnerability to extinction. As contemporary insect conservation management is broadly based on species- and habitat-targeted approaches, ecological data will be central to integrating management strategies into a broader, landscape scale of dynamic, interconnected habitats capable of delivering bee conservation in the context of global environmental change.

Measuring bee diversity in different European habitats and biogeographical regions

Bee pollinators are currently recorded with many different sampling methods. However, the relative performances of these methods have not been systematically evaluated and compared. In response to the strong need to record ongoing shifts in pollinator diversity and abundance, global and regional pollinator initiatives must adopt standardized sampling protocols when developing large-scale and long-term monitoring schemes. We systematically evaluated the performance of six sampling methods (observation plots, pan traps, standardized and variable transect walks, trap nests with reed internodes or paper tubes) that are commonly used across a wide range of geographical regions in Europe and in two habitat types (agricultural and seminatural). We focused on bees since they represent the most important pollinator group worldwide. Several characteristics of the methods were considered in order to evaluate their performance in assessing bee diversity: sample coverage, observed species richness, species richness estimators, collector biases (identified by subunit-based rarefaction curves), species composition of the samples, and the indication of overall bee species richness (estimated from combined total samples). The most efficient method in all geographical regions, in both the agricultural and seminatural habitats, was the pan trap method. It had the highest sample coverage, collected the highest number of species, showed negligible collector bias, detected similar species as the transect methods, and was the best indicator of overall bee species richness. The transect methods were also relatively efficient, but they had a significant collector bias. The observation plots showed poor performance. As trap nests are restricted to cavity-nesting bee species, they had a naturally low sample coverage. However, both trap nest types detected additional species that were not recorded by any of the other methods. For large-scale and long-term monitoring schemes with surveyors with different experience levels, we recommend pan traps as the most efficient, unbiased, and cost-effective method for sampling bee diversity. Trap nests with reed internodes could be used as a complementary sampling method to maximize the numbers of collected species. Transect walks are the principal method for detailed studies focusing on plant-pollinator associations. Moreover, they can be used in monitoring schemes after training the surveyors to standardize their collection skills.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.