【作者】 李艳伟；

【导师】 黄大卫；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 榕树(桑科:榕属)与榕小蜂(昆虫纲:膜翅目:小蜂总科)在形态学、生理学和行为学上的协同适应使其成为协同进化研究的经典模型。根据是否为榕树传粉可以将榕小蜂分为传粉榕小蜂和非传粉榕小蜂。榕树为榕小蜂提供食物和栖息场所来保证小蜂的生长、发育及繁衍后代,同样传粉榕小蜂又为榕树传粉以保证其有性繁殖,形成了典型的互惠共生现象。通常情况下,榕树与传粉榕小蜂之间是专性共生关系。但是随着大量分子研究的深入,逐步瓦解了这种严格意义上的“一对一”关系。同样,作为榕树——榕小蜂系统中的重要组成部分——非传粉榕小蜂也受到越来越多的关注,其寄主专一性问题也成为热点。然而,关于榕树与榕小蜂关系的大量研究的进行,都必需建立在准确有效的分类系统之上。令人堪忧的是,榕小蜂的分类面临着传统分类难以逾越的难题。绝大部分榕小蜂种存在极端的雌雄异形和雄性多型现象,使仅依靠形态的分类很难正确鉴定这些小蜂的身份,更不用说进一步解决种内雌雄配对问题。除此之外,通常非传粉榕小蜂的几个亚科或属的物种同时寄生一种榕树,使整个系统更加复杂。有时几种形态上相似的近缘种共存于同一种榕果,以及隐蔽的交配行为等都大大加剧了榕小蜂(特别是非传粉榕小蜂)物种鉴定和同种雌雄配对的难度。这也是非传粉榕小蜂分类研究相对滞后的原因之一。这些榕小蜂的特殊现象直击传统分类的弱点,让我们不得不寻求新的有效的方法来解决问题,同时也给我们提供了很好的机会来探讨分类学上令人头痛的问题。因此,榕小蜂分类系统急需结合分子手段来帮助传统分类的发展。2003年,由Hebert等提出的DNA条形码技术,根据标准基因序列(线粒体基因COI)的种间差异大于种内差异的原理实现准确快速的物种鉴定,并且在昆虫类、鱼类、鸟类、贝类、植物、菌类等物种中得到了较好地应用。目前,COI基因在动物中的广泛使用,不仅快速补充了已命名物种的分子数据,而且加速了新物种的发现;COI序列甚至有时成为至关重要不可比拟的“分类特征”,并在一些类群中发现了大量的隐存种,让分类学家、生态学家需要重新估计生物多样性。尽管DNA条形码也受到年轻种、近缘种、祖先多型现象、Wolbachia感染等多个因素的制约,但大量的事实证明它的鉴定能力和效果是不容忽视的。目前世界范围内,对传粉榕小蜂的研究较为深入;相比较而言,非传粉榕小蜂的研究只是刚刚开始。尤其是中国,小蜂的研究更是刚刚起步,目前为止还没有系统地将中国境内的榕小蜂进行大规模的形态分类及分子鉴定的研究。本研究选择了来自中国南方5个省市的雌雄同株和雌雄异株的榕树共38种上生活的约388个临时形态种共2256个标本进行试验。预计解决榕小蜂的身份鉴定、种内雌雄配对、寄主专一性、COI基因多样性格局等方面的问题。结果显示:DNA条形码能成功将榕小蜂鉴定到种级水平,共得到189个种,并解决95%以上榕小蜂雌雄配对问题,大于一半以上的榕小蜂种内差异小于2%。未解决的种主要表现在:本身未采集到的同种雌虫或雄虫,同一个形态种出现2个及以上的单体群。形态数据和分子数据存在约90%的一致性,包括需要改正的形态鉴定错误和不确定的需要增加基因的分子数据。同时发现:中国榕小蜂隐存种的发生主要由于地理差异而产生,传粉榕小蜂产生的几率高于非传粉榕小蜂。由地理差异产生的两种传粉榕小蜂和同域物种形成的两种传粉榕小蜂为一种榕树传粉的现象打破了“一对一”理论。本研究首次发现了非传粉榕小蜂中Walkerella, Philotrypesis, Sycoscapter三个属,种级水平寄生不同榕树种,部分种存在寄主转移现象。DNA条形码应用于榕小蜂时需要强调的问题有:结合通用引物和特异小蜂引物共同使用避免非特异性扩增。注意结合榕小蜂寄主榕树的分类系统,利于检测榕树及榕小蜂中出现的分类错误。面对一些不确定因素必要时应采用多个基因联合分析。作为非传粉榕小蜂的一种,Sycophila属具有广泛的寄主偏好性,同时与竹子、橡树、草莓和榕树等植物相关。本文则研究一些与榕树密切相关的Sycophila种。尽管一些报道显示Sycophila没有很高的寄主专一性,但是不同种雌虫的形态特征差别微小,令我们很难确定它们是否是一个种寄生多个寄主。尽管关于Sycophila与其多样寄主的关系问题的研究非常稀少,但是Sycophila丰富的物种多样性和广泛的寄主范围都使其成为研究物种形成和寄主专一性的理想系统。但是由于雌雄异型、形态的可塑性和贫瘠的生物学知识都使Sycophila的鉴定及雌雄配对都面临非常大的挑战,更不用说进一步的寄主专一性等问题的探讨。本研究通过选取线粒体基因COI和核基因ITS2来共同鉴定来自中国海南六种榕树上154个Sycophila属的标本。研究的主要目的是区分单个榕树种上的Sycophila种,并进一步确定来自中国南方六个雌雄同株榕树上寄主专一性问题。结果显示:25个形态临时种被分为15种,其中13个种具有寄主专一性,而另外2个种同时寄生两种寄主。四种榕树上分别只有1个Sycophila种寄生,而垂叶榕和小叶榕上则分别同时寄生6个Sycophila种。分子数据显示三个雄虫形态型属于同一种。并证明COI和ITS2是在对Sycophila进行物种鉴定时起到了相同的作用,解决了形态学的可塑性问题。更多还原

【Abstract】 Figs (Moraceae, Ficus) and pollinating wasps (Hymenoptera, Agaoninae) are usually considered as the classic model of co-evolution due to their morphology, physiology and behavioural co-adaptation. According to whether pollinate figs, fig wasps can be divided into pollinating wasps and non-pollinating wasps. Pollinator wasps pollinate the figs, and figs provide the resources and habits for fig wasps’development, that is the well-known mutualism relationship. In most cases, each fig tree species is pollinated by one special pollinator wasp species, however, more and more studies has ruined the strict“one-to-one”rule which make the mutualism system more complex. Similarly, non-pollinators, major components of the figs-wasps system, attract much attention, especially for their host specificity.As we know, all kinds of researches on fig and fig wasps must be base on correct and valid taxonomy studies. However, the taxonomy of fig wasps faces the big problem such as extreme sexual dimorphism and male polymorphism which are not easily fixed just on morphological character. In addition, the fact that many species of non-pollinating wasps, from various subfamilies or families, exist in one fig species complicates the fig wasp system. All these extraordinary phenomena hinder scientists from correctly recognizing the different species and matching sexual individuals of cospecies, especially for non-pollinating wasps. Therefore, it is urgent to combine effective molecular method and morphology to fix the problem of fig wasps taxonomy system.Paul (2003) raised the point that we should choose one standard gene fragment as new“taxonomy character”to help traditional taxonomy. The mtDNA COI fragment is a core gene employed by DNA barcoding initiatives and has the potential to facilitate both the identification of known species and the discovery of new ones. Although there are some limitations like young species, close species, ancient polymorphism and Wolbachia infection, many studies have demonstrated that DNA barcodes are effective in diagnosing most species, such as insects, fishes, birds, plants etc.In this study, we DNA barcoded 2256 wasp samples belonging to 24 genera of 7 subfamilies and 6 families reared from 38 different fig species in 5 provinces of Southern China. We asked whether COI barcode could correctly distinguish species, match sexual individuals of cospecies with extreme dimorphism, identify host-specificity etc. We got 189 species out of 388 provisional morphology morphs by gene sequences, and 95% sexual female individuals match male individuals. Barcoding analysis revealed that the cryptic species of Chinese fig wasps mostly due to geographic area differences, pollinator wasps have more geographic cryptic species than non-pollinator wasps. Moreover, two geographic pollinators pollinate one fig species and two sympatric pollinators exist in one fig species respectively were found. Some species from non-pollinator genera, Walkerella, Philotrypesis, Sycoscapter, can attack several fig species, which indicating host-switch for these wasp species. In addition, it is necessary to combine common primer and specific primer to avoid contaminative species gene amplification in DNA bacoding program.Although the genus Sycophila has broad host preferences, some species are specifically associated with figs as non-pollinator wasps. Because of their sexual dimorphism, morphological plasticity, cryptic mating behaviour and poorly known biology, species identifications are often uncertain. It is particularly difficult to match con-specific females and males. Although some studies have suggested that Sycophila are not highly host-specific, the morphological characters differentiating females are very subtle, making it difficult to be certain that a single species uses multiple hosts. Nevertheless, its high species diversity and broad host use make Sycophila an excellent potential model system for the investigation of speciation patterns and host specificity. In this study, we employed two molecular markers, mitochondrial COI and nuclear ITS2, to identify Sycophila from six Chinese fig species. The primary goal of this study was to clarify the diversity of Sycophila species on single fig species and the extent of host specialization in the community found in association with six monoecious Ficus species in southern China. Morphological studies revealed 25 female and male morphs, while sequence results for both genes were consistent in supporting the presence of 15 species, of which 13 were host specialists and two used dual hosts. A single species of Sycophila was respectively found on four fig species, but six species were isolated from Ficus benjamina and a same number was reared from Ficus microcarpa. Sequence results revealed three male morphs in one species and detected two species that were overlooked by morphological analysis.更多还原