【作者】 杨春燕；

【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， 2012， 博士

【摘要】 榕树-榕小蜂是典型的协同进化系统,是研究物种形成,种群遗传与动态,适应性进化,共生体系协调稳定机制的理想材料。榕果(隐头花序)是榕小蜂生存的场所。垂叶榕果是多花层的,生态位丰富,小蜂物种多样。果内小蜂存在共取食和寄生关系,生态关系非常密切。果腔狭小,雄虫打斗激烈,存在多种交配和打斗机制。与此同时,榕果提供一个密闭的系统,榕小蜂从受精卵到成虫的发育过程完全在榕果内进行,只有很少的机会与外界接触,这与自然界中绝大多数的昆虫与植物都处于开放或半开放系统中不同。紧密的生态学关系,相对隔绝的生存环境使垂叶榕果成为一个独特而理想的实验材料。Wolbachia作为一种内共生菌,具有生殖调控作用,一般认为是垂直传播的母系遗传。它感染约上百万种昆虫,其感染的广泛性和引起的作用不容忽视。之前的研究发现膜翅目有较高的Wolbachia感染率,而榕小蜂更是如此。那么垂叶榕果内小蜂Wolbachia的感染状况是怎样的呢?特殊的密闭榕果环境中,是否与开放环境中Wolbachia的感染格局不同?系统内是否因为紧密的生态学联系有更多的水平传播现象?Wolbachia是如何传入密闭榕果环境的?Wolbachia感染与宿主所处的密闭环境和紧密的生态学关系相结合,使这些问题变得十分有趣。通过对垂叶榕果内4科、8属、17种、870头小蜂个体的Wolbachia感染状况研究发现：1)垂叶榕密闭榕果系统内榕小蜂Wolbachia感染率很高；2)果内榕小蜂感染wsp序列具有高重组现象；3)Wolbachia在果内多水平传播；4)结合垂叶榕小蜂生态学和水平传播状况的研究,我们发现广泛存在的非重组序列由果外传入,而有翅型雄性与雌虫的果外交配是可能的传入途径。在垂叶榕小蜂Wolbachia感染研究中发现,榕果内不同的物种感染状态是不同的。有的物种感染单一wsp序列,浓度高,种内感染率高；有的物种感染多种wsp序列,浓度低,种内感染率适中或较低,这就形成了两种极端不同的感染模式。不同感染模式形成的原因十分值得思考。Wolbachia感染格局(包括浓度、株系、感染率)的形成,不仅与Wolbachia株系本身特点有关,还与感染的宿主,及宿主的生存环境有关。之前关于感染状态的研究,大多集中在感染率,感染株系及株系传播途径的推断上,很少从宿主角度对感染格局的形成原因进行研究。本研究从宿主系统发生,物种形成时间,宿主群落特点等方面对Wolbachia感染格局的形成进行分析,帮助我们全面深入了解榕小蜂Wolbachia的感染格局。经过垂叶榕小蜂感染调查,我们发现了两种典型的感染格局,即高浓度、单株系、高感染率格局和低浓度、多株系和低感染率格局。为了深入了解这种感染格局形成的原因,我们选取了垂叶榕Acophila, Walkerella, Sycoscapter和它们的近缘物种进行研究。又联合本实验室之前在中国榕小蜂DNA条形码研究中偶然扩增的Wolbachia的coxA在宿主中的分布情况分析发现：1)亲缘关系近,感染状态不一定相似,感染具有一定的随机性；2)物种形成年代久远,易形成高浓度单株系的稳定感染,例如造瘿小蜂物种间分化时间长,一旦感染易形成单一株系感染；3)物种形成事件频繁的寄生性榕小蜂,对于多株系感染的忍耐力强,一旦感染容易形成多株系、低浓度感染格局；4)雌雄异株小蜂因为更多的近缘交配,更大的窝数,更低的性比,更少的有翅雄蜂种类,利于形成Wolbachia单一株系、高浓度、高感染率的稳定感染状态。本部分研究,有助于我们理解小蜂感染格局的形成原因,预测小蜂的感染状况,寻找Wolbachia与宿主相互作用的实验材料组,进行下一步试验。榕小蜂有很高的物种丰富度,总量很大,近缘种也很多,姊妹种和隐存种现象较为多见。榕小蜂具有高的Wolbachia感染率,对宿主具有生殖调控作用,尤其是其双向胞质不亲和作用,很可能导致物种的形成。那么,某些榕小蜂活跃的物种形成是否与Wolbachia有关呢?确定物种形成的因素十分复杂,我们不能轻易下结论。但是我们首次选取了同域共存的,系统发育关系最为相近的垂叶榕传粉榕小蜂,检测它们的Wolbachia感染状态,用以探索共生榕小蜂物种形成的可能线索。或可能寻找形态分化的选择压力。经过垂叶榕传粉小蜂线粒体c0I和核基因ITS2序列建树比较发现,1)垂叶榕传粉榕小蜂共有4个种,1个海南地理种,3个云南同域共存种；2)联合该属近缘物种建树发现,这4个物种聚在一起,不与其它榕树小蜂物种混合,说明在现有的分子数据下,这4个物种没有发现寄主转移；3)云南3个物种可以在同批次收蜂中找到,说明这3个物种可以同域共存；4)4个物种中除了海南物种感染特有的wben-3,云南物种没有与系统发育对应的wsp感染状态；5)感染个体和不感染个体能共享相同的线粒体单体型；6)四种传粉榕小蜂线粒体基因均偏离中性选择；7)海南和云南地域物种的分化时间大概是500-600万年前。垂叶榕上的四种传粉小蜂是否由同域物种形成,Wolbachia是否在其中起关键作用我们目前还不得知,下一步工作将对四个物种的形态分化和Wolbachia感染株系进行深入而细致研究。更多还原

【Abstract】 Fig and fig wasps are thought to be a classical model of coevolved system. They are ideal materials in studies of speciation, population genetics and dynamics, adaptive evolution, and mutualism stabilization etc. Syconium provides different fig wasp species with various niches modulated by using the different stylus lengths or syconium thicknesses through its developing stages. Fig wasp species phylogenetically closely related or highly diverged can coexist within the same syconium, and they are parasitic correlated and have intimate spatial relationship time continue, space intimate and food associate. Males usually fight with their brothers, which enhances the possibility of physical contact and ecological association. Meanwhile, compared with the other insects living outside of fig syconia, fig wasps have much few chances to communicate with the outside world except when mature female emerge out from the syconia and try to find other appropriate figs for eggs-laying or some winged males come out and mate with female in the outside. That is different from common plant-insect systems which are in open or semi-open conditions. All these above make syconium of Ficus benjamina a good model to study the infection pattern and transferring routes of Wolbachia.Wolbachia, maternally inherited endosymbiotic bacteria that widely infect a variety of arthropod and nematode hosts, can manipulate host reproduction. As to the pandemic distribution, there are about1million infected species in insects alone. Researchers have proved that fig wasps have a high infection incidence of Wolbachia, even significantly higher than the average estimate based on a broad collection of insects. We are thus very interested in the following questions:1) what is the Wolbachia infection pattern in all of the fig wasps associated with the same fig tree;2) if there is a decrease in Wolbachia exchange between the relative compact world within syconia (fig wasps) and the outside (other insects);3) if there are more than common frequent horizontal transfer events of Wolbachia among these wasp species;4) how can the Wolbachia be introduced from outside into syconia;5) whether the Wolbachia infected within the syconia have unique evolutionary characters.In the present study, by focusing on the17species of chalcidoid wasps living on Ficus benjamina, covering4families,6subfamilies, and8genera, we make a thorough survey on the Wolbachia infection pattern with wsp (Wolbachia Surface Protein) sequences. We also investigate the biological characteristics of these fig wasps, based on which we try to presume the putative horizontal transferring route of Wolbachia.1) the enclosed syconium with small cavity and multiple wasp species have high infection incidence of Wolbachia;2) frequent recombination events of wsp;3) prevailing horizontal transfer of wsp; Though the fig wasps have relatively less contact with outside world, Wolbachia may be introduced inside horizontally via the fig wasps species which have winged males and ecologically visit the syconia earlier.In the screening experiments, we detected two divergent infection patterns of Wolbachia in fig wasps. For some species infected with single wsp type, the infection within one individual is often with high concentration. However, for some species infected with multiple wsp sequence types, one individual often has low infection concentration. We are interested in the cause of the different patterns. As we know, the formation of Wolbachia infection patterns is associated with not only Wolbachia strains themselves but also their hosts and circumstance. Previous studies mostly focused on Wolbachia infection incidence, strains and transfer routes, yet rarely on why the infection patterns are formed. We here try to explore the reasons from the following aspects:host phylogeny, time of speciation, and host population trait such as sex ration and brood sizes.We chose fig wasp species of Acophila, Walkerella, Sycoscapter in Ficus benjamina and their phylogenetically closely related species (from the same genus or near genus) from other ficus, and analyzed the infection patterns with the occasionally amplified coxA sequences of Wolbachia in our previous studies on host col amplification. Our results are as following:1) Phylogenetically closely related fig wasp species do not always have similar infection patterns;2) old species, if infected, are prone to be stably infected by single wsp sequence type with high concentration;3) the fig wasp species from genera with multiple recent speciation events often bear more wsp sequences types, mostly each with low infection prevalence and low titre;4) fig wasps living in dioecious figs have more chance of inbreeding, and often have big brood sizes, low sex ratio, and few winged males, which makes them more possible to be infected by single wsp sequence type with high concentration.Fig wasps have a large number of species, and the existence of sister or cryptic species is very common. Previous studies have proved that bidirectional cytoplasmic incompatibility can cause speciation of Nasonia. Fig wasps have high infection incidence of Wolbachia which can probably manipulate host reproduction. May Wolbachia play a part in fig wasp speciation? Though we need much evidence to answer this question, we can firstly try to associate the Wolbachia infection patterns with some biological charaters on the species coexisted and phylogenetically closely related. We here take the pollinators on Ficus benjamina as examples. Both col and ITS2NJ trees present that1) four clades are detected, which may imply the coexistence of four pollinator species in Ficus benjamina;2) the four species make a single lineage on the tree constructed with all the available sequences from the genus, which suggests no host shifting of the four spcecies;3) samples from the three Yunnan species can be collected from the same crop, which may be further evidence of their coexistence;4) except that samples in Hainan province were infected by unique wsp sequence (wben-3), the infection pattern of wsp sequences do not have clear correlation with the three species from Yunnan province;5) infect and uninfect samples can share the same col haplotype;6) col sequences of the four species deviate from natural selection;7) Hainan and Yunnan spcies may have been separated5-6million years before. The present results can not clearly tell us whether the four species came from sympatric speciation, and whether Wolbachia was associated with the separation of the four species. Further studies will focus on the detailed divergence of the morphological characters among the four species, and more detailed information on the Wolbachia strains.更多还原