Wolbachia Infection Patterns And The Evolution Of Wsp Genes In The Fig Wasps Associated With Ficus Benjamina

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.