The Role of Wolbachia in Rhagoletis Fruit Fly Speciation

Speciation is a fundamental evolutionary process responsible for the great diversity of life on Earth. Main factors contributing to the evolution of reproductive isolation are geographic and ecological isolation. However, oft- forgotten and controversially discussed players in the speciation process of insects are reproduction-manipulating endosymbiotic bacteria of the genus Wolbachia that can form pre- and postzygotic barriers in infected insect populations. The multiply infected Rhagoletis fruit flies are an excellent system to study such effects as the evolutionary history of this genus involves a number radiations with different geographic modes, ranging from classic allopatric divergence to sympatric speciation via host plant shifting. Aim of this proposal is to evaluate the contribution of Wolbachia to speciation in different Rhagoletis species utilizing on-site research at the highly complex structured North American fruit fly populations. The research objective will be accomplished by analyzing Wolbachia strains from different Rhagoletis species groups that have undergone allopatric (e.g., R. suavis group) vs. sympatric (e.g., R. pomonella group) speciation. Following the broadly accepted theory that bacterial reproductive isolation almost always evolves in allopatry, I hypothesize that sympatrically derived species with extant gene flow will share the same strains of Wolbachia while allopatric species will possess different strains. I will test this hypothesis by the characterization of different Rhagoletis populations. Further, Wolbachia strains in parasitoid wasps attacking Rhagoletis flies will be characterized as possible vector of horizontal Wolbachia transmission between Rhagoletis species. This will give new insights into routes of horizontal transmission between different species and the evolution of Wolbachia in geographically separated populations with possible influences into speciation. Going beyond the capabilities of the established MLST system for strain discrimination, a combination of Sanger and next generation high throughput sequencing will be adapted to perform a high resolution characterization of this bacteria. Additionally the Rhagoletis species and their parasitoids will be screened for other bacterial endosymbionts influencing the host`s reproduction. Verifying an active role of Wolbachia in reproductive isolation and Rhagoletis speciation will ultimately require breeding of flies that are depleted of the bacteria to determine the extent that fertility is restored between flies. Thus, a secondary objective of the current proposal is to develop and establish protocols for curing Rhagoletis of Wolbachia, allowing follow-up manipulative studies in the future to confirm a direct connection of Wolbachia with fly reproductive isolation.

Bacterial endosymbionts have been hypothesized to play an important contributing role in the process of speciation. Cytoplasmic incompatibility between populations of organisms possessing different bacterial strains can contribute to postmating reproductive isolation. However, there is currently little direct evidence for this in nature, and the role of endosymbionts in speciation is still controversial. The goal of the Erwin Schrödinger Fellowship was to compare the genetic variation of Wolbachia among sympatric and allopatric Rhagoletis species to understand the potential role of the bacterial endosymbiont Wolbachia in speciation of Rhagoletis fruit flies. Specifically we focused on the characterization of different species and host races of the Rhagoletis pomonella species group and cherry infesting members of the Rhagoletis cingulata species group. We adapted the restriction site associated DNA sequencing (RADseq) approach to characterize Wolbachia (and potentially other endosymbionts) more efficiently. The enrichment of specific Wolbachia fragments, followed by ddRADseq and Illumina MiSeq sequencing enables the high-resolution characterization of Wolbachia in multiple individuals. With this approach we characterized Wolbachia in different Rhagoletis species. We characterized Wolbachia in Rhagoletis pomonella populations from the Midwestern US, Pacific Northwest and Mexico. Additionally we sequenced the endosymbiont from the sister taxa Rhagoletis mendax, Rhagoletis zephyria, the undescribed flowering dogwood fly and Rhagoletis cornivora. Based on the six different genes all members of the R. pomonella species group share the same Wolbachia strain. Populations from the central highlands of Mexico, however, are infected by a second Wolbachia strain. Similar, different populations of the two cherry attacking sister species Rhagoletis cingulata and Rhagoletis indifferens are all infected by the same Wolbachia strain. Populations from Southwestern US, however, are infected by an additional Wolbachia strain, while R. indifferens populations from Central Washington are additionally infected by a third Wolbachia strain. Crossings of individuals infected by different Wolbachia strains revealed unidirectional incompatibilities between males from Southwestern US with females from Eastern and Western US suggesting Wolbachia-induced cytoplasmic incompatibility. In this project we further described the Wolbachia spread in the European cherry fruit fly Rhagoletis cerasi, we characterized Wolbachia in parasitoids attacking different Rhagoletis species and we performed an initial characterization of the microbial community in different Rhagoletis species. We also described an additional Endosymbiont Spiroplasma in the R. pomonella species group and studied Wolbachia in the gall wasp Belonocnema traetae.