Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe HANNES SCHULER,* CORALIE BERTHEAU,* SCOTT P. EGAN, † JEFFREY L. FEDER, † MARKUS RIEGLER, ‡ BIRGIT C. SCHLICK-STEINER, § FLORIAN M. STEINER, § JES JOHANNESEN, ¶ PETER KERN,* ‡ KATALIN TUBA,** FERENC LAKATOS,** KIRSTEN K € OPPLER, †† WOLFGANG ARTHOFER § 1 and CHRISTIAN STAUFFER* 1 *Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection, Boku, University of Natural Resources and Life Sciences, Hasenauerstr, 38, 1190 Vienna, Austria, †Department of Biological Sciences, Galvin Life Sciences Building, University of Notre Dame, Notre Dame, IN 46556, USA, ‡Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia, §Institute of Ecology, Molecular Ecology Group, University of Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria, ¶Zoological Institute, Department of Ecology, University of Mainz, Johann-Joachim-Becherweg 13, 55128 Mainz, Germany, **Institute of Silviculture and Forest Protection, University of West-Hungary, Bajcsy-Zs. u. 4, 9400 Sopron, Hungary, ††Center for Agricultural Technology Augustenberg, Nesslerstr. 23-31, 76227 Karlsruhe, Germany Abstract The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species bound- aries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well-characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolba- chia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in sev- eral European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some popu- lations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hun- gary. In summary, our study provides strong evidence for a very recent inter-specific Wol- bachia transmission with a subsequent spatial spread in field populations. Keywords: horizontal transmission, invasive species, multilocus sequence typing, Rhagoletis cerasi, Rhagoletis cingulata, Wolbachia Received 13 August 2012; revision received 17 April 2013; accepted 18 April 2013 Introduction The endosymbiotic a-Proteobacterium Wolbachia is prob- ably the most common intracellular symbiont, infecting approximately 40% of all insect species (Zug & Ham- merstein 2012). In the majority of cases, maternally Correspondence: Hannes Schuler, Fax: +43-1-3686352-97; E-mail: hannes.schuler@boku.ac.at 1 Equally contributing senior authors. © 2013 John Wiley & Sons Ltd Molecular Ecology (2013) 22, 4101–4111 doi: 10.1111/mec.12362