Similar to old laboratory strains of AM, CA and OR semispecies, both AB lines obtained from more recent collections showed statistically significant reduction in their assortative mating behavior after partial symbiont depletion in combinations with untreated CA semispecies (Table S2,z=3.89,P<0.0001). and if so, to what extent. Here we have reevaluated this classic case of infectious speciation by means of present day molecular approaches and artificial symbiont depletion experiments. We have isolated the -proteobacteriaWolbachiaas the maternally transmitted core endosymbionts of allD. paulistorumsemispecies that have coevolved towards obligate mutualism with their respective native hosts. In hybrids, however, these mutualists transform into pathogens by overreplication causing embryonic inviability and male sterility. We show that experimental reduction in nativeWolbachiatiter causes alterations in sex ratio, fecundity, and mate discrimination. Our results indicate that formerly designatedMycoplasma-like organisms are most likelyWolbachiathat have evolved by becoming essential mutualistic symbionts in their respective natural hosts; they have the potential to trigger pre- and postmating isolation. Furthermore, in light of our new findings, we revisit the concept of infectious speciation and discuss potential mechanisms that can restrict or promote symbiont-induced speciation Norepinephrine hydrochloride at post- and prezygotic levels in nature and under artificial laboratory conditions. == Author Summary == TheDrosophila paulistorumspecies complex serves as a well-studied model system for evaluating the impact of symbiosis on host speciation since they evolve rapidly and comprise an ancestral, but highly dynamic, reservoir of microbial symbionts. Theory and some experimental evidence suggest that in evolutionary longterm host-symbiont interactions, reproductive parasites might evolve a more benign lifestyle towards mutualism, manipulate sexual mating behavior, and foster host speciation. However, it is an ongoing debate as SP-II to whether or not microbial symbionts are capable of driving host speciation in nature and if so, to what extent. Prime candidates areWolbachia, inherited, endosymbiotic bacteria of many arthropods, presently attracting attention as potential biocontrol agents since they affect host reproductive biology. Here we document that allD. paulistorumsemispecies harborWolbachiathat provide significant fitness benefits to their natural hosts. In semispecies hybrids, however, mutualisticWolbachiaturn into pathogens, triggering embryonic lethality and male sterility via overreplication. Besides their impacts on post-mating isolation, we show that in their nativeD. paulistorumhostsWolbachiamanipulate sexual behavior by triggering pre-mating isolation via selective mate avoidance,i.e.avoiding mates harboring another, incompatible symbiont variant. Our study reveals that endosymbionts can coevolve rapidly with their native hosts and play a significant role in driving natural host speciation. == Introduction == == Nuclearvs.Symbiotic Speciation Mechanisms == In contrast to prezygotic reproductive isolating mechanisms acting before fertilization via Norepinephrine hydrochloride mating behavior, postzygotic isolation arises after mating when hybrids are less fit than their parents[1]. In the latter case, the Dobzhansky-Muller model proposes that hybrid incompatibilities, contributing to speciation, are caused by the interaction between nuclear genes that have functionally diverged over time in their respective hybridizing species[2],[3]. Initial direct molecular and genetic evidence for the existence of such Dobzhansky-Muller incompatibility genes recently came from the two sister-species,D. melanogasterandD. simulans[4]. In this case one of the two incompatibility genes Norepinephrine hydrochloride localizes to centromeric heterochromatin – an inherently dynamic part of all eukaryotic genomes known for its accelerated genomic turnover and molecular drive[5],[6]. Genes located within or functionally interacting with hyperdynamic genomic regions, such as nuclear heterochromatin, evolve rapidly and so contribute to hybrid incompatibilities and thereby to speciation[4][6]. In this speciation model, however, incompatibility genes are both nuclear genes that have evolved differently after an ancestral populations split. A second class of rapidly evolving genes possessive of high potential for driving accelerated functional divergence are those genes that are under antagonistic coevolution versus parasites,i.e. the reciprocal evolution of host defense and parasite counter-defense mechanisms[7][10]. Accordingly, one or more incompatibility genes is encoded by the host nucleus, and the second set by a parasite. In a consistent arms race between.