Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA, USA). wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the Wolbachia tree, indicating parallel evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon analogous to the wPip cinAB operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though fleas in wild populations mostly harbor wCfeT alone. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.

Importance 44Many arthropod and certain nematode species are infected with wolbachiae which are 45 intracellular bacteria well known for reproductive parasitism (RP). Like other RP 46 strategies, Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and 47 frequency in host populations. Mutualism is another strategy employed by wolbachiae 48 to maintain host infection, with some strains synthesizing and supplementing certain B 49 vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novel 50Wolbachia strains that co-infect cat fleas (Ctenocephalides felis): wCfeT carries biotin 51 synthesis genes, while wCfeJ carries a CI-inducing toxin-antidote operon. Our analyses 52 of these genes highlight their mobility across the Wolbachia phylogeny and source to 53 other intracellular bacteria. Remarkably, the C. felis genome also carries two CI-like 54 antidote genes divergent from the wCfeJ antidote gene, indicating episodic RP in cat 55 fleas. Collectively, wCfeT and wCfeJ inform on the rampant dissemination of diverse 56 factors that mediate Wolbachia strategies for persisting in invertebrate host populations. 57 58 Key words 59Wolbachia, Ctenocephalides felis, cat flea, reproductive parasitism, mutualism, lateral 60 gene transfer, cytoplasmic incompatibility, biotin operon 61 killing have been characterized (24-29) and occur predominantly in the eukaryotic 87 association module (EAM) of Wolbachia prophage genomes (30). These genes 88 highlight the role of LGT in providing wolbachiae with factors facilitating mutualism or 89 RP, both of which are highly successful strategies for increasing infection frequency in 90 invertebrate host populations. 91Compared to reproductive parasites, Wolbachia mutualists appear to form more 92 stable, long-term relationships with their hosts, as supported by Wolbachia-host 93 codivergence in certain filarial nematodes (31) and Nomada bees (32). In contrast to 94 the stability of mutualists, relationships of reproductive parasites appear more 95 ephemeral. RP can be a strong mechanism to increase infection frequency, and CI 96 inducing strains can replace populations without infections (33, 34). Despite this, CI is 97 prone to neutralization through the evolution of host suppression (23, 35, 36) and 98 purifying selection on the host doesn't preserve CI (36). A weakened CI background 99 might be a ripe setting for invasions to begin. Whether invasion occurs at the level of 100 alternate wolbachiae, WO-phages, or CI operons themselves is an area of active 101 evolutionary research, but a clear result of this evolutionary complexity is that RP 102inducing Wolbachia phylogenies are discordant with those of their hosts (37-39). 103Horizontal transmissions, which can occur through direct host interactions, 104 environmental contacts (shared habitat, food sources, etc.) or predator/parasitoid 105 delivery, contribute to the discordance (40) and possibly drive episodic invasions and 106 replacements. 107Horizontal transmission is also evident from...