bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 Evolution of Wolbachia Mutualism and Reproductive Parasitism:

2 Insight from Two Novel Strains that Co-infect Cat Fleas

3

4 Timothy P. Driscoll a, Victoria I. Verhoeve b, Cassia Brockway a, Darin L. Shrewsberry a,

5 Mariah L. Plumer b, Spiridon E. Sevdalis b, John F. Beckmann c, Laura M. Krueger

6 Prelesnik d, Kevin R. Macaluso e, Abdu F. Azad b, Joseph J. Gillespie b,#

7

8 a Department of Biology, West Virginia University, Morgantown, West Virginia.

9 b Department of Microbiology and Immunology, University of Maryland School of

10 Medicine, Baltimore, Maryland.

11 c Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama.

12 d Orange County Mosquito and Vector Control District, Garden Grove, California.

13 e Department of Microbiology and Immunology, University of South Alabama, Mobile,

14 Alabama.

15

16 Running Head: Mechanisms of Evolution in Wolbachia-host associations

17

18 # Address correspondence to: Joe Gillespie, [email protected]

19 Timothy P. Driscoll and Victoria I. Verhoeve contributed equally to this work. Author

20 order is alphabetical. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 2

21 Abstract

22 Wolbachiae are obligate intracellular bacteria that infect arthropods and certain

23 nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or

24 alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the

25 assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-

26 infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA).

27 wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to

28 supergroups C, D and F. Both genomes contain laterally transferred genes that inform

29 on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis

30 Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five

31 independent acquisitions of BOOM across the Wolbachia tree, indicating parallel

32 evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon

33 analogous to the wPip cinAB operon recently characterized as an inducer of

34 cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and immediate-5’ end genes are

35 syntenic to large modular toxins encoded in CI-like operons of certain Wolbachia strains

36 and Rickettsia species, signifying that CI toxins streamline by fission of larger toxins.

37 Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent

38 from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing

39 mobility of CI loci independent of WO-phage. Additional screening revealed

40 predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though occasionally

41 wCfeJ singly infects fleas in wild populations. Collectively, genomes of wCfeT, wCfeJ,

42 and their cat flea host supply instances of lateral gene transfers that could drive

43 transitions between parasitism and mutualism. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 3

44 Importance

45 Many arthropod and certain nematode species are infected with wolbachiae which are

46 intracellular bacteria well known for reproductive parasitism (RP). Like other RP

47 strategies, Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and

48 frequency in host populations. Mutualism is another strategy employed by wolbachiae

49 to maintain host infection, with some strains synthesizing and supplementing certain B

50 vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novel

51 Wolbachia strains that co-infect cat fleas (Ctenocephalides felis): wCfeT carries biotin

52 synthesis genes, while wCfeJ carries a CI-inducing toxin-antidote operon. Our analyses

53 of these genes highlight their mobility across the Wolbachia phylogeny and source to

54 other intracellular bacteria. Remarkably, the C. felis genome also carries two CI-like

55 antidote genes divergent from the wCfeJ antidote gene, indicating episodic RP in cat

56 fleas. Collectively, wCfeT and wCfeJ inform on the rampant dissemination of diverse

57 factors that mediate Wolbachia strategies for persisting in invertebrate host populations.

58

59 Key words

60 Wolbachia, Ctenocephalides felis, cat flea, reproductive parasitism, mutualism, lateral

61 gene transfer, cytoplasmic incompatibility, biotin operon

62

63 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 4

64 Introduction

65 Wolbachiae (Alphaproteobacteria: Rickettsiales: Anaplasmataceae) comprise Gram-

66 negative, obligate intracellular bacteria that infect over half the world’s described insect

67 species as well as certain parasitic nematodes (1). Unlike other notable rickettsial

68 genera that contain human pathogens (e.g., Rickettsia, Orientia, Neorickettsia,

69 Anaplasma, and Ehrlichia), wolbachiae do not infect vertebrates (2). A single species,

70 Wolbachia pipientis, is formally recognized with numerous members designated as

71 strains within 15 reported supergroups (3–9). Genomic divergence indicates further

72 species names are warranted (10), though increasing diversity and community

73 consensus suggest caution regarding further Wolbachia classification at the species

74 level (11, 12).

75 Like other obligate intracellular microbes, wolbachiae are metabolic parasites that

76 complement a generally reduced metabolism with pilfering of host metabolites (13, 14)).

77 Their ability to survive and flourish is also heavily influenced by the acquisition of key

78 functions through lateral gene transfer (LGT). Several described Wolbachia strains

79 demonstrate characteristics of limited mutualism with their invertebrate partner (15, 16),

80 through the synthesis and provisioning of riboflavin (17) and biotin (18, 19). While

81 riboflavin biosynthesis genes are highly conserved in wolbachiae (20), biotin

82 biosynthesis genes are rare and likely originated via LGT with taxonomically divergent

83 intracellular bacteria (21). Still other strains of Wolbachia exert varying degrees of

84 reproductive parasitism (RP) on their insect host (22), influencing host sexual

85 reproduction via processes such as male-killing, feminization, parthenogenesis and

86 cytoplasmic incompatibility (CI) (23). Wolbachia genes underpinning CI and male bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 5

87 killing have been characterized (24–29) and occur predominantly in the eukaryotic

88 association module (EAM) of Wolbachia prophage genomes (30). These genes

89 highlight the role of LGT in providing wolbachiae with factors facilitating mutualism or

90 RP, both of which are highly successful strategies for increasing infection frequency in

91 invertebrate host populations.

92 Compared to reproductive parasites, Wolbachia mutualists appear to form more

93 stable, long-term relationships with their hosts, as supported by Wolbachia-host

94 codivergence in certain filarial nematodes (31) and Nomada bees (32). In contrast to

95 the stability of mutualists, relationships of reproductive parasites appear more

96 ephemeral. RP can be a strong mechanism to increase infection frequency, and CI

97 inducing strains can replace populations without infections (33, 34). Despite this, CI is

98 prone to neutralization through the evolution of host suppression (23, 35, 36) and

99 purifying selection on the host doesn’t preserve CI (36). A weakened CI background

100 might be a ripe setting for invasions to begin. Whether invasion occurs at the level of

101 alternate wolbachiae, WO-phages, or CI operons themselves is an area of active

102 evolutionary research, but a clear result of this evolutionary complexity is that RP

103 inducing Wolbachia phylogenies are discordant with those of their hosts (37–39).

104 Horizontal transmissions, which can occur through direct host interactions,

105 environmental contacts (shared habitat, food sources, etc.) or predator/parasitoid

106 delivery, contribute to the discordance (40) and possibly drive episodic invasions and

107 replacements.

108 Horizontal transmission is also evident from hosts infected with multiple Wolbachia

109 strains (41–43), though little is known about the dynamics and stability of Wolbachia co- bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 6

110 infections (44). For instance, in host populations showing variability in single- versus

111 double-infections, does co-infection reflect a transitory phase (i.e. one strain replacing

112 another) or does it reflect stability of both strains in a population? A single infection may

113 be a consequence of closely related strains carrying different arsenals of RP-inducing

114 genes, and hence battling for control of host reproduction (38). A stable co-infection

115 may reflect more divergent strains that utilize different strategies (i.e. nutrient

116 provisioning versus CI) to inhabit different micro-niches in the same host. Such a case

117 is in part witnessed in doubly-infected cochineals (Dactylopius coccus), wherein a

118 Supergroup B strain (wDacB) carries an arsenal of RP-inducing genes but a

119 Supergroup A strain (wDacA) carries none (41). Unfortunately, few systems with

120 divergent co-infecting Wolbachia have been characterized sufficiently to dissect

121 contrasting mechanisms that enable long-term host co-infections.

122 We recently sequenced the genome of the cat flea (Ctenocephalides felis) and

123 concurrently closed the genomes of two novel wolbachiae, wCfeT and wCfeJ, that co-

124 infect fleas of the Elward Laboratory (Soquel, California; hereafter EL fleas) (45).

125 Genome-based phylogeny estimation placed wCfeT on an ancestral divergent branch,

126 while wCfeJ was found to subtend Wolbachia Supergroup C. Both genomes show

127 evidence of past WO phage infection, with LGT hotspots indicating recent acquisition of

128 factors predicted to either underpin mutualism (wCfeT) or mediate CI (wCfeJ).

129 Additionally, we describe two loci within the C. felis genome itself that support repeated

130 acquisitions of CI anti-toxin genes by the host. Infection surveys of cat flea populations

131 and colonies from across the US indicate extensive spread of wCfeT among wild

132 populations, and a strong bias for wCfeT/wCfeJ co-infection in colonies, highlighting the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 7

133 complex ecological relationship that drives this system. Finally, we apply an informatics

134 approach including phylogenomics, synteny analysis, and functional alignment to

135 construct a comprehensive picture revealing the recurrent evolution of nutritional

136 symbiosis and RP across the Rickettsiales.

137

138 Results and Discussion

139 Identification of novel Wolbachia parasites.

140 Recently, we assembled and annotated closed genomes from two divergent

141 Wolbachia strains, named wCfeT and wCfeJ, that were captured during genome

142 sequencing of the cat flea (EL Colony) (45). The 16S rDNA sequences of these two

143 strains are identical to those previously identified in another cat flea colony (Louisiana

144 State University) (46–48), which historically has been replenished with EL colony fleas.

145 wCfeT and wCfeJ are broadly similar to other closed Wolbachia genomes in terms of

146 length, GC content, number of protein coding genes, and coding density (Table 1),

147 although it is interesting to note that wCfeT exhibits one of the highest pseudogene

148 counts (second only to wCle) while wCfeJ boasts one of the lowest (second only to

149 wOv). Coupled with other recently sequenced genomes for non-Supergroup A and B

150 strains (e.g., wFol and wVulC), these cat flea-associated wolbachiae blur the lines

151 demarcating genomic traits previously used to distinguish ancestral Wolbachia lineages

152 from those of Supergroups A and B strains (44).

153 Prior reports have indicated the presence of multiple Wolbachia “types” infecting C.

154 felis, with phylogenies inferred from partial gene sequences placing these unnamed

155 Wolbachia in supergroups B or F, or in ancestral lineages (49–51). Our robust genome- bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 8

156 based phylogeny estimation reveals that both wCfeT and wCfeJ are early branching

157 wolbachiae (Fig. 1; Table S1). wCfeT is similar to undescribed C. felis-associated

158 strains that branch ancestrally to most other Wolbachia lineages (51–53), while wCfeJ is

159 similar to undescribed C. felis-associated strains closely related to Wolbachia

160 supergroups C, D and F (49, 51). The substantial divergence of wCfeT and wCfeJ from

161 a Wolbachia supergroup B strain associated with C. felis from Germany (wCte)

162 indicates a diversity of wolbachiae that are capable of infecting cat fleas. Importantly,

163 the nature of host-microbe relationships for any of these C. felis-associated wolbachiae

164 is unknown.

165

166 wCfeT is equipped with biotin synthesis genes.

167 The enzymatic steps for bacterial biosynthesis of biotin from malonyl-CoA are largely

168 conserved (54) and usually involve six bio enzymes and the fatty acid biosynthesis

169 machinery (55) (Fig. 2A). We previously identified the first plasmid-borne bio gene

170 operon, which occurs on plasmid pREIS2 of Rickettsia buchneri, an endosymbiont of

171 the black-legged tick (21). We observed a rare gene order for the pREIS2 bio operon

172 shared only with bio operons encoded on the chromosomes of obligate intracellular

173 pathogens Neorickettsia spp. and Lawsonia intracellularis (Deltaproteobacteria). These

174 unique bio operons formed a clade in estimated phylogenies, indicating LGT of the

175 operon across divergent intracellular species. Subsequent studies, using our same

176 dataset for phylogeny estimation, discovered this bio operon in certain Wolbachia (19,

177 32, 56), Cardinium (57, 58) and Legionella (59) species. We refer hereafter to this bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 9

178 intriguing LGT of bio genes as the Biotin synthesis Operon of Obligate intracellular

179 Microbes (BOOM).

180 wCfeT carries a complete BOOM with greater similarity to Wolbachia BOOM than

181 those of other bacteria (Fig. 2B). Phylogeny estimation of BOOM and other diverse bio

182 gene sets indicates wCfeT BOOM is ancestral to other Wolbachia BOOM (Fig. 2C, Fig.

183 S1). However, as the divergence pattern for other Wolbachia BOOM is discordant with

184 genome-based phylogeny (Fig. 1), it is clear that multiple independent BOOM

185 acquisitions have occurred in wolbachiae irrespective of lineage divergences. This is

186 supported by a previous study hypothesizing recent BOOM acquisition in wNfla and

187 wNleu (32), as well as the nature of BOOM flanking regions in the genomes of wCfeT,

188 wVulC, wCle, wStr, wLug, which collectively lack synteny and are riddled with

189 transposases, recombinases and phage-related elements (Table S3). Furthermore,

190 analysis of wCfeT BOOM flanking genes reveals hotspots for LGT, exemplified by an S-

191 adenosylmethionine importer (EamA) gene (Fig. S1B) that is a signature among many

192 diverse obligate intracellular bacteria (21, 60–62).

193 As some BOOM-containing Wolbachia strains have been shown to provide biotin to

194 their insect hosts (18, 19), we posit that wCfeT has established an obligate mutualism

195 with C. felis mediated by biotin-provisioning. This is consistent with a WO prophage in

196 the wCfeT genome that is highly colinear to the WOVitA1 prophage (30), except for the

197 absence of a EAM and hence no RP-inducing loci (Fig. S2). Wolbachia strains

198 associated with bedbug (wCle), planthopper (wStr, wLug), and termites (wCtub) with

199 evidence for BOOM (63) reside in bacteriocytes of their insect hosts, structures

200 associated with nutritional symbiosis in many arthropods (64). Interestingly, C. felis is bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 10

201 not known to contain bacteriocytes, and the ability of wCfeT to synthesize and

202 supplement biotin to cat fleas awaits experimentation.

203

204 wCfeJ lends insight on the evolution of reproductive parasitism.

205 wCfeJ carries a toxin-antidote (TA) operon that is architecturally similar to the

206 wPip_Pel CinA/B TA operon (Fig. 3A), which was previously characterized as a CI

207 inducer in flies (24). Grouped into three types (26, 65), CinB toxins harbor dual

208 nuclease (NUC) domains in place of the deubiquitinase (DUB) domain of CidB,

209 another CI inducer in flies (26–28). Chimeric NUC-DUB toxins, termed CndB toxins

210 (66), also occur as operons (cndAB) in certain Wolbachia and Rickettsia genomes (27,

211 67). Although their functions remain unknown, these larger CndB toxins belong to an

212 extraordinarily diverse array of toxins with highly modular architectures that occur in

213 divergent intracellular bacteria (67). Curiously, despite a size similar to the CinB toxins,

214 wCfeJ’s CinB toxin has much higher sequence identity to larger CndB and NUC-

215 containing toxins found mostly in Wolbachia Supergroups A and B (Fig. 3B). A similar

216 pattern was found for wCfeJ’s CinA antidote, indicating the genes were likely acquired

217 as an operon.

218 In contrast to wCfeT, wCfeJ harbors a highly degraded WO prophage genome with

219 only a partial EAM, the CinA/B operon, and several other genes encoding hypothetical

220 proteins and transposases (Fig. 3C). This is reminiscent of the wRec parasite, which

221 lacks a functional WO phage yet possesses TA modules within remnant EAMs (68).

222 Synteny analysis with other Wolbachia phage revealed wCfeJ cinB and three

223 downstream genes are colinear with a large (3707 aa) wDacB cndB toxin (Fig. 3D). bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 11

224 This provides strong evidence for the fission of a large modular CndB toxin into a

225 smaller CinB toxin. This interpretation is also consistent with our prior hypothesis

226 stating that more streamlined RP-inducing toxins (i.e., cinB and cidB) originate from

227 larger modular toxins (i.e., cndB and others) that are widespread in the intracellular

228 mobilome, particularly Wolbachia phage and Rickettsia plasmids and integrative and

229 conjugative elements (ICEs) (48, 67).

230 wCfeJ is unique among described early-branching wolbachiae by containing a CI-

231 like TA operon. In fact, the Wolbachia strains in closely related Supergroups C, F and D

232 are considered mutualists with their nematode and arthropod hosts. wFol, another

233 ancestral Wolbachia lineage (Fig. 1), does carry a myriad of large modular candidate

234 RP-inducing toxins but does not have cin, cid or cnd TA operons (69, 70). Collectively

235 this indicates acquisition of a WO prophage by wCfeJ, possibly from another

236 Supergroup A or B Wolbachia strain that also infects cat fleas (e.g., wCte), with viral or

237 Wolbachia level selection streamlining a cndAB locus into a cinAB operon. Given that

238 the genomes of many Wolbachia reproductive parasites harbor diverse arrays of

239 CinA/B-and CidA/B-like operons (66, 67), we posit that wCfeJ’s CinA/B TA operon might

240 function in CI or some other form of RP; however, further study is required to evaluate

241 wCfeJ as a cat flea reproductive parasite. Furthermore, an intriguing speculation is that

242 functions of the larger modular toxins, exemplified by wDacB CndB, whatever they be,

243 are likely the primal functions that gave rise to CI.

244

245 Lateral transfer of Wolbachia CI-like antidotes to the cat flea genome. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 12

246 Our previous work identified CI-like toxin and antidote genes on scaffolds in several

247 arthropod genome assemblies, although antidote genes were found more frequently

248 possibly due to utility in suppressing microbial invaders implementing RP (67). Many of

249 these genomes contain multiple divergent antidote genes, though none were found to

250 harbor large-scale Wolbachia gene transfer that is known to occur in certain

251 invertebrates (71–77). In light of this observation, we originally speculated that CI-like

252 genes are mobilized from wolbachiae to host genomes possibly through WO phage

253 introgression; however, the absence of other WO phage genes in these genomes made

254 this assertion tenuous. Instead, more recent work describing the first WO prophage

255 carrying two sets of CI loci provides compelling evidence for transposon-mediated LGT

256 of CI loci between divergent wolbachiae (38). This phage-independent mechanism for

257 RP gene purging and replenishment in wolbachiae may result in the inadvertent capture

258 of these genes by host genomes.

259 When we analyzed the cat flea genome for CI-like genes, we discovered that the

260 largest C. felis scaffold (185.5 Mb) was found to harbor two CI-like antidote genes (Fig.

261 4A). Remarkably, neither antidote has any significant similarity to CinA of wCfeJ (Fig.

262 4B), indicating that these CI antidotes originated from other, as yet undescribed

263 wolbachiae. The C. felis CinA-like CDS (XP_026474240.1) has greater similarity to

264 CinA proteins than CidA proteins, while the CidA-like CDS (not annotated in the current

265 C. felis assembly) has much greater similarity to CidA antidotes (27, 28, 78–80). The C.

266 felis cinA-like gene is found as an exon within a larger flea gene model

267 (LOC113377978), and is well supported (18X coverage, 7.2 TPM) by transcripts from

268 the 1KITE project (81). Since these transcripts were generated in fleas from a different bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 13

269 colony (Dr. Michael Dryden, Kansas State University), we conclude that this gene

270 transfer occurred during an historical infection predating the establishment of these two

271 colonies. Finally, the C. felis CinA- and CidA-like antidotes each possess the conserved

272 motifs we previously defined for all CI antidotes (67), with the exception of a truncation

273 of motifs 5 and 6 in the CinA-like protein (Fig. 4D). As other arthropod-encoded

274 antidotes share this C-terminal truncation, it may signify an expendable domain in the

275 host (i.e., a bacterial secretion signal, a motif involved in cognate toxin activation or

276 delivery, etc.).

277 The incorporation of a Wolbachia antidote into an exon of a host gene provides one

278 explanation for how such a laterally transferred gene would remain functional in the

279 absence of the native regulatory elements encoded on WO prophage genomes. We

280 previously hypothesized that CI antidote genes would be under strong selection in

281 arthropod genomes if their expression could result in rescuing CI induction by

282 reproductive parasites (67). In support of this supposition, it has been shown that: host

283 genotypes modulate CI (68, 82–84); some hosts show weak (Dmel and D. suzukii (35,

284 85, 86)) or strong (Culex pipiens and D. simulans (87, 88)) CI; and a Wolbachia strain’s

285 CI can be suppressed when transfected across different hosts (84). While none of the

286 arthropod-encoded CI antidotes have been characterized, we propose they impart

287 immunity to toxins secreted by intracellular parasites, curtailing chronic parasite drive

288 into populations. At least four independent evolutionary mechanisms might lead to

289 suppression of CI. Gene family expansion of, and/or overexpression of key factors

290 capable of counteracting CI mechanisms is possible; similarly, it was postulated that

291 evolution in Cid interacting proteins like karyopherin-α or P32 could lead to target site bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 14

292 resistance (25). A third possibility contributing to weakening CI, though not suppression

293 per se, is simply the mutational collapse of the operons themselves due to lack of

294 purifying selection (89). We supply evidence of a fourth possibility whereby CI might be

295 counteracted by germline expression of acquired CI antidotes. Significantly, our finding

296 raises awareness of a potential barrier to Wolbachia-mediated pathogen biocontrol

297 measures.

298

299 Characterization of wCfeT and wCfeJ infection of cat fleas.

300 Coupled with earlier reports indicating multiple early-branching wolbachiae lineages

301 infecting C. felis (49, 51), our characterization of two divergent Wolbachia strains

302 infecting cat fleas in the EL colony (45) as well as fleas maintained for a decade at LSU

303 (46–48) prompted us to test individual fleas for double infection. In addition to EL fleas,

304 three other colony strains and three geographically diverse wild cat flea populations

305 were also surveyed (Fig. 5A). Aside from the Modesto Wild strain, which is historically

306 replenished with wild-caught cat fleas (Dr. Bill Donahue, personal communication),

307 strong wCfeJ-wCfeT co-infection was found in colony fleas. Conversely, single wCfeT

308 infection dominated wild cat fleas, with only a few fleas from a large sampling

309 throughout Orange Co. CA harboring wCfeJ. While far greater sampling of both colony

310 and wild populations is necessary, our results hint at a strong selection for wCfeT over

311 wCfeJ in nature. The ability of wCfeJ to highly infect colony fleas may arise as a

312 consequence of artificial bottlenecks within colonies, which are known to strengthen CI

313 and maternal transmission (36). Nonetheless, the ability of wCfeT and wCfeJ to co-

314 infect individual cat fleas provides a system to study contrasting forces (nutritional bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 15

315 symbiosis and RP) that potentially drive multiple Wolbachia infections in a single

316 invertebrate host.

317 To gain more insight into the nature of the wCfeT-wCfeJ co-infection, we analyzed

318 the distribution of wCfeT and wCfeJ in EL flea tissues (Fig. 5B). Surmising that both

319 Wolbachia strains are maternally transmitted, we dissected out the ovaries of unfed

320 virgin female fleas and compared Wolbachia density in these tissues versus others

321 (primarily midgut). Counter to what we expected, wCfeT predominantly localizes in flea

322 ovaries, while the CI harbinger, wCfeJ, was predominantly in somatic tissues. Although

323 the two are not mutually exclusive and they do co-localize to some extent. The

324 significance of this distribution is unclear. wCfeJ may relocate to ovaries upon a mating

325 cue, or perhaps exert CI via some undefined somatic cell mechanism. Transmission of

326 wCfeJ may also be predominantly horizontal via environmental or direct flea contact

327 (per (40)). Sampling co-infected fleas at specific timepoints during development, as well

328 as determining wCfeJ tissue localization in the absence of wCfeT infection, will be

329 required to unravel the intertwined transmission dynamics of these Wolbachia strains in

330 cat fleas.

331

332 Evolution of Wolbachia Mutualism and Reproductive Parasitism

333 Order Rickettsiales is a highly diverse group of obligate intracellular bacteria (2, 62,

334 90). A rapid pace of newly identified lineages (60, 91–104) provides a rich source for

335 evaluating mechanisms of evolution within constraints of the eukaryotic cell. Phage,

336 plasmids, transposons, ICEs, and other insertion sequences are variably present across

337 rickettsial genomes and mobilize numerous genes to offset reductive genome evolution bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 16

338 and provide lifestyle altering traits (21). Our analyses of Wolbachia BOOM and RP

339 genes prompted us to evaluate the recurrent evolution of nutritional-mediated mutualism

340 and reproductive parasitism across diverse rickettsial lineages (Fig. 6).

341 Nutritional-mediated mutualism. Phylogenetic estimation supports three

342 independent acquisitions of biotin biosynthesis genes by rickettsial species (Fig. 6A,

343 Fig. S1A). The largest clade (group 1) contains all BOOM-containing species, though

344 only Neorickettsia spp., certain wolbachiae, R. buchneri, and the Peranema

345 trichophorum endosymbiont carry the conserved BOOM gene arrangement (highlighted

346 yellow in Fig. 6A). Other species in this group, as well as Groups 2 and 3, show

347 different gene arrangements indicating recombination and/or possible replacement of

348 specific bio genes through additional LGT (Fig. S3). BOOM and other bio gene clusters

349 have repeatedly lost bioH, which encodes the methyl esterase generating pimeloyl-CoA,

350 the last step in the synthesis of the pimelate moiety of biotin. Bacteria have evolved

351 numerous strategies for catalyzing this reaction (54, 105–112). Interestingly, a novel

352 methyl esterase of Ehrlichia chaffeensis (BioU) was recently shown to complement an

353 E. coli DbioH mutant (113); however, we determined that BioU orthologs are present in

354 many rickettsial species, including some that harbor a BioH already and others with no

355 bio synthesis genes at all (Fig. S4A-C). This indicates that while BioU may generate

356 pimeloyl-CoA in certain rickettsial species – namely, those with complete bio synthesis

357 modules except for BioH – it is likely to have a broad range of functions in bacteria (Fig.

358 S4D). Furthermore, some species lack the genes for both BioH and BioU, indicating

359 that alternative pathways for generating pimeloyl-CoA await discovery. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 17

360 Analysis of the genomic distribution of biotin synthesis genes, the biotin transporter

361 BioY, the biotin ligase BirA, and the enzymes involved in malonyl-CoA synthesis (biotin-

362 dependent propionyl-CoA carboxylase complex and FabD) revealed several strategies

363 for biotin utilization in Rickettsiales (Fig. 6A). Species that harbor a complete set of bio

364 genes (as discussed above) also contain ligase and utilization enzymes, although (not

365 surprisingly) most do not carry the BioY importer. Genera Wolbachia and Rickettsia

366 largely rely on parasitic scavenging of host biotin to fuel fatty acid biosynthesis: they are

367 dominated by species that lack bio genes but carry the BioY importer, BirA ligase, and

368 utilization enzymes. Two species of Transitional Group Rickettsiae (R. felis and R.

369 hoogstraalii) may be able to utilize imported dethiobiotin as well, as they retain the biotin

370 synthase BioB; alternatively, this may represent the frayed end of a decaying metabolic

371 pathway similar to the glycerol phospholipid and folate biosynthesis pathways in

372 Rickettsiae (13, 114). While characterized as two-component transporters (with

373 BioM/N), BioY subunits alone can efficiently transport biotin into cells (115) and also

374 participate with host transporters to relocate cytosolic biotin to vacuoles occupied by

375 pathogens (116). However, certain rickettsial species lacking synthesis genes also lack

376 BioY importers yet retain the ligase and utilization enzymes, indicating the presence of

377 alternative transport mechanisms. Remarkably, Orientia spp. lack all genes involved in

378 biotin synthesis, import, ligation and utilization, exhibiting the ultimate form of parasitism

379 for host-dependent fatty acid synthesis: these Rickettsiae must pilfer malonyl-CoA from

380 the host (117), using a transport system yet to be described.

381 The acquisition of BOOM by certain wolbachiae and R. buchneri provides a

382 theoretical framework for how LGT facilitates a transition from metabolite thievery to bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 18

383 nutritional-mediated symbiosis. However, it is important to consider that these

384 endoparasites may be utilizing the BOOM primarily for selfish purposes, as almost all

385 bacteria require biotin. For example, Neorickettsia spp. and the deltaproteobacterium

386 Lawsonia intracellularis are pathogens that also harbor the BOOM, suggesting a more

387 complex ecological role surrounding biotin acquisition. If Neorickettsia spp. do indeed

388 actively provision biotin to their trematode and insect vectors, as has been shown for

389 wCle (19), such host-specific supplementation would necessitate as-yet unidentified

390 genes encoding export factors as well as stringent regulation of the BOOM.

391 Canonical regulation of biotin synthesis is accomplished via a repressor, fused to the

392 N-terminal domain of the biotin ligase gene, which down-regulates transcription of the

393 bio gene cluster in response to high cellular biotin (118, 119). Interestingly, all rickettsial

394 BirA biotin ligases lack this repressor domain, indicating that alternative mechanisms for

395 regulating biotin synthesis and host supplementation are utilized by these bacteria.

396 “Candidatus Aquarickettsia rohweri” and the Trichoplax sp. H2 endosymbiont

397 (Midichloriaceae) lack BirA ligases altogether (Fig. 6A), and no other biotin ligases were

398 found in these genomes (data not shown). Regarding the Trichoplax sp. H2

399 endosymbiont, our prior analysis of the closely related Rickettsiales endosymbiont of

400 Trichoplax adhaerens (RETA) discovered two biotin-protein ligase N-terminal domain-

401 containing proteins (BPL_N) encoded in the T. adhaerens genome (62). These host-

402 encoded genes contain introns and eukaryotic regulatory elements yet are strongly

403 supported as rickettsial in origin. Homologs of these T. adhaerens proteins are also

404 present in the Trichoplax sp. H2 genome (120) (NCBI acc. nos. RDD42697 and

405 RDD42436), supporting their cross-domain LGT. This raises the possibility for a bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 19

406 metabolic interdependence involving biotin synthesis and utilization in placozoans and

407 their rickettsial symbionts, similar to the mosaic metabolic networks underpinning

408 nutritional symbioses in other systems (121–123) . For example, the nested tripartite

409 symbiosis between Planococcus citri mealybugs, Tremblaya princeps, and Moranella

410 endobia shows evidence for LGT of several bio genes to the mealybug genome (124);

411 these genes share highest similarity to Wolbachia BOOM counterparts (data not

412 shown). This is reminiscent of the pea aphid-Buchnera symbiosis, wherein several

413 rickettsial LGTs have been implicated in facilitating metabolic interdependence between

414 host and symbiont (125). For rickettsial endosymbionts of placozoans, host acquisition

415 of BPL_N genes (among others (62)) may have established an inextricable obligatory

416 relationship (126, 127) and may hint at the evolutionary fate of other BOOM-containing

417 rickettsiae. However, while our analyses of the BOOM and other bio genes in rickettsial

418 genomes are suggestive, in the absence of experimental validation we caution against

419 implicating these genes as factors underpinning nutritional-mediated mutualism.

420 Reproductive parasitism. Among other bacterial reproductive parasites of

421 arthropods (e.g., species of Cardinium, Arsenophonus and Spiroplasma) wolbachiae

422 are perhaps the best-studied for their roles in RP induction (23). Yet within the

423 Rickettsiales, wolbachiae do not walk alone in inducing RP in their hosts. Based on a

424 survey of the literature and analysis of genomic data for the ever-growing pool of RP-

425 inducing genes, we identify at least five strategies for RP induction that have evolved

426 throughout rickettsial evolution, with four independent gains in the Rickettsiaceae

427 coupled to the widespread RP seen in wolbachiae (Fig. 6B). We posit that this model is

428 more parsimonious than RP as an ancestral rickettsial trait, given that RP-inducing bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 20

429 genes are mostly found embedded within mobile genetic elements (e.g., WO prophage

430 in Wolbachia, RAGE ICEs and plasmids in Rickettsia spp.).

431 RP-inducing genes are found in greater numbers and diversity in wolbachiae,

432 undoubtedly due to their strong association with EAMs of WO prophage genomes and

433 mobile elements (30, 65). In our previous work, we uncovered a link between the

434 characterized CI-inducing CinA/B and CidA/B operons of wolbachiae and a putative TA

435 operon encoded on the pLbAR plasmid of Rickettsia felis str. LSU-Lb, an obligate

436 endosymbiont of the booklouse Liposcelis bostrychophila (48). We posited that this TA

437 operon induces parthenogenesis in the booklouse: pLbAR is unknown in other strains of

438 R. felis which do not induce parthenogenesis, in booklice or other arthropods (48), and

439 booklice lacking R. felis str. LSU-Lb reproduce sexually (128, 129). Aside from being a

440 CndB toxin (harboring both the NUC and DUB domains of wolbachiae CinB and CidB

441 toxins, respectively) the large pLbAR toxin (3,241 aa) was found to carry several other

442 domains present in other Wolbachia proteins, most of which are also EAM-associated.

443 These domains, together with the large central domain (LCD) of the pLbAR toxin,

444 were utilized to identify an extraordinarily diverse array of candidate RP-inducing toxins

445 in a wide-range of intracellular bacteria, some of which are known reproductive

446 parasites (67). Importantly, because this large assemblage of proteins was identified

447 using sequence characteristics of the pLbAR toxin, we consider the collective family

448 linked within the portion of the intracellular mobilome driving the recurrent evolution of

449 RP in invertebrate-borne bacteria.

450 Aside from CI induction by CinA/B and CidA/B operons (24–27), virtually nothing is

451 known about the functions of related candidate RP-inducing genes. The function of the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 21

452 CinA/B-like operon of O. tsutsugamushi is unclear. O. tsutsugamushi is the pathogen

453 causing scrub typhus and is capable of inducing parthenogenesis in Leptotrombidium

454 mites (130, 131). Given this fact, exploration of its corresponding CinA/B module is

455 warranted. Though the connection to RP in mites is unclear given that CinA/B only

456 occurs in the Boryong strain; a myriad of other proteins encoding the pLbAR LCD are

457 amplified in all Orientia genomes. Genome sequences are needed (but not yet

458 available) to determine toxin profiles for Rickettsia species either in ancestral lineages

459 (132–134) or Transitional Group rickettsiae (132, 135, 136) that induce parthenogenesis

460 in eulophid wasps or trogiid booklice. Genome sequences are also lacking for

461 Rickettsia species from ancestral lineages known to induce male-killing in buprestid and

462 ladybird beetles (137–140). We previously mined an unpublished genome of the male

463 killer Rickettsia parasite of the ladybird beetle Adalia bipunctata (141) and found a toxin

464 highly similar in architecture to the Spiroplasma male killer toxin (142). While both

465 toxins carry an ovarian tumor (OTU) cysteine protease domain (143), we found a

466 hodgepodge of toxin architectures carrying OTU domains from bacteria known to induce

467 other RP phenotypes, such as parthenogenesis (wFol (69)) and feminization (wVulC

468 (144)). Thus, it is clear that additional high-quality (closed) genomes are sorely needed

469 to correlate RP-inducing protein domains and RP phenotypes. New correlations will

470 lead to downstream functional analyses of target’s abilities to induce RP.

471 A large number of candidate proteins from the pLbAR-derived pool occur in species

472 that are not known as reproductive parasites. These include CndB toxins, NUC- and

473 DUB-domain containing proteins that are much larger than characterized CinB and CidB

474 toxins, diverse OTU-domain containing toxins, and especially proteins harboring the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 22

475 LCD that may or may not exhibit toxin-like profiles (67). Some of these candidate toxins

476 appear to have cognate antidotes, and some genomes carry antidotes only, highlighting

477 the complex diversity of this biological function. Recently, an EAM-encoded

478 transcriptional factor in wMel termed WO-mediated killing (wmk) was shown to induce

479 male-specific lethality during early embryogenesis when expressed transgenically in

480 fruit flies (29). This male-killer gene was not detected as part of our pLbAR-derived

481 pool, indicating that RP-inducing gene arsenals are probably much larger than have

482 been described thus far, particularly considering that cin, cid and cnd loci are not

483 present in the genomes of Cardinium and Arsenophonus reproductive parasites (66, 67,

484 145) for which the mechanisms for RP induction remain unknown (146, 147).

485 Candidate RP-inducing toxins with similar features to the pLbAR toxin (large,

486 modular, and associated with mobile genetic elements) are found in several Wolbachia

487 genomes (e.g., wFol, wStr, wDacB, wVulC, wCon, wCed, wCauA). They are often in

488 conjunction with other cin and/or cid loci, yet not always associated with WO prophage.

489 Consistent with prior reports identifying LGT between Rickettsia and Wolbachia

490 genomes (21, 48, 148, 149), similar toxins in Rickettsia genomes (R. felis str. LSU-Lb,

491 R. gravesii, male killer of Adalia bipunctata) indicate a dynamic platform for

492 disseminating RP-inducing genes across diverse rickettsial species that includes

493 Rickettsia plasmids and WO prophage. Growing numbers of identified Rickettsia

494 plasmids (48), proliferative RAGE ICEs in Rickettsia and Orientia genomes (21, 117,

495 150–153), and the recent discovery of the first Wolbachia plasmid (154), attest to a

496 highly dynamic mobilome not previously realized for obligate intracellular bacteria. The

497 wCfeJ fission of a CndB-like toxin into a CinB toxin described in the current study (Fig. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 23

498 3D) supports the premise that larger toxins recombine into WO prophage and undergo

499 streamlining into discrete inducers of CI or other RP phenotypes. As with the evolution

500 of biotin utilization, Rickettsiales provides a framework to study the nature of RP in a

501 diverse array of bacteria that enjoy different relationships with their invertebrate and

502 vertebrate hosts.

503

504 Conclusion

505 Fleas are understudied vectors of several human and animal diseases (155). While

506 wolbachiae (mostly unnamed strains) have been detected in many flea species (50, 51,

507 156), it is pressing from a biocontrol perspective to know if these bacteria affect the

508 ability of fleas to vector pathogens. The cat flea transmits pathogenic bacteria such as

509 Rickettsia typhi (murine typhus), R. felis (murine typhus-like illness), Bartonella spp.

510 (e.g., cat-scratch disease and various other bartonelloses), and to a lesser extent the

511 plague agent Yersinia pestis (157–160). Cat fleas are also intermediate hosts of the

512 tapeworm Dipylidium caninum and the filarial nematode Acanthocheilonema

513 reconditum, both of which can infect humans (161–163). Other organisms observed in

514 C. felis include undescribed Baculoviridae, amoebae, trypanosomatids, cephaline

515 gregarines (Apicomplexa), and microsporidia (164). However, nearly all epidemiological

516 studies have focused on determining the frequency and distribution of only certain

517 pathogens (e.g., Bartonella spp., R. typhi, R. felis), with few documenting wolbachiae

518 co-occurrence with these pathogens (165, 166).

519 The remarkable opposing features of wCfeT and wCfeJ offer testable hypotheses for

520 contrasting host relationships (nutritional symbiosis versus RP, respectively). They also bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 24

521 provide a framework to study the dynamics of Wolbachia co-infections, both in colonies

522 and wild populations. The wCfeT and wCfeJ genomes yield lessons on the role of LGT

523 in shaping host associations, highlighting the ephemeral nature of nutritional-based

524 mutualism and RP over a general rickettsial platform of obligate intracellular parasitism.

525 Host genome sequences provide clues to on-going mutualism and historical RP, and

526 our hypothesis for host CI-gene capture leading to suppression of RP illuminates

527 possible limitations for deployment of Wolbachia reproductive parasites to combat

528 arthropod transmission of human pathogens. In summary, our robust characterization

529 of two divergent strains of C. felis-associated wolbachiae, as well as the C. felis genome

530 itself (45), provides essential information for determining the impact of these bacteria on

531 cat flea biology and pathogen vectoral dynamics.

532

533 Materials and Methods

534 Assembly and annotation of wCfeT and wCfeJ. Our recent report detailed 1) the

535 assembly of Wolbachia reads generated from C. felis genome sequencing, 2) genome

536 circularization and naming of two novel strains (wCfeT and wCfeJ), and 3) complete

537 genome annotation (45). All relevant information pertaining to wCfeT and wCfeJ is

538 available at NCBI under Bioproject PRJNA622233.

539

540 Wolbachiae Phylogenomics. Protein sequences (n=84,836) for 66 sequenced

541 Wolbachia genomes plus 5 additional Anaplasmataceae (Neorickettsia helminthoeca

542 str. Oregon, Anaplasma centrale Israel, A. marginale Florida, Ehrlichia chaffeensis

543 Arkansas, and E. ruminantium Gardel) were either downloaded directly from NCBI bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 25

544 (n=48), retrieved as genome sequences from the NCBI Assembly database (n=13),

545 contributed via personal communication (n=8; Michael Gerth, Oxford Brookes

546 University), or sequenced here (n=2) (Table S1). For genomes lacking functional

547 annotations (n=15), gene models were predicted using the RAST v2.0 server (n=12) or

548 GeneMarkS-2 v1.10_1.07 (n=3; (167)). Ortholog groups (n=3,038) were subsequently

549 constructed using FastOrtho, an in-house version of OrthoMCL (168), using an expect

550 threshold of 0.01, percent identity threshold of 30%, and percent match length threshold

551 of 50% for ortholog inclusion. A subset of single-copy families (n=12) conserved across

552 at least 60 of the 66 genomes were independently aligned with MUSCLE v3.8.31 (169)

553 using default parameters, and regions of poor alignment were masked with trimal

554 v1.4.rev15 (170) using the "automated1" option. All modified alignments were

555 concatenated into a single data set (2,803 positions) for phylogeny estimation using

556 RAxML v8.2.4 (171), under the gamma model of rate heterogeneity and estimation of

557 the proportion of invariant sites. Branch support was assessed with 1,000 pseudo-

558 replications. Final ML optimization likelihood was -52350.085098.

559

560 BOOM characterization. Phylogeny estimation. Genomes (n=93) containing at

561 least 4 of the 6 biotin synthesis genes were identified using Blastp searches against the

562 NCBI nr database (e-value < 0.01; accessed January 9, 2020). Protein sequences for

563 these loci were downloaded from NCBI, aligned with MUSCLE v3.8.31 (169) using

564 default parameters, and regions of poor alignment masked with trimal v1.4.rev15 (170)

565 using the "automated1" option. All modified alignments were concatenated into a single

566 data set (1,457 positions) for phylogeny estimation using RAxML v8.2.4 (171), under the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 26

567 gamma model of rate heterogeneity and estimation of the proportion of invariant sites.

568 Branch support was assessed with 1,000 pseudo-replications. Final ML optimization

569 likelihood was -127140.955066. See Table S2 for NCBI protein accession IDs for all

570 sequences included in this analysis.

571 Gene neighborhood analysis of wCfeT BOOM. Genes in a 25-gene window around

572 the wCfeT BOOM cluster were used to query (Blastp) the NCBI nr database (accessed

573 January 9, 2020). The top 50 matches to each sequence (e-value < 1, BLOSUM45

574 matrix) were binned by taxonomic assignment as "Wolbachia", "other Rickettsiales", or

575 "other (non-Rickettsiales) taxa." Other (non-Rickettsiales) taxa were further binned by

576 major taxonomic group to assess the boundaries and diversity of the wCfeT BOOM

577 MGE. Synteny analysis of biotin genes in wolbachiae. Rickettsiales genomes with at

578 least 5 of the 6 biotin synthesis genes (n=28) were downloaded from NCBI and ortholog

579 groups (n=2,527) constructed with FastOrtho, an in-house version of OrthoMCL (168),

580 using an expect threshold of 0.01, percent identity threshold of 30%, and percent match

581 length threshold of 50% for ortholog inclusion. OGs containing biotin synthesis genes

582 were identified using Rickettsia buchneri str. Wikel sequences as seeds. Genome

583 locations for all genes were retrieved from NCBI in gene file format (gff) and used to

584 order the orthologs in each genome.

585 Phylogenetic estimation of the EamA protein of wCfeT. Taxa were selected from

586 Blastp searches against the NCBI Rickettsiales and nr (excluding Rickettsiales)

587 databases using the wCfeT EamA protein (WP_168464633.1) as a query. Proteins

588 were aligned with MUSCLE v3.8.31 (169) using default parameters, with regions of poor

589 alignment masked using Gblocks (172). Phylogeny was estimated with the WAG bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 27

590 substitution model using RAxML v8.2.4 (171) under the gamma model of rate

591 heterogeneity and estimation of the proportion of invariant sites.

592

593 CI gene characterization. Wolbachiae. wCfeJ CinA and CinB were compared to

594 other RP-inducing TA operons following our previous approach (67). Another novel

595 CndAB operon was identified on a small scaffold (NCBI Nucleotide ID

596 CABPRJ010001055.1) in the aphid Cinara cedri, which has an associated Wolbachia

597 symbiont, wCed (173), and was also characterized in a similar manner. Briefly, EMBL’s

598 Simple Modular Architecture Research Tool (SMART) (174) and /or the Protein

599 Homology/analogY Recognition Engine V 2.0 (Phyre2) (175) were used to predict the

600 following domains: OTU cysteine protease (Pfam OTU, PF02338) (143), endoMS-like

601 (176) and NucS-like (177) PD-(D/E)XK nuclease (Pfam PDDEXK_1, PF12705) (178),

602 CE clan protease (Pfam Peptidase_C1, PF00112) (179), Latrotoxin-CTD (180), and

603 ankyrin repeats. Individual protein schemas were generated using Illustrator of

604 Biological Sequences (181) with manual adjustment.

605 C. felis. A cidA-like gene was identified in the Ctenocephalides felis genome during

606 initial genome decontamination (45). Briefly, a contig composed of a mosaic of flea-

607 and Wolbachia-like sequence was identified by our comparative BLAST-based pipeline

608 and flagged for manual inspection. Gene models on this contig were predicted using

609 the RAST v2.0 server. The resulting proteins were used to create a custom Blast

610 database that was queried using Blastp with CinAB sequences from Wolbachia

611 endosymbiont of Culex quinquefasciatus Pel (CAQ54390, CAQ54391), and CidAB

612 sequences Wolbachia pipientis wAlbB (CCE77512, CCE77513). The contig was bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 28

613 subsequently confirmed as belonging to the largest scaffold (NW_020538040) in the C.

614 felis assembly. A second, cinA-like gene was identified by querying all CDS in the

615 published C. felis assembly (NCBI accession ASM342690v1) with the same set of

616 Wolbachia CinAB and CidAB sequences.

617

618 Screening colonies and wild populations of cat fleas for wCfeT and wCfeJ.

619 Upon receipt from collaborators, cat fleas were stored at -20°C in EtOH until processing

620 for DNA extraction and qPCR. Fleas were sexed under a stereomicroscope and

621 surfaced sterilized for 5 min with 1% bleach solution, followed by 5 min with 70% EtOH,

622 and finally with three washes using molecular grade water. Individual fleas were flash-

623 frozen in liquid N2 and ground to powder with sterile pestle in a 1.5ml microcentrifuge

624 tube. DNA was extracted from dissected tissues using the Zymo Quick-DNA Miniprep

625 plus kit following the solid tissue protocol (Zymo Research; Irvine, CA). The presence

626 of wCfeT, wCfeJ, and C. felis DNA was determined via qPCR using the following primer

627 sets: Cfe#18S|179, 5’-TGCTCACCGTTTGACTTGG-3’ and 5’-

628 GTTTCTCAGGCTCCCTCTCC-3’ (182); wCfeT#ApaG|75: 5’-

629 GCCGTCACTGGCAGGTAATA-3’ and 5’-GCTGTTCTCCAATAACGCCA-3’,

630 wCfeJ#CinA|76, 5’-AGCAACACCAACATGCGATT-3’ and 5’-

631 GAACCCCAGAGTTGGAAGGG-3’. Each primer set amplicon was sequenced before

632 use to ensure specificity. qPCR was performed using a QuantStudio 3 with the

633 PowerUp SYBR green mastermix in a 20µl reaction volume containing 2µl of sample

634 DNA with 400nM of each primer. Cycling parameters were consistent with the “fast

635 cycling mode” including a 2 min cycle at 50°C, a 2 min cycle at 95°C, 40 cycles with 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 29

636 sec at 95°C and 30 seconds at 60°C followed by a melt curve analysis. Standard

637 curves were generated to quantify the number of DNA copies present in each reaction.

638 Results were expressed as the ratio of Wolbachia amplicons to C. felis 18S rDNA

639 copies to normalize for varying efficiency of DNA extractions. Fleas were considered

640 positive for wolbachiae if the flea 18S rDNA Ct value is 30 or lower and the Wolbachia

641 Ct value is 37 or lower.

642

643 Determining wCfeT and wCfeJ localization in C. felis tissues. Elward

644 Laboratories (EL; Soquel, CA) cat fleas stored at -80°C were used to assay tissue

645 localization of wCfeT and wCfeJ. Ovaries and midguts of individual female fleas were

646 dissected under a stereomicroscope. DNA extraction and qPCR were performed as

647 described above.

648

649 Data availability. All relevant information pertaining to wCfeT and wCfeJ is

650 available at NCBI under Bioproject PRJNA622233. Complete genome sequences are

651 available in the NCBI RefSeq database at accession identifiers NZ_CP051156.1

652 (wCfeT) and NZ_CP051157.1 (wCfeJ).

653

654

655 SUPPLEMENTAL MATERIAL

656 Data as well as custom analysis scripts generated for this project that are not published

657 elsewhere are provided on GitHub in the "wCfe_genomes" repository available at

658 https://www.github.com/wvuvectors/wCfe_genomes. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 30

659

660 TABLE S1. Information supporting the Wolbachia genome-based phylogeny estimation.

661

662 TABLE S2. Information supporting the phylogeny estimation of biotin genes.

663

664 TABLE S3. Information supporting the comparative analysis of BOOM gene

665 neighborhoods in wolbachiae genomes.

666

667 FIG S1. Phylogeny estimations of biotin synthesis enzymes and EamA transporters.

668 (A) Complete phylogeny estimation of BOOM and other bio gene sets from diverse

669 bacteria. Tree was estimated from the concatenation of six bio enzymes (BioC, BioH,

670 BioF, BioA, BioD, and BioB) or subsets in certain cases (see Table S2 for all sequence

671 information and Materials and Methods for details on dataset processing and tree

672 estimation). Branch support was assessed with 1,000 pseudo-replications. Final ML

673 optimization likelihood was -127140.955066. In taxon color scheme, [R] denotes

674 Rickettsiales, with Holosporaceae [H] as a revised family of Rhodospirillales (101). The

675 families of Rickettsiales are similarly noted: Anaplasmataceae [A], Midichloriaceae [M],

676 Rickettsiaceae [R], and “Candidatus Deianiaeaceae” [D], with the latter considered

677 provisional (91).

678 (B) Estimated phylogeny of EamA transporters. See Materials and Methods for details

679 on dataset processing and tree estimation. Branch support was assessed with 1,000

680 pseudo-replications. Final ML optimization likelihood was -23058.797227. Taxon color

681 scheme as described in panel A. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 31

682

683 FIG S2. In silico characterization of the wCfeT prophage genome.

684 Schema shows the comparison of the wCfeT and WOVitA1 prophage genomes, with

685 gene descriptions following prior studies (26, 30, 148, 183). wCfeT genes within the

686 typical Eukaryotic Association Module (EAM) were used as queries in Blastp searches

687 against the NCBI nr protein database, with summary statistics provided for top subjects

688 (dashed box).

689

690 FIG S3. Synteny analysis of biotin synthesis genes in Rickettsiales genomes.

691 Rickettsiales taxa with at least 5 of 6 biotin synthesis genes (n=28) were analyzed for

692 co-linearity of their biotin genes. Ortholog groups (n=2,527) were constructed from

693 these genomes as described for Fig. 1 (see Materials and Methods) and OGs

694 containing biotin synthesis genes identified. Genome locations were retrieved from

695 NCBI in gene file format (gff) and used to order the orthologs in each genome. For

696 unclosed genomes, all contigs were included in OG construction; only a single genome

697 (Candidatus Aquarickettsia rohweri) contained biotin synthesis genes on multiple

698 contigs. Biotin genes are colored according to the ideal BOOM shown at the top. Black

699 boxes indicate stretches of unrelated genes. Taxa are grouped into 3 classes according

700 to the synteny of biotin genes in their genomes: Class I taxa demonstrate completely

701 conserved gene order; Class II taxa contain only a single gene out of order (Class IIA:

702 BioA; Class IIB: BioB); Class III taxa exhibit little or no conservation of gene order. All

703 blocks are anchored on BioB for ease of comparison, except Class IIB which are

704 anchored on BioF instead. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 32

705

706 FIG S4. Bioinformatics analysis of BioU and related methyl esterases.

707 (A) Alignment of Ehrlichia chaffeensis and Anaplasma centrale BioU proteins with

708 orthologs from other Rickettsiales species. BioU orthologs were retrieved from Blastp

709 searches against the NCBI Rickettsiales databse using E. chaffeensis str. Sapulpa BioU

710 (EAM85518.1) as a query. The catalytic residues (Ser–Asp–His) characteristic of

711 methyl esterases are highlighted yellow (184). Sequences were aligned with MUSCLE

712 v3.8.31 (169) using default parameters. NCBI protein accession numbers are listed in

713 panel C.

714 (B) Percent identity matrix calculated from the alignment shown in panel A.

715 (C) Estimated phylogeny of proteins shown in panel A. Two alphaproteobacterial

716 outgroups and two hymenopteran mitochondrial methyl esterases are included.

717 Phylogeny was estimated using RAxML v8.2.4 (171) under the gamma model of rate

718 heterogeneity and estimation of the proportion of invariant sites. Branch support was

719 assessed with 1,000 pseudo-replications. Final ML optimization likelihood was -

720 5448.767799.

721 (D) Analysis of eight rickettsial genomes that contain biotin synthesis genes except for

722 the BioH methyl esterase. Blastp searches against the rickettsial genomes were

723 conducted using the following queries, all of which have been shown to orthogonally

724 substitute for BioH: BioV of Helicobacter sp. L8b (TSA87024.1); BioG of Haemophilus

725 influenzae str. CGSHiCZ412602 (AIB45959.1); BtsA of Moraxella catarrhalis str. CCUG

726 353 (KZR94829.1); BioJ of Francisella marina str. E103-15 (QEO56965.1); BioZ of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 33

727 Mesorhizobium japonicum str. R7A (AAG47795.1); EstN1 of Nitrososphaera viennensis

728 str. EN76 (AIC14436.1); BioK of Prochlorococcus marinus str. SS51 (KGG31655.1).

729

730 ACKNOWLEDGMENTS

731 We acknowledge Dr. Michael Gerth (University of Liverpool) for sharing unpublished

732 contigs from sequencing projects for Wolbachia endosymbiont of Zootermopsis

733 nevadensis (wZoo), Wolbachia endosymbiont of Mengenilla moldrzyki (wMen), and

734 Wolbachia endosymbiont of Ctenocephalides felis (wCte). We are grateful to Dr.

735 Michael Dryden (Kansas State University), Dr. Bill Donahue (Sierra Research

736 Laboratories, Modesto, CA), Dr. Lance Durden (Georgia Southern University), and Eric

737 Dotseth (Division of Infectious Disease Epidemiology, West Virginia Department of

738 Health and Human Resources) for contributing cat fleas.

739 This work was supported with funds from the National Institutes of Health National

740 Institute of Allergy and Infectious Diseases grants (R21AI26108 and R21AI146773 to

741 JJG, R01AI017828 and R01AI126853 to AFA, and R01AI122672 to KRM). TPD and

742 VIV were supported by start-up funding provided to TPD by West Virginia University.

743 JFB was supported by start-up funds from Auburn University, USDA Hatch Grant

744 (1015922) and an Alabama Agricultural Experiment Station SEED grant. The content is

745 solely the responsibility of the authors and does not necessarily represent the official

746 views of the funding agencies. The funders had no role in study design, data collection

747 and analysis, decision to publish, or preparation of the manuscript.

748

749 REFERENCES bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 34

750 1. Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH. 2008.

751 How many species are infected with Wolbachia? - A statistical analysis of current

752 data. FEMS Microbiol Lett 281:215–220.

753 2. Gillespie JJ, Nordberg EK, Azad AA, Sobral BW. 2012. Phylogeny And

754 Comparative Genomics: The Shifting Landscape In The Genomics Era, p. 84–

755 141. In A.F. Azad and G.H. Palmer (ed.), Intracellular Pathogens II: Rickettsiales.

756 American Society of Microbiology, Boston.

757 3. Augustinos AA, Santos-Garcia D, Dionyssopoulou E, Moreira M, Papapanagiotou

758 A, Scarvelakis M, Doudoumis V, Ramos S, Aguiar AF, Borges PA V, Khadem M,

759 Latorre A, Tsiamis G, Bourtzis K. 2011. Detection and characterization of

760 Wolbachia infections in natural populations of aphids: is the hidden diversity fully

761 unraveled? PLoS One 6:e28695.

762 4. Baldo L, Werren JH. 2007. Revisiting Wolbachia supergroup typing based on

763 WSP: spurious lineages and discordance with MLST. Curr Microbiol 55:81–7.

764 5. Bing XL, Xia WQ, Gui JD, Yan GH, Wang XW, Liu SS. 2014. Diversity and

765 evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae)

766 whiteflies. Ecol Evol 4:2714–2737.

767 6. Glowska E, Dragun-Damian A, Dabert M, Gerth M. 2015. New Wolbachia

768 supergroups detected in quill mites (Acari: Syringophilidae). Infect Genet Evol

769 30:140–146.

770 7. Haegeman A, Vanholme B, Jacob J, Vandekerckhove TTM, Claeys M, Borgonie

771 G, Gheysen G. 2009. An endosymbiotic bacterium in a plant-parasitic nematode:

772 member of a new Wolbachia supergroup. Int J Parasitol 39:1045–54. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 35

773 8. Lo N, Casiraghi M, Salati E, Bazzocchi C, Bandi C. 2002. How Many Wolbachia

774 Supergroups Exist? Mol Biol Evol 19:341–346.

775 9. Ros VID, Fleming VM, Feil EJ, Breeuwer JAJ. 2009. How diverse is the genus

776 Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia

777 supergroup recovered from spider mites (Acari: Tetranychidae). Appl Environ

778 Microbiol 75:1036–43.

779 10. Chung M, Munro JB, Tettelin H, Dunning Hotopp JC. 2018. Using Core Genome

780 Alignments To Assign Bacterial Species. mSystems 3.

781 11. Lindsey ARI, Bordenstein SR, Newton ILG, Rasgon JL. 2016. Wolbachia pipientis

782 should not be split into multiple species: A response to Ramírez-Puebla et al. Syst

783 Appl Microbiol 39:220–222.

784 12. Ramírez-Puebla ST, Servín-Garcidueñas LE, Ormeño-Orrillo E, Vera-Ponce de

785 León A, Rosenblueth M, Delaye L, Martínez J, Martínez-Romero E. 2015.

786 Species in Wolbachia? Proposal for the designation of ‘Candidatus Wolbachia

787 bourtzisii’, ‘Candidatus Wolbachia onchocercicola’, ‘Candidatus Wolbachia

788 blaxteri’, ‘Candidatus Wolbachia brugii’, ‘Candidatus Wolbachia taylori’,

789 ‘Candidatus Wolbachia collembol. Syst Appl Microbiol 38:390–399.

790 13. Driscoll TP, Verhoeve VI, Guillotte ML, Lehman SS, Rennoll SA, Beier-Sexton M,

791 Rahman MS, Azad AF, Gillespie JJ. 2017. Wholly Rickettsia ! Reconstructed

792 Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells. MBio

793 8:e00859-17.

794 14. Jiménez NE, Gerdtzen ZP, Olivera-Nappa Á, Salgado JC, Conca C. 2019. A

795 systems biology approach for studying Wolbachia metabolism reveals points of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 36

796 interaction with its host in the context of arboviral infection. PLoS Negl Trop Dis

797 13:e0007678.

798 15. Zug R, Hammerstein P. 2015. Bad guys turned nice? A critical assessment of

799 Wolbachia mutualisms in arthropod hosts. Biol Rev Camb Philos Soc.

800 16. Hosokawa T, Koga R, Kikuchi Y, Meng XY, Fukatsu T. 2010. Wolbachia as a

801 bacteriocyte-associated nutritional mutualist. Proc Natl Acad Sci U S A 107:769–

802 774.

803 17. Moriyama M, Nikoh N, Hosokawa T, Fukatsu T. 2015. Riboflavin Provisioning

804 Underlies Wolbachia’s Fitness Contribution to Its Insect Host. MBio 6:e01732-15.

805 18. Ju J-F, Bing X-L, Zhao D-S, Guo Y, Xi Z, Hoffmann AA, Zhang K-J, Huang H-J,

806 Gong J-T, Zhang X, Hong X-Y. 2019. Wolbachia supplement biotin and riboflavin

807 to enhance reproduction in planthoppers. ISME J 1–12.

808 19. Nikoh N, Hosokawa T, Moriyama M, Oshima K, Hattori M, Fukatsu T. 2014.

809 Evolutionary origin of insect-Wolbachia nutritional mutualism. Proc Natl Acad Sci

810 U S A 111:10257–62.

811 20. Newton ILG, Rice DW. 2020. The Jekyll and Hyde symbiont: Could Wolbachia be

812 a nutritional mutualist? J Bacteriol. American Society for Microbiology.

813 21. Gillespie JJ, Joardar V, Williams KP, Driscoll TP, Hostetler JB, Nordberg E,

814 Shukla M, Walenz B, Hill CA, Nene VM, Azad AF, Sobral BW, Caler E. 2012. A

815 Rickettsia genome overrun by mobile genetic elements provides insight into the

816 acquisition of genes characteristic of an obligate intracellular lifestyle. J Bacteriol

817 194:376–94.

818 22. Werren JH, Baldo L, Clark ME. 2008. Wolbachia: master manipulators of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 37

819 invertebrate biology. Nat Rev Microbiol 6:741–51.

820 23. Hurst GDD, Frost CL. 2015. Reproductive parasitism: maternally inherited

821 symbionts in a biparental world. Cold Spring Harb Perspect Biol 7:a017699.

822 24. Chen H, Ronau JA, Beckmann JF, Hochstrasser M. 2019. A Wolbachia Nuclease

823 and Its Binding Partner Comprise a Novel Mechanism for Induction of

824 Cytoplasmic Incompatibility.

825 25. Beckmann JF, Sharma GD, Mendez L, Chen H, Hochstrasser M. 2019. The

826 wolbachia cytoplasmic incompatibility enzyme CIDB targets nuclear import and

827 protamine-histone exchange factors. Elife 8.

828 26. LePage DP, Metcalf JA, Bordenstein SR, On J, Perlmutter JI, Shropshire JD,

829 Layton EM, Funkhouser-Jones LJ, Beckmann JF, Bordenstein SR. 2017.

830 Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic

831 incompatibility. Nature 543:243–247.

832 27. Beckmann JF, Ronau JA, Hochstrasser M. 2017. A Wolbachia deubiquitylating

833 enzyme induces cytoplasmic incompatibility. Nat Microbiol 2:17007.

834 28. Beckmann JF, Fallon AM. 2013. Detection of the Wolbachia protein WPIP0282 in

835 mosquito spermathecae: Implications for cytoplasmic incompatibility. Insect

836 Biochem Mol Biol 43:867–878.

837 29. Perlmutter JI, Bordenstein SR, Unckless RL, LePage DP, Metcalf JA, Hill T,

838 Martinez J, Jiggins FM, Bordenstein SR. 2019. The phage gene wmk is a

839 candidate for male killing by a bacterial endosymbiont. PLoS Pathog 15.

840 30. Bordenstein SR, Bordenstein SR. 2016. Eukaryotic association module in phage

841 WO genomes from Wolbachia. Nat Commun 7:13155. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 38

842 31. Bandi C, Anderson TJC, Genchi C, Blaxter ML. 1998. Phylogeny of Wolbachia in

843 filarial nematodes. Proc R Soc B Biol Sci 265:2407–2413.

844 32. Gerth M, Bleidorn C. 2017. Comparative genomics provides a timeframe for

845 Wolbachia evolution and exposes a recent biotin synthesis operon transfer. Nat

846 Microbiol 2:16241.

847 33. Turelli M, Hoffmann AA. 1991. Rapid spread of an inherited incompatibility factor

848 in California Drosophila. Nature 353:440–442.

849 34. Schmidt TL, Barton NH, Rašić G, Turley AP, Montgomery BL, Iturbe-Ormaetxe I,

850 Cook PE, Ryan PA, Ritchie SA, Hoffmann AA, O’Neill SL, Turelli M. 2017. Local

851 introduction and heterogeneous spatial spread of dengue-suppressing Wolbachia

852 through an urban population of Aedes aegypti. PLoS Biol 15.

853 35. Cooper BS, Ginsberg PS, Turelli M, Matute DR. 2017. Wolbachia in the

854 Drosophila yakuba Complex: Pervasive Frequency Variation and Weak

855 Cytoplasmic Incompatibility, but No Apparent Effect on Reproductive Isolation.

856 Genetics 205:333–351.

857 36. Turelli M. 1994. EVOLUTION OF INCOMPATIBILITY-INDUCING MICROBES

858 AND THEIR HOSTS. Evolution 48:1500–1513.

859 37. Raychoudhury R, Baldo L, Oliveira DCSG, Werren JH. 2009. Modes of acquisition

860 of Wolbachia: Horizontal transfer, hybrid introgression, and codivergence in the

861 Nasonia species complex. Evolution (N Y) 63:165–183.

862 38. Cooper BS, Vanderpool D, Conner WR, Matute DR, Turelli M. 2019. Wolbachia

863 Acquisition by Drosophila yakuba -Clade Hosts and Transfer of Incompatibility

864 Loci Between Distantly Related Wolbachia. Genetics 212:1399–1419. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 39

865 39. Turelli M, Cooper BS, Richardson KM, Ginsberg PS, Peckenpaugh B, Antelope

866 CX, Kim KJ, May MR, Abrieux A, Wilson DA, Bronski MJ, Moore BR, Gao JJ,

867 Eisen MB, Chiu JC, Conner WR, Hoffmann AA. 2018. Rapid Global Spread of

868 wRi-like Wolbachia across Multiple Drosophila. Curr Biol 28:963-971.e8.

869 40. Pietri JE, DeBruhl H, Sullivan W. 2016. The rich somatic life of Wolbachia.

870 Microbiologyopen. Blackwell Publishing Ltd.

871 41. Ramirez-Puebla ST, Ormeno-Orrillo E, Vera-Ponce de Leon A, Lozano L,

872 Sanchez A, Rosenblueth M, Martinez-Romero E. 2016. Genomes of Candidatus

873 Wolbachia Bourtzisii wDacA and Candidatus Wolbachia Pipientis wDacB from the

874 Cochineal Insect Dactylopius coccus (Hemiptera: Dactylopiidae). G3&#58;

875 Genes|Genomes|Genetics 6:3343–3349.

876 42. Gotoh T, Noda H, Ito S. 2007. Cardinium symbionts cause cytoplasmic

877 incompatibility in spider mites. Heredity (Edinb) 98:13–20.

878 43. Chu C, Hoffmann M, Braswell WE, Pelz-Stelinski KS. 2019. Genetic variation and

879 potential coinfection of Wolbachia among widespread Asian citrus psyllid (

880 Diaphorina citri Kuwayama) populations. Insect Sci 26:671–682.

881 44. Landmann F. 2019. The Wolbachia Endosymbionts. Microbiol Spectr 7.

882 45. Driscoll TP, Verhoeve VI, Gillespie J, Johnston JS, Guillotte ML, Rennoll-Bankert

883 KE, Rahman MS, Hagen D, Elsik CG, Macaluso K, Azad A. 2020. Cat fleas in

884 flux: Rampant gene duplication, genome size plasticity, and paradoxical

885 Wolbachia infection. BMC Biol.

886 46. Pornwiroon W, Kearney MT, Husseneder C, Foil LD, Macaluso KR. 2007.

887 Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 40

888 fleas, Ctenocephalides felis. ISME J 1:394–402.

889 47. Sunyakumthorn P, Bourchookarn A, Pornwiroon W, David C, Barker SA,

890 Macaluso KR. 2008. Characterization and growth of polymorphic Rickettsia felis in

891 a tick cell line. Appl Environ Microbiol 74:3151–8.

892 48. Gillespie JJ, Driscoll TP, Verhoeve VI, Utsuki T, Husseneder C, Chouljenko VN,

893 Azad AF, Macaluso KR. 2015. Genomic Diversification in Strains of Rickettsia

894 felis Isolated from Different Arthropods. Genome Biol Evol 7:35–56.

895 49. Casiraghi M, Bordenstein SR, Baldo L, Lo N, Beninati T, Wernegreen JJ, Werren

896 JH, Bandi C. 2005. Phylogeny of Wolbachia pipientis based on gltA, groEL and

897 ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F

898 supergroup, and evidence for further diversity in the Wolbachia tree. Microbiology

899 151:4015–4022.

900 50. Dittmar K, Whiting MF. 2004. New Wolbachia endosymbionts from Nearctic and

901 Neotropical fleas (Siphonaptera). J Parasitol 90:953–7.

902 51. Gorham CH, Fang QQ, Durden LA. 2003. Wolbachia endosymbionts in fleas

903 (Siphonaptera). J Parasitol 89:283–9.

904 52. González-Álvarez VH, de Mera IGF, Cabezas-Cruz A, de la Fuente J, Ortega-

905 Morales AI, Almazán C. 2017. Molecular survey of Rickettsial organisms in

906 ectoparasites from a dog shelter in Northern Mexico. Vet Parasitol Reg Stud

907 Reports 10:143–148.

908 53. Vasconcelos EJR, Billeter SA, Jett LA, Meinersmann RJ, Barr MC, Diniz PPVP,

909 Oakley BB. 2018. Assessing Cat Flea Microbiomes in Northern and Southern

910 California by 16S rRNA Next-Generation Sequencing. Vector-Borne Zoonotic Dis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 41

911 18:491–499.

912 54. Lin S, Cronan JE. 2011. Closing in on complete pathways of biotin biosynthesis.

913 Mol Biosyst.

914 55. Lin S, Hanson RE, Cronan JE. 2010. Biotin synthesis begins by hijacking the fatty

915 acid synthetic pathway. Nat Chem Biol 6:682–688.

916 56. Balvín O, Roth S, Talbot B, Reinhardt K. 2018. Co-speciation in bedbug

917 Wolbachia parallel the pattern in nematode hosts. Sci Rep 8:8797.

918 57. Penz T, Schmitz-Esser S, Kelly SE, Cass BN, Müller A, Woyke T, Malfatti SA,

919 Hunter MS, Horn M. 2012. Comparative Genomics Suggests an Independent

920 Origin of Cytoplasmic Incompatibility in Cardinium hertigii. PLoS Genet

921 8:e1003012.

922 58. Zeng Z, Fu Y, Guo D, Wu Y, Ajayi OE, Wu Q. 2018. Bacterial endosymbiont

923 Cardinium cSfur genome sequence provides insights for understanding the

924 symbiotic relationship in Sogatella furcifera host. BMC Genomics 19:688.

925 59. Ríhová J, Nováková E, Husník F, Hypša V. 2017. Legionella Becoming a

926 Mutualist: Adaptive Processes Shaping the Genome of Symbiont in the Louse

927 Polyplax serrata. Genome Biol Evol 9:2946–2957.

928 60. Klinges JG, Rosales SM, McMinds R, Shaver EC, Shantz AA, Peters EC, Eitel M,

929 Wörheide G, Sharp KH, Burkepile DE, Silliman BR, Vega Thurber RL. 2019.

930 Phylogenetic, genomic, and biogeographic characterization of a novel and

931 ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus

932 Aquarickettsia rohweri, gen. nov., sp. nov. ISME J 13:2938–2953.

933 61. Haferkamp I, Penz T, Geier M, Ast M, Mushak T, Horn M, Schmitz-Esser S. 2013. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 42

934 The Endosymbiont Amoebophilus asiaticus Encodes an S-Adenosylmethionine

935 Carrier That Compensates for Its Missing Methylation Cycle. J Bacteriol

936 195:3183–3192.

937 62. Driscoll TP, Gillespie JJ, Nordberg EK, Azad AF, Sobral BW. 2013. Bacterial DNA

938 sifted from the Trichoplax adhaerens (Animalia: Placozoa) genome project

939 reveals a putative rickettsial endosymbiont. Genome Biol Evol 5:621–45.

940 63. Hellemans S, Kaczmarek N, Marynowska M, Calusinska M, Roisin Y, Fournier D.

941 2019. Bacteriome-associated Wolbachia of the parthenogenetic termite

942 Cavitermes tuberosus. FEMS Microbiol Ecol 95.

943 64. Douglas AE. 2015. Multiorganismal insects: diversity and function of resident

944 microorganisms. Annu Rev Entomol 60:17–34.

945 65. Lindsey ARI, Rice DW, Bordenstein SR, Brooks AW, Bordenstein SR, Newton

946 ILG. 2018. Evolutionary genetics of cytoplasmic incompatibility genes cifA and

947 cifB in prophage WO of Wolbachia. Genome Biol Evol.

948 66. Beckmann JF, Bonneau M, Chen H, Hochstrasser M, Poinsot D, Merçot H, Weill

949 M, Sicard M, Charlat S. 2019. The Toxin–Antidote Model of Cytoplasmic

950 Incompatibility: Genetics and Evolutionary Implications. Trends Genet.

951 67. Gillespie JJ, Driscoll TP, Verhoeve VI, Rahman MS, Macaluso KR, Azad AF.

952 2018. A Tangled Web: Origins of Reproductive Parasitism. Genome Biol Evol

953 10:2292–2309.

954 68. Metcalf JA, Jo M, Bordenstein SR, Jaenike J, Bordenstein SR. 2014. Recent

955 genome reduction of Wolbachia in Drosophila recens targets phage WO and

956 narrows candidates for reproductive parasitism. PeerJ 2:e529. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 43

957 69. Faddeeva-Vakhrusheva A, Kraaijeveld K, Derks MFL, Anvar SY, Agamennone V,

958 Suring W, Kampfraath AA, Ellers J, Le Ngoc G, van Gestel CAM, Mariën J, Smit

959 S, van Straalen NM, Roelofs D. 2017. Coping with living in the soil: the genome of

960 the parthenogenetic springtail Folsomia candida. BMC Genomics 18:493.

961 70. Kampfraath AA, Klasson L, Anvar SY, Vossen RHAM, Roelofs D, Kraaijeveld K,

962 Ellers J. 2019. Genome expansion of an obligate parthenogenesis-associated

963 Wolbachia poses an exception to the symbiont reduction model. BMC Genomics

964 20.

965 71. Leclercq S, Thézé J, Chebbi MA, Giraud I, Moumen B, Ernenwein L, Grève P,

966 Gilbert C, Cordaux R. 2016. Birth of a W sex chromosome by horizontal transfer

967 of Wolbachia bacterial symbiont genome. Proc Natl Acad Sci U S A 113:15036–

968 15041.

969 72. Hotopp JCD, Clark ME, Oliveira DCSG, Foster JM, Fischer P, Torres MCM,

970 Giebel JD, Kumar N, Ishmael N, Wang S, Ingram J, Nene R V., Shepard J,

971 Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko BE, Tettelin H, Werren JH.

972 2007. Widespread Lateral Gene Transfer from Intracellular Bacteria to

973 Multicellular Eukaryotes. Science (80- ) 317:1753–1756.

974 73. Aikawa T, Anbutsu H, Nikoh N, Kikuchi T, Shibata F, Fukatsu T. 2009. Longicorn

975 beetle that vectors pinewood nematode carries many Wolbachia genes on an

976 autosome. Proc R Soc B Biol Sci 276:3791–3798.

977 74. Kondo N, Nikoh N, Ijichi N, Shimada M, Fukatsu T. 2002. Genome fragment of

978 Wolbachia endosymbiont transferred to X chromosome of host insect. Proc Natl

979 Acad Sci U S A 99:14280–14285. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 44

980 75. Dhaygude K, Nair A, Johansson H, Wurm Y, Sundström L. 2019. The first draft

981 genomes of the ant Formica exsecta, and its Wolbachia endosymbiont reveal

982 extensive gene transfer from endosymbiont to host. BMC Genomics 20.

983 76. McNulty SN, Foster JM, Mitreva M, Hotopp JCD, Martin J, Fischer K, Wu B, Davis

984 PJ, Kumar S, Brattig NW, Slatko BE, Weil GJ, Fischer PU. 2010. Endosymbiont

985 DNA in endobacteria-free filarial nematodes indicates ancient horizontal genetic

986 transfer. PLoS One 5.

987 77. Werren JH, Richards S, Desjardins CA, Niehuis O, Gadau J, Colbourne JK,

988 Beukeboom LW, Desplan C, Elsik CG, Grimmelikhuijzen CJP, Kitts P, Lynch JA,

989 Murphy T, Oliveira DCSG, Smith CD, Van Zande L De, Worley KC, Zdobnov EM,

990 Aerts M, Albert S, Anaya VH, Anzola JM, Barchuk AR, Behura SK, Bera AN,

991 Berenbaum MR, Bertossa RC, Bitondi MMG, Bordenstein SR, Bork P, Bornberg-

992 Bauer E, Brunain M, Cazzamali G, Chaboub L, Chacko J, Chavez D, Childers CP,

993 Choi JH, Clark ME, Claudianos C, Clinton RA, Cree AG, Cristino AS, Dang PM,

994 Darby AC, De Graaf DC, Devreese B, Dinh HH, Edwards R, Elango N, Elhaik E,

995 Ermolaeva O, Evans JD, Foret S, Fowler GR, Gerlach D, Gibson JD, Gilbert DG,

996 Graur D, Gründer S, Hagen DE, Han Y, Hauser F, Hultmark D, Hunter Iv HC,

997 Hurst GDD, Jhangian SN, Jiang H, Johnson RM, Jones AK, Junier T, Kadowaki

998 T, Kamping A, Kapustin Y, Kechavarzi B, Kim J, Kim J, Kiryutin B, Koevoets T,

999 Kovar CL, Kriventseva E V., Kucharski R, Lee H, Lee SL, Lees K, Lewis LR,

1000 Loehlin DW, Logsdon JM, Lopez JA, Lozado RJ, Maglott D, Maleszka R,

1001 Mayampurath A, Mazur DJ, McClure MA, Moore AD, Morgan MB, Muller J,

1002 Munoz-Torres MC, Muzny DM, Nazareth L V., Neupert S, Nguyen NB, Nunes bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 45

1003 FMF, Oakeshott JG, Okwuonu GO, Pannebakker BA, Pejaver VR, Peng Z, Pratt

1004 SC, Predel R, Pu LL, Ranson H, Raychoudhury R, Rechtsteiner A, Reese JT,

1005 Reid JG, Riddle M, Hugh M Robertson II, Romero-Severson J, Rosenberg M,

1006 Sackton TB, Sattelle DB, Schlüns H, Schmitt T, Schneider M, Schüler A, Schurko

1007 AM, Shuker DM, Simões ZLP, Sinha S, Smith Z, Solovyev V, Souvorov A,

1008 Springauf A, Stafflinger E, Stage DE, Stanke M, Tanaka Y, Telschow A, Vattathil

1009 CTS, Eveline C Verhulst II, Viljakainen L, Wanner KW, Waterhouse RM, Whitfield

1010 JB, Wilkes TE, Williamson M, Willis JH, Wolschin F, Wyder S, Yamada T, Yi S V.,

1011 Zecher CN, Zhang L, Gibbs RA. 2010. Functional and evolutionary insights from

1012 the genomes of three parasitoid nasonia species. Science (80- ) 327:343–348.

1013 78. Bonneau M, Landmann F, Labbé P, Justy F, Weill M, Sicard M. 2018. The cellular

1014 phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB

1015 diversity. PLOS Pathog 14:e1007364.

1016 79. Madhav M, Parry R, Morgan JAT, James P, Asgari S. 2020. Wolbachia

1017 Endosymbiont of the Horn Fly (Haematobia irritans irritans): a Supergroup A

1018 Strain with Multiple Horizontally Acquired Cytoplasmic Incompatibility Genes. Appl

1019 Environ Microbiol 86.

1020 80. Morrow JL, Schneider DI, Klasson L, Janitz C, Miller WJ, Riegler M. 2020. Parallel

1021 sequencing of Wolbachia wCer2 from donor and novel hosts reveals multiple

1022 incompatibility factors and genome stability after host transfers. Genome Biol

1023 Evol.

1024 81. Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB,

1025 Ware J, Flouri T, Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 46

1026 Wappler T, Rust J, Aberer AJ, Aspock U, Aspock H, Bartel D, Blanke A, Berger S,

1027 Bohm A, Buckley TR, Calcott B, Chen J, Friedrich F, Fukui M, Fujita M, Greve C,

1028 Grobe P, Gu S, Huang Y, Jermiin LS, Kawahara AY, Krogmann L, Kubiak M,

1029 Lanfear R, Letsch H, Li Y, Li Z, Li J, Lu H, Machida R, Mashimo Y, Kapli P,

1030 McKenna DD, Meng G, Nakagaki Y, Navarrete-Heredia JL, Ott M, Ou Y, Pass G,

1031 Podsiadlowski L, Pohl H, von Reumont BM, Schutte K, Sekiya K, Shimizu S,

1032 Slipinski A, Stamatakis A, Song W, Su X, Szucsich NU, Tan M, Tan X, Tang M,

1033 Tang J, Timelthaler G, Tomizuka S, Trautwein M, Tong X, Uchifune T, Walzl MG,

1034 Wiegmann BM, Wilbrandt J, Wipfler B, Wong TKF, Wu Q, Wu G, Xie Y, Yang S,

1035 Yang Q, Yeates DK, Yoshizawa K, Zhang Q, Zhang R, Zhang W, Zhang Y, Zhao

1036 J, Zhou C, Zhou L, Ziesmann T, Zou S, Li Y, Xu X, Zhang Y, Yang H, Wang J,

1037 Wang J, Kjer KM, Zhou X. 2014. Phylogenomics resolves the timing and pattern

1038 of insect evolution. Science (80- ) 346:763–767.

1039 82. Hornett EA, Charlat S, Duplouy AMR, Davies N, Roderick GK, Wedell N, Hurst

1040 GDD. 2006. Evolution of male-killer suppression in a natural population. PLoS

1041 Biol 4:e283.

1042 83. Hornett EA, Moran B, Reynolds LA, Charlat S, Tazzyman S, Wedell N, Jiggins

1043 CD, Hurst GDD. 2014. The Evolution of Sex Ratio Distorter Suppression Affects a

1044 25 cM Genomic Region in the Butterfly Hypolimnas bolina. PLoS Genet

1045 10:e1004822.

1046 84. Bordenstein SR, Uy JJ, Werren JH. 2003. Host genotype determines cytoplasmic

1047 incompatibility type in the haplodiploid genus Nasonia. Genetics 164:223–33.

1048 85. Conner WR, Blaxter ML, Anfora G, Ometto L, Rota-Stabelli O, Turelli M. 2017. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 47

1049 Genome comparisons indicate recent transfer of wRi-like Wolbachia between

1050 sister species Drosophila suzukii and D. subpulchrella. Ecol Evol 7:9391–9404.

1051 86. Hamm CA, Begun DJ, Vo A, Smith CCR, Saelao P, Shaver AO, Jaenike J, Turelli

1052 M. 2014. Wolbachia do not live by reproductive manipulation alone: infection

1053 polymorphism in Drosophila suzukii and D. subpulchrella. Mol Ecol 23:4871–

1054 4885.

1055 87. Poinsot D, Charlat S, Merçot H. 2003. On the mechanism of Wolbachia -induced

1056 cytoplasmic incompatibility: Confronting the models with the facts. BioEssays

1057 25:259–265.

1058 88. Merçot H, Charlat S. 2004. Wolbachia Infections in Drosophila Melanogaster and

1059 D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility. Genetica

1060 120:51–59.

1061 89. Meany MK, Conner WR, Richter S V., Bailey JA, Turelli M, Cooper BS. 2019.

1062 Loss of cytoplasmic incompatibility and minimal fecundity effects explain relatively

1063 low Wolbachia frequencies in Drosophila mauritiana. Evolution (N Y) 73:1278–

1064 1295.

1065 90. Darby AC, Cho N-H, Fuxelius H-H, Westberg J, Andersson SGE. 2007.

1066 Intracellular pathogens go extreme: genome evolution in the Rickettsiales. Trends

1067 Genet 23:511–520.

1068 91. Castelli M, Sabaneyeva E, Lanzoni O, Lebedeva N, Floriano AM, Gaiarsa S,

1069 Benken K, Modeo L, Bandi C, Potekhin A, Sassera D, Petroni G. 2019.

1070 Deianiraea, an extracellular bacterium associated with the ciliate Paramecium,

1071 suggests an alternative scenario for the evolution of Rickettsiales. ISME J. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 48

1072 92. Tashyreva D, Prokopchuk G, Votýpka J, Yabuki A, Horák A, Lukeš J. 2018. Life

1073 Cycle, Ultrastructure, and Phylogeny of New Diplonemids and Their

1074 Endosymbiotic Bacteria. MBio 9.

1075 93. Kamm K, Osigus HJ, Stadler PF, DeSalle R, Schierwater B. 2019. Genome

1076 analyses of a placozoan rickettsial endosymbiont show a combination of

1077 mutualistic and parasitic traits. Sci Rep 9:17561.

1078 94. Gruber-Vodicka HR, Leisch N, Kleiner M, Hinzke T, Liebeke M, McFall-Ngai M,

1079 Hadfield MG, Dubilier N. 2019. Two intracellular and cell type-specific bacterial

1080 symbionts in the placozoan Trichoplax H2. Nat Microbiol. Nature Publishing

1081 Group.

1082 95. Martijn J, Schulz F, Zaremba-Niedzwiedzka K, Viklund J, Stepanauskas R,

1083 Andersson SGE, Horn M, Guy L, Ettema TJG. 2015. Single-cell genomics of a

1084 rare environmental alphaproteobacterium provides unique insights into

1085 Rickettsiaceae evolution. ISME J 9:2373–85.

1086 96. Schulz F, Martijn J, Wascher F, Lagkouvardos I, Kostanjšek R, Ettema TJG, Horn

1087 M. 2016. A Rickettsiales symbiont of amoebae with ancient features. Environ

1088 Microbiol 18:2326–2342.

1089 97. Yurchenko T, Ševčíková T, Přibyl P, El Karkouri K, Klimeš V, Amaral R,

1090 Zbránková V, Kim E, Raoult D, Santos LMA, Eliáš M. 2018. A gene transfer event

1091 suggests a long-term partnership between eustigmatophyte algae and a novel

1092 lineage of endosymbiotic bacteria. ISME J 12:2163–2175.

1093 98. Schrallhammer M, Ferrantini F, Vannini C, Galati S, Schweikert M, Görtz H-D,

1094 Verni F, Petroni G. 2013. “Candidatus Megaira polyxenophila” gen. nov., sp. nov.: bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 49

1095 considerations on evolutionary history, host range and shift of early divergent

1096 rickettsiae. PLoS One 8:e72581.

1097 99. Montagna M, Sassera D, Epis S, Bazzocchi C, Vannini C, Lo N, Sacchi L,

1098 Fukatsu T, Petroni G, Bandi C. 2013. “Candidatus Midichloriaceae” fam. Nov.

1099 (Rickettsiales), an ecologically: Widespread clade of intracellular

1100 alphaproteobacteria. Appl Environ Microbiol 79:3241–3248.

1101 100. Boscaro V, Husnik F, Vannini C, Keeling PJ. 2019. Symbionts of the ciliate

1102 Euplotes : diversity, patterns and potential as models for bacteria–eukaryote

1103 endosymbioses . Proc R Soc B Biol Sci 286:20190693.

1104 101. Muñoz-Gómez SA, Hess S, Burger G, Franz Lang B, Susko E, Slamovits CH,

1105 Roger AJ. 2019. An updated phylogeny of the alphaproteobacteria reveals that

1106 the parasitic rickettsiales and holosporales have independent origins. Elife 8.

1107 102. Mediannikov O, Nguyen T-T, Bell-Sakyi L, Padmanabhan R, Fournier P-E, Raoult

1108 D. 2014. High quality draft genome sequence and description of Occidentia

1109 massiliensis gen. nov., sp. nov., a new member of the family Rickettsiaceae.

1110 Stand Genomic Sci 9:9.

1111 103. Szokoli F, Castelli M, Sabaneyeva E, Schrallhammer M, Krenek S, Doak TG,

1112 Berendonk TU, Petroni G. 2016. Disentangling the Taxonomy of Rickettsiales and

1113 Description of Two Novel Symbionts ("Candidatus Bealeia

1114 paramacronuclearis" and "Candidatus Fokinia cryptica") Sharing

1115 the Cytoplasm of the Ciliate Protist Paramecium biaurelia. Appl Environ Microbiol

1116 82:7236–7247.

1117 104. Prokopchuk G, Tashyreva D, Yabuki A, Horák A, Masařová P, Lukeš J. 2019. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 50

1118 Morphological, Ultrastructural, Motility and Evolutionary Characterization of Two

1119 New Hemistasiidae Species. Protist 170:259–282.

1120 105. Shapiro MM, Chakravartty V, Cronan JE. 2012. Remarkable Diversity in the

1121 Enzymes Catalyzing the Last Step in Synthesis of the Pimelate Moiety of Biotin.

1122 PLoS One 7.

1123 106. Bi H, Zhu L, Jia J, Cronan JE. 2016. A Biotin Biosynthesis Gene Restricted to

1124 Helicobacter. Sci Rep 6.

1125 107. Feng Y, Napier BA, Manandhar M, Henke SK, Weiss DS, Cronan JE. 2014. A

1126 Francisella virulence factor catalyses an essential reaction of biotin synthesis. Mol

1127 Microbiol 91:300–314.

1128 108. Zeng Q, Yang Q, Jia J, Bi H. 2020. A Moraxella Virulence Factor Catalyzes an

1129 Essential Esterase Reaction of Biotin Biosynthesis. Front Microbiol 11:148.

1130 109. Chow J, Danso D, Ferrer M, Streit WR. 2018. The Thaumarchaeon N. gargensis

1131 carries functional bioABD genes and has a promiscuous E. coli ΔbioH-

1132 complementing esterase EstN1. Sci Rep 8.

1133 110. Shi J, Cao X, Chen Y, Cronan JE, Guo Z. 2016. An Atypical α/β-Hydrolase Fold

1134 Revealed in the Crystal Structure of Pimeloyl-Acyl Carrier Protein Methyl Esterase

1135 BioG from Haemophilus influenzae. Biochemistry 55:6705–6717.

1136 111. Estrada P, Manandhar M, Dong SH, Deveryshetty J, Agarwal V, Cronan JE, Nair

1137 SK. 2017. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-

1138 forming enzymes. Nat Chem Biol 13:668–674.

1139 112. Sullivan JT, Brown SD, Yocum RR, Ronson CW. 2001. The bio operon on the

1140 acquired symbiosis island of Mesorhizobium sp. strain R7A includes a novel gene bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 51

1141 involved in pimeloyl-CoA synthesis. Microbiology 147:1315–1322.

1142 113. Hang X, Zeng Q, Zeng L, Jia J, Bi H. 2019. Functional Replacement of the BioC

1143 and BioH Proteins of Escherichia coli Biotin Precursor Biosynthesis by Ehrlichia

1144 chaffeensis Novel Proteins. Curr Microbiol 76:626–636.

1145 114. Audia JP. 2012. Rickettsial Physiology And Metabolism In The Face Of Reductive

1146 Evolution, p. 221–242. In Palmer, GH, Azad, AF (eds.), Intracellular Pathogens II:

1147 Rickettsiales. American Society for Microbiology (ASM).

1148 115. Hebbeln P, Rodionov DA, Alfandega A, Eitinger T. 2007. Biotin uptake in

1149 prokaryotes by solute transporters with an optional ATP-binding cassette-

1150 containing module. Proc Natl Acad Sci 104:2909–2914.

1151 116. Fisher DJ, Fernández RE, Adams NE, Maurelli AT. 2012. Uptake of Biotin by

1152 Chlamydia Spp. through the Use of a Bacterial Transporter (BioY) and a Host-Cell

1153 Transporter (SMVT). PLoS One 7:e46052.

1154 117. Nakayama K, Yamashita A, Kurokawa K, Morimoto T, Ogawa M, Fukuhara M,

1155 Urakami H, Ohnishi M, Uchiyama I, Ogura Y, Ooka T, Oshima K, Tamura A,

1156 Hattori M, Hayashi T. 2008. The Whole-genome sequencing of the obligate

1157 intracellular bacterium Orientia tsutsugamushi revealed massive gene

1158 amplification during reductive genome evolution. DNA Res 15:185–99.

1159 118. Barker DF, Campbell AM. 1981. Genetic and biochemical characterization of the

1160 birA gene and its product: evidence for a direct role of biotin holoenzyme

1161 synthetase in repression of the biotin operon in Escherichia coli. J Mol Biol

1162 146:469–92.

1163 119. Satiaputra J, Shearwin KE, Booker GW, Polyak SW. 2016. Mechanisms of biotin- bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 52

1164 regulated gene expression in microbes. Synth Syst Biotechnol. KeAi

1165 Communications Co.

1166 120. Kamm K, Osigus HJ, Stadler PF, DeSalle R, Schierwater B. 2018. Trichoplax

1167 genomes reveal profound admixture and suggest stable wild populations without

1168 bisexual reproduction. Sci Rep 8:11168.

1169 121. Bublitz DAC, Chadwick GL, Magyar JS, Sandoz KM, Brooks DM, Mesnage S,

1170 Ladinsky MS, Garber AI, Bjorkman PJ, Orphan VJ, McCutcheon JP. 2019.

1171 Peptidoglycan Production by an Insect-Bacterial Mosaic. Cell 179:703-712.e7.

1172 122. McCutcheon JP. 2010. The bacterial essence of tiny symbiont genomes. Curr

1173 Opin Microbiol.

1174 123. Wilson ACC, Duncan RP. 2015. Signatures of host/symbiont genome coevolution

1175 in insect nutritional endosymbioses. Proc Natl Acad Sci U S A 112:10255–10261.

1176 124. Husnik F, Nikoh N, Koga R, Ross L, Duncan RP, Fujie M, Tanaka M, Satoh N,

1177 Bachtrog D, Wilson ACC, von Dohlen CD, Fukatsu T, McCutcheon JP. 2013.

1178 Horizontal gene transfer from diverse bacteria to an insect genome enables a

1179 tripartite nested mealybug symbiosis. Cell 153:1567–78.

1180 125. Nikoh N, McCutcheon JP, Kudo T, Miyagishima SY, Moran NA, Nakabachi A.

1181 2010. Bacterial genes in the aphid genome: Absence of functional gene transfer

1182 from Buchnera to its host. PLoS Genet 6:e1000827.

1183 126. McCutcheon JP. 2016. From microbiology to cell biology: When an intracellular

1184 bacterium becomes part of its host cell. Curr Opin Cell Biol. Elsevier Ltd.

1185 127. McCutcheon JP, Boyd BM, Dale C. 2019. The Life of an Insect Endosymbiont

1186 from the Cradle to the Grave. Curr Biol. Cell Press. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 53

1187 128. Yang Q, Kučerová Z, Perlman SJ, Opit GP, Mockford EL, Behar A, Robinson WE,

1188 Stejskal V, Li Z, Shao R. 2015. Morphological and molecular characterization of a

1189 sexually reproducing colony of the booklouse Liposcelis bostrychophila

1190 (Psocodea: Liposcelididae) found in Arizona. Sci Rep 5:10429.

1191 129. Mockford LE, Krushelnycky DP. 2008. New species and records of Liposcelis

1192 Motschulsky (Psocoptera: Liposcelididae) from Hawaii with first description of the

1193 male of Liposcelis bostrychophila Badonnel. Zootaxa 1766:53–68.

1194 130. Roberts LW, Rapmund G, Cadigan FC. 1977. Sex ratios in rickettsia

1195 tsutsugamushi-infected and noninfected colonies of Leptotrombidium (Acari:

1196 trombiculidae). J Med Entomol 14:89–92.

1197 131. Takahashi M, Urakami H, Yoshida Y, Furuya Y, Misumi H, Hori E, Kawamura A,

1198 Tanaka H. 1997. Occurrence of high ratio of males after introduction of

1199 minocycline in a colony of Leptotrombidium fletcheri infected with Orientia

1200 tsutsugamushi. Eur J Epidemiol 13:79–86.

1201 132. Perotti MA, Clarke HK, Turner BD, Braig HR. 2006. Rickettsia as obligate and

1202 mycetomic bacteria. FASEB J 20:2372–4.

1203 133. Gualtieri L, Nugnes F, Nappo AG, Gebiola M, Bernardo U. 2017. Life inside a gall:

1204 closeness does not favour horizontal transmission of Rickettsia between a gall

1205 wasp and its parasitoid. FEMS Microbiol Ecol 93.

1206 134. Giorgini M, Bernardo U, Monti MM, Nappo AG, Gebiola M. 2010. Rickettsia

1207 Symbionts Cause Parthenogenetic Reproduction in the Parasitoid Wasp Pnigalio

1208 soemius (Hymenoptera: Eulophidae). Appl Environ Microbiol 76:2589–2599.

1209 135. Hagimori T, Abe Y, Date S, Miura K. 2006. The first finding of a Rickettsia bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 54

1210 bacterium associated with parthenogenesis induction among insects. Curr

1211 Microbiol 52:97–101.

1212 136. Nugnes F, Gebiola M, Monti MM, Gualtieri L, Giorgini M, Wang J, Bernardo U.

1213 2015. Genetic Diversity of the Invasive Gall Wasp Leptocybe invasa

1214 (Hymenoptera: Eulophidae) and of its Rickettsia Endosymbiont, and Associated

1215 Sex-Ratio Differences. PLoS One 10:e0124660.

1216 137. Werren JH, Hurst GD, Zhang W, Breeuwer JA, Stouthamer R, Majerus ME. 1994.

1217 Rickettsial relative associated with male killing in the ladybird beetle (Adalia

1218 bipunctata). J Bacteriol 176:388–94.

1219 138. von der Schulenburg JH, Habig M, Sloggett JJ, Webberley KM, Bertrand D, Hurst

1220 GD, Majerus ME. 2001. Incidence of male-killing Rickettsia spp. (alpha-

1221 proteobacteria) in the ten-spot ladybird beetle Adalia decempunctata L.

1222 (Coleoptera: Coccinellidae). Appl Environ Microbiol 67:270–7.

1223 139. Lawson ET, Mousseau TA, Klaper R, Hunter MD, Werren JH. 2001. Rickettsia

1224 associated with male-killing in a buprestid beetle. Heredity (Edinb) 86:497–505.

1225 140. Majerus TM, Majerus ME. 2010. Discovery and identification of a male-killing

1226 agent in the Japanese ladybird Propylea japonica (Coleoptera: Coccinellidae).

1227 BMC Evol Biol 10:37.

1228 141. Murray GGR, Weinert LA, Rhule EL, Welch JJ. 2016. The Phylogeny of Rickettsia

1229 Using Different Evolutionary Signatures: How Tree-Like is Bacterial Evolution?

1230 Syst Biol 65:265–79.

1231 142. Harumoto T, Lemaitre B. 2018. Male-killing toxin in a bacterial symbiont of

1232 Drosophila. Nature 557:252–255. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 55

1233 143. Makarova KS, Aravind L, Koonin E V. 2000. A novel superfamily of predicted

1234 cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends

1235 Biochem Sci 25:50–2.

1236 144. Badawi M, Moumen B, Giraud I, Grève P, Cordaux R. 2018. Investigating the

1237 Molecular Genetic Basis of Cytoplasmic Sex Determination Caused by Wolbachia

1238 Endosymbionts in Terrestrial Isopods. Genes (Basel) 9:290.

1239 145. Gebiola M, Giorgini M, Kelly SE, Doremus MR, Ferree PM, Hunter MS. 2017.

1240 Cytological analysis of cytoplasmic incompatibility induced byCardiniumsuggests

1241 convergent evolution with its distant cousinWolbachia. Proceedings Biol Sci

1242 284:20171433.

1243 146. Siozios S, Pilgrim J, Darby AC, Baylis M, Hurst GDD. 2019. The draft genome of

1244 strain cCpun from biting midges confirms insect Cardinium are not a monophyletic

1245 group and reveals a novel gene family expansion in a symbiont. PeerJ 2019.

1246 147. Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstädter J, Hurst GD.

1247 2008. The diversity of reproductive parasites among arthropods: Wolbachia do

1248 not walk alone. BMC Biol 6.

1249 148. Ishmael N, Hotopp JCD, Loanidis P, Biber S, Sakamoto J, Siozios S, Nene V,

1250 Werren J, Boutriz K, Bordenstein SR, Tettelin H. 2009. Extensive genomic

1251 diversity of closely related wolbachia strains. Microbiology 155:2211–2222.

1252 149. Baldridge GD, Markowski TW, Witthuhn BA, Higgins L, Baldridge AS, Fallon AM.

1253 2016. The Wolbachia WO bacteriophage proteome in the Aedes albopictus

1254 C/wStr1 cell line: evidence for lytic activity? Vitr Cell Dev Biol - Anim 52:77–88.

1255 150. Batty EM, Chaemchuen S, Blacksell S, Richards AL, Paris D, Bowden R, Chan C, bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 56

1256 Lachumanan R, Day N, Donnelly P, Chen S, Salje J. 2018. Long-read whole

1257 genome sequencing and comparative analysis of six strains of the human

1258 pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis 12:e0006566.

1259 151. Cho N-H, Kim H-R, Lee J-H, Kim S-Y, Kim J, Cha S, Kim S-Y, Darby AC, Fuxelius

1260 H-H, Yin J, Kim JH, Kim J, Lee SJ, Koh Y-S, Jang W-J, Park K-H, Andersson

1261 SGE, Choi M-S, Kim I-S. 2007. The Orientia tsutsugamushi genome reveals

1262 massive proliferation of conjugative type IV secretion system and host cell

1263 interaction genes. Proc Natl Acad Sci 104:7981–7986.

1264 152. Nakayama K, Kurokawa K, Fukuhara M, Urakami H, Yamamoto S, Yamazaki K,

1265 Ogura Y, Ooka T, Hayashi T. 2010. Genome comparison and phylogenetic

1266 analysis of Orientia tsutsugamushi strains. DNA Res 17:281–91.

1267 153. Hagen R, Verhoeve VI, Gillespie JJ, Driscoll TP. 2018. Conjugative transposons

1268 and their cargo genes vary across natural populations of Rickettsia buchneri

1269 infecting the tick Ixodes scapularis. Genome Biol Evol 10:3218–3229.

1270 154. Reveillaud J, Bordenstein SR, Cruaud C, Shaiber A, Esen ÖC, Weill M,

1271 Makoundou P, Lolans K, Watson AR, Rakotoarivony I, Bordenstein SR, Eren AM.

1272 2019. The Wolbachia mobilome in Culex pipiens includes a putative plasmid. Nat

1273 Commun 10.

1274 155. Rust M. 2017. The Biology and Ecology of Cat Fleas and Advancements in Their

1275 Pest Management: A Review. Insects 8:118.

1276 156. Lawrence AL, Hii S-F, Chong R, Webb CE, Traub R, Brown G, Šlapeta J. 2015.

1277 Evaluation of the bacterial microbiome of two flea species using different DNA-

1278 isolation techniques provides insights into flea host ecology. FEMS Microbiol Ecol bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 57

1279 91:fiv134.

1280 157. Glickman LT, Moore GE, Glickman NW, Caldanaro RJ, Aucoin D, Lewis HB.

1281 2006. Purdue University-Banfield National Companion Animal Surveillance

1282 Program for emerging and zoonotic diseases. Vector Borne Zoonotic Dis 6:14–23.

1283 158. Nogueras MM, Pons I, Ortuño A, Miret J, Pla J, Castellà J, Segura F. 2013.

1284 Molecular detection of Rickettsia typhi in cats and fleas. PLoS One 8:e71386.

1285 159. Angelakis E, Mediannikov O, Parola P, Raoult D. 2016. Rickettsia felis: The

1286 Complex Journey of an Emergent Human Pathogen. Trends Parasitol 32:554–

1287 564.

1288 160. Leulmi H, Socolovschi C, Laudisoit A, Houemenou G, Davoust B, Bitam I, Raoult

1289 D, Parola P. 2014. Detection of Rickettsia felis, Rickettsia typhi, Bartonella

1290 Species and Yersinia pestis in Fleas (Siphonaptera) from Africa. PLoS Negl Trop

1291 Dis 8.

1292 161. Fourie JJ, Crafford D, Horak IG, Stanneck D. 2012. Prophylactic treatment of flea-

1293 infested cats with an imidacloprid/flumethrin collar to forestall infection with

1294 Dipylidium caninum. Parasit Vectors 5:151.

1295 162. Brianti E, Gaglio G, Napoli E, Giannetto S, Dantas-Torres F, Bain O, Otranto D.

1296 2012. New insights into the ecology and biology of Acanthocheilonema

1297 reconditum (Grassi, 1889) causing canine subcutaneous filariosis. Parasitology

1298 139:530–6.

1299 163. Traversa D. 2013. Fleas infesting pets in the era of emerging extra-intestinal

1300 nematodes. Parasit Vectors 6:59.

1301 164. Beard CB, Butler JF, Hall DW. 1990. Prevalence and biology of endosymbionts of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 58

1302 fleas (Siphonaptera: Pulicidae) from dogs and cats in Alachua County, Florida. J

1303 Med Entomol 27:1050–61.

1304 165. Tay ST. 2013. Wolbachia endosymbionts, Rickettsia felis and Bartonella species,

1305 in Ctenocephalides felis fleas in a tropical region. J Vector Ecol 38:200–202.

1306 166. Oteo JA, Portillo A, Portero F, Zavala-Castro J, Venzal JM, Labruna MB. 2014.

1307 `Candidatus Rickettsia asemboensis’ and Wolbachia spp. in Ctenocephalides felis

1308 and Pulex irritans fleas removed from dogs in Ecuador. Parasit Vectors 7:455.

1309 167. Lomsadze A, Gemayel K, Tang S, Borodovsky M. 2018. Modeling leaderless

1310 transcription and atypical genes results in more accurate gene prediction in

1311 prokaryotes. Genome Res 28:1079–1089.

1312 168. Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for

1313 eukaryotic genomes. Genome Res 13:2178–89.

1314 169. Edgar RC. 2004. MUSCLE: Multiple sequence alignment with high accuracy and

1315 high throughput. Nucleic Acids Res 32:1792–1797.

1316 170. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool for

1317 automated alignment trimming in large-scale phylogenetic analyses.

1318 Bioinformatics 25:1972–3.

1319 171. Stamatakis A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-

1320 analysis of large phylogenies. Bioinformatics 30:1312–1313.

1321 172. Talavera G, Castresana J. 2007. Improvement of phylogenies after removing

1322 divergent and ambiguously aligned blocks from protein sequence alignments.

1323 Syst Biol 56:564–77.

1324 173. Gómez-Valero L, Soriano-Navarro M, Pérez-Brocal V, Heddi A, Moya A, García- bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 59

1325 Verdugo JM, Latorre A. 2004. Coexistence of Wolbachia with Buchnera

1326 aphidicola and a secondary symbiont in the aphid Cinara cedri. J Bacteriol

1327 186:6626–33.

1328 174. Letunic I, Bork P. 2017. 20 years of the SMART protein domain annotation

1329 resource. Nucleic Acids Res.

1330 175. Kelley LA, Sternberg MJE. 2009. Protein structure prediction on the Web: a case

1331 study using the Phyre server. Nat Protoc 4:363–371.

1332 176. Nakae S, Hijikata A, Tsuji T, Yonezawa K, Kouyama K-I, Mayanagi K, Ishino S,

1333 Ishino Y, Shirai T. 2016. Structure of the EndoMS-DNA Complex as Mismatch

1334 Restriction Endonuclease. Structure 24:1960–1971.

1335 177. Ren B, Kühn J, Meslet-Cladiere L, Briffotaux J, Norais C, Lavigne R, Flament D,

1336 Ladenstein R, Myllykallio H. 2009. Structure and function of a novel endonuclease

1337 acting on branched DNA substrates. EMBO J 28:2479–89.

1338 178. Kinch LN, Ginalski K, Rychlewski L, Grishin N V. 2005. Identification of novel

1339 restriction endonuclease-like fold families among hypothetical proteins. Nucleic

1340 Acids Res 33:3598–3605.

1341 179. Pruneda JN, Durkin CH, Geurink PP, Ovaa H, Santhanam B, Holden DW,

1342 Komander D. 2016. The Molecular Basis for Ubiquitin and Ubiquitin-like

1343 Specificities in Bacterial Effector Proteases. Mol Cell 63:261–76.

1344 180. Zhang D, de Souza RF, Anantharaman V, Iyer LM, Aravind L. 2012. Polymorphic

1345 toxin systems: Comprehensive characterization of trafficking modes, processing,

1346 mechanisms of action, immunity and ecology using comparative genomics. Biol

1347 Direct 7:18. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 60

1348 181. Liu W, Xie Y, Ma J, Luo X, Nie P, Zuo Z, Lahrmann U, Zhao Q, Zheng Y, Zhao Y,

1349 Xue Y, Ren J. 2015. IBS: an illustrator for the presentation and visualization of

1350 biological sequences. Bioinformatics 31:3359–61.

1351 182. Reif KE, Stout RW, Henry GC, Foil LD, Macaluso KR. 2008. Prevalence and

1352 infection load dynamics of Rickettsia felis in actively feeding cat fleas. PLoS One

1353 3:e2805.

1354 183. Kent BN, Funkhouser LJ, Setia S, Bordenstein SR. 2011. Evolutionary genomics

1355 of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia).

1356 PLoS One 6.

1357 184. Polgár L. 2005. The catalytic triad of serine peptidases. Cell Mol Life Sci.

1358 185. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K,

1359 Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL,

1360 Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich

1361 C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST

1362 Server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75.

1363 186. Perlman SJ, Hunter MS, Zchori-Fein E. 2006. The emerging diversity of

1364 Rickettsia. Proc Biol Sci 273:2097–106.

1365

1366

1367 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 61

1368 Table 1. Summary statistics of selected Wolbachia genomes.

Protein Length GC Pseudo- Mean gene Coding density Taxon RefSeq ID SG coding (nt) (%) genes length (nt) (coding/total nt) genes

wFol NZ_CP015510.2 E 1801626 34.4 1537 85 976 0.880

wCfeT NZ_CP051156.1 ? 1495538 35.2 1424 201 890 0.727 wOv NZ_HG810405.1 C 960618 32.1 650 45 967 0.700

wOo NC_018267.1 C 957990 32.1 639 73 968 0.719 wCfeJ NZ_CP051157.1 ? 1201647 35.6 1116 51 917 0.813

wCle NZ_AP013028.1 F 1250060 36.3 1012 210 818 0.829 wBm NZ_CP034333.1 D 1080064 34.2 803 178 868 0.789

wBm NC_006833.1 D 1080084 34.2 839 167 854 0.795 wMau NZ_CP034334.1 B 1273527 34.0 1045 136 944 0.876

wMau NZ_CP034335.1 B 1273530 34.0 1047 131 946 0.876 wNo NC_021084.1 B 1301823 34.0 1066 133 952 0.878 wAlbB NZ_CP041923.1 B 1482279 34.5 1190 195 917 0.858 FL2016 wAlbB NZ_CP041924.1 B 1483853 34.4 1200 187 917 0.858 HN2016 wAlbB NZ_CP031221.1 B 1484007 34.4 1197 191 916 0.858 wMeg NZ_CP021120.1 B 1376868 34.0 1136 125 948 0.869

wHa NC_021089.1 A 1295804 35.1 1123 104 899 0.852 wCauA NZ_CP041215.1 A 1449344 35.0 1260 134 861 0.860

wAna NZ_CP042904.1 A 1401460 35.2 1218 107 893 0.857 wRi NC_012416.1 A 1445873 35.2 1245 122 892 0.859

wAu NZ_LK055284.1 A 1268461 35.2 1099 125 878 0.855 wMel NC_002978.6 A 1267782 35.2 1116 129 863 0.857

wMel I23 NZ_CP042444.1 A 1269137 35.2 1119 128 862 0.857 wMel N25 NZ_CP042446.1 A 1267781 35.2 1120 125 863 0.857

wMel ZH26 NZ_CP042445.1 A 1267436 35.2 1117 125 865 0.857 wIrr NZ_CP037426.1 A 1352354 35.3 1232 115 827 0.855 1369

1370

1371 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 62

1372 Figure Legends

1373

1374 Figure 1. Wolbachia genome-based phylogeny estimation.

1375 Wolbachia supergroups are within colored ellipses. Ctenocephalides felis-associated

1376 Wolbachiae are within black boxes, with red (wCfeT) and blue (wCfeJ) stars depicting

1377 the two novel Wolbachia parasites infecting C. felis. Gray inset: wCfeT and wCfeJ

1378 genome sequences and assembly statistics. White inset: color scheme for nematode

1379 and arthropod hosts. Phylogeny was estimated for 66 Wolbachia and five outgroup

1380 genomes (see Materials and Methods for more details). Branch support was assessed

1381 with 1,000 pseudoreplications. Final ML optimization likelihood was -52350.085098.

1382 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Neorickettsia helminthoeca str. Oregon Anaplasma centrale str. Israel branch lengths Anaplasma marginale str. Florida other Anaplasmataceae reduced by 200% Ehrlichia chaffeensis str. Arkansas Ehrlichia ruminantium str. Gardel wPpe (Pratylenchus penetrans) wPni (Pentalonia nigronervosa) wFol (Folsomia candida) wCfeT (Ctenocephalidis felis) B wOv (Onchocerca volvulus) wOo (Onchocerca ochengi) C wDim (Dirofilaria immitis) wCfeJ (Ctenocephalidis felis) bootstrap wCle (Cimex lectularius) B .91-1 wMen (Mengenilla moldrzyki) F .81-.90 wOc (Osmia caerulescens) .71-.80 wLs (Litomosoides sigmodontis) Fig. 1 .61-.70 wBm (Brugia malayi) .51-.60 D < .50 wWb (Wucheria bancrofti) wVulC-f (Armadillidium vulgare) B wCon (Cylisticus convexus) wStr (Laodelphax striatella) B wLug (Nilaparvata lugens) B wDi (Diaphorina citri) 0.1 sub./site wBta (Bemisia tabaci) (Wolbachiae only) wMau (Drosophila mauritiana) wNo (Drosophila simulans) wDacB (Dactylopius coccus) wCoc1 (Dactylopius coccus) wVitB (Nasonia vitripennis) wLcl (Leptopilina clavipes) wAlbB (Aedes albopictus) Biotin synthesis Operon wAlbB-2 (Aedes albopictus) B of Obligate intracellular wCte (Ctenocephalidis felis) wTpre (Trichogramma pretiosum) Microbes (BOOM) wPip_Pel (Culex quinquefasciatus) wPip_Mol (Culex molestus) wPip_JHB (Culex quinquefasciatus) lineages outside of wOb_Wba (Operophtera brumata) Supergroups A and B wAus (Plutella australiana) wBol1-b (Hypolimnas bolina) lacking complete wMeg (Chrysomya megacephala) B Cid or Cin operons wZoo (Zootermopsis nevadensis) wDacA (Dactylopius coccus) wFex (Formica exsecta) wNfe (Nomada ferruginata) wNfla (Nomada flava) B wNleu (Nomada leucophthalma) B Arthropoda wNpa (Nomada panzeri) wVitA (Nasonia vitripennis) Isopoda Crustacea wUni_MUJL (Muscidifurax uniraptor) wUni_ACFP (Muscidifurax uniraptor) Collembola wGmm (Glossina morsitans morsitans) Hemiptera wNon (Nasonia oneida) wHa (Drosophila simulans) Blattodea wCauA (Carposina sasakii) Hymenoptera wSuzi (Drosophila suzukii) Hexapoda wSpc (Drosophila subpulchrella) Strepsiptera wAna (Drosophila ananassae) Lepidoptera wSim (Drosophila simulans) wRi (Drosophila simulans) Siphonaptera wAu (Drosophila simulans) wWil (Drosophila willistoni) Diptera wRec (Drosophila recens) wInc_Cu (Drosophila incompta) Nematoda wInc_SM (Drosophila incompta) wMel (Drosophila melanogaster) Tylenchoidea (plant pathogen) wMelPop (Drosophila melanogaster) Filaroidea (Spirurida) wDya (Drosophila yakuba) wDsa (Drosophila santomea) wDte (Drosophila teissieri) A bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 64

1384 FIG 2. wCfeT carries the Biotin synthesis Operon of Obligate intracellular

1385 Microbes (BOOM).

1386 (A) Metabolism of biotin from malonyl-CoA (55).

1387 (B) Comparisons of wCfeT BOOM to equivalent loci in Cardinium endosymbiont of

1388 Encarsia pergandiella (cEper1, CCM10336-CCM10341) and Wolbachia endosymbiont

1389 of Cimex lectularius (wCle, BAP00143-BAP00148). Red shading and numbers indicate

1390 % identity across pairwise protein alignments (Blastp). Gene colors correspond to

1391 enzymatic steps depicted in panel A, with all proteins drawn to scale (as a reference,

1392 wCfeT BioB is 316 aa).

1393 (C) Phylogeny of BOOM and other bio gene sets from diverse bacteria estimated from

1394 the concatenation of six bio enzymes (BioC, BioH, BioF, BioA, BioD, and BioB); see

1395 Table S2 for all sequence information, and Figure S1A for complete tree estimated

1396 under Maximum Likelihood. In taxon color scheme, [R] denotes Rickettsiales, with

1397 Holosporaceae as a revised family of Rhodospirillales (101).

1398 (D) Analysis of genes flanking wCfeT BOOM. See Table S3 for comparison with other

1399 BOOM-containing wolbachiae. Graph depicts top 50 Blastp subjects (e-value < 1,

1400 BLOSUM45 matrix) using the wCfeT proteins as queries against the NCBI nr database

1401 (January 9, 2020). Colors distinguish three taxonomic groups (see inset). Black

1402 columns: no significant similarity (nss) in the NCBI nr database. Tnp, predicted

1403 transposase. Brown, pseudogenization. See Figure S1B for EamA phylogeny

1404 estimation. Pie charts across the bottom delineate taxonomic diversity of hits to “Other

1405 taxa,” for wCfeT proteins with significant Blastp matches outside of the Rickettsiales.

1406 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NCBioB 4.0 InternationalBioF licenseBioH. BioC BioD BioA A Malonyl-CoA B cEper1 .83 .74 .60 .60 .67 .71 BioC SAM SAH wCfeT .86 .85 .69 .71 .80 .83 Malonyl-CoA methyl ester wCle

fatty acid Fig. 2 synthesis (2 cycles) C “Candidatus Paracaedibacter_symbiosus” Pimeloyl-CoA methyl ester “Candidatus Odyssella thessalonicensis” “Candidatus Deianiraea vastatrix” “Candidatus Midichloria mitochondrii” str. IricVA BioH H 2 O CH3OH Occidentia massiliensis Neorickettsia spp. (5) Pimeloyl-CoA Rickettsia buchneri (2) Lawsonia intracellularis (2) “Candidatus Fokinia solitaria” BioF L-Ala ACP + CO2 Rickettsiales endosymbiont (Peranema trichophorum) Holospora spp. (4) KAPA Legionella endosymbiont (Polyplax serrata) Cadinium endosymbionts (3) AMTOB SAH wCfeT BioA bootstrap wLug .91-1 wStr DAPA .81-.90 wNfla .71-.80 wNleu .61-.70 wVulC CO2 + ATP AMP + PPi BioD .51-.60 sp. KTCN < .50 sp. SYDL Cimex- Dethiobiotin sp. TIH associated 0.1 sub./site wCle BioB SAM 5’-DOA sp. TUA “Cand. Deianiaeaceae” [R] Midichloriaceae [R] Holosporaceae Legionellales Biotin Anaplasmataceae [R] Rickettsiaceae [R] Deltaproteobacteria Bacteroidetes

D EamA BOOM nsstnp tnp nsstnp tnp tnp nss bf h c d a nss 1.0

0.8

0.6

Wolbachia Other Rickettsiales Normalized bitscore 0.4 Other taxa

0.2 -25 -20 -15 -10 -5 5 10 15 20

Alphaproteobacteria Gammaproteobacteria PVC Group Other Bacteria non-Bacteria

Holosporaceae Chromotiales Chlamydiae Actinobacteria Arthropoda Rhizobiales Legionellales Others Cyanobacteria Rhodospirillales Oceanospirillales Others Others Thiotrichales Other taxa Others (non-Rickettsiales) bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 66

1408 FIG 3. wCfeJ lends insight on the evolution of reproductive parasitism.

1409 (A) Comparison of known and predicted RP-inducing toxin-antidote operons of diverse

1410 rickettsial species. Light green: CidA/B of wPip_Pel (CAQ54390, CAQ54391); grey:

1411 CndA/B of pLbAR_36/38 (KHO02168, KHO02170), Rickettsia gravesii

1412 (WP_017442886, WP_024547315), wDacB (OAM06111, OAM06112), wStr

1413 (OAB79364, OAB79365), wVulC (OJH31528, OJH31527), wCon (RDD34163,

1414 RDD34164); light brown, CinA/B of wCfeJ (blue star, WP_168456002 &

1415 WP_168456001), Orientia tsutsugamushi (CAM80637, CAM80639), wRi (ACN95432,

1416 ACN95431), wVita (ONI57866, ONI57867), and wPip_Pel (CAQ54402, CAQ54403).

1417 Type I-IV CI operons of Wolbachia parasites (26) are distinguished. Protein domains as

1418 follows: green, CE clan protease; brown, PD-(D/E)XK nuclease; orange, OTU cysteine

1419 protease. Red shading and numbers indicate % identity across pairwise alignments.

1420 Proteins are drawn to scale, with additional C-terminal sequence for some CndB toxins

1421 depicted in dark gray boxes.

1422 (B) Sequence similarity profiles for wCfeJ’s CinA antidote (top) and CinA toxin

1423 (bottom). Subjects are from a Blastp search against the NCBI ‘Wolbachia’ database.

1424 Size, amino acids. Antidotes with cognate toxins (operon partners) also recovered in

1425 searches are noted with asterisks. Top seven hits are shown, with matches (where

1426 significant) to proteins of types II-IV TA operons shown below dashed lines. Toxin

1427 domains are from our prior report or predicted newly here using the EMBL’s Simple

1428 Modular Architecture Research Tool (SMART) (174). Only NUC and DUB domains are

1429 listed, though larger toxins contain many other predicted domains. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 67

1430 (C) Comparison of wCfeJ and wDacB degraded WO prophage genomes captures

1431 streamlining of a CI-like TA operon from a Cnd operon. WO prophage coordinates:

1432 wCfeJ, WP_168455994-WP_168456003; wDacB, OAM06111-OAM06118

1433 (LSYY01000098). Schema follows the description of the WOVitA prophage (30).

1434 (D) wCfeJ’s CinB-like toxin evolved from gene fission of a larger CndB-like toxin. NCBI

1435 protein accession numbers: wCfeJ (WP_168455999, WP_168456000, and

1436 WP_168456001); wDacB (OAM06111, OAM06112). Red shading and numbers

1437 indicate % identity across pairwise alignments. Proteins are drawn to scale; as a

1438 reference, wDacB CndB is 3707 aa.

1439 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Cognate A wPip_Pel CidA CidB type I B Subject Acc. no. Size %ID toxin 22 22 36 Cid R. felis 1387 wCed VVC35096 434 77.0 VVC35097 * wDacB OAM06474 437 70.0 None 19 23 26 wStr OAB79341 430 63.1 OAB79342 * R. gravesii 900 wCauA QDH18678 451 53.0 QDH18679 * 24 26 wVulC-f OJH31528 415 49.3 OJH31527 * wDacB 1784 wCon RDD34511 424 48.0 None

82 73 CinA antitode wCon RDD34438 421 48.0 None Cnd wStr wRi ACN95432 456 NS ACN95431

57 45 38 64 CfeJ wVita ONI57866 490 NS ONI57867 wVulC 939 w wPip_Pel CAQ54402 445 34.0 CAQ54403 74 77 wCon Subject Acc. no. Size %ID Domains 54 48 wCfeJ wCed VVC35097 2500 65.0 NUC (2) 27 25 wCauA QDH18679 3644 49.0 NUC (2) O. tsutsugamushi wStr OAB79342 3941 50.0 NUC (2) wCon RDD34164 1351 48.0 DUB-NUC (2) 21 30 35 wCed VVC35051 1964 46.0 NUC (2) wRi type II

Cin wVulC-f OJH31527 2770 47.8 OTU-NUC (2) CinB toxin OTU 33 36 wCon RDD33817 875 45.0 NUC (2) DUB wVita type III

CfeJ wRi ACN95431 456 26.0 NUC (2) 29 31 NUC w wVita ONI57867 490 27.0 NUC (2) wPip_Pel CinA CinB type IV wPip_Pel CAQ54403 445 34.0 NUC (2)

C toxin gene fission Syntenic with WOVitA1 Hypothetical Protein (HP) wCfeJ Transposase CndA/B and fission products Eukaryotic Association Module wDacB Recombinase MutL HTH RadC HP HTH HTH Pat D CinA CinB HP HP HP (Latrotoxin-CTD) wCfeJ

32 55 37 65 35 38 34

wDacB CndA CndB Ankyrin-repeat region Fig. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 69

1441 FIG 4. Identification of two CI-like antidote genes in the cat flea genome.

1442 (A) The largest C. felis scaffold (NW_020538040) contains a CidA-like gene, predicted

1443 using RAST (185) (left), and a CinA-like exon encoded within a larger gene model with

1444 transcriptional support (right). Red, CI-like antidote genes. Blue, C. felis genes:

1445 113377302 (RING finger and SPRY domain-containing protein 1-like); 113377491

1446 (negative elongation factor B); 113377978 (uncharacterized). The CinA-like exon

1447 (XP_026474240) contains a corresponding transcript (dark blue, XM_026618455)

1448 generated from a study (81) independent of the cat flea genome sequencing (45).

1449 (B) Sequence similarity profiles for the C. felis CidA-like (left) and CinA-like (right)

1450 antidotes. Subjects above the black dashed line are the top hits from a Blastp search

1451 against the NCBI nr protein database; subjects below are weaker matches to CinA (left)

1452 and CidA (right) proteins; matches to wCfeJ CinA are below the orange line. Size,

1453 amino acids.

1454 (C) Alignment of C. felis CidA-like and CinA-like antidotes to CidA, CndA and CinA

1455 proteins from selected Rickettsiaceae species and Wolbachia strains. Schema at top

1456 depicts the wCfeJ CinA antidote and location of six conserved regions shown in the

1457 alignment below. Amino acid coloring as follows: black, hydrophobic; red, negatively

1458 charged; green, hydrophilic; purple, aromatic; blue, positively charged. Alignment

1459 generated using MUSCLE, default parameters (169).

1460

1461 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A XM_026618455 Predicted with RAST XP_026474240

73 Mb 113377302 113377491 113377978

B C. felis CidA-like C. felis CinA-like

Subject Acc. no. Size %ID Supergroup Subject Acc. no. Size %ID Supergroup

wCer2 NGZ19963 480 85.1 A wNleu, wNfla WP_065095361 463 76.3 B wIrr QGT16094 483 76.8 A wCon RDD34742 467 75.3 ? wPip (Buckeye) AGR50404 491 76.3 B wAna EAL58408 443 67.9 A Cid Cin wPip_Pel (type I) CAQ54390 451 75.6 B wRi (type II) ACN95432 456 67.9 B wPipI_Utique QED12394 415 75.3 B wCon RDD34842 464 62.9 ?

wRi (type II) ACN95432 456 45.6 A wRi ACN95317 474 56.4 A wVita (type III) ONI57866 490 35.9 A wMel (WD0631) AAS14330 490 55.9 A Cid

Cin wPip_Pel (type IV) CAQ54402 445 29.0 B wPip_Pel (type I) CAQ54390 451 52.0 B

wCfeJ no acc. ID 431 24.1 - Cin wCfeJ XP_026474239 431 32.1 -

1 2 3 4 5 6 C CinA 431 aa (wCfeJ)

1 2 3 4 5 6 C. felis CidA-like [ 24] W [27] KYEMACSYCVVNKI [45] VFDFW [28] AEYFYNHL [27] ILDFCVRN [63] Y [176] 427

Cid wPip_Pel [ 69] W [27] KYEMACSYCVVDKI [45] VFDFW [28] VEYFYNHL [27] ILDFCVNK [61] Y [197] 491 R. felis [132] W [29] LYHIACQFCLEDEI [26] LKIFW [49] VEFFWNKL [26] IAAFCVEQ [82] Y [255] 614 R. gravesii [104] W [25] LYKFACNYCLEEYT [43] LIKYF [33] VKYFWNRL [33] IFAFLIEE [46] Y [454] 775 wDacB [ 60] W [37] RYKIACWCCFEDDI [27] LMIYW [34] LEFFWDKL [31] MVDFCLRY [51] Y [134] 411

Cnd wStr [ 60] W [37] RYRIACWCCFEDEI [27] LMIYW [34] LEFFWNKL [31] MVDFCLRH [51] Y [135] 412 wVulC [ 59] W [30] SYDIACRYCLEEDI [33] LIVFW [34] VEFFWNKI [34] IFEFCFSQ [51] Y [141] 419 wCon [ 14] - [ 6] -YDHLVRCCGFDLL [ 4] LAQFW [34] VEFFWNKI [34] IFEFCFGQ [51] Y [146] 320 wCfeJ [ 56] W [37] RYQIACWCCLEEDI [36] MMQFW [37] VEFFWDKV [32] VFEFCLSQ [51] Y [145] 431 O. tsutsugamushi [185] W [27] KYRLACNYCLEDSI [22] MVYFW [57] IEYIWNRL [20] FRSLLLSQ [48] F [466] 863 wRi [ 65] W [27] IYDMACRYCVIDKI [47] VFSFW [29] VEYFYSLL [29] TLDFCLSK [59] Y [163] 456

Cin wVita [ 87] W [24] IYKIACIECVESEI [47] VFNFW [29] VEHFYNRL [33] TLSSLLGK [63] Y [170] 490 wPip_Pel [ 66] W [31] RYYLACKYCLEDKI [38] IEAFW [35] IEFFYNKL [30] TIEFCLSK [49] Y [159] 445 C. felis CinA-like [105] W [27] RYNMACRYCVTDKI [47] VFTFW [29] VEYFYRRM [ 2] ------[ 0] - [ 0] 239

Fig. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 71

1463 FIG 5. Characterization of wCfeJ and wCfeJ in cat fleas.

1464 (A) Screening for wolbachiae in geographically diverse cat flea colonies and wild

1465 populations. Cat fleas from six locations (including two strains from Sierra Research

1466 Laboratories) were assessed for the presence of wCfeT and wCfeJ. Wolbachia

1467 infection of individual fleas was assessed by qPCR (see Materials and Methods for

1468 details).

1469 (B) Localization of wCfeT and wCfeJ in cat flea tissues. Dissected ovaries and other

1470 tissues (mostly midguts) from individual cat fleas (EL Colony) were assayed by qPCR

1471 see Materials and Methods for details). Five fleas were assayed for each tissue type.

1472

1473 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A Colonies B 125 Elward Kansas St. Sierra Research Labs Two-way ANOVA Laboratory University (Riner) (Modesto Wild) Factor Pr (n = 20) (n = 20) (n = 20) (n = 20) 100 Target (apaG, cinA) 0.0789 Tissue (ovary, non-ovary) 0.0389 Target:Tissue 0.0001 75 flea 18S rDNA 5 50 Wild Populations

Statesboro Monroe Orange Co. wCfeT Georgia West Virginia California 25

wCfeJ Copies/10 (n = 38) (n = 13) (n = 216) Both None 0 Ovarian Other Ovarian Other

wCfeT wCfeJ

Fig. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 73

1475 FIG 6. Recurrent evolution of nutritional-mediated mutualism and reproductive

1476 parasitism across diverse rickettsial lineages.

1477 (A) Diverse strategies for biotin biosynthesis, acquisition and utilization in Rickettsiales.

1478 Top, biotin synthesis genes (BOOM arrangement), the BioY biotin transporter, the biotin

1479 ligase BirA, and biotin utilization enzymes (biotin-dependent propionyl-CoA carboxylase

1480 complex and FabD) involved in malonyl-CoA synthesis. The distribution of these genes

1481 across major rickettsial lineages is summarized, with phylogeny of Rickettsiales drawn

1482 as a consensus from recent studies (13, 91, 101, 103). The acquisition of biotin

1483 synthesis operons is depicted by yellow, blue and orange highlighting over branches

1484 (see estimated phylogeny in Figure 1A). Taxa highlighted yellow indicate the

1485 conservation of BOOM gene arrangement (see Figure S3). Question marks indicate

1486 missing BioH enzymes in otherwise complete biotin synthesis pathways. These

1487 genomes were scanned for other functionally equivalent methyl esterases and found to

1488 contain none (Figure S4D). Inset highlights the gatekeeper role of BioH in shunting

1489 Pimeloyl-CoA methyl ester (metabolite 2) away from fatty acid biosynthesis for the

1490 production of biotin. All other metabolites in the biotin synthesis pathway are described

1491 in Figure 2A. X, complete loss of biotin utilization in O. tsutsugamushi.

1492 (B) Phylogenomic analysis of RP in Rickettsiales. Phylogeny estimation is further

1493 simplified relative to the tree in panel A. RP is considered an acquired trait, with origins

1494 shown as yellow circles. Spotted Fever Group rickettsiae are shown in gray since RP is

1495 unknown, though CI-like genes are present in some species. Information on rickettsial

1496 hosts/vectors (2) and known RP (67, 186) are compiled from numerous reports. Check

1497 marks reflect the compilations of groups of RP-inducing genes from our prior report, or bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Mechanisms of Evolution in Wolbachia-host associations 74

1498 the recent identification of the WO-mediated killing (wmk) gene implicated in Wolbachia

1499 male-killing (29). Helix-turn-helix domains of certain XRE-family like proteins across

1500 Rickettsiales were not considered related to wmk-encoded proteins. ‘Other’ refers to

1501 additional proteins presented in our prior report (67). bioRxiv preprint doi: https://doi.org/10.1101/2020.06.01.128066; this version posted June 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A SYNTHESIS (BOOM) IMPORT LIGATION UTILIZATION BioB BioF BioH BioC BioD BioA BioY BirA2 PccA PccB FabD

BioB 6 Biotin BioD “Cand. Deianiraea vastatrix” ? PccA BirA A “Cand. Xenolissoclinum pacificiensis” BCCP Neorickettsia spp. 5 ATP “Cand. Neoehrlichia lotoris” Anaplasma spp. U BioA Biotin- Ehrlichia spp. U PccABCCP other Wolbachia 4 ATP Wolbachia (w/ BOOM; see Fig. 2B) - PccA HCO “Cand. Fokinia solitaria” BioF 3 Trichoplax sp. H2 endosymbiont ? 3 “Cand. Aquarickettsia rohweri” ? Carboxy-biotin- M “Cand. Midichloria mitochondrii” PccA BCCP Peranema trichophorum endosymbiont BioH “Cand. Jidaibacter acanthamoeba” Acetyl- PccB Stachyamoeba lipophora endosymbiont 2 CoA Rickettsiales bacterium str. Ac37b ? BioC R “Cand. Arcanobacter lacustris” Malonyl-CoA “Cand. Phycorickettsia trachydisci” 1 “Cand. Occidentia massiliensis” ? Holo- FabD X Orientia spp. ACP “Cand. Megaira polyxenophila” Group 1 “Cand. Sneabacter namystus” Malonyl-ACP Group 2 R. felis and R. hoogstraalii Group 3 R. buchneri (two BOOM copies) fatty acid all other Rickettsia spp. synthesis FabH

BioCBioHBioFBioABioDBioBBioY BPLXccAXccBFabD

B Hosts/vectors RP RP-inducing genes phenotypes cinAB cidAB cndAB other wmk “Cand. Deianiraeaceae” A Xenolyssoclinum ~66% of insect species; male killing; Neorickettsia many other arthropods; CI; PI; Wolbachia some filarial worms feminization M other Anaplasmataceae Midichloriaceae ancient Rickettsiaceae Mites (chiggers) PI; CI? Occidentia Orientia Beetles; wasps; male killing; R Megaira / Sneabacter booklice PI; CI? ancestral lineages Transitional Group Wasps; booklice PI; CI? Typhus Group Rickettsia Spotted Fever Group Ticks unknown Fig. 6