# Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters.

Thomas Boivin, Hélène Henri, Fabrice Vavre, Cindy Gidoin, Philippe Veber, Jean-Noël Candau, Emmanuelle Magnoux, Alain Roques, Marie-Anne Auger-Rozenberg

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## To cite this version:

Thomas Boivin, Hélène Henri, Fabrice Vavre, Cindy Gidoin, Philippe Veber, et al.. Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters.. Molecular Ecology Notes, 2014, 23 (9), pp.2362-2375. 10.1111/mec.12737. hal-01092614

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1 **Title:** Epidemiology of asexuality induced by the endosymbiotic *Wolbachia* across

2 phytophagous wasp species: host plant specialization matters

3

4 **Authors:** Boivin T¹, Henri H², Vavre F², Gidoin C¹, Veber P², Candau J-N¹,³, Magnoux E⁴,

5 Roques A⁴ and Auger-Rozenberg M-A⁴

6 **Addresses:**

7

1. INRA, UR629 Ecologie des Forêts Méditerranéennes, URFM, F-84914 Avignon, France

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2. Université de Lyon, F-69000, Lyon ; Université Lyon 1 ; CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France.

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3. National Resources Canada, Canadian Forest Service, Great Lakes Forestry Ctr, Sault Ste Marie, ON P6A 2E5, Canada

11

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4. INRA, UR633 Unité de Recherche de Zoologie Forestière, 45075 Orléans, France

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**Keywords:** parthenogenesis, ecological specialization, thelytoky, multilocus sequence typing,
*Megastigmus*

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17 **Word count:** 7770

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**Corresponding author:** Thomas Boivin, INRA, UR629 Ecologie des Forêts Méditerranéennes, URFM, Domaine Saint Paul, F-84914 Avignon, France, fax: +33 (0)4 32 72 29 02, boivin@avignon.inra.fr

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21

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23

**Running title:** Spread of thelytoky in phytophagous wasps
24 Abstract

Among eukaryotes, sexual reproduction is by far the most predominant mode of
reproduction. However, some systems maintaining sexuality appear particularly labile and
raise intriguing questions on the evolutionary routes to asexuality. Thelytokous
parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex
ratio towards females and resulting from mutation, hybridization or infection by bacterial
endosymbionts. We investigated whether ecological specialization is a likely mechanism of
spread of thelytoky within insect communities. Focusing on the highly-specialized genus
*Megastigmus* (Hymenoptera: Torymidae), we first performed a large literature survey to
examine the distribution of thelytoky in these wasps across their respective obligate host
plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15
arrhenotokous and 10 thelytokous species for *Wolbachia*, *Cardinium*, *Arsenophonus* and
*Rickettsia* endosymbionts and by performing antibiotic treatments. Finally, we performed
phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the
evolution of endosymbiont-mediated thelytoky in *Megastigmus* and its possible connections
to host plant specialization. We demonstrate that thelytoky evolved from ancestral
arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis-
inducing *Wolbachia*. We find that ecological specialization in *Wolbachia's* hosts was
probably a critical driving force for *Wolbachia* infection and spread of thelytoky, but also a
constraint. Our work further reinforces the hypothesis that community structure of insects is
a major driver of the epidemiology of endosymbionts and that competitive interactions
among closely related species may facilitate their horizontal transmission.
46

47 **Introduction**

48 Sex is by far the most predominant mode of reproduction among eukaryotes, such that only
49 one out of a thousand animal species shows some type of asexual reproduction
50 (Suomalainen *et al.*, 1987). Among the diverse mechanisms involved in asexual reproduction,
51 parthenogenesis is defined *sensus stricto* as the development of an egg without fertilization.

52 The origin of parthenogenesis is polyphyletic in both invertebrates and vertebrates,
53 suggesting that systems maintaining sexuality are labile and stimulating elucidation of the
54 evolutionary routes to parthenogenesis (reviewed in Simon et al., 2003). Spontaneous loss of
55 sexuality may occur through mutations or intra- and interspecific hybridization (Simon et al.,
56 2003; Dedryver et al., 2001), but it can also result from infection by bacterial endosymbionts
57 that are predominantly transmitted vertically through the female egg cytoplasm and distort
58 the host sex ratio towards females to their own advantage (Engelstädter and Hurst, 2009).

59 In the insect order Hymenoptera, several forms of parthenogenesis have been
60 defined according to the sex of offspring produced by a virgin female (Cook, 1993; Cook and
61 Crozier, 1995). The dominant and ancestral form of parthenogenesis is arrhenotoky, in which
62 fertilized eggs develop as diploid females and unfertilized eggs develop as haploid males.
63 However, numerous species display thelytoky, which is a form of complete parthenogenesis
64 where unfertilized eggs develop into diploid females. Thelytoky can be under the control of
65 the insect itself or its endosymbionts (Stouthamer et al, 1990; Vavre et al., 2004).
66 Parthenogenesis inducers include bacteria of the genera *Wolbachia* (Werren, 1997),
67 *Cardinium* (Zchori-Fein and Perlman, 2004) and *Rickettsia* (Hagimori et al, 2006). While
induction of male production is impossible in genetically-based thelytoky, endosymbiotic
thelytoky can be characterized by its reversibility, as antibiotic or heat treatment of females
leads to bacteria elimination and production of males in their progeny (Stouthamer *et al.*,
1990). Generally, infection by parthenogenesis-inducing (PI) *Wolbachia* is fixed within
species or populations and results in an obligate parthenogenesis associated with a loss of
sexual function in females (Jeong and Stouthamer 2005; Kremer *et al.*, 2009), or in both
sexes (Gottlieb and Zchori-Fein, 2001), making *Wolbachia* indispensable. Only in very rare
cases, polymorphism of infected and uninfected females is maintained (REF).

Despite vertical transmission via the egg cytoplasm being the predominant
transmission mode of PI endosymbionts (Werren, 1997), phylogenetic incongruence
between host and endosymbiont histories strongly indicates occasional horizontal
transmission events (O'Neill *et al.*, 1992; Stouthamer *et al.*, 1993; Baldo *et al.* 2006; Perlman
*et al.*, 2010). Horizontal transmissions of *Wolbachia* have been successfully demonstrated
experimentally by injection (Braig *et al.*, 1994; Grenier et al, 1998), introgression (Jaenike,
2007), maintenance of close contacts between conspecifics (Rigaud and Juchault, 1995) or in
host-parasitoid associations (Vavre *et al.* 1999). Effective horizontal transmission of
endosymbionts depends (i) on intimate ecological associations which provide within-
community horizontal transmission opportunities (Vavre *et al.*, 1999; Sintupachee *et al.*,
2006; Stahlhut *et al.*, 2010) and (ii) on the phylogenetic similarity of donor and recipient host
species because internal defence mechanisms against infections are likely to be more similar
between closely related hosts (Stahlhut *et al.*, 2010). Studies addressing how insect
phylogeny and ecology affect patterns of similarity between strains of PI endosymbionts
would help gain critical insights on how these two forces affect shifts in reproduction modes.
In particular, study systems involving several groups of closely related thelytokous species but displaying different levels of ecological proximity due to habitat specialization can be of critical interest to define the possible ecological boundaries to the evolutionary trajectory of thelytoky in insects.

In this paper, we address the potential for phylogeny, ecology and infection by PI endosymbionts to drive the spread of asexuality within insects. For this purpose, we focused on the genus *Megastigmus* (Hymenoptera: Torymidae), which counts more than 125 arrhenotokous and thelytokous wasp species worldwide (Grissell, 1999). Half of these species are specialist seed feeders, the other half are presumed to be parasitoids, gall-makers or to have unknown hosts. Within the seed-specialized group, two thirds of species exploit gymnosperm hosts (Pinaceae and Cupressaceae families), whereas one third exploits preferentially angiosperms, especially the Rosaceae and Anacardiaceae families (Roques & Skrzypczynska, 2003). Being highly specialized in the seed resource, several wasp species exploiting the same host strictly require the same ecological niche, which is then particularly propitious to direct intra- or interspecific interactions within this insect group (Boivin *et al.*, 2008; Auger-Rozenberg and Roques, 2012). In the genus *Megastigmus*, the dominant and ancestral form of parthenogenesis is by far arrhenotoky as thelytoky characterizes only a minority of species (Grissell, 1999), but both arrhenotokous and thelytokous species can occur in sympatry on gymnosperm or on angiosperm hosts (Boivin *et al.*, 2008; Auger-Rozenberg and Roques, 2012). Thelytoky has only been established from records of highly female-biased sex ratios in field populations, but neither its determinism nor its evolutionary trajectory have been elucidated yet. We first performed a large literature survey to examine the distribution of thelytoky in *Megastigmus* across their known obligate host-plant families.
Second, we tested for thelytoky caused by endosymbionts by screening in a large sample of arrhenotokous and thelytokous species for endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions to examine the evolution of endosymbiont-mediated thelytoky in *Megastigmus* and its possible connections to host-plant specialization. This study strongly suggests that thelytoky is caused by *Wolbachia* endosymbionts, and interestingly, we show that host plant specialization is a key determinant of *Wolbachia* infection and thelytoky.

121

## 122 Material and Methods

Distribution of thelytoky among phytophagous *Megastigmus* species

Parthenogenesis, i.e. arrhenotoky or thelytoky, is the only mode of reproduction in the *Megastigmus* genus. It has never been formally studied and it was generally deduced from sex ratio estimates in field sampled wasp populations. Here, we used extensive catalogs of seed-feeding *Megastigmus* species to gather the current knowledge on the relative prevalence of arrhenotoky and thelytoky in the genus *Megastigmus* (Grissell, 1999; Roques & Skrzypczynska, 2003; Roques *et al.*, 2003; Auger-Rozenberg *et al.*, 2006). A bibliographic search of non-redundant articles in the Web of Science (2003-2012) and CAB abstracts (2003-2012) was also performed on key terms '*Megastigmus* and seed*' to check for potential new species descriptions or increase knowledge in the biology of the species described in the above catalogs. We considered only wasp species for which arrhenotoky or thelytoky were ascertained by authors, or for which male frequencies estimated at emergence from sampled seed lots provided unambiguous support for arrhenotoky (high male abundance) or thelytoky (absence or <1%). Species for which the parthenogenesis
mode was not mentioned or for which sample sizes were insufficient (<10) to estimate sex ratios were not included. The prevalence of thelytoky was assessed as the percentage of thelytokous species in all of the selected wasp species. The distributions of both arrhenotoky and thelytoky relative to host plant specialization was assessed by assorting both arrhenotokous and thelytokous wasp species according to their host plant group (gymnosperm or angiosperm), family and genus.

143

144 *Wasp sampling*

145 *Megastigmus* species were collected across the Northern Hemisphere (Nearctic and Palearctic) by seed samplings on diverse host plants. Insect-infested seeds were separated from non-infested ones by X-ray radiography (using Faxitron-43855®, 15 kV, 3 mA, 3'30" to 4'30" depending on seed species with X-ray-sensitive films Kodak 'Industrex M', and Faxitron-MX20®, 20 kV, 0.3 mA, 1'45" with an EZ20 digital scanner). The insect-infested seeds were placed in individual rearing boxes stored in outdoor insectaries located at INRA, Orléans, France and at INRA, Avignon, France. Adult emergence was recorded over the 3 years following seed maturation because of a possible prolonged diapause (Roques & Skrzypczynska, 2003). After emergence, insect species determination was ascertained using the morphological keys of Grissell (1999) and Roques and Skrzypczynska (2003). Emerged insects were then sorted by species and collection site and preserved in 100% alcohol at -20°C. A total of 25 *Megastigmus* species were used in this study, 10 were thelytokous and 15 were arrhenotokous. These species, their reproduction mode, their host-plants, collecting localities and distributions are summarized in Table 1.

159

160 DNA extraction
Total genomic DNA was isolated by crushing individually whole adult *Megastigmus* females using two different procedures, depending on sample size and further DNA use. Screening for PI endosymbiont infection, which involves large sample sizes, was performed using the Chelex method. This fast and cheap method consisted of digesting tissues kept for 2 h at 56°C in a 200 µl 10% Chelex solution (Biorad). After 30 min at 100°C, samples were centrifuged and supernatants were used as DNA sources. For sequencing and further phylogenetic analyses, which requires smaller sample sizes and long term conservation of DNA, we used the DNeasy extraction Tissue Kit (Qiagen). Total genomic DNA was eluted in 200 µl of AE buffer in this case. In addition, a PCR was systematically carried out on Qiagen extractions with *Wolbachia* primers in order to confirm the lack of bias in the detection of the symbiont due to different methods of DNA extraction.

172

## Wasp sequences

We used Sigma RedTaq for PCR amplification. The forward and reverse primers used were 1775-COI-F (Clyde, 5'-CGAATAAATAATATAAGATTTTG-3'), and 2773-COI-R (Bonnie, 5'-GGATAATCTCTATATCGACGAGGTAT-3') (Scheffer & Grissell 2003) for the segment of the cytochrome oxidase I (COI) gene. Cycling programs were as follows: a denaturation step at 94°C for 1 min, annealing for 1 min at 48°C, and extension at 72°C for 1 min with 30 cycles being performed. All PCR products were then purified with a QIAquick PCR purification kit (Qiagen) and were directly sequenced with the amplification primers. Sequencing was performed using the big-dye terminator sequencing kit (PE Applied Biosystem) and carried out with an ABI 3100 automatic sequencer. The gene fragment was sequenced on two to four individuals for each species, except for cryptic and rare species because of the small number of specimens available. When individuals were identified as
originating from a different biogeographic region (i.e. when invasive specimens were found), additional specimens from native areas were sequenced in order to ensure genetic similarity between native and introduced individuals. Sequences were aligned using Clustal W (Thompson et al. 1994) as implemented in BioEdit 7.05 (Hall 1999).

In addition to the COI gene, we studied a nuclear fragment, the D2 region of the 28S ribosomal subunit (rDNA), to build a phylogenetic tree of the studied *Megastigmus* species.

Nuclear primers previously used for reconstructing a molecular phylogeny of *Megastigmus* spp. on conifers (Auger-Rozenberg et al., 2006) were chosen due to their utility for molecular identification at intrageneric taxonomic level. However, the 28S data were not used further because their resolution was insufficient for the goal of this study (results not shown).

Population screenings for PI-endosymbiont infection

In a wide range of insect species, sex ratio distortion has been associated with infection by diverse bacterial symbionts such as *Wolbachia* (Werren, 1997), *Cardinium* (Zchori-Fein and Perlman, 2004), *Arsenophonus* (Thao and Baumann, 2004) and *Rickettsia* (Hagimori et al, 2006). As a first test for an association between such endosymbionts and thelytoky in the *Megastigmus* genus, we screened for their presence in both arrhenotokous and thelytokous species (listed in Table 1). Separate Polymerase Chain Reactions (PCR) were performed on each *Megastigmus* female using specific primers for *Wolbachia* surface protein (wsp) (Braig et al., 1998), *Cardinium* 16S rRNA (Zchori-Fein and Perlman, 2004), *Arsenophonus* 23S rDNA (Thao and Baumann, 2004) and *Rickettsia* 16S rRNA (Hagimori et al, 2006) gene fragments.

PCR reactions were done in a 25-µl final volume reaction containing 200 µM dNTP, 10 pM primers, 0.5 IU HotStarTaq® DNA polymerase, and 1.5 µl DNA solution. All reactions were
209 run in a ABI thermal cycler (PE Applied Biosystems PCR System 9700) with an initial
denaturing step at 94°C for 4 min, an annealing step for 1 min, an elongation step at 72°C for
210
211 1 min 30s and a final extension step of 72°C for 1 min 30 s. Annealing temperatures and
212 primer pairs used for *Wolbachia*, *Cardinium*, *Arsenophonus* and *Rickettsia* PCR screening are
detailed in Table S2. All PCR reactions included a positive control of *Asobara tabida*
213
214 (*Wolbachia* infected) and *Bemisia tabaci* biotype Q (*Cardinium*, *Arsenophonus* or *Rickettsia*
215 infected) samples. For each bacterial gene, 5 µl of amplified reaction product was run on a
216 1% agarose gel stained with ethidium bromide after the PCRs in order to determine the
217 presence of an amplified DNA fragment. If a sample of a *Megastigmus* species was negative
218 for any bacterial-specific amplification, but COI amplification succeeded, the insect was
219 considered uninfected. When amplification with a bacterial-specific primer yielded visible
220 bands, the insect was considered infected, and samples were kept for sequencing and strain
221 characterization.

222
223 *Wolbachia strain characterization*

224 Only *Wolbachia* was detected in our samples (see Results section). We used multilocus
sequence typing (MLST) (Baldo *et al.*, 2006) to characterize *Wolbachia* strain similarity
225
226 among *Megastigmus* host species. A *wsp* sequence was also obtained from each infected
227 insect. MLST was based on the methods of Baldo *et al.* (2006) using the standard primers
228 that amplify the five conserved *Wolbachia* genes *hcpA*, *ftsZ*, *gatB*, *coxA* and *fbpA*. These are
229 degenerate primers designed to amplify sequences from diverse *Wolbachia* strains. Despite
230 repeated attempts, we could not amplify the *fbpA* gene. Thus, four MLST loci (*hcpA*, *ftsZ*,
231 *gatB* and *coxA*), plus the *wsp* gene, were sequenced in both directions to provide double
232 coverage of the region of interest, with the exception of both *M. borriesi* and *M. rosae* var.

233 *alba*, which could be sequenced for the *wsp* gene only.

234

235 *Phylogenetic analyses*

236 *Wolbachia*. Two samples of each species were sequenced and revealed no polymorphism

237 between individuals. A single sequence was therefore chosen for alignment to a set of

238 database sequences for phylogenetic analysis. Our multi-locus gene phylogeny was based on

239 the four successfully amplified loci (*coxA*, *gatB*, *hcpA*, *ftsZ*) used by Baldo *et al.* (2006) and

240 includes many *Wolbachia* strains identified as from A and B supergroups. Sequences are

241 accessible from the GenBank database under accession numbers KF531859 to KF531890.

242 *Wolbachia* from *Brugia malayi* was used as an outgroup. As more sequences are available

243 for *wsp* gene (in our sampling as well as in Genbank database) the analysis was conducted

244 separately for *wsp* sequences for fine scale genotyping of *Wolbachia* harboured by

245 *Megastigmus* species since this gene often evolves rapidly (Genbank accession numbers

246 KF531891 to KF531900).

247 For all datasets, alignments were initially generated using the MUSCLE software (Edgar,

248 2004) implemented in CLC Main Workbench v6.7.1 (CLC Bio). Phylogenetic analyses were

249 performed using maximum likelihood (ML) inference with PhyML v3.0 (Guindon *et al.*, 2010).

250 The appropriate model of evolution was evaluated with jModeltest v0.1.1 (Posada, 2008).

251 The models selected were TIM1+G for *coxA* and *gatB*, TIM3+G for *ftsZ* and *hcpA* and

252 GTR+I+G for the concatenated MLST data set (*coxA* + *gatB* + *ftsZ* + *hcpA*) and for the *wsp*

253 gene. The robustness of the nodes was assessed with 100 bootstrap replicates. Additionally,

254 bayesian inferences were also used to reconstruct phylogenies with MrBayes v3.1.2
255 (Ronquist & Huelsenbeck, 2003) using appropriate settings leading to convergence between
256 two independent runs. Finally, trees were edited with Figtree v1.4.0 (A. Rambaut,
257 http://tree.bio.ed.ac.uk/software/figtree).

258 *Megastigmus*. The same procedure as for *Wolbachia* data was conducted on *COI* gene
259 sequences. Sequences are accessible from the GenBank database under accession numbers
260 KF531833 to KF531858. Models of substitution selected were GTR+I+G for the *COI* gene.

261 *Analysis of congruence*. The Shimodaira-Hasegawa (SH) and approximately unbiased (AU) 262 tests were used to evaluate the significance of topological incongruence among trees. First, 263 to check for some methodological artifacts, topologies obtained with the two methods (ML 264 and bayesian inferences) were compared and showed no significant difference. Second, for 265 *Wolbachia* phylogenies, we compared topologies obtained with the different loci and also 266 with the concatenated data set. These comparisons were performed on the entire dataset 267 (i.e. the *Wolbachia* from *Megastigmus* species and other sequences retrieved from 268 Genbank) and for the *Wolbachia* infecting *Megastigmus* only. Differences in log likelihood 269 between the best ML topology and an alternative topology were used as values of the AU 270 test and the SH test and then p-values were calculated with Consel program (v0.1i) 271 (Shimodaira & Hasegawa, 2001).

272
273 *Analysis of host-symbiont associations*
274 Parafit statistic was used to test whether hosts and symbionts evolution were independent 275 or if there was a pattern of co-cladogenesis between them (Legendre *et al.*, 2002). The 276 AxParafit program implemented in the Copycat software v2.00.02 (Meier-Kolthoff *et al.*, 277 2007) was used with patristic distances as input matrix and 999 permutations both for *COI*
278 sequences data and for 28S sequences compared with the *Wolbachia* sequences
279 concatenated data set.

280
281 Antibiotic treatments

282 When thelytoky is induced by symbionts, antibiotic curing of infected thelytokous females is
283 expected to show reversion to male production with elimination of the bacteria (Stouthamer
284 et al., 1990; Zchori-Fein et al, 1992). Because it was not possible to rear and treat all of the
285 *Wolbachia* positive *Megastigmus* species due to the variety of their ecological requirements,
286 we chose to focus antibiotic treatments on one model species, *M. pinsapinis*, to ascertain
287 the association between thelytoky and *Wolbachia* infection. Antibiotic treatments were also
288 performed on a closely related arrhenotokous species, *M. schimitscheki*, to exclude any toxic
289 effects of antibiotics on *M. pinsapinis* reproduction.

290 Two hundred *M. pinsapinis* females within 24h of emergence from seeds of *Cedrus atlantica*
291 (Pinaceae) were split into two groups of 100, one dedicated to antibiotic treatment and the
292 other used as a control. The 100 females to be treated with antibiotics were placed in
293 ventilated plastic tubes and were provided with cotton soaked in a sugared solution
294 containing tetracycline hydrochloride (Sigma) at a final 0.2% concentration in the diet. These
295 females were referred to as being cured. The 100 control females were provided with
296 sugared water only. Both cured and control females were left for 5 days at 19°C and natural
297 daylight. Fifty females from each of the cured and control groups were then removed and
298 stored at -20°C in 100% ethanol for subsequent DNA tests for the presence of *Wolbachia*
299 with PCR tests using the *wsp* gene. The remaining females of each group were allowed to lay
300 eggs on young conelets of *C. atlantica* after their introduction in insect-proof bags. Because
emergence of a cohort of this species may require up to 5 years due to prolonged diapause
and because sex is indistinguishable at the larval stage using morphological characters,
proportions of males and females in the progeny of both cured and control females were
estimated directly on larvae using flow cytometry (Boivin and Candau, 2007). Antibiotic
treatments, *Wolbachia* detection and male proportion estimations in the *M. schimitscheki*
progeny strictly followed the same procedure as that in *M. pinsapinis*, using 160 newly
emerged females to constitute two groups of 80 for the establishment of both cured and
control groups, and 30 and 50 females of each group for PCR and progeny production,
respectively.

310

## 311 **Results**

### 312 *Distribution of thelytoky among phytophagous Megastigmus species*

A review of the current knowledge of parthenogenesis in the seed-feeding *Megastigmus*
313 group showed a low prevalence of thelytoky (15%) as only 10 out of 69 species for which the
314 reproduction mode could be unambiguously established are thelytokous (Table S1).

315
316
317
318
319
320
321
322

Thelytoky tends to be more frequently associated with species developing on angiosperms (26%) than on gymnosperms (9%), but the difference was not statistically significant (Fisher's exact test, P=0.0729). Seed wasps of gymnosperms count 4 thelytokous species, all specialized on Pinaceae, specifically in the genus *Abies*, *Larix* and *Cedrus* (Table S1).

Arrhenotokous species of *Megastigmus* are also found on the same host plant genus. Among seed wasp of angiosperms, thelytoky occurs almost exclusively in species specialized on the *Rosa* genus (Rosaceae) except for one on Anacardiaceae (Table S1).
In order to study the evolutionary history of thelytoky in *Megastigmus*, thelytokous species must be placed on the phylogenetic tree of the *Megastigmus* genus. COI reconstruction was performed on sequences obtained from the 25 species sampled in this study (Figure 1).

There was an ancestral split between the species related to Anacardiaceae and the others.

Wasp species related to Rosaceae form two clades, supported by high bootstrap values, with an initial split between the *aculeatus* group on the one hand and all others species associated with Rosaceae on the other hand. This latter clade is sister to a monophyletic clade comprising all species exploiting gymnosperms (posterior probability 0.73). In accordance with previous studies, *Megastigmus* developing on gymnosperms also show specialization, with for example, species exploiting the Pinaceae forming a monophyletic group (except for *M. thyoides*, the only species related to Cupressaceae in the Nearctic region, which could rather reflect a secondary change of host family than an ancestral association).

Replacing species reproducing through thelytoky on that tree does not show any strong phylogenetic signal: thelytokous species occur sporadically on the tree. While the ancestral mode of reproduction is difficult to assess, one clear result is that transitions from one mode of reproduction to the other have regularly occurred during the evolutionary history of seed-specialized *Megastigmus*.

## Screening for PI-endosymbiont infection

A total of 25 arrhenotokous and thelytokous *Megastigmus* species (15 and 10 species, respectively) were sampled and screened for the presence of *Wolbachia*, *Cardinium*, *Arsenophonus* and *Rickettsia* using specific primers. None of the arrhenotokous species was found infected by any of the targeted endosymbionts (Table 2). On the contrary, *Wolbachia*
346 was found fixed in all of the thelytokous samples tested, while *Cardinium*, *Arsenophonus* and
347 *Rickettsia* were not detected in any of these species (Table 2). The correct amplification of
348 both positive controls and arthropod COI indicated true lack of infection and not a failure in
349 the set-up of the PCR reactions. These results demonstrate the strong association of
350 *Wolbachia* infection with thelytokous parthenogenesis in *Megastigmus* (Fisher's Exact Test's
351 p-value<0.001).

352
353 Antibiotic treatments
354 Tetracycline treatments led to an almost complete loss of *Wolbachia* infection in *M.*
355 *pinsapinis* females and a significant recovery of male production, whereas control ones
356 remained infected and produced only females (Table 3). As expected for arrhenotokous
357 species, both control and treated *M. schimitscheki* females were not infected by *Wolbachia*
358 and produced exclusively males. In addition, for each species, the mean brood size was not
359 significantly altered by tetracycline treatment (Table 3). These data suggest that arrhenotoky
360 restoration in treated *M. pinsapinis* was due to symbiont curing rather than direct toxicity of
361 antibiotics to insects, and that *Wolbachia* is probably the causative agent of thelytoky.

362
363 Diversity and distribution of Wolbachia strains in Megastigmus
364 Topologies obtained with the four MLST genes were not congruent among each other on the
365 entire dataset (34 taxa; all p-values <0.01 for SH test, <3.10⁻⁵ with AU test), as already shown
366 (Baldo et al. 2006). In contrast, for the eight *Wolbachia* infecting *Megastigmus* for which
367 sequences have been obtained on the four MLST genes, topologies were globally congruent
368 as all SH tests and most AU were not significant. The only consistent exception was for the
369 *coxA* gene, for which topology was significantly different from the other genes based on AU
tests. In relation to that, a closer look at the *Wolbachia* strain infecting *M. pistaciae* suggests
370 this strain has undergone a recombination event between A and B *Wolbachia*. Indeed, this
371 strain belongs to the B supergroup based on all sequenced genes, except for *coxA* for which
372 it falls within the A supergroup (Figure S1). In summary, despite the complex history of
373 *Wolbachia* strains at a global scale, the tree based on the concatenated dataset is a good
374 mean representation of the history of *Wolbachia* infection in *Megastigmus* (Figure 2) and is
375 also consistent with the tree obtained on the wsp gene (Figure S2).
376

377 Three highly homogeneous lineages of *Wolbachia* infecting *Megastigmus* species were
378 identified (namely WA1, WA2 and WB). We refer to these as lineages as opposed to strains,
379 because although they are closely related, some substitutions were observed. A strict
380 association between *Wolbachia* lineage and host plant is observed. The WB lineage is
381 restricted to *M. pistaciae*, the only thelytokous species developing on Anacardiaceae. A
382 second lineage, named WA1, was only found in thelytokous species developing on Rosaceae,
383 i.e. *M. brevicaudis*, *M. rosae* (both subtypes) and *M. aculeatus* (both subtypes). The third
384 lineage, named WA2, is closely related to WA1 and grouped the *Wolbachia* infecting *M.
385 borriesi*, *M. pictus*, *M. pinsapinis* and *M. suspectus*, which all develop on Pinaceae.
386

386 Importantly, and despite this strict association between host plant and *Wolbachia* lineage,
387 no significant association was detected between *Wolbachia* and host lineages using Parafit
388 test. In addition, *Wolbachia* (concatenated dataset) and *Wolbachia*-infected *Megastigmus*
389 (COI) topologies were not congruent (SH test, p<0.014 and AU test, p<0.003). These analyses
390 exclude co-cladogenesis, suggesting horizontal transfers have played a major role in the
391 distribution of the *Wolbachia* strains.
The distribution of Wolbachia strains among Megastigmus species suggests that horizontal transmission routes are constrained by the plant family, with only one plant family per strain. Our tree in Fig. 1 shows no exception to this rule, but this could nevertheless be obtained by chance, at least in principle. To evaluate how likely this is, we performed a simple test, stating as our null hypothesis that a strain can appear in an insect irrespective of the plant it parasites. Because infection with *PI-Wolbachia* rapidly leads to the irreversible loss of sexual reproduction, rendering *Wolbachia* indispensable for the insect, infection loss is highly improbable (Stouthamer et al. 2010).

Under this hypothesis, from the 10 infected species, at least 7 independent *Wolbachia* acquisitions have occurred during Megastigmus evolution. Indeed, in three cases, it is not possible to distinguish between independent acquisition or ancestral infection (e.g. in the two M. rosae). In all other cases, the presence of an uninfected species within the clade excludes ancestral acquisition. Using the constraint solver clingo [1], we first enumerated all possible scenarios with 7 independent Wolbachia acquisitions (at least one by strain) possibly occurring at each node of the actual Megastigmus phylogeny and leading to 10 infected extant species. Among all these scenarios, we reported the proportion p of cases where a strain is strictly specific of a plant family and obtained a value for p of 0.0051. This result indicates that the probability of getting such a plant specific distribution by chance is exceptional, which constitutes a reasonable evidence that the events of horizontal transmission are not occurring randomly, but are rather influenced by the host plant.

413

414 Discussion
Distribution and endosymbiotic origin of thelytoky in phytophagous *Megastigmus* species

A few hundred species in all of the major animal groups are characterized by thelytokous parthenogenesis; its patchy taxonomic distribution in Hymenoptera is consistent with several independent evolutionary origins from ancestral arrhenotokous species (Cook, 1993). The relatively high prevalence of thelytoky in Hymenoptera might reflect that some mechanisms of their haplodiploid sex determination are rather easily redirected to thelytoky by mutations (Lattorff et al. 2005) or bacteria (Cordaux et al., 2011). Our literature review of parthenogenesis in the *Megastigmus* genus estimated the prevalence of thelytoky to 15%, which supported the assumption that arrhenotoky is the dominant and ancestral form of parthenogenesis in these phytophagous wasps. Thelytoky was unambiguously described in species exploiting diverse habitats, although it appears to occur in only three out of the ten host plant families currently known to host *Megastigmus* species in both gymnosperms and angiosperms (Pinaceae, and Rosaceae and Anacardiaceae, respectively). Interestingly, several thelytokous species share congeneric host plants (e.g. on the *Rosa* genus), but each groups of thelytokous species associated with Pinaceae, Rosaceae or Anacardiaceae remains confined at the host family taxonomic level. This observation supports the potential for intimate ecological connections within a given host plant family, but not between different host plant families.

Thelytoky may be under the control of the insect itself or its endosymbionts (Stouthamer et al, 1990; Vavre et al., 2004). Strict association between thelytoky and *Wolbachia* infection and reversibility of thelytoky through antibiotic treatments provide a particularly strong support for an endosymbiotic origin of thelytoky in *Megastigmus* wasps. Within the Hymenoptera, *Wolbachia*-induced thelytoky is found in three super-families: the Cynipoidea
438 (Rokas *et al.*, 2002), the Braconidae (Kremer *et al.*, 2009) and the Chalcidoidea (Stouthamer 1997), within which only entomophagous families were concerned. To our knowledge, we document here the first case of *Wolbachia*-induced thelytoky in phytophagous chalcids.

441

442 *Host plant specialization matters in the spread of thelytoky*

443 The phylogenetic tree of the *Megastigmus* genus including all main ecological groups (angiosperm- and gymnosperm-specialized seed wasps) indicated that thelytokous *Megastigmus* species do not form a monophyletic group, but occur sporadically on the COI phylogenetic tree (Figure 1). This pattern is consistent with the hypothesis of independent evolutionary origins of thelytokous species from ancestral arrhenotokous species (Cook, 1993), and thus that transitions from arrhenotoky to thelytoky have repeatedly occurred.

449 While transition from arrhenotoky to thelytoky is possible, the reverse is much more constrained and even impossible after sexual traits have decayed (Jeong and Stouthamer 2005; Kremer *et al.*, 2009).

452 The phylogenetic tree obtained on the concatenated MLST dataset provided critical insights on the history of *Wolbachia* infection in *Megastigmus*. Three homogeneous lineages of *Wolbachia* (WA1, WA2 and WB) were identified and shown to be associated with the host plant families of their hosts (Figure 1). Although both lineages were found closely related, WA1 infected exclusively thelytokous wasp species exploiting the Rosaceae while WA2 was detected only in those exploiting the Pinaceae. The third lineage WB was restricted to the only currently known thelytokous wasp species exploiting Anacardiaceae (*M. pistaciae*).

459 More interestingly, there was no statistical support for an association between *Wolbachia* and host lineages. For example, WA1 is found in five species developing on Rosaceae, but
belonging to highly divergent clades (10% COI). Similarly, WA2 is only found in four species exploiting Pinaceae, despite a divergence of as high as 5-6% on COI among these species. On the opposite, we found a non-random association between *Wolbachia* strains and the host plant used by the insects. Altogether, this led us to postulate on the one hand that thelytoky may have spread across the *Megastigmus* genus through horizontal transmission of *Wolbachia* among wasp species, and on the other hand that these transfers occurred preferentially between species exploiting similar host plants.

Within-community horizontal transmission of *Wolbachia* is likely to result from ecologically mediated pathways such as host-parasitoid associations (Vavre *et al.* 1999; Huigens *et al.*, 2004). Shared feeding and breeding sites may also act as a route through which *Wolbachia* can be transmitted from one host species to another (Sintupachee *et al.* 2006). Primary infection by *Wolbachia* has to face two main filters (Vavre *et al.* 2003). First, *Wolbachia* must come into contact with the recipient species (i.e. has to pass the encounter filter). Second, *Wolbachia* has to evade the host immune system and to develop in the new host (i.e. has to pass the compatibility filter), which is facilitated by the relatedness of the donor and recipient species. Community structure may affect both filters and thus impact the epidemiology of *Wolbachia* across species, as recently showed theoretically using small-world networks (Zug *et al.*, 2012). Whether communities are composed of generalist or specialist species, and of distantly or closely related species will clearly impact these two filters. On the one hand, interspecific horizontal transmission of *Wolbachia* may be favoured in particular habitats that are attractive for a wide set of generalist host species, which will simultaneously feed and develop on it and/or which can be connected by shared parasitoids (Stahlhut *et al.*, 2010). The situation encountered in *Trichogramma* may be representative of
such case with the existence of a specialized PI-*Wolbachia* clade infecting these generalist wasps, but within which horizontal transmission is rampant (Schilthuizen and Stouthamer, 1997). On the other hand, intimate interspecific connections between host species may also arise from host plant specialization, which may favour *Wolbachia* transmission due to narrow shared ecological niches. This situation opens widely the encounter filter, but also restricts the host spectrum to the few species exploiting this particular resource. When species within the community are moreover closely related species exploiting the same resource, the compatibility filter will also be open and facilitates symbiont infection. This latter situation is probably the one encountered in the seed-feeding *Megastigmus* analysed here, revealing two essential features of the spread of thelytoky in the context of insect ecological specialization.

First, the COI reconstruction of the present 25 *Megastigmus* species strongly confirmed host plant specialization in this genus (Figure 1). Within the species on angiosperms, the clade of species attacking Anacardiaceae first diverged. Within one of the sister groups, wasp species exploiting gymnosperms form a monophyletic group and even show specialization, as depicted by the monophyletic group developing on the Pinaceae (except for *M. thyoides*).

The existence of a common *Wolbachia* lineage in all wasps exploiting the Rosaceae (WA1) and another one in all those developing on the Pinaceae (WA2) suggests that host ecological specialization can promote the spread of thelytoky through seed use. All *Megastigmus* females oviposit during a rather narrow period of the development of the host reproductive structures and progeny develop exclusively within seeds at a final density of one larva per seed, while several eggs can be found within the same seed (Turgeon *et al.*, 1994; Rouault *et al.* 2004; T. Boivin, personal observation). On both wild roses and Pinaceous trees, many
507 *Megastigmus* species show some restricted behavioral plasticity allowing shifts onto 508 different congeneric host species or even onto a new host genus (Grissell 1999; Auger-509 Rozenberg & Roques 2012). The possibility of short-range host shifts may have favoured 510 novel wasp assemblages and novel opportunities for them to interact and potentially 511 exchange symbionts. Although shared parasitoids between phytophagous *Megastigmus* spp. 512 can not be ruled out (Mailleux *et al.*, 2008; M.-A. Auger-Rozenberg, personal observation), 513 host-parasitoid associations in this genus remain too poorly documented to formally invoke 514 this *Wolbachia* horizontal route at this point. We rather suggest that if *Wolbachia* infection 515 occurs mainly via ingestion during the early larval stages, both seed tissues and/or 516 cannibalism by larvae competing for the seed tissues may facilitate interspecific horizontal 517 transfers of the endosymbiont. A similar pattern of endosymbionts sharing among sibling 518 species competing for a common resource have recently been shown in weevils (Merville *et 519 al.* 2013).

520 Another key result of this study is that *Megastigmus* ecological specialization at the host 521 plant family level probably constrained the invasion of *Wolbachia* lineages throughout the 522 whole host genus. Indeed, diverging strategies in the use of angiosperms and gymnosperms 523 may be an essential feature of the strict host radiation depicted here in the evolutionary 524 history of *Megastigmus*. For this reason, intimate ecological connections promoting 525 horizontal transfers of *Wolbachia* (see above) are thus unlikely to arise at this community 526 scale. More interestingly, thelytoky appears to have still not invaded wasps developing on 527 both the Cupressaceae (Fig. 1, Table S1). According to Auger-Rozenberg *et al.* (2006), wasps 528 developing on the Cupressaceae exhibit a higher level of host specificity, because they seem 529 host species specific, while wasps on Pinaceae or Rosaceae are specialized to particular host
genera, but frequently attack several congeneric species if they occur in sympatry. This is
consistent with a taxonomic radiation following initial host adaptation, which might have
constituted an efficient barrier to the spread of thelytoky in this group, due to unlikely
opportunities for interspecific horizontal transfers of *Wolbachia*.

Altogether, our results show that ecological specialization can be a driving force of the
spread of endosymbiotic thelytoky, but also a constraint. This further reinforces the
hypothesis that community structure of insects is a major driver of the epidemiology of
endosymbionts (Ferrari and Vavre, 2011) and that competitive interactions among closely
related species may facilitate horizontal transmission (Merville *et al.*, 2013).

539

540 Acknowledgements

541 We are grateful to Amber Paulson and Dr Steve Perlman for insightful discussions on earlier
542 drafts of this manuscript. We acknowledge Anne Roig and Bruno Jouaud for their help on
543 DNA extraction and endosymbiont PCR screenings, and Marion Sondo and Alain Chalon for
544 their help in wasp sampling and antibiotic treatments. This work was supported by grants
545 from INRA-EFPA, the E.U. project FP6-ALARM 2005-2008, the MEDD project Invagraines
546 2003-2006, and the ANR-10-INTB-1705-04-MACBI (French Agence Nationale pour la
547 Recherche, http://www.agence-nationale-recherche.fr).

548

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708

709 **Data accessibility**

710 *Wolbachia* and wasp DNA sequences: GenBank accession numbers KF531859-KF531890 and

711 KF531891-KF531900, and KF531833-KF531858, respectively.

712

713

714 **Author contributions**

715 M.-A.A.R., F.V. and T.B. conceived and designed the study. M.-A.A.R, A.R. and T.B. acquired

716 the samples and provided funding support. E.M., C.G., J.-N., M.-A.A.R and T.B. performed the

717 experiments and produced the data. H.H., M.-A.A.R, F.V. and T.B. analyzed the data and

718 wrote the paper. All authors have checked and approved the final version of the manuscript.

719

720 **Figure Legends**

721 **Figure 1.** Bayesian likelihood inference phylogeny based on COI sequences in seed-

722 specialized wasps of the *Megastigmus* genus (26 taxa, 962 bp). *Torymus azureus* was used as

723 an outgroup. Posterior probability values are indicated at each node. Host plant families are

724 indicated at each branch forming a monophyletic group (excepted for the Cupressaceae).

725 Anarcadiaceae and Rosaceae are angiosperms, and Pinaceae, Cupressaceae and Taxodiaceae

726 are gymnosperms. *Megastigmus* species infected by *Wolbachia* are indicated by arrowheads

727 followed by the name of the *Wolbachia* lineage.

728

729 **Figure 2.** Phylogenetic placement of the *Wolbachia* strains infecting the seed-specialized

730 wasps *Megastigmus* spp. among other *Wolbachia* belonging to A and B supergroups.
731 Bayesian likelihood inference phylogenies are shown, while maximum likelihood analyses gave substantially the same results. *Wolbachia* sequences are labelled with the name of their host. *Wolbachia* of *Brugia malayi* was used as an outgroup. Posterior probability values 732 are indicated at each node. This phylogeny is based on concatenated sequences data set for 733 the four MLST loci *coxA*, *gatB*, *ftsZ* and *hcpA* (34 strains, 1650 bp). 734

736

737

738 **Supplementary material**

739 **Figure S1.** Phylogenetic placement of *Wolbachia* infecting the seed-specialized wasps 740 *Megastigmus* spp., among other *Wolbachia* strains belonging to A and B supergroups, based 741 on the sequences of each of the four MLST genes used in this study (*coxA*, *gatB*, *ftsZ* and 742 *hcpA*). 743

743

744 **Figure S2.** Phylogenetic placement of the *Wolbachia* strains infecting the seed-specialized wasps *Megastigmus* spp., among other *Wolbachia* strains belonging to A and B supergroups, based on wsp sequences of 49 strains (530 bp). 745 746

747

748 **Table S1.** Literature review of the distribution of arrhenotoky, thelytoky and host-plant 749 specialization among the seed-feeding wasps of the *Megastigmus* genus. 750

750

751 **Table S2.** The primer pairs used for endosymbiont PCR screening in *Megastigmus* spp.
<table><caption>**Table 1.** Collection data for the specimens used in the study of 15 arrhenotokous and all the currently known thelytokous species (N=10) in the *Megastigmus* genus. A: arrhenotokous parthenogenesis. T: thelytokous parthenogenesis. Note that *Torymus azureus* was used as an outgroup for building the COI phylogenetic tree of the *Megastigmus* genus presented in Fig. 1. Specimens collected outside of their native range belonged to invasive populations.</caption><thead><tr><th>Species name</th><th>Reproduction</th><th>Host-plant group</th><th>Host-plant family</th><th>Host-plant species</th><th>Collection site</th><th>Native range</th></tr></thead><tbody><tr><td>M. aculeatus</td><td>T</td><td>Angiosperm</td><td>Rosaceae</td><td>Rosa rugosa</td><td>Krasnoyarsk, Russia</td><td>Palearctic</td></tr><tr><td>M. aculeatus nigroflavus</td><td>T</td><td>Angiosperm</td><td>Rosaceae</td><td>Rosa sp.</td><td>Iowa, USA</td><td>Nearctic</td></tr><tr><td>M. borriesi</td><td>T</td><td>Gymnosperm</td><td>Pinaceae</td><td>Abies koreana</td><td>Rold skov, Denmark</td><td>East-Asia</td></tr><tr><td>M. brevicaudis</td><td>T</td><td>Angiosperm</td><td>Rosaceae</td><td>Sorbus sp.</td><td>Ojcow, Poland</td><td>Palearctic</td></tr><tr><td>M. pictus</td><td>T</td><td>Gymnosperm</td><td>Pinaceae</td><td>Larix gmelini</td><td>Loiret, France</td><td>Palearctic</td></tr><tr><td>M. pinsapinis</td><td>T</td><td>Gymnosperm</td><td>Pinaceae</td><td>Cedrus atlantica</td><td>Vaucluse, France</td><td>Palearctic</td></tr><tr><td>M. pistaciae</td><td>T</td><td>Angiosperm</td><td>Anacardiaceae</td><td>Pistacia terebenthus</td><td>Exocori, Greece</td><td>Palearctic</td></tr><tr><td>M. rosae</td><td>T</td><td>Angiosperm</td><td>Rosaceae</td><td>Rosa sp.</td><td>Hautes-Alpes, France</td><td>Palearctic</td></tr><tr><td>M. rosae alba</td><td>T</td><td>Angiosperm</td><td>Rosaceae</td><td>Rosa majalis</td><td>Vilnius, Lithuania</td><td>Palearctic</td></tr><tr><td>M. suspectus</td><td>T</td><td>Gymnosperm</td><td>Pinaceae</td><td>Abies alba</td><td>Gard, France</td><td>Palearctic</td></tr><tr><td>M. amicorum</td><td>A</td><td>Gymnosperm</td><td>Cupressaceae</td><td>Juniperus phoenicea</td><td>Erica, Portugal</td><td>Palearctic</td></tr><tr><td>M. atedius</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Picea sp.</td><td>British Columbia, Canada</td><td>Nearctic</td></tr><tr><td>M. atlanticus</td><td>A</td><td>Gymnosperm</td><td>Cupressaceae</td><td>Cupressus atlantica</td><td>Idni, Morocco</td><td>Afro-tropical</td></tr><tr><td>M. bipunctatus</td><td>A</td><td>Gymnosperm</td><td>Cupressaceae</td><td>Juniperus communis</td><td>Hautes-Alpes, France</td><td>Palearctic</td></tr><tr><td>M. cryptomeriae</td><td>A</td><td>Gymnosperm</td><td>Cupressaceae</td><td>Cryptomeria fortunei</td><td>Zeihjang, China</td><td>East-Asia</td></tr><tr><td>M. hoffmeyeri</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Tsuga canadensis</td><td>Ontario, Canada</td><td>Nearctic</td></tr><tr><td>M. lasiocarpae</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Abies amabilis</td><td>British Columbia, Canada</td><td>Nearctic</td></tr><tr><td>M. nigrovariegatus</td><td>A</td><td>Angiosperm</td><td>Rosaceae</td><td>Rosa sp.</td><td>British Columbia, Canada</td><td>Nearctic</td></tr><tr><td>M. pinus</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Abies procera</td><td>California, USA</td><td>Nearctic</td></tr><tr><td>M. rafni</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Abies alba</td><td>Aude, France</td><td>Nearctic</td></tr><tr><td>M. schimitscheki</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Cedrus atlantica</td><td>Luberon, France</td><td>Palearctic</td></tr><tr><td>M. spermotrophus</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Pseudotsuga mensiezii</td><td>California, USA</td><td>Nearctic</td></tr></tbody></table>
<table><tr><td>M. thyoides</td><td>A</td><td>Gymnosperm</td><td>Cupressaceae</td><td>Chamaecyparis sp.</td><td>North Carolina, USA</td><td>Nearctic</td></tr><tr><td>M. transvaalensis</td><td>A</td><td>Angiosperm</td><td>Anacardiaceae</td><td>Schinus molle</td><td>Marrakech, Morocco</td><td>Afro-tropical</td></tr><tr><td>M. tsugae</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Tsuga heterophylla</td><td>British Columbia, Canada</td><td>Nearctic</td></tr><tr><td>Torymus azureus</td><td>A</td><td>Gymnosperm</td><td>Pinaceae</td><td>Picea abies</td><td>Suchora, Poland</td><td>Palearctic</td></tr></table>
<table><caption>**Table 2.** *Megastigmus* species screened for bacterial infection (+) or non-infection (-) using PCR with *Wolbachia*, *Cardinium*, *Arsenophonus* and *Rickettsia*-specific primers. Each female was screened for all target endosymbionts. The mitochondrial cytochrome oxidase-I (COI) gene of the *Megastigmus* host was amplified when no bacterial-specific primers yielded PCR products to test for correct DNA extraction in the procedure.

N: number of females tested for infection. +: amplification of the primer. -: no amplification of the primer tested. NA: non available data.</caption><thead><tr><th>Parthenogenesis and species (N)</th><th>Wolbachia<br>(infection %)</th><th>Cardinium</th><th>Arsenophonus</th><th>Rickettsia</th><th>Arthropod COI</th></tr></thead><tbody><tr><td colspan="6"><strong>Thelytoky</strong></td></tr><tr><td><em>M. aculeatus</em> (43)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. aculeatus nigroflavus</em> (21)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. brevicaudis</em> (1)</td><td>+ (100)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td><em>M. borriesi</em> (1)</td><td>+ (100)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td><em>M. pictus</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. pinsapinis</em> (60)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. pistaciae</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. rosae</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. rosae alba</em> (5)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. suspectus</em> (65)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="6"><strong>Arrhenotoky</strong></td></tr><tr><td><em>M. amicorum</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. atlanticus</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. cryptomeriae</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. pinus</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. rafni</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. spermotrophus</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. schimitscheki</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td><em>M. transvaalensis</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr></tbody></table>
<table><caption>**Table 3.** Wolbachia infection rates in control and tetracycline-treated females of *M. pinsapinis* (thelytokous) and *M. schimitscheki* (arrhenotokous), and brood size and proportions of males produced by control and tetracycline-treated females of these species.</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">Wolbachia infection</th><th colspan="3">Progeny produced by treated females</th></tr><tr><th>Treatment (N*)</th><th>% of wsp positives**</th><th>Number of mothers</th><th>Mean brood size***</th><th>Mean % of males</th></tr></thead><tbody><tr><td rowspan="2">M. pinsapinis</td><td>control (44)</td><td>100</td><td>44</td><td>21.4 ± 5.3<sup>a</sup></td><td>0</td></tr><tr><td>tetracycline (42)</td><td>4.7</td><td>42</td><td>18.7 ± 7.8<sup>a</sup></td><td>92 ± 1.1</td></tr><tr><td rowspan="2">M. schimitscheki</td><td>control (30)</td><td>0</td><td>30</td><td>30.3 ± 4.5<sup>a</sup></td><td>100</td></tr><tr><td>tetracycline (28)</td><td>0</td><td>28</td><td>32.1 ± 7.5<sup>a</sup></td><td>100</td></tr></tbody></table>

<footnote>*Number of treated females</footnote>

<footnote>**Infection rates were determined by PCR using Wolbachia-specific primers of the wsp gene.</footnote>

<footnote>***For each species, similar letters indicate that the mean number of larvae (±SE) produced do not differ significantly using a Kruskal-Wallis test (P<0.05).</footnote>
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<table><caption>**Table S1.** Distribution of arrhenotoky, thelytoky and host-plant specialization among the seed-feeding wasps of the *Megastigmus* genus. Data were compiled from reviews of Grissell (1999), Roques and Skrzypczynska (2003), Roques et al. (2003) and Auger-Rozenberg et al. (2006). Species in bold were specifically studied in this paper. A: arrhenotokous parthenogenesis. T: thelytokous parthenogenesis. * Ratio of males to females; balanced: consideration of similar frequencies of females and males by authors; NA: no specific mention on sex ratio by authors but numbers of males mentioned in samples (thelytoky being associated with “males unknown” or “males are scarce” specific mentions).</caption><thead><tr><th>Host-plant taxon</th><th>Species</th><th>Reproduction</th><th>Sex ratio*</th><th>Main host-plant genus</th><th>Native area</th></tr></thead><tbody><tr><td rowspan="17"><u>Gymnosperms</u></td><td><u>Cupressaceae</u></td><td></td><td></td><td></td><td></td></tr><tr><td><b>M. chamaecyparidis</b></td><td>A</td><td>balanced</td><td><i>Chamaecyparis</i></td><td>Palaearctic</td></tr><tr><td><b>M. thyoides</b></td><td>A</td><td>balanced</td><td><i>Chamaecyparis</i></td><td>Nearctic</td></tr><tr><td><b>M. atlanticus</b></td><td>A</td><td>balanced</td><td><i>Cupressus</i></td><td>Palaearctic</td></tr><tr><td><b>M. carinus</b></td><td>A</td><td>NA</td><td><i>Cupressus</i></td><td>Palaearctic</td></tr><tr><td><b>M. cupressi</b></td><td>A</td><td>NA</td><td><i>Cupressus</i></td><td>Oriental</td></tr><tr><td><b>M. duclouxiana</b></td><td>A</td><td>NA</td><td><i>Cupressus</i></td><td>Palaearctic</td></tr><tr><td><b>M. watchli</b></td><td>A</td><td>0.5-1.7</td><td><i>Cupressus</i></td><td>Palaearctic</td></tr><tr><td><b>M. amicorum</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. bipunctatus</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. certus</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. formosana</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>East-Asia</td></tr><tr><td><b>M. fidus</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. gravis</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. juniperi</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. pingii</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>East-Asia</td></tr><tr><td><b>M. procerae</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. rigidae</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. sabinae</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. somaliensis</b></td><td>A</td><td>NA</td><td><i>Juniperus</i></td><td>Afrotropical</td></tr><tr><td><b>M. thuriferana</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. validus</b></td><td>A</td><td>balanced</td><td><i>Juniperus</i></td><td>Palaearctic</td></tr><tr><td><b>M. thuyopsis</b></td><td>A</td><td>NA</td><td><i>Thuyopsis</i></td><td>Palaearctic</td></tr><tr><td><u>Pinaceae</u></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><b>M. firmae</b></td><td>A</td><td>NA</td><td><i>Abies</i></td><td>Palaearctic</td></tr><tr><td><b>M. lasiocarpae</b></td><td>A</td><td>balanced</td><td><i>Abies</i></td><td>Nearctic</td></tr><tr><td><b>M. milleri</b></td><td>A</td><td>balanced</td><td><i>Abies</i></td><td>Nearctic</td></tr><tr><td><b>M. pinus</b></td><td>A</td><td>0.4-0.5</td><td><i>Abies</i></td><td>Nearctic</td></tr><tr><td><b>M. rafni</b></td><td>A</td><td>0.4</td><td><i>Abies</i></td><td>Nearctic</td></tr><tr><td><b>M. specularis</b></td><td>A</td><td>balanced</td><td><i>Abies</i></td><td>Nearctic</td></tr><tr><td><b>M. schimitscheki</b></td><td>A</td><td>0.4-0.5</td><td><i>Cedrus</i></td><td>Palaearctic</td></tr><tr><td><b>M. laricis</b></td><td>A</td><td>balanced</td><td><i>Larix</i></td><td>Nearctic</td></tr><tr><td><b>M. atedius</b></td><td>A</td><td>0.19-0.66</td><td><i>Picea</i></td><td>Palaearctic</td></tr><tr><td><b>M. ezomatsuanus</b></td><td>A</td><td>0.5</td><td><i>Picea</i></td><td>Palaearctic</td></tr><tr><td><b>M. likiangensis</b></td><td>A</td><td>NA</td><td><i>Picea</i></td><td>East-Asia</td></tr><tr><td><b>M. strobilobius</b></td><td>A</td><td>0.4-0.6</td><td><i>Picea</i></td><td>Palaearctic</td></tr><tr><td><b>M. albifrons</b></td><td>A</td><td>balanced</td><td><i>Pinus</i></td><td>Nearctic</td></tr><tr><td><b>M. strobiformis</b></td><td>A</td><td>NA</td><td><i>Pinus</i></td><td>Nearctic</td></tr></tbody></table>
<table><tbody><tr><td></td><td>M. pseudotsugae</td><td>A</td><td>NA</td><td>Pseudotsuga</td><td>East-Asia</td></tr><tr><td></td><td>M. spermotrophus</td><td>A</td><td>balanced</td><td>Pseudotsuga</td><td>Nearctic</td></tr><tr><td></td><td>M. hoffmeyeri</td><td>A</td><td>balanced</td><td>Tsuga</td><td>Nearctic</td></tr><tr><td></td><td>M. tsugae</td><td>A</td><td>balanced</td><td>Tsuga</td><td>Nearctic</td></tr><tr><td></td><td>M. tsugaphilus</td><td>A</td><td>NA</td><td>Tsuga</td><td>Palaearctic</td></tr><tr><td></td><td>M. borriesi</td><td>T</td><td>♂<0.1%</td><td>Abies</td><td>East-Asia</td></tr><tr><td></td><td>M. suspectus</td><td>T</td><td>♂<0.2%</td><td>Abies</td><td>Palaearctic</td></tr><tr><td></td><td>M. pinsapinis</td><td>T</td><td>♂<0.1%</td><td>Cedrus</td><td>Palaearctic</td></tr><tr><td></td><td>M. pictus</td><td>T</td><td>♂<1%</td><td>Larix</td><td>Palaearctic</td></tr><tr><td>Taxodiaceae</td><td>M. cryptomeriae</td><td>A</td><td>balanced</td><td>Cryptomeria</td><td>East-Asia</td></tr><tr><td colspan="6"><strong>Angiosperms</strong></td></tr><tr><td>Anacardiaceae</td><td>M. thomseni</td><td>A</td><td>NA</td><td>Heeria</td><td>Afrotropical</td></tr><tr><td></td><td>M. ozoroae</td><td>A</td><td>NA</td><td>Ozoroae</td><td>Afrotropical</td></tr><tr><td></td><td>M. rhusi</td><td>A</td><td>NA</td><td>Rhus</td><td>Afrotropical</td></tr><tr><td></td><td>M. transvaalensis</td><td>A</td><td>0.5</td><td>Schinus</td><td>Afrotropical</td></tr><tr><td></td><td>M. pistaciae</td><td>T</td><td>♂<0.1%</td><td>Pistacia</td><td>Palaearctic</td></tr><tr><td>Fabaceae</td><td>M. albizziae</td><td>A</td><td>NA</td><td>Albizia</td><td>Oriental</td></tr><tr><td>Hamameliaceae</td><td>M. distylii</td><td>A</td><td>NA</td><td>Distylium</td><td>Palaearctic</td></tr><tr><td>Myrtaceae</td><td>M. ophelinii</td><td>A</td><td>NA</td><td>Eucalyptus</td><td>Australasia</td></tr><tr><td>Proteaceae</td><td>M. hakeae</td><td>A</td><td>NA</td><td>Hakea</td><td>Australasia</td></tr><tr><td>Rhamnaceae</td><td>M. helianthae</td><td>A</td><td>NA</td><td>Helinius</td><td>Afrotropical</td></tr><tr><td>Rosaceae</td><td>M. amelanchieris</td><td>A</td><td>NA</td><td>Amelanchier</td><td>Nearctic</td></tr><tr><td></td><td>M. cotoneastri</td><td>A</td><td>NA</td><td>Cotoneaster</td><td>Palaearctic</td></tr><tr><td></td><td>M. mali</td><td>A</td><td>NA</td><td>Malus</td><td>Palaearctic</td></tr><tr><td></td><td>M. pourthiaceae</td><td>A</td><td>0.6</td><td>Photinia</td><td>Palaearctic</td></tr><tr><td></td><td>M. physocarpi</td><td>A</td><td>NA</td><td>Physocarpus</td><td>Nearctic</td></tr><tr><td></td><td>M. fangii</td><td>A</td><td>NA</td><td>Rosa</td><td>East-Asia</td></tr><tr><td></td><td>M. nigrovariegatus</td><td>A</td><td>balanced</td><td>Rosa</td><td>Nearctic</td></tr><tr><td></td><td>M. yunnanensis</td><td>A</td><td>NA</td><td>Rosa</td><td>East-Asia</td></tr><tr><td></td><td>M. aculeatus</td><td>T</td><td>♂<4%</td><td>Rosa</td><td>Palaearctic</td></tr><tr><td></td><td>M. aculeatus nigroflavus</td><td>T</td><td>♂<1%</td><td>Rosa</td><td>Palaearctic</td></tr><tr><td></td><td>M. rosae</td><td>T</td><td>♂<1%</td><td>Rosa</td><td>Palaearctic</td></tr><tr><td></td><td>M. rosae var. alba</td><td>T</td><td>♂<1%</td><td>Rosa</td><td>Palaearctic</td></tr><tr><td></td><td>M. brevicaudis</td><td>T</td><td>♂<1%</td><td>Sorbus</td><td>Palaearctic</td></tr></tbody></table>
<table><caption>**Table S2.** Primer pairs used for endosymbiont PCR screening in *Megastigmus* spp.</caption><thead><tr><th>Primer pair (5'-3')</th><th>Target group</th><th>Target gene (fragment size)</th><th>Annealing temperature (°C)</th></tr></thead><tbody><tr><td>wsp81F TGGTCCAATAAGTGATGAAGAAAC<sup>a</sup><br>wsp691R AAAAATTAAACGCTACTCCA<sup>a</sup></td><td>Wolbachia pipientis</td><td>wsp<br>(610 bp)</td><td>59</td></tr><tr><td>Ch-F TACTGTAAGAATAAGCACCGGC<sup>b</sup><br>Ch-R GTGGATCACTTAACGCTTTCG<sup>b</sup></td><td>Cardinium sp.</td><td>16S rRNA<br>(~900 bp)</td><td>57</td></tr><tr><td>Ars23S-1 CGTTTGATGAATTCATAG TCAAA<sup>c</sup><br>Ars23S-2 GGTCCTCCAGTTAGTGTTACCCAAC<sup>c</sup></td><td>Arsenophonus nasoniae</td><td>23S rDNA<br>(650 bp)</td><td>60</td></tr><tr><td>27f GAGAGTTTGATCCTGGCTCAG<sup>d</sup><br>1495r CTACGGCTACCTTGTTACGA<sup>d</sup></td><td>Rickettsia sp.</td><td>16S rRNA<br>(1500 bp)</td><td>50</td></tr><tr><td>LCO1490 GGTCAACAAATCATAAAGATATTGG<sup>e</sup><br>HCO2198 TAAACTTCAGGGTGACCAAAAAATCA<sup>e</sup></td><td>Arthropods</td><td>COI<br>(658 bp)</td><td>48</td></tr></tbody></table>

<footnote><sup>a</sup>Braig *et al.* (1998)</footnote>

<footnote><sup>b</sup>Zchori-Fein and Perlman (2004)</footnote>

<footnote><sup>c</sup>Thao and Baumann (2004)</footnote>

<footnote><sup>d</sup>Hagimori *et al.* (2006)</footnote>

<footnote><sup>e</sup>Folmer *et al.* (1994)</footnote>
**Figure S1.** Phylogenetic placement of *Wolbachia* infecting the seed-specialized wasps *Megastigmus* spp., among other *Wolbachia* strains belonging to A and B supergroups, based on the sequences of each of the four MLST genes used in this study (*coxA*, *gatB*, *ftsZ* and *hcpA*). Bayesian likelihood inference phylogenies are shown, while maximum likelihood analyses gave substantially the same results. *Wolbachia* sequences are labelled with the name of their host. *Wolbachia* of *Brugia malayi* was used as an outgroup. Posterior probability values are indicated at each node. Each tree represents phylogenetic reconstruction based on 34 Wolbachia strains for *coxA* (402 bp), *gatB* (369 bp), *ftsZ* (435 bp) and *hcpA* (444 bp). Three lineages are revealed (green, red and light brown), two belonging to the A supergroup while the position of the *Wolbachia* infecting *M. pistaciae* suggests a recombination event between A and B *Wolbachia* supergroups. Indeed, this strain belongs to the B supergroup based on all sequences except for *coxA* for which it is placed in the B supergroup.

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /><figcaption>ftsZ</figcaption></figure>

<figure><img src="image_6.png" /></figure>
hcpA

A

B

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>**Figure S2.** Phylogenetic placement of the *Wolbachia* strains infecting the seed-specialized wasps *Megastigmus* spp., among other *Wolbachia* strains belonging to A and B supergroups, based on wsp sequences of 49 strains (530 bp). Bayesian likelihood inference phylogenies are shown, while maximum likelihood analyses gave substantially the same results. *Wolbachia* sequences are labelled with the name of their host. *Wolbachia* of *Brugia malayi* was used as an outgroup. Posterior probability values are indicated at each node.</figcaption></figure>
References

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Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoeck R (1994) DNA primers for amplification of mitochondrial cytochrome *c* oxydase subunit I from diverse metazoan invertebrates. *Molecular Marine Biology and Biotechnology*, **3**, 294-299.

Hagimori T, Abe Y, Date S, Miura K (2006) The first finding of a *Rickettsia* bacterium associated with parthenogenesis induction among insects. *Current Microbiology*, **52**, 97-101.

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