Molecular Ecology (2014) 23, 2105–2117

doi: 10.1111/mec.12704

Origin, acquisition and diversification of heritable bacterial endosymbionts in louse flies and bat flies OLIVIER DURON,* ULRICH E. SCHNEPPAT,† ARNAUD BERTHOMIEU,‡ STEVEN M. GOODMAN,§ BORIS DROZ,¶ CHRISTOPHE PAUPY,* ** € L O B A M E N K O G H E , * * * N I L R A H O L A * * * and P A B L O T O R T O S A † † ‡ ‡ JUDICAE *Laboratoire MIVEGEC, UMR 5290-224 CNRS-IRD-UM1-UM2, Centre de Recherche IRD, 34090 Montpellier, France, €ndner Naturmuseum, Amt fu €r Kultur Graubu €nden, Masanserstrasse 31, CH-7000 Chur, Switzerland, ‡Institut des Sciences †Bu de l’Evolution, CNRS - Universite Montpellier 2 (UMR5554), Place Eugene Bataillon, 34095 Montpellier Cedex 05, France, §Science and Education, Field Museum of Natural History, 1400 South Lake Shore Drive, Chicago, IL 60605, USA, ¶Faculty of Geoscience and Environment, University of Lausanne, Geopolis, 1015 Lausanne, Switzerland, **Unite d’Ecologie des Systemes Vectoriels, Centre International de Recherches Medicales de Franceville (CIRMF), BP 769, Franceville, Gabon, ††Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Ocean Indien, 2 rue Maxime Riviere, 97490 Ste Clotilde, France, ‡‡Universite de La Reunion, 15 Avenue Rene Cassin, 97715 Saint Denis Messag Cedex 9, France

Abstract The c-proteobacterium Arsenophonus and its close relatives (Arsenophonus and like organisms, ALOs) are emerging as a novel clade of endosymbionts, which are exceptionally widespread in insects. The biology of ALOs is, however, in most cases entirely unknown, and it is unclear how these endosymbionts spread across insect populations. Here, we investigate this aspect through the examination of the presence, the diversity and the evolutionary history of ALOs in 25 related species of blood-feeding flies: tsetse flies (Glossinidae), louse flies (Hippoboscidae) and bat flies (Nycteribiidae and Streblidae). While these endosymbionts were not found in tsetse flies, we identify louse flies and bat flies as harbouring the highest diversity of ALO strains reported to date, including a novel ALO clade, as well as Arsenophonus and the recently described Candidatus Aschnera chinzeii. We further show that the origin of ALO endosymbioses extends deep into the evolutionary past of louse flies and bat flies, and that it probably played a major role in the ecological specialization of their hosts. The evolutionary history of ALOs is notably complex and was shaped by both vertical transmission and horizontal transfers with frequent host turnover and apparent symbiont replacement in host lineages. In particular, ALOs have evolved repeatedly and independently close relationships with diverse groups of louse flies and bat flies, as well as phylogenetically more distant insect families, suggesting that ALO endosymbioses are exceptionally dynamic systems. Keywords: Arsenophonus, bat fly, co-evolution, endosymbiosis, heritable bacteria, louse fly Received 13 November 2013; revision received 10 February 2014; accepted 12 February 2014

Introduction Insects commonly have close associations with maternally inherited bacterial endosymbionts (Moran et al. 2008; Engelstadter & Hurst 2009; Wernegreen 2012). While in some cases, these organisms are obligate Correspondence: Olivier Duron, Fax: +33 467 416299; E-mail: [email protected] © 2014 John Wiley & Sons Ltd

endosymbionts essential for the survival of the host; in many other cases, they are facultative either providing ecologically contingent advantages or manipulating the reproduction of their host (Haine 2008; Moran et al. 2008; Werren et al. 2008; Engelstadter & Hurst 2009; Saridaki & Bourtzis 2010). Each of these effects is of ecological and evolutionary importance, broadening host trophic interactions, facilitating adaptation to changing environments, driving changes in sex determinism or

2106 O . D U R O N E T A L . assisting speciation (Oliver et al. 2010; Cordaux et al. 2011; Ferrari & Vavre 2011). Remarkably, the acquisition of endosymbionts can serve as a source of evolutionary innovations in insects: endosymbionts change host phenotypes and, because they are heritable, may facilitate host adaptation to habitats (Oliver et al. 2010; Ferrari & Vavre 2011; Duron & Hurst 2013). Obligate endosymbionts typically evolve towards exclusive and irreversibly specialized interactions with particular insect lineages as well exemplified with many insect species having restricted diets (Moran et al. 2008; Wernegreen 2012). In these cases, endosymbionts tend to supplement the hosts’ dietary intake with vitamins or amino acids that are rare or absent in available food resources. Their acquisition by hosts may extend into the deep evolutionary past, dating to the origins of major groups such as aphids, leafhoppers or carpenter ants, and associated with exclusive vertical transmission that tracks host speciation, which in turn results in codiversification between host and endosymbiont, as well illustrated in aphids (Moran et al. 2005; Jousselin et al. 2009) or tsetse flies (Chen et al. 1999). Interestingly, a few case studies, involving sap-feeding insects (Fukatsu & Ishikawa 1992, 1996; Moran et al. 2005) and weevils (Conord et al. 2008; Toju et al. 2013), have shown that an obligate endosymbiont may occasionally be lost and replaced by another one recently acquired from a different host species following lateral transfer. It has been further hypothesized that a switch of the host habitat may have favoured a replacement of ancient endosymbionts by novel organisms facilitating adaptation to the new host habitat (Conord et al. 2008; Toju et al. 2013). Flies from the Hippoboscoidea superfamily (Diptera) are obligate blood feeders allied with a diverse assemblage of bacterial endosymbionts. There are four recognized family-level taxa within Hippoboscoidea: the Glossinidae or tsetse flies (22 species, feeding on mammals and, to a lesser extent, on reptiles and birds), the Hippoboscidae or louse flies (>150 species, feeding on birds and mammals) and two families of bat flies (exclusively feeding on bats): the Streblidae (>230 species) and the Nycteribiidae (>270 species) (Dittmar et al. 2006; Petersen et al. 2007). Among them, the endosymbionts of tsetse flies, consisting of the obligate symbiont Wigglesworthia glossinidia (Aksoy 1995) and two facultative symbionts, Sodalis glossinidius (Aksoy et al. 1997; Dale & Maudlin 1999) and Wolbachia (Cheng et al. 2000; Doudoumis et al. 2012), are the best studied, reflecting the medical importance of tsetse flies as vectors of trypanosomes. By contrast, much less attention has been paid to louse flies and bat flies. However, recently, another endosymbiont, Arsenophonus, was found exceptionally common in members of

these two hippoboscoid families (Dale et al. 2006; Trowbridge et al. 2006; Duron et al. 2008; Novakova et al. 2009; Lack et al. 2011; Morse et al. 2013). Arsenophonus has emerged within recent years as one of the most diverse symbiotic lineages known to date: widespread infections were found in numerous insect groups, including aphids, parasitoid wasps and triatomine bugs (Duron et al. 2008, 2010; Novakova et al. 2009; Taylor et al. 2011; Russell et al. 2012; Jousselin et al. 2013). Some Arsenophonus strains (formerly known as Phlomobacter sp.) were also detected in the phloem of plants on which infected phytophagous insects fed and were assumed opportunistic plant pathogens (Bressan et al. 2012). Further surveys have revealed that Arsenophonus is a part of an emerging novel clade of endosymbionts within c-proteobacteria: bacteria phylogenetically allied to Arsenophonus (Arsenophonus and like organisms, ALOs hereafter) have been characterized as Cand. Riesia pediculicola in lice (SasakiFukatsu et al. 2006; Allen et al. 2007) and Cand. Aschnera chinzeii, as well as two unnamed bacterial groups, in bat flies (Hosokawa et al. 2011; Morse et al. 2012, 2013). How ALO infections have spread within the Hippoboscoidea is still unclear, and very few studies have addressed this question: while some authors have presented evidence of codiversification of Arsenophonus within the Hippoboscidae and Streblidae (Trowbridge et al. 2006) and of Cand. Aschnera chinzeii within Nycteribiidae (Hosokawa et al. 2011), two other studies have revealed a very different evolutionary scenario with disagreement between fly and ALO phylogenies, a result suggestive of symbiont horizontal transfers (Novakova et al. 2009; Morse et al. 2013). Here, we reconsider these issues by (i) undertaking an extensive screening for the presence of ALOs, (ii) measuring their diversity and (iii) retracing their evolutionary history in the Hippoboscoidea superfamily. Although the 16S rRNA gene sequence has been commonly used as an exclusive bacterial taxonomic marker, it was recently shown to be inadequate for inferring a reliable intrageneric phylogeny in diverse c-proteobacteria, including Arsenophonus: the intrageneric topology is typically poorly resolved and usually unstable because of insufficient sequence polymorphism and intragenomic variability between divergent 16S rRNA copies (Sorfova et al. 2008; Novakova et al. 2009; Husnik et al. 2011). Therefore, in the present study, we characterized ALO strains using the GroEL locus and, for Arsenophonus, a recently developed multilocus typing scheme allowing intrageneric phylogeny to be resolved (Duron et al. 2010; Mouton et al. 2012; Jousselin et al. 2013). Finally, we used this data set to infer the evolutionary processes that have shaped the diversity of ALOs in louse flies and bat flies. © 2014 John Wiley & Sons Ltd

E N D O S Y M B I O N T S I N L O U S E F L I E S A N D B A T F L I E S 2107

Materials and methods Fly collection One hundred and fifty-six specimens representing the four Hippoboscoidea families, seven subfamilies, 12 genera and 25 species were examined (Table 1). Specimens were collected in the field between 1995 and 2012 in Africa, Europe and western Indian Ocean islands, on taxonomically diverse bird and mammal hosts. Specimens from laboratory colonies of tsetse flies Glossina f. fuscipes and G. m. morsitans, and of the pigeon louse fly Pseudolynchia canariensis were also used. In this latter species, an Arsenophonus strain has been formally named, A. arthropodicus, by Dale et al. (2006). Within each examined species, 1–20 individuals were analysed separately. All samples were preserved in 70–90% ethanol at room temperature until laboratory use.

Screening and sequencing for bacterial endosymbionts Fly DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) following the instructions of the manufacturer. The quality of fly DNA was systematically tested by PCR amplification of a conserved region of the mitochondrial CO1gene (Table S1, Supporting information). The ALO infections were then screened and genotyped using a nested PCR assay based on the GroEL gene, using two sets of specific primers as detailed in Table S1 (Supporting information). For Arsenophonus intrageneric phylogenetic analyses, ftsK, yaeT and fbaA bacterial sequences were obtained from infected fly specimens. Gene features and primers are listed in Table S1 (Supporting information). All the PCR amplifications were performed under the following conditions: initial denaturation at 93 °C for 3 min, 30 cycles of denaturation (93 °C, 30 s), annealing (50–52 °C, depending on primers, cf. Table S1, Supporting information 30 s), extension (72 °C, 1 min) and a final extension at 72 °C for 5 min. The PCR products were electrophoresed in a 1.5% agarose gel. PCR products were purified with the QIAquick gel extraction kit (Qiagen, Venlo, Netherlands) and directly sequenced on an ABI Prism 3130 sequencer using the BigDye Terminator Kit (Applied Biosystems, Foster City, California) after purification. The chromatograms were manually edited with CHROMAS LITE (http://www.technelysium. com.au/chromas_lite.html), and sequence alignment was performed using CLUSTALW (Thompson et al. 2002) implemented in MEGA (Kumar et al. 2004). The sequences were deposited in GenBank (Table S2, Supporting information). © 2014 John Wiley & Sons Ltd

Molecular and phylogenetic analyses Phylogenetic relationships were evaluated using fly mitochondrial CO1 sequences (421 bp aligned nucleotide sites) and bacterial GroEL (679 bp), fbaA (363 bp), ftsK (214 bp) and yaeT (430 bp) sequences obtained from louse flies and bat flies. We also used additional CO1 and bacterial sequences, including sequences from ALOs found in various arthropod hosts and from closest ALO relatives available in GenBank (Table S2, Supporting information). Using this data set, we first estimated the phylogenetic relationships between all ALO endosymbionts using the GroEL sequences and, in the case of Arsenophonus, we refined our analyses using independent and concatenated fbaA, ftsK and yaeT sequences. We then reconstructed the phylogenetic relationships between louse flies and bat flies using CO1 sequences and finally contrasted the CO1 phylogeny of Hippoboscoidea and the GroEL phylogeny of their ALO endosymbionts. Phylogenetic analyses were based on maximum-likelihood (ML) analyses and Bayesian inference. A ML heuristic search using a starting tree obtained by neighbourjoining was conducted in MEGA (Kumar et al. 2004). Clade robustness was assessed by bootstrap analysis using 500 replicates. Bayesian phylogenetic analyses were conducted in MrBayes (Ronquist & Huelsenbeck 2003). Two replicate analyses were run for 5 million generations. For each replicate, we ran one cold chain and three hot chains of the Markov Chain Monte Carlo method, using a random starting tree and sampling trees every 100 generations. The point of stationarity was determined as when the likelihood distribution reached a plateau and trees prior to stationarity were discarded (5000 trees). The remaining trees were used to calculate 50% majority rule consensus trees. Posterior probabilities (pp) were summarized accordingly. The evolutionary models most closely fitting the sequence data were determined using Akaike information criterion with the program MEGA (Kumar et al. 2004). For each data set, the best-fit approximation was the general time reversible model with gamma distribution and invariant sites (GTR + G + I). All sequence alignments were checked for putative recombinant regions using the GENECONV (Sawyer 1989) and RDP (Martin & Rybicki 2000) methods available in the RDP3 computer analysis package (Martin et al. 2010). The ML phylogenies of host CO1 and ALO GroEL were then used to conduct cophylogenetic analyses using TREEMAP, which is based on algorithms for computing and displaying reconstructions of host–parasite evolution (Charleston & Page 2002). A pairwise distance Spearman correlation test was performed to compare the significance of correlations of pairwise distances

2108 O . D U R O N E T A L . Table 1 List and origin of louse flies (Hippoboscidae), bat flies (Nycteribiidae and Streblidae) and tsetse flies (Glossinidae) used in this study n

Origin

Vertebrate host

15 3 5

France (2006) France (2012) France (2012)

Domestic horse (Equus caballus, Equidae) Cattle (Bos taurus, Bovidae) Domestic cat (Felis silvestris catus, Felidae)

2 5

Greece (2012) Switzerland (2012)

5

Switzerland (2008–2012)

Lipoptena fortisetosa (Maa, 1965)

1

Austria (2012)

Lipoptena sp.

1 2

Switzerland (2012) Austria (2012)

Human (Homo sapiens, Hominidae) European red deer (Cervus elaphus hippelaphus, Cervidae) European roe deer (Capreolus capreolus capreolus, Cervidae) European mouflon (Ovis orientalis musimon, Bovidae) Human (Homo sapiens, Hominidae) Alpine chamois (Rupicapra rupicapra rupicapra, Bovidae)

Host taxa Hippoboscidae Hippoboscinae Hippobosca equina (Linnaeus, 1758) Hippobosca longipennis (Fabricius, 1805) Lipopteninae Lipoptena capreoli (Rondani, 1878) Lipoptena cervi (Linnaeus, 1758)

Ornithomyinae Crataerina melbae (R ondani, 1879) Crataerina pallida (Latreille, 1812) Ornithomya avicularia (Linnaeus, 1758)

Ornithomya biloba (Dufour, 1827) Ornithomya chloropus (Bergroth, 1901) Ornithomya fringillina (Curtis, 1836) Pseudolynchia canariensis (Macquart, 1839)

5 20 1

Switzerland (2004–2006) Switzerland (2004–2006) Switzerland (2005)

6 1 1

Switzerland (2005) Austria (2012) Austria (2010)

2 5 5

Austria (2010, 2012) Switzerland (2003) Switzerland (2005–2007)

4 1 5

Switzerland (2005) Switzerland (2005) Laboratory colony derived from field specimens collected in Utah, USA Switzerland (2003, 2012)

Alpine swift (Apus melba, Apodidae) Common swift (Apus apus, Apodidae) Eurasian sparrowhawk (Accipiter nisus, Accipitridae) Undetermined bird species Brown Owl (Strix aluco, Strigidae) Eurasian sparrowhawk (Accipiter nisus, Accipitridae) Eurasian eagle-owl (Bubo bubo, Strigidae) Barn swallow (Hirundo rustica, Hirundinidae) Diverse passerine bird species (Passeriformes) Coal tit (Periparus ater, Paridae) Eurasian nuthatch (Sitta europaea, Sittidae) Unknown

Common house martin (Delichon urbica, Hirundinidae)

Stenepteryx hirundinis (Linnaeus, 1758)

12

Nycteribiidae Cyclopodiinae Eucampsipoda africana (Theodor, 1955)

7

Gabon (2012)

Egyptian fruit bat (Rousettus aegyptiacus, Pteropodidae)

1

France (2002)

5

Gabon (2012)

1

Gabon (2012)

Nycteribia stylidiopsis (Speicer, 1908)

6

Comoros Islands (2012)

Nycteribia kolenatii (Theodor & Moscona, 1954) Basilia nov. sp.

1

France (2012)

2

Madagascar (2012)

Penicillidia conspicua (Speiser, 1901)

1

France (1995)

Schreibers’ long-fingered bat (Miniopterus schreibersi, Miniopteridae) Greater long-fingered bat (Miniopterus inflatus, Miniopteridae) Sundevall’s round-leaf bat (Hipposideros caffer, Hipposideridae) Long-winged bat (Miniopterus spp., Miniopteridae) Daubenton’s bat (Myotis daubentonii, Vespertilionidae) Malagasy large house bat (Scotophilus robustus, Vespertilionidae) Schreiber’s long-fingered bat (Miniopterus schreibersi, Miniopteridae)

Nycteribiinae Nycteribia schmidlii schmidlii (Schiner, 1853) Nycteribia schmidlii scotti (Falcoz, 1923)

© 2014 John Wiley & Sons Ltd

E N D O S Y M B I O N T S I N L O U S E F L I E S A N D B A T F L I E S 2109 Table 1 Continued Host taxa Penicillidia fulvida (Bigot, 1885) Penicillidia leptothrinax (Speiser, 1908) Streblidae Brachytarsininae Brachytarsina allaudi (Falcoz, 1923) Glossinidae Glossininae Glossina fuscipes fuscipes (Newstead, 1911) Glossina morsitans morsitans (Westwood, 1850)

n 4 10

Origin

Vertebrate host

Gabon (2012)

Greater long-fingered bat (Miniopterus inflatus, Miniopteridae) Long-winged bat (Miniopterus spp., Miniopteridae)

Madagascar (2012)

3

Gabon (2012)

Giant round-leaf bat (Hipposideros gigas, Hipposideridae)

4

Laboratory colony derived from field specimens collected in Central African Republic Laboratory colony derived from field specimens collected in Zimbabwe

Unknown

4

between taxa, for associated clades in the two phylogenies, against a distribution of such measures estimated by randomizing subtrees of the ALO phylogeny. Each P-value was estimated with 1000 Monte Carlo replicates, and the 95% confidence upper bound was calculated for each using Wilson’s score interval for binomial proportions. We further conducted a phylogenetic network analysis based on uncorrected P distances using the Neighbour-net algorithm (Bryant & Moulton 2004) implemented in SPLITSTREE (Huson & Bryant 2006). The resulting phylogenetic networks generalize the trees by allowing cross-connections between branches, which might display conflicting signals in phylogenetic data sets because of lateral gene transfers.

Results Distribution of ALO endosymbionts One hundred and fifty-six specimens from 25 species, including louse flies (Hippoboscidae: 14 species), bat flies (Nycteribiidae: 8; Streblidae: 1) and tsetse flies (Glossinidae: 2) were screened for the presence of ALOs with a GroEL-based nested PCR approach on DNA templates with quality being assessed by CO1 PCR amplification (Table 1). Of the 25 species examined, GroEL-nested PCR assay indicated the presence of ALOs in 23 species: infection was found in all taxa of Hippoboscidae, Nycteribiidae and Streblidae, while no Glossinidae was detected to be ALO-positive. Within each infected species, endosymbionts were observed in all tested individuals (n = 1–20, depending on species; Table 1), suggesting that infection by ALOs is ubiquitous in louse flies and bat flies. © 2014 John Wiley & Sons Ltd

Unknown

Diversity of ALO endosymbionts The diversity of ALO was examined using sequences from one to four bacterial markers, as detailed below. First, bacterial sequences from the GroEL gene were taken from a subsample of 67 specimens that were positive for ALO infection (one to three specimens per infected species were examined). The sequences were easily readable without double peaks, indicating that there was no coinfection of ALO strains in any specimen. Overall, 21 distinct GroEL alleles with 0.1–21.4% nucleotide divergence were characterized from infected specimens. Twenty-one ALO strains were identified based on GroEL alleles. Each of the 23 infected species of louse flies and bat flies harboured a different ALO infection with the exception of two species pairs, Hippobosca equina/H. longipennis and Lipoptena cervi/Lipoptena sp., which were infected by genetically identical ALO strains. No sequence variation was observed within each of the infected species, even between specimens from different locations or from different vertebrate hosts, suggesting that only one ALO strain may be present within each of louse fly and bat fly species. Second, we performed an additional analysis using the multilocus typing approach employed for recent genotyping of Arsenophonus strains (Duron et al. 2010; Jousselin et al. 2013). Efforts were made to amplify three bacterial genes (fbaA, ftsK and yaeT) from each louse fly and bat fly sample. We obtained PCR products only from host species infected by Arsenophonus (Table S2, Supporting information), indicating that currently available genotyping tools of Arsenophonus cannot be universally applied to all ALO strains. The 3 Arsenophonus genes were successfully amplified for nine host species, while only one gene (fbaA) was amplified for three

2110 O . D U R O N E T A L . other species (Lipoptena sp., L. cervi and L. fortisetosa; Table S2, Supporting information). Overall, nine, seven and eight distinct alleles were identified from the fbaA, ftsK and yaeT gene sequences, respectively. The allelic diversity at these loci was consistent with the results inferred from the GroEL sequences: each of the Arsenophonus-infected species harboured a distinct bacterial strain, with the exception of the same two species pairs, H. equina/H. longipennis and L. cervi/Lipoptena sp., which were infected by the same Arsenophonus strains. As observed with GroEL sequences, no fbaA, ftsK and yaeT sequence variation was observed within each of the Arsenophonus-infected species.

Phylogeny of ALO endosymbionts The phylogenetic relationships between the endosymbionts of louse flies and bat flies were first estimated using the GroEL sequences from 48 fly species: included in the analyses were the GroEL sequences obtained from louse flies (14 species) and bat flies (nine species) isolated in this study, as well as GroEL sequences from another 25 bat fly species (Hosokawa et al. 2011; Morse et al. 2012, 2013) available in GenBank (Table S2, Supporting information). In addition, GroEL sequences from Arsenophonus strains found in other insects, from Cand. Riesia pediculicola and from five close ALO relatives, were also included (Table S2, Supporting information). No recombination events were detected using the GENECONV and RDP methods for the GroEL data set (all P > 0.30). The ML analysis and Bayesian inferences produced congruent phylogenetic trees with minor differences in branch support (Fig. 1), which are also consistent with results from the network analysis (Fig. 2). All phylogenetic analyses based on the GroEL sequences showed that the ALO clade consists of at least six distinct groups: Arsenophonus spp., Cand. Aschnera chinzeii, Cand. Riesia pediculicola and three unnamed bacterial groups (hereafter ALO-1, ALO-2 and ALO-3). While the ALO-1 and ALO-2 groups have no formal names, they have been observed in previous studies (‘clade B’ and ‘clade D’, respectively, in Morse et al. 2013). To our knowledge, the ALO-3 group was not previously recognized before this current study and is a novel member of the ALO clade. Of the six ALO groups, three (Arsenophonus spp., Cand. Aschnera chinzeii and ALO-3) were retrieved from our sampling, the three other groups (Cand. Riesia pediculicola, ALO-1 and ALO-2) were only represented by sequences already available in GenBank (Table S2, Supporting information). On the basis of GroEL sequences, the bacterial strains found in louse flies and bat flies proved to be phylogenetically closely related to the ALOs strains found in

other insect species and all clearly fall within the ALO clade (Figs 1 and 2). However, they have different evolutionary origins: infections in louse flies and bat flies did not cluster together and were found in five of the six ALO groups (Cand. Riesia pediculicola was not found in louse flies or bat flies). Of the 48 fly species examined, Arsenophonus was found in 13 (Hippoboscidae: 9 species; Nycteribiidae: 3; Streblidae: 1); Cand. Aschnera chinzeii in 18 (all from the Nycteribiidae subfamily of Nycteribiidae); ALO-1 in six (all from the Nycterophiliinae and Trichobiinae subfamilies of Streblidae); ALO-2 in five (all from the Trichobiinae subfamily of Streblidae); and ALO-3 in five (all from the Ornithomyinae subfamily of Hippoboscidae) (Table S2, Supporting information). Overall, ALO infections found in each host family are thus polyphyletic: Hippoboscidae were infected by two distinct ALO groups, Nycteribiidae by two groups and Streblidae by three groups (Figs 1 and 2). Of important note, (i) the ALO-3 group is specific to louse flies feeding exclusively on birds belonging to the orders Passeriformes (Ornithomya biloba, O. chloropus, O. fringillina and Stenepteryx hirundinis; see Table 1), and Strigiformes and Accipitriformes (O. avicularia); (ii) Arsenophonus infections found in louse flies and bat flies are closely related to strains found in phylogenetically distant insect species (Figs 1 and 2), such as A. nasoniae (Gherna et al. 1991), the male-killer of the parasitoid wasp Nasonia vitripennis and A. triatominarum (Hypsa & Dale 1997), a strain infecting triatomine bugs. A second analysis was performed to refine the intrageneric phylogeny of Arsenophonus. For this, we used the Arsenophonus fbaA, ftsK and yaeT sequences obtained in the present work from louse flies (nine species) and bat flies (three species), as well as Arsenophonus sequences from 16 other insect species (mainly from aphids, parasitoid wasps and triatomine bugs) available from GenBank (Table S2, Supporting information). When the sequences were examined separately for each gene, ML analyses and Bayesian inferences produced congruent phylogenetic trees with minor differences in branch support and indicated that these infections belonged to the genus Arsenophonus (Figs S1, S2 and S3, Supporting information). We next compared the fbaA, ftsK and yaeT phylogenies, but because neither ftsK nor yaeT could be amplified from three Lipoptena species, the comparison between Arsenophonus phylogenies was then restricted to six species of louse flies and three of bat flies (Table S2, Supporting information). This multilocus comparison revealed the same phylogenetic pattern for three species of louse flies (Pseudolynchia canariensis, Crataerina melbae and C. pallida) and three of bat flies (Basilia nov. sp., Brachytarsina allaudi and Eucampsipoda africana), while there was incongruence for three louse flies (Hippobosca equina, H. longipennis © 2014 John Wiley & Sons Ltd

E N D O S Y M B I O N T S I N L O U S E F L I E S A N D B A T F L I E S 2111 S Basilia rybini S S Basilia truncata S

79/0.69

S Basilia nattereri S S Phthiridium hindlei S Phthiridium cf. tonk inensis

100/1.00 100/1.00

S Nycteribia pleuralis S Nycteribia pygmaea

100/1.00

S Nycteribia allotopa

S Nycteribia kolenatii

100/1.00 82/0.76

100/1.00

84/ 0.79

SS Nycteribia Nycteribia stylidiopsis styliopsis S Nycteribia schmidlii scotti 100/1.00 S Nycteribia schmidlii schmidlii S Penicillidia monoceros 100/1.00 S Penicillidia dufourii

Cand. Aschnera chinzeii

S Penicillidia fulvida S Penicillidia leptothrinax S S Penicillidia jenynsii

88/0.90 100/1.00

S Penicillidia oceanica S Penicillidia conspicua 100/1.00

S Pediculus humanus (Cand. S (Cand. Riesia pediculicola) *

S Pediculus capitis (Cand. (Cand. Riesia pediculicola) *

Cand. Riesia pediculicola

S S Trichobius intermedius S Megistopoda aranea

62/0.60

S Paratrichobius longicrus S Trichobius neotropicus

ALO-2

S Trichobius parasiticus

84/0.70

S Ornithomya chloropus

65/0.66

S Ornithomya avicularia

67/0.64

S Ornithomya biloba

100/1.00 71/0.80 86/0.84

ALO-3

S Ornithomya fringillina S Stenepteryx hirundinis 76/0.69 100/1.00

S Lipoptena sp. Lipoptena cervi cervi SS Lipoptena S Lipoptena fortisetosa

99/0.99

98/0.95

S Eucampsipoda africana S Eucampsipoda inermis S Lipoptena capreoli

78/0.72

100/1.00

S Hippobosca equina S Hippobosca longipennis

100/1.00

93/0.94 85/0.80

S Crataerina pallida S Crataerina melbae S Trichobius nov. nov. sp. S Trichobius cf. cf yunk eri

S Nycterophylia parnelli

100/1.00

S Nycterophylia nov. nov. sp. 100/1.00 78/0.78

Arsenophonus spp.

S Pseudolynchia canariensis (Arsenophonus arthropodicus) 100/1.00 85/0.90

100/1.00

SS Basilia Paracyclopodia nov. sp.nov. sp.

S Nasonia vitripennis (Arsenophonus nasoniae) * S Brachytarsina allaudi S Triatoma infestans (Arsenophonus triatominarum) * S

ALO-1

S Nycterophylia coxata

cf. coxata S Nycterophylia cf. Proteus mirabilis

100/1.00

Proteus penneri 100/1.00

100/1.00 100

94/0.90

Providencia rustigianii Providencia stuartii Photorhabdus luminescens

0.05

Fig. 1 Arsenophonus and like organisms (ALO) phylogeny constructed using maximum-likelihood (ML) estimations based on GroEL gene sequences. Insect symbionts have the prefix S followed by the scientific name of their host; all other names refer to bacteria. Closest ALO relatives (Proteus mirabilis, P. penneri, Providencia rustigianii, P. stuartii and Photorhabdus luminescens) were used to delineate the ALO clade. The six major ALO groups are named in the right portion of the figure. New sequences from this study are in bold and underlined. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. The arrow denotes a novel group of ALO endosymbionts, ALO-3. Branch numbers indicate percentage bootstrap support for major branches (500 replicates; only bootstrap values >60% are shown) followed by Bayesian posterior probabilities (Bayesian topology not shown) The scale bar is in units of substitution/site.

© 2014 John Wiley & Sons Ltd

2112 O . D U R O N E T A L . Fig. 2 Phylogenetic network obtained from Arsenophonus and like organisms (ALO) GroEL gene sequences using the Neighbour-net method. The six major ALO groups are presented. The arrow denotes a novel group of ALO endosymbionts, ALO-3. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. Closest ALO relatives (Proteus mirabilis, P. penneri, Providencia rustigianii, P. stuartii and Photorhabdus luminescens) were used to delineate the ALO clade.

and Lipoptena capreoli). The analysis of concatenated sequences showed significant recombination events in the Arsenophonus strains from H. equina (as previously shown by Jousselin et al. 2013) and H. longipennis through the whole region encompassing the fbaA gene, as well as in yaeT in the strain infecting L. capreoli (all P > 10 3 for the GENECONV and RDP recombinationdetection tests).

Host–symbiont codivergence We reconstructed the phylogenetic relationships between louse flies and bat flies using ML methods and Bayesian inference based on nucleotide sequences from mitochondrial CO1 fragments, including sequences produced from this study, as well as bat fly sequences available on GenBank (Table S2, Supporting information). Although the consensus CO1 phylogenetic tree shows some polytomies due to insufficient phylogenetic information, it was globally congruent with the louse fly and bat fly classification: we recovered partitioning of the three families (Hippoboscidae, Nycteribiidae and Streblidae; Fig. 3A), in accordance with previous phylogenetic investigations (Dittmar et al. 2006; Petersen et al. 2007). At the genus level, however, (i) Basilia (Nycteribiidae) was paraphyletic given that other genera (e.g.

Penicillidia and Nycteribia) clustered within the clade delineated by Basilia species (Fig. 3A), as previously reported (Dittmar et al. 2006); (ii) Ornithomya (Hippoboscidae) was also paraphyletic with Stenepteryx hirundinis within the clade delineated by Ornithomya spp. (but with low support values; see Fig. 3A), as previously reported (Petersen et al. 2007). We further contrasted the CO1 phylogeny of Hippoboscoidea and the GroEL phylogeny of their ALO endosymbionts (Fig. 3A and B). Because there was no published CO1 sequence from host species allied to the ALO-1 and ALO-2 groups, we conducted this analysis using louse flies (14 species) and bat flies (17 species) infected either by Arsenophonus (12 strains), Cand. Aschnera chinzeii (14 strains) or ALO-3 (five strains) (Table S2, Supporting information). The cophylogenetic analyses suggest that, in most cases, there are more codivergence events between fly hosts and ALO endosymbionts than would be expected by chance if their phylogenies were independent. Despite the presence of polytomies in the CO1 tree, the pairwise distance correlation test showed significant congruence at the roots of host and ALO phylogenies (P < 5.10 4; 95% confidence upper bound, 1.10 3) suggesting that the phylogenies have been highly dependent on each other throughout their history. Reconciling the phylogeny of Cand. © 2014 John Wiley & Sons Ltd

E N D O S Y M B I O N T S I N L O U S E F L I E S A N D B A T F L I E S 2113 Penicillidia monoceros

A

S Penicillidia monoceros S Penicillidia jenynsii

Penicillidia jenynsii Penicillidia conspicua 90/0.88

96/0.91

Penicillidia leptothrinax

S Penicillidia leptothrinax

Penicillidia fulvida Nycteribia schmidlii scotti 90/0.89

Nycteribia schmidlii schmidlii Nycteribia stylidiopsis

76/0.76

S Penicillidia fulvida S Nycteribia schmidlii scotti

96/0.94

S Nycteribia allotopa

Nycteribia pleuralis

S Nycteribia pleuralis

Nycteribia pygmaea

S Nycteribia pygmaea

Basilia rybini

S Basilia rybini

S Ornithomya biloba

Eucampsipoda africana

S Ornithomya avicularia

Brachytarsina allaudi

S Ornithomya chloropus

Ornithomya y biloba

S Ornithomya y fringillina g

Ornithomya avicularia

S Stenepteryx hirundinis

100/0.98 93/0.91

Ornithomya fringillina

S Eucampsipoda africana

Stenepteryx hirundinis 69/0.68

Crataerina melbae

100/1.00

Hippobosca longipennis

S Crataerina melbae

81/0.70

84/0.80

95/0.92

S Crataerina pallida S Pseudolynchia canariensis S Hippobosca longipennis

Hippobosca equina

100/1.00

81/0.80

Arsenophonus

100/1.00

S Hippobosca equina S Lipoptena cervi

Lipoptena cervi 100/1.00

88/0.90

S Lipoptena capreoli

Lipoptena capreoli Pseudolynchia canariensis

ALO-3

100/1.00

S Brachytarsina allaudi

Crataerina pallida 97/0.95

67/0.63 67/0.66

S Basilia nov. sp.

Ornithomya chloropus

Hippoboscidae

89/0.90

S Basilia truncata

Basilia truncata

77/ 0.74

100/1.00

100/1.00

S Phthiridium hindlei

Basilia nov. sp.

Streblidae

100/1.00

87/0.80

S Nycteribia kolenatii

Phthiridium hindlei 100/ 1.00

Cand. Aschenra chinzeii

95/0.96 100/1.00

S Nycteribia stylidiopsis

Nycteribia allotopa

70/0.67

B

99/0.97

89/0.80

S Nycteribia schmidlii schmidlii

Nycteribia kolenatii

Nyteribiidae

72/0.66

S Penicillidia conspicua

Lipoptena fortisetosa

100/1.00

S Lipoptena fortisetosa S Lipoptena sp.

Lipoptena sp.

78/0.69

Fig. 3 Congruence between host and Arsenophonus and like organisms (ALO) phylogenies. (A) host tree constructed using maximumlikelihood (ML) estimations based on CO1 sequences from louse flies (Hippoboscidae) and bat flies (Nycteribiidae and Streblidae); (B) ALO tree constructed using ML estimations based on GroEL sequences from Arsenophonus, Cand. Aschnera chinzeii and ALO-3. New sequences from this study are in bold and underlined. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. Numbers on branches indicate percentage bootstrap support for major branches (500 replicates; only bootstrap values >60% are shown) followed by Bayesian posterior probabilities (Bayesian topology not shown).

Aschnera chinzeii with that of bat flies indicates topological congruence, with clear codivergence events: a monophyletic clade within the Nycteribiinae subfamily of Nycteribiidae is specifically allied to Cand. Aschnera chinzeii (Fig. 3A and B), as reported by Hosokawa et al. (2011), and within many genera, such as Nyteribia and Penicillidia, closely related species always host closely related bacterial strains. Interestingly, the ALO-3 phylogeny may also show complete concordance with that of a monophyletic clade within the Ornithomyinae subfamily of Hippoboscidae, but low support values in the CO1 tree prevent any definitive conclusion. However, the exclusive association of ALO-3 endosymbionts with the Ornithomyinae subfamily strongly suggests that ALO-3 endosymbionts may have been also stably maintained over long evolutionary periods in louse flies. The evolutionary history of Arsenophonus with their louse fly and bat fly hosts is somewhat different: although the pairwise distance correlation test showed significant congruence at the roots of Arsenophonus and its host phylogenies (P = 10 3; 95% confidence upper bound, 10 2), it is clear that related Arsenophonus strains did not always infect related host species (Fig. 3A and B). In some cases, related host species, such as Hippobosca equina and H. longipennis or Crataerina pallida and C. melbae, are infected by closely related Arsenophonus strains, suggesting that codivergence events may have © 2014 John Wiley & Sons Ltd

occurred. In other cases, however, codivergence is very unlikely: for example, the Arsenophonus strain of the bat fly Brachytarsina allaudi is closely related to some strains found in louse fly (Pseudolynchia canariensis, C. pallida and C. melbae), which suggests they recently diverged. However, phylogenetic analyses based on CO1 sequences did not support this hypothesis, with no close relationship between B. allaudi and these louse fly species. Overall, these results indicate that Arsenophonus rarely codiverged with its hosts, showing that louse flies and bat flies only recently acquired these infections through horizontal transfers from other species.

Discussion We sampled 25 species of the Hippoboscoidea superfamily for ALOs, an emerging clade of bacterial endosymbionts, and found that louse flies and bat flies always engage in associations with ALO endosymbionts. In all cases, ALO infection frequency was observed at fixation in host populations. We confirmed the presence of Arsenophonus infections in louse flies and bat flies, as showed by recent studies (Dale et al. 2006; Trowbridge et al. 2006; Duron et al. 2008; Novakova et al. 2009; Lack et al. 2011; Morse et al. 2013), as well as of Cand. Aschnera chinzeii in bat flies (Hosokawa et al. 2011; Morse et al. 2013). Further, we found wider endosymbiont diversity than previously known, which

2114 O . D U R O N E T A L . included a novel ALO group, ALO-3, which is specifically allied to a subclade of louse flies. Overall, this strongly suggests that ALOs are obligate endosymbionts in louse flies and bat flies, possibly provisioning their hosts with essential nutrients that are absent from vertebrate blood. Although we demonstrate here that ALO endosymbionts are ubiquitous in louse flies and bat flies, they have never been identified from tsetse flies (Aksoy 1995; Aksoy et al. 1997; Dale & Maudlin 1999; Cheng et al. 2000; Doudoumis et al. 2012). This pattern is suggestive of the evolutionary importance of ALOs in the ecology of their hosts. Interestingly, within the superfamily Hippoboscoidea, the Hippoboscidae, Nycteribiidae and Streblidae families form a monophyletic group known as ‘Pupipara’ that is a sister group to the Glossinidae (Petersen et al. 2007). Hence, the presence of ALOs in all Pupipara suggests that acquisition of these endosymbionts may coincide with the evolution of this fly group. Fossils provide indicators of emergence for Pupipara: the only known fossils of louse flies and bat flies have been recorded in the Oligocene entomofauna (Prokop & Fikacek 2007; Poinar & Brown 2012), which implies an approximate minimal date of 20 million years for the original ALO infection in Pupipara. Thus, the origin of ALOs endosymbiosis extends deep into the evolutionary past of the Pupipara, and it probably played a major role in the evolutionary history of these flies. The acquisition of ALO endosymbionts coincided with a major evolutionary transition within the Hippoboscoidea as specialization towards true ectoparasitism originated in the common ancestor of the Pupipara (Petersen et al. 2007). While tsetse flies are free-living and blood-feeding flies, louse flies and bat flies have evolved towards an obligate and close association with their vertebrate hosts. This evolutionary trajectory led to the specialization of morphological traits, including in most extreme cases, the lack of wings, reduced head and eyes, dorsoventrally flattened thorax, dorsally inserted legs, resulting in a crab-like (louse fly) or a spider-like (bat fly) appearance. However, whether the emergence of Pupipara was favoured by ALO acquisition or whether specialization itself allowed the acquisition of ALO through horizontal transfer is still an open question. Interesting in this regard, herein we found that ALO diversity relates to ecological niche specificity of some louse flies, as shown with ALO-3 endosymbionts that are only found in flies feeding on birds. In bat flies, Morse et al. (2012) recently found that the acquisition of ALO-1 endosymbionts coincides with an ecological shift towards higher temperatures found in some cases. Overall, these observations are suggestive of the potential importance of ALO in shaping

evolutionary transitions of ecological characters within Pupipara. The ALO evolutionary history in louse flies and bat flies was notably complex, showing that it has been shaped by both vertical transmission and horizontal transfers with frequent host turnover, as shown by the incongruence between ALO and host phylogenies. Most striking examples are found within the genus Arsenophonus: the close relatedness between Arsenophonus strains from louse flies and bat flies and those from very diverse insect species (aphids, parasitoid wasps) clearly indicates that horizontal transfers within insect communities are recent and likely ongoing. However, congruent patterns of codivergence are also apparent within some ALO groups, as shown by ALO-3 within the Ornithomyinae subfamily of Hippoboscidae (this study), by Cand. Aschnera chinzeii and the Nycteribiinae subfamily of Nycteribiidae (Hosokawa et al. 2011; this study) and by some ALO-1 strains with the Nycterophiliinae subfamily of Streblidae (Morse et al. 2012). Two distinct evolutionary strategies are thus found in ALO endosymbionts: some are highly specialized concerning their hosts, with ancient acquisition followed by codiversification, while others are more generalists and acquired through recent horizontal transfers. It is likely that these different strategies are also illustrated by the localization of ALO endosymbionts within their hosts. The ‘specialized’ ALO endosymbionts of bat flies are typically found in dedicated organs (bacteriomes) as shown for Cand. Aschnera chinzeii (Hosokawa et al. 2011), ALO-1 and ALO-2 (clade B and D in Morse et al. 2013). Remarkably, Zacharias (1928) reported that the endosymbionts of the louse fly O. avicularia (a species found in our study infected by ALO-3) are also hosted in bacteriomes. By contrast, ‘generalist’ ALO endosymbionts may be not hosted within bacteriomes. For instance, Arsenophonus arthropodicus was found to be widespread in a variety of tissues (hemocytes, gut, fat body and reproductive tissues) from the louse fly P. canariensis (Dale et al. 2006). The localization pattern of ALO infection within fly hosts may be thus a key factor in these symbiotic systems. The polyphyly of louse fly and bat fly ALO endosymbionts therefore sustains the hypothesis that symbiont replacement may be common within the Hippoboscoidea superfamily, as also recently suggested in bat flies by Morse et al. (2012). This is well exemplified by examining the distribution of Arsenophonus: most infections were established more recently in louse flies and bat flies by other ALO groups, probably by endosymbiont replacement. The factors favouring this process remain speculative but some endosymbionts, such as Arsenophonus, have characteristics making them potentially successful invaders. For instance, the © 2014 John Wiley & Sons Ltd

E N D O S Y M B I O N T S I N L O U S E F L I E S A N D B A T F L I E S 2115 Arsenophonus strain of the bat fly Basilia nov. sp. is genetically very close to the type species, A. nasoniae, which is unique among insect endosymbionts because it is able to grow outside the host cells (Huger et al. 1985; Werren et al. 1986), an aspect that enhances the likelihood of successful horizontal transfer (Skinner 1985; Duron et al. 2010). Remarkably, the rarity of codivergence events within Arsenophonus suggests that its evolutionary maintenance in louse flies and bat flies is mainly possible through repeated interspecies transfers. To conclude, we would emphasize that the ALO clade is emerging as a diverse group of insect endosymbionts. While diverse insect lineages harbour ALO endosymbionts, it is obvious that louse flies and bat flies host the widest ALO diversity known to date. Most importantly, given that some ALO strains have repeatedly established endosymbiotic relationships with diverse groups of louse flies and bat flies, with apparent symbiont replacement, this suggests that their acquisition may confer an ecological advantage to infected species. This aspect in turn indicates that the ALO endosymbiotic system in louse flies and bat flies is dynamic and may rapidly evolve through horizontal transfers between distantly related host species. Worthy of note is that many louse flies and bat flies may be simultaneously infected with more than one endosymbiont because Wolbachia and Sodalis have been previously detected in some of these species (Dale et al. 2006; Novakova & Hypsa 2007; Morse et al. 2013). In this larger context, the dynamic ALO endosymbiotic systems in louse flies and bat flies are also likely to be affected by other coinfecting endosymbionts.

Acknowledgements We are grateful to J. Barabas, J.C. Beaucournu, T. Disca, N. Dsouli, B. Godelle, E. Legadec, S. Marcoux, J.F. Noblet, B. Ramasindrazana, M. Raymond and the Musee d’Histoire Naturelle de Neuch^atel (Switzerland) for providing specimens, and to P. Makoundou and S. Unal for technical help. All sequence and morphological data were obtained on the Environmental Genomic Platform of the IFR Montpellier-EnvironnementBiodiversite. This work was supported by FEDER POCT Reunion, Pathogenes associes a la Faune Sauvage Ocean Indien #31189.

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Experiments were designed and performed by O.D., A.B. and P.T. Specimens were collected and identified by U.S., S.G., B.D., C.P., J.O.N. and N.R. A.B. and O.D. analysed data. O.D. and P.T. wrote the paper.

Data accessibility New sequence data have been submitted to GenBank (Accession nos KF453413-KF453489; see Table S2 (Supporting information) for details). The sequence alignments and the tree files are available in Dryad (doi:10. 5061/dryad.7n344).

Supporting information Additional supporting information may be found in the online version of this article. Table S1 Genes and primers for screening and sequencing. The ALO infections were screened and genotyped using a

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nested PCR assay based on the GroEL gene, using a first pair of primers (*) for a first round of PCR amplification and a second pair of primers (nested primers, **) for a second round of PCR amplification. Table S2 List of host species, ALO groups and GenBank Accession nos. Underlined Accession nos represent new sequence data generated for this study. Sequences of closest Enterobacteriaceae members (outgroup) were included in the analysis to delineate the ALO clade: Proteus mirabilis (GenBank Accession no AM942759), P. penneri (ABVP01000000), Providencia rustigianii (ABXV01000000), P. stuartii (CP003488) and Photorhabdus luminescens (BX571864). Fig. S1 Arsenophonus phylogeny constructed using maximumlikelihood (ML) estimations based on fbpA gene sequences. Insect symbionts have the prefix S followed by the scientific name of their host; all other names refer to bacteria. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. The arrows denote Arsenophonus strains with recombinant haplotypes. Numbers on branches indicate percentage bootstrap support for major branches (500 replicates; only bootstrap values >60% are shown) followed by Bayesian posterior probabilities (Bayesian topology not shown). The scale bar is in units of substitutions/site. Fig. S2 Arsenophonus phylogeny constructed using maximumlikelihood (ML) estimations based on ftsK gene sequences. New sequences from this study are in bold and underlined. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. The arrows denote Arsenophonus strains with recombinant haplotypes. Numbers on branches indicate percentage bootstrap support for major branches (500 replicates; only bootstrap values >60% are shown) followed by Bayesian posterior probabilities (Bayesian topology not shown). The scale bar is in units of substitutions/site. Fig. S3 Arsenophonus phylogeny constructed using maximumlikelihood (ML) estimations based on yaeT gene sequences. New sequences from this study are in bold and underlined. White circles: endosymbionts from Hippoboscidae; black squares: endosymbionts from Nycteribiidae; black diamond: endosymbionts from Streblidae; asterisks: endosymbionts from other insect hosts. The arrows denote Arsenophonus strains with recombinant haplotypes. Numbers on branches indicate percentage bootstrap support for major branches (500 replicates; only bootstrap values >60% are shown) followed by Bayesian posterior probabilities (Bayesian topology not shown). The scale bar is in units of substitutions/site.