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Research article Open Access Arsenophonus, an emerging clade of intracellular symbionts with a broad host distribution Eva Nováková*1, Václav Hypša2 and Nancy A Moran3

Address: 1Faculty of Science, University of South Bohemia, Branišovská 31, Жeské BudЕjovice 37005, Czech Republic, 2Faculty of Science, University of South Bohemia and Institute of Parasitology, Biology Centre of ASCR, Branišovská 31, Жeské BudЕjovice 37005, Czech Republic and 3Department of Ecology and Evolutionary Biology, The University of Arizona, 1041 E. Lowell St, Tucson, Arizona 85721-0088, USA Email: Eva Nováková* - [email protected]; Václav Hypša - [email protected]; Nancy A Moran - [email protected] * Corresponding author

Published: 20 July 2009 Received: 25 June 2009 Accepted: 20 July 2009 BMC Microbiology 2009, 9:143 doi:10.1186/1471-2180-9-143 This article is available from: http://www.biomedcentral.com/1471-2180/9/143 © 2009 Nováková et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: The genus Arsenophonus is a group of symbiotic, mainly insect-associated with rapidly increasing number of records. It is known from a broad spectrum of hosts and symbiotic relationships varying from parasitic son-killers to coevolving mutualists. The present study extends the currently known diversity with 34 samples retrieved mainly from hippoboscid (Diptera: Hippoboscidae) and nycteribiid (Diptera: Nycteribiidae) hosts, and investigates phylogenetic relationships within the genus. Results: The analysis of 110 Arsenophonus sequences (incl. Riesia and Phlomobacter), provides a robust monophyletic clade, characterized by unique molecular synapomorphies. On the other hand, unstable inner topology indicates that complete understanding of Arsenophonus evolution cannot be achieved with 16S rDNA. Moreover, taxonomically restricted Sampling matrices prove sensitivity of the phylogenetic signal to sampling; in some cases, Arsenophonus monophyly is disrupted by other symbiotic bacteria. Two contrasting coevolutionary patterns occur throughout the tree: parallel host-symbiont evolution and the haphazard association of the symbionts with distant hosts. A further conspicuous feature of the topology is the occurrence of monophyletic symbiont lineages associated with monophyletic groups of hosts without a co-speciation pattern. We suggest that part of this incongruence could be caused by methodological artifacts, such as intragenomic variability. Conclusion: The sample of currently available molecular data presents the genus Arsenophonus as one of the richest and most widespread clusters of insect symbiotic bacteria. The analysis of its phylogenetic lineages indicates a complex evolution and apparent ecological versatility with switches between entirely different life styles. Due to these properties, the genus should play an important role in the studies of evolutionary trends in insect intracellular symbionts. However, under the current practice, relying exclusively on 16S rRNA sequences, the phylogenetic analyses are sensitive to various methodological artifacts that may even lead to description of new Arsenophonus lineages as independent genera (e.g. Riesia and Phlomobacter). The resolution of the evolutionary questions encountered within the Arsenophonus clade will thus require identification of new molecular markers suitable for the low-level phylogenetics.

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Background important changes in knowledge of Arsenophonus evolu- The bacterial genus Arsenophonus corresponds to a group tion and roles in hosts. First, the known host spectrum has of insect intracellular symbionts with a long history of been considerably extended with diverse insect groups investigation. Although many new Arsenophonus and even non-insect taxa. So far, Arsenophonus has been sequences have been published in the last several years, identified from parasitic wasps, triatomine bugs, psyllids, along with documentation of diverse evolutionary pat- whiteflies, , ticks, ant lions, hippoboscids, stre- terns in this group (Figure 1), the first records of these bac- blids, bees, lice, and two plant species [4,7-23]. Second, teria date to the pre-molecular era. Based on these recent studies have revealed an unsuspected diver- ultrastructural features, several authors described a trans- sity of symbiotic types within the genus. This dramatically ovarially transmitted infection associated with son-killing changes the original perception of Arsenophonus as a bio- in the parasitoid wasp Nasonia vitripennis [1-3]. Later, they nomically homogeneous group of typical secondary ("S- were formally assigned to a new genus within the family ") symbionts undergoing frequent horizontal transfers with a single species, Arsenophonus among phylogenetically distant hosts. For example, recent nasoniae [4]. The same authors proposed a close relation- findings indicate that some insect groups harbor mono- ship of Arsenophonus to free-living bacteria of the genus phyletic clusters of Arsenophonus, possibly playing a role of Proteus. Independently, other microscopic studies typical primary ("P-") symbionts. These groups were revealed morphologically similar symbionts from various reported from the dipteran families Hippoboscidae and tissues of blood-sucking triatomine bugs [5,6]; a decade Streblidae [20] and most recently from several lice species later these bacteria were determined on molecular [18,24,25]. Such a close phylogenetic relationship of dif- grounds to belong to the same clade and were named ferent types of symbiotic bacteria is not entirely unique Arsenophonus triatominarum [7]. Interestingly, the next among insect symbionts. With the increasing amount of record on symbiotic bacteria closely related to A. nasoniae knowledge on the heterogeneity and evolutionary dynam- was from a phytopathological study investigating mar- ics of symbiotic associations, it is becoming clear that no ginal chlorosis of strawberry [8]. Since available sequence distinct boundaries separate the P- and S-symbionts. data were insufficient for reliable phylogenetic placement, Thus, in their strict meaning, the terms have recently the phloem-inhabiting pathogen was described as a new become insufficient, especially for more complex situa- genus, Phlomobacter, with a single species P. fragariae [8]. tions, such as studies exploring bacterial diversity within a single host species [14,17]. Furthermore, these terms have Since these descriptions, the number of Arsenophonus been shown not to reflect phylogenetic position; remark- records has steadily been increasing, resulting in two able versatility of symbiotic associations can be observed

AnFigure increase 1 of records on Arsenophonus bacteria from various insect groups An increase of records on Arsenophonus bacteria from various insect groups. The bars show cumulative numbers of sequences deposited into GenBank; dark tops represent new records added in the given year. The sequences are identified with the following accession numbers: 1991 – M90801; 1997 – U91786; 2000 – AF263561, AF263562, AF286129, AB038366; 2001 – AF400474, AF400480, AF400481, AF400478, AY057392; 2002 – AY136168, AY136153, AY136142; 2003 – AY265341– AY265348, Y264663–AY264673, AY264677; 2004 – AY587141, AY587142, AY587140; 2005 – DQ068928, DQ314770– DQ314774, DQ314777, DQ314768, DQ115536; 2006 – DQ538372–DQ538379, DQ508171–DQ508186, DQ517447, DQ508193, DQ837612, DQ837613; 2007 – EU039464, EU043378, EF110573, EF110574, DQ076660, DQ076659, EF110572, EF647590, AB263104.

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in the overall, as well as within the degree of similarity (0.1–7.3% divergence) and exhibit individual clusters, such as Arsenophonus or Sodalis GC content and sequence length similar to those found in [16,26]. free-living enterobacteria. The set also includes several sequences with modifications typical for many proteobac- The genus Arsenophonus is striking in the diversity of sym- terial symbionts, particularly the presence of long inser- biont types represented. Apart from many lineages with tions within the variable regions and decreased GC typical S-symbiont features, this genus has given rise to content. Sequence distances among these taxa range up to several clusters of P-symbionts [18,20,24]. Unfortunately, 17.8%. this heterogeneity introduced an annoying degree of phy- logenetic instability and nomenclatory confusion. Phylogeny Because P-symbionts show accelerated evolutionary rates, All phylogenetic analyses of the Basic matrix yielded a they form long branches in phylogenies, leading to unsta- monophyletic Arsenophonus clade (Figure 2). The new 34 ble patterns of clustering as observed for P-symbionts sequences (Figure 2, arrows), identified by BLAST as puta- within Enterobacteriaceae [27]. The same behavior can be tive members or relatives of the genus Arsenophonus, seen in the louse-specific clade of Arsenophonus, which are always clustered within the Arsenophonus clade. Their pre- consequently originally described as a new bacterial genus cise position was only partially correlated with host taxon. Riesia [25]. In addition, the Arsenophonus cluster is the Some of the Arsenophonus sequences from hippoboscoid only monophyletic group of symbiotic bacteria currently hosts clustered within monophyletic host-specific groups known to possess at least four highly different pheno- (Figure 2, printed in red) while others were scattered types, including son-killing [4], phytopathogenicity [8], across the tree as isolated lineages (Figure 2, printed in obligate association with bacteriocytes in the host dark orange). Two distinct sequences were determined [18,20,24], and apparently non-specific horizontally from each individual specimen of O. avicularia; these clus- transmitted bacteria that are possibly mutualistic [15]. tered at distant positions within the tree (Figure 2, num- These characteristics indicate that the genus Arsenophonus bers with asterisks). represents an important and widespread lineage of symbi- otic bacteria that serves as a valuable model for examining The most typical lineages display short-branches with low molecular evolution of bacteria-arthropod associations. divergence and unstable positions within the Arseno- phonus clade (Figure 2, printed in dark orange). At the In this study, we add 34 new records on symbionts to the opposite extreme are well supported host-specific clusters known spectrum of Arsenophonus lineages. We explore and exhibiting long branches, such as the louse symbiont summarize the current picture of Arsenophonus evolution Riesia or the symbionts described from several streblid by analyzing all sequences available for this clade. To species. An intermediate situation is found in putatively investigate the phylogenetic position, stability and evolu- host-specific but less robust clusters, such as the Arseno- tionary trends of the Arsenophonus cluster, we complete phonus lineages from triatomine bugs, some hippobos- the sample with related symbionts and free-living bacte- coids or homopterans (Figure 2). In an analogy to ria. Finally, we explore molecular characteristics and previously analyzed symbiotic bacteria [e.g. [28,29]], the informative value of the 16S rRNA gene as the most fre- phylogenetic properties of the sequences were also quently used phylogenetic marker. reflected in their GC contents. In the short-branched taxa, the GC content of the 16S rRNA sequence varies from Results 51.72 to 54.84%, the values typical for S-symbionts and Sequences and alignments free-living bacteria [30]. In contrast, the 16S rRNA From 15 insect taxa, we obtained 34 sequences of 16S sequences with low GC content, varying between 46.22 rDNA that exhibited a high degree of similarity to and 51.93%, were found in the long-branched taxa clus- sequences from the bacterial genus Arsenophonus when tering within the host-specific monophyletic lineages (e.g. identified by BLAST. The length of the PCR-amplified frag- the symbionts from Ornithomyia, Lipoptena, Trichobius, ments varied from 632 to 1198 bp, with the guanine-cyto- and the Riesia clade). sine (GC) content ranging from 46.22 to 54.84% (Figure 2, bars). For three specimens of the hippoboscid Orni- Considerable loss of phylogenetic information was thomya avicularia, two different sequences were obtained observed in the Conservative matrix. In this case, the rela- from each single individual. After combining with all Arse- tionships among individual Arsenophonus lineages were nophonus 16S rDNA sequences currently available in the highly unstable, resulting in large polytomies of many GenBank, and several additional free-living and symbiotic short-branched taxa within the consensus trees (see Addi- bacteria, the dataset produced a 1222 bp long Basic matrix. tional file 1). Also the relationships among the long- The alignment has a mosaic structure, discussed below. branched lineages, although resolved, differ sharply from Within the set, a large group of sequences show a high those derived from the Basic matrix data, and the genus

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Chromatium okenii Pseudomonas aeruginosa luminescens Proteus mirabilis Symb- Aphis spiraecola () Arsenophonus insecticola Symb- Diaphorina citri (psyllid) Symb- Australiococcus greville (whitefly) Symb- Neomaskellia andropogonis (whitefly) Symb- Dialeurodes hongkongensis (whitefly) 73 Symb- Glycaspis brimblecombei 2 (psyllid) Symb- Glycaspis brimblecombei 1 (psyllid) 50 Symb- Acanthaleyrodes styraci (whitefly) Symb- Tetraleurodes acaciae (whitefly) Symb- Heteropsylla texana (psyllid) Symb- Wahlgreniella nervata (aphid) Symb- Myzocallis sp. 1 (aphid) Symb- Myzocallis sp. 2 (aphid) 86 Symb- Ornithomyia biloba 1 (hippoboscid) Symb- Ornithomyia avicularia 2 (hippoboscid) Symb- Hippobosca camelina 1 (hippoboscid) Symb- Hippobosca camelina 2 (hippoboscid) Symb- Hippobosca camelina 3 (hippoboscid) Symb- Myzocallis sp. 3 (aphid) Symb- Aleyrodes proletella (whitefly) Symb- Dermacentor variabilis 4 99 Symb- Dermacentor variabilis 1 Symb- Dermacentor variabilis 3 Symb- Dermacentor variabilis 2 ticks Symb- Dermacentor variabilis 5 Symb- Dermacentor variabilis 6 Symb- Dermacentor variabilis 7 Symb- Dermacentor variabilis 8 Symb- Aleuroplatus gelatinosus (whitefly) Symb- Aleyrodes elevatus (whitefly) Phlomobacter fragariae 8 59 Phlomobacter fragariae 6 Symb- Beta vulgaris Phlomobacter fragariae 7 plants Phlomobacter fragariae 5 Phlomobacter fragariae 4 Phlomobacter fragariae 2 62 Phlomobacter fragariae 1 Phlomobacter fragariae 3 Symb- Nycteribia sp. (hippoboscid) Symb- Nycteribia kolenatii 2 (hippoboscid) Symb- Trialeurodes vaporariorum (whitefly) Symb- Trialeurodes hutchingsi (whitefly) Symb- Tetraleurodes mori (whitefly) Symb- Bemisia tabaci 2 (whitefly) Symb- Bemisia tabaci 1 (whitefly) 91 Symb- Bemisia tabaci 3 (whitefly) Symb- Bemisia tabaci 4 (whitefly) 99 Arsenophonus triatominarum 2 Symb- Triatoma rubrofasciata 2 Arsenophonus triatominarum 1 53 Symb- Meccus mazzottii 2 Symb- Triatoma melanosoma 2 triatomine bugs Symb- Eratyrus mucronatus 2 Symb- Eratyrus mucronatus 1 Symb- Triatoma melanosoma 1 Symb- Meccus mazzottii 1 Symb- Triatoma rubrofasciata 1 Symb- Aleurodicus dugesii 2 Symb- Bemisia tabaci 5 100 Symb- Aleurodicus dispersus whiteflies Symb- Aleurodicus dugesii 1 Symb- Siphoninus phillyreae (whitefly) Arsenophonus nasoniae Symb- Myrmeleon mobilis (antlion) Symb- Apis mellifera 1 (honeybee)* 85 Symb- Apis mellifera 2 (honeybee) Symb- Ornithomyia avicularia 8 £ ¨ 80 Symb- Ornithomyia avicularia 7 hippoboscids

Symb- Ornithomyia avicularia 5 ¦ Symb- Ornithomyia avicularia 3 98 Symb- Aenictus huonicus Symb- Technomyrmex albipes 1 ants Symb- Technomyrmex albipes 2 Symb- Melophagus ovinus 100 Symb- Lipoptena cervi 1 96 Symb- Lipoptena cervi 2 Symb- Lipoptena fortisetosa 1 100 Symb- Lipoptena fortisetosa 2 Symb- Lipoptena fortisetosa 3 Symb- Lipoptena sp. Riesia pthiripubis Riesia pediculischaeffi 99 Riesia pediculicola 1 lice 92 Riesia pediculicola 2 Riesia pediculicola 4 hippoboscoids 88 Riesia pediculicola 5 Riesia pediculicola 3 Symb- Ornithomyia chloropus 100 Symb- Ornithomyia biloba 3 100 Symb- Ornithomyia biloba 2

Symb- Ornithomyia avicularia 1 ¥ 99

Symb- Ornithomyia avicularia 6 §

68 Symb- Ornithomyia avicularia 4 ¤ Symb- Ornithomyia avicularia 9 93 Symb- Nycteribia kolenatii 1 98 Symb- Penicillidia monoceros Symb- Penicilidia sp. 83 Symb- Trichobius caecus 1 Symb- Trichobius caecus 2 51 100 Symb- Trichobius parasiticus 2 Symb- Trichobius parasiticus 1 79 Symb- Trichobius sp. Symb- Trichobius longipes 1 Symb- Trichobius longipes 4 100 Symb- Trichobius longipes 3 Symb- Trichobius longipes 2 100 Symb- Lipoptena cervi 4 Symb- Lipoptena cervi 3 99 Regiella insecticola 2 Regiella insecticola 1 Pasteurella multocida 100 2 Hamiltonella defensa 1 Symb- Cacopsylla pyri (psyllid) 100 Symb- Vryburgia amaryllidis (mealybug) 73 Symb- Erium globosum (mealybug) 99 Symb- Dysmicoccus neobrevipes (mealybug) Symb- Dysmicoccus brevipes (mealybug) Symb- Eulachnus pallidus (aphid) Symb- Paracoccus nothofagicola (mealybug) Symb- Cyphonococcus alpinus (mealybug) 100 Symb- Blastopsylla occidentalis 2 (psyllid) Symb- Blastopsylla occidentalis 1 (psyllid) Sodalis glossinidius Symb- Psylla floccosa (psyllid) Symb- Bactericera cockerelli (psyllid) Symb- Heteropsylla cubana (psyllid) 99 Symb- Antonina pretiosa (mealybug) Symb- Antonina crawii (mealybug) Symb- Amonostherium lichtensioides (mealybug) 87 Symb- Melanococcus albizziae (mealybug) Symb- Australicoccus grevilleae (mealybug) Symb- Calophya schini (psyllids) Symb- Haematopinus apri (pig louse) Nardonella sp. 100 Symb- Psylla pyricola (psyllid) Symb- Cacopsylla myrthi (psyllid) Blochmannia fellah Symb- Trioza magnoliae (psyllid) Symb- Haematomyzus elephantis (elephant louse) Symb- Pseudolynchia canariensis (hippoboscid) Wigglesworthia glossinidia 99 Symb- Planococcus ficus (mealybug) Symb- Planococcus citri 2 (mealybug) 99 Symb- Planococcus citri 1 (mealybug) Baumannia cicadellinicola Symb- Aphalaroida inermis (psyllids) Buchnera aphidicola Ishikawaella capsulata 100 Portiera aleyrodidarum 2 Portiera aleyrodidarum 1 100 Symb- Anomoneura mori (psyllids)

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PhylogeneticFigure 2 tree derived from the Basic matrix (1222 positions) under ML criterion Phylogenetic tree derived from the Basic matrix (1222 positions) under ML criterion. Names of the taxa clustering within the Arsenophonus clade are printed in colour: red for the long-branched taxa, dark orange for the short-branched taxa. The arrows point to the new sequences obtained in our study. Different types of sequences determined from the specimens of O. avicularia are designated by the numbers with asterisks. The type species A. nasoniae is designated by the orange asterisk. Solid circles on branches label the clusters strictly concordant with the host phylogenies. Open circles designate host-specific lineages without coevolutionary signal. Solid vertical lines indicate reciprocally monophyletic groups of symbionts and hosts. Dashed lines show paraphyletic symbiont clades restricted to monophyletic host groups. Names in the brackets indicate host taxa. "Symb-" in the taxon designation stands for "Symbiotic bacteria of". Bars represent GC content of each taxa. Complete information on the sequences is provided in the Additional file 5.

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Proteus was not positioned as the closest relative of Arsen- many phylogenetic analyses. Interestingly, the mono- ophonus. Thus, the information contained in the Conserva- phyletic nature of Arsenophonus was preserved even in this tive matrix (restricted to one fourth of Basic dataset, i.e., highly Conservative matrix. This indicates that within the 284 bp) is insufficient for reliable phylogenetic placement complete data set, the phylogenetic information underly- of closely related taxa. ing the Arsenophonus monophyly is sufficiently strong and is contained in the conservative regions of the sequences. The analyses of taxonomically restricted Sampling matrices In accordance with this presumption, several molecular confirmed the expected dependence of the phylogenetic synapomorphies can be identified in the Basic and Con- conclusions on the taxon sampling (examples of topolo- servative matrices. The most pronounced is the motif GTC/ gies obtained are provided in Figures 3, 4 and Additional GTT located in positions 481–483 and 159–161 of Basic file 2). The highest degree of susceptibility was observed matrix and Conservative matrix, respectively. with MP, particularly under Tv:Ts ratio set to 1. The most fundamental distortion occurred with the matrix Relevance of the sampling Sampling3, where one lineage (composed of Buchnera, To test an effect of sampling on the phylogenetic inference Wigglesworthia, Blochmannia, and S-symbiont from Trioza within Arsenophonus, we examined five Sampling matrices magnoliae) clustered either as a sister group of Riesia clade with different taxa compositions (see the section Meth- or together with Sodalis. Thus, the consensus tree did not ods). In addition to the MP, ML, and Bayesian analyses, we preserve the monophyly of an Arsenophonus clade performed an ML calculation under the nonhomogene- (Figure 3). ous model of the substitutions, designated as T92 [31,32]. This model was previously used to test the monophyly/ The calculation of divergence times yielded substantially polyphyly of the P-symbiotic lineages and brought the different results depending on the choice of calibration first serious evidence for a possible independent origin of points. Use of the Riesia diversification as a reference major P-symbiotic taxa [27]. We were not able to apply point suggested a recent origin of the triatomine-associ- the same approach to the Basic and Conservative matrices ated Arsenophonus branch; the median value of the esti- since the program Phylowin failed to process these large mate distribution was 2.6 mya. In contrast, the calibration datasets under the ML criterion. The analyses of several by Escherichia-Salmonella returned considerably higher taxonomically restricted Sampling matrices proved the dates with the median at 24.5 mya. sensitivity of phylogenetic signal to the sampling. In the most extreme case, shown in Figure 3A, even the mono- Discussion phyly of the Arsenophonus clade was disrupted by other Phylogenetic patterns and the stability of the information lineages of symbiotic bacteria. Considering the results of Phylogenetic relationships of the Arsenophonus symbionts the extensive analysis of the Basic matrix, this arrangement display a remarkably complex arrangement of various is clearly a methodological artifact. Since both Riesia and types of symbiosis and evolutionary patterns. Moreover, a the P-symbiont lineage are long-branched taxa with rapid comparison of the branch ordering within each of these evolution of 16S rDNA, their affinity is very likely caused subclusters to the host phylogeny indicates a cospeciation by Long Branch Attraction (LBA; for review see [33]) process within several lineages (discussed below). From within the taxonomically compromised matrix. It is the phylogenetic perspective, no clearcut boundary symptomatic that this topology was inferred by MP, the divides the set of Arsenophonus sequences into the ecolog- method known to be particularly prone to the LBA. To fur- ically distinct types. The position of the long-branched ther test this distortion, one of the long-branched taxa was subclusters within the topology is not stable. Under the removed from the data set (matrix Sampling4). This MP criterion with transition rate 1:3 and under the ML cri- approach restored the Arsenophonus monophyly and con- terion they form a unique monophyletic cluster (Figure firmed the effect of LBA phenomenon (see Additional 5A), while in other analyses the individual host-specific file 2). subclusters were scattered among the short-branched lin- eages (Figure 5B, Figure 6). The aim of these taxonomically restricted analyses was to "simulate" phylogenetic placement of newly determined The low resolution and instability of the trees inferred symbionts. In such casual studies, the symbiotic lineages from the Conservative matrix suggest that a substantial part are rarely represented by all available sequences in the way of the phylogenetic information is located within the we composed the Basic matrix. Rather, each symbiotic lin- "ambiguously" aligned regions that were removed by the eage is represented by few randomly selected sequences. GBlocks procedure. This fact is particularly important Under such circumstances, incorrect topologies (e.g. the when considering the frequent occurrence of insertions/ Sampling5-derived topology on the Figure 4) can be deletions within the sequences (see Additional file 3). obtained due to various methodological artifacts. This sit- This may lead to deletion of these critical fragments in uation can be illustrated by empirical data: at least in two

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Chromatium okenii A) Chromatium okenii A1 Pseudomonas aeruginosa Pasteurella multocida Pseudomonas aeruginosa Sodalis glossinidius Pasteurella multocida Photorhabdus luminescens Proteus mirabilis Sodalis glossinidius Symb- Melophagus ovinus Symb- Lipoptena fortisetosa 2 Photorhabdus luminescens Symb- Lipoptena cervi 2 Symb- Lipoptena cervi 4 Proteus mirabilis Symb- Trichobius longipes 1 Symb- Trichobius longipes 4 Symb- Melophagus ovinus (hippoboscid) Symb-Trichobius caecus 1 Symb- Lipoptena fortisetosa 2 (hippoboscid) Symb- Trichobius caecus 2 Symb- Ornithomyia biloba 2 Symb- Lipoptena cervi 2 (hippoboscid) Symb- Ornithomyia avicularia 4 Symb- Nycteribia kolenatii 1 Symb- Ornithomyia biloba 2 (hippoboscid) Symb- Penicilidia sp. Buchnera aphidicola Symb- Ornithomyia avicularia 4 (hippoboscid) Wigglesworthia glossinidia Blochmannia fellah Symb- Nycteribia kolenatii 1 (bat fly) Symb- Trioza magnoliae Symb- Penicilidia sp. (bat fly) Riesia pediculischaeffi Riesia pediculicola 3 Buchnera aphidicola Riesia pediculicola 2 Riesia pediculicola 4 Wigglesworthia glossinidia Arsenophonus nasoniae Symb- Technomyrmex *albipes 2 Blochmannia fellah Symb- Australiococcus greville Symb-Trioza magnoliae (psyllid) Phlomobacter fragariae 1 Symb- Ornithomyia avicularia 2 Riesia pediculischaeffi (louse) Symb- Myzocallis sp. 3 Symb- Hippobosca camelina 3 Riesia pediculicola 3 (louse) Symb- Nycteribia sp. Symb- Bemisia tabaci 4 Riesia pediculicola 2 (louse) Symb- Bemisia tabaci 5 Riesia pediculicola 4 (louse) Symb- Meccus mazzottii 2 Symb- Triatoma melanosoma 2 Symb- Lipoptena cervi 4 (hippoboscid) Symb- Aleurodicus dugesii 2 Symb- Dermacentor variabilis 8 Symb- Trichobius longipes 1 (streblid bat fly) Symb- Ornithomyia avicularia 8 Symb- Myrmeleon mobilis Symb- Trichobius longipes 4 (streblid bat fly) Symb- Apis mellifera 2 Symb- Trichobius caecus 1 (streblid bat fly) Symb- Trichobius caecus 2 (streblid bat fly) Symb- Australiococcus greville (whitefly) Phlomobacter fragariae 1 A2 Chromatium okenii Symb- Nycteribia sp. (bat fly) Pseudomonas aeruginosa Symb- Meccus mazzottii 2 (triatomine bug) Pasteurella multocida Sodalis glossinidius Symb- Triatoma melanosoma 2 (triatomine bug) Buchnera aphidicola Arsenophonus nasoniae Wigglesworthia glossinidia Blochmannia fellah Symb- Technomyrmex albipes* 2 (ant) Symb- Trioza magnoliae Photorhabdus luminescens Symb- Bemisia tabaci 4 (whitefly) Proteus mirabilis Symb- Melophagus ovinus Symb- Bemisia tabaci 5 (whitefly) Symb- Lipoptena fortisetosa 2 Symb- Aleurodicus dugesii 2 (whitefly) Symb- Lipoptena cervi 2 Symb- Lipoptena cervi 4 Symb- Dermacentor variabilis 8 (tick) Symb- Trichobius longipes 1 Symb- Trichobius longipes 4 Symb- Ornithomyia avicularia 2 (hippoboscid) Symb-Trichobius caecus 1 Symb- Myzocallis sp. 3 (aphid) Symb- Trichobius caecus 2 Symb- Ornithomyia biloba 2 Symb- Hippobosca camelina 3 (hippoboscid) Symb- Ornithomyia avicularia 4 Symb- Nycteribia kolenatii 1 Symb- Ornithomyia avicularia 8 (hippoboscid) Symb- Penicilidia sp. Symb- Myrmeleon mobilis (antlion) Riesia pediculischaeffi Riesia pediculicola 3 Symb- Apis mellifera 2 (honeybee) Riesia pediculicola 2 Riesia pediculicola 4 Arsenophonus nasoniae Symb- Technomyrmex albipes* 2 B) Symb- Australiococcus greville Phlomobacter fragariae 1 Symb- Ornithomyia avicularia 2 Symb- Myzocallis sp. 3 Chromatium okenii Symb- Hippobosca camelina 3 Pseudomonas aeruginosa Symb- Nycteribia sp. Symb- Bemisia tabaci 4 Pasteurella multocida Symb- Bemisia tabaci 5 Buchnera aphidicola Symb- Meccus mazzottii 2 Symb- Triatoma melanosoma 2 Photorhabdus luminescens Symb- Aleurodicus dugesii 2 Symb- Dermacentor variabilis 8 Sodalis glossinidius Symb- Ornithomyia avicularia 8 Blochmannia fellah Symb- Myrmeleon mobilis Symb-Trioza magnoliae (psyllid) Symb- Apis mellifera 2 Wigglesworthia glossinidia Proteus mirabilis Symb- Melophagus ovinus (hippoboscid) Symb- Lipoptena fortisetosa 2 (hippoboscid) Symb- Lipoptena cervi 2 (hippoboscid) Symb- Lipoptena cervi 4 (hippoboscid) Symb- Trichobius longipes 1 (streblid bat fly) Symb- Trichobius longipes 4 (streblid bat fly) Symb- Trichobius caecus 1 (streblid bat fly) Symb- Trichobius caecus 2 (streblid bat fly) Symb- Ornithomyia biloba 2 (hippoboscid) Symb- Ornithomyia avicularia 4 (hippoboscid) Symb- Nycteribia kolenatii 1 (bat fly) Symb- Penicilidia sp. (bat fly) Riesia pediculischaeffi (louse) Riesia pediculicola 3 (louse) Riesia pediculicola 2 (louse) Riesia pediculicola 4 (louse) Symb- Australiococcus greville (whitefly) Symb- Nycteribia sp. (bat fly) Phlomobacter fragariae 1 Symb- Bemisia tabaci 4 (whitefly) Symb- Ornithomyia avicularia 2 (hippoboscid) Symb- Myzocallis sp. 3 (aphid) Symb- Hippobosca camelina 3 (hippoboscid) Symb- Technomyrmex albipes 2 (ant) Symb- Aleurodicus dugesii 2 (whitefly) Symb- Dermacentor variabilis 8 (tick) Symb- Meccus mazzottii 2 (triatomine bug) Symb- Triatoma melanosoma 2 (triatomine bug) Symb- Bemisia tabaci 5 (whitefly) Symb- Apis mellifera 2 (honey bee) Symb- Ornithomyia avicularia 8 (hippoboscid) Symb- Myrmeleon mobilis (antlion) Arsenophonus nasoniae * FigureTopologies 3 derived from Sampling3 matrix (851 positions) Topologies derived from Sampling3 matrix (851 positions). A) consensus of the trees and two tree examples A1 and A2, obtained under the MP criterion with Tv/Ts ratio set to 1:1 B) consensus of the trees obtained under the MP criterion with Tv/Ts ratio set to 1:3. The type species A. nasoniae is designated by the orange asterisk.

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Chromatium okenii within the long-branched clusters of hippoboscoid-asso- Sodalis glossinidius ciated taxa are in agreement with the host phylogeny (the Symb- Blastopsylla occidentalis (psyllid) Arsenophonus clusters strictly reflecting the host phylogeny Symb- Planococcus citri (mealybug) Symb- Aphalaroida inermis (psyllid) are designated by solid circle in the Figure 2). Interest- Regiella insecticola ingly, a coevolutionary pattern was also identified for stre- Hamiltonella defensa blids of the genus Trichobius and their symbionts. In the Symb- Calophya schini (psyllid) original study published by Trowbridge et al. [20], the dis- Symb- Haematopinus apri (pig louse) tribution of Trichobius symbionts was apparently not con- Wigglesworthia glossinidia Blochmania fellah sistent with the host phylogeny. Our analysis in a broad Buchnera aphidicola context indicates that this discrepancy might have been Symb- Antonina crawii (mealybug) caused by different bacterial sampling and particularly by Symb- Amonostherium lichtensioides (mealybug) Symb- Trioza magnoliae (psyllid) aberrant behavior of the sequence from Trichobius yunkeri Symb- Haematomyzus elephantis (elephant louse) [GenBank: DQ314776]. This sequence is likely to be an Proteus mirabilis artificial chimerical product of at least two distant line- Photorhabdus luminescens ages; according to our BLAST tests it shares 100% identity Arsenophonus insecticola Arsenophonus nasoniae * with S-symbiont of Psylla pyricola [GenBank: AF286125] Riesia pediculischaeffi (louse) along a 1119 bp long region. Removal of this sequence Riesia pediculicola 1 (louse) from the dataset restored a complete phylogenetic congru- Riesia pediculicola 3 (louse) Riesia pediculicola 2 (louse) ence between Trichobius, based on the phylogeny of this Riesia pediculicola 4 (louse) genus published by Dittmar et al. [35], and its symbionts. Riesia pediculicola 5 (louse) This finding exemplifies the danger of chimeric sequences TreematrixFigure consensus under 4 the derived MP criterion from Sampling5 (936 positions) in studies of symbiotic bacteria, obtained by the PCR on Tree consensus derived from Sampling5 (936 posi- the sample containing DNA mixture from several bacteria. tions) matrix under the MP criterion. Transversion/ The presence of several symbiotic lineages within a single transition ratio was set to 1:1. The type species A. nasoniae is host is well known [e.g. [14,36-38]]. In this study, we designated by the orange asterisk. demonstrate a possible such case in O. avicularia. From three individuals of this species we obtained pairs of dif- ferent sequences branching at two distant positions studies, the louse-associated lineage of Arsenophonus was (labelled by the numbers 1* to 3* in Figure 2). The iden- not recognized as a member of the Arsenophonus clade tical clustering seen in all three pairs within the tree shows [25,34]. Consequently, when more recent studies, based that they are not chimeric products but represent two dif- on better sampling, proved the position of Riesia within ferent sequences. the Arsenophonus cluster [18,24] the genus Arsenophonus became paraphyletic (see the section Conclusion for more While the identity between symbiont relationships and details). the host phylogeny is apparently a consequence of host- symbiont cophylogeny, the interpretation of the ran- Interestingly, topologies inferred by likelihood analyses domly scattered symbionts is less obvious. Usually, such using the T92 evolution model [31] were influenced nei- an arrangement is explained as result of transient infec- ther by the compromised sampling nor by the removal of tions and frequent horizontal transfers among distant unreliably aligned regions. host taxa. This is typical, for example, of the Wolbachia symbionts in wide range of insect species [39]. Generally, Cophylogeny vs. horizontal transfers: possible sources of the capability to undergo inter-host transfers is assumed phylogenetic incongruence for several symbiotic lineages and has even been demon- The phylogenetic tree of all Arsenophonus sequences exhib- strated under experimental conditions [40,41]. Since the its both patterns, the parallel evolution of symbionts and Arsenophonus cluster contains bacteria from phylogeneti- their hosts and the haphazard association of symbionts cally distant insect taxa and also bacteria isolated from from different host taxa. Coincidentally, both arrange- plants, it is clear that horizontal transfers and/or multiple ments can be demonstrated on the newly sequenced sym- establishments of the symbiosis have occurred. However, bionts from various hippoboscoid species. Some of part of the incongruence could be caused by methodolog- hippoboscoid-associated Arsenophonus show possible ical artifacts. A conspicuous feature of the Arsenophonus host specificity; in a few analyses they cluster within sev- topology is the occurrence of monophyletic symbiont lin- eral monophyletic short-branched groups. Since relation- eages associated with monophyletic groups of insect host ships among the short-branched taxa are generally not but without a co-speciation pattern. Although our study well resolved, these lineages are scattered throughout the cannot present an exhaustive explanation of such a pic- whole topology (Figure 2). In contrast, relationships ture, we want to point out two factors that might in theory

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Chromatium okenii Chromatium okenii Pseudomonas aeruginosa A Pseudomonas aeruginosa B Pasteurella multocida Portiera aleyrodidarum 2 Hamiltonella defensa 2 Portiera aleyrodidarum 1 Hamiltonella defensa 1 Symb- Anomoneura mori (psyllids) Symb- Cacopsylla pyri (psyllid) Symb- Psylla pyricola (psyllids) Regiella insecticola 2 Pasteurella multocida Regiella insecticola 1 Sodalis glossinidius Sodalis glossinidius Ishikawaella capsulata Symb- Psylla floccosa (psyllid) Buchnera aphidicola Symb- Bactericera cockerelli (psyllid) Symb- Psylla floccosa (psyllid) Symb- Eulachnus pallidus (aphid) Symb- Bactericera cockerelli (psyllid) Symb- Heteropsylla cubana (psyllid) Symb- Eulachnus pallidus (aphid) Symb- Blastopsylla occidentalis 2 (psyllid) Symb- Heteropsylla cubana (psyllid) Symb- Blastopsylla occidentalis 1 (psyllid) Photorhabdus luminescens Symb- Paracoccus nothofagicola (mealybug) Symb- Cacopsylla myrthi (psyllid) Symb- Cyphonococcus alpinus (mealybug) Symb- Psylla pyricola (psyllid) Symb- Planococcus ficus (mealybug) Baumannia cicadellinicola Symb- Planococcus citri 2 (mealybug) Symb- Aphalaroida inermis (psyllids) Symb- Planococcus citri 1 (mealybug) Symb- Planococcus ficus (mealybug) Symb- Vryburgia amaryllidis (mealybug) Symb- Planococcus citri 2 (mealybug) Symb- Erium globosum (mealybug) Symb- Planococcus citri 1 (mealybug) Symb- Dysmicoccus neobrevipes (mealybug) Symb- Blastopsylla occidentalis 2 (psyllid) Symb- Dysmicoccus brevipes (mealybug) Symb- Blastopsylla occidentalis 1 (psyllid) Baumannia cicadellinicola Symb- Paracoccus nothofagicola (mealybug) Symb- Aphalaroida inermis (psyllids) Symb- Cyphonococcus alpinus (mealybug) Symb- Amonostherium lichtensioides (mealybug) Symb- Vryburgia amaryllidis (mealybug) Symb- Melanococcus albizziae (mealybug) Symb- Erium globosum (mealybug) Symb- Australicoccus grevilleae (mealybug) Symb- Dysmicoccus neobrevipes (mealybug) Symb- Antonina pretiosa (mealybug) Symb- Dysmicoccus brevipes (mealybug) Symb- Antonina crawii (mealybug) Regiella insecticola 2 Ishikawaella capsulata Regiella insecticola 1 Buchnera aphidicola Hamiltonella defensa 2 Symb- Haematopinus apri (pig louse) Hamiltonella defensa 1 Nardonella sp. Symb- Cacopsylla pyri (psyllid) Symb- Calophya schini (psyllids) Symb- Amonostherium lichtensioides (mealybug) Symb- Cacopsylla myrthi (psyllid) Symb- Melanococcus albizziae (mealybug) Symb- Psylla pyricola (psyllid) Symb- Australicoccus grevilleae (mealybug) Blochmannia fellah Symb- Antonina pretiosa (mealybug) Symb- Pseudolynchia canariensis (hippoboscid) Symb- Antonina crawii (mealybug) Wigglesworthia glossinidia Symb- Haematopinus apri (pig louse) Symb- Trioza magnoliae (psyllid) Nardonella sp. Symb- Haematomyzus elephantis (elephant louse) Symb- Calophya schini (psyllids) Portiera aleyrodidarum 2 Blochmannia fellah Portiera aleyrodidarum 1 Symb- Trioza magnoliae (psyllid) Symb- Anomoneura mori (psyllids) Symb- Haematomyzus elephantis (elephant louse) Symb- Psylla pyricola (psyllids) Symb- Pseudolynchia canariensis (hippoboscid) Photorhabdus luminescens Wigglesworthia glossinidia Proteus mirabilis Proteus mirabilis Symb- Melophagus ovinus Symb- Melophagus ovinus Symb- Lipoptena cervi 1 Symb- Lipoptena cervi 1

Symb- Lipoptena cervi 2 Symb- Lipoptena cervi 2 hippoboscoids Symb- Lipoptena fortisetosa 1 Symb- Lipoptena fortisetosa 1 Symb- Lipoptena fortisetosa 2 Symb- Lipoptena fortisetosa 2 Symb- Lipoptena fortisetosa 3 Symb- Lipoptena fortisetosa 3 Symb- Lipoptena sp. hippoboscoids Symb- Lipoptena sp. Symb- Lipoptena cervi 4 Symb- Lipoptena cervi 4 Symb- Lipoptena cervi 3 Symb- Lipoptena cervi 3 Symb- Trichobius caecus 1 Symb- Trichobius caecus 1 Symb- Trichobius caecus 2 Symb- Trichobius caecus 2 Symb- Trichobius parasiticus 2 Symb- Trichobius parasiticus 2 Symb- Trichobius parasiticus 1 Symb- Trichobius parasiticus 1 Symb- Trichobius sp. Symb- Trichobius sp. Symb- Trichobius longipes 1 Symb- Trichobius longipes 1 Symb- Trichobius longipes 4 Symb- Trichobius longipes 4 Symb- Trichobius longipes 3 Symb- Trichobius longipes 3 Symb- Trichobius longipes 2 Symb- Trichobius longipes 2 Symb- Ornithomyia chloropus Symb- Aenictus huonicus Symb- Ornithomyia biloba 3 Symb- Technomyrmex albipes 2 Symb- Ornithomyia biloba 2 Symb- Technomyrmex albipes 1 Symb- Ornithomyia avicularia 1 Symb- Diaphorina citri (psyllid) Symb- Ornithomyia avicularia 6 Symb- Australiococcus greville (whitefly) Symb- Ornithomyia avicularia 4 Arsenophonus insecticola Symb- Ornithomyia avicularia 9 Symb- Wahlgreniella nervata (aphid) Symb- Nycteribia kolenatii 1 Symb- Neomaskellia andropogonis (whitefly) Symb- Penicillidia monoceros Symb- Aleyrodes elevatus (whitefly) Symb- Penicilidia sp. Symb- Dialeurodes hongkongensis (whitefly) Riesia pthiripubis Symb- Aleuroplatus gelatinosus (whitefly) Riesia pediculischaeffi Symb- Heteropsylla texana (psyllid) Riesia pediculicola 1 lice Symb- Trialeurodes vaporariorum (whitefly) Riesia pediculicola 2 Symb- Aphis spiraecola (aphid) Riesia pediculicola 4 Symb- Trialeurodes hutchingsi (whitefly) Riesia pediculicola 5 Symb- Myzocallis sp. 2 (aphid) Riesia pediculicola 3 Symb- Myzocallis sp. 1 (aphid) Symb- Aenictus huonicus Symb- Nycteribia sp. (hippoboscid) Symb- Technomyrmex albipes 2 ants Symb- Nycteribia kolenatii 2 (hippoboscid) Symb- Technomyrmex albipes 1 Symb- Glycaspis brimblecombei 2 (psyllid) Symb- Myrmeleon mobilis (antlion) Symb- Glycaspis brimblecombei 1 (psyllid) Arsenophonus nasoniae Symb- Acanthaleyrodes styraci (whitefly) Symb- Siphoninus phillyreae (whitefly) Symb- Tetraleurodes acaciae (whitefly) Symb- Apis mellifera 1 (honeybee)* Symb- Tetraleurodes mori (whitefly) Symb- Apis mellifera 2 (honeybee) Symb- Bemisia tabaci 2 (whitefly) Symb- Ornithomyia avicularia 8 Symb- Bemisia tabaci 1 (whitefly) Symb- Ornithomyia avicularia 7 Symb- Aleyrodes proletella (whitefly) Symb- Ornithomyia avicularia 5 hippoboscids Symb- Bemisia tabaci 3 (whitefly) Symb- Ornithomyia avicularia 3 Symb- Bemisia tabaci 4 (whitefly) Symb- Dermacentor variabilis 4 Symb- Hippobosca camelina 1 (hippoboscid) Symb- Dermacentor variabilis 1 Symb- Hippobosca camelina 2 (hippoboscid) Symb- Dermacentor variabilis 3 Symb- Hippobosca camelina 3 (hippoboscid) Symb- Dermacentor variabilis 2 ticks Symb- Myzocallis sp. 3 (aphid) Symb- Dermacentor variabilis 5 Symb- Ornithomyia biloba 1 (hippoboscid) Symb- Dermacentor variabilis 6 Symb- Ornithomyia avicularia 2 (hippoboscid) Symb- Dermacentor variabilis 7 Symb- Beta vulgaris Symb- Dermacentor variabilis 8 Phlomobacter fragariae 8 Symb- Aleurodicus dugesii 2 Phlomobacter fragariae 6 Symb- Bemisia tabaci 5 Phlomobacter fragariae 7 Symb- Aleurodicus dispersus whiteflies Phlomobacter fragariae 5 plants Symb- Aleurodicus dugesii 1 Phlomobacter fragariae 4 Arsenophonus triatominarum 2 Phlomobacter fragariae 2 Symb- Triatoma rubrofasciata 2 Phlomobacter fragariae 1 Symb- Triatoma rubrofasciata 1 Phlomobacter fragariae 3 Symb- Meccus mazzottii 2 Symb- Aleurodicus dugesii 2 Symb- Triatoma melanosoma 2 Symb- Bemisia tabaci 5 Symb- Eratyrus mucronatus 2 triatomine bugs Symb- Aleurodicus dispersus whiteflies Symb- Eratyrus mucronatus 1 Symb- Aleurodicus dugesii 1 Symb- Triatoma melanosoma 1 Arsenophonus triatominarum 2 Symb- Meccus mazzottii 1 Symb- Triatoma rubrofasciata 2 Arsenophonus triatominarum 1 Symb- Triatoma rubrofasciata 1 Symb- Diaphorina citri (psyllid) Symb- Meccus mazzottii 2 Symb- Australiococcus greville (whitefly) Symb- Triatoma melanosoma 2 Arsenophonus insecticola Symb- Eratyrus mucronatus 2 triatomine bugs Symb- Wahlgreniella nervata (aphid) Symb- Eratyrus mucronatus 1 Symb- Neomaskellia andropogonis (whitefly) Symb- Triatoma melanosoma 1 Symb- Aleyrodes elevatus (whitefly) Symb- Meccus mazzottii 1 Symb- Dialeurodes hongkongensis (whitefly) Arsenophonus triatominarum 1 Symb- Aleuroplatus gelatinosus (whitefly) Symb- Myrmeleon mobilis (antlion) Symb- Heteropsylla texana (psyllid) Symb- Apis mellifera 1 (honeybee) Symb- Trialeurodes vaporariorum (whitefly) Symb- Apis mellifera 2 (honeybee) Symb- Aphis spiraecola (aphid) Symb- Ornithomyia avicularia 8 Symb- Trialeurodes hutchingsi (whitefly) Symb- Ornithomyia avicularia 7 Symb- Myzocallis sp. 2 (aphid) Symb- Ornithomyia avicularia 5 hippoboscids Symb- Myzocallis sp. 1 (aphid) Symb- Ornithomyia avicularia 3 Symb- Nycteribia sp. (hippoboscid) Arsenophonus nasoniae Symb- Nycteribia kolenatii 2 (hippoboscid) Symb- Siphoninus phillyreae (whitefly) Symb- Glycaspis brimblecombei 2 (psyllid) Symb- Dermacentor* variabilis 4 Symb- Glycaspis brimblecombei 1 (psyllid) Symb- Dermacentor variabilis 1 Symb- Acanthaleyrodes styraci (whitefly) Symb- Dermacentor variabilis 3 ticks Symb- Tetraleurodes acaciae (whitefly) Symb- Dermacentor variabilis 2 Symb- Tetraleurodes mori (whitefly) Symb- Dermacentor variabilis 5 Symb- Bemisia tabaci 2 (whitefly) Symb- Dermacentor variabilis 6 Symb- Bemisia tabaci 1 (whitefly) Symb- Dermacentor variabilis 7 Symb- Aleyrodes proletella (whitefly) Symb- Dermacentor variabilis 8 hippoboscoids Symb- Bemisia tabaci 3 (whitefly) Symb- Ornithomyia chloropus Symb- Bemisia tabaci 4 (whitefly) Symb- Ornithomyia biloba 3 Symb- Hippobosca camelina 1 (hippoboscid) Symb- Ornithomyia biloba 2 Symb- Hippobosca camelina 2 (hippoboscid) Symb- Ornithomyia avicularia 1 Symb- Hippobosca camelina 3 (hippoboscid) Symb- Ornithomyia avicularia 6 Symb- Myzocallis sp. 3 (aphid) Symb- Ornithomyia avicularia 4 Symb- Ornithomyia biloba 1 (hippoboscid) Symb- Ornithomyia avicularia 9 Symb- Ornithomyia avicularia 2 (hippoboscid) Symb- Nycteribia kolenatii 1 Symb- Beta vulgaris Symb- Penicillidia monoceros Phlomobacter fragariae 8 Symb- Penicilidia sp. Phlomobacter fragariae 6 Riesia pthiripubis Riesia pediculischaeffi Phlomobacter fragariae 7 plants lice Phlomobacter fragariae 5 Riesia pediculicola 1 Phlomobacter fragariae 4 Riesia pediculicola 2 Phlomobacter fragariae 2 Riesia pediculicola 4 Phlomobacter fragariae 1 Riesia pediculicola 5 Phlomobacter fragariae 3 Riesia pediculicola 3

FigureTopologies 5 derived from the Basic matrix (1222 positions) Topologies derived from the Basic matrix (1222 positions). A) consensus of the trees obtained under the MP criterion with transversion/transition ratio set to 1:3 and the ML criterion; B) consensus of the MP trees obtained with the transversion/ transition ratio 1:1. The type species A. nasoniae is designated by the orange asterisk.

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Chromatium okenii Pseudomonas aeruginosa Photorhabdus luminescens Proteus mirabilis Symb- Nycteribia sp. (hippoboscid) 0,98 Symb- Nycteribia kolenatii 2 (hippoboscid) Symb- Australiococcus greville (whitefly) Symb- Diaphorina citri (psyllid) Symb- Glycaspis brimblecombei 2 (psyllid) 1 Symb- Glycaspis brimblecombei 1 (psyllid) Arsenophonus insecticola Symb- Hippobosca camelina 1 (hippoboscid) 0,96 Symb- Hippobosca camelina 2 (hippoboscid) Symb- Ornithomyia biloba 1 (hippoboscid) 1 Symb- Ornithomyia avicularia 2 (hippoboscid) Symb- Hippobosca camelina 3 (hippoboscid) 1 Symb- Myzocallis sp. 3 (aphid) Symb- Neomaskellia andropogonis (whitefly) 1 Symb- Tetraleurodes acaciae (whitefly) 0,99 Symb- Acanthaleyrodes styraci (whitefly) Symb- Aleyrodes proletella (whitefly) Symb- Dialeurodes hongkongensis (whitefly) Symb- Aleyrodes elevatus (whitefly) Phlomobacter fragariae 8 Phlomobacter fragariae 6 Symb- Beta vulgaris 1 Phlomobacter fragariae 7 Phlomobacter fragariae 5 plants Phlomobacter fragariae 4 0,88 Phlomobacter fragariae 2 Phlomobacter fragariae 1 Phlomobacter fragariae 3 Symb- Wahlgreniella nervata (aphid) 0,51 Symb- Heteropsylla texana (psyllid) Symb- Bemisia tabaci 2 (whitefly) 1 0,92 1 Symb- Bemisia tabaci 3 (whitefly) Symb- Bemisia tabaci 1 (whitefly) 0,51 Symb- Bemisia tabaci 4 (whitefly) 0,5 Symb- Trialeurodes vaporariorum (whitefly) Symb- Trialeurodes hutchingsi (whitefly) Symb- Aphis spiraecola (aphid) Symb- Myrmeleon mobilis (antlion) 1 Symb- Apis mellifera 1 (honeybee) Symb- Apis mellifera 2 (honeybee) Symb- Ornithomyia avicularia 8 0,82 1 Symb- Ornithomyia avicularia 7 Symb- Ornithomyia avicularia 5 hippoboscids Symb- Ornithomyia avicularia 3 Arsenophonus nasoniae Symb- Siphoninus phillyreae (whitefly) Symb- Dermacentor* variabilis 4 0,53 Symb- Dermacentor variabilis 1 Symb- Dermacentor variabilis 3 1 0,51 Symb- Dermacentor variabilis 2 ticks Symb- Dermacentor variabilis 5 Symb- Dermacentor variabilis 6 1 Symb- Dermacentor variabilis 7 Symb- Dermacentor variabilis 8 hippoboscoids 1 0,95 Symb- Ornithomyia avicularia 9 1 Symb- Ornithomyia avicularia 6 Symb- Ornithomyia avicularia 4 0,61 Symb- Ornithomyia avicularia 1 1 1 Symb- Ornithomyia biloba 3 Symb- Ornithomyia biloba 2 Symb- Ornithomyia chloropus 0,53 1 1 Symb- Nycteribia kolenatii 1 1 Symb- Penicilidia sp. 1 1 Symb- Penicillidia monoceros 1 Riesia pthiripubis 0,58 Riesia pediculicola 1 lice 1 Riesia pediculicola 2 1 Riesia pediculicola 4 0,84 Riesia pediculicola 5 Riesia pediculicola 3 Riesia pediculischaeffi Symb- Triatoma rubrofasciata 1 Symb- Triatoma melanosoma 1 0,66 Symb- Eratyrus mucronatus 1 Symb- Meccus mazzottii 1 Symb- Meccus mazzottii 2 triatomine bugs 0,63 Symb- Triatoma melanosoma 2 0,77 Symb- Eratyrus mucronatus 2 Arsenophonus triatominarum 1 1 Arsenophonus triatominarum 2 0,97 Symb- Triatoma rubrofasciata 2 1 Symb- Aleurodicus dugesii 1 0,87 Symb- Aleurodicus dispersus 0,59 Symb- Aleurodicus dugesii 2 whiteflies Symb- Bemisia tabaci 5 Symb- Aleyrodes proletella (whitefly) Symb- Myzocallis sp. 2 (aphid) Symb- Myzocallis sp. 1 (aphid) 0,53 Symb- Technomyrmex albipes 1 1 Symb- Technomyrmex albipes 2 ants Symb- Aenictus huonicus Symb- Tetraleurodes mori (whitefly) Symb- Melophagus ovinus Symb- Lipoptena sp. 1 Symb- Lipoptena fortisetosa 1

Symb- Lipoptena fortisetosa 2 hippoboscoids 1 0,78 1 Symb- Lipoptena fortisetosa 3 1 Symb- Lipoptena cervi 2 Symb- Lipoptena cervi 1 1 1 Symb- Lipoptena cervi 4 Symb- Lipoptena cervi 3 Symb- Trichobius sp. 1 Symb- Trichobius longipes 1 1 1 Symb- Trichobius longipes 4 0,97 Symb- Trichobius longipes 3 Symb- Trichobius longipes 2 1 1 Symb- Trichobius parasiticus 1 Symb- Trichobius parasiticus 2 1 Symb- Trichobius caecus 1 Symb- Trichobius caecus 2 1 Regiella insecticola 1 1 Regiella insecticola 2 0,74 Hamiltonella defensa 1 1 Symb- Cacopsylla pyri (psyllid) Hamiltonella defensa 2 Sodalis glossinidius 1 Symb- Blastopsylla occidentalis 2 (psyllid) Symb- Blastopsylla occidentalis 1 (psyllid) 0,71 Symb- Bactericera cockerelli (psyllid) 0,57 Symb- Psylla floccosa (psyllid) Symb- Eulachnus pallidus (aphid) 1 Symb- Planococcus ficus (mealybug) 1 1 Symb- Planococcus citri 2 (mealybug) Symb- Planococcus citri 1 (mealybug) 1 Symb- Dysmicoccus brevipes (mealybug) 1 Symb- Dysmicoccus neobrevipes (mealybug) 1 Symb- Erium globosum (mealybug) Symb- Vryburgia amaryllidis (mealybug) 0,5 1 Symb- Cyphonococcus alpinus (mealybug) Symb- Paracoccus nothofagicola (mealybug) 1 Symb- Pseudolynchia canariensis (hippoboscid) Wigglesworthia glossinidia 0,53 Symb- Amonostherium lichtensioides (mealybug) 1 Symb- Australicoccus grevilleae (mealybug) 1 Symb- Melanococcus albizziae (mealybug) 1 Symb- Antonina crawii (mealybug) 0,55 Symb- Antonina pretiosa (mealybug) 1 Symb- Aphalaroida inermis (psyllids) Baumannia cicadellinicola Symb- Calophya schini (psyllids) 0,86 Symb- Haematopinus apri (pig louse) 0,5 0,78 Blochmannia fellah 0,58 Symb- Trioza magnoliae (psyllid) 0,97 Symb- Haematomyzus elephantis (elephant louse) 0,79 Nardonella sp. 1 Symb- Cacopsylla myrthi (psyllid) Symb- Psylla pyricola (psyllid) Symb- Heteropsylla cubana (psyllid) 0,56 Ishikawaella capsulata Buchnera aphidicola 1 Symb- Anomoneura mori (psyllids) Symb- Psylla pyricola (psyllids) 1 Portiera aleyrodidarum 2 Portiera aleyrodidarum 1

0.2 PhylogeneticFigure 6 tree derived from Basic matrix (1222 positions) using Bayesian analysis Phylogenetic tree derived from Basic matrix (1222 positions) using Bayesian analysis. Names of the taxa clustering within the Arsenophonus clade are printed in colour: red for the long-branched taxa, dark orange for the short-branched taxa. Names in the brackets designate the host family. Numbers represent Bayesian posterior probability for each node. The type species A. nasoniae is designated by the orange asterisk.

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take part in shaping the relationships among Arsenophonus as a guide for deciding between different coevolutionary sequences, lateral gene transfer (LGT) and intragenomic scenarios. They used the Escherichia-Salmonella divergence heterogeneity. Both have previously been determined as [54,55] as a calibration point for calculating the diver- causes of phylogenetic distortions and should be consid- gence time among various Arsenophonus lineages from tri- ered in coevolutionary studies at a low phylogenetic level. atomine bugs. Applying the Multdiv method [56], they placed the ancestor of triatomine-associated symbionts Incongruence due to LGT and intragenomic heterogeneity into a broad range of approx. 15 – 40 mya and concluded An apparently "mosaic" structure of the Arsenophonus that this estimate is compatible or even exceeds the age alignment (for example see Additional file 4) raises the estimates available for the tribe triatomine (according to question of whether various regions of this sequence Gaunt and Miles [57]). Here, we took advantage of a new could have undergone different evolutionary histories. age-estimate for closely related bacteria, namely the louse- Recombination of 16S rDNA genes were previously iden- associated symbionts of the genus Riesia [18]. Comparing tified in some other bacteria [42-44]. In actinomycetes, the estimates based on the two calibration methods the occurrence of short rDNA segments with high number (Escherichia-Salmonella and Riesia), we found that due to of non-random variations was attributed to the lateral the variability of evolutionary rates among the lineages, transfer as the most parsimonious explanation [45]. Later, the results may differ by an order of magnitude. Such Gogarten et al. [46] suggested that, analogously to an marked variance among different bacterial lineages entire bacterial genome, 16S rDNA possesses a mosaic (including different symbiotic bacteria from the same character originated by LGT, respectively by transfer of host species) was previously reported for many bacterial gene subunits. groups [29,30,37,39,58-63]. Most recently, Allen et al. [64] reported an extremely high evolutionary rate for the As bacterial genomes often carry more than one rRNA young symbiotic lineage Riesia, and suggested that the operon, intragenomic heterogeneity of the rDNA copies is evolutionary tempo changes with the age of the symbiotic occasionally found to blur the phylogenetic picture [47- lineage. We therefore conclude that this method cannot 50]. Although there is no direct information on the be directly used to assess the effect of intragenomic heter- number of rRNA gene copies in Arsenophonus genomes, ogeneity on our reconstruction of Arsenophonus relation- Stewart and Cavanaugh [51] showed bacterial genomes to ships. encode in average five rRNA operons. The most closely related bacterium of which the complete genome has Conclusion recently been sequenced, Proteus mirabilis, carries seven With more than one hundred records, the genus Arseno- copies [GenBank: AM942759]. Arsenophonus-focused phonus represents one of the richest and most widespread studies indicate that two different forms of the rRNA clusters of insect symbiotic bacteria. Considering its broad operon are present in its genome, as is typical for Entero- host spectrum and apparent ecological versatility, Arseno- bacteraceae [23,52]. Furthermore, Šorfová et al. [23] sug- phonus should play an important role in studies of evolu- gest that the variability among individual copies may tionary trends in insect intracellular symbionts. Due to cause the incongruence observed between triatomines this fact, Arsenophonus is likely to attract a growing atten- and their Arsenophonus lineages. They point out that this tion, and the number of the records may rapidly be process could, in principle, explain an otherwise problem- increasing during the next years. For example, 7 new atic observation: in some hosts, such as triatomines or sequences were deposited into the GenBank since the some homopterans, the hosts and the Arsenophonus bacte- completion of this study [[65], and unpublished record ria create reciprocally monophyletic clusters but do not FJ388523]. However, since these new Arsenophonus show any cospeciation pattern. In the symbionts of grain records originated in screening rather than phylogenetic weevils, divergence between rRNA sequences within a study, they are only represented by short DNA fragments genome was shown sometimes to exceed divergence of (approx. 500 bp). Preliminary analyses of these fragments orthologous copies from symbionts from different hosts; together with our complete datasets confirmed a limited this unusual situation was hypothesized to reflect loss of informative value of such short sequences and they were recombinational repair mechanisms from these symbiont not included into the more exhaustive phylogenetic pro- genomes [53]. cedures.

Estimates of the divergence time The analysis of 110 available sequences of Arsenophonus With the present incomplete knowledge of the Arseno- 16S rDNA from 54 host taxa revealed several interesting phonus genome, it is difficult to assess whether and how evolutionary patterns. In particular, this clade includes at deeply rRNA heterogeneity affects phylogenetic recon- least two transitions from S-symbiont, with ability to struction. Trying to find alternative solution, Šorfová et al. invade new host lineages, to P-symbiont, showing obli- [23] attempted to use the estimation of divergence times gate relationship to hosts and a strict pattern of maternal

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transmission. Thus, it is a promising system for exploring approx. 1020 bp of 16S rDNA, particularly within Entero- the genomic and biological changes that accompany the bacteriaceae [34], were used for all samples. PCR was per- shift from facultative to obligate symbiont. Arsenophonus formed under standard conditions using HotStart Taq is also one of the few groups of insect symbionts for which polymerase (HotStarTaqi DNA Polymerase, Qiagen). The strains have been grown in pure culture [4,7,16], a feature PCR products were analyzed by gel electrophoresis and that further enhances its potential as a model for symbi- cloned into pGEM-T Easy System 1 vector (Promega). ont research. Inserts from selected colonies were amplified using T7 and SP6 primers and sequenced in both directions, with Our results also indicate that a complete understanding of the exception of 3 fragments sequenced in one direction the Arsenophonus phylogeny cannot be achieved with 16S only (sequences from Aenictus huonicus and Myzocalis sp.). rDNA genes alone. A similar situation is, for example, DNA sequencing was performed on automated sequencer found in another large symbiotic group, the genus Wol- model 310 ABI PRISM (PE-Biosystems, Foster City, Cali- bachia, where other genes are often used as alternative fornia, USA) using the BigDye DNA sequencing kit (PE- sources of phylogenetic information [66,67]. Identifica- Biosystems). For each sample, five to ten colonies were tion of suitable low-level-phylogeny marker(s) is thus one screened on average. The contig construction and of the most crucial steps in the further research on Arseno- sequence editing was done in the SeqMan program from phonus evolution. The sequencing of the complete Arseno- the DNASTAR platform (Dnastar, Inc. 1999). Identifica- phonus genome, which is currently under the process tion of the sequences was done using BLAST, NCBI http:/ http://genomesonline.org/gold.cgi?want=Bacte /www.ncbi.nlm.nih.gov/blast/Blast.cgi. rial+Ongoing+Genomes&pubsort=Domain, will provide a valuable background for such enterprise. Alignments To analyze thoroughly the behavior of Arsenophonus 16S Based on the presented analyses, we also want to point rDNA and assess its usefulness as a phylogenetic marker, out that the genus Arsenophonus is currently paraphyletic we prepared several matrices and performed an array of due to the two lineages described as separate genera Riesia phylogenetic analyses on each of them. and Phlomobacter but clustering within the Arsenophonus group (e.g. Figure 2). Two procedures can, in principle, The Basic matrix was composed of the 34 new sequences, solve this undesirable situation, splitting of the Arseno- all Arsenophonus sequences available in the GenBank and phonus cluster into several separate genera or classification additional 45 sequences of various P-symbionts, S-symbi- of all its members within the genus Arsenophonus. Taking ont and 5 free living bacteria (see Additional file 5). To into account the phylogenetic arrangement of the individ- show the impact of random or restricted sampling on the ual lineages, the first approach would inevitably lead to resulting topology, five different matrices labelled Sam- establishment of many genera with low sequence diver- plingi (i.e. Sampling1, Sampling2, etc.) were prepared from gences and very similar biology. The second option has Basic matrix by removing various taxa and including addi- been previously mentioned in respect to the genus Phlo- tional/alternative outgroups. The matrices Sampling1 to mobacter [68], and we consider this approach (i.e. reclassi- Sampling4 were composed of various numbers of non- fication of all members of the Arsenophonus clade within a Arsenophonus symbiotic taxa (ranging from 3 to 35), three single genus) a more appropriate solution of the current sequences of free-living bacteria, and an arbitrarily situation within the Arsenophonus clade. selected set of all Arsenophonus lineages. Matrix designated as Sampling5 was restricted to a lower number of taxa, Methods including 5 ingroup sequences and alternative lineages of Samples symbiotic and free-living bacteria. The host species used in this study were acquired from several sources. All of the nycteribiid samples were All matrices were aligned in the server-based program obtained from Radek Lučan. Most of the hippoboscids MAFFT http://align.bmr.kyushu-u.ac.jp/mafft/online/ were provided by Jan Votýpka. Ant species were collected server/, using the E-INS-i algorithm with default parame- by Milan Janda in Papua New Guinea. All other samples ters. The program BioEdit [69] was used to manually cor- are from the authors' collection. List of the sequences rect the resulting matrices and to calculate the GC content included in the Basic matrix is provided in the Additional of the sequences. file 5. To test an effect of unreliably aligned regions on the phy- DNA extraction, PCR and sequencing logenetic analysis, we further prepared the Conservative The total genomic DNA was extracted from individual matrix, by removing variable regions from the Basic matrix. samples using DNEasy Tissue Kit (QIAGEN; Hilden, Ger- For this procedure, we used the program Gblocks [70] many). Primers F40 and R1060 designed to amplify available as server-based application on the web page

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http://molevol.cmima.csic.es/castresana/ Additional material Gblocks_server.html.

Finally, the Clock matrix, composed of 12 bacterial Additional file 1 Consensus tree derived from the Conservative matrix (284 positions) sequence (see Additional file 5), was designed to calculate under MP criterion. Transversion/transition ratio was set to 1:3. Names time of divergence for several nodes within the Arseno- of the taxa clustering within the Arsenophonus clade derived from Basic phonus topology. matrix are printed in colour: red for the long-branched taxa, dark orange for the short-branched taxa. Phylogenetic analyses Click here for file The matrices were analyzed using maximum parsimony [http://www.biomedcentral.com/content/supplementary/1471- 2180-9-143-S1.eps] (MP), maximum likelihood (ML) and Bayesian probabil- ity. For analyses, we used the following programs and pro- Additional file 2 cedures. The GTR+Γ+inv model of molecular evolution Tree consensus derived from Sampling4 (1107 positions) matrix was determined as best fitting by the program Modeltest under the MP criterion. Transversion/transition ratio was set to 1:1. The [71] and was used in all ML-based analyses. MP analysis type species A. nasoniae is designated by the orange asterisk. was carried out in TNT program [72] using the Traditional Click here for file search option, with 100 replicates of heuristic search, [http://www.biomedcentral.com/content/supplementary/1471- 2180-9-143-S2.eps] under the assumptions of Ts/Tv ratio 1 and 3. ML analysis was done in the Phyml program [73] with model param- Additional file 3 eters estimated from the data. Bayesian analysis was per- Insertions within the sequences of P-like symbionts. formed in Mr. Bayes ver. 3.1.2. with following parameter Click here for file settings: nst = 6, rates = invgamma, ngen = 3000000, sam- [http://www.biomedcentral.com/content/supplementary/1471- plefreq = 100, and printfreq = 100. The program Phylowin 2180-9-143-S3.eps] [74] was employed for the ML analysis under the nonho- mogeneous model of substitution [31]. Additional file 4 The 41 bp long motif inconsistently distributed among distinct bacte- rial taxa. Position of the sequence in alignment and 16S rDNA secondary A calculation of divergence time was performed in the structure is indicated by the arrows. Following records are not included in program Beast [75] which implements MCMC procedure the Additional file 1: Sitophilus rugicollis [GenBank: AY126639], to sample target distribution of the posterior probabili- Drosophila paulistorum [GenBank: U20279, U20278], Polyrhachis ties. The gamma distribution coupled with the GTR+inv- foreli [GenBank: AY336986], Haematopinus eurysternus [GenBank: gamma model was approximated by 6 categories of DQ076661]. substitution rates. Relaxed molecular clock (uncorrelated Click here for file [http://www.biomedcentral.com/content/supplementary/1471- lognormal option) was applied to model the rates along 2180-9-143-S4.eps] the lineages. To obtain a time-framework for the tree, we used the estimate on louse divergence (approximately 5.6 Additional file 5 mya [18]). Since the resulting estimate was considerably List of sequences included in Basic matrix. Dashed line separates mem- lower that that reported previously with Escherichia-Salmo- bers of the Arsenophonus clade from the outgroup taxa. Sequences nella calibration [23], we prepared an additional matrix included into the Clock matrix are underlined. and used the Escherichia-Salmonella split [54,55] as an Click here for file [http://www.biomedcentral.com/content/supplementary/1471- alternative calibration; taxa included according to Šorfová 2180-9-143-S5.doc] et al. [23]. All analyses were performed in three independ- ent runs, each taking 5 million generations.

Authors' contributions Acknowledgements EN obtained the sequence data, compiled alignments and This work was supported by Ministry of Education, Czech Republic (grants participated in the study design, phylogenetic inference, LC06073 and MSM 60076605801), the Grant Agency of Academy of Sci- interpretation of the results, and preparation of the man- ences of the Czech Republic (Grant IAA601410708) and a National Science uscript. VH conceived of the study and participated in Foundation grant (0626716) to N.A. Moran (University of Arizona). We conduction of the phylogenetic inference. Both, VH and thank all of our collaborators for providing the samples. NAM participated in the study design, evolutionary inter- pretation of the results and preparation of the manuscript. References 1. Huger AM, Skinner SW, Werren JH: Bacterial infections associ- All authors read and approved the final manuscript. ated with the son-killer trait in the parasitoid wasp, Nasonia (= Mormoniella) vitripennis (Hymenoptera, Pteromalidae). J Invertebr Pathol 1985, 46:272-280.

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