Phylogeny of Wolbachia pipientis based on gltA, groEL and ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F supergroup, and evidence for further diversity in the Wolbachia tree

M. Casiraghi1,{dagger}, S. R. Bordenstein2, L. Baldo1,{ddagger}, N. Lo3, T. Beninati1, J. J. Wernegreen2, J. H. Werren4 and C. Bandi1

1 Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Patologia Generale e Parassitologia, Università degli Studi di Milano, Milano, Italy
2 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, The Marine Biological Laboratory, Woods Hole, MA, USA
3 School of Biological Sciences, The University of Sydney, NSW 2006, Australia
4 Department of Biology, University of Rochester, Rochester, NY, USA

Correspondence
N. Lo
nathan{at}usyd.edu.au


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Current phylogenies of the intracellular bacteria belonging to the genus Wolbachia identify six major clades (A–F), termed ‘supergroups’, but the branching order of these supergroups remains unresolved. Supergroups A, B and E include most of the wolbachiae found thus far in arthropods, while supergroups C and D include most of those found in filarial nematodes. Members of supergroup F have been found in arthropods (i.e. termites), and have previously been detected in the nematode Mansonella ozzardi, a causative agent of human filariasis. To resolve the phylogenetic positions of Wolbachia from Mansonella spp., and other novel strains from the flea Ctenocephalides felis and the filarial nematode Dipetalonema gracile, the authors generated new DNA sequences of the Wolbachia genes encoding citrate synthase (gltA), heat-shock protein 60 (groEL), and the cell division protein ftsZ. Phylogenetic analysis confirmed the designation of Wolbachia from Mansonella spp. as a member of the F supergroup. In addition, it was found that divergent lineages from Dip. gracile and Cte. felis lack any clear affiliation with known supergroups, indicating further genetic diversity within the Wolbachia genus. Finally, although the data generated did not permit clear resolution of the root of the global Wolbachia tree, the results suggest that the transfer of Wolbachia spp. from arthropods to nematodes (or vice versa) probably occurred more than once.


Abbreviations: BI, Bayesian inference; ML, maximum-likelihood; TBR, tree bisection and reconnection

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are listed in Table 1.

The phylogeny inferred from the concatenated dataset (gltA, groEL, ftsZ), rooted with two outgroup species (Anaplasma marginale and Ehrlichia ruminantium), is shown in Supplementary Fig. S1, available with the online version of this paper.

{dagger}Present address: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano Bicocca, Milano, Italy.

{ddagger}Present address: Department of Biology, University of California, 900 University Avenue, Riverside, CA, USA.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The genus Wolbachia encompasses obligately intracellular bacteria that are cytoplasmically transmitted in arthropods and filarial nematodes (Werren, 1997; Bandi et al., 2001; Bordenstein et al., 2003). Unlike most other obligately intracellular bacteria, the genus Wolbachia forms a monophyletic clade comprising both mutualistic and parasitic lineages that showcase the diversity of symbiotic associations. In arthropods, Wolbachia spp. are commonly known as reproductive parasites, since they distort host reproductive strategies to selfishly enhance their maternal transmission into the next generation (Werren, 1997). Wolbachia spp. have a remarkably high prevalence in some arthropod groups (Jeyaprakash & Hoy, 2000). In contrast, the Wolbachia lineages infecting filarial nematodes are beneficial symbionts required for host fertility and larval viability (Bandi et al., 2001).

The gene phylogenies of the genus Wolbachia have shown the existence of six major clades (A–F), which, in the absence of a formal species description, have been named ‘supergroups' (see Lo et al., 2002, and references therein). The monophyly of some of these supergroups is not firmly established (i.e. supergroup F; Lo et al., 2002), and their relationships are not resolved. Supergroups A and B include most of the parasitic Wolbachia spp. thus far found in arthropods (Werren et al., 1995). Supergroups C and D include the majority of the Wolbachia spp. found in filarial nematodes (Bandi et al., 1998) (though some nematodes have been found to lack Wolbachia spp.; Bordenstein et al., 2003). The E supergroup encompasses Wolbachia spp. from primitively wingless insects, the springtails (Collembola) (Vandekerckhove et al., 1999; Czarnetzki & Tebbe, 2004). Members of supergroup F are known to infect arthropods (termites), and recent studies suggest that they also infect the filarial parasite Mansonella ozzardi (Casiraghi et al., 2001a; Lo et al., 2002). More recently, a new supergroup, named G, has been proposed for Wolbachia spp. of certain Australian spiders (Rowley et al., 2004). Divergent lineages have been detected in fleas (Fischer et al., 2002; Gorham et al., 2003), and in the filarial nematode Dipetalonema gracile (Casiraghi et al., 2004); however, they have not been labelled new supergroups, since only 16S rRNA has been obtained. In summary, the overall diversity and phylogeny of the genus Wolbachia is increasingly informative, but also incomplete, with possible new hosts (Mansonella spp.) and new lineages (i.e. in Dip. gracile and fleas) still requiring confirmation.

In this report, we address two key issues facing the molecular taxonomy of the genus Wolbachia. First, we verify the presence of supergroup F in the genus Mansonella, and in termites with additional host species and Wolbachia genes. Supergroup F is of particular interest for understanding the origins and evolutionary relationships of Wolbachia spp. because it is the only supergroup reported to infect both nematodes and arthropods (Lo et al., 2002). If the supergroup's dual host range is confirmed, it could represent a clear and relatively recent transfer of Wolbachia spp. between the nematode and arthropod phyla. The apparent novelty of this group's host range is tentative, and based upon only a single gene analysis of 16S rDNA sequences spanning a few termite species, and microfilariae from one nematode species, M. ozzardi. With the small possibility of a false-positive result in Mansonella due to cross-contamination with Wolbachia-infected microarthropods (i.e. mites), it is necessary to expand the taxon and gene sampling for this supergroup. We do so by developing new primers and PCR protocols for the genes encoding citrate synthase (gltA), heat-shock protein 60 (groEL) and the cell division protein ftsZ of Wolbachia. Second, we investigate the phylogenetic placement of Wolbachia from the cat flea (Ctenocephalides felis) and the nematode Dip. gracile – two lineages that may be genetically distinct from the other supergroups. In investigating both these issues, we contribute to the reconstruction of the overall phylogeny of the Wolbachia genus, and extend the available Wolbachia gene dataset (28 new gltA sequences; 30 groEL; 2 ftsZ).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
DNA isolation, amplification and sequencing.
A total of 25 insect and 15 filarial nematode species harbouring Wolbachia pipientis were included in the study (see Table 1). DNA extraction from arthropod specimens was performed using either a standard phenol/chloroform procedure (Sambrook & Russell, 2001), or the DNeasy Tissue Kit (Qiagen). For nematode specimens, crude DNA preparations were obtained through proteinase K treatment, according to Bandi et al. (1994). All arthropod and nematode specimens used in this study were infected with a single strain of Wolbachia.


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Table 1. Host taxonomic details and Wolbachia sequence accession numbers

Twenty-five arthropod and 15 nematode host species are listed in the table. Accession numbers in bold represent new sequence data generated for this study.

 
gltA was amplified and sequenced using the following primers: WgltAF1, 5'-TAC GAT CCA GGG TTT GTT TCT AC-3'; WgltARev1, 5'-CTC ATT AGC TCC ACC GTG TG-3'; WgltARev2, 5'-CAT TTC ATA CCA CTG GGC AA-3'. These primers were designed on the basis of homologous regions of gltA among Anaplasma marginale (AF304146), Ehrlichia ruminantium (AF304140) and W. pipientis from Drosophila melanogaster (AE017260). groEL was amplified and sequenced using the following primers: WgroF1, 5'-GGTGAGCAGTTGCAAGAAGC-3'; WgroRev1, 5'-AGATCTTCCATCTTGATTCC-3'; WgroF1deg, 5'-GGT GAG CAG TT(GA) CA(GA) (CG)AA GC-3'; WgroR1d, 5'-AG(GA) TCT TCC AT(CT) TT(AG) ATT CC-3'; WgroR2d, 5'-GGT ATT (AG)TC TTT AGT (AG)A(TC) TTT AAC-3'; WgroR4d, 5'-TTT (AG)AC AGC ATC AGC AAT-3'. Primers were designed on the basis of groEL regions conserved among the Wolbachia spp. from Onchocerca volvulus, Litomosoides sigmodontis, Dirofilaria immitis, Brugia malayi and Dro. melanogaster (Y09416; AF409113; AJ558023; AF373870; AE017257). ftsZ and 16S rDNA were amplified and sequenced using the primers and conditions described in Casiraghi et al. (2001b, 2004).

PCR amplifications were performed in 20–50 µl volumes, under the following final conditions: 1x Eppendorf buffer including 1·5 mM MgCl2, 0·2 µM of each dNTP, 1 µM each of forward and reverse primers, and 0·5 units MasterTaq (Eppendorf). The thermal profiles we used were: (1) gltA, 94 °C 45 s, 52 °C 45 s, and 72 °C 90 s, for 40 cycles; (2) groEL, 94 °C 45 s, 60 °C 45 s, 72 °C 80 s, for 5 cycles, and 94 °C 45 s, 55 °C 45 s, and 72 °C 80 s, for 34 cycles; (3) ftsZ, 94 °C 30 s, 60 °C 45 s, 72 °C 90 s, for 5 cycles, and 94 °C 30 s, 57 °C 45 s, and 72 °C 90 s, for 34 cycles.

PCR products were purified and sequenced bidirectionally on an ABI 3730 or 3310 automated sequencer using Big Dye v2.0 or v3.0 (Applied Biosystems). The 60 new gene sequences generated in this study were deposited in the EMBL database (see Table 1 for accession numbers).

The gltA, groEL and ftsZ sequences were not obtained from all the taxa included in this study, mainly due to the scarcity of certain specimens, and amplification/sequencing problems for some of the species examined. For example, despite attempts with various primer combinations and PCR conditions, we were unable to amplify Wolbachia groEL and ftsZ sequences from Folsomia candida and Dip. gracile, respectively. Based on their high divergence at other loci (see Results), these genes may have accumulated substitutions in the regions of the primers tested.

Sequence alignment and phylogenetic analysis.
DNA alignments were based on translated proteins, and were performed manually in MacClade 4.05. A total of four straightforward alignments were generated, with very few gaps. Total lengths of alignments were 639 bp for gltA, 876 bp for groEL, 735 bp for ftsZ, and 2250 bp for a concatenated alignment that included taxa for which two out of three gene sequences were available. Each of the alignments has been deposited in the EBI alignment databases, with the following accession numbers: ALIGN_000922 (gltA), ALIGN_000923 (groEL) and ALIGN_000920 (ftsZ).

Phylogenetic analyses were conducted using maximum-likelihood (ML) and Bayesian inference (BI) methods. For ML, the appropriate models of sequence evolution for each dataset were estimated via likelihood ratio tests in the program modeltest 3.06 (Posada & Crandall, 1998). The models selected were GTR+I+G for gltA, GTR+I+G for groEL, GTR+G for ftsZ, and GTR+I+G for the combined dataset. ML heuristic searches were performed using 100 random taxon addition replicates, with tree bisection and reconnection (TBR) branch swapping. ML bootstrap support was determined using 100 bootstrap replicates, each using 10 random taxon addition replicates with TBR branch swapping. Searches were performed in parallel on a Beowulf cluster using custom software with PAUP version 4.0b10 (Swofford, 2002). ML analysis of the combined dataset was performed with and without the outgroup taxa A. marginales and E. ruminantium to estimate rooted and unrooted phylogenies, respectively.

Initial BI analyses were performed using MrBayes 3.0 (Ronquist & Huelsenbeck 2003), with simultaneous estimation of genealogies and DNA sequence parameters. In the analyses of the gltA, groEL, ftsZ and combined datasets, these initial analyses ran for 100 000 generations, with trees sampled every 100 generations. The first 500 of the 1000 sampled trees were considered the ‘burn-in’, and were discarded. From the remaining 500 trees, 50 % majority-rule consensus trees were generated. For each single-gene dataset, topological agreement and overall similarity of posterior probabilities among five independent runs indicated adequate convergence and mixing. As for the ML analyses, the combined dataset was analysed with and without the presence of outgroups.

Final BI analyses for each single-gene dataset and the combined dataset (without outgroups) consisted of two independent runs with 3 000 000 generations, and four chains per run, using Mr Bayes version 3.1.1 (Ronquist & Huelsenbeck, 2003). The likelihood model was set to the GTR, with a proportion of the sites invariable, and the rest drawn from a gamma distribution (‘lset Nst=6 rates=invgamma’). When applicable (see below), the site-specific rates model was allowed to vary among data partitions (prset ratepr=variable). Trees were sampled every 100 generations, resulting in 30 000 trees per run (60 000 per analysis consisting of two independent runs). The first third of these trees was considered the ‘burn-in’, and was discarded. Posterior probabilities were estimated from the consensus of the remaining 40 000 trees. Partitioning of datasets by codon positions or (in the case of combined dataset) by gene did not affect the resulting topology, and had minimal effects on posterior probabilities. Posterior probabilities presented here (Figs 1 and 2) are those from the 3 000 000-generation BI analyses performed as described above, in which data were partitioned by first and second positions in one partition, and third positions in a second partition.




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Fig. 1. Phylogenetic reconstructions based on individual analyses of (a) gltA, (b) groEL and (c) ftsZ. Taxa include representatives of supergroups A–F, Wolbachia spp. of Cte. felis, and Wolbachia spp. of Dip. gracile. Data for groEL of F. candida-derived isolates and ftsZ of Dip. gracile-derived isolates were not obtained (see text). For each dataset, phylogenies based on ML and BI were topologically identical. The phylogenies presented reflect ML branch lengths. Nodes are marked by their ML bootstrap values (top number, in bold) estimated from 100 bootstrap replicates, each of which was based on 10 random starting trees, and Bayesian posterior probabilities (bottom number) based on two independent runs of three million generations each. For clarity of presentation, the figure excludes confidence values for some recent divergences within supergroups.

 


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Fig. 2. W. pipientis phylogeny based on the concatenated datasets of gltA, groEL and ftsZ gene sequences (2250 bp). This tree topology was inferred using both ML and BI, and the phylogeny presented reflects ML branch lengths. Nodes are marked by their ML bootstrap values (top number, in bold) estimated from 100 bootstrap replicates, each of which was based on 10 random starting trees, and Bayesian posterior probabilities (bottom number) based on two independent runs of three million generations each (bottom number). For clarity of presentation, the figure excludes confidence values for some recent divergences within subgroups.

 

   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Host range and taxonomic positioning of supergroup F
The filarial nematode Mansonella sp. included in this study represents a different species than the human parasite M. ozzardi sampled previously (Casiraghi et al., 2001a). We found that Wolbachia spp. of the two Mansonella species group together and, as shown previously for M. ozzardi (Casiraghi et al., 2001a), also group with Wolbachia spp. from termites (see Figs 1 and 2, and text below). In addition, new Wolbachia groEL sequences from the termites Coptotermes lacteus and Coptotermes acinaciformis confirmed both the presence of Wolbachia spp. in termites, and their phylogenetic proximity to Wolbachia spp. of Mansonella spp. In all analyses, Wolbachia sequences from termites and the two Mansonella spp. consistently clustered together, forming a separate clade from the other supergroups. This pattern confirms the existence of the F supergroup of the genus Wolbachia and its dual host range spanning nematodes (Mansonella) and arthropods (termites). In addition to Mansonella spp. and termites, the F supergroup appears to encompass Wolbachia spp. of other hosts, including the human bed bug (Cimex lectulatis), the cliff swallow bug (Oeciacus vicarius), and the beetle Rhinocyllus conicus (Rasgon & Scott, 2004; Bandi et al., 1997; Lo et al., 2002).

In previous PCR screens for Wolbachia spp. in Mansonella spp. (Casiraghi et al., 2001a; Lo et al., 2002), we used microfilariae of M. ozzardi (i.e. 250-µm-long juveniles) collected in South America from human blood. To rule out the possibility that previously identified Wolbachia spp. arose from some other contaminant of the blood, here we used adult specimens of Mansonella spp. (3–5 cm long) from Sika deer (Cervus nippon). To rule out the possibility that termite Wolbachia spp. actually infect nematodes that might infest the termites, we performed a PCR screen of the four termite species using general primers for nematode 18S rDNA. No evidence was found for the presence of nematodes within the termites (data not shown).

Phylogeny of Wolbachia genus supergroups
Below we present phylogenetic relationships among Wolbachia genus supergroups, and discuss the implications of the F supergroup's association with both insects and nematodes. Phylogenies based on single-gene datasets were generally well resolved (Fig. 1a–c). For any given gene, ML and BI analyses gave identical topologies. Multiple BI runs for each dataset resulted in identical trees, and similar posterior probabilities, indicating convergence among runs. The gltA tree clearly distinguishes supergroups A–F (Fig. 1a), in agreement with previous studies (Werren et al., 1995; Bandi et al., 1998; Lo et al., 2002). Supergroups A and B include Wolbachia spp. from arthropods only, while known members of supergroups C and D are restricted to filarial nematodes. Wolbachia spp. from the Collembolan F. candida represent a divergent lineage, named supergroup E by Vandekerckhove et al. (1999). As discussed above, supergroup F comprises representatives of filarial nematodes (Mansonella spp.) and the termite Kalotermes flavicollis. Notably, bacteria from the filarial nematode Dip. gracile and the flea Cte. felis represent two divergent branches. The long branch leading to the Wolbachia spp. of Dip. gracile is the sister lineage to supergroup C. Phylogenies based on groEL (Fig. 1b) and ftsZ (Fig. 1c) also distinguish major supergroups (A–D and F in the groEL tree, and A–F in the ftsZ tree). As in the gltA tree, the groEL and ftsZ phylogenies indicate that Wolbachia spp. from Dip. gracile and Cte. felis are two separate branches that are quite divergent from known subgroups, and termites are found to group with Mansonella spp.

Because there is only one representative in each of the two novel lineages, there are insufficient data to determine whether Wolbachia spp. of Dip. gracile and Cte. felis represent new supergroups. Further sampling and gene sequencing within taxonomic groups related to these two organisms (e.g. Kikuchi & Fukatsu, 2003; Czarnetzki & Tebbe, 2004) may help to decide the status of these lineages. In addition to Cte. felis, numerous other flea species also host Wolbachia spp. However, based on a relatively short fragment of 16S rDNA (Gorham et al., 2003), these Wolbachia spp. do not form a single cluster signifying a coherent supergroup of flea associates. The results found by Gorham et al. (2003) require further investigations involving other gene sequences to determine their supergroup status.

The results obtained using the concatenated dataset are consistent with those based on single-gene analyses, with the Wolbachia spp. from Dip. gracile showing a distant but well-supported grouping with members of supergroup C (Fig. 2). In addition, most datasets support the position of Wolbachia spp. from Cte. felis just outside the Dip. gracile + supergroup C clade, within a major clade also encompassing the F and D supergroups. The exception is the groEL phylogeny, in which Cte. felis apparently falls just outside the F subgroup, rather than the D. gracile + supergroup C clade. It is possible this discrepancy is an artefact of excluding F. candida from the groEL analysis. In sum, with respect to the relationships among the main lineages, the phylogeny of the unrooted tree of the concatenated dataset was found to be identical to that of gltA and ftsZ, and, with the exception noted above, to that of groEL phylogeny.

The phylogeny inferred from the concatenated dataset was rooted with two outgroup species (A. marginale and E. ruminantium) provides no clear insight into the root of the global tree of diverse Wolbachia pipientis strains (see Supplementary Fig. S1 with the online version of this paper). While the ‘best’ rooted tree under ML and BI criteria suggests that supergroup B is the earliest-branching lineage within the genus Wolbachia, support for this hypothesis is very weak (e.g. 54 % bootstrap value). Using a Shimodaira–Hasegawa test (Shimodaira & Hasegawa, 1999), we compared the relative support for 18 topologies in which the root of the tree (leading to the two outgroups) was placed as a sister group to each of the different supergroups, as well as to Wolbachia spp. from Dip. gracile and Cte. felis (see Lo et al., 2002). These phylogenies were statically indistinguishable, with likelihood values ranging from –14621·82 to –14627·88, and P values ranging from 0·916 to 0·148. That is, in no case was one root placement significantly more likely than any other. The rooting of the Wolbachia pipientis tree remains problematic. Since outgroups are extremely divergent from Wolbachia, they have not been useful in resolving the basal relationships among supergroups (for details see Lo et al., 2002).

As stated above, supergroup F is of particular interest regarding the macroevolution of Wolbachia. This clustering of Wolbachia spp. from arthropod and nematode hosts suggests that an independent horizontal transfer of the bacterium between these host phyla might have occurred much more recently than ~100 million years ago, a proposed date for the first transfer based on the number of substitutions between supergroups A versus D (Bandi et al., 1998). In the absence of a stable root for the overall tree of the Wolbachia genus, the original host group and interaction type (mutualistic or parasitic) of Wolbachia remains uncertain, as does the direction of host switching between phyla.


   ACKNOWLEDGEMENTS
 
We thank Odile Bain and Coralie Martin (Muséum National d'Histoire Naturelle France) for samples of Dip. gracile, Litomosoides hamletti and Litomosoides brasiliensis, Shigeniko Uni (Osaka City University Medical School) for Mansonella sp., Vittorio Parisi (Università degli Studi di Parma) for F. candida, and John W. McCall (The University of Georgia) for Cte. felis. We are most grateful to C. Bazzocchi for technical assistance. We thank P. H. Degnan for superb technical assistance, including DNA sequencing. This work was partially supported by grants to C. B. (MIUR-FIRB RBAU01TA3W), J. J. W. from the National Institutes of Health (R01 GM62626-01) and the NASA Astrobiology Institute (NNA04CC04A), and J. H. W. and J. J. W. from the National Science Foundation (EF-0328363). This work was performed while S. R. B. held a National Research Council Research Associateship Award at the Marine Biological Laboratory. N. L. was supported by the Australian Research Council.


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RESULTS AND DISCUSSION
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Received 30 June 2005; revised 12 September 2005; accepted 12 September 2005.



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