Molecular Ecology (2013) 22, 6149–6162
doi: 10.1111/mec.12549
Tracing horizontal Wolbachia movements among bees (Anthophila): a combined approach using multilocus sequence typing data and host phylogeny € T H E and C H R I S T O P H B L E I D O R N MICHAEL GERTH, JULIANE RO Molecular Evolution and Systematics of Animals, Institute for Biology, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany
Abstract The endosymbiotic bacterium Wolbachia enhances its spread via vertical transmission by generating reproductive effects in its hosts, most notably cytoplasmic incompatibility (CI). Additionally, frequent interspecific horizontal transfer is evident from a lack of phylogenetic congruence between Wolbachia and its hosts. The mechanisms of this lateral transfer are largely unclear. To identify potential pathways of Wolbachia movements, we performed multilocus sequence typing of Wolbachia strains from bees (Anthophila). Using a host phylogeny and ecological data, we tested various models of horizontal endosymbiont transmission. In general, Wolbachia strains seem to be randomly distributed among bee hosts. Kleptoparasite-host associations among bees as well as other ecological links could not be supported as sole basis for the spread of Wolbachia. However, cophylogenetic analyses and divergence time estimations suggest that Wolbachia may persist within a host lineage over considerable timescales and that strictly vertical transmission and subsequent random loss of infections across lineages may have had a greater impact on Wolbachia strain distribution than previously estimated. Although general conclusions about Wolbachia movements among arthropod hosts cannot be made, we present a framework by which precise assumptions about shared evolutionary histories of Wolbachia and a host taxon can be modelled and tested. Keywords: bees (Anthophila), codivergence, horizontal transfer, multilocus sequence typing, Wolbachia Received 18 March 2013; revision received 25 September 2013; accepted 27 September 2013
Introduction Wolbachia are a group of maternally inherited intracellular alphaproteobacteria that are found in many arthropods and some nematode taxa. Infection frequencies in terrestrial arthropods have recently been estimated to be around 40% (Zug & Hammerstein 2012), and Wolbachia may thus be the most common endosymbiont on earth. A large variety of Wolbachia strains is known, and the classification on a higher level (‘supergroups’) is mainly based on sequence variation in the 16S rRNA gene (Ros et al. 2009) with new supergroups regularly being detected (Augustinos et al. 2011). To differentiate Correspondence: Michael Gerth, Fax: +493419736789; E-mail:
[email protected] © 2013 John Wiley & Sons Ltd
among closely related strains (e.g. within supergroups), a multilocus sequence typing system (MLST, Maiden et al. 1998) similar to that of other microorganisms was established (Baldo et al. 2006). Wolbachia has repeatedly been reported to induce diverse effects in its hosts, such as sex-ratio-distortion via various mechanisms (Werren et al. 1981; Rigaud et al. 1999), cytoplasmic incompatibility resulting in enhanced Wolbachia spread in populations (CI, Werren 1997; Stouthamer et al. 1999), interference with mitochondrial inheritance patterns (Whitworth et al. 2007; Gompert et al. 2008; Raychoudhury et al. 2010; Dyer et al. 2011), nutritional mutualism (Hosokawa et al. 2010), conferring resistance to pathogens (Hedges et al. 2008; Teixeira et al. 2008; Walker et al. 2011) and others (reviewed in Werren et al. 2008). Most of those
€ T H E and C . B L E I D O R N 6150 M . G E R T H , J . R O phenomena have evolved as a consequence of vertical transmission of Wolbachia through the maternal line over evolutionary timescales. However, theory predicts that infections are lost eventually (Koehncke et al. 2009), and evidence exists that Wolbachia strains are transmitted horizontally between hosts on a frequent basis. Specifically, the lack of phylogenetic congruence between Wolbachia strains and arthropod hosts (O’Neill et al. 1992; Schilthuizen & Stouthamer 1997; Vavre et al. 1999; Baldo et al. 2008; Raychoudhury et al. 2009) has led to this conclusion. Nevertheless, the underlying mechanisms are only understood from a few model systems dealing with parasitoid wasps (Heath et al. 1999; Huigens et al. 2000, 2004) and no common patterns describing Wolbachia’s distribution could be identified. In general, Wolbachia strains seem to be specialized at least on higher phylogenetic levels; for example, lepidopterans usually carry supergroup B Wolbachia, whereas within hymenopterans and dipterans, supergroup A strains are more common (Russell et al. 2009; Stahlhut et al. 2010). Furthermore, geographical specialization on a large scale (old vs. new world) has been demonstrated (Russell et al. 2009). Still, in most Wolbachia–host systems, transmission routes shaping the endosymbiont distribution patterns remain unclear. In the present study, we aimed to investigate the evolutionary history of Wolbachia strains infecting bees (Anthophila). Bees are a group of aculeate Hymenoptera with about 17 500 described species (Michener 2007). As important pollinators, they contribute significantly not only to ecosystem functioning, but also to human food supply (Greenleaf & Kremen 2006; Garibaldi et al. 2013). Many aspects of bee biology are comparatively well studied (Westrich 1989; Michener 2007) and especially phenomena such as the evolution of eusociality and kleptoparasitism have received much attention (Schwarz et al. 2007; Cardinal et al. 2010). For these reasons, and because Wolbachia is widespread among bees (Gerth et al. 2011), they present an ideal study system to investigate Wolbachia transmission among hosts. We sampled Wolbachia-infected bees over a small geographical scale to identify major routes of Wolbachia movements. Particularly, we were interested whether Wolbachia may be transmitted from bees to their kleptoparasites (brood parasites, cuckoos). These bees have lost the ability to build nests and collect provisions for their brood but rather lay their eggs into the nest of a host bee. The cuckoo larva ‘steals’ the provisions provided by the host bee, whereas the host larva is killed by either the kleptoparasite or its larva. In many cases, kleptoparasites are specialized on a single host species only (Westrich 1989). Given that provisions provided by bees contain secretions of salivary glands (Westrich 1989), these glands may contain Wolbachia and Wolbachia
cells may survive extracellular phases (Rasgon et al. 2006), the requirements for a transmission from host bees to their kleptoparasites via the pollen are fulfilled. We classified Wolbachia strains from bee hosts by MLST and specifically tested for the scenario that Wolbachia is horizontally transmitted from bees to their kleptoparasites. Furthermore, by including a bee phylogeny into our analyses, we tested the two prevailing (not mutually exclusive) assumptions of how Wolbachia are primarily transmitted: between closely related hosts (Werren et al. 2008; Zug et al. 2012) or between ecologically linked hosts (Sintupachee et al. 2006; Stahlhut et al. 2010). Finally, we tested whether codivergence between bees and corresponding Wolbachia strains has occurred and whether it contributes significantly to strain distribution and diversity among bees.
Materials and methods Insect collection and DNA extraction Bees were collected between 2009 and 2012 from various locations in Germany (Table S1, Supporting information) with hand nets directly from their nest entrances or pollen sources. All specimens were killed, pinned with insect needles, prepared and dried before being identified to species level. For DNA extraction, one or two individuals per species were used. Due to the fact that Wolbachia usually infects ‘most-or-few’ individuals within a species (Hilgenboecker et al. 2008), sampling one or two host individuals allows us to draw general conclusions about Wolbachia strain distributions in bees. However, in species with low infection frequencies, this strategy may miss the presence of Wolbachia infections. Our sampling focused on kleptoparasites and the corresponding hosts, information on these associations was taken from Westrich (1989). The final data set included 23 kleptoparasite-host associations which are not mutually exclusive, that is, some kleptoparasites are linked to more than one host and vice versa (Table 1). DNA was isolated from flight muscle tissue or, for smaller individuals, from the whole thorax. DNA extraction was performed either by digestion with proteinase K and subsequent chloroform extraction or with NucleoSpin© Tissue Kit (Macherey-Nagel), following the manufacturers protocol. The remains of the animals were stored in 96% ethanol as vouchers.
Analysed gene fragments, PCR conditions and sequencing As a control for the success of the DNA extractions, a fragment of the single copy nuclear gene long wavelength © 2013 John Wiley & Sons Ltd
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6151 Table 1 Bee hosts and their respective included in the data set of this study
kleptoparasites
Host
Kleptoparasite
Andrena barbilabris (Kirby 1802)
Nomada alboguttata Herrich-Sch€affer 1839 Sphecodes pellucidus Smith 1845 Nomada fabriciana (Linnaeus 1767) N. fabriciana (Linnaeus 1767)
Andrena bicolor Fabricius 1775 Andrena chrysosceles (Kirby 1802) Andrena clarkella (Kirby 1802) Andrena fulva (M€ uller 1766) Andrena fuscipes (Kirby 1802) Andrena nigroaenea (Kirby 1802) Andrena nitida (M€ uller 1776) Andrena praecox (Scopoli 1763) Andrena proxima (Kirby 1802) Andrena ruficrus Nylander 1848 Andrena tibialis (Kirby 1802) Andrena ventralis Imhoff 1832 Colletes cunicularius (Linnaeus 1761) Colletes nasutus Smith 1853 Eucera nigrescens Perez 1879 Halictus quadricinctus (Fabricius 1776) Lasioglossum albipes (Fabricius 1781) Lasioglossum calceatum (Scopoli 1763) Lasioglossum malachurum (Kirby 1802) Lasioglossum pauxillum (Schenck 1853) Macropis europaea Warncke 1973 Macropis fulvipes (Fabricius 1804)
Nomada leucophthalma (Kirby 1802) Nomada signata Jurine 1807 Nomada rufipes Fabricius 1793 Nomada flava Panzer 1798 Nomada goodeniana (Kirby 1802) Nomada succincta Panzer 1798 N. flava Panzer 1798 N. goodeniana (Kirby 1802) N. succincta Panzer 1798 Nomada ferruginata (Linnaeus 1767) Nomada conjugens Herrich–Sch€affer 1839 Nomada obscura Zetterstedt 1838 Nomada fulvicornis Fabricius 1793 N. alboguttata Herrich–Sch€affer 1839 Sphecodes albilabris (Fabricius 1793) Epeolus schummeli Schilling 1849 Nomada sexfasciata Panzer 1799 Sphecodes gibbus (Linnaeus 1758) Sphecodes monilicornis (Kirby 1802) S. monilicornis (Kirby 1802) S. monilicornis (Kirby 1802)
Sequence editing, alignment and phylogenetic analyses Sphecodes crassus Thomson 1870 Epeoloides coecutiens (Fabricius 1775) E. coecutiens (Fabricius 1775)
rhodopsin (LW rhodopsin) was amplified with the primers OpsFor and OpsRev from Danforth et al. (2004), using the PCR conditions described therein. All samples were screened for the presence of Wolbachia DNA by performing a PCR on the ftsZ gene, utilizing primers and methodology as described in Baldo et al. (2006). Wolbachia strains from bee hosts were subject to MLST, that is, the conserved bacterial housekeeping © 2013 John Wiley & Sons Ltd
genes gatB, coxA, hcpA, ftsZ and fbpA were amplified and sequenced, which is a standard method for Wolbachia strain discrimination (Baldo et al. 2006). For cophylogenetic analysis of Wolbachia and five closely related kleptoparasitic Nomada species sharing almost identical Wolbachia strains, a part of the faster evolving genes, Wolbachia surface protein gene (wsp) and its paralog wspB, was sequenced as an additional source of discrimination. We used the primers from Baldo et al. (2006) and Wu et al. (2004) to amplify wsp and wspB, respectively. Altogether, 58 unique combinations of bee hosts and Wolbachia strains were included in subsequent analyses. To obtain a phylogeny of the bees used in this study, we used LW rhodopsin. The addressed fragment of this gene is conserved enough to resolve family-level relationships among bees but also contains highly variable intronic positions (Danforth et al. 2004). We chose not to include mitochondrial genes in our analyses because the presence of endosymbionts such as Wolbachia may hamper the interpretation of mitochondrial phylogenies for a variety of reasons (Hurst & Jiggins 2005). All PCRs were carried out in a total volume of 20 lL, containing 1 lL of each primer (10 pM), 0.5 lL of each dNTP (2 mM), 2.5 lL of 109 DreamTaqTM Green Buffer (Fermentas), 0.5 Units of DreamTaqTM Green DNA Polymerase (Fermentas), 1 lL of genomic DNA and 12.4 lL of ddH2O. Negative controls containing water instead of template DNA were included in all PCRs. Sanger sequencing in both forward and reverse directions for all fragments was performed by GATC Biotech AG (Germany). All sequences were submitted to GenBank under Accession nos KC798065–KC798382 and KC812731 (Table S1, Supporting information). New MLST alleles were submitted to Wolbachia PubMLST Database (http://pubmlst.org/wolbachia).
All sequences were viewed in BIOEDIT 7.0.5.3 (Hall 1999) or CLC MAIN WORKBENCH 6.7 (CLC bio) and aligned with the online tool MAFFT 6, applying the L-INS-i algorithm (Katoh et al. 2002). We used template alignments from the Wolbachia MLST website (Jolley et al. 2004, http://pubmlst.org/wolbachia/) as a profile to align the five MLST genes. Subsequent analyses of Wolbachia MLST genes were performed on a small data set containing only Wolbachia strains from bee hosts plus two supergroup B strains (ST-20 and ST-21) as outgroups and one large data set containing all available Wolbachia strains from PubMLST as well as Wolbachia strains from bee hosts. Analyses of the large data set containing altogether 548 strains were conducted to identify the Wolbachia strains from bee hosts that are
€ T H E and C . B L E I D O R N 6152 M . G E R T H , J . R O unique and those that are related to strains from other hosts. A phylogeny of Wolbachia strains was inferred with CLONALFRAME (Didelot & Falush 2007) from both data sets. CLONALFRAME uses information of substitution as well as recombination events and is therefore suitable to reconstruct bacterial evolution based on multilocus data (Didelot & Falush 2007). We performed 10 independent runs, each with 250 000 MCMC iterations as burnin period and 750 000 as sampling period and a sampling frequency of 100. We chose the two runs with the best mean log likelihoods and compared these for convergence of the chains using the implemented methods of Gelman & Rubin (1992). The trees of the posterior samples of the converged runs were then combined to compute a majority rule consensus tree. A maximum-likelihood tree was calculated with the Pthread version of RAXML 7.6.3 (Stamatakis 2006) under the GTR + G + I model. Clade robustness was estimated with 1000 bootstrap replicates (Felsenstein 1985). Additionally, we estimated relationships among Wolbachia strains with a Bayesian approach using MRBAYES 3.1.2 (Ronquist & Huelsenbeck 2003), again choosing the GTR + G + I model. Two runs with four chains were each run for 1 million MCMCMC steps (Altekar 2004), sampling every 500th generation. The first 250 000 generations were discarded as burnin after a convergence of chains was diagnosed by a deviation of split frequencies below 5%. Posterior probabilities were inferred from clade frequencies of the majority rule consensus tree constructed from the remaining trees. Some intron positions of the LW rhodopsin data set could not be aligned unambiguously. Consequently, we conducted an alignment masking with the server version of GBLOCKS 0.91b (Castresana 2000), resulting in a reduced alignment of 603 bp length. As outgroup taxa, we used the three crabronid wasps Philanthus triangulum, Crabro scutellatus and Astata nr. bakeri (GenBank Accession nos: AY995812, AY995811 and JN374890, respectively), as Crabronidae are most likely the closest living relatives of bees (Lohrmann et al. 2008; Debevec et al. 2012). We partitioned the data set into intron and exon positions, as an analysis with PARTITIONFINDER 1.1.1 (Lanfear et al. 2012), and RAXML revealed this to be favourable over using a single partition. For both partitions, a GTR + G + I model was selected via PARTITIONFINDER analysis. A phylogeny of bees was inferred with RAXML and MRBAYES as described above. We used a constrained topology for those analyses which was designed to fit the splits between Nomada flava, Nomada leucophthalma, Nomada panzeri, Nomada ferruginata and Nomada signata as inferred from multi gene analysis (Fig. 3, see below).
Test for directionality in Wolbachia movements among bees To examine whether Wolbachia strains move horizontally between kleptoparasites and their hosts more frequently than expected by chance, we employed BayesMultiState as implemented in BAYESTRAITS, a method to reconstruct models of trait evolution (Pagel et al. 2004). To account for phylogenetic uncertainties, we used a set of 100 best trees (those with highest likelihoods) of Wolbachia strains from bee hosts taken from the posterior sample of the Bayesian analysis. Each Wolbachia host was coded as a different trait. Transitions between traits can thus be interpreted as horizontal movements of Wolbachia strains between hosts. A model with one uniform transition rate (i.e. random Wolbachia transfers) between all traits was assumed as null hypothesis. Against this null hypothesis, we tested a model with two fixed transition rates: one between strains of kleptoparasites and their corresponding hosts and one between all other pairs. If Wolbachia strains are predominantly transmitted horizontally between bees and their kleptoparasites, one would expect that these two fixed rates explain the distribution of hosts on the Wolbachia tree significantly better than a uniform rate between all hosts. In likewise manner, we set up other models implementing two or three rates, testing for the influence of ecological traits (nesting substrate, lifestyle) as well as for the influence of phylogenetic relation on the trait distribution (Table 2). BayesMultiState was run in MCMC mode for 10 million generations, discarding the first 1 000 000 as burnin. The acceptance rate for newly proposed rate parameters was set to be between 20% and 40% by choosing an appropriate value for the ‘ratedev’ parameter. Two independent runs were performed for each model; the resulting harmonic mean log likelihoods were averaged, compared and ranked with Akaike information criterion (AIC, Akaike 1974), after stability of parameters in the post-burnin period was ascertained with TRACER 1.4 (Rambaut & Drummond 2007).
Detection of vertical Wolbachia transfer over evolutionary timescales If Wolbachia strains are inherited over evolutionary timescales in bees (and at the same time, horizontal transfers are absent), this vertical transfer should be traceable by mapping Wolbachia infections as traits onto the host tree and testing if these traits predict host phylogeny, that is, testing for phylogenetic signal. We used our maximum-likelihood phylogeny of the bees, excluded duplicate taxa and created a corresponding matrix with Wolbachia strain groups coded as traits. © 2013 John Wiley & Sons Ltd
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6153 Table 2 Models employed in BayesMultiState analyses and results ranked according to their Akaike information criterion (AIC) values
No.
Number of fixed transition rates
Description of model
AIC value
1 2 3 4 5 6 7
1 2 2 2 2 2 3
All rates equal Equal rates between Equal rates between Equal rates between Equal rates between Equal rates between Equal rates between
476.94 479.96 480.24 480.25 480.27 481.75 488.03
strains infecting Nomada species strains infecting Andrena species all kleptoparasitic species strains of hosts and their corresponding kleptoparasites all ground nesting species all kleptoparasitic species and equal rates between all ground nesting species
Wolbachia strain groups were defined with the software EBURST 3 (Feil et al. 2004) as strains sharing three or more MLST alleles with at least one other member of the strain group. The resulting matrix was composed of 12 different traits: five groups and seven singletons (i.e. groups comprising only one strain, Fig. 2). To test for phylogenetic signal in the traits with respect to the host tree, we used the function ‘phylosignal’ as implemented in the R package picante (Kembel et al. 2010; R Development Core Team 2012). This test uses K statistics to compare observed signals in a trait to the signal under a Brownian motion (neutral) model of trait evolution (Blomberg et al. 2003). We performed the test with 1 million replicates for the entire host tree and separately for pruned trees comprising only Andrena, Nomada and Halictidae, respectively (Fig. 2). Moreover, we tested for phylogenetic signal using Pagel’s lambda (Pagel 1999) as implemented in the function ‘FitDiscrete’ of the R package geiger (Harmon et al. 2008). Three models were calculated as follows: one with a fitted lambda value and one each for lambda = 0 (which can be interpreted as random distribution of traits on the phylogeny) and lambda = 1 (perfect fit of traits). The log likelihoods of the models were then ranked using the AIC.
Estimation of divergence times and cophylogenetic analysis To estimate the age of Wolbachia–host associations within Nomada species, we aimed to date the splits of five closely related Nomada species (N. flava, N. leucophthalma, N. ferruginata, N. panzeri, N. signata), all of which carry very similar Wolbachia strains. We based our molecular clock analysis on a recently published data set on apid bee phylogeny including 160 ingroup (Apidae) and 30 outgroup (Megachilidae) taxa (Cardinal et al. 2010). Consistent with the methodology of this study, we amplified and sequenced six nuclear gene fragments (in addition to LW rhodopsin) for these taxa: the ribosomal genes 18S and 28S, elongation factor © 2013 John Wiley & Sons Ltd
1-alpha (EF-1a), sodium potassium adenosine triphosphatase (Nak), RNA polymerase II (pol II) and wingless; primers were taken from Cardinal et al. (2010). The sequences were submitted to NCBI GenBank under Accession nos KF512682–KF512710. The loci were then aligned separately with MAFFT 6 as described above. Ambiguously aligned positions were excluded from the data set. For all subsequent analyses, the concatenated data set was partitioned into four partitions: (1) nuclear ribosomal genes (2–4) single codon positions of nuclear genes, thereby following the argumentation of Cardinal et al. (2010). The combined data set of five Nomada species plus the data set of Cardinal et al. (2010; 195 taxa, seven nuclear loci, 6195 bp altogether) was then used to estimate divergence times. We employed a relaxed molecular clock model as implemented in the program BEAST 1.7 (Drummond et al. 2012). All priors were set in accordance to the BEAST analysis performed by Cardinal et al. (2010). In brief, lognormal distributed prior ages for 10 nodes were constrained, information on which was taken from the fossil record. A GTR + G + I substitution model was applied to all partitions, and the Yule tree prior was used, as appropriate for species-level phylogenies. The analysis was run twice for 100 million generations with a random starting tree, a sampling frequency of 2500 and a burnin of 10 million generations. After convergence of runs was diagnosed with TRACER 1.4, post-burnin samples of both runs were combined to compute a maximum clade credibility tree. Details on the reasoning behind fossil calibration points and all other priors are given in Cardinal et al. (2010). Five closely related Wolbachia strains that all infect Nomada hosts were chosen to be tested for codivergence with their hosts. Using JANE 4 (Conow et al. 2010), we mapped the CLONALFRAME phylogeny based on five MLST genes plus wsp and wspB onto a phylogeny of five Nomada species. We reconstructed codivergence patterns with default cost values: cospeciation: 1, duplication: 1, duplication and host switch: 2, loss: 1, failure to diverge: 1. The Nomada phylogeny was based on
€ T H E and C . B L E I D O R N 6154 M . G E R T H , J . R O Fig. 1 Majority rule CLONALFRAME genealogy of Wolbachia strains from bee hosts, based on two independent runs. Labels correspond to host names. Support values represent the percentage of trees from the posterior sample in which each node was present. Bootstrap values from maximum-likelihood analysis based on 1000 pseudoreplicates are also given. Supergroup B strains 20 and 21 were used as outgroups. ≥ 90 / 80 / 70 CLONALFRAME support values Bootstrap values St
St
seven genes (see above) and inferred with RAXML as well as MRBAYES as described above. Three Epeolus outgroup taxa were taken from Cardinal et al. (2010).
Results Phylogenetic relationships among Wolbachia strains from bee hosts Altogether, of 75 host individuals (62 species), we amplified and analysed MLST genes from 58 unique Wolbachia–host combinations, all classified as supergroup A strains. Individuals carrying multiple infections were not detected. In some cases, amplification or sequencing of loci failed (Table S1, Supporting information). CLONALFRAME and maximum-likelihood analyses as well as Bayesian analysis yielded mostly congruent topologies (Figs 1 and S1, Supporting information), indicating that the role of recombination might be negligible. Also, the ratio of nucleotide changes resulting from recombination compared with those resulting from point mutation (r/m value) on average as calculated with CLONALFRAME was 1.33 (0.85–1.94, 95% credibility region), which is considerably lower than a standard value for Wolbachia MLST data sets (3.5, Vos & Didelot 2009). Some strongly supported clades of this
analysis were also recovered in the CLONALFRAME analysis of the large data set including all currently available MLST profiles (Fig. S2, Supporting information). However, Wolbachia strains from bee hosts do not form a monophyletic clade and many strains cluster with strains from Lepidoptera, Diptera and other hosts. Still, the proportion of new and thus potentially bee-specific alleles is large: we found eight of 14, six of 12, eight of 15, six of 10 and two of nine new alleles for gatB, coxA, hcpA, ftsZ and fbpA, respectively (Fig. 2).
Wolbachia movements among bee hosts By setting up different models of trait transition (i.e. host transition of Wolbachia) in the BayesMultiState analyses, we tested whether any directionality in Wolbachia movements is traceable. We found no evidence that Wolbachia moves horizontally between species of the genus Nomada or Andrena more frequently than expected by chance. Also, kleptoparasitic lifestyle or ground nesting does not seem to shape Wolbachia’s distribution patterns. Furthermore, a model assuming equal rates of transmission between kleptoparasites and their corresponding hosts was not supported with statistical significance (Table 2). In summary, all of the models tested performed similar and none of the © 2013 John Wiley & Sons Ltd
fbpA
N4 32 32 32 32 33 33 32 N6 N3 N3 32 32 32 32 N6 N6 32 33 33 33 33 32 32 33 32 32 6 6 32 N5 15 32 N2 N1 N1 23 N3 32 N3 37 23 23 23 33 N3 33
N2 103 103 103 103 42 42 103 N5 60 60 103 103 N6 N7 N6 N6 103 42 42 42 42 103 103 42 103 103 7 7 103 N3 N4 103 X N8 N8 24 N1 108 X 41 24 24 24 42 N1 42
72 3 3 3 3 32 154 3 N3 N5 N5 3 3 N4 N4 N4 N4 X 154 154 154 154 3 3 X 3 3 3 3 3 N1 N2 3 3 3 3 3 3 3 N1 3 3 3 3 154 3 154
44 N1 N1 N1 N1 N1 122 N1 15 120 120 N1 N1 15 15 15 15 N1 122 122 122 122 N1 N1 X N1 N1 120 120 X N2 17 N1 120 147 147 23 120 N1 120 120 23 23 120 122 120 122
Nomada
ftsZ
32 87 87 87 87 32 32 87 N3 1 1 87 87 N3 N3 N3 N3 87 N8 N8 32 32 87 87 32 87 87 X X 87 N6 N7 87 N2 N4 N4 22 1 N1 X N5 22 22 22 32 1 32
Halictidae
hcpA
7 5 5 5 5 2 2 5 1 4 4 5 5 1 1 1 1 5 2 2 2 2 5 5 2 5 5 11 11 5 8 6 5 12 10 10 3 4 5 4 9 3 3 3 2 4 2
Andrena
Group
coxA
Anthophora plumipes Epeoloides coecutiens Epeoloides coecutiens Epeolus schummeli Nomada rufipes Nomada succincta Nomada goodeniana Nomada fabriciana Nomada conjugens Nomada alboguttata Nomada alboguttata Nomada fulvicornis Nomada signata Nomada ferruginata Nomada panzeri Nomada flava Nomada leucophtalma Melitta haemorrhoidalis Macropis fulvipes Macropis fulvipes Macropis europaea Macropis europaea Sphecodes pellucidus Sphecodes albilabris Sphecodes rufiventris Specodes gibbus Halictus quadricinctus Lasioglossum pauxillum Lasioglossum pauxillum Lasioglossum calceatum Colletes cunicularius Colletes daviesanus Colletes nasutus Andrena fuscipes Andrena fulva Andrena fulva Andrena praecox Andrena ventralis Andrena barbilabris Andrena proxima Andrena minutula Andrena dorsata Andrena dorsata Andrena bicolor Andrena gravida Andrena nigroaenea Andrena nitida
gatB
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6155
Fig. 2 Maximum-likelihood phylogeny of bees based on LW rhodopsin. Tree was pruned to include only taxa for which information on Wolbachia strains is available. For each Wolbachia strain, the allele numbers for MLST genes were assigned from Wolbachia PubMLST database. New alleles are marked with N and numbered consecutively. For alleles marked with X, amplification or sequencing was not successful. Group numbers correspond to Wolbachia strain groups as defined by eBURST. For the taxa shaded in grey, separate analyses for the detection of phylogenetic signal were conducted. MLST, multilocus sequence typing.
models performed better than the model with equal (random) transition rates (assuming DAIC ≤ 2 indicates substantial support, Burnham & Anderson 2002). To assess how host phylogeny predicts Wolbachia distribution, we obtained a robust phylogeny of bees (Fig. S3, Supporting information). Family and genus level relationships are consistent with recent estimates of bee phylogeny based on multilocus data (Danforth et al. 2013). Although some nodes are not strongly supported, © 2013 John Wiley & Sons Ltd
Bayesian inference and maximum-likelihood analyses yielded mostly congruent topologies, with five nodes of the former not recovered in Bayesian analysis, but rather resolved in polytomies of the majority rule consensus of post-burnin trees (Fig. S3, Supporting information). For subsequent analyses, a fully resolved tree was required and we therefore used the maximum-likelihood tree. The tree was pruned to exclude taxa not carrying Wolbachia (Fig. 2). Coding Wolbachia strain
€ T H E and C . B L E I D O R N 6156 M . G E R T H , J . R O Table 3 Results of tests for phylogenetic signal within Wolbachia strain groups using the phylosignal function Phylogeny used in analysis
p-value
Complete host tree Nomada phylogeny Andrena phylogeny Halictidae phylogeny
0.055 0.262 0.187 0.572
Table 4 Akaike information criterion values for models of trait (i.e. Wolbachia strain group) distribution on host phylogeny Model specifications
groups as traits (Fig. 2), we tested for phylogenetic signal within those traits. Whereas K statistics did not reveal phylogenetic signal in any of the analyses (Table 3), we were able to identify some support for phylogenetic signal within the Nomada clade using Pagel’s lambda (Table 4). Only within the genus, Nomada did the model specified with k = 1 perform best, suggesting that the Nomada phylogeny explains the distribution of Wolbachia strain groups better than a random arrangement (DAIC 2, Table 4).
Phylogeny used in analysis
k=1
k=0
Fitted k
Complete host tree Nomada phylogeny Andrena phylogeny Halictidae phylogeny
176.43 30.73 48.70 15.93
178.49 34.22 48.41 13.15
170.12 32.73 50.70 15.15
with its Nomada hosts. The phylogeny of the five Nomada species based on seven genes was strongly supported in maximum-likelihood and Bayesian analysis (Fig. 3). Although there is not a perfect match of Wolbachia and Nomada phylogenies, some degree of codivergence could be detected. The hypothetical coevolutionary scenario includes two independent horizontal Wolbachia transfers (both to other Nomada species) and two cases of codivergence (Fig. 3). This scenario suggests that a Wolbachia infection was present in the last common ancestor of Nomada leucophthalma, N. flava and N. panzeri and that this infection was vertically transmitted over considerable timescales.
Cophylogenetic analyses and age estimation To trace the evolutionary history of Wolbachia strain group 1 (Fig. 2), we performed a test for codivergence
wNCon Nomada conjugens
Cospeciation Duplication & host switch
wNLeu Nomada leucophthalma 59
56 76 1
wNFla Nomada flava
100 71 0.99 100
wNPan Nomada panzeri
76 0.79
wNFer Nomada ferruginata
95 1
Nomada signata
7.25
2
1.7
1
Mya
0
Fig. 3 Maximum clade credibility tree (in black) as estimated from seven nuclear genes with a relaxed molecular clock model, pruned to include only taxa investigated in this study. Outgroups are not shown. Numbers on nodes correspond to bootstrap values from 1000 pseudoreplicates in maximum-likelihood analysis (above) and to Bayesian posterior probabilities (below). Wolbachia CLONALFRAME genealogy (in grey, not drawn to scale) based on five MLST genes plus wsp and wspB was mapped onto the host phylogeny with JANE 4. Numbers correspond to CLONALFRAME support values as percentages. The hypothetical coevolutionary scenario includes two incidences of cocladogenesis and two horizontal transfers, one of which to a distantly related host (N. conjugens). The thick grey line indicates the last common ancestor of Nomada leucophthalma, Nomada flava and Nomada panzeri and thus the age of the Wolbachia infection in this lineage to be 1.7 million years old (0.86–2.61, 95% HPD). HPD, highest posterior density; MLST, multilocus sequence typing. © 2013 John Wiley & Sons Ltd
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6157 Divergence time estimations of a data set including 195 bee taxa based on a dated relaxed molecular clock model yielded consistent results to the corresponding analysis performed by Cardinal et al. (2010, Fig. S4, Supporting information). The age of the last common ancestor of N. leucophthalma, N. flava and N. panzeri, and thus, the age of the Wolbachia infection was estimated to be 1.7 million years old (0.86–2.61, 95% highest posterior density, HPD).
Discussion We studied the evolutionary history of Wolbachia strains within bees, an ecologically diverse group of important pollinators. By employing an MLST approach to classify Wolbachia strains in combination with a robust phylogeny of bee hosts, we were able to specifically test for and potentially identify pathways of Wolbachia transfer. Furthermore, we found evidence for codivergence between bees and their Wolbachia endosymbionts on a small scale.
Wolbachia strains in bees and mechanisms of horizontal transmission Wolbachia strains investigated in this study show specialization at the level of supergroups: we found only supergroup A Wolbachia in the studied bee taxa. This result corroborates previous studies in which hymenopteran hosts were shown to carry mainly supergroup A strains (Stahlhut et al. 2010; Russell 2012). On a smaller scale, patterns are less obvious. Wolbachia strains seem to be scattered randomly among bees (Figs 1 and 2) and also among other hosts (Fig. S1, Supporting information). However, some strains do seem bee specific and the proportion of new MLST alleles found in this study is large (30 of 60 altogether). Given that taxonspecific Wolbachia screenings have often yielded the discovery of new strains (Baldo et al. 2008; Russell et al. 2009), the question arises whether this indicates unique Wolbachia lineages or is merely an artefact due to the potentially huge diversity of Wolbachia strains. Without more exhaustive screens, this question will remain unresolved. However, with model-based testing one may identify patterns of Wolbachia distribution in some taxa. Our BayesMultiState analyses could not identify any of the tested models as a common mechanism of horizontal transmission. The models specified with fixed-transition rates between ecologically linked Wolbachia–hosts and those with fixed rates between related hosts performed similar (Table 2). Notably, the model specified with fixed transition rates between host-kleptoparasite pairs did not perform significantly worse than any of the other models. Transmission from bees © 2013 John Wiley & Sons Ltd
to their kleptoparasites requires passage of Wolbachia from salivary glands to provisions provided for the offspring, enduring an extracellular phase and another passage from gut to ovaries for permanent establishment of the infection. Wolbachia invades salivary glands of bees (Fig. S5, Supporting information) and was further shown to survive extracellular phases (Rasgon et al. 2006). Wolbachia’s active passage through cell membranes was also demonstrated (Frydman et al. 2006), rendering the proposed pathway plausible. Plants act as reservoirs for other endosymbionts (Caspi-Fluger et al. 2011), and inflorescences attract many different arthropod taxa. Flowers might thus act as platforms for the exchange of Wolbachia strains among pollinators. However, transmission via pollen or pollen provisions has not been documented yet and empirical measurement of horizontal transfer events through plant usage might be infeasible. Experimental testing is also lacking for other proposed mechanisms for the interspecific transmission of Wolbachia, such as common food sources (Mitsuhashi et al. 2002; Sintupachee et al. 2006; Stahlhut et al. 2010), predation (Kittayapong et al. 2003), phoresis (Covacin & Barker 2007), ectoparasites (Noda et al. 2001) and social parasites (Dedeine et al. 2005). However, these scenarios require careful interpretation, as some of the underlying studies are based on a single marker or very limited taxon sampling. The role of parasitoids in horizontal Wolbachia transmission has been demonstrated experimentally (Heath et al. 1999; Huigens et al. 2000, 2004) but cannot explain distribution patterns in all host systems. One well-documented mechanism by which Wolbachia spreads between species is hybrid introgression (Charlat et al. 2009; Raychoudhury et al. 2009; Dyer et al. 2011; Xiao et al. 2012). However, because hybridization is restricted to closely related species (Dowling & Secor 1997; Mallet 2005), it may be hard to distinguish from strictly vertical transmission by molecular methods.
The importance of ecological vs. phylogenetic relatedness of hosts for horizontal Wolbachia transmission Irrespective of the underlying mechanisms, it repeatedly was reported that Wolbachia strains move predominantly between closely related hosts (Baldo et al. 2008; Zug et al. 2012). This seems plausible when assuming that genetic distance correlates negatively with physiological similarity, and consequently, Wolbachia’s invasion into related species is facilitated in comparison to species in which costly adaption to novel physiological parameters (e.g. host defence mechanisms) is required. However, transfection experiments were successful in moving and establishing Wolbachia strains between closely (Boyle
€ T H E and C . B L E I D O R N 6158 M . G E R T H , J . R O et al. 1993; Charlat et al. 2002) and more distantly related hosts (Braig et al. 1994) although transfection does not work in all donor-recipient systems (Riegler et al. 2004) and is more likely to succeed when closely related species are involved (Rigaud et al. 2001). In bees, we find no strong evidence for frequent horizontal Wolbachia transmission between closely related hosts. Neither within the genus Nomada nor within Andrena do Wolbachia strains move more frequently than expected by chance, that is, the model with random transitions performed significantly better (Table 2). Although this pattern may be specific for the investigated system and not true for all hosts, we want to stress the importance of physical contact as a prerequisite for horizontal Wolbachia transmission. For physical contact, the involved host species must have sympatric distribution ranges and similar ecological requirements. Many bee species are highly specialized in terms of their pollen source and their nesting substrate; kleptoparasites in turn depend on the presence of their bee hosts, as they are needed for reproduction. As a consequence, ecological speciation via host plant specialization or specialization on hosts for kleptoparasites seems to be common in bees (Mazzucco & Mazzucco 2007). Furthermore, bees show a pronounced phenology, many species emerging only a for a few weeks per year (Westrich 1989). Ecological constraints such as the ones described are likely to exist for other arthropod species as well. Therefore, in some systems, ecological rather than genetic relatedness of hosts may be a major determining factor for Wolbachia strain distribution (Engelst€adter & Hurst 2006).
Codivergence of Wolbachia strains and bee hosts We found evidence for one Wolbachia strain having codiverged with its Nomada hosts (Table 3, Fig. 3). The strain (strain group 1, Fig. 2) that infects closely related Nomada species is only present within these species and Melecta luctuosa (Fig. 1). When analysing all available MLST profiles, this strain still forms a monophyletic clade (Fig. S1, Supporting information). Consequently, it is more parsimonious to expect the Wolbachia strain to have been present in an ancestral Nomada lineage instead of it having moved independently there various times from a host not identified so far. We also think an artefact due to incomplete sampling (see above) is unlikely due to the close relationship of the host species (Fig. S2, Supporting information). As no transmission experiments of Wolbachia were conducted for bees yet, the costs and frequency of horizontal transmissions of Wolbachia are hard to evaluate. However, for the reasons given above, the horizontal transmissions reported here for a clade of closely related Nomada species are likely to
be results of ancient hybridizations events. Recent hybridization or physical contact is relatively unlikely as the species involved are specialized on different hosts which differ in habitat requirements and phenology (Westrich 1989). Several studies have reported that codivergence contributes little to overall diversity of Wolbachia strains infecting arthropods (Baldo et al. 2008; Stahlhut et al. 2010; Zug et al. 2012). Nevertheless, due to frequent random loss and horizontal transfers, the effects of vertical transmission over large timescales may be blurred and difficult to trace. The evaluation of the role of vertical transfer therefore requires complete host taxon sampling as well as a robust host phylogeny. Our analyses suggest that Wolbachia has been present within the investigated Nomada lineage for 1.7 million years (0.86–2.61, 95% HPD, Fig. 3). This estimate based on a large data set with a relaxed molecular clock model can be considered as robust. Interestingly, even when taking a potential uncertainty into account, this is one of the oldest Wolbachia infections reported so far. In other Wolbachia–host systems, infection ages have been estimated to be under 21 000 years (Duplouy et al. 2010; Atyame et al. 2011), 15 000 to ≥750 000 years (Jaenike & Dyer 2008) and 410 000 to 500 000 years (Raychoudhury et al. 2009). Nomada species may thus be unique Wolbachia hosts evolving relatively stable relationships with their endosymbionts. In some systems, long-term stable host–Wolbachia relationships have evolved towards mutalism (Hosokawa et al. 2010; Taylor et al. 2013). In the light of recent data, we are not able to test if this is also true for Nomada species. On the other hand, there may also be a bias in existing studies as recent Wolbachia-induced mitochondrial sweeps resulting in homogeneous haplotypes are easier to spot as older Wolbachia invasions. Estimations of the age of Wolbachia infections were performed largely in host systems in which Wolbachia was linked to a mitochondrial sweep (Jaenike & Dyer 2008; Duplouy et al. 2010; Atyame et al. 2011). It is therefore unclear whether Wolbachia can persist within bees for an exceptionally long time or the mean age of Wolbachia infections is generally older than estimated from these relatively recent sweeps.
Conclusions Combining MLST and phylogenetic methods, we show how precise assumptions about Wolbachia movements among a group of hosts can be tested. We present an approach that is readily transferrable to other Wolbachia–host groups for which ecological and phylogenetic data are available. So far, knowledge of Wolbachia’s evolutionary history is fragmentary and limited to few host taxa. This may partly be due to the fact that MLST methods have only limited resolution, especially when © 2013 John Wiley & Sons Ltd
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6159 closely related strains are involved. Strain typing using complete genomes is a promising approach to reconstruct recent evolutionary histories of Wolbachia strains. As next-generation sequencing technologies are becoming affordable standard techniques in molecular ecology (Tautz et al. 2010; Ekblom & Galindo 2011), the sequencing of bacterial genomes to address ecological questions is feasible. Already, Wolbachia genomes are being sequenced as by-products of arthropod sequencing projects (Richardson et al. 2012) and many more are likely to be available soon (Robinson et al. 2011). Using complete Wolbachia genomes will therefore broaden our understanding of the mechanisms that contribute to the spread of this ubiquitous endosymbiont.
Acknowledgements We thank public authorities in Germany for permitting the collection of protected species. We acknowledge Annemarie Geißler for laboratory assistance and Detlef Bernhard, Franziska A. Franke, Robert Mayer, Stefan Schaffer, Christian Venne and Ronny Wolf for collecting animals. Susann Kauschke and Georg Mayer aided in antibody staining and CLSM imaging. We thank Seraina Klopfstein for fruitful discussions and assistance with statistical analyses. Four anonymous reviewers are acknowledged for providing helpful comments and suggestions on earlier versions of this manuscript. We thank George Papafotiou and Kostas Bourtzis for providing wsp antibody. We acknowledge financial support by the University of Leipzig and Martin Schlegel for providing laboratory facilities.
References Akaike H (1974) A new look at the statistical model identification. IEEE Transactions on Automatic Control, 19, 716–723. Altekar G (2004) Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics, 20, 407–415. Atyame CM, Delsuc F, Pasteur N, Weill M, Duron O (2011) Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito. Molecular Biology and Evolution, 28, 2761–2772. Augustinos AA, Santos-Garcia D, Dionyssopoulou E et al. (2011) Detection and characterization of Wolbachia infections in natural populations of aphids: is the hidden diversity fully unraveled? PLoS ONE, 6, e28695. Baldo L, Dunning Hotopp JC, Jolley KA et al. (2006) Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Applied and Environmental Microbiology, 72, 7098–7110. Baldo L, Ayoub NA, Hayashi CY et al. (2008) Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity. Molecular Ecology, 17, 557–569. Blomberg SP, Garland T Jr, Ives AR (2003) Testing for phylogenetic signal in comparative data: behavioral traits are more labile. Evolution, 57, 717–745. Boyle L, O’Neill SL, Robertson HM, Karr TL (1993) Interspecific and intraspecific horizontal transfer of Wolbachia in Drosophila. Science, 260, 1796–1799. © 2013 John Wiley & Sons Ltd
Braig HR, Guzman H, Tesh RB, O’Neill SL (1994) Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart. Nature, 367, 453–455. Burnham KP, Anderson DR (2002) Model Selection and MultiModel Inference: A Practical Information-Theoretic Approach, 2nd edn. Springer, New York, New York. Cardinal S, Straka J, Danforth BN (2010) Comprehensive phylogeny of apid bees reveals the evolutionary origins and antiquity of cleptoparasitism. Proceedings of the National Academy of Sciences of the USA, 107, 16207–16211. Caspi-Fluger A, Inbar M, Mozes-Daube N et al. (2011) Horizontal transmission of the insect symbiont Rickettsia is plantmediated. Proceedings of the Royal Society B-Biological Sciences, 279, 1791–1796. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540–552. Charlat S, Nirgianaki A, Bourtzis K, Mercot H (2002) Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia. Evolution, 56, 1735–1742. Charlat S, Duplouy A, Hornett EA et al. (2009) The joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina. BMC Evolutionary Biology, 9, 64. Conow C, Fielder D, Ovadia Y, Libeskind-Hadas R (2010) Jane: a new tool for the cophylogeny reconstruction problem. Algorithms for Molecular Biology, 5, 16. Covacin C, Barker SC (2007) Supergroup F Wolbachia bacteria parasitise lice (Insecta: Phthiraptera). Parasitology Research, 100, 479–485. Danforth BN, Brady S, Sipes S, Pearson A (2004) Single-copy nuclear genes recover cretaceous-age divergences in bees. Systematic Biology, 53, 309–326. Danforth BN, Cardinal S, Praz C, Almeida EAB, Michez D (2013) The impact of molecular data on our understanding of bee phylogeny and evolution. Annual Review of Entomology, 58, 57–78. Debevec AH, Cardinal S, Danforth BN (2012) Identifying the sister group to the bees: a molecular phylogeny of Aculeata with an emphasis on the superfamily Apoidea. Zoologica Scripta, 41, 527–535. Dedeine F, Ahrens M, Calcaterra L, Shoemaker DD (2005) Social parasitism in fire ants (Solenopsis spp.): a potential mechanism for interspecies transfer of Wolbachia. Molecular Ecology, 14, 1543–1548. Didelot X, Falush D (2007) Inference of bacterial microevolution using multilocus sequence data. Genetics, 175, 1251–1266. Dowling TE, Secor CL (1997) The role of hybridization and introgression in the diversification of animals. Annual Review of Ecology and Systematics, 28, 593–619. Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29, 1969–1973. Duplouy A, Hurst GDD, O’Neill SL, Charlat S (2010) Rapid spread of male-killing Hypolimnas bolina. Journal of Evolutionary Biology, 23, 231–235. Dyer KA, Burke C, Jaenike J (2011) Wolbachia-mediated persistence of mtDNA from a potentially extinct species. Molecular Ecology, 20, 2805–2817. Ekblom R, Galindo J (2011) Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity, 107, 1–15.
€ T H E and C . B L E I D O R N 6160 M . G E R T H , J . R O Engelst€adter J, Hurst G (2006) The dynamics of parasite incidence across host species. Evolutionary Ecology, 20, 603–616. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG (2004) eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. Journal of Bacteriology, 186, 1518–1530. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791. Frydman HM, Li JM, Robson DN, Wieschaus E (2006) Somatic stem cell niche tropism in Wolbachia. Nature, 441, 509–512. Garibaldi LA, Steffan-Dewenter I, Winfree R et al. (2013) Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science, 339, 1608. Gelman A, Rubin DB (1992) Inference from iterative simulation using multiple sequences. Statistical Science, 7, 457–472. Gerth M, Geißler A, Bleidorn C (2011) Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding. Systematics and Biodiversity, 9, 319–327. Gompert Z, Forister ML, Fordyce JA, Nice CC (2008) Widespread mito-nuclear discordance with evidence for introgressive hybridization and selective sweeps in Lycaeides. Molecular Ecology, 17, 5231–5244. Greenleaf SS, Kremen C (2006) Wild bees enhance honey bees’ pollination of hybrid sunflower. Proceedings of the National Academy of Sciences of the USA, 103, 13890–13895. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series, 41, 95–98. Harmon LJ, Weir JT, Brock CD, Glor RE, Challenger W (2008) GEIGER: investigating evolutionary radiations. Bioinformatics, 24, 129–131. Heath BD, Butcher RD, Whitfield WG, Hubbard SF (1999) Horizontal transfer of Wolbachia between phylogenetically distant insect species by a naturally occurring mechanism. Current Biology, 9, 313–316. Hedges LM, Brownlie JC, O’Neill SL, Johnson KN (2008) Wolbachia and virus protection in insects. Science, 322, 702. Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH (2008) How many species are infected with Wolbachia?- a statistical analysis of current data. FEMS Microbiology Letters, 281, 215–220. Hosokawa T, Koga R, Kikuchi Y, Meng X-Y, Fukatsu T (2010) Wolbachia as a bacteriocyte-associated nutritional mutualist. Proceedings of the National Academy of Sciences of the USA, 107, 769–774. Huigens ME, Luck RF, Klaassen RHG et al. (2000) Infectious parthenogenesis. Nature, 405, 178–179. Huigens ME, de Almeida RP, Boons PAH, Luck RF, Stouthamer R (2004) Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Trichogramma wasps. Proceedings of the Royal Society of London B-Biological Sciences, 271, 509–515. Hurst GDD, Jiggins FM (2005) Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. Proceedings of the Royal Society of London B-Biological Sciences, 272, 1525–1534. Jaenike J, Dyer KA (2008) No resistance to male-killing Wolbachia after thousands of years of infection. Journal of Evolutionary Biology, 21, 1570–1577.
Jolley K, Chan M-S, Maiden M (2004) mlstdbNet—distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics, 5, 86. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30, 3059–3066. Kembel SW, Cowan PD, Helmus MR et al. (2010) Picante: R tools for integrating phylogenies and ecology. Bioinformatics, 26, 1463–1464. Kittayapong P, Jamnongluk W, Thipaksorn A, Milne JR, Sindhusake C (2003) Wolbachia infection complexity among insects in the tropical rice-field community. Molecular Ecology, 12, 1049–1060. Koehncke A, Telschow A, Werren JH, Hammerstein P (2009) Life and death of an influential passenger: Wolbachia and the evolution of CI-modifiers by their hosts. PLoS ONE, 4, e4425. Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution, 29, 1695–1701. Lohrmann V, Ohl M, Bleidorn C, Podsiadlowski L (2008) Phylogenie der “Sphecidae” (Hymenoptera: Apoidea) basierend auf molekularen Daten. Mitteilungen der Deutschen Gesellschaft fuer Allgemeine und Angewandte Entomologie, 16, 99–102. Maiden MCJ, Bygraves JA, Feil E et al. (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proceedings of the National Academy of Sciences of the USA, 95, 3140–3145. Mallet J (2005) Hybridization as an invasion of the genome. Trends in Ecology & Evolution, 20, 229–237. Mazzucco K, Mazzucco R (2007) Wege der Mikroevolution und Artbildung bei Bienen (Apoidea, Hymenoptera): Populationsgenetische und empirische Aspekte. Denisia, 20, 617–686. Michener CD (2007) The Bees of the World, 2nd edn. Johns Hopkins University Press, Baltimore, Maryland. Mitsuhashi W, Saiki T, Wei W, Kawakita H, Sato M (2002) Two novel strains of Wolbachia coexisting in both species of mulberry leafhoppers. Insect Molecular Biology, 11, 577–584. Noda H, Miyoshi T, Zhang Q et al. (2001) Wolbachia infection shared among planthoppers (Homoptera: Delphacidae) and their endoparasite (Strepsiptera: Elenchidae): a probable case of interspecies transmission. Molecular Ecology, 10, 2101–2106. O’Neill SL, Giordano R, Colbert AM, Karr TL, Robertson HM (1992) 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proceedings of the National Academy of Sciences of the USA, 89, 2699–2702. Pagel M (1999) Inferring the historical patterns of biological evolution. Nature, 401, 877–884. Pagel M, Meade A, Barker D (2004) Bayesian estimation of ancestral character states on phylogenies. Systematic Biology, 53, 673–684. R Development Core Team (2012) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Rambaut A, Drummond A (2007) Tracer v1.4. Available from http://beast.bio.ed.ac.uk/Tracer. Rasgon JL, Gamston CE, Ren X (2006) Survival of Wolbachia pipientis in cell-free medium. Applied and Environmental Microbiology, 72, 6934–6937.
© 2013 John Wiley & Sons Ltd
H O R I Z O N T A L W O L B A C H I A T R A N S M I S S I O N I N B E E S 6161 Raychoudhury R, Baldo L, Oliveira DCSG, Werren JH (2009) Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex. Evolution, 63, 165–183. Raychoudhury R, Grillenberger BK, Gadau J et al. (2010) Phylogeography of Nasonia vitripennis (Hymenoptera) indicates a mitochondrial-Wolbachia sweep in North America. Heredity, 104, 318–326. Richardson MF, Weinert LA, Welch JJ et al. (2012) Population genomics of the Wolbachia endosymbiont in Drosophila melanogaster. PLoS Genetics, 8, e1003129. Riegler M, Charlat S, Stauffer C, Mercßot H (2004) Wolbachia transfer from Rhagoletis cerasi to Drosophila simulans: investigating the outcomes of host-symbiont coevolution. Applied and Environmental Microbiology, 70, 273–279. Rigaud T, Moreau J, Juchault P (1999) Wolbachia infection in the terrestrial isopod Oniscus asellus: sex ratio distortion and effect on fecundity. Heredity, 83, 469–475. Rigaud T, Pennings PS, Juchault P (2001) Wolbachia bacteria effects after experimental interspecific transfers in terrestrial isopods. Journal of Invertebrate Pathology, 77, 251–257. Robinson GE, Hackett KJ, Purcell-Miramontes M et al. (2011) Creating a buzz about insect genomes. Science, 331, 1386. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574. Ros VID, Fleming VM, Feil EJ, Breeuwer JAJ (2009) How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae). Applied and Environmental Microbiology, 75, 1036–1043. Russell JA (2012) The ants (Hymenoptera: Formicidae) are unique and enigmatic hosts of prevalent Wolbachia (Alphaproteobacteria) symbionts. Myrmecological News, 16, 7–23. Russell JA, Goldman-Huertas B, Moreau CS et al. (2009) Specialization and geographic isolation among Wolbachia symbionts from ants and lycaenid butterflies. Evolution, 63, 624–640. Schilthuizen M, Stouthamer R (1997) Horizontal transmission of parthenogenesis-inducing microbes in Trichogramma wasps. Proceedings of the Royal Society of London B-Biological Sciences, 264, 361–366. Schwarz MP, Richards MH, Danforth BN (2007) Changing paradigms in insect social evolution: insights from halictine and allodapine bees. Annual Review of Entomology, 52, 127–150. Sintupachee S, Milne JR, Poonchaisri S, Baimai V, Kittayapong P (2006) Closely related Wolbachia strains within the pumpkin arthropod community and the potential for horizontal transmission via the plant. Microbial Ecology, 51, 294–301. Stahlhut JK, Desjardins CA, Clark ME et al. (2010) The mushroom habitat as an ecological arena for global exchange of Wolbachia. Molecular Ecology, 19, 1940–1952. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688–2690. Stouthamer R, Breeuwer JAJ, Hurst GDD (1999) Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annual Review of Microbiology, 53, 71–102. Tautz D, Ellegren H, Weigel D (2010) Next generation molecular ecology. Molecular Ecology, 19, 1–3.
© 2013 John Wiley & Sons Ltd
Taylor MJ, Voronin D, Johnston KL, Ford L (2013) Wolbachia filarial interactions. Cellular Microbiology, 15, 520–526. Teixeira L, Ferreira A, Ashburner M (2008) The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biology, 6, e2. Vavre F, Fleury F, Lepetit D, Fouillet P, Bouletreau M (1999) Phylogenetic evidence for horizontal transmission of Wolbachia in host-parasitoid associations. Molecular Biology and Evolution, 16, 1711–1723. Vos M, Didelot X (2009) A comparison of homologous recombination rates in bacteria and archaea. The ISME Journal, 3, 199–208. Walker T, Johnson PH, Moreira LA et al. (2011) The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature, 476, 450–453. Werren JH (1997) Biology of Wolbachia. Annual Review of Entomology, 42, 587–609. Werren JH, Skinner SW, Charnov EL (1981) Paternal inheritance of a daughterless sex ratio factor. Nature, 293, 467–468. Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nature Reviews Microbiology, 6, 741–751. Westrich P (1989) Die Wildbienen Baden-W€ urttembergs I & II. Eugen Ulmer GmbH & Co. Stuttgart. Whitworth TL, Dawson RD, Magalon H, Baudry E (2007) DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae). Proceedings of the Royal Society of London B-Biological Sciences, 274, 1731–1739. Wu M, Sun LV, Vamathevan J et al. (2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biology, 2, e69. Xiao J-H, Wang N-X, Murphy RW et al. (2012) Wolbachia infection and dramatic intraspecific mitochondrial DNA divergence in a fig wasp. Evolution, 66, 1907–1916. Zug R, Hammerstein P (2012) Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS ONE, 7, e38544. Zug R, Koehncke A, Hammerstein P (2012) Epidemiology in evolutionary time: the case of Wolbachia horizontal transmission between arthropod host species. Journal of Evolutionary Biology, 25, 2149–2160.
C.B. and M.G. designed the experiments. C.B., J.R. and M.G. performed sampling, J.R. and M.G. performed laboratory work. M.G. analysed the data and C.B. and M.G. wrote the manuscript.
Data accessibility DNA sequences: GenBank Accession nos KC798065– KC798382, KC812731, KF512682–KF512710. Phylogenetic trees and alignments: Data Dryad DOI: 10.5061/dryad.bj00s. Sampling locations: uploaded as online supporting information (Table S1, Supporting information).
€ T H E and C . B L E I D O R N 6162 M . G E R T H , J . R O
Supporting information Additional supporting information may be found in the online version of this article. Fig. S1 Majority rule consensus tree of Wolbachia strains as inferred from the posterior sample of Bayesian analysis. Fig. S2 CLONALFRAME genealogy based on MLST genes for Wolbachia strains from this study (marked in blue) and profiles from Wolbachia PubMLST database.
Fig. S3 Maximum likelihood tree of bees based on LW rhodopsin data from this study. Fig. S4 Maximum clade credibility tree of 135 apid bees and 60 megachilid outgroup taxa as estimated in BEAST with a relaxed molecular clock model. Fig. S5 Antibody staining of Wolbachia cells within salivary gland of a single Anthophora plumipes female. Table S1 Sampling localities for bees and GenBank accession nos for each analysed gene fragment.
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