Wolbachia dynamics and host effects: what has (and has not) been demonstrated?

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Wolbachia dynamics and host effects: what has (and has not) been demonstrated? Andrew R. Weeks, K. Tracy Reynolds and Ary A. Hoffmann The vertically transmitted endoparasitic bacterium Wolbachia infects a wide range of invertebrates, including insects, crustaceans, mites and nematodes. These bacteria have received increasing attention over the past decade because they have been implicated in causing an array of effects in their hosts. But how convincing is the evidence that supports these conjectures and are we overemphasizing the importance of Wolbachia as an evolutionary force in host evolution? We argue that, although Wolbachia effects are widespread in arthropods, other factors, including nuclear effects and different endosymbionts might have been overlooked. In addition, factors that can influence the expression of Wolbachia effects are rarely considered in laboratory studies and this makes extrapolation to the field difficult. If we are to understand the role that Wolbachia plays in host evolution, a more thorough approach to documenting their effects is required. Published online: 3 April 2002

Andrew R. Weeks Centre for Environmental Stress and Adaptation Research, Monash University, Clayton, VIC 3800, Australia. K. Tracy Reynolds Ary A. Hoffmann* Centre for Environmental Stress and Adaptation Research, La Trobe University, Bundoora, VIC 3083, Australia. *e-mail: A.Hoffmann@ latrobe.edu.au

The intracellular bacterium Wolbachia infects between 20% and 76% of all insects and has also been found in mites (Acari), crustaceans and nematodes [1–3]. These bacteria have attracted considerable attention over the past decade because they are assumed to account for many different phenomena in their hosts (Box 1), including cytoplasmic incompatibility (CI), parthenogenesis induction, male-killing, feminization, sex determination, sperm competition, speciation and even local adaptation (reviewed in [1]). Wolbachia are also thought to have a marked impact on evolutionary divergence in mitochondrial DNA [4,5] and their host effects could have potential for pest control [6]. But how convincing is the evidence that supports these conjectures? Is Wolbachia an all-important powerful group of intracellular bacteria that often drives host evolution, or has its importance been oversold and are we in danger of ignoring alternatives? What type of data is needed to demonstrate the ubiquity and impact of such host effects? We argue that, although Wolbachia effects are widespread, there is a risk that other factors causing similar effects have been overlooked. Moreover, factors that can mediate the expression of Wolbachia effects on hosts, such as male age, remating frequency and temperature, have not been considered in laboratory studies of most Wolbachia–host systems. This makes extrapolation to the field situation difficult. http://tree.trends.com

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We advocate a more thorough and cautious approach for documenting Wolbachia effects so that the role that the bacterium plays in host evolution can be clarified. Incompatibility: is it always Wolbachia induced?

Perhaps the most well-known effect of Wolbachia is its ability to generate cytoplasmic incompatibility (CI) in its hosts. When a Wolbachia-infected host line is crossed with an uninfected line, a unidirectional incompatibility often results. Crosses between infected males and uninfected females produce non-viable embryos, whereas the reciprocal cross is unaffected. In addition, two-way (bidirectional) incompatibility can occur when two host lines are infected by different Wolbachia strains [7]. This phenomenon appears widespread, having been described in six insect orders as well as in isopods (Crustacea) and mites (Box 1). However, the presence of Wolbachia does not always imply incompatibility and, conversely, the presence of incompatibility does not always imply that Wolbachia is the causative agent. This linkage is sometimes assumed in the absence of other data [8,9]. A demonstration of CI requires a rigorous set of crosses with appropriate replication (Box 2), yet researchers often undertake a few simple crosses without adequate controls. Factors, such as male age effects, nuclear background and random genetic drift, are rarely taken into consideration when describing CI. There are other factors that cause incompatibility among populations. Nuclear genes are often involved. For instance, crosses among lines of the egg parasitoid Trichogramma deion display different degrees of incompatibility, and backcrosses show the incompatibility to be of nuclear origin rather than because of cytoplasmic factors [10]. Tetranychidae mites also exhibit incompatibilities between populations that are not necessarily because of Wolbachia. In the spider mite Tetranychus urticae, both Wolbachia-induced and nuclear-induced incompatibilities are known [11,12], highlighting the complexity of some systems and the need for extensive crossing schemes to determine what causes incompatibility in particular hosts. Cytoplasmic factors other than Wolbachia can also cause incompatibility. In Drosophila paulistorum, for instance, a cytoplasmic factor thought to be a streptococcal-like bacterium causes sterility in male progeny from crosses between infected females of one strain with infected males from another [13] and results in male hybrid breakdown comparable to that found in a line of Wolbachia-infected spider mites [11]. However, it does appear that the common form of CI that arises in crosses between infected males and uninfected females is strictly associated with Wolbachia. Male-killers, parthenogenesis induction, feminization and alternative bacteria

The phenomenon of male killing is well known in ladybirds, and has also been found in some Lepidoptera, Diptera, Hymenoptera and Hemiptera as well as other

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Box 1. Host phenomena associated with Wolbachia Reproductive manipulations Wolbachia can only be transmitted through females (maternal transmission); therefore, the four reproductive phenotypes discussed below can result in Wolbachia spreading through host populations.

Cytoplasmic incompatibility Wolbachia-induced cytoplasmic incompatibility (CI) is a reproductive incompatibility that results in zygote mortality in diploid species, and either zygote mortality or haploid male production in haplodiploid species. Unidirectional incompatibility arises when Wolbachia-infected males mate with uninfected females (the reciprocal cross is compatible). Bidirectional incompatibility occurs when a male and a female are infected with different strains of Wolbachia that are incompatible. The incompatibility typically results in the paternal chromosomes being eliminated, which renders the developing embryo haploid. Parthenogenesis induction Parthenogenesis-inducing (PI) Wolbachia cause infected female hosts to produce infected daughters without fertilization by males. This reproductive phenotype seems to be strictly associated with haplodiploidy. Unfertilized infected eggs, which would normally develop as haploid males, develop as diploid females. PI Wolbachia restore diploidy either through gamete duplication, which results in complete homozygosity throughout the genome [a], or with an unknown cytogenetic mechanism that results in heterozygosity being maintained [b]. Feminization of genetic males Wolbachia can alter the normal pattern of sex determination in their host. In some isopod (Crustacean) species and two lepidopteran species, Wolbachia cause genetic male embryos to develop phenotypically as functional females. In the isopod Armadillidium vulgare, Wolbachia induces feminization by blocking the formation of the androgenic gland, which produces the androgenic hormone responsible for male differentiation [c]. Feminized infected males still require fertilization by phenotypic males to produce progeny. Male-killing Secondary female-biased sex ratios are found in several arthropods. As seen in many other bacteria (Table 1, main text), Wolbachia induce this secondary female bias in some hosts by killing male embryos. Male-killing is thought to benefit sibling females, either by eliminating competition or by providing resources to females that cannibalize dead brothers. Other phenomena Wolbachia have been associated with other phenomena, but in most cases the evidence is equivocal.

Speciation The concept that Wolbachia could induce host speciation through CI is appealing; however, conclusive data have not yet been obtained.

Sperm competition Indirect tests for sperm competition between Wolbachia infected and uninfected sperm have shown a fitness advantage to infected sperm in the beetle Tribolium confusum [d]. However, direct tests in two Drosophila species have failed to find evidence of sperm effects associated with infection [e,f]. Longevity A strain of Wolbachia called popcorn, which reduces the longevity of its host, has been isolated from a laboratory strain of Drosophila melanogaster [g]. This strain over-replicates in host tissues and results in host mortality. The popcorn infection is thought to be a CI nonexpresser; however, recent data on male age effects (Box 3) raise doubts about this status. Temperature and nuclear background effects on the longevity phenotype remain to be investigated. Host fitness effects Both positive and negative fitness effects have been associated with Wolbachia infections. An extreme positive fitness effect has been found in Wolbachia infected filarial nematodes; nematodes produce no microfilarial progeny if Wolbachia is eliminated by antibiotics [h]. An example of a negative fitness effect has been found in Trichogramma deion and T. pretiosum, where PI Wolbachia cause a reduction in fecundity compared to uninfected females [i]. References a Stouthamer, R. and Kazmer, D.J. (1994) Cytogenetics of microbe-associated parthenogenesis and its consequences for gene flow in Trichogramma wasps. Heredity 73, 317–327 b Weeks,A.R. and Breeuwer, J.A.J. (2001) Wolbachia-induced parthenogenesis in a genus of phytophagous mites. Proc. R. Soc. London B Biol. Sci. 268, 2245–2251 c Martin, G. et al. (1990) Purification and characterization of androgenic hormone from the terrestrial isopod Armadillidium vulgare. Gen. Comp. Endocrinol. 80, 349–354 d Wade, M.J. and Chang, N.W. (1995) Increased male fertility in Tribolium confusum beetles after infection with the intracellular parasite Wolbachia. Nature 373, 72–74 e Hoffmann, A.A. et al. (1990) Factors affecting the distribution of cytoplasmic incompatibility in Drosophila simulans. Genetics 126, 933–948 f Hoffmann, A.A. et al. (1998) Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster. Genetics 148, 221–231 g Min, K.T. and Benzer, S. (1997) Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death. Proc. Natl. Acad. Sci. U. S. A. 94, 10792–10796 h Hoerauf, A. et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J. Clin. Invest. 103, 11–17 i Stouthamer, R. and Luck, R.F. (1993) Influence of microbe-associated parthenogenesis on the fecundity of Trichogramma deion and T. pretiosum. Entomol. Exp. Appl. 67, 183–192

Coleoptera [14]. Wolbachia appear to be responsible for the death of male progeny in the ladybird Adalia bipunctata, the butterflies Acraea encedon and A. encedana [15], the beetle Tribolium madens [16] and Drosophila bifasciata [17]. However, Wolbachia is only one of five different bacteria known to cause malekilling (Table 1). In addition nuclear genes, or meiotic drive genes, can also cause sex-ratio distortion [18]. However, if nuclear genes are the cause of the sex-ratio distortion, this shift towards females will occur without the elevated mortality associated with male killing. Two other manipulations of host reproduction that Wolbachia can cause are feminization [19] and parthenogenesis [20]. As with male killing, these effects are not unique to Wolbachia. Several species of microsporidia have been shown to cause feminization [21] http://tree.trends.com

and, recently, Weeks et al. [22] found that an undescribed bacterium from the Cytophaga–Flexibacter– Bacteroides (CFB) phylum causes feminization in the mite Brevipalpus phoenicis. A similar CFB bacterium has been associated with parthenogenesis in several parasitoid species from the genus Encarsia [23], although the evidence for CFB causing parthenogenesis in this group is still circumstantial. This undescribed bacterium also occurs in the tick Ixodes scapularis, where its effects are unknown [24]. Wolbachia has been considered to be unique in its ability to alter the reproduction of its hosts in more than one way. However, the discovery of the CFB bacteria that can also cause feminization, male killing and possibly parthenogenesis in their hosts (Table 1) demonstrates that other bacteria can also have more

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Box 2. Testing for Wolbachia-induced cytoplasmic incompatibility When testing for Wolbachia-induced cytoplasmic incompatibility (CI), an experimental design is needed that limits potential confounding effects. These effects can be caused by (1) the nuclear background of the host; (2) drift between antibiotic-treated and untreated lines after treatment but before testing for CI; (3) the infection status of the antibiotic-treated lines, and (4) male age effects. Figure I outlines a crossing strategy that allows these effects to be separated, where U represents uninfected individuals and I represents infected individuals. Where uninfected lines can be generated readily, the multiple line scheme should be followed (a). Where only one uninfected line is available (b), reciprocal crosses or backcrosses can be used to minimize potential confounding effects of the nuclear background.

Infected strain (status unknown) (a)

(b)

Potential to generate multiple uninfected strains (heat, antibiotics, etc.)

Single uninfected strain available, difficult to generate uninfecteds

Generate multiple uninfected strains, maintain independently

Cross uninfected strain reciprocally to infected strain (or, if cytoplasmic incompatibility strong and not possible, backcross both strains to a third strain) U

×I

I

F1(U)

×U F1(I)

Confirm Wolbachia status

Confirm Wolbachia status Crosses to determine cytoplasmic incompatibility: U

×U

U

×I

I

Fig. I

×U

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than one effect on the reproduction of their host. Even if Wolbachia is present it might not be responsible for a particular phenomenon if another bacterium also infects the host. Unfortunately, researchers often screen for the presence of Wolbachia first and then document its effects on the host, ignoring other bacteria that might be present. It is worth noting that most vertically transmitted male-killing microorganisms were identified first by phenotype and that Wolbachia is the most recently discovered of these male-killers. Other phenomena

Wolbachia is thought to interfere with sperm competition. This is based on an indirect test in the flour beetle Tribolium confusum [25]. Other attempts to demonstrate sperm competition mediated by Wolbachia have failed. In a more direct test in Drosophila simulans, Wolbachia did not influence sperm competition [26]. Similarly, experiments with a D. melanogaster mutant also failed to find a sperm competition effect [27]. No other Wolbachia sperm competition effects have been reported, although Wolbachia can affect the production of sperm cysts [28]. http://tree.trends.com

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Wolbachia dynamics

The presence of CI promotes the spread of Wolbachia into a population. Wolbachia can rapidly invade a population consisting of uninfected individuals once an infection frequency representing an unstable equilibrium is exceeded. This equilibrium occurs because of deleterious effects of the infection on its host and/or imperfections in its vertical transmission rate [26,29]. Once enough individuals are infected, any disadvantages associated with infection are outweighed by the presence of CI-induced mortality. In other words, the more common the infection, the more likely it is that uninfected females will encounter infected males and hence suffer reproductive losses. To date, the spread of Wolbachia in a population has been documented in D. simulans [30] and in the planthopper Laodelphax striatellus [31]. Where there are two or more different Wolbachia infections in a population the outcome is similar, although the unstable equilibrium point will be different. For instance, if two infections that are incompatible are present, the most common infection will spread to fixation, all else being equal. This is because females infected with the common strain are more likely to mate with males infected with the same strain (i.e. engage in compatible matings) and produce fertile offspring. Because Wolbachia is passed onto offspring via the maternal cytoplasm, the spread of Wolbachia is also associated with changes in the frequency of mitochondrial genes. Where an infected population encounters an uninfected population, Wolbachia is expected to spread into the uninfected population carrying along with it the mitochondrial genes of the infected population [4]. The resulting population will thus contain the mitochondrial genome(s) of the original infected population but a mix of nuclear genes from both. Complications in predicting Wolbachia dynamics and phenotypes

CI expression that drives the spread of Wolbachia in populations can depend on several factors, including the age of the host [26], density at which the host is reared [32], remating status of the host [33], temperature [26,32] and antibiotics [32]. Unfortunately, when researchers undertake crosses to establish CI, they rarely consider such factors. Yet these can mean the difference between the expression of strong CI and weak or even no CI. For instance, it was recently found that male age is important for the expression of CI (Box 3). Although this was previously known for D. simulans [26], in at least two lines of D. melanogaster CI expression can be undetectable when infected males four-days post-eclosion are mated to uninfected females. However, if infected males 24-h post-eclosion are used then strong CI expression is found. The male age effect is more prolonged in Wolbachia-infected D. simulans from Riverside, California (the other organism for which male age effects have been investigated), where CI is still detectable up to three-weeks post-eclosion [26]. One of

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Table 1. Vertically transmitted microorganisms with known effects on the reproduction of their hosts Phenotypic effect on reproduction

a

Microorganism

Host

Refs

Male-killing

Spiroplasma poulsonii (Mollicutes) Drosophila willistoni (Diptera) Spiroplasma sp. Adalia bipunctata, Harmonia axyridis (Coleoptera); Danaus chrysippus (Lepidoptera) Unnamed CFB species 1 Coleomegilla maculata, Adonia variegata (Coleoptera) Rickettsia (␣-proteobacteria) Adalia bipunctata, A. decempunctata, Brachys tessellatus (Coleoptera) Wolbachia (␣-proteobacteria) Adalia bipunctata, Tribolium madens (Coleoptera); Acraea encedon, A. encedana (Lepidoptera); Drosophila bifasciata (Diptera) Arsenophonus nasoniae Nasonia vitripennis (Hymenoptera) (␥-proteobacteria) Feminization Wolbachia Isopoda (Crustacea); Ostrinia furnacalis, O. scapulalis (Lepidoptera) Microsporidia sp. (four different Gammarus duebeni, Orchestia gammarellus, O. mediterranea, species) O. aestuarensis (Crustacea) Unnamed CFB species 2 Brevipalpus phoenicis (Acari) Parthenogenesis Wolbachia Parasitoids (Hymenoptera); Franklinothrips vespiformis (Thysanoptera); induction Bryobia praetiosa, B. sp. x (Acari) Unnamed CFB species 3 Encarsia pergandiella (Hymenoptera) Cytoplasmic Wolbachia Diptera, Lepidoptera, Hymenoptera, Coleoptera, Orthoptera, incompatibility (including Hemiptera; Isopoda (Crustacea); mites (Acari) F1 hybrid breakdown) Streptococcal-like bacterium Drosophila paulistorum

[14] [14] [14] [47,48] [14,16,17] [14] [40] [21] [22] [1,49,50] [23] [1,11] [13]

a

Abbreviation: CFB, Cytophaga–Flexibacter–Bacteriodes.

the D. melanogaster lines (Canton-S) was previously described as a CI nonexpressor [34], which is clearly not the case (Box 3). This highlights the importance of assessing male age and other factors that can influence CI expression before making inferences about Wolbachia phenotypes and Wolbachia dynamics. Factors other than CI expression can also influence the likelihood of Wolbachia invading a population. For instance, maternal transmission rates are imperfect in some systems, but estimates are rare, particularly under field conditions. Field estimates are necessary, because transmission rates in laboratory populations might not be the same as those in nature. In D. simulans, for example, Turelli and Hoffmann [35] found maternal transmission to be 100% in the laboratory, whereas in the field it was variable, with an average of 95–97%. Unfortunately, such estimates are not easy to obtain, because accurate estimates of transmission require large field samples, preferably from multiple sites. As for CI, factors such as age, temperature, natural antibiotics, diapause and density that influence incompatibility in the field are likely to affect maternal transmission and account for differences among laboratory and field transmission estimates. The effects of Wolbachia on host fitness has been considered in detail in only a few cases, although there is some evidence from laboratory studies that Wolbachia can influence host fitness. Perhaps the most extreme case is in some nematode species in which Wolbachia appears to be an obligate endosymbiont: nematodes cannot produce microfilarial progeny once Wolbachia is removed [36]. Unfortunately, as in the case of CI and transmission, field fitness effects can bear little resemblance to those detected in the laboratory. Under field conditions, Wolbachia effects might depend on interactions with the host nuclear background [37]. http://tree.trends.com

Modification–rescue and host effects

Recent studies have emphasized a modification–rescue model to explain CI Wolbachia effects (Box 3). However, when interpreting results in terms of this model, researchers often ignore the influence of the host nuclear genome. Boyle et al. [38] and Poinsot et al. [39] have shown host effects through transinfection experiments of Wolbachia from D. melanogaster into D. simulans (low CI and high CI, respectively), suggesting that host genes modify the effect of Wolbachia via changes such as a reduction in density or host-tissue distribution. Recently, a more extreme case of host effects has been shown by Fujii et al. [40] in two lepidopteran species. These authors transferred a strain of Wolbachia that causes feminization in Ostrinia scapulalis to Ephestia kuehniella, where it resulted in male-killing. Host effects, which are not part of the modification–rescue system, can play an important role in the dynamics of Wolbachia infections. The modification–rescue model therefore cannot solely explain CI expression as a Wolbachia strain effect and a more realistic model awaits elucidation of the mechanism of CI. Wolbachia and speciation

Recently, Bordenstein et al. [41] showed that Wolbachia-mediated reproductive isolation between two closely related infected parasitoid Nasonia spp. pre-dates other postzygotic isolation mechanisms (such as hybrid inviability and hybrid sterility). This suggests that Wolbachia causing bidirectional CI can play a role in reproductive isolation. In another study, Shoemaker et al. [42] suggested that Wolbachiainduced unidirectional CI between two closely related species of Drosophila (D. recens and D. subquinaria) contributed to reproductive isolation. These studies have been promoted as supporting the notion that

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Box 3. Male age and modification–rescue Wolbachia strains

References a Hurst, L.D. and McVean, G.T. (1996) Clade selection, reversible evolution and the persistence of selfish elements: the evolutionary dynamics of cytoplasmic incompatibility. Proc. R. Soc. London B Biol. Sci. 263, 97–104

1.0 Proportion of unhatched eggs

Following Hurst and McVean [a], Werren [b] proposed that the mechanism of Wolbachia-induced cytoplasmic incompatibility (CI) involves a twocomponent system, comprising bacterial (Wolbachia) modification (mod) of sperm and bacterial rescue (resc) in the fertilized egg. Incompatibility occurs when a modified sperm cannot be rescued in the fertilized egg (completing fertilization). Therefore, crosses involving Wolbachia-infected males (modified sperm) and uninfected females are incompatible. Crosses between different strains of Wolbachia are also incompatible because these strains have different modification–rescue systems. Under the mod–resc system, there are four possible classes; mod+resc+, mod−resc+, mod+resc− and mod−resc−. Before this system was proposed, Prout [c] and Turelli [d] modelled the dynamics of CI expression and found that, under certain conditions, Wolbachia variants that reduce CI and are compatible with their counterparts can invade and spread within a population. Using Werren’s terminology, they predicted the evolution of mod−resc+ variants, provided that these variants also have increased vertical transmission and/or fecundity. Although mod+resc+, mod−resc+ and mod−resc− have previously been reported from natural populations, it was recently found that so-called ‘mod−’ strains might in fact be mod+ when male age is carefully controlled. Two lines of Wolbachia-infected Drosophila melanogaster, Canton-S [e] and a line from Innisfail, Australia, were retested for CI expression using males ranging in age from 24-h post-eclosion up to six days post-eclosion. The Canton-S line was previously described as a CI nonexpressor [e], whereas the line from Innisfail was described as a weak CI expresser [f]. When uninfected females from both lines were mated with infected males from their respective lines 24-h post-eclosion, strong CI was found for both lines (Fig. I). CI expression decreased rapidly with increasing male age, and no CI was detected when males were four–five days post-eclosion. Male age effects were previously characterized in infected D. simulans populations from Riverside, California [g] and in this system CI was still detectable when males were up to three-weeks post-eclosion (Fig. I). Host male age effects have not been tested for mod−resc− or mod−resc+ strains and studies on these often use males up to five days post-eclosion to test for CI [h]. Their modification status therefore remains in doubt.

0.6 0.4 0.2 0.0

1

2

3 Male age (days)

4

5

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Fig. I. Fraction of Drosophila melanogaster eggs that hatch from infected male × uninfected female crosses set up with males of different ages. The green squares (dark-blue squares) indicate the means for incompatible (compatible) crosses for D. melanogaster from Innisfail, Australia. The red circles (light-blue circles) indicate the means for incompatible (compatible) crosses for the Canton-S D. melanogaster line. The purple diamonds indicate the means from incompatible crosses for D. simulans Riverside line (data taken from [g]). Error bars are 95% confidence intervals.

b Werren, J.H. (1997) Biology of Wolbachia. Annu. Rev. Entomol. 42, 587–609 c Prout, T. (1994) Some evolutionary possibilities for a microbe that causes incompatibility in its host. Evolution 48, 909–911 d Turelli, M. (1994) Evolution of incompatibility-inducing microbes and their hosts. Evolution 48, 1500–1513 e Holden, P.R. et al. (1993) Evidence for a Wolbachia symbiont in Drosophila melanogaster. Genet. Res. 62, 23–29 f Hoffmann, A.A. et al. (1994) Cytoplasmic incompatibility in Australian populations of Drosophila melanogaster. Genetics 136, 993–999 g Hoffmann, A.A. et al. (1990) Factors affecting the distribution of cytoplasmic incompatibility in Drosophila simulans. Genetics 126, 933–948 h Giordano, R. et al. (1995) Wolbachia infection and the expression of cytoplasmic incompatibility in Drosophila sechellia and D. mauritiana. Genetics 140, 1307–1317

Wolbachia commonly have a role in mediating speciation [43,44] and thereby contribute significantly to invertebrate diversity. However, issues related to population dynamics make speciation based largely on Wolbachia effects unlikely. The main problem is that Wolbachia infections causing strong CI are expected to sweep through populations once an unstable equilibrium has been exceeded. Any postmating isolation associated with Wolbachia is therefore expected to be transient, preventing the selection of mechanisms leading to the evolution of pre-mating reproductive isolation between taxa. Moreover, high transmission and strong CI are needed to prevent gene flow among populations. To counter this problem, advocates of Wolbachiainduced speciation have proposed alternative models that allow some degree of CI to persist between two populations, promoting the development of pre- and postmating isolation [45]. In the simplest case, one population infected by a strain of Wolbachia is geographically isolated from another population infected by a different strain (bidirectional CI), but there is some gene flow between the populations. Because of limited gene flow, different Wolbachia strains persist in each of the populations (because they are the most common in http://tree.trends.com

0.8

their own region). In this case, nuclear genes arising in each population that promote pre-mating reproductive isolation would be favoured, because matings between populations have low fitness because of CI. What is the evidence for these conditions? In the case of Nasonia longicornis and N. giraulti, these species are allopatric so there is no gene flow between them [41]. In crosses between N. giraulti and another closely related Wolbachia-infected Nasonia species (N. vitripennis), which do have an overlapping distribution, other preand postmating mechanisms have evolved that are not attributable to Wolbachia [46]. In the case of D. recens and D. subquinaria, the populations are likely to have diverged significantly in allopatry before secondary contact, making it difficult to estimate the impact of Wolbachia in the speciation process. Concluding remarks

There is no doubt that Wolbachia have many interesting effects on organisms that have helped to raise awareness of the potential contribution of endosymbionts to host evolution. However, it is important not to overemphasize Wolbachia and discard other alternatives. Host effects can be important and interact with Wolbachia. CI expression has not been documented adequately in most

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Acknowledgements We thank Carla Sgrò, Brad Rundle, Linda Thomson, Richard Stouthamer, Michael Turelli, John Werren and two anonymous reviewers for helpful comments. This project was supported by the Australian Research Council via their Special Research Centre program.

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studies, because factors influencing expression have not been considered. There are other bacterial systems that can cause phenotypes normally associated with Wolbachia and, in the case of male-killing, these might be more common than Wolbachia. The behaviour of Wolbachia infections in the field are likely to be much more complicated than is apparent from laboratory studies, and it is difficult to predict Wolbachia dynamics from studies that ignore the many factors influencing the expression of reproductive effects, maternal transmission and host fitness effects.

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