Wolbachia infection reduces sperm competitive ability in an insect

The maternally inherited bacterium Wolbachia pipientis imposes significant fitness costs on its hosts. One such cost is decreased sperm production resulting in reduced fertility of male Drosophila simulans infected with cytoplasmic incompatibility (CI) inducing Wolbachia. We tested the hypothesis that Wolbachia infection affects sperm competitive ability and found that Wolbachia infection is indeed associated with reduced success in sperm competition in non-virgin males. In the second male role, infected males sired 71% of the offspring whereas uninfected males sired 82% of offspring. This is the first empirical evidence indicating that Wolbachia infection deleteriously affects sperm competition and raises the possibility that polyandrous females can utilize differential sperm competitive ability to bias the paternity of broods and avoid the selfish manipulations of Wolbachia. This suggests a relationship between Wolbachia infection and host reproductive strategies. These findings also have important consequences for Wolbachia population dynamics because the transmission advantage of Wolbachia is likely to be undermined by sperm competition.     

Increased male mating rate in Drosophila is associated with Wolbachia infection

The maternally inherited bacterium Wolbachia pipientis infects 25-75% of arthropods and manipulates host reproduction to improve its transmission. One way Wolbachia achieves this is by inducing cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females are inviable. Infected males suffer reduced fertility through CI and reduced sperm production. However, Wolbachia induce lower levels of CI in nonvirgin males. We examined the impact of Wolbachia on mating behaviour in male Drosophila melanogaster and D. simulans, which display varying levels of CI, and show that infected males mate at a higher rate than uninfected males in both species. This may serve to increase the spread of Wolbachia, or alternatively, may be a behavioural adaptation employed by males to reduce the level of CI. Mating at high rate restores reproductive compatibility with uninfected females resulting in higher male reproductive success thus promoting male promiscuity. Increased male mating rates also have implications for the transmission of Wolbachia.     

Wolbachia infection lowers fertile sperm transfer in a moth

The endosymbiotic bacterium Wolbachia pipientis manipulates host reproduction by rendering infected males reproductively incompatible with uninfected females (cytoplasmic incompatibility; CI). CI is believed to occur as a result of Wolbachia-induced modifications to sperm during maturation, which prevent infected sperm from initiating successful zygote development when fertilizing uninfected females' eggs. However, the mechanism by which CI occurs has been little studied outside the genus Drosophila. Here, we show that in the sperm heteromorphic Mediterranean flour moth, Ephestia kuehniella, infected males transfer fewer fertile sperm at mating than uninfected males. In contrast, non-fertile apyrene sperm are not affected. This indicates that Wolbachia may only affect fertile sperm production and highlights the potential of the Lepidoptera as a model for examining the mechanism by which Wolbachia induces CI in insects.     

Overcoming cytoplasmic incompatibility in Drosophila.

The endocellular microbe Wolbachia pipientis infects a wide variety of invertebrate species, in which its presence is closely linked to a form of reproductive failure termed cytoplasmic incompatibility (CI). CI renders infected males unable to father offspring when mated to uninfected females. Because CI can dramatically affect fitness in natural populations, mechanisms that abate CI can have equally large impacts on fitness. We have discovered that repeated copulation by Wolbachia-infected male Drosophila simulans significantly diminishes CI. Repeated copulation does not prevent Wolbachia from populating developing spermatids, but may reduce the time during spermatogenesis when Wolbachia can express CI. This restoration of fertility in premated infected males could have important implications for Wolbachia transmission and persistence in nature and for its exploitation as an agent of biological pest control.     

The impact of Wolbachia,male age and mating history on cytoplasmic incompatibility and sperm transfer in Drosophila simulans

Most insects harbour a variety of maternally inherited endosymbionts, the most widespread being Wolbachia pipientis that commonly induce cytoplasmic incompatibility (CI) and reduced hatching success in crosses between infected males and uninfected females. High temperature and increasing male age are known to reduce the level of CI in a variety of insects. In Drosophila simulans, infected males have been shown to mate at a higher rate than uninfected males. By examining the impact of mating rate independent of age, this study investigates whether a high mating rate confers an advantage to infected males through restoring their compatibility with uninfected females over and above the effect of age. The impact of Wolbachia infection, male mating rate and age on the number of sperm transferred to females during copulation and how it relates to CI expression was also assessed. As predicted, we found that reproductive compatibility was restored faster in males that mate at higher rate than that of low mating and virgin males, and that the effect of mating history was over and above the effect of male age. Nonvirgin infected males transferred fewer sperm than uninfected males during copulation, and mating at a high rate resulted in the transfer of fewer sperm per mating irrespective of infection status. These results indicate that the advantage to infected males of mating at a high rate is through restoration of reproductive compatibility with uninfected females, whereas uninfected males appear to trade off the number of sperm transferred per mating with female encounter rate and success in sperm competition. This study highlights the importance Wolbachia may play in sexual selection by affecting male reproductive strategies.     

Cross-species comparison of Drosophila male accessory gland protein genes

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.     

Effects of Wolbachia on sperm maturation and architecture in Drosophila simulans Riverside

Wolbachia is an intracellular obligate symbiont, that is relatively common in insects and also found in some nematodes. Cytoplasmic incompatibility (CI) is the most commonly expressed form, of several sex altering phenotypes caused by this rickettsial-like bacterium. CI is induced when infected males mate with uninfected females, and is likely the result of bacterial-induced modification of sperm grown in a Wolbachia-infected environment. Several studies have explored the dynamics of Wolbachia bacteria during sperm development in Drosophila. This study confirms and extends these earlier investigations of Wolbachia's distribution and proliferation in male germ cell lines. We examined Wolbachia population dynamics during testis development of Drosophila simulans (Riverside) by studying their distribution during the early mitotic divisions of secondary spermatogonial and subsequent meiotic cyst cells. Wolbachia are found in lower concentration in spermatogonial than in spermatocyte cells. Cytoplasmically incompatible crosses result in low levels of viable embryos despite the occurrence of fairly high levels of uninfected cysts. During meiotic divisions Wolbachia organize themselves at the poles during prophase and telophase but arrange themselves in equatorial bands during metaphase and anaphase. Moreover, during meiosis Wolbachia are asymmetrically divided between some daughter cells. There is no strong relationship between the fusome and Wolbachia and we have not found evidence that bacteria cross the ring canals. Wolbachia were observed at the distal and proximal sides of individualization complexes. Multiple altered sperm structures were observed during the process of individualization of infected sperm.     

Wolbachia transfer from Drosophila melanogaster into D. simulans: Host effect and cytoplasmic incompatibility relationships.

Wolbachia are maternally transmitted endocellular bacteria causing a reproductive incompatibility called cytoplasmic incompatibility (CI) in several arthropod species, including Drosophila. CI results in embryonic mortality in incompatible crosses. The only bacterial strain known to infect Drosophila melanogaster (wDm) was transferred from a D. melanogaster isofemale line into uninfected D. simulans isofemale lines by embryo microinjections. Males from the resulting transinfected lines induce >98% embryonic mortality when crossed with uninfected D. simulans females. In contrast, males from the donor D. melanogaster line induce only 18-32% CI on average when crossed with uninfected D. melanogaster females. Transinfected D. simulans lines do not differ from the D. melanogaster donor line in the Wolbachia load found in the embryo or in the total bacterial load of young males. However, >80% of cysts are infected by Wolbachia in the testes of young transinfected males, whereas only 8% of cysts are infected in young males from the D. melanogaster donor isofemale line. This difference might be caused by physiological differences between hosts, but it might also involve tissue-specific control of Wolbachia density by D. melanogaster. The wDm-transinfected D. simulans lines are unidirectionally incompatible with strains infected by the non-CI expressor Wolbachia strains wKi, wMau, or wAu, and they are bidirectionally incompatible with strains infected by the CI-expressor Wolbachia strains wHa or wNo. However, wDm-infected males do not induce CI toward females infected by the CI-expressor strain wRi, which is found in D. simulans continental populations, while wRi-infected males induce partial CI toward wDm-infected females. This peculiar asymmetrical pattern could reflect an ongoing divergence between the CI mechanisms of wRi and wDm. It would also confirm other results indicating that the factor responsible for CI induction in males is distinct from the factor responsible for CI rescue in females.     

Wolbachia distribution and cytoplasmic incompatibility during sperm development: the cyst as the basic cellular unit of CI expression

The growth and distribution of the intracellular microbe Wolbachia pipientis during spermatogenesis in several different host/symbiont genetic combinations in Drosophila melanogaster and Drosophila simulans is described. Considerable intra- and inter-strain variation in Wolbachia density and tissue distribution was observed. Wolbachia were found inside spermatocytes and spermatids or within the somatic cyst cells surrounding the germ cells. Some strains displayed both tissue distributions. High rates of cytoplasmic incompatibility (CI) are correlated with high levels of Wolbachia only when spermatocytes and/or spermatids harbor the microbe. Wolbachia infection of somatic cyst cells, although sometimes present at high levels, did not result in significant CI expression. CI-inducing Wolbachia strains within D. simulans showed no distinguishable differences in distribution or density within infected spermatids. To dissect the relative contribution of host and symbiont to the expression of CI, Wolbachia from various host strains known to exhibit varying levels of CI were introgressed into new uninfected host genetic backgrounds. These introgression experiments confirm that the mod(+)/mod(-) phenotype is an intrinsic Wolbachia trait and is not determined by host factors. The level of sperm modification in those lines harboring Wolbachia capable of modifying sperm, however, is influenced by host genetic background. These results form the basis of the Wolbachia Infected Spermatocyte/Spermatid Hypothesis (WISSH). According to WISSH, Wolbachia infection in spermatocytes and then spermatids during sperm development is required for CI expression.     

A genetic test of the mechanism of Wolbachia-induced cytoplasmic incompatibility in Drosophila

Cytoplasmic bacteria of the genus Wolbachia are best known as the cause of cytoplasmic incompatibility (CI): many uninfected eggs fertilized by Wolbachia-modified sperm from infected males die as embryos. In contrast, eggs of infected females rescue modified sperm and develop normally. Although Wolbachia cause CI in at least five insect orders, the mechanism of CI remains poorly understood. Here I test whether the target of Wolbachia-induced sperm modification is the male pronucleus (e.g., DNA or pronuclear proteins) or some extranuclear factor from the sperm required for embryonic development (e.g., the paternal centrosome). I distinguish between these hypotheses by crossing gynogenetic Drosophila melanogaster females to infected males. Gynogenetic females produce diploid eggs whose normal development requires no male pronucleus but still depends on extranuclear paternal factors. I show that when gynogenetic females are crossed to infected males, uniparental progeny with maternally derived chromosomes result. This finding shows that Wolbachia impair the male pronucleus but no extranuclear component of the sperm.     

Cytoplasmic Incompatibility in Drosophila Simulans: Dynamics and Parameter Estimates from Natural Populations

In Drosophila simulans, cytoplasmically transmitted Wolbachia microbes cause reduced egg hatch when infected males mate with uninfected females. A Wolbachia infection and an associated mtDNA variant have spread northward through California since 1986. PCR assays show that Wolbachia infection is prevalent throughout the continental US and Central and South America, but some lines from Florida and Ecuador that are PCR-positive for Wolbachia do not cause incompatibility. We estimate from natural populations infection frequencies and the transmission and incompatibility parameter values that affect the spread of the infection. On average, infected females from nature produce 3-4% uninfected ova. Infected females with relatively low fidelity of maternal transmission show partial incompatibility with very young infected laboratory males. Nevertheless, crosses between infected flies in nature produce egg-hatch rates indistinguishable from those produced by crosses between uninfected individuals. Incompatible crosses in nature produce hatch rates 30-70% as high as those from compatible crosses. Wild-caught infected and uninfected females are equally fecund in the laboratory. Incompatibility decreases with male age, and age-specific incompatibility levels suggest that males mating in nature may often be 2 or 3 weeks old. Our parameter estimates accurately predict the frequency of Wolbachia infection in California populations.     

Characterization of non-cytoplasmic incompatibility inducing Wolbachia in two continental African populations of Drosophila simulans

Wolbachia is an endocellular bacterium infecting arthropods and nematodes. In arthropods, it invades host populations through various mechanisms, affecting host reproduction, the most common of which being cytoplasmic incompatibility (CI). CI is an embryonic mortality occurring when infected males mate with uninfected females or females infected by a different Wolbachia strain. This phenomenon is observed in Drosophila simulans, an intensively studied Wolbachia host, harbouring at least five distinct bacterial strains. In this study, we investigate various aspects of the Wolbachia infections occurring in two continental African populations of D. simulans: CI phenotype, phylogenetic position based on the wsp gene and associated mitochondrial haplotype. From the East African population (Tanzania), we show that (i) the siIII mitochondrial haplotype occurs in continental populations, which was unexpected based on the current views of D. simulans biogeography, (ii) the wKi strain (that rescues from CI while being unable to induce it) is very closely related to the CI-inducing strain wNo, (iii) wKi and wNo might not derive from a unique infection event, and (iv) wKi is likely to represent the same entity as the previously described wMa variant. In the West African population (Cameroon), the Wolbachia infection was found identical to the previously described wAu, which does not induce CI. This finding supports the view that wAu might be an ancient infection in D. simulans.     

Dynamics of double and single Wolbachia infections in Drosophila simulans from New Caledonia

The bacterial symbiont Wolbachia can cause cytoplasmic incompatibility in Drosophila simulans flies: if an infected male mates with an uninfected female, or a female with a different strain of Wolbachia, there can be a dramatic reduction in the number of viable eggs produced. Here we explore the dynamics associated with double and single Wolbachia infections in New Caledonia. Doubly infected females were compatible with all males in the population, explaining the high proportion of doubly infected flies. In this study, males that carry only wHa or wNo infections showed reduced incompatibility when mated to uninfected females, compared with previous reports. These data suggest that either the DNA of these bacterial isolates have diverged from those previously collected, or the genetic background of the host has lead to a reduction in the phenotype of incompatibility. Mitochondrial sequence polymorphism at two sites within the host genome was assayed to investigate population structure related to infection types. There was no correlation between sequence polymorphism and infection type suggesting that double infections are the stable type, with singly infected and uninfected flies arising from stochastic segregation of bacterial strains. Finally, we discuss the nomenclature of Wolbachia strain designation.     

Wolbachia Infections in Drosophila Melanogaster and D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.     

The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster

A crucial factor determining sperm fertilization success in multiply mated Drosophila melanogaster females is the efficiency with which sperm are stored. This process is modulated by the accessory gland protein Acp36DE. In this study, we show that the effect of Acp36DE on sperm storage itself alters the outcome of sperm competition. As second-mating males, Acp36DE1 (null) males had significantly lower P2-values than Acp36DE2 (truncation) or Acp36DE+ (control) males, as might be expected as the null males' sperm are poorly stored. We used spermless males, which are null for Acp36DE, to show that, in the absence of sperm co-transfer, Acp36DE itself could not displace first-male sperm. The results therefore suggest that males null for Acp36DE suffer in sperm displacement because fewer sperm are stored or retained, not because Acp36DE itself displaces sperm. Acp36DE1 (null) males also gained significantly fewer fertilizations than controls when they were the first males to mate. Using spermless males, we also showed that significantly more second-male offspring were produced following the transfer of Acp36DE by spermless first-mating males. This implies that the transfer of Acp36DE itself by the first male facilitated the storage or use of the second male's sperm and that co-transfer with sperm is not necessary for Acp36DE effects on second-male sperm storage. Acp36DE may persist in the reproductive tract and aid the storage of any sperm including those of later-mating males or prime the female for future efficient sperm storage. Our results indicate that mutations in genes that affect sperm storage can drastically affect the outcome of sperm competition.     

Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus

Abstract.  Cytoplasmic incompatibility (CI) induced by maternally inherited Wolbachia bacteria is a potential tool for the suppression of insect pest species with appropriate patterns of infection. The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) is known to be infected by two strains of Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), w Alb A and w Alb B, throughout its geographical distribution. This infection pattern theoretically restricts the application of CI-based control strategies. However, Wolbachia can be horizontally transferred using embryonic microinjection to generate incompatible transfected lines harbouring a single new strain of Wolbachia. In order to assess the feasibility of this approach, the effects of Wolbachia removal on mosquito fitness need to be clearly evaluated as the removal of natural superinfection is an inescapable step of this approach. Previous research has shown that uninfected females, produced by antibiotic treatment, showed a decrease in fitness compared with those infected with Wolbachia. In this study, the effect of Wolbachia removal on male fitness was investigated. Longevity and reproductive potential (mating competitiveness and sperm capacity) were assessed in both laboratory cages and greenhouses. No differences were observed between uninfected and infected males with respect to longevity, mating rate, sperm capacity and mating competitiveness in either laboratory conditions or greenhouses. The preservation of fitness in males of Ae. albopictus deprived of natural Wolbachia infection is discussed in relation to the development of incompatible insect technique suppression strategies. Finally, the potential application of aposymbiotic males in mark–release–recapture studies is suggested.     

From parasite to mutualist: rapid evolution of Wolbachia in natural populations of Drosophila

Wolbachia are maternally inherited bacteria that commonly spread through host populations by causing cytoplasmic incompatibility, often expressed as reduced egg hatch when uninfected females mate with infected males. Infected females are frequently less fecund as a consequence of Wolbachia infection. However, theory predicts that because of maternal transmission, these "parasites" will tend to evolve towards a more mutualistic association with their hosts. Drosophila simulans in California provided the classic case of a Wolbachia infection spreading in nature. Cytoplasmic incompatibility allowed the infection to spread through individual populations within a few years and from southern to northern California (more than 700 km) within a decade, despite reducing the fecundity of infected females by 15%-20% under laboratory conditions. Here we show that the Wolbachia in California D. simulans have changed over the last 20 y so that infected females now exhibit an average 10% fecundity advantage over uninfected females in the laboratory. Our data suggest smaller but qualitatively similar changes in relative fecundity in nature and demonstrate that fecundity-increasing Wolbachia variants are currently polymorphic in natural populations.     

Within- and between-population variation for Wolbachia-induced reproductive incompatibility in a haplodiploid mite

Wolbachia pipientis is a bacterium that induces cytoplasmic incompatibility (CI), the phenomenon in which infected males are reproductively incompatible with uninfected females. CI spreads in a population of hosts because it reduces the fitness of uninfected females relative to infected females. CI encompasses two steps: modification (mod) of sperm of infected males and rescuing (resc) of these chromosomes by Wolbachia in the egg. Infections associated with CI have mod+ resa+ phenotypes. However, mod- resc+ phenotypes also exist; these do not result in CI. Assuming mod/resc phenotypes are properties of the symbiont, theory predicts that mod- resc+ infections can only spread in a host population where a mod+ resc+ infection already occurs. A mod- resc+ infection spreads if the cost it imposes on the infected females is lower than the cost inflicted by the resident (mod+ resc+) infection. Furthermore, introduction of a mod- Wolbachia eventually drives infection to extinction. The uninfected population that results can be recolonized by a CI-causing Wolbachia. Here, we investigated whether variability for induction of CI was present in two Tetranychus urticae populations. In one population all isofemale lines tested were mod-. In the other, mod+ resc+ and mod- resc+ isofemale lines coexisted. We found no evidence for a cost difference to females expressing either type (mod-/-). Infections in the two populations could not be distinguished based on sequences of two Wolbachia genes. We consider the possibility that mod- is a host effect through a population dynamics model. A mod- host allele leads to infection extinction in the absence of fecundity differences. Furthermore, the uninfected population that results is immune to reestablishment of the (same) CI-causing Wolbachia.     

The distribution and proliferation of the intracellular bacteria Wolbachia during spermatogenesis in Drosophila.

Wolbachia is a cytoplasmically inherited alpha-proteobacterium found in a wide range of host arthropod and nematode taxa. Wolbachia infection in Drosophila is closely associated with the expression of a unique form of post-fertilization lethality termed cytoplasmic incompatibility (CI). This form of incompatibility is only expressed by infected males suggesting that Wolbachia exerts its effect during spermatogenesis. The growth and distribution of Wolbachia throughout sperm development in individual spermatocysts and elongating sperm bundles is described. Wolbachia growth within a developing cyst seems to begin during the pre-meiotic spermatocyte growth phase with the majority of bacteria accumulating during cyst elongation. Wolbachia are predominantly localized in the proximal end of the immature cyst, opposite the spermatid nuclei, and throughout development there appears little movement of Wolbachia between spermatids via the connecting cytoplasmic bridges. The overall number of new cysts infected as well as the number of spermatids/cysts infected seems to decrease with age and corresponds to the previously documented drop in CI with age. In contrast, in one CI expressing line of Drosophila melanogaster, fewer cysts are infected and a much greater degree of variation in numbers is observed between spermatids. Furthermore, the initiation and extent of the fastest period of Wolbachia growth in the D. melanogaster strain lags behind that of Drosophila simulans. The possible implications on the as yet unexplained mechanism of CI are discussed.     

Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia

Abstract.— The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI). CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains. The present study focuses on this phenomenon in two Drosophila species: D. simulans and D. sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including w Ha and w No. Drosophila sechellia is infected by only two Wolbachia : w Sh and w Sn. In both Drosophila species, double infections with Wolbachia are found. As indicated by several molecular markers, w Ha is closely related to w Sh, and w No to w Sn. Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely si I (associated with w Ha and w No in D. simulans ) and se (associated with w Sh and w Sn in D. sechellia ). To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected w Sh and w Sn into D. simulans , to compare their CI properties to those of their sister strains w Ha and w No, respectively, in the same host genetic background. We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible. We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.