Multiple long-range host shifts of major Wolbachia supergroups infecting arthropods

Wolbachia is the most widespread endosymbiotic organism in arthropods. One of the main features thought to be responsible for its successful long-term persistence in nature is its ability to manipulate host physiology and specifically host reproductive biology, conferring fitness benefits to Wolbachia and eventually to its host, including, for instance, increased pathogen resistance29. Maternal transmission, or vertical transfer, is the main process used by Wolbachia to infect a new host offspring, which, through evolutionary time, may allow these bacteria to prevail in different host species. Additionally, Wolbachia infection also can occur via hybridization and introgression of similarly related species, or by HS between closely and distantly related species30.

Although Wolbachia HS is a well-documented phenomenon6,7,18,31,32,33,34a large amount of the literature depicts it as a rare event19.20. Our comparative genomic analyzes of several Wolbachia strains and their hosts reinforce the occurrence of HS in these bacteria, showing many cases in which different host species share Wolbachia more similar than would be expected by long-term coevolution of vertically transmitted endosymbionts with their hosts. However, the novel finding of our data is that HS, at least for Wolbachia supergroups A and B, seems to be more frequent than expected.

Six out of 17 host species bearing Wolbachia supergroups A and B showed Wolbachia similarity higher than 95%, pointing out that this Wolbachia was shared by HS very recently, even between phylogenetically distant host taxa as Hymenoptera, Coleoptera and Diptera (Fig. 3a). Additionally, for supergroup B, four host species as phylogenetically distant as Acari, Diptera and Hemiptera share a Wolbachia lineage that is more than 93% similar at the nucleotide level (Fig. 3b). Therefore, from the 17 host species analysed, at least 10 (58.8%) shared Wolbachia lineages by HS. Thus, we ask: is HS a rare phenomenon in Wolbachia evolution?

Figure 3
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Wolbachia similarity between different hosts. The high Wolbachia similarity between distant related hosts is a strong evidence of HS since there is no feasible way of vertical transfer of Wolbachia between those hosts. ws, Wolbachia similarity.

HS depends on specific environmental conditions to happen, alongside the ability of a Wolbachia strain to infect a new host and maintain the infection7. It has been hypothesized that the closer the phylogenetic relationship of the hosts, the more likely HS is to occur34which may induce novel phenotypes in the new host18. The underlying mechanisms of HS are not yet fully understood, leading it to be overlooked on many occasions.

Wolbachia migrates from somatic tissues to germline cells during the host’s development, transferred by cell-to-cell contact via phagocytic/endocytic machinery. Yet, in cell culture, Wolbachia can infected Wolbachia-free cells independently of cell contact through the culture medium31. infection by Wolbachiawhich is present in the haemolymph, can occur by contact with excretions or injuries of an infected host to an uninfected host34; thus, shared food sources and feeding habits are plausible pathways for Wolbachia HS between different hosts35. Another factor contributing to Wolbachia HS is predation, where ingested larvae contaminate the uninfected host, crossing the digestive system epithelium and colonizing the future ovarian stem cells36. Parasitoid-host interactions are well documented as another route Wolbachia uses to move between species12,15,18. Among the organisms analyzed in the present study, some already showed previous evidence of HS, and are either parasitoids, eg, Diachasma alleum11or parasitized by a parasitoid, for example in Drosophila melanogaster and other Drosophila species4. HS through such interactions reinforce them as a viable mechanisms of direct Wolbachia transfer on a short time scale. It is important to note that, in field samples, the Wolbachia detected on a host may be due to sequencing reads derived from another species that are closely associated with the primary investigated host such as endoparasitoids. Par exemple, Wolbachia detected in Ixodes ricinuswhich were actually from its endoparasitoid Ixodiphagus hookeri37and the detection of Wolbachia from Strepsiptera found in the Australian tephritid fruit flies38. Although this may occur, it should not affect the general HS pattern identified, since there is no evidence that most of the host species analyzed have endoparasitoids. Also, by the amount of data analyzed in our work and the detection of high similarity between many different species as we present here, it would be very unlikely that it is the case here, thus causing any sort of analysis bias.

The phylogenetic patterns of Wolbachia and its hosts usually show incongruences, indicating recent HS events and successful infection of new host species30. We found several instances of incongruences in the phylogenetic trees of Wolbachia and its hosts (Supplementary Fig. 1), reinforcing the presence of HS. Moreover, our similarity analysis showed that different Wolbachia show high levels of similarity within the group for both supergroup A and B (Supplementary Tables 2 and 3), whilst host similarity was lower, indicating that HS is very likely to occur in natural environments, as previously suggested32.

The order Coleoptera dates from more than 250 million years ago (mya), and the Diptera order around 200 mya39. In our analysis, the supergroup A of Wolbachia from both the Coleoptera D. virgifera and Diptera D. melanogaster showed very high similarity (Fig. 3a), considering that supergroup A dates from 76 mya40; HS presents itself as a strong hypothesis to explain the high similarity of Wolbachia from remotely related hosts. The same rationale is applied when comparing the Hemiptera (an order dating from nearly 350 mya) D.citri and A. albopictus (Diptera), in which their respective Wolbachia from supergroup B (dating from around 112 mya) also shows high similarity (Fig. 3b).

In the process of genome assembly of eukaryotic organisms, a common step is the removal of bacterial sequences. This process, although important for these studies, reduces the possibility of a proper assessment of symbionts HS18, which may be related to claims of HS not being a common event. In our study, using publicly available data, we calculated the within groups similarity of Wolbachia from supergroups A and B, tracing a parallel with their hosts’ similarity. The data showed that many Wolbachia from distantly related hosts share high similarity, while their hosts’ core gene similarity is significantly lower, alongside a divergence between host and Wolbachia phylogenetic trees. We found that 58.8% of host species analyzed share two particular Wolbachia lineages, indicating that these lineages have been acquired by HS recently and suggesting that HS events may be more frequent than previously thought. This is evidence for the HS hypothesis being a common outcome of different ecological interactions, explaining at least partially how Wolbachia became such a ubiquitous organism across multiple clades. In addition, epidemiological modeling of Wolbachia transmission demonstrated that it would not be possible to explain Wolbachia incidence in a broad range of clades only considering it as vertically transmitted41thus it is necessary to take host shift into account to explain the spread of Wolbachia in phylogenetically distant hosts.

Wolbachia HS is a known event described by a wide range of literature4,6,7,14,15,32,33yet it is still somewhat overlooked and sometimes disbelieved as a more common mechanism19,20,30as it is still not very clear how it is established in some cases13. Nevertheless, Wolbachia has an arsenal of well described methods to thrive when first encountering a new host, which may explain its success jumping across clades by HS6. This arsenal consists of the facts that Wolbachia has no problem adapting to new environments7can, without much effort, move across cells and tissues, as it is a proficient manipulator of its hosts physiology6.42. Even though Wolbachia may cause reduced host fitness, the opposite is also true, as Wolbachia may alter pathogen susceptibility conferring viral protection for its hosts43. Also, Wolbachia can survive for a limited time in an extracellular environment, albeit being an obligatory intracellular endosymbiont12.35.

By using gene similarity of over 1000 reconstructed genomes21alongside a phylogenetic reconstruction, we were able to bring focus to Wolbachia HS, estimate the event and compare it in Wolbachia supergroups A and B of close and distant related hosts and their Wolbachiashedding more light on the importance of HS as a major player in Wolbachia pervasiveness on very distinctive branches of the Arthropoda tree.

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