Ecological Contacts and Host Specificity Promote Replacement of Nutritional Endosymbionts in Ticks

Symbiosis with vitamin-provisioning microbes is essential for the nutrition of animals with some specialized feeding habits. While coevolution favors the interdependence between symbiotic partners, their associations are not necessarily stable: Recently acquired symbionts can replace ancestral symbionts. In this study, we demonstrate successful replacement by Francisella-like endosymbionts (-LE), a group of B-vitamin-provisioning endosymbionts, across tick communities driven by horizontal transfers. Using a broad collection of Francisella-LE-infected tick species, we determined the diversity of Francisella-LE haplotypes through a multi-locus strain typing approach and further characterized their phylogenetic relationships and their association with biological traits of their tick hosts. The patterns observed showed that Francisella-LE commonly transfer through similar ecological networks and geographic distributions shared among different tick species and, in certain cases, through preferential shuffling across congeneric tick species. Altogether, these findings reveal the importance of geographic, ecological, and phylogenetic proximity in shaping the replacement pattern in which new nutritional symbioses are initiated.


Introduction
Mutualisms with microbes are at the origin of animal lineages feeding on nutritionally incomplete food resources [1][2][3]. Microbes can synthesize essential amino acids and vitamins that animals cannot and thus have vital roles in compensating for nutritional deficiencies. Through this mechanism, symbiont acquisition enabled the emergence and expansion of many animal lineages, such as aphids, bed bugs, and leeches, which would otherwise not exist [1,3,4]. Over evolutionary times, hosts and nutritional symbionts coevolve traits that stabilize their interactions, often leading to strict host-symbiont co-cladogenesis [2,4]. However, nutritional symbioses can be much more dynamic: Ancestral nutritional symbionts can be replaced by recently acquired symbionts able to provide similar or additional benefits to the hosts [1,[4][5][6]. Indeed, sap-feeding insects display a mosaic of nutritional symbiont combinations, reflecting repeated symbiont acquisitions, replacements, and losses [4]. These novel symbioses typically originate either following horizontal transfer from one host species to another or from uptake of novel symbionts from the environment and thus primarily depend on the symbiont's ability to successfully shift hosts across species boundaries [4,5]. In arthropods, most of the novel nutritional symbionts evolve from intracellular or extracellular bacteria already adapted to the arthropod cell or gut environment, but not essential to host survival [4][5][6]. Yet, it remains unclear how symbionts are primarily acquired by novel host species.
Recent surveys suggest that Francisella-LE are spreading through tick communities at the expense of Coxiella-LE [7,8]. These endosymbionts rarely coexist together and a tick species hosting one usually does not harbor the other one. Phylogenetic reconstructions revealed that Coxiella-LE, but not Francisella-LE, are ancestral endosymbionts in most tick lineages: Coxiella-LE commonly form evolutionarily stable associations lasting for million years and leading to strict co-cladogenesis [7,8,18]. However, repeated replacements of Coxiella-LE by Francisella-LE are apparent across the tick phylogeny, with recent acquisitions of Francisella-LE through horizontal transfers and extinctions of ancestral Coxiella-LE in several tick lineages [7,8]. Thanks to these dynamics, at least 20% of tick species may be infected either by obligate or by facultative Francisella-LE [8].
In this study, we explored the routes of Francisella-LE horizontal transmission to determine their impact on the stability of tick nutritional symbioses. We tested for the existence of two distinct mechanisms: (1) ecological contacts and (2) specificity towards the current tick hosts. Tick species may be involved in ecological contacts either directly (i.e., feeding on same vertebrate types) or indirectly (i.e., inhabiting the same geographic region). These contacts may create primary opportunities for symbiont horizontal transfers that can result in the distribution of related Francisella-LE in unrelated tick species. Specificity towards current tick hosts may be the consequence of coevolution between the symbiotic partners and imposes a certain Francisella-LE distribution that is restricted to the certain tick species and their close relatives with which they have coevolved. Each of these mechanisms have been reported in common facultative endosymbionts of arthropods (e.g. [25][26][27][28][29], but none has been examined in the context of nutritional symbioses in ticks. To investigate these routes of transmission, we first characterized Francisella-LE diversity using a multi-locus typing system, which was recently developed to study Francisella-LE evolution in the Amblyomma tick genus [7]. Here, we extended this multi-locus analysis to a more cosmopolitan tick collection and used phylogenetic reconstructions to estimate the proximity of Francisella-LE haplotypes, including obligate and facultative endosymbiotic forms, and retraced their evolutionary histories across tick species. We also compared this diversity with other members of the Francisella genus, including virulent intracellular pathogens of vertebrates such as the agent of tularemia, F. tularensis. We next traced the network of movements of Francisella-LE among tick species by examining the association of Francisella-LE haplotypes with tick phylogeny and their geographical and ecological traits.

Tick Collection
A collection of 51 individual DNA templates obtained from 51 specimens of 14 tick species was used (Table S1). For each DNA template, tick identification and infection by Francisella-LE have been formally characterized in previous studies by molecular and/or morphological characteristics (for ticks) and single-locus DNA sequencing (for Francisella-LE) (Table S1). Of the 14 tick species examined, one belongs to the Argasidae family (soft ticks), and to the Ornithodoros genus. The 13 other species belong to the Ixodidae family (hard ticks), and to the Amblyomma (one species), Dermacentor (three species), Hyalomma (seven species), Ixodes (two species), and Rhipicephalus (one species) genera. Most specimens were collected on vegetation or on taxonomically diverse bird and mammal hosts while a few others were from a laboratory colony (Table S1). The 14 tick species were infected either by putative obligate Francisella-LE (11 species) or by facultative Francisella-LE (three species) (Table S1). The use of the genetic resources was declared to the French Ministry of the Environment (reference TREL19028117S/156).  [7]. Primers and PCR conditions are detailed in Table S2. Following visualization via electrophoresis in 1.5% agarose gel, positive PCR products were sequenced by Eurofins. Sequence chromatograms were cleaned with Chromas Lite (http:// www. techn elysi um. com. au/ chrom as_ lite. html), and alignments were performed using ClustalW, implemented in the MEGA software package [30].

Multi-locus Typing of Francisella-LE
Alleles of Francisella-LE were determined on the basis of sequence identity in nucleotide alignments. Analyses of allelic profiles included multi-locus sequences of (1) the Francisella-LE of the 51 specimens belonging to the 14 tick species characterized in this study, (2) the Francisella-LE of 25 additional specimens belonging to 12 Amblyomma species and available on GenBank from Binetruy et al.  (Table 1). Overall, this dataset included Francisella-LE multi-locus sequences from 76 tick specimens and four Francisella-LE genomes. It included data from 29 tick species (three belonging to the Argasidae family and 26 to the Ixodidae family), including 22 species for which Francisella-LE were previously identified as putative obligate endosymbionts, six species with Francisella-LE as facultative endosymbionts, and one species with a Francisella-LE of undetermined status (Table 1). Key traits of the 29 tick species (family, geographic distribution, and vertebrate types on which they usually feed) are detailed in Table S3.

Molecular Phylogenetic Analyses
Phylogenetic analyses were based on sequence alignments of single or concatenated Francisella-LE gene sequences obtained for analyses of allelic profiles. Sequences of other Francisella species (F. opportunistica: CP022377; F. tularensis: AJ749949; F. novicida: CP009633; F. hispaniensis: CP018093) obtained from GenBank were also included in the analyses. The Gblocks program with default parameters was used to obtain non-ambiguous sequence alignments [32]. All sequence alignments were also checked for putative recombinant regions using the RDP3 analysis package [33]. Tree-based phylogenetic analyses were performed using maximum-likelihood (ML) analyses using MEGA. The evolutionary models that best fit the sequence data were determined using the Akaike information criterion. Clade robustness was assessed by bootstrap analysis using 1000 replicates.
The tick phylogeny was constructed as a simplified cladogram of tick genera adapted from Burger et al. [34]. This tree was used as for some tick species used in the Francisella-LE phylogeny, there was no available tick sequence in GenBank and lack of DNA for certain ticks prevents novel sequencing. To test for associations between Francisella-LE and tick phylogenies, we used the Procrustean Approach to Cophylogeny (PACo) method [35] implemented in R (http:// www.r-proje ct. org) using the APE [36] and VEGAN [37] packages. PACo is a global-fit method that assesses phylogenetic congruence with the explicit aim to test the dependency of one phylogeny on another. For Francisella-LE, the phylogeny was obtained with the 16S rRNA, rpoB, groEL, ftsZ, and gyrB concatenated dataset as described above. To avoid spurious clustering because of multiple Francisella-LE haplotypes per host species, we conducted cophylogenetic analyses by using only one representative Francisella-LE haplotype per host species. Because all Francisella-LE haplotypes found in a same tick species are always closely related (see "Results"), we randomly sampled one Francisella-LE haplotype per host species and used it in further cophylogenetic analyses. The significance of cophylogenetic tests was established by 10,000 random permutations of the association matrix.

Statistical Analyses
We explored the association of the phylogenetic distribution of Francisella-LE haplotypes with key traits, including endosymbiosis type (obligate vs. facultative endosymbiosis), geographic distribution (Eastern Hemisphere vs. Western Hemisphere, i.e., Europe, Africa, and Asia vs. North, Central, and South Americas), tick family (Argasidae vs. Ixodidae), and feeding preferences (i.e., vertebrate classes on which the tick host species usually feed: mammals, birds, and reptiles) (Table S3). There is a great variation of feeding preference in ticks: Some tick species (A. latum and Hy. aegyptium) feed exclusively on reptiles while other species (O. moubata and D. nitens) only feed on mammals. Some species (A. maculatum and I. scapularis) are more generalist species feeding on birds and mammals, but never on reptiles. A few tick species (I. ricinus) are also generalist, feeding on birds, mammals, and reptiles (Table S3). We used the D metric [38] to estimate the overall degree of clustering of these traits on the Francisella-LE phylogeny. If a trait shows a phylogenetic signal, it may be assumed that Francisella-LE haplotypes sharing this trait are not randomly distributed over the phylogeny: They are more phylogenetically clustered than expected by chance. The D metric provides an estimate of phylogenetic conservatism for binary traits that can be compared either with a random shuffle of trait values at the tips of a phylogeny or with a Brownian motion model of evolution that allows one to depict evolutionary diversification processes along a topology [38]. A D value of 1 indicates a phylogenetically random distribution, whereas a D value inferior to 1 indicates phylogenetic clustering. A D value of 0 indicates that the trait is clustered as if it had evolved under Brownian motion of evolution (i.e., in a random dispersal with constant trait variance over time [39]), whereas a D value less than 0 indicates an extremely clustered trait [38]. To assess the significance of the D metric estimates, two p-values were furthermore calculated using permutation tests: p(D < 1) indicates whether the D metric is significantly smaller than 1, meaning that the trait is not randomly distributed over the phylogeny, and p(D > 0) indicates whether the D metric is significantly greater than 0, meaning that the trait has a significantly different distribution on the phylogeny from the Brownian threshold model of evolution. We calculated the D metric implemented by the function "phylo.d" in the R package caper [40] with the default parameter of 1000 permutations. Multiple testing was taken into account using the sequential Bonferroni procedure, according to Hochberg (1988) [41]. As done for cophylogenetic analyses to avoid spurious clustering, we calculated the D metric using only one representative Francisella-LE haplotype per host species.
We applied a second approach to examine the phylogenetic clustering of Francisella-LE haplotypes from the same tick genus and thus to test for the level of specificity of Francisella-LE. To this aim, we computed the pairwise distances between all Francisella-LE haplotypes from the concatenated ML phylogenetic tree using its branch lengths (using only one representative Francisella-LE haplotype per host species). We further partitioned this dataset into (1) pairwise phylogenetic distances between Francisella-LE haplotypes from a same tick genus (intrageneric pairwise phylogenetic distances) and (2) pairwise phylogenetic distances between Francisella-LE haplotypes from different tick genera (intergeneric pairwise phylogenetic distances). Intrageneric pairwise phylogenetic distances for a given tick genus that are lower than intergeneric pairwise phylogenetic distances indicate specificity of these Francisella-LE haplotypes. We compared intrageneric vs. intergeneric pairwise phylogenetic distances using Wilcoxon signed-rank tests.

Characterization of Francisella-LE Haplotypes
We amplified and sequenced the Francisella-LE 16S rRNA, rpoB, groEL, ftsZ, and gyrB gene sequences from the 51 DNA templates belonging to 14 tick species (Table 1). We supplemented these data with additional sequences from 15 other tick species that include genomic and multi-locus typing of Francisella-LE datasets available on GenBank (listed in

Phylogenetic and Statistical Analyses
ML analyses based on 16S rRNA, rpoB, groEL, ftsZ, and gyrB gene sequences were conducted to examine the Francisella-LE phylogeny (Fig. S1-S5). We observed no sign of recombination in the dataset (all p > 0.17 for the GENE-CONV and RDP recombination detection tests) and we thus further conducted a new ML analysis based on the 16S rRNA, rpoB, groEL, ftsZ, and gyrB concatenated dataset (Fig. 1). All but one phylogenetic reconstruction showed that the Francisella-LE, including F. persica, delineate a robust monophyletic clade within the Francisella genus (Fig. 1,  Fig. S2-S5). Only the topology of the16S rRNA gene tree is poorly resolved due to insufficient sequence polymorphism (Fig. S1). The closest relative of Francisella-LE is an opportunistic Francisella pathogen (F. opportunistica [42]), as well as other Francisella pathogens, including the agent of tularemia, F. tularensis (Fig. 1, Fig. S1-S5).
Phylogenetic reconstructions showed that the different Francisella-LE haplotypes found in the same tick species always cluster together (Fig. 1). Indeed, the four Francisella-LE haplotypes of Hy. excavatum are more closely related to each other than to any other Francisella-LE haplotype. A similar pattern was observed for the six other tick species hosting more than one Francisella-LE haplotype.
It is noteworthy that the tick phylogeny, while resolved at the genus level, lacks resolution at the level of species. To adjust for this, all congeneric tick species were arbitrarily considered here as equally distant because of the lack of data for some tick species (see "Methods"). The detection of a cophylogenetic signal, thus, implies that it is driven by congruent branching patterns at the tick genus level and higher but not necessarily with tick species. However, the diagram of the interaction network shows some major phylogenetic incongruences (Fig. 2). As can be seen, no tick genus harbors a specific and monophyletic Francisella-LE subclade: The Francisella-LE of Amblyomma are scattered among Francisella-LE of other tick genera, as best shown with the Francisella-LE of A. sculptum and A. paulopunctatum that are more closely related to the Francisella-LE of the soft ticks O. moubata and O. porcinus than to the Francisella-LE of other Amblyomma species (Fig. 1). Hence, the Francisella-LE of Amblyomma belonged to a minimum of three distinct phylogenetic clusters (Figs. 1 and 2). Similarly, the Francisella-LE of Dermacentor tick species, as well as for Ixodes species, were each scattered among two different Francisella-LE branches ( Figs. 1 and 2). In addition, we found a non-significant clustering signal for tick families (Argasidae and Ixodidae) on the phylogeny of Francisella-LE haplotypes (D = − 0.43): Their distribution on the tree is significantly random (p(D < 1) = 0.04, non-significant after sequential Bonferroni correction) and statistically distinguishable from a clustered distribution expected by Brownian motion (p(D > 0) = 0.67) (Fig. 2). Overall, these patterns are the signatures of repeated horizontal transfer events, revealing the ability of Francisella-LE to extensively move among tick species.
We found no signal of phylogenetic clustering for Francisella-LE endosymbiosis types (D = 1.81): The distribution of obligate and facultative Francisella-LE on the tree is random (p(D < 1) = 0.92) and facultative Francisella-LE are scattered along the phylogeny among obligate Francisella-LE (Fig. 2). The Francisella-LE haplotype #6 illustrates this pattern well, as it was associated with obligate endosymbiosis in A. dissimile but with facultative endosymbiosis in A. geayi and A. latepunctatum (Table S3).
In contrast to the latter comparison, the geographic origin of Francisella-LE haplotypes showed a significant phylogenetic signal (D = − 0.08, p(D < 1) = 0.008, p(D > 0) = 0.59) with a clear non-random distribution of Francisella-LE haplotypes between ticks from the Eastern and Western Hemispheres (Fig. 2). The best examples include Francisella-LE haplotypes of American Dermacentor and Ixodes species that cluster together within the same subclade, while Francisella-LE haplotypes of European Dermacentor and Ixodes species cluster together in another subclade. There are only a few exceptions to this geographical pattern as shown with the Francisella-LE haplotype of an African Amblyomma species, A. latum, which clusters within a clade otherwise only composed of American Amblyomma species (Fig. 2). We found no significant signal of phylogenetic clustering for certain vertebrates on which ticks feed (Fig. 2): Francisella-LE haplotypes do not cluster with tick species feeding on birds (D = 0.14, p(D < 1) = 0.02, non-significant after sequential Bonferroni correction, p(D > 0) = 0.39), on mammals (D = 0.78, p(D < 1) = 0.36, p(D > 0) = 0.10), or on reptiles (D = 0.99, p(D < 1) = 0.51, p(D > 0) = 0.06). However, although globally non-significant, some tick species that exclusively feed on mammals (e.g., O. moubata, O porcinus, A. sculptum, and A. paulopunctatum) harbor closely related Francisella-LE haplotypes (Fig. 2). Similarly, A. rotundatum and A. dissimile, which both feed on reptiles, harbor closely related Francisella-LE haplotypes. Exceptions are also observed on the haplotype-based tree as shown with A. longirostre: This species feeds on birds but harbors a Francisella-LE haplotype more closely related to haplotypes of Amblyomma species feeding on mammals and reptiles than to haplotypes of other tick species feeding on birds (Fig. 2).

Discussion
We identified 38 distinct Francisella-LE haplotypes, including obligate and facultative forms, from a broad collection of 29 tick species. All Francisella-LE haplotypes were clustered in a monophyletic clade nested within the Francisella genus among virulent intracellular pathogens of vertebrates. This confirms early studies showing that Francisella-LE emerged from a pathogenic Francisella ancestor of vertebrates that had evolved a specialized endosymbiotic lifestyle with ticks [9,13,14]. The distribution of current Francisella-LE haplotypes reveals how these endosymbionts spread in tick communities presumably at the expense of Coxiella-LE [7,8].

Francisella-LE Commonly Move Along Ecological Networks Connecting Tick Species
The cophylogenetic analysis revealed that some Francisella-LE haplotype groups are consistently associated with certain  Fig. 2 Cladogram depicting the 50% majority-rule consensus of Francisella-LE haplotype phylogenetic trees (left part), association with key biological traits (middle) and network association with the tick phylogeny (right part). The Francisella-LE tree was constructed with the 29 Francisella-LE haplotypes characterized in the 29 tick species used in this study and using maximum-likelihood (ML) estimations based on concatenated 16S rRNA, rpoB, groEL, ftsZ, and gyrB nucleotide sequences (3232 unambiguously aligned bp; best-fit approximation for the evolutionary model: GTR + G). Branch numbers indicate percentage bootstrap support for major branches (1000 replicates). Only bootstrap support values > 70% are shown. The tick tree is a simplified cladogram of tick genera adapted from Burger et al. [34], and all congeneric tick species were arbitrarily considered as equally distant  [13,24,43,44], suggesting that ticks could inject these endosymbionts during feeding. Ticks, unlike other arthropod vectors, often attach and aggregate on vertebrates for several days to obtain a blood Fig. 3 Intrageneric and intergeneric pairwise phylogenetic distances of Francisella-LE haplotypes. Comparisons were conducted for each tick genus harboring more than one Francisella-LE haplotype. **, p < 0.005; ***, p < 0.001; NS, not significant (p > 0.05) meal, a process termed "co-feeding". The spatiotemporal proximity of ticks during co-feeding may favor the horizontal transfers of Francisella-LE between different tick species, as commonly observed for tick-borne pathogens [45,46]. This process could also potentially lead to opportunistic infections in vertebrates, but such infection by Francisella-LE was only documented once and under laboratory conditions: The Francisella-LE of the soft tick A. arboreus, F. persica, was isolated following injection of a tick crushed into chick embryo yolk sacs, suggesting that it may be an opportunistic pathogen able to grow in vertebrate cells [47]. Using artificial infection protocols, F. persica was also shown to be slightly moderately pathogenic for the guinea pig, mouse, and newborn chick, but not for the cotton rat, adult chicken, or rabbit [47]. Interestingly, the genome of F. persica, but not those of other sequenced Francisella-LE, contains genes of the type VI secretion system (T6SS), and its associated Francisella pathogenicity island (FPI) [13] which are used for Francisella pathogenic species such as F. tularensis to infect macrophages of vertebrates [48,49]. Certain Francisella-LE may thus induce opportunistic infections in vertebrates through their T6SS and FPI virulence genes and use vertebrates as an ecological arena for transfer across tick species.

Specificity of Francisella-LE in Some Tick Genera
We observed related Francisella-LE haplotypes in Hyalomma tick species and, to a lesser extent, Amblyomma genera. Because of maternal inheritance, co-divergence between Francisella-LE and ticks may explain this pattern, as recently observed in the Hyalomma genus [24]. However, multiple lines of evidence indicate that in Amblyomma, it was the consequence of multiple horizontal transfers between congeneric species rather than of co-divergence. For instance, A. goeldi and A. humerale harbored closely related Francisella-LE haplotypes although they are not closely related species within the Amblyomma genus [7]. A similar pattern is also observed with the Francisella-LE haplotype #6 found in A. dissimile, A. geayi, and A. latepunctatum that are not related tick species although they belong to the same genus [7]. Co-divergence alone is thus insufficient to explain why related Francisella-LE haplotypes are present in certain tick genera such as Amblyomma. The pattern observed suggests instead that some Francisella-LE are specific to related tick species, preferentially moving horizontally among congeneric species. Under this scenario, Francisella-LE may be preadapted to infect related tick species because they share similar physiological traits with their current tick hosts, a pattern also observed in endosymbionts of other arthropod species [26,27]. Thus, certain Francisella-LE may have the capacity to maintain infections in only a limited range of related tick species. This level of specificity seems variable depending on Francisella-LE haplotypes: High in some tick genera (Hyalomma, Ornithodoros, Amblyomma), but not in others (Dermacentor, Ixodes). As such, this diversity of specificity levels should impact movements of Francisella-LE across tick communities. However, it is also important to point out that association of some Francisella-LE haplotypes restrictively to a particular tick genus may not imply specificity, as suggested by the presence of a Francisella-LE haplotype in R. decoloratus closely related to haplotypes found in Hyalomma species. Instead, it could simply reflect a higher chance of Francisella-LE transfer and successful establishment of the endosymbiosis among interconnected tick species: Hyalomma species examined here were from the Eastern Hemisphere, as R. decoloratus. Hence, the pattern of Francisella-LE diversity observed in Hyalomma could reflect geographical structuring (as discussed above) rather than tick specificity.

Underestimation of Francisella-LE Diversity
It is our prediction that, despite recent genotyping efforts (including the present study), the diversity of Francisella-LE is underestimated. Our sampling was highly biased towards obligate Francisella-LE since they are fixed in tick populations, and thus more easily sampled. Inversely, facultative Francisella-LE have more variable infection frequencies in tick populations [7,8], and they are rarely detected. For instance, surveys of 91 specimens of the African blue tick R. decoloratus from five distinct populations detected the presence of Francisella-LE in only one specimen [8]. This means that we have probably typed the diversity of obligate Francisella-LE well, but only of a small fraction of facultative Francisella-LE. Accounting for these missed facultative Francisella-LE, a large diversity of facultative Francisella-LE may be widely circulating, but at low infection frequencies, within tick communities.
Facultative Francisella-LE are probably pivotal to establishing novel endosymbioses. In arthropods, obligate endosymbionts commonly enter into an evolutionary route that leads to irreversible codependence with hosts and the secondary loss of the capability to move horizontally between host species [4,5]. Conversely, facultative endosymbionts have more labile interactions with their hosts and undergo occasional horizontal transfers across arthropod species [4,5], as commonly observed for diverse endosymbionts in ticks [8,50,51]. This suggests that novel nutritional Francisella-LE symbioses are initiated by facultative forms that further evolved to obligate forms. Once established as obligate endosymbionts, Francisella-LE may, however, enter into the same evolutionary route that limits any further horizontal transfers. The mixed facultative and obligate forms Ecological Contacts and Host Specificity Promote Replacement of Nutritional Endosymbionts… 785 on the Francisella-LE tree with no clear phylogenetic signal of clustering suggests that the transition from facultative to obligate forms is a common feature for these endosymbionts.

Concluding Remarks
This study confirmed that nutritional symbiosis in ticks is not a stable evolutionary state, but rather a dynamic system impacted by repeated acquisition of novel potential nutritional endosymbionts through horizontal transfers. Such an evolutionary succession of nutritional symbionts is more widespread than previously appreciated. In louse flies, it was observed with the recent acquisition of a Sodalis endosymbiont [52]. In sucking lice, there are independent lineages of B-vitamin-provisioning endosymbionts [53][54][55][56], suggesting several replacement events. Such pattern was also commonly observed in sap-feeding insects as in Cinara aphids [57] or Coccoidea scale insects [58] which display varied nutritional symbioses, reflecting repeated symbiont acquisitions, replacements, and losses. However, while it is clear that horizontal transfers create opportunity for new nutritional endosymbiosis to emerge, the biological features favoring the transfers are unclear. In this study, we have determined that ecological networks within tick communities, along with variable levels of Francisella-LE specificity to their current tick hosts, are important drivers of this symbiont's dynamics. These endosymbionts notably combine maternal inheritance with infectious transmission between tick species in a number of cases but also potentially using vertebrates as occasional hosts. The overall probability that such tick-to-vertebrate transfers of Francisella-LE occur may be high because ticks are found worldwide and feed on many different hosts. However, apart from cases obtained in artificial conditions with F. persica [47], all other Francisella-LE described to date seem to be confined to ticks. Nonetheless, future research will be necessary to describe the global diversity of Francisella-LE, to characterize the presence of virulence genes in their genomes, and then to assess the potential infection risk to vertebrates. Further investigation on endosymbiont genomes may also reveal the mechanisms providing the advantage to Francisella-LE over Coxiella-LE. Beyond B vitamins, the replacement of Coxiella-LE suggests that Francisella-LE could supply an additional benefit that Coxiella-LE was unable to supply to ticks, thereby outcompeting them. The genomes of some Coxiella-LE have lost most of their gene contents, being usually small in size and dense in gene content but also suffering Muller's ratchet, with fixation of deleterious mutations through genetic drift [1]. Coxiella-LE may have evolved overly reduced (degraded) genomes and become maladapted, opening the road to replacement by a Francisella-LE.