key: cord-0257374-l0z79udh
authors: McKee, Clifton D.; Bai, Ying; Webb, Colleen T.; Kosoy, Michael Y.
title: Bats are key hosts in the radiation of mammal-associated Bartonella bacteria
date: 2020-04-05
journal: bioRxiv
DOI: 10.1101/2020.04.03.024521
sha: 3b0b0f730fbd22ac5857143b28950a28e7b458e1
doc_id: 257374
cord_uid: l0z79udh

Bats are notorious reservoirs of several zoonotic diseases and may be uniquely tolerant of infection among mammals. Broad sampling has revealed the importance of bats in the diversification and spread of viruses and eukaryotes to other animal hosts. Vector-borne bacteria of the genus Bartonella are prevalent and diverse in mammals globally and recent surveys have revealed numerous Bartonella lineages in bats. We assembled a sequence database of Bartonella strains, consisting of nine genetic loci from 209 previously characterized lineages and 121 new cultured strains from bats, and used these data to perform the most comprehensive phylogenetic analysis of Bartonella to date. This analysis included estimation of divergence dates using a molecular clock and ancestral reconstruction of host associations and geography. We estimate that Bartonella began infecting mammals 62 million years ago near the Cretaceous-Paleogene boundary. Additionally, the radiation of particular Bartonella clades correlate strongly to the timing of diversification and biogeography of mammalian hosts. Bats were inferred to be the ancestral hosts of all mammal-associated Bartonella and appear to be responsible for the early geographic expansion of the genus. We conclude that bats have had a deep influence on the evolutionary radiation of Bartonella bacteria and their spread to other mammalian orders. These results support a ‘bat seeding’ hypothesis that could explain similar evolutionary patterns in other mammalian parasite taxa. Application of such phylogenetic tools as we have used to other taxa may reveal the general importance of bats in the ancient diversification of mammalian parasites. Significance statement Discovering the evolutionary history of infectious agents in animals is important for understanding the process of host adaptation and the origins of human diseases. To clarify the evolution of the Bartonella genus, which contains important human pathogens, we performed phylogenetic analysis on a broad diversity of Bartonella strains, including novel strains from bats. Our results indicate that Bartonella clades diversified along with their mammal hosts over millions of years. Bats appear to be especially important in the early radiation and geographic dispersal of Bartonella lineages. These patterns are consistent with research indicating a chiropteran origin of important human viruses and eukaryotic parasites, suggesting that bats may play a unique role as historical sources of infections to other hosts.

A central part of the work done by disease ecologists is to understand the host range of infectious agents. However, host ranges must be understood in a coevolutionary context, specifically how agents have adapted to and diversified in hosts over time. Only by considering both ecological and evolutionary context can we understand how agents come to infect and adapt to new hosts. While cophylogeny is a common tool for studying the codiversification of hosts and parasites, few studies have examined the relative timing of the diversification of parasite lineages in parallel with that of hosts (1, 2).

The genus Bartonella is an excellent study system for disease ecology and evolution because it is common and diverse in many mammalian hosts (3) . These alphaproteobacteria are facultative intracellular pathogens that can cause persistent, hemotropic infections in their hosts. Transmission between hosts occurs through a variety of hematophagous arthropod vectors, wherein bartonellae colonize the midgut and are then shed in arthropod feces (4) . Clades of Bartonella species tend to be host-specific (5) , so it could be hypothesized that the genus diversified along with its mammalian hosts millions of years ago. However, there have been few comprehensive phylogenies of this genus and limited research on the influence of particular host groups on Bartonella evolution.

Bats are a group of special interest because they have traits that are amenable to parasite transmission, including their global distribution, ability to fly, seasonal migration, dense aggregations and high sociality in some species, long life spans, and the use of torpor and hibernation (6) . There is also evidence that chiropteran immune systems are highly tolerant of infections, especially of viruses (7) . Thus, their role as reservoirs for Bartonella bacteria may be uniquely influential among mammals.

Bats are also an ancient clade of mammals (8) , providing ample time for diversification of bacterial parasites and transitions from bats to other mammals. Research has concluded that bats are potentially ancestral hosts that influenced the diversification and spread of coronaviruses (9) , lyssaviruses (10), paramyxoviruses (11) , trypanosomes (12) , and haemosporidia (13) among other mammalian orders.

Drawing from Hamilton et al. (14) , who developed the 'bat-seeding' hypothesis to explain the geographic and host distribution of Trypanosoma lineages related to the agent of Chagas disease, T.

cruzi, we hypothesize that bats may have also been influential in the ancient diversification and spread of Bartonella.

Successful amplification of Bartonella DNA from recent fossils points to a prolonged history of Bartonella infection in some hosts, such as humans and domestic cats (15) . However, it is unlikely that DNA could be successfully amplified from more ancient fossils to test hypotheses about the origin of bartonellae in mammals. Instead, a molecular clock approach can be used to estimate the rate at which substitutions accumulate in Bartonella DNA and then extrapolate divergence dates of clades.

Considering new research has shown that mammal-associated bartonellae evolved from arthropod symbionts (16) , we rely on a molecular clock for the 16S ribosomal RNA (rRNA) gene based on sequence divergence data from bacterial symbionts of arthropod hosts separated for millions of years (17) . We perform a multi-locus analysis using the most comprehensive database of Bartonella strains to date, including a greater number of loci than a recent time tree analysis (18) and broader taxon sampling than previous genomic analyses (19) . Many new Bartonella strains have recently been discovered in bats (20) , so we have included 121 novel strains of bats in this study to amend current delineation of Bartonella clades (19) and to determine the influence of bats on the diversification and spread of Bartonella bacteria to other mammalian orders.

Using this molecular clock approach, we extrapolate when the genus Bartonella diversified and compare the timing of Bartonella clade diversification along with their hosts. We hypothesize that mammal-infecting bartonellae evolved with their hosts starting in the late Cretaceous or early Paleogene when many eutherian and metatherian taxa diversified (21) . We expect to see clustering of lineages associated by host orders and correlation between diversification dates of hosts and Bartonella clades. Using ancestral state reconstruction and network analysis, we discern which orders of mammals were highly influential in the diversification and spread of Bartonella to other host orders and geographic regions. We predict that the speciose orders of bats (Chiroptera) and rodents (Rodentia) are important in the historical expansion of the Bartonella genus, however bats may have a more profound influence in this process because of their ability to fly and quickly disperse over wide areas. This study provides a more complete understanding of Bartonella evolution and biogeography and the role of bats as important hosts of pathogens through a suite of phylogenetic methods that can be adapted to understand these processes in other host-specific parasites and symbionts. Such investigations could lead to a deeper evolutionary understanding of symbiosis and parasitism and the identification of key host groups in the diversification and spread of these organisms. 

To balance the need for increased taxon sampling and adequate sequence data to produce a well-supported phylogeny, we assembled a database of Bartonella sequences from published genomes on GenBank, previous studies using multi-locus sequence analysis (MLSA), and archived cultures from bats. We targeted nine genetic markers (SI Appendix, Table S1 ) commonly used for Bartonella detection and phylogenetic analysis (22) . Data from MLSA studies and genomes published as of 2018 were collected from GenBank via accession numbers or strain numbers from 74 studies (SI Dataset 1), including recent publications that have isolated bartonellae or related bacterial symbionts in arthropods and past studies characterizing bat-associated Bartonella strains from Asia, Africa, and North America.

We excluded any strains that were noted in the studies as showing evidence of homologous recombination between Bartonella species to prevent issues with incomplete lineage sorting in phylogenetic analysis. Additional molecular data collection of Bartonella strains from bats included a subset of cultures archived in our laboratory from previous studies in Africa, North and South America, 

Bayesian phylogenetic analysis was performed using BEAST v1.8.4 (23) via the CyberInfrastructure for Phylogenetic RESearch (CIPRES) Science Gateway portal v3.3 (24) . The nine loci were analyzed separately using GTR+I+G sequence evolution models, estimated base frequencies, four gamma rate categories, an uncorrelated relaxed clock with an exponential distribution of clock rates along branches for each locus, and a birth-death speciation model with incomplete sampling (25) .

Brucella abortus was set as the outgroup in all analyses. To determine a clock prior for the 16S rRNA locus, we analyzed published 16S rRNA sequence divergence and host divergence times for bacterial symbionts of arthropods (17) . A linear regression model was fit to the data in R (26) and a lognormal prior was estimated by moment matching to the normal distribution for the fitted mean and standard error of the slope (SI Appendix, Fig. S5 ). The prior distribution for the exponential clock rate for 16S rRNA was set to this lognormal distribution while prior distributions for the exponential clocks of the remaining eight loci were set to an approximate reference prior for continuous-time Markov chain (CTMC) rates (27) . Thus, the 16S rRNA clock acts a strong prior and the rates for the other eight loci are estimated relative to the 16S rRNA rate. This approach allows for external validation of Bartonella diversification events based on host diversification dates without explicitly using host diversification dates as calibration points for the parasite tree. Extensive testing using alternative substitution (with or without codon partitioning), clock, and tree models and subsets of genetic data determined that model choice or the exclusion of the ITS locus had little influence on tree topology and estimated divergence dates (SI Appendix, Table S5 ). Additional details regarding model priors and run settings can be found in SI Appendix. 

In addition to divergence time estimation, we performed ancestral state reconstruction in BEAST. We assigned discrete traits to each tip based on the taxonomic order of the host and the ecozone (28) that includes the majority of the host's geographic range. The association of some Bartonella lineages with arthropods and not mammals are justified in SI Appendix. Ancestral state reconstruction was performed using a symmetrical rate model to reduce the number of state transitions that needed to be inferred.

We performed tip-association tests using the Bayesian Tip-association Significance testing (BaTS) program v1 to assess the clustering of traits along tips of the phylogenetic tree (29) . We performed four sets of simulations using the same assignments of host orders and geographic ecozones used in the ancestral state reconstruction above. The two sets of traits were simulated on 1000 posterior sampled trees from the final BEAST run and on the single maximum likelihood (ML) tree. Clustering of traits was measured by the association index (AI) and parsimony score (PS), producing a distribution for the 1000 Bayesian trees and a single value for the ML tree. Null distributions for these measures were generated using 100 randomizations of traits onto tips of the trees. The significance of clustering was evaluated based on the overlap between observed values or distributions of AI and PS and their null distributions. For both measures, small values indicate a stronger phylogeny-trait association (29) .

We defined host-associated Bartonella clades a posteriori based on high posterior support (>0.9) and clustering by host orders from the ancestral state reconstruction (Fig. 1A) . Previous analyses of Bartonella host associations have shown that host-switching is common (30) , so a calibration approach that assumes strict cospeciation across the tree would not accurately reflect the evolutionary history of these bacteria. However, Bartonella lineages are broadly host-specific within orders (18) and host-switching is more frequent between closely related hosts (31) . We defined 15 host-associated Bartonella clades (Tables S5-S6 ) at relevant taxonomic scales below the order level to test the hypothesis that Bartonella lineages diversified with their hosts while accounting for frequent hostswitching that could occur within a host clade. We collated divergence dates for the most recent common ancestor uniting the host taxa of interest within each clade from available studies in the TimeTree database (http://timetree.org/), summarized by the estimated mean, 95% confidence intervals, and range of dates across studies (32) . We then correlated these mean host divergence dates with our estimated median divergence date of the associated Bartonella clade (Table S7) . A significant linear fit between these dates would support the hypothesis that Bartonella diversified within their hosts after colonization. To validate measurement of the divergence time for mammal-associated Bartonella clades with the ultrametric tree produced in BEAST, we also generated a calibrated timed phylogeny with the ML tree. Using the RelTime relative rate framework (33) within MEGA v10.0.5 (34) we generated a timed phylogeny using host clade divergence dates from TimeTree (Table S7) . We used confidence intervals (or ranges in the case of clade J) for the 15 host clade divergence dates as minimum and maximum divergence dates in RelTime. The program then calculated divergence dates on the tree using a maximum likelihood approach (33), producing mean estimates and 95% confidence intervals for clade dates that we could compare with the eubartonellae date estimated in BEAST. This analysis can confirm that the date estimation is robust to different approaches by comparing a calibration-based method on an existing tree to a method that relies on relaxed clock priors during tree estimation. 

To determine the inferred ancestral host order and ecozone of mammal-infecting eubartonellae, we initially inspected the results of the ancestral state reconstruction on the maximum clade credibility (MCC) tree. Specifically, we inspected the posterior support for the node and the posterior probability of the host order and ecozone at the node across all posterior trees. However, due to the large number of Bartonella lineages associated with Chiroptera in the database (n = 160) relative to those in other diverse orders (Rodentia, 87; Artiodactyla, 32; Carnivora, 21), we tested the influence of this sampling bias on uncertainty about ancestral states using stochastic character mapping of host orders and ecozones onto trees (35) . We wrote a custom R function to resample tips from the phylogenetic tree and perform stochastic character mapping on the pruned tree using the packages ape and phytools (36, 37) assuming an equal-rates model. The function ran 100 mapping simulations on each pruned tree and calculated the probability that Chiroptera and Palearctic were the inferred host order and ecozone at the node uniting eubartonellae. These states were chosen based on initial reconstructions from BEAST indicating them as ancestral traits. We performed this simulation using three resampling schemes: equalizing the number of tips associated with bats and rodents (n = 87), equalizing tips associated with bats, rodents, and artiodactyls (n = 32), and equalizing tips associated with bats, rodents, artiodactyls, and carnivores (n = 21). Resampling schemes were run with 100 resampling iterations on the MCC tree and 10 resampling steps on 10 randomly sampled posterior trees. We summarized the resulting probability distributions by the mean and interquartile range (SI Appendix, Table S11 224 S12). Separate host order and ecozone networks were then built from these median transitions, and node-level properties including degree, out-degree, and betweenness centrality were calculated using the R package igraph (38) .

Using molecular data from nine genetic loci sequenced from 331 Bartonella strains (SI Appendix, Table S1 ), we produced a well-supported Bayesian phylogeny ( Fig. 1; SI Appendix, Fig. S8) that confirmed monophyletic clades of Bartonella species identified in past studies (19) . These during the Paleogene. Estimates of divergence dates using alternative substitution, tree, and clock models placed the origin of mammal-infecting eubartonellae between 57-70 mya (SI Appendix, Table   S4 ).

Following the hypothesis that the Bartonella genus radiated with their mammal hosts, we performed tip-association tests to analyze the clustering of host taxonomic traits and geographic origin along the tips of the tree. Simulations using 1000 posterior sampled trees showed significant clustering of host orders and geographic ecozones across the phylogeny according to association indices (AI) and parsimony scores (PS). Observed distributions for both measures did not overlap their respective null distributions based on random associations of traits to tips (SI Appendix, Table S10). Host orders had smaller values for AI and PS than geographic origin, indicating a stronger phylogeny-trait association with host taxonomy than geographic origin. This phylogeny-trait association with host taxonomy is illustrated in Fig. 1A through strong support for monophyletic groups associated with host orders.

We clarified this association with host taxonomy by describing 15 Bartonella clades (SI Appendix, Tables S5 and S6) predominantly associated with marsupials (B), ruminants (C), carnivores (F), rodents (E, H, I, J, K, M, O), and bats (Fig. 1A) . We then compared divergence dates of each Bartonella clade with divergence dates of the associated hosts within each clade (SI Appendix, Table   S7 ) collated from TimeTree (32). We found a strong correlation between Bartonella and host clade divergence times (R 2 = 0.72, F = 36.4, P < 0.0001). However, most (13/15) Bartonella clades were younger than their associated host clades; on average, the age of Bartonella clades was 76% that of their associated host clades (Fig. 2) .

The Bayesian tree used in these analyses was similar to a maximum likelihood (ML) tree produced from concatenated sequences of all nine loci, with only minor differences in topology for Fig. S7 ). Tipassociation tests using the ML tree showed similar results to the Bayesian tree (SI Appendix, Table   S10 ). Using confidence intervals for host clade divergence dates provided from TimeTree as calibration dates on the ML tree within the RelTime relative rate framework (39), we estimated the origin of mammal-infecting eubartonellae at 66.3 mya (95% CI: 63.5-69.1). This separate analysis validates the Bayesian relaxed clock estimate (SI Appendix, Table S5 ) and further supports the inference that Bartonella began diversifying with mammals near the Cretaceous-Paleogene boundary.

Bats appear to be highly influential in the diversification and spread of Bartonella geographically and to other host orders. Bat-associated clades (A, D, G, L, N) are broadly distributed across the tree and form external branches to clades associated with other mammalian orders (Fig. 1A ).

This contrasts with clades associated with marsupials, ruminants, carnivores, and rodents, which are less dispersed on the tree and stem from more internal branches. Based on ancestral state analysis using host orders as states, bats were inferred to be the ancestral host of all mammal-infecting eubartonellae with a posterior probability of 0.99. Due to the large number of bat-associated strains in the database (n = 160), this inference of the ancestral host may have been biased towards bats. Yet in all resampling scenarios, the median posterior probability that bats are the ancestral hosts of mammal-infecting eubartonellae exceeded 0.9 (SI Appendix, Table S11 ). In further support of this inference, the diversification of mammal-infecting Bartonella started almost exactly when bats began their evolutionary radiation around 62 mya (95% CI: 59-64, range: 51.9-74.9) according to compiled studies from TimeTree (32) .

In addition to ancestral host associations, we also inferred the ancestral biogeography of Table S11 ). Regardless of the exact geographical origin, it is probable that bats have been influential in the ancient geographic spread of Bartonella infections ( Fig. 1) .

We explored the influence of particular hosts on the spread of Bartonella among mammalian orders and across ecozones using stochastic character mapping and network analysis. After mapping the number of host and ecozone transitions across 1000 posterior sampled trees, we built a network consisting of host and ecozones as nodes and the median number of transitions between nodes as edges ( Fig. 3 ; SI Appendix, Table S12 ). In general, the ecozone network was more highly connected than the host network (Fig. 3) . The higher number of connections in the ecozone network corresponds with the results of the tip-association tests (SI Appendix, Table S10), which showed that clustering of traits was stronger for host taxonomy than geographic origin. That is, the high frequency of transitions between ecozones leads to lower levels of geographical clustering on the tree.

Examining the network properties of the nodes, we find that certain host orders are influential in the spread of Bartonella among host orders (SI Appendix, Table S13 ). In particular, we considered degree (the number of edges connected to a node), out-degree (the number of edges originating from a node), and betweenness (the number of shortest paths that connect any two nodes in the network that pass through the node in question) because these measures describe how each node serves as a source of Bartonella to other nodes. Bats and rodents were a source to other mammalian orders (Fig. 3A) , with the highest degree and out-degree of all host orders and high betweenness (SI Appendix, Table S13 ).

Transitions between ecozones show that the historical movement of Bartonella by hosts led to the present global distribution of these bacteria (Fig. 1B) through bidirectional exchange (Fig. 3B ).

Palearctic and Indo-Malayan ecozones showed the highest degree, out-degree, and betweenness. Thus, these two regions may have played an important role as geographic hubs for Bartonella diversification and movement of hosts to other ecozones ( Fig. 1B; SI Appendix, Fig. S8B ).

Bartonella is a broadly distributed bacterial genus associated with mammals and arthropod vectors globally. Patterns of host-specificity and phylogenetic diversity in this genus reflect general trends in other zoonotic pathogens. Thus, Bartonella serves as a model system for understanding the evolution and ecology of zoonotic agents. Specifically, this system could inform theory about how agents adapt to and diversify in hosts over time and the ecological conditions that lead to accidental infections and host-switching. Using a multi-faceted analytical approach, this study answered several key questions about the evolution of Bartonella bacteria. First, we found that the Bartonella genus began diversifying with mammals around the Cretaceous-Paleogene boundary. Our novel approach used a strong relaxed clock prior on the 16S rRNA locus based on substitution rates observed in bacterial symbionts of arthropods (17) while accounting for rate variation at eight other genetic loci to yield a highly supported phylogenetic tree with estimated divergence dates. Second, we showed that Bartonella clades diversified along with their mammalian hosts. Ancestral state reconstruction on the phylogenetic tree showed that Bartonella lineages tend to cluster by host taxonomic orders and this clustering was found to be significantly higher than random expectations using tip-association tests.

Additionally, we found a significant correlation between the divergence times of 15 Bartonella clades and their associated host clades. A separate time tree estimation approach calibrated using these host divergence dates confirmed the dating of eubartonellae diversification. The use of ancestral state reconstruction or stochastic character mapping of host traits paired with network analysis is a nascent approach in the study of infectious agents that can provide additional insights from phylogenies (41) (42) (43) . These analyses demonstrated that bats have been key to both the origin and spread of Bartonella among other mammals and geographic regions, while rodents were responsible for additional spread.

This work elucidates key aspects of the ecology and evolution of Bartonella, yet there are several avenues of research to be explored in future studies.

One necessity is to thoroughly catalog Bartonella diversity. While description of Bartonella species was slow through the 20 th century, the advent of genetic sequencing has brought about an explosion of Bartonella diversity with over 40 named and likely many other unnamed species. Our phylogenetic analysis used the most comprehensive sequence database to date, including broad taxon sampling of Bartonella strains characterized from 10 mammalian orders. These data, along with a relaxed clock approach, have reshaped the Bartonella phylogeny, defining five new clades of batassociated Bartonella strains and reorganizing the relationships of deeply branching clades. Attempts to culture and characterize novel Bartonella strains from undersampled mammalian orders or other potential vertebrate hosts (e.g., birds (44) ) are needed to further improve taxon sampling. This continued work will undoubtedly reshape the Bartonella tree further and may lead to new hypotheses about ancient associations with hosts.

Our results also provide context to the biological changes that are associated with the shift of Bartonella bacteria from an arthropod symbiont to a mammal parasite. Our phylogeny reaffirms work demonstrating this shift (16, 18) and provides an estimated time for when it occurred, suggesting that an existing bacterial population colonized a new niche in mammals shortly after their emergence as potential hosts. Some of the molecular machinery that could have facilitated this colonization was (16, 19, 45, 46) . Secretion systems have only been detected and characterized in a few Bartonella species across the phylogeny, so our revision of Bartonella tree topology highlights a need for future work regarding the machinery (e.g., flagella, T4SS) shared between bat-associated lineages and their relatives.

Given that current mammal-associated bartonellae are vectored by blood-feeding arthropods and ancestral bartonellae were likely arthropod symbionts, it is probable that early adaptation to bloodfeeding arthropods facilitated the colonization of the mammalian bloodstream. Hematophagous arthropods frequently harbor endosymbionts to cope with their nutritionally deficient diet (47) Bartonella lineages in arthropods and confirm potential transmission routes between mammal hosts and arthropod vectors will clarify the evolution of host-vector-Bartonella relationships.

As apparent in Figs. 1 and 3 , the evolutionary history of Bartonella has involved several hostswitching events. Thus, calibrating divergence dates by relying on codivergence between host taxa would poorly reflect this history. Instead we initially avoided a calibration approach in favor of using a relaxed clock prior, then validated estimated divergence dates based on 15 radiation events within particular bat, rodent, ruminant, and marsupial host taxa. The Bartonella divergence dates correlate strongly with the host divergence dates, although with a widespread delay in the colonization of Bartonella within a clade (Fig. 2) . While it is possible that this delay in Bartonella colonization is associated with the divergence date estimation approach and bacteria diverged immediately along with their hosts, we suspect the delay reflects some biological reality. According to Manter's rules (56, 57) , parasites evolve more slowly than their hosts due to the relatively uniform environments they experience within a host. This slow evolution may help to explain rampant Bartonella host-switching between related hosts in the tree, since from a parasite's perspective the intracellular environments of phylogenetically similar hosts are unlikely to have significantly changed. Despite these inherent delays, the clustering of Bartonella strains with host orders and particular clades within those orders along with the correlation of divergence times strongly suggest a shared evolutionary history between Bartonella strains and their hosts, although a more complicated one than simple cospeciation.

Beyond patterns of codiversification, it is clear from this study that Bartonella evolution has been shaped by certain hosts, particularly rodents and bats. As the two most speciose groups of mammals, they could be expected to host diverse parasites according to Eichler's rule (58) , which predicts positive covariance between host and parasite diversity. While more studies will need to be done to explicitly test patterns of host and Bartonella diversity while accounting for sampling biases, it associated clades in the growing diversity of trypanosomes, Hamilton and others hypothesized that bats may have been highly influential in the geographic spread of the T. cruzi clade and host-switching to other mammals (14) . This 'bat-seeding' hypothesis has continued to gain support since it was proposed with the discovery of diverse lineages in the T. cruzi clade in bats globally (12, 41) . Similar patterns have been noted in malarial parasites (Haemosporida), wherein the transition from sauropsids into mammals likely occurred only once, with bats being a possible bridge to other mammals (13, 65) . In light of the results of this study and the patterns in other systems, we contend that the 'bat-seeding' hypothesis may apply more widely among mammalian parasites. Our approach using comprehensive phylogenetic analysis, estimation of divergence times, and ancestral reconstruction of host associations could be applied to understand the evolutionary radiation and host-switching patterns of these parasites, and potentially the role that bats have played in their diversification. between the outgroup (Brucella abortus) and between mammalian orders and arthropods have been removed for clarity. All transition counts with a median above zero are shown in SI Appendix, Table   S9 . 

Fleas (Siphonaptera) are Cretaceous, and evolved with Theria

Bedbugs evolved before their bat hosts and did not co-speciate with ancient humans

Ecological associations between bacteria of the genus Bartonella and mammals

Intruders below the radar: molecular pathogenesis of Bartonella spp

Insights in Bartonella host specificity

Bats: important reservoir hosts of emerging viruses

Novel insights into immune systems of bats

Speciation dynamics during the global radiation of extant bats

Origin and evolution of pathogenic coronaviruses

Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders

Bats host major mammalian paramyxoviruses

Trypanosoma rangeli is phylogenetically closer to Old World trypanosomes than to Trypanosoma cruzi

The polyphyly of Plasmodium: comprehensive phylogenetic analyses of the malaria parasites (order Haemosporida) reveal widespread taxonomic conflict

The evolution of Trypanosoma cruzi: the 'bat seeding' hypothesis

Paleomicrobiology of Bartonella infections

Genomic changes associated with the evolutionary transition of an insect gut symbiont into a blood-borne pathogen

Inferring clocks when lacking rocks: the variable rates of molecular evolution in bacteria

Global fingerprint of humans on the distribution of Bartonella bacteria in mammals

Evolutionary dynamics of pathoadaptation revealed by three independent acquisitions of the VirB/D4 type IV secretion system in Bartonella

Phylogenetic and geographic patterns of bartonella host shifts among bat species

Geomolecular dating and the origin of placental mammals

Genotyping of Bartonella bacteria and their animal hosts: current status and perspectives

BEAST: Bayesian evolutionary analysis by sampling trees

Creating the CIPRES Science Gateway for inference of large phylogenetic trees

On incomplete sampling under birth-death models and connections to the sampling-based coalescent

R: a language and environment for statistical computing

Bayesian analysis of elapsed times in continuous-time Markov chains

Terrestrial ecoregions of the world: a new map of life on Earth

Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty

Contrasting patterns in mammal-bacteria coevolution: Bartonella and Leptospira in bats and rodents

Host phylogeny, geographic overlap, and roost sharing shape parasite communities in European bats

TimeTree: a resource for timelines, timetrees, and divergence times

Estimating divergence times in large molecular phylogenies

MEGA X: molecular evolutionary genetics analysis across computing platforms

Stochastic mapping of morphological characters

APE: analyses of phylogenetics and evolution in R language

phytools: an R package for phylogenetic comparative biology (and other things)

The igraph software package for complex network research

Theoretical foundation of the RelTime method for estimating divergence times from variable evolutionary rates

A molecular phylogeny for bats illuminates biogeography and the fossil record

Out of Africa: the origins of the protozoan blood parasites of the Trypanosoma cruzi clade found in bats from Africa

Transmission pathways and spillover of an erythrocytic bacterial pathogen from domestic cats to wild felids

Simultaneously reconstructing viral cross-species transmission history and identifying the underlying constraints

Expanding our view of Bartonella and its hosts: Bartonella in nest ectoparasites and their migratory avian hosts

Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella

Role of distinct type-IV-secretion systems and secreted effector sets in host adaptation by pathogenic Bartonella species

Host-symbiont-pathogen interactions in blood-feeding parasites: nutrition, immune cross-talk and gene exchange

Bartonella species and their ectoparasites: selective host adaptation or strain selection between the vector and the mammalian host?

Survey of parasitic bacteria in bat bugs, Colorado

Competence of Cimex lectularius bed bugs for the transmission of Bartonella quintana, the agent of trench fever

A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies

Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura)

Implications of a dating analysis of Hippoboscoidea (Diptera) for the origins of phoresis in feather lice (Psocodea: Phthiraptera: Philopteridae)

Detection of Bartonella tamiae, Coxiella burnetii and rickettsiae in arthropods and tissues from wild and domestic animals in northeastern Algeria

Human exposure to novel Bartonella species from contact with fruit bats

The zoogeography of trematodes of marine fishes

Parasites of fishes as biological indicators of recent and ancient conditions. Host-Parasite Relationships

Die Entfaltungsregel und andere Gesetzmäßigkeiten in den parasitogenetischen Beziehungen der Mallophagen und anderer ständiger Parasiten zu ihren Wirten

A nuclear DNA phylogenetic perspective on the evolution of echolocation and historical biogeography of extant bats (Chiroptera)

Bats as 'special' reservoirs for emerging zoonotic pathogens

Bat tolerance to viral infections

Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host

Immunological control of viral infections in bats and the emergence of viruses highly pathogenic to humans

Comparative analysis of bat genomes provides insight into the evolution of flight and immunity

Diverse sampling of East African haemosporidians reveals chiropteran origin of malaria parasites in primates and rodents

We thank the members of the Webb laboratory for their suggestions on early drafts of this manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of CDC.