Predatory fireflies and their toxic firefly prey have evolved distinct toxin resistance strategies.

Toxic cardiotonic steroids (CTSs) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). Although most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies and gene editing in Drosophila, we find that the initial steps toward resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. By contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as a predator of toxic firefly prey.

Granulocytic anaplasmosis in cats from central Europe and molecular characterization of feline Anaplasma phagocytophilum strains by ankA gene, groEL gene and multilocus sequence typing.

BACKGROUND: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats. METHODS: To address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed. RESULTS: Fever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance. CONCLUSIONS: The genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats.

Genomic analyses indicate the North American Ap-ha variant of the tick-vectored bacterium Anaplasma phagocytophilum was introduced from Europe.

BACKGROUND: Anaplasma phagocytophilum is a tick-vectored, obligately intracellular bacterium that infects a diversity of vertebrate hosts. In North America, the Ap-ha variant of A. phagocytophilum can cause dangerous infections in humans, whereas symptomatic human infections in Europe are rare. Conversely, the European host-generalist ecotype of A. phagocytophilum frequently causes illness in domestic ruminants while no comparable infections have been recorded from North America. Despite these differences in pathogenicity, the Ap-ha variant is closely aligned phylogenetically with the European host-generalist ecotype. Furthermore, North American populations of A. phagocytophilum are less genetically diverse than those in Europe. Taken together, these observations suggest that the North American Ap-ha variant may represent an introduced population of this bacterium. METHODS: Data from publicly available whole genomes of A. phagocytophilum were used to compare phylogeographic patterns and the extent of genetic divergence between the North American Ap-ha variant and the European host-generalist ecotype. RESULTS: The results confirm that North American Ap-ha samples are phylogenetically nested within the diversity of the European host-generalist ecotype, and that Ap-ha likely radiated within the last 100 years. As expected, the Ap-ha variant also exhibited relatively low genetic diversity levels compared to the European host-generalist ecotype. Finally, North American Ap-ha harbored significantly more derived alleles than the European host-generalist A. phagocytophilum population. CONCLUSIONS: Collectively, these results support the hypothesis that the Ap-ha variant was recently introduced to North America from Europe and underwent a strong genetic bottleneck during this process (i.e. a 'founder event'). Adaptation to novel vectors may have also played a role in shaping genetic diversity and divergence patterns in these pathogenic bacteria. These findings have implications for future studies aimed at understanding evolutionary patterns and pathogenicity variation within A. phagocytophilum.

Patterns of cytonuclear discordance and divergence between subspecies of the scarlet macaw (Ara macao) in Central America.

The scarlet macaw, Ara macao, is a neotropical parrot that contains two described subspecies with broadly discrete geographical distributions. One subspecies, A. m. macao, is found from South America north into southwestern Costa Rica, while the second subspecies, A. m. cyanoptera, is found from eastern Costa Rica north into central Mexico. Our previous research using mitochondrial data to examine phylogeographical divergence across the collective range of these two subspecies concluded that they represent distinct evolutionary entities, with minimal contemporary hybridization between them. Here we further examine phylogenetic relationships and patterns of genetic variation between these two subspecies using a dataset of genetic markers derived from their nuclear genomes. Our analyses show clear nuclear divergence between A. m. macao and A. m. cyanoptera in Central America. Collectively however, samples from this region appear genetically more similar to one another than they do to the examined South American (Brazilian) A. m. macao sample. This observation contradicts our previous assessments based on mitochondrial DNA analyses that A. m. macao in Central and South America represent a single phylogeographical group that is evolutionarily distinct from Central American A. m. cyanoptera. Nonetheless, in agreement with our previous findings, ongoing genetic exchange between the two subspecies appears limited. Rather, our analyses indicate that incomplete lineage sorting is the best supported explanation for cytonuclear discordance within these parrots. High-altitude regions in Central America may act as a reproductive barrier, limiting contemporary hybridization between A. m. macao and A. m. cyanoptera. The phylogeographic complexities of scarlet macaw taxa in this region highlight the need for additional evolutionary examinations of these populations.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates change at the macroevolutionary level is a major question in evolutionary biology. With >66,000 described species, rove beetles (Staphylinidae) comprise the largest metazoan family. Their exceptional radiation has been coupled to pervasive biosynthetic innovation whereby numerous lineages bear defensive glands with diverse chemistries. Here, we combine comparative genomic and single-cell transcriptomic data from across the largest rove beetle clade, Aleocharinae. We retrace the functional evolution of two novel secretory cell types that together comprise the tergal gland-a putative catalyst behind Aleocharinae's megadiversity. We identify key genomic contingencies that were critical to the assembly of each cell type and their organ-level partnership in manufacturing the beetle's defensive secretion. This process hinged on evolving a mechanism for regulated production of noxious benzoquinones that appears convergent with plant toxin release systems, and synthesis of an effective benzoquinone solvent that weaponized the total secretion. We show that this cooperative biosynthetic system arose at the Jurassic-Cretaceous boundary, and that following its establishment, both cell types underwent ∼150 million years of stasis, their chemistry and core molecular architecture maintained almost clade-wide as Aleocharinae radiated globally into tens of thousands of lineages. Despite this deep conservation, we show that the two cell types have acted as substrates for the emergence of adaptive, biochemical novelties-most dramatically in symbiotic lineages that have infiltrated social insect colonies and produce host behavior-manipulating secretions. Our findings uncover genomic and cell type evolutionary processes underlying the origin, functional conservation and evolvability of a chemical innovation in beetles.

Predatory fireflies and their toxic firefly prey have evolved distinct toxin resistance strategies.

Toxic cardiotonic steroids (CTS) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). While most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly-resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies, and gene-editing in Drosophila, we find that the initial steps towards resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. In contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as predator of toxic firefly prey.

A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes.

Understanding patterns of diversification, genetic exchange, and pesticide resistance in arthropod disease vectors is necessary for effective population management. With the availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for a large number of genetic markers. To this end, the targeting of gene sequences of known function can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens such as avian malaria parasites that are detrimental to birds. Here we describe the development of a targeted, gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we sequenced samples from several members of the complex, as well as from distinct populations of the relatively under-studied Culex quinquefasciatus. The data generated was then used to examine taxonomic divergence and population clustering between and within these mosquitoes. We also used this data to investigate genetic variants present in our samples that had previously been shown to correlate with insecticide-resistance. Broadly, our gene capture approach successfully enriched the genomic regions of interest, and proved effective for facilitating examinations of taxonomic divergence and geographic clustering within the Cx. pipiens complex. It also allowed us to successfully survey genetic variation associated with insecticide resistance in Culex mosquitoes. This enrichment protocol will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of these ubiquitous and increasingly damaging disease vectors.

Demographic Expansions and the Emergence of Host Specialization in Genetically Distinct Ecotypes of the Tick-Transmitted Bacterium Anaplasma phagocytophilum.

In Europe, genetically distinct ecotypes of the tick-vectored bacterium Anaplasma phagocytophilum circulate among mammals in three discrete enzootic cycles. To date, potential ecological factors that contributed to the emergence of these divergent ecotypes have been poorly studied. Here, we show that the ecotype that predominantly infects roe deer (Capreolus capreolus) is evolutionarily derived. Its divergence from a host generalist ancestor occurred after the last glacial maximum as mammal populations, including roe deer, recolonized the European mainland from southern refugia. We also provide evidence that this host specialist ecotype's effective population size (Ne) has tracked changes in the population of its roe deer host. Specifically, both host and bacterium have undergone substantial increases in Ne over the past 1,500 years. In contrast, we show that while it appears to have undergone a major population expansion starting ~3,500 years ago, in the past 500 years, the contemporary host generalist ecotype has experienced a substantial reduction in genetic diversity levels, possibly as a result of reduced opportunities for transmission between competent hosts. IMPORTANCE The findings of this study reveal specific events important for the evolution of host specialization in a naturally occurring, obligately intracellular bacterial pathogen. Specifically, they show that host range shifts and the emergence of host specialization may occur during periods of population growth in a generalist ancestor. Our results also demonstrate the close correlation between demographic patterns in host and pathogen for a specialist system. These findings have important relevance for understanding the evolution of host range diversity. They may inform future work on host range dynamics, and they provide insights for understanding the emergence of pathogens that have human and veterinary health implications.

Persistence and expansion of cryptic endangered red wolf genomic ancestry along the American Gulf coast.

Admixture and introgression play a critical role in adaptation and genetic rescue that has only recently gained a deeper appreciation. Here, we explored the geographical and genomic landscape of cryptic ancestry of the endangered red wolf that persists within the genome of a ubiquitous sister taxon, the coyote, all while the red wolf has been extinct in the wild since the early 1980s. We assessed admixture across 120,621 single nucleotiode polymorphism (SNP) loci genotyped in 293 canid genomes. We found support for increased red wolf ancestry along a west-to-east gradient across the southern United States associated with historical admixture in the past 100 years. Southwestern Louisiana and southeastern Texas, the geographical zone where the last red wolves were known prior to extinction in the wild, contained the highest and oldest levels of red wolf ancestry. Further, given the paucity of inferences based on chromosome types, we compared patterns of ancestry on the X chromosome and autosomes. We additionally aimed to explore the relationship between admixture timing and recombination rate variation to investigate gene flow events. We found that X-linked regions of low recombination rates were depleted of introgression, relative to the autosomes, consistent with the large X effect and enrichment with loci involved in maintaining reproductive isolation. Recombination rate was positively correlated with red wolf ancestry across coyote genomes, consistent with theoretical predictions. The geographical and genomic extent of cryptic red wolf ancestry can provide novel genomic resources for recovery plans targeting the conservation of the endangered red wolf.

Genomic signatures of spatially divergent selection at clownfish range margins.

Understanding how evolutionary forces interact to drive patterns of selection and distribute genetic variation across a species' range is of great interest in ecology and evolution, especially in an era of global change. While theory predicts how and when populations at range margins are likely to undergo local adaptation, empirical evidence testing these models remains sparse. Here, we address this knowledge gap by investigating the relationship between selection, gene flow and genetic drift in the yellowtail clownfish, Amphiprion clarkii, from the core to the northern periphery of the species range. Analyses reveal low genetic diversity at the range edge, gene flow from the core to the edge and genomic signatures of local adaptation at 56 single nucleotide polymorphisms in 25 candidate genes, most of which are significantly correlated with minimum annual sea surface temperature. Several of these candidate genes play a role in functions that are upregulated during cold stress, including protein turnover, metabolism and translation. Our results illustrate how spatially divergent selection spanning the range core to the periphery can occur despite the potential for strong genetic drift at the range edge and moderate gene flow from the core populations.

The urban-adapted underground mosquito Culex pipiens form molestus maintains exogenously influenceable circadian rhythms.

Genes known to affect circadian rhythms (i.e. 'clock genes') also influence the photoperiodic induction of overwintering reproductive diapause in the northern house mosquito, Culex pipiens f. pipiens. This suggests that molecular changes in one or more clock genes could contribute to the inability to diapause in a second form of this mosquito, Culex pipiens f. molestus. Temperate populations of Cx. pipiens f. molestus inhabit underground locations generally devoid of predictable photoperiods. For this reason, there could be limited fitness consequences if the hypothesized molecular changes to its clock genes also eliminated this mosquito's ability to regulate circadian rhythms in response to photoperiod variation. Here, we demonstrate that in contrast to this prediction, underground derived Cx. pipiens f. molestus retain exogenously influenceable circadian rhythms. Nonetheless, our genetic analyses indicate that the gene Helicase domino (dom) has a nine-nucleotide, in-frame deletion specific to Cx. pipiens f. molestus. Previous work has shown that splice variants in this gene differentially influence circadian behavior in Drosophila melanogaster. We also find derived, non-synonymous single nucleotide polymorphisms (SNPs) in eight genes that may also affect circadian rhythms and/or diapause induction in Cx. pipiens f. molestus. Finally, four putative circadian genes were found to have no quantifiable expression during any examined life stage, suggesting potential regulatory effects. Collectively, our findings indicate that the distinct, but molecularly interconnected life-history traits of diapause induction and circadian rhythms are decoupled in Cx. pipiens f. molestus and suggest this taxon may be a valuable tool for exploring exogenously influenced phenotypes in mosquitoes more broadly.

Correction: Can public online databases serve as a source of phenotypic information for Cannabis genetic association studies?

[This corrects the article DOI: 10.1371/journal.pone.0247607.].

Can public online databases serve as a source of phenotypic information for Cannabis genetic association studies?

The use of Cannabis is gaining greater social acceptance for its beneficial medicinal and recreational uses. With this acceptance has come new opportunities for crop management, selective breeding, and the potential for targeted genetic manipulation. However, as an agricultural product Cannabis lags far behind other domesticated plants in knowledge of the genes and genetic variation that influence plant traits of interest such as growth form and chemical composition. Despite this lack of information, there are substantial publicly available resources that document phenotypic traits believed to be associated with particular Cannabis varieties. Such databases could be a valuable resource for developing a greater understanding of genes underlying phenotypic variation if combined with appropriate genetic information. To test this potential, we collated phenotypic data from information available through multiple online databases. We then produced a Cannabis SNP database from 845 strains to examine genome wide associations in conjunction with our assembled phenotypic traits. Our goal was not to locate Cannabis-specific genetic variation that correlates with phenotypic variation as such, but rather to examine the potential utility of these databases more broadly for future, explicit genome wide association studies (GWAS), either in stand-alone analyses or to complement other types of data. For this reason, we examined a very broad array of phenotypic traits. In total, we performed 201 distinct association tests using web-derived phenotype data appended to 290 uniquely named Cannabis strains. Our results indicated that chemical phenotypes, such as tetrahydrocannabinol (THC) and cannabidiol (CBD) content, may have sufficiently high-quality information available through web-based sources to allow for genetic association inferences. In many cases, variation in chemical traits correlated with genetic variation in or near biologically reasonable candidate genes, including several not previously implicated in Cannabis chemical variation. As with chemical phenotypes, we found that publicly available data on growth traits such as height, area of growth, and floral yield may be precise enough for use in future association studies. In contrast, phenotypic information for subjective traits such as taste, physiological affect, neurological affect, and medicinal use appeared less reliable. These results are consistent with the high degree of subjectivity for such trait data found on internet databases, and suggest that future work on these important but less easily quantifiable characteristics of Cannabis may require dedicated, controlled phenotyping.

Genomic Analysis of the Only Blind Cichlid Reveals Extensive Inactivation in Eye and Pigment Formation Genes.

Trait loss represents an intriguing evolutionary problem, particularly when it occurs across independent lineages. Fishes in light-poor environments often evolve "troglomorphic" traits, including reduction or loss of both pigment and eyes. Here, we investigate the genomic basis of trait loss in a blind and depigmented African cichlid, Lamprologus lethops, and explore evolutionary forces (selection and drift) that may have contributed to these losses. This species, the only known blind cichlid, is endemic to the lower Congo River. Available evidence suggests that it inhabits deep, low-light habitats. Using genome sequencing, we show that genes related to eye formation and pigmentation, as well as other traits associated with troglomorphism, accumulated inactivating mutations rapidly after speciation. A number of the genes affected in L. lethops are also implicated in troglomorphic phenotypes in Mexican cavefish (Astyanax mexicanus) and other species. Analysis of heterozygosity patterns across the genome indicates that L. lethops underwent a significant population bottleneck roughly 1 Ma, after which effective population sizes remained low. Branch-length tests on a subset of genes with inactivating mutations show little evidence of directional selection; however, low overall heterozygosity may reduce statistical power to detect such signals. Overall, genome-wide patterns suggest that accelerated genetic drift from a severe bottleneck, perhaps aided by directional selection for the loss of physiologically expensive traits, caused inactivating mutations to fix rapidly in this species.

Updating the Bibliography of Interbreeding among Canis in North America.

This bibliography provides a collection of references that documents the evolution of studies evidencing interbreeding among Canis species in North America. Over the past several decades, advances in biology and genomic technology greatly improved our ability to detect and characterize species interbreeding, which has significance for understanding species in a changing landscape as well as for endangered species management. This bibliography includes a discussion within each category of interbreeding, the timeline of developing evidence, and includes a review of past research conducted on experimental crosses. Research conducted in the early 20th century is rich with detailed records and photographs of hybrid offspring development and behavior. With the progression of molecular methods, studies can estimate historical demographic parameters and detect chromosomal patterns of ancestry. As these methods continue to increase in accessibility, the field will gain a deeper and richer understanding of the evolutionary history of North American Canis.

Global evaluation of taxonomic relationships and admixture within the Culex pipiens complex of mosquitoes.

BACKGROUND: Within the Culex pipiens mosquito complex, there are six contemporarily recognized taxa: Cx. quinquefasciatus, Cx. pipiens f. pipiens, Cx. pipiens f. molestus, Cx. pipiens pallens, Cx. australicus and Cx. globocoxitus. Many phylogenetic aspects within this complex have eluded resolution, such as the relationship of the two Australian endemic taxa to the other four members, as well as the evolutionary origins and taxonomic status of Cx. pipiens pallens and Cx. pipiens f. molestus. Ultimately, insights into lineage relationships within the complex will facilitate a better understanding of differential disease transmission by these mosquitoes. To this end, we have combined publicly available data with our own sequencing efforts to examine these questions. RESULTS: We found that the two Australian endemic complex members, Cx. australicus and Cx. globocoxitus, comprise a monophyletic group, are genetically distinct, and are most closely related to the cosmopolitan Cx. quinquefasciatus. Our results also show that Cx. pipiens pallens is genetically distinct, but may have arisen from past hybridization. Lastly, we observed complicated patterns of genetic differentiation within and between Cx. pipiens f. pipiens and Cx. pipiens f. molestus. CONCLUSIONS: Two Australian endemic Culex taxa, Cx. australicus and Cx. globocoxitus, belong within the Cx. pipiens complex, but have a relatively older evolutionary origin. They likely diverged from Cx. quinquefasciatus after its colonization of Australia. The taxon Cx. pipiens pallens is a distinct evolutionary entity that likely arose from past hybridization between Cx. quinquefasciatus and Cx. pipiens f. pipiens/Cx. pipiens f. molestus. Our results do not suggest it derives from ongoing hybridization. Finally, genetic differentiation within the Cx. pipiens f. pipiens and Cx. pipiens f. molestus samples suggests that they collectively form two separate geographic clades, one in North America and one in Europe and the Mediterranean. This may indicate that the Cx. pipiens f. molestus form has two distinct origins, arising from Cx. pipiens f. pipiens in each region. However, ongoing genetic exchange within and between these taxa have obscured their evolutionary histories, and could also explain the absence of monophyly among our samples. Overall, this work suggests many avenues that warrant further investigation.

Prosthetic Valve Endocarditis with Bartonella washoensis in a Human European Patient and Its Detection in Red Squirrels (Sciurus vulgaris).

Members of the genus Bartonella are fastidious Gram-negative facultative intracellular bacteria that are typically transmitted by arthropod vectors. Several Bartonella spp. have been found to cause culture-negative endocarditis in humans. Here, we report the case of a 75-year-old German woman with prosthetic valve endocarditis due to Bartonella washoensis The infecting agent was characterized by sequencing of six housekeeping genes (16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB), applying a multilocus sequence typing (MLST) approach. The 5,097 bp of the concatenated housekeeping gene sequence from the patient were 99.0% identical to a sequence from a B. washoensis strain isolated from a red squirrel (Sciurus vulgarisorientis) from China. A total of 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensisgltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Genetic characterization of Anaplasma phagocytophilum strains from goats (Capra aegagrus hircus) and water buffalo (Bubalus bubalis) by 16S rRNA gene, ankA gene and multilocus sequence typing.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results.