Controversies in bacterial taxonomy: The example of the genus Borrelia

Journal Pre-proof Controversies in bacterial taxonomy: The example of the genus Borrelia Gabriele Margos, Volker Fingerle, Sally Cutler, Alexander Gofton, ˜ Brian Stevenson, Agust´ın Estrada-Pena

PII:

S1877-959X(19)30409-1

DOI:

https://doi.org/10.1016/j.ttbdis.2019.101335

Reference:

TTBDIS 101335

To appear in:

Ticks and Tick-borne Diseases

Received Date:

24 September 2019

Revised Date:

15 November 2019

Accepted Date:

15 November 2019

Please cite this article as: Margos G, Fingerle V, Cutler S, Gofton A, Stevenson B, ˜ A, Controversies in bacterial taxonomy: The example of the genus Borrelia, Estrada-Pena Ticks and Tick-borne Diseases (2019), doi: https://doi.org/10.1016/j.ttbdis.2019.101335

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Controversies in bacterial taxonomy: The example of the genus Borrelia

Gabriele Margosa*, Volker Fingerlea, Sally Cutlerb, Alexander Goftonc, Brian Stevensond, Agustín Estrada-Peñae

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Bavarian Health and Food Safety Authority, German National Reference Center for Borrelia,

Veterinärstr. 2, Oberschleissheim, Germany b

School of Health, Sport and Bioscience, University of East London, London E15 4LZ, UK

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Australia d

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Australian National Insect Collection, CSIRO, Black Mountain, Clunies Ross St, Acton, ACT, 2901,

Department of Microbiology, Immunology, and Molecular Genetics, and Department of Entomology,

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University of Kentucky, Lexington, Kentucky, 40502, USA

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Department of Animal Pathology, Faculty of Veterinary Medicine, Miguel Servet, 177, 50013 Zaragoza,

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Spain

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Corresponding author: [email protected]; [email protected]

Key words: Bacterial Systematics; Taxonomy; Borrelia; Borreliella; relapsing-fever; Lyme borreliosis

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Abstract In this paper we survey key issues in bacterial taxonomy and review the literature regarding the recent genus separation proposed for the genus Borrelia. We discuss how information on members of the genus Borrelia is increasing but detailed knowledge on the relevant features is available only for a small subset of species. The data accumulated here show that there is considerable overlap in ecology, clinical aspects and molecular features between clades that argue against splitting of the

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genus Borrelia.

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Introduction The introduction of next generation sequencing has considerably decreased the cost and time of producing and analyzing whole bacterial genomes. With increasing quantities of sequence data available in online databases, bacterial systematics should benefit through better comparability of isolates, strains and species. However, bacterial taxonomy remains complicated, and the lack of general consensus of how to resolve key issues remains, particularly regarding genus or higher taxa classifications (Philippot et al., 2010). In the absence of a general concept for bacterial species delineation (Cohan, 2002; Gevers et al., 2005; Stackebrandt and Ebers, 2006; Achtman and Wagner, 2008) and no general rule for classification of taxonomic ranks higher than species, efforts are being

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made to modernize bacterial taxonomy by maximizing the additional resources provided by whole bacterial genomes e.g. (Konstantinidis and Tiedje, 2005; Thompson et al., 2013; Garrity, 2016; Parks et al., 2018).

Taxonomy is a fundamental scientific discipline that underpins many other scientific, economic and

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administrative endeavors. As Garrity and Lyons eloquently described in their contribution “Futureproofing biological nomenclature” (Garrity and Lyons, 2003): “To most biologists, it seems

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inconceivable that the simple act of naming a biological entity has any more significance than identifying a personal achievement or staking a claim to a territory of research interest, akin to carving one’s initials into the tree-of-life. However, this simple act has potentially far-reaching and long-lived

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consequences. Names, especially those ascribed to organisms, serve as a primary entry point into the scientific, medical, and technical literature and figure prominently in countless laws and regulations governing various aspects of commerce, public safety and public health. These names also serve as a

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primary entry point into many of the central databases that the scientific community and the general public now rely upon.”

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Bacterial taxonomy is concerned with characterization, classification and nomenclature of bacterial isolates (Tindall et al., 2010). Whilst rules for nomenclature are well developed, characterization and

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classification are less progressed. Bacterial taxonomy Characterization and classification of bacteria were initially founded upon phenotypic analyses, but today bacterial systematics is largely based on molecular data. Despite the many advantages molecular data have over conventional taxonomic techniques there are still major shortfalls in the appropriate analysis and interpretation of such complex data. Methods utilized for bacterial species delineation, their advantages and disadvantages have been the subject of several reviews e.g. (Gevers et al., 2006; 3

Konstantinidis and Tiedje, 2007; Tindall et al., 2010; Thompson et al., 2013). Here, we only briefly recapitulate these methods for the benefit of the reader. Methods in bacterial taxonomy Since the 1960s, phenotypic methods were complemented by genetic methods including DNA-DNA hybridization, 16S rRNA sequence analysis, DNA G+C content, multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA). For more than a decade, DNA-DNA hybridization has been the gold standard procedure in bacterial taxonomy (Stackebrandt and Ebers, 2006). Isolates showing >70 % DNA-DNA hybridization and differences in their melting temperature (Tm) of <5 °C were considered to belong to the same species. In addition, 16S rRNA sequence analyses have been and are still widely

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used for taxonomic purposes. In that context, it is worth noting that only full length 16S rRNA sequences give reliable results for taxonomic purposes (Yarza et al., 2014). However, whilst 16S rRNA sequence analysis are valuable to get a first indication on the taxonomic position of a strain, the lack of general threshold values for all bacterial taxa, for species, or genus designation, clearly

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demonstrates that additional methods are required to obtain meaningful answers. As a rule of thumb, identity values of 97 % or higher for 16S rRNA have been proposed for species assignment, whilst

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values of <95 % were suggested for genus assignment of bacterial strains (Yarza et al., 2014). However, these values may not be generally applicable to all taxa due to the presence of multiple copies of 16S rRNA or lack of resolution in some taxa, e.g. Burkholderia or Bacillus (Janda and Abbott, 2007). Progress

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in molecular techniques such as PCR and sequencing allowed the evolution of other methods such as MLST and MLSA to be used for taxonomic purposes (Gevers et al., 2005; Stackebrandt and Ebers, 2006;

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Maiden et al., 2013).

More recently, methods that are based on whole genome sequencing have been proposed as valuable methods for taxonomic analysis (reviewed by (Garrity, 2016)). In the genomic era new approaches

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including average nucleotide identity (ANI) (Konstantinidis et al., 2006), genomic average nucleotide identity (gANI), alignment fraction (AF) (Varghese et al., 2015), amino acid identity (AAI), Karlin genome

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signature, in silico genome-genome hybridization (isGGH) (Thompson et al., 2013) or the maximal unique matches (MUM) (Deloger et al., 2009) have been proposed for bacterial species delineation. But even using indices such as ANI or AAI for species delineation, it has been challenging to agree upon a general threshold for all bacterial taxa. It has been proposed that ANI values of 95-96% correspond to 70% DDH similarity in many bacterial taxa (Goris et al., 2007) although for some bacteria the value may be higher. Examples include the genera Mycobacterium, Escherichia, and Borrelia (Jain et al., 2018; Talagrand-Reboul et al., 2018).

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Methods for delineation of genera or higher taxa have also been proposed, amongst them the percentage of conserved proteins (POCP) combined with genome size (Qin et al., 2014), 16S rRNA analyses (Yarza et al., 2014; Yilmaz et al., 2014), conserved signature insertions/deletion (CSI), conserved signature proteins (CSP) combined with phylogeny (Gupta and Griffiths, 2002), and a genome taxonomy database for all ranks of taxonomy (Parks et al., 2018). In spite of these proposed approaches, a general consensus how to revise taxonomy has not yet been reached (Garrity, 2016). It is unlikely that a single approach can be used for all bacterial taxa as there are issues to distinguish some taxa at the species level (e.g. Yersinia pseudotuberculosus/Yersinia pestis, Burkholderia pseudomallei/B. mallei, Bacillus sp.) or genus level (e.g. Escherichia/Shigella) (Logan and Turnbull,

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1999; Godoy et al., 2003; Achtman, 2012; Devanga Ragupathi et al., 2018). The Bacteriological Code – rules and regulations for bacterial nomenclature

The International Code of Nomenclature of Bacteria, in short The Bacterial Code, regulates bacterial nomenclature. A new starting point for bacterial nomenclature was initiated in the 1970s. An Ad Hoc

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Committee was selected to review the bacterial names in use at that time and to keep only those names for taxa that were adequately described. The names of the bacteria to be retained were to be

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published in the Journal of Systematic Bacteriology as “Approved List of Bacterial Names”. In addition, it was decided that types or reference strains should be available for cultivable bacterial taxa. Authors of new species, new subspecies and new combinations need to deposit type strains of cultivable

needs to be provided.

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organisms in at least two recognized culture collections in two different countries and proof of this

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What was also introduced was the valid publication of taxon names. In the International Code of Nomenclature of Bacteria (1990 Revision) (Lapage et al., 1992) it is stated that “the name of a taxon is validly published, and therefore has standing in nomenclature, if one of the following criteria is met:

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(i) The name is cited in the Approved Lists of Bacterial Names. (ii) The name is published in papers in the International Journal of Systematic Bacteriology (IJSB) or in the International Journal of Systematic

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and Evolutionary Microbiology (IJSEM) and conforms to requirements laid down in the Bacteriological Code. [….] (iii) The name is validly published by announcement in a Validation List.” (cited from Introduction of LPSN, (Parte, 2018)). The latter may happen if a paper describing a new taxon is published in a different peer-reviewed journal than the two mentioned above. Within a year of publication the authors can apply for the species being published in a validation list. This emphasizes the role that reviewers (and perhaps editors) have in the taxonomic process and it is their responsibility to judge the sense (or non-sense) of proposed taxon changes.

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Valid publication of new names, however, does not mean that only the last published name is correct for the taxon; names that have been given previously to a species, genus, etc. are not invalidated by that act. Rather, they remain as synonyms in the taxonomic records. Very few names have been rejected (nomina rejicienda); reasons for this include names that are ambiguous (i.e. have more than one meaning), confusing (e.g. based on mixed culture), perilous (i.e. cause danger to health or economy), doubtful and perplexing names (i.e. names that cause uncertainty). A prominent example being the rejection of the name Yersinia pseudotuberculosis subsp. pestis given to Yersinia pestis (Bacteriology, 1985). This example highlights the important consideration that needs to be given to pathogenic organisms, especially when core genomic differences between strains/subspecies may not contribute to disease manifestations (Achtman, 2012). In such cases taxonomic revisions (such as the

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example above) can lead to misdiagnoses and may detrimentally affect public health efforts.

An important online instrument for researchers seeking information is the List of Prokaryotic names with Standing in Nomenclature (LPSN; www.bacterio.net) (Parte, 2014). It contains the nomenclature of prokaryotes in an alphabetical and chronological order. Nomenclatural changes including names of

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novel taxa as cited in the Approved Lists of Bacterial Names or validly published in the International Journal of Systematic Bacteriology (IJSB) or in the International Journal of Systematic and Evolutionary

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Microbiology (IJSEM) are added and updated on a regular basis.

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Current taxonomic challenges

With a large majority of bacterial diversity still remaining to be characterized and classified (Yarza et al., 2014), attempts to group or separate bacterial taxa can only be hypothetical. Additionally, using

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different analytical approaches to delineate bacterial taxa can often produce competing taxonomic hypotheses which cannot be easily resolved. When such cases relate to important pathogenic organisms it cannot be the sole role of bacterial taxonomists to judge whether bacterial classifications

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are correct or incorrect. In this respect it is very important that authors, reviewers and editors of scientific journals should be aware of the impact their taxonomic decisions have on other scientific

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disciplines where such taxonomic revisions may not have practical or biological relevance.

Different taxonomical hypotheses: the genus Borrelia The genus Borrelia was first described by Swellengreb 1907 (Skerman et al., 1989) with Borrelia anserina chosen as the type species. The genus currently comprises 42 named species (LPSN bacterio.net) including 21 species within the relapsing fever-associated group (RF), 20 species within

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the Lyme borreliosis-associated group (LB), and one species (B. turcica) within the novel reptileassociated group. However, there is a wide diversity of borreliae not represented by official named species (Mitani et al., 2004; Lin et al., 2005; Takano et al., 2011; Fedorova et al., 2014; Ivanova et al., 2014; Fingerle et al., 2016; Loh et al., 2017; Kumagai et al., 2018). Some of these novel Borrelia species phylogenetically cluster within previously characterized borreliae lineages such as Candidatus Borrelia texasensis, Candidatus Borrelia kalaharica, and Borrelia sp. from Tanzania in the argasid-transmitted RF clade, and B. chilensis in the Ixodes-transmitted LB clade (Fig. 1) (Mitani et al., 2004; Lin et al., 2005; Ivanova et al., 2014; Fingerle et al., 2016). However, more significant is the extensive diversity of Borrelia being

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described from metastriate ticks that form deeply branching unique monophyletic lineages within the genus. Such species include Candidatus Borrelia tachyglossi from echidnas (Loh et al., 2017), several novel species from Testudines (Takano et al., 2011), lizards (Panetta et al., 2017; Kaenkan et al., 2019; Supriyono et al., 2019), and snakes (Takano et al., 2010), and two putative species associated with Haemaphysalis spp. and Asian deer (Kumagai et al., 2018). The diversity of these ‘unofficial’ borreliae

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the true phylogenetic diversity of genus Borrelia.

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is illustrated in Fig. 1, where it is apparent that officially recognized species significantly underrepresent

Adeolu and Gupta published a paper in 2014 proposing to split the genus Borrelia into two, Borrelia (relapsing-fever group spirochaetes) and a novel genus Borreliella (Lyme borreliosis group

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spirochaetes) (Adeolu and Gupta, 2014). Metastriate-transmitted Borrelia species were largely absent from their genomic analyses. The concept developed by Gupta and co-authors was based on CSI and CSP to evaluate the phylogenetic patterns of these bacterial groups. The authors described 53 CSIs and

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25 CSPs that could distinguish between the LB Borrelia species and the RF Borrelia group. Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) analyses (usually considered as marker

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for species definition (Konstantinidis and Tiedje, 2005; Konstantinidis et al., 2006; Konstantinidis et al., 2006)) were also included. Differences in pathogenicity profiles and arthropod vectors of Borrelia spp. were also included as arguments to show distinctiveness between groups. Based on these data a

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division of the genus Borrelia into two separate genera was proposed and 14 Borrelia species were proposed in the new genus Borreliella (Adeolu and Gupta, 2014). Proposing taxonomical changes, it was unfortunate that the authors did not include type material for all species included in the study. The inclusion of “non-type material” is problematic for taxonomic purposes as this can complicate data presented and may be ambiguous (Konstantinidis and Tiedje, 2005; Tindall et al., 2010; Varghese et al., 2015; Garrity, 2016). Following publication, the authors requested to include the novel genus Borreliella in the validation list of named prokayotic species which went largely unnoticed by scientist and medical professionals working in the field. This was not 7

widely noticed until early 2016, when NCBI GenBank started to use Borreliella for some species of the LB group. Confusingly, NCBI GenBank changed the names of only eight species that belong to the LB group although the paper published by Adeolu and Gupta included 14 species and in total there are >20 species known to belong to the LB group (B. burgdorferi s.l. species complex). Letters were submitted to the editors of IJSEM and ‘Ticks- and Tick-Borne Diseases’ to point out inadequacies in the proposal for Borrelia genus separation, to raise awareness about the consequences for patients and to ask for reversion of the genus split (Margos et al., 2017; Stevenson et al., 2019). A reply letter that argued for validity of the genus separation followed, which presented analyses similar to the original paper, with inclusion of some additional genome sequences, yet still omitting the group of reptile-

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associated species (Barbour et al., 2017). In 2018 and 2019, two independent studies further evaluated justification of the genus separation (Margos et al., 2018; Estrada-Peña and Cabezas-Cruz, 2019). One used a different concept of genus delineation (Margos et al., 2018), the percentage of conserved proteins (POCP), published by Qin et al. (2014). The second used a phyloproteomics approach (Estrada-Peña and Cabezas-Cruz, 2019). Both

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reports independently concluded that genus separation of Borrelia was not supported by their results.

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The POCP is based on pairwise comparison of conserved proteins. Proteins were considered conserved if BLAST matches had an E-value of < 1e-5, >40% sequence identity and >50% coverage of the query sequence in each of the reciprocal searches. The authors tested their approach on a number of

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bacterial genera, concluding its advantage over 16S rRNA analyses to determine genus designation of bacterial species (Qin et al., 2014). This approach has been applied to genera of the order Chlamydiales and to Amycolatopsis species (Pannekoek et al., 2016; Adamek et al., 2018). It was used for a re-

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evaluation of the genus designation of Borrelia species and included two species that belong to the reptile-associated clade (B. turcica IST7T) and the novel echidna-associated Borrelia species detected

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in Australia, Candidatus Borrelia tachyglossi (Margos et al., 2018). For inter-genera measures closely related taxa were used (i.e. Brachyspira, Spirochaeta, Treponema, Leptospira). These data unambiguously supported the view that all taxa, RF species, LB species, reptile- and echidna-associated

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species belonged to a single genus (Fig. 2). Apart from POCP analyses, the 53 CSIs suggested by Adeolu and Gupta (Adeolu and Gupta, 2014) to be molecular markers for either LB group or RF group spirochaetes were re-evaluated in this study. Importantly, 20% of the CSIs in the reptile- and echidnaassociated groups did not cluster with the RF group of spirochaetes as had been predicted (Barbour et al., 2017) but with the LB group of spirochetaes (Table 2) (Margos et al., 2018). Using the definition given by Gupta and co-workers (Adeolu et al., 2016), CSIs that are shared between B. burgdorferi sensu lato, B. turcica and Candidatus Borrelia tachyglossi point to a common ancestor of these groups, thus, arguing for common ancestry between all Borrelia clades. 8

Additionally, independent support for a retention of all groups within the genus Borrelia has recently been presented in a study that investigated the phyloproteomic functional relationships among these spirochaetes based on the biological process of over 40,000 proteins shared among 41 proteomes including species not transmitted by vectors (i.e. Treponema spp., Leptospira spp.) and free-living species (Estrada-Peña and Cabezas-Cruz, 2019). The hypothesis was “functional” testing whether proteins with the same function are shared. A network framework was used to capture the relationships among species. Only reference proteomes were included in the study to avoid the use of partially sequenced proteomes. These authors identified that only marginal functional differences exist among Borrelia species, including RF and LB species: the overall difference between well-accepted separated genera (i.e. Treponema and Leptospira) were far greater than among the species of the RF

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and LB species. The phyloproteomic networks evaluated in the study conducted by Estrada-Peña and Cabezas-Cruz (2019) are more effective than other methods (including POCP and CSI/CSPs) for assessing the evolutionary relationship among taxa and concluded independently that there is little support to split the genus Borrelia. In spite of all the arguments summarized in this review, the author

‘Ticks- and Tick-Borne Diseases’ (Margos et al., 2019).

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reiterated his claim (Gupta, 2019) to which we have specifically commented in a letter to the editor of

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The claim that clinical, ecological and molecular differences support the differences between clades (Barbour et al., 2017) include data only for a subset of species, because they do not exist for all species.

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When the available literature is taken into consideration, it does not support the proposed genus split (Table 1, Figure 2):

(i) The clinical symptoms described for B. miyamotoi, a member of the RF clade, are somewhere in

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between RF and LB (Telford et al., 2015). Symptoms resembling Lyme neuroborreliosis have been described in a B. miyamotoi infected patient (Boden et al., 2016). Tissue invasion has been reported

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for several RF species that have been demonstrated to readily invade the brain, heart and breach placental barriers (Jongen et al., 1997; Gebbia et al., 1999; Cadavid et al., 2001; Larsson et al., 2006;

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Larsson et al., 2008).

(ii) The type species of the genus, B. anserina, though not a human pathogen, seems to be tissue associated in infected poultry (McNeil et al., 1948). Moreover, it is not clear whether the disease caused by B. anserina, avian spirochaetosis, manifests as a relapsing fever in its bird hosts. (iii) For reptile-associated or echidna-associated species it remains unclear whether they can cause any (human) disease.

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(iv) Members of both groups, RF and LB, may utilize prostriate ticks as vectors, thus demonstrating ecological overlap (Fig. 1) (see below about the possible co-evolution of ticks and Borrelia spp.)

(v) The average number of flagella is known only for a subset of species (five RF, five LB species, one reptile-associated). Taken into consideration all reports on flagella numbers, the value ranges from 820 in the RF group, 4-14 in the LB group and 10 in the reptile-associated species B. turcica (HovindHougen, 1974; Karimi et al., 1978; Hovind-Hougen, 1984; Barbour and Hayes, 1986; Hovind-Hougen, 1995; Cutler et al., 1997; Yano et al., 1997; Masuzawa et al., 2001; Guner et al., 2003; Kudryashev et al., 2009). For most named species (16 RF and 15 LB), information is lacking regarding the number of

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flagella present. Taken together, different taxonomic hypotheses have been proposed regarding the genus Borrelia. Diversity of borreliae is far greater than currently recognized as new strains and species are consistently being described around the world, especially within the metastriate-transmitted borreliae

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which sit phylogenetically between the well-defined RF and LB clades (Fig. 1). Many of these recently described species, strains and genotypes have unique vertebrate host- and tick-associations that

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challenge long-held paradigms regarding Borrelia evolution and biology. The biology and evolutionary history of these metastriate-transmitted Borrelia have yet to be studied in detail and there remain several phylogenetic and taxonomic hypotheses concerning these Borrelia and their relationship to the

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RF clade that requires additional genomic data to resolve. Furthermore, the understanding of vector competence is being increasingly challenged by the finding of more RF species in multiple vector species. Although we acknowledge that finding Borrelia or its DNA in a vector does not necessarily

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equal vector competence, this topic certainly deserves further investigation. Possibly the most diverse example being B. theileri that has been found in various metastriate tick species, but also among

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Ornithodoros ticks and a single detection in head lice from the Republic of Congo (Amanzougaghene et al., 2016; Kleinerman and Baneth, 2017)

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Estrada-Peña et al. (Estrada-Peña et al., 2016) proposed a scenario of co-evolution between the main tick lineages (following data by (Mans et al., 2016)). However, genomes for the metastriatetransmitted species B. turcica and Candidatus Borrelia tachyglossi were not yet available for analysis. Molecular clocks calculated by Mans et al. (Mans et al., 2016) placed the origin of ticks in what is now Australia or South Africa, and some species of Borrelia remain associated with some of the oldest genera of ticks (Amblyomma and Bothriocroton). After the split of Argasidae and Ixodidae (calculated in having taken place about 290 My ago), these founder species of Borrelia remained associated with both clades of ticks, resulting in RF (present in both Argasidae and Ixodidae, Fig. 1) and LB (present only in ticks of the genus Ixodes even in the southern Hemisphere). Thus, it is conceivable that a 10

speciation event led to the formation/evolution of LB species when the prostriate ticks evolved (Estrada-Peña et al., 2016). Certainly, it appears that at least some metastriate-transmitted borrelial groups (such as the reptile-associate species, and the B. theileri group) form unique monophyletic lineages; however, the inclusion of additional taxa and more genetic information is needed to substantiate the taxonomic standing of these novel Borrelia. Additionally, within the RF clade there appear to be at least two separate linages that are associated with argasid ticks as vectors and that remain poorly delineated (Fig. 1). Furthermore, there are taxonomical problems with examinations of the genus Borrelia such as the absence of many type strains in culture collections especially among the RF clade. Of great importance,

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cultures of the type species, B. anserina, have not been deposited. This is likely to reflect difficulties in cultivation of these fastidious organisms, though today, there are improved culture methods, so these problems could be rectified (Teegler et al., 2014; Margos et al., 2015; Marosevic et al., 2017). Without those essential strains, legitimate evaluation of the genus Borrelia cannot truly be performed.

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A key question that arises from the above is whether all sister clades should become different genera. Systematics is a man-made system to serve the community, thus the impact of decisions must be fully

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evaluated. Given this and the increasing diversity being recognized among Borrelia, division of this

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genus at this conjecture may not be justified at all, but is certainly premature.

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Acknowledgements

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The authors gratefully acknowledge the Robert Koch-Institute Berlin for funding the German National Reference Center of Borrelia.

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References:

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Figure legends:

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Figure 1. Phylogenetic reconstruction of Borrelia based on near full-length 16S rRNA gene sequences performed with MrBayes using the K2P+G+I substitution model. All posterior probabilities are >0.7 except where indicated. Parentheses indicate GenBank accessions. T indicates type strains. Main vector tick species or host species have been included. * louse- or Ixodidae-transmitted RF species. † Ixodes-transmitted species. Tree is rooted with Leptospira spp. and Spirochaeta spp. 16S rRNA sequences (root branch not shown).

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ro of -p re lP na ur Jo Figure 2. POCP values of intra- and inter-genus relationships in Borrelia. Notably, for all species within the (original) genus Borrelia (i.e. RF, LB, reptile- and echidna-associates) POCP values much higher than 50 % were observed. From Margos et al. 2018 PlosOne 13(12): e0208432, https://doi.org/10.1371/journal.pone.0208432.

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Table 1 Borrelia – key features of clades Clade

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Morphology*

Borrelia burgdorferi Relapsing fever group Reptile- and echidnasensu lato of spirochaetes associated spirochaetes motile spirochaetal motile spirochaetal motile spirochaetal bacteria, helically bacteria, helically bacteria, helically shaped with tapered shaped with tapered shaped with tapered ends; ends; ends; diderm membrane diderm membrane periplasmic flagellae architecture (outer architecture (outer (n=10); surface membrane, surface membrane, 0.2 – 0.3 μm diameter periplasmic space, 10-25 μm in length periplasmic space, peptidoglycanpeptidoglycancytoplasmic cytoplasmic membrane); membrane); periplasmic flagellae periplasmic flagellae (n=4-14); (n=8-20); 0.2 – 0.3 μm diameter 0.2-0.5 μm diameter 10-30 μm in length 10-40 μm in length Fragmented genome, Fragmented genome, Fragmented genome, Linear chromosome, linear chromosome, linear chromosome, plasmids (5-70 kb) plasmids (5-165 kb), Plasmids (30-130) Size 1.5 Mb Size 1.5 Mb Size 1.5 Mb GC content 28%; GC content 27-30%; GC content 30% common ancestry of common ancestry of plasmids cp26 (all LB) plasmids cp26 (all LB) and lpB (B. miyamotoi) and lpB (B. miyamotoi) hard tick genus Ixodes hard-ticks of genera hard-tick genera Ixodes, Rhipicephalus, Hyalomma, Amblyomma, Amblyomma, Ixodes, Haemaphysalis Bothriocroton Soft tick genera Ornithodoros, Argas; body louse Pediculus humanus#; Commonly tissue Commonly found in Unknown pathogens, transient blood before/during blood phase except in febrile periods but B. mayonii (blood colonize also tissue. 5 densities up to 10 – For many unknown. B. 6 10 cells / ml). duttonii is well known to cross the placenta

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Genomic features*

Pathogenicity profile*

20

causing peri-natal mortality; B. miyamotoi infection may resemble Lyme (neuro)borreliosis. common

transovarial rare, shown for B. transmission afzelii§ *information is available only for a subset of species, not for all species.

unknown

#

the louse may not be considered a proper vector because transmission occurs when the louse is

crushed and gut content is smeared into the skin

Jo

ur

na

lP

re

-p

ro of

§ (van Duijvendijk et al., 2016)

21

Table 2 CSIs in reptile- and echidna-associated Borrelia species (from Margos et al. 2018, PLoS One 13(12): e0208432, https://doi.org/10.1371/journal.pone.0208432) ‘Ca. B. tachyglossi’

B. turcica

RecA

RF

RF

Nicotinamide-nucleotide adenylyltransferase

LB

LB

Hypothetical protein (BB0838)

LB

LB

Trigger factor Tig

RF

RF

Chemotaxis protein CheY

RF

RF

DNA polymerase III subunit beta

RF

RF

Translation factor Sua5

N/A

RF

Ferrous iron transporter

RF

RF

Glucose-6-phosphate isomerase

RF

Hypothetical protein (BRE16)

RF

Hypothetical protein (BDU327)

RF

Hypothetical protein (BT0471)

LB

L-latcate permease

RF

1-phosphofructokinase

RF

GTP-binding protein

RF

Sodium/panthothenate symporter

LB

Hypothetical protein (BRE32)

RF

Hypothetical protein (Q7M33)

RF

RF RF RF LB

-p

RF RF

lP

re

RF LB

RF RF

RF

RF

L-proline transport system ATP-binding protein RF

RF

Penicillin-binding protein

RF

RF

Hypothetical protein (Q7M131)

RF

RF

Hypothetical protein (BT0110)

RF

RF

Hypothetical protein (BB0110)

RF

RF

Glutamate racemase

RF

RF

16S ribosomal RNA methyltransferase RsmE

RF

RF

DNA mismatch repair protein mutL

RF

RF

Putative lipoprotein

LB

LB

Membrane protein

LB

RF

Hypothetical protein (BRE314)

RF

RF

Methylgalactoside ABC transporter ATP-binding protein

RF

RF

Hypothetical protein (BRE355)

LB

LB

Sensor transduction histidine kinase

RF

RF

Jo

ur

na

Hypothetical protein (BRE47)

ro of

Gene with CSI

22

LB

RF

Hypothetical protein (Q7M860)

RF

RF

Hypothetical protein (KK90081)

RF

RF

Hypothetical protein (Q7M140)

LB

LB

Hypothetical protein (BG0159)

LB

LB

Outer membrane protein

RF

RF

Transglycosylase SLT domaincontaining protein

RF

RF

Cell division protein FtsZ

RF

RF

Excinuclease ABC subunit C

RF

RF

Hypothetical protein (BG0519)

RF

RF

Hypothetical protein (BBIDN1270545)

RF

RF

Hypothetical protein (BBUN400354)

RF

RF

Hypothetical protein (BBUZS70553)

RF

Hypothetical protein (BB0554)

RF

Hypothetical protein (BB0554)

RF

Hypothetical protein (BBUCA803285)

RF

Methyl-accepting chemotaxis protein

LB

Chemotaxis protein

RF

Chemotaxis protein

RF

Hypothetical protein (L14403475)

RF

ro of

DNA polymerase III subunit delta

RF RF RF RF

-p

LB

RF RF

Jo

ur

na

lP

re

RF

23