Discussion
The expansion of vector-borne pathogens is inherently linked to their
ability to infect and transmit through reservoir host and vector
populations. This fact can be observed as the current major etiological
agents of human Lyme borreliosis (LB) in Eurasia (B. afzelii ,B. bavariensis , and B. garinii ) are vectored mainly by two
different tick species: I. persulcatus in Asia, and I.
ricinus in Europe. This means that each of these genospecies has, at
least once during its evolution, successfully invaded a novel tick
species and consequently a local population of vertebrate hosts. Yet how
this invasion occurred and in which order is currently not known due to
a lack of data. Here we report a reconstructed phylogeny of 142 Eurasian
isolates belonging to the genospecies B. afzelii , B.
bavariensis , and B. garinii . All three genospecies appear to
share an Asian origin, suggesting a repeated expansion into Europe in
relation to successfully invading a novel tick vector, I.
ricinus . However, all three genospecies display unique sub-structuring
which could be linked to ecological variability in their specific
reservoir hosts. The results further show that the observed bottleneck
in European B. bavariensis isolates argued to be in connection to
invading I. ricinus (Becker et al., 2020; Gatzmann et al., 2015;
Gabriele Margos et al., 2019), is not shared by the other two
genospecies. This all suggests that Eurasian Lyme borreliosis agents
were all capable of geographic expansion through vector shifts but
differ in their capacity as emergent pathogens in relation to potential,
future expansions into novel transmission cycles.
Borrelia bavariensis was already argued to have an Asian origin
due to the deep branching observed between European and Asian isolates
and that the majority of diversity exists in the Asian population
(Becker et al., 2020; Gatzmann et al., 2015; Gabriele Margos et al.,
2019). This finding is supported by the expanded analysis reported here.
One point that warrants consideration is that all European B.
bavariensis isolates come from humans and that the observed pattern in
diversity could potentially be an artifact of sampling only a low
diversity sub-set of European B. bavariensis better adapted to
human infection. As our study includes European human isolates for two
additional genospecies, we were able to disprove that this pattern is
due to sampling bias through showing that human B. garinii orB. afzelii isolates do not display a similar reduction in genetic
diversity (Text S2). In addition to this, a search of theBorrelia MLST (multiple locus sequencing typing) database
(Gabriele Margos et al., 2008) shows eight B. bavariensis samples
coming from I. ricinus DNA which do not differ from patient
isolates on the eight MLST loci, which can roughly proxy the full
chromosome diversity (Figure S3). These data support that the observed
pattern in B. bavariensis is genuine. Compared to B.
bavariensis , no research has focused on the geographic origin ofB. afzelii or B. garinii . Previous work raised the
hypothesis of an Asian origin for B. afzelii but based on very
few samples (Takano et al., 2011), whereas for B. garinii only
partial structuring between continents was previously reported (Ana
Cláudia Norte et al., 2020). Here though, we show that both B.
afzelii and B. garinii are characterized by a basal node which
splits a fully Asian clade from a clade of mixed geographic origin,
suggesting for the first time that all three of these pathogenic
genospecies originate in Asia and that through successful colonization
of I. ricinus were able to expand into Europe. MDS clustering
based on plasmid profiles further supported this by suggesting that the
plasmid profiles present in Europe are a subset of available profiles
present in Asia (Figure 3A). This could further show that the European
population stems from the Asian population. Borrelia afzelii was
the only genospecies which showed a step-wise colonization pattern from
far-east Asia through Russia and into Europe (Figure S1) which has been
observed in other tick-borne pathogens (Kovalev & Mukhacheva, 2014)
suggesting differences in migratory patterns between species. We further
hypothesized about which genospecies colonized Europe first through
calculating absolute divergence (DXY ) and
Tajima’s D (Tajima, 1989). Borrelia bavariensis shows the
highest DXY suggesting that this colonization is
the oldest of the three with B. afzelii then the youngest with
the lowest value of DXY . Additionally, as
expected from bacterial populations (Gatzmann et al., 2015; Tajima,
1989), Tajima’s D values are consistent with population expansion
(negative Tajima’s D ) but the European expansion for each species
is younger (more negative values). The magnitude of difference in
Tajima’s D mirrors that of DXY withB. bavariensis showing the lowest difference in Tajima’s D(less recent) and B. afzelii showing the highest difference (most
recent).
It is apparent from our analysis that, after the colonization of Europe,
each genospecies experienced variable levels of gene flow which we argue
can be related back to their host associations. The fact that B.
garinii showed little to no geographic structuring is in accordance
with previous results (Pär Comstedt et al., 2009, 2011; Ana Cláudia
Norte et al., 2020). Borrelia garinii utilizes birds as reservoir
hosts and exists in overlapping terrestrial and marine transmission
cycles, where it is vectored by different tick species (terrestrial:I. ricinus and I. persulcatus ; marine: I. uriae )
(Pär Comstedt et al., 2006, 2011; Gómez-Díaz et al., 2011; A. C. Norte,
Ramos, Gern, Núncio, & Lopes de Carvalho, 2013) (Figure 1). The lack of
geographic structure observed in B. garinii is thought to be a
result of this, as birds could aid in the migration of this genospecies
(Pär Comstedt et al., 2009, 2011; Ana Cláudia Norte et al., 2020). This
would explain why we cannot differentiate between distinct geographic
locations. This pattern for B. garinii was already observed on a
European level (Ana Cláudia Norte et al., 2020), but we are now able to
show that it occurs over the whole distribution range of the
genospecies. Borrelia afzelii and B. bavariensisdisplayed structured populations in our analysis. Within-continent
structuring for European B. afzelii was previously attributed to
utilizing rodents as reservoir hosts (Gallais et al., 2018; Vollmer et
al., 2011), which we now propose to also occur in Asian B.
afzelii populations. Even though our analysis does show some level of
migration is possible along the geographic scale of this project as one
Hokkaido isolate does cluster within the Honshu clade (Figure 2). AsB. bavariensis also associates with rodents (Gabriele Margos et
al., 2009, 2013), we would expect to also observe geographic
structuring. As previously reported, there does not appear to be gene
flow between the European and Asian populations suggesting genetic
isolation (high FST andDXY ; Table 1), but within Asia B.
bavariensis is not structured as expected for a rodent adapted
genospecies (Gabriele Margos et al., 2009, 2013). Our analysis builds
upon previous work which observed migration between Asian regions (i.e.
Japan, China, Russia) (Becker et al., 2020), but by further adding
randomly sampled isolates from distinct Japanese islands: Honshu and
Hokkaido. Unlike B. afzelii , where we observe lower migration
between the islands (FST =0.379; Table S2),B. bavariensis isolates do not seem to have the same barrier to
migration (FST =0.057; Table S2). This brings
forward the question, what mechanism(s) could result in this unexpected
migration of Asian B. bavariensis isolates? One suggestion could
be that Asian B. bavariensis utilize secondary hosts besides
rodents which increase effective dispersal rate. Recently, B.
bavariensis DNA was found far afield of its Eurasian range in seabird
associated ticks (I. uriae ) in Canada (Munro et al., 2017). As
there are similarities in the structuring of Asian B. bavariensisto B. garinii from our results (low FST ,
high π , AMOVA with low σ due to geography; Table 1 & 2), it
could be that in rare cases B. bavariensis may successfully
transmit through avian hosts although rodent adapted. This fact had been
previously observed where rodent-associated genospecies (i.e. B.
afzelii ) appeared to transmit through avian hosts in Europe (Heylen,
Matthysen, Fonville, & Sprong, 2014). Although the extent of
transmission appears to be different between B. bavariensis andB. afzelii based on our analyses. Until 2009 (Gabriele Margos et
al., 2009), B. bavariensis was considered a sub-type of B.
garinii which utilized rodents as reservoir hosts (Masuzawa, 2004;
Takano et al., 2011). This association with rodents was experimentally
shown for two isolates (PBi, European; NT29, Asian) where they were
exposed to rodent or avian immune sera and were susceptible to avian
sera only (Kurtenbach et al., 2002, 1998). In this case, as in many
studies, immune serum resistance is taken as a proxy of reservoir host
associations (Kurtenbach et al., 2002, 1998). This result was used to
support that B. bavariensis is not able to transmit through avian
hosts. As the Asian population is quite diverse (Becker et al., 2020;
Gatzmann et al., 2015) it is possible that a single isolate will not be
representative for the entire population. Previous work did indeed
suggest that similar genotypes of B. bavariensis (described as
rodent adapted B. garinii ) which were isolated from infected mice
in Japan shared unique sequence components to a bird isolated strain
from the Korean Peninsula, suggesting that B. bavariensis could
spread from mainland Asia to Japan through migratory birds (Ishiguro,
Takada, & Masuzawa, 2005), as we are proposing. Additionally, a study
based on restriction fragment length polymorphism (RFLP) analysis
described a novel RFLP type (type IVa) (Nakao, Miyamoto, & Fukunaga,
1994) which is now known to belong to B. bavariensis (Dr. Minoru
Nakao & Dr. Hiroki Kawabata, personal communication). The isolates
belonging to this RFLP type were isolated from rodents, humans, but also
birds (Nakao et al., 1994). Whole genome sequencing of these isolates
would allow us to confirm if these bird isolates truly belong toB. bavariensis .
The results presented here suggest some answers to how LB spirochetes
(B. afzelii , B. bavariensis , and B. garinii )
expanded across Eurasia, through showing that all currently known
pathogenic Eurasian Borrelia genospecies expanded into Europe
from an ancestral Asian population through successful colonizing a novel
tick vector (I. ricinus ). Recently, B. garinii was found
in I. uriae ticks in seabird colonies along the Atlantic coast of
North America (Pär Comstedt et al., 2009, 2011; Smith et al., 2006). AsB. garinii was shown to be rarely transmitted through the North
American tick vector (I. scapularis ) in lab based studies (Eisen,
2020) and here we show that B. garinii expanded into Europe
through colonization of I. ricinus , potentially another expansion
into the North American transmission cycle is possible if other
requirements, such as reservoir host availability, are met. Outside of
this, we further observed that post-colonization gene flow appears to
relate to host association and were able to make further testable
hypotheses regarding the ecology of the populations. Our analysis
provides novel information to the spread of LB-causing spirochetes
across Eurasia with applications to how adaptation to novel vector
species can facilitate geographic expansion and thus potentially aid in
the spread of emergent human pathogens.