Ticks and the city - are there any differences between city parks and natural forests in terms of tick abundance and prevalence of spirochaetes?
Kowalec et al. Parasites & Vectors
Ticks and the city - are there any differences between city parks and natural forests in terms of tick abundance and prevalence of spirochaetes?
Maciej Kowalec 0
Tomasz Szewczyk 1
Renata Welc-Falęciak 0
Edward Siński 0
Grzegorz Karbowiak 1
Anna Bajer 0
0 Department of Parasitology, Institute of Zoology, Faculty of Biology, University of Warsaw , 1 Miecznikowa Street, 02-096 Warsaw , Poland
1 W. Stefański Institute of Parasitology of the Polish Academy of Sciences , 51/55 Twarda Street, 00-818 Warsaw , Poland
Background: Ixodes ricinus ticks are commonly encountered in either natural or urban areas, contributing to Lyme disease agents Borreliella [(Borrelia burgdorferi (sensu lato)] spp. and Borrelia miyamotoi enzootic cycles in cities. It is an actual problem whether urbanization affects pathogen circulation and therefore risk of infection. The aim of the study was to evaluate main tick-borne disease risk factors in natural, endemic areas of north-east (NE) Poland (Białowieża) and urban areas of central Poland (Warsaw), measuring tick abundance/density, prevalence of infection with spirochaetes and diversity of these pathogens in spring-early summer and late summer-autumn periods between 2012 and 2015. Methods: Questing I. ricinus ticks were collected from three urban sites in Warsaw, central Poland and three natural sites in Białowieża, NE Poland. A total of 2993 ticks were analyzed for the presence of Borreliella spp. and/or Borrelia miyamotoi DNA by PCR. Tick abundance was analyzed by General Linear Models (GLM). Prevalence and distribution of spirochaetes was analyzed by Maximum Likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). Species typing and molecular phylogenetic analysis based on the sequenced flaB marker were carried out. Results: Overall 4617 I. ricinus ticks were collected (2258 nymphs and 2359 adults). We report well established 2 population of ticks in urban areas (10.1 ± 0.9 ticks/100 m ), as in endemic natural areas with higher mean 2 tick abundance (16.5 ± 1.5 ticks/100 m ). Tick densities were the highest in spring-early summer in both types of areas. We observed no effect of the type of area on Borreliella spp. and B. miyamotoi presence in ticks, resulting in similar prevalence of spirochaetes in urban and natural areas [10.9% (95% CI: 9.7-12.2%) vs 12.4% (95% CI: 10.1-15.1%), respectively]. Prevalence of spirochaetes was significantly higher in the summer-autumn period than in the spring-early summer [15.0% (95% CI: 12.8-17.5%) vs 10.4% (95% CI: 9.2-11.6%), respectively]. We have detected six species of bacteria present in both types of areas, with different frequencies: dominance of B. afzelii (69.3%) in urban and B. garinii (48.1%) in natural areas. Although we observed higher tick densities in forests than in maintained parks, the prevalence of spirochaetes was significantly higher in the latter [9.8% (95% CI: 8.6-11.0%) vs 17.5% (95% CI: 14.4-20.5%)]. Conclusions: Surprisingly, a similar risk of infection with Borreliella spp. and/or B. miyamotoi was discovered in highlyand low-transformed areas. We suggest that the awareness of presence of these disease agents in cities should be raised.
Ixodes ricinus; Borreliella; Borrelia miyamotoi; City; Urban; Natural; Borreliosis; Risk factors
Borrelia burgdorferi (sensu lato) spirochaetes are a
complex of Lyme disease (LD) causative agents transmitted
by ticks. Among the 21 species of these spirochaetes
registered worldwide [
], three are responsible for
almost all cases of human borreliosis (LD) in Europe: B.
burgdorferi (sensu stricto), B. afzelii and B. garinii .
Recently, the genus Borrelia was divided into two
genera: Borrelia, comprising all relapsing fever (RF)
spirochaetes and new genus Borreliella [
] including all
species of the B. burgdorferi (sensu lato) complex. The
division was supported by the wide molecular analyses
of either selected molecular markers or whole genomes,
as well as on the basis of ecological features of species
and their pathogenicity [
]. Because the type species
for Borrelia is B. anserina, belonging to the RF group,
LD-spirochaetes were excluded from the genus Borrelia
and obtained a new name which replaces the term ‘B.
burgdorferi (s.l.)’. For these reasons, in present paper we
will use Borreliella for LD-causative bacteria and
Borrelia when referring to RF-causative agents, including
In Poland, as in the whole of central and western
Europe, Ixodes ricinus ticks constitute the main vector
of LD-spirochaetes. Eight Borreliella species were
detected to date in I. ricinus ticks and vertebrate hosts in
Poland: B. afzelii, B. bavariensis, B. burgdorferi, B.
garinii, B. lusitaniae, B. spielmani, B valaisiana and B. turdii
]. These species exhibit different pathogenicity and
host specificity, e.g. B. lusitaniae is commonly found in
lizards, B. garinii and B. turdii are associated with birds,
while B. afzelii and B. burgdorferi are detected mainly in
rodents . However, there are a limited number of
studies on the particular species prevalence in Poland,
providing information only on B. burgdorferi (s.l.)
complex, current genus Borreliella. Nevertheless, during last
15 years the incidence of LD in Poland has risen from
1850 cases in the year 2000, to 4407 in 2005 and 9011 in
2010, to almost 14,000 cases in 2014. In 2016 the
number of cases reached 21,000 [
LDspirochaetes, Ixodes ticks in the whole of the northern
hemisphere transmit the RF-causative agent B.
miyamotoi. It was isolated for the first time from I. persulcatus
ticks in Japan in 1995 [
], later also found in ticks in
], and recently has been recognized as a
human pathogen. First, 46 cases of B. miyamotoi
infection in humans were described in Russia in 2011 .
Two years later, B. miyamotoi was found in 50 patients
with symptoms of relapsing fever with high temperature
in the USA, Netherlands and Japan [
Manifestation of B. miyamotoi infection is similar to human
granulocytic anaplasmosis (HGA) or tick-borne
encephalitis, and was recently referred to as ‘B. miyamotoi
disease’ (BMD) [
Co-occurrence of Borreliella spp. and B. miyamotoi in
I. ricinus ticks may also have affected the previously
conducted studies on prevalence of LD-causative agents in
ticks, particularly the results published before wide
recognition of B. miyamotoi in ticks in Europe in 2002 [
as B. miyamotoi were not differentiated from other
borreliae (Borreliella spp.). Molecular resemblance of B.
miyamotoi and Borreliella spp. may also have caused
misinterpretation of the results of sero-prevalence
studies in tick-bitten persons [
]. It is plausible that the
same mechanism was responsible for quite late
recognition of B. miyamotoi infection in patients with clinical
symptoms of a disease after a tick bite [
Importantly, Ixodes ticks presence is commonly
reported in urbanized areas such as suburban forests and
city parks [
]. While progressive environmental
changes and urbanization process increase human
exposure to ticks, we do not know how these affect
tick-borne pathogens circulation and transmission [
Fragmentation of forests is discussed as a factor limiting
biodiversity and therefore tick abundance; however no
effect of fragmentation on prevalence of LD-spirochaetes
was observed in recent study [
]. Despite relatively low
biodiversity of ticks and mammals in urban areas, it is
possible that LD risk in these habitats is not much
different than that in natural areas and could be quite high
in cities [
] and within urban space which is not
commonly associated with tick-borne disease risk [
The risk of acquiring tick-borne disease depends on
pathogen, reservoir and vector presence in the
environment. All three factors may be affected by urbanization,
other environmental transformations and direct human
]. Therefore, it is important to monitor
these risk factors in both natural and urbanized areas.
Investigation on whether B. miyamotoi is present in ticks
in frequently visited foci is equally important as study on
detection of LD-agents, both actions aiming at focusing
attention of physicians and diagnosticians on new
possible disease/pathogen diagnosis, in concordance with
contemporary conception of ‘One Health’ [
The aim of our study was to assess and compare risk
factors, i.e. tick abundance and prevalence of infection
with Borreliella spp. and/or B. miyamotoi spirochaetes,
in natural areas of north-east (NE) Poland and an
agglomeration area in central Poland. An additional aim of
our study was to evaluate the diversity of spirochaetes in
these two ecologically different types of areas.
Tick collection and research areas
Ixodes ricinus ticks were collected in 4-year period,
between 2012 and 2015, by flagging in selected
seminatural areas of NE Poland and urban areas of central
Poland. Six sites were monitored, three in urban forests
or city park in Warsaw and three in forests and city park
in Białowieża area. Flagging was performed on surfaces
of 50–600 m2 with a 1 m2 flag in two tick-activity
periods: spring-early summer and late summer-autumn.
The first season of tick activity, spring-early summer
peak, involved collections from March 21st (earliest
sampling) to July 31st, the second comprised period
between August 1st and October 31st (latest sampling).
The length of the whole sampling period reflects the
length of vegetation period in Poland. Designated
sampling seasons take into account tick summer
diapause (hot and dry continental summer in Poland)
followed by changes in vegetation structure. Ticks
were not collected during and shortly after rainfall.
Ticks were identified to species and stage level [
], counted, and tick densities were calculated per
100 m2 for each individual flagging event (each visit
at specific site). Two types of areas were compared in
the study: urban forests/park in Warsaw
agglomeration (central Poland) and semi-natural forest/park
areas near Primeval Białowieża Forest in NE Poland.
Selected urban and natural sites differed, particularly
in level of human impact as expressed by the level of
human-derived landscape transformation. The matrix
 of semi-natural areas involved natural and
managed forests and low-transformed settlement foci
(Fig. 1a). The matrix of urban areas involved highly
transformed areas of urban infrastructure, residential
areas, streets or arable land (Fig. 1b). In each type of
area, 3 study sites were selected, two forest sites
(Subtype 1: forest) and one park (Subtype 2: park),
representing a gradient of human impact among each
area: from undisturbed or moderate (forests) to
relatively high (parks).
The main difference (beside localization in urban or
natural areas) noted between urban and semi-natural
sites could be expressed by everyday activity of humans
at each site. All three urban sites are characterized by
high numbers of people performing different activities in
the forests/parks, especially walking dogs several times a
day, walking with children, cycling, etc. Among urban
sites, only Kabacki Forest is large enough to avoid
human presence in every part of the forest, and in this case
human activities may be particularly increased during
the weekend period. On the contrary, forest sites around
Białowieża town have much lower level of
every-dayactivity of humans, and are visited mainly by forestry
workers, tourists or mushroom pickers, during selected
periods of the year.
Natural and semi-natural areas near Białowieża town
Białowieża Forest (Białowieża National Park; BNP) (52°
46′20″N, 23°50′60″E) (10,517.27 ha) is a residual
primeval forest. In 1972 it was added to UNESCO
World Heritage List. This region of NE Poland is
considered as borreliosis endemic area. Ticks were collected at
3 selected sites distant from each other (Fig. 1a).
The first site, Białowieża Palace Park (BPP) (52°42′
24.6″N, 23°50′42.6″E), is a fenced, maintained, regularly
mowed park in Białowieża town. The park history dates
to the eighteenth century. It was founded by a Russian
tsar. Now it is frequently visited by tourists as one of
local touristic attractions. It constitutes the island of
transformed, wooded environment in this matrix.
However, the park is situated in a close vicinity of forest
nature reserve (BNP).
The second forest site, Białowieża, North-West
(BNW) (52°43′41.0″N, 23°47′19.0″E) is comprising a
forest ecotone adjacent to recreational area, localized
north-west from Białowieża, mowed partially from the
east side and occasionally visited by tourists or forestry
The third natural site, Białowieża, South-West (BSW)
(52°39′40.1″N, 23°46′06.2″E) is a forest path inside
protected forest area, rarely visited by humans, though
distant from nature reserve.
BNP and surrounding forests are known for being
inhabited by numerous large mammals, particularly a
free-living population of the European bison (Bison
bonasus), as well as roe (Capreolus capreolus) and red
deer (Cervus elaphus) or wild boar (Sus scrofa). Wolves
(Canis lupus lupus), elks (Alces alces) and lynxes (Lynx
rufus) are permanent inhabitants of the forest, which is
also the habitat of many rodents, insectivores, numerous
bird species and reptiles [
Urban, highly transformed areas
Central Poland, Warsaw capital city. Urban and highly
transformed areas constitute the matrix of urban sites in
the study (Fig. 1b). All selected urban forest habitats are
inhabited by rodent and avian hosts, as well as
synanthropic carnivores such as red foxes (Vulpes vulpes),
martens (Martes foina) and hedgehogs (Erinaceus
europaeus). Larger mammals such as roe deer may also
The first urban site, Bielański Forest (WBF) (52°17′32″N,
20°57′36″E), is a small urban forest (152 ha) in north
districts of Warsaw, close to Vistula River (Fig. 1b). It
comprises the paths of primeval deciduous forest and is under
law protection as a nature reserve. Two medium size
academic centers (Cardinal Stefan Wyszyński University and
Józef Piłsudski University of Physical Education) are placed
within the area of WBF. WBF is a place of recreation and
physical activities of capital residents. The forest is rich with
small mammal species like red squirrels (Sciurus vulgaris),
Apodemus mice (A. agrarius and A. flavicollis). Large
mammals like elk and roe deer were recorded, probably due to
proximity of Kampinoski National Park in the North-West.
The second urban site, Kabacki Forest (WKF) (52°6′
58″N, 21°3′26″E), is a relatively large managed forest
(903 ha) at the south border of the Polish capital city
(Fig. 1b). It is a place of recreation and physical activities
of residents of the adjacent highly populated residential
areas (Ursynów, Kabaty quarters). It is under protection
as a landscape reserve. Besides common hosts, it is
dwelled by wild boars, roe deer and lizards.
The third urban site, Royal Łazienki Park (WLP)
(52°12′53″N, 21°01′58″E), is a vast city park (76 ha),
placed near the centre of the capital city (Fig. 1b), the
second most frequently visited park in Poland; the
attendance of visitors in the palace-park complex is estimated at
over 2 million people per year [
]. WLP is carefully
managed by a municipality, characterized also by open mowed
areas. Classical music concerts are a regular part of
summer activities in the park. The park is fenced,
protected by park guards and no dogs are allowed inside. It is
populated by potential tick hosts such as striped field
mouse (A. agrarius), red squirrels, hedgehogs, dozen bird
]. Even the presence of roe deer has been
A representative number of collected ticks (65%) were
subjected to the molecular study. Genomic DNA from
ticks was isolated with Genomic Tissue Spin-Up kit (AA
Biotechnology, Gdynia, Poland) according to the
manufacturer’s protocol, from individual adults and from
pools of 10 nymphs. Genomic DNA was used for
molecular screening for spirochaetes by amplification of
pathogen 16S rDNA with published primers [
], but in
modified reaction conditions as follows: initial
denaturation in 95 °C for 5 min, 40 cycles of denaturation in
95 °C for 30 s, 30 s of primers annealing in 53 °C and
elongation in 72 °C for 30 s. Subsequently, positive
samples were analyzed by nested PCR with the use of
Borreliella spp. and B. miyamotoi flagellin gene (flaB)
marker, with published primers [
]. Initial PCR
conditions were modified as follows: initial denaturation in
95 °C for 5 min, 35 cycles of denaturation in 95 °C for
30 s, 30 s of primers annealing in 52 °C and elongation
in 72 °C for 80 s, with final elongation in 72 °C for 7 min.
Nested PCR was performed with minor modification:
denaturation in 95 °C for 20 s and annealing in 55 °C for 20 s,
elongation in 72 °C for 60 s. PCR products were visualized
on 1.5% agarose gels stained with Midori Green Stain
(Nippon Genetics Europe, Düren, Germany). Primers used
in both PCR protocols amplify DNA of both Borreliella
spp. and B. miyamotoi. A representative number of positive
samples was subsequently sequenced. Additionally, a gene
fragment of outer surface protein A (ospA) was amplified
and sequenced for confirmation of genotype, with primers
and PCR conditions already described [
For B. miyamotoi detection among positive samples,
specific primers for flaB marker were designed for the
nested reaction: forward BmF (5′-AAC TTG CTG TTC
AGT CTG GT-3′) and reverse BmR (5′-TTA ACT CCA
CCT TGA ACT GG-3′) (424 bp product). Nested PCR
conditions remained unmodified.
In silico analysis
Statistical analysis was performed using IBM SPSS
Statistics v. 20.0 software. Differences in tick densities
(arithmetic means) were evaluated by ANOVA using
models with normal errors. General Linear Model
(GLM) of One Variable was used to test main effects of
Year (2012, 2013, 2014 or 2015), Season (spring-summer
or summer-autumn), Type of area (urban or natural),
Subtype of area (forest or park) and Site (Białowieża,
natural: BSW, BNW and BPP; Warsaw, urban: WBF,
WKF and WLP).
Prevalence of Borreliella spp. and/or B. miyamotoi
infection (percentage of ticks infected) was analyzed by
Maximum Likelihood techniques based on log-linear
analysis of contingency tables (HILOGLINEAR). For
analysis of the prevalence of Borreliella spp. and/or B.
miyamotoi in ticks, we fitted prevalence of bacteria as a
binary factor (infected = 1, uninfected = 0) and then
Year (3 levels: 2013–2015), Season (spring-summer or
summer-autumn), Type of area (urban or semi-natural)
or Subtype of area (forest or park) or Site (1–6; BSW,
BNW, BPP, WBF, WKF and WLP). A minimum
sufficient model was then obtained, for which the likelihood
ratio of χ2 was not significant, indicating that the model
was sufficient in explaining the data.
Additionally, the distribution of Borreliella spp. and B.
miyamotoi species among positive samples (frequencies
or ratio) was compared between Years, Seasons and
Types or Subtypes of areas or Sites by adding Species
criterion for positive samples in prevalence analysis using
the same method (HILOGLINEAR). The Species ratio
was tested for each main species detected
(species-infected = 1, other species infected = 0) or for each
detected species (7 levels). For analysis of distribution of
species among natural and urban areas, a Jaccard Index
of similarity (JI) was calculated as the number of shared
variants of each species present in both urban and
natural areas, divided by total number of variants of each
Minimum Infection Rate (MIR) was calculated for
pools of nymphs; if a sample was positive it was assumed
that only one tick specimen in the pool was infected.
Additionally, NIP value; Nymphal Infection Prevalence
(as acknowledged human disease risk-measure [
estimated. NIP was calculated as follows: π = 1-(1-P)1/k,
where π stands for NIP value, P is the ratio of number
of infected samples (including pools; n) to total number
of samples in analysis (Q), and k is the number of
specimens in the pool (Hauckl’s equation as published before,
taking into account possibility of more than one
specimen being infected in a pool [
Borreliella spp. and B. miyamotoi sequences obtained
were analysed using BLAST-NCBI and MEGA v.6.06
] was used for sequence alignment and
further species typing.
Molecular phylogenetic analyses were performed using
Maximum Likelihood method of tree-construction. The
evolutionary model was chosen with accordance to the
data (following implemented model test in MEGA v.
6.06) and bootstrapped over 1000 randomly generated
sample trees. Identical sequences obtained in the study
were pooled for analysis.
The new nucleotide sequences have been deposited in
the GenBank database under the accession numbers
MF150046–MF150082 and KT948321–KT948324.
Tick abundance (2012-2015)
During four years of study, 4617 I. ricinus ticks were
collected: 2258 nymphs, 1164 females and 1195 males, in
total 296 collection events: 82 in natural and 214 in
urban areas. The overall mean abundance (± standard
deviation, SD) was 13.2 ± 0.8, 3.5 ± 2.0, 3.8 ± 2.0 and
6.0 ± 0.5/100 m2 for total ticks, females, males and
GLM model for total tick abundance
Year × season × type of area
Abundance of ticks (nymphs and adults combined) by
year and season of the study, and by site and area is
presented in Table 1. In tested models, Year had an
independent strong effect on tick density (main effect of
Year: F(3,295) = 6.9, P < 0.001). The highest tick
abundance was recorded in 2015, while abundance in
the years 2012–2014 was half of 2015 (Table 1). Also,
Season had a strong effect on tick density (main effect
of Season on tick density: F(1,295) = 57.3, P < 0.001).
Generally, higher tick abundance was recorded in the
first season of tick activity (spring-summer) in
comparison to the second one, however it was similar in
both seasons in 2013 (Table 1, Fig. 2a).
Type of area (urban or natural) had an independent
effect on the abundance of ticks (main effect of Type of area
on tick density: F(1,295) = 15.2, P < 0.001) and was involved
in two effect interactions. Mean abundance of ticks was
significantly higher in the natural areas near Białowieża,
while in city forests and parks of Warsaw the density was
about 40% lower (Table 1, Fig. 2a). We have obtained
significant effect interaction between Year and Type of area
influencing tick density (Year × Type of area on tick
density: F(3,295) = 8.4, P < 0.001). Although tick densities were
generally higher in natural areas (Fig. 2a), in 2013 similar
tick abundance was recorded in both types of areas
(Table 1). Another significant effect interaction
incorporated three factors (Year × Season × Type of area
on tick density: F(3,295) = 6.9, P < 0.001). Interestingly,
although tick abundance was higher in natural habitats in
the first season of tick activity, it was very similar both in
urban and natural areas in the second season of tick
activity (summer-autumn) (Table 1, Fig. 2a).
Year × season × subtype of area
Subtype of area (forest or park) had an independent
effect on the abundance of ticks (main effect of
Subtype of area: F(1,295) = 11.7, P = 0.001). Tick
abundance in forests was almost 2 times higher than
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in parks in the study (Fig. 2b, Additional file 1: Table S1).
Although abundance in urban and rural parks is similar, it
is generally higher in natural forests in comparison to
urban forests (Table 1).
Year × season × site
There were also significant differences in tick abundance
between individual study sites (main effect of Site on tick
density: F(5,295) = 18.9, P < 0.001). Both the highest
(27.2 ± 2.0 ticks/100 m2) and the lowest (5.2 ± 2.2 ticks/
100 m ) tick abundance was noted among sites near
Białowieża town, at the natural forest sites BSW and in
BPP, respectively. With the exception of WBF, densities
of ticks were generally halved at urban sites, in
comparison to natural ones (Table 1). Very similar patterns were
observed in the abundance of nymphs and adult ticks.
The abundance of nymphs and adults (females and
males) is presented in Additional file 2: Table S2 and
Additional file 3: Table S3, respectively. Statistical
outcomes of GLM analyses are presented in Additional file 4
Prevalence of spirochaetes (2013-2015)
A total of 4124 I. ricinus ticks were collected, of which
2993 specimens (1535 adults and 1458 nymphs) in 1685
samples (860 females, 675 males and 150 pools of
nymphs) were screened for spirochaetes with general
primers detecting both Borreliella spp. and B. miyamotoi.
Prevalence of Borreliella spp. and/or B. miyamotoi in
I. ricinus ticks (combined, adults, nymphs) in urban and
natural sites by Year of the study, and by Site and Type/
Subtype of area is presented in Table 2.
Among five factors implemented into log linear analyses
of prevalence in ticks, only Season was associated with
infection status (Season × presence/absence of Borreliella
spp. and/or B. miyamotoi: χ2 = 4.3, df = 1, P = 0.039). A
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higher percentage of positive ticks was detected in the
second season of tick activity (late summer-autumn), 10.4%
(95% CI: 9.2–11.6%) vs 15.0% (12.8–17.5%).
Interestingly, differences in Borreliella spp. and/or B.
miyamotoi prevalence between years of the study and
the two types of areas were not significant (Table 2).
Overall prevalence of Borreliella spp. and/or B.
miyamotoi infection was very similar in both urban (11%) and
natural areas (12.4%) for all ticks combined (NS). Overall
prevalence was almost identical in urban and natural
areas, both for adults [about 19% (17.0–21.0%)] and
nymphs [MIR about 3% (2.3–4.2%)] (Table 2).
Differences in Borreliella spp. and/or B. miyamotoi
prevalence between years in adult ticks were about 5%,
differences in MIR in nymphs ranged 2–5% (NS).
Additionally, for Borreliella spp. and/or B. miyamotoi
infection in nymphs, identical NIP was calculated both in
natural (3.5%; n = 9; Q = 31) and urban (3.8%; n = 37;
Q = 119) areas. The differences between NIPs and MIRs
were not significant. Although there were some
differences in Borreliella spp. and/or B. miyamotoi prevalence
between individual sites, they were not significant
(Table 2). The highest percentage of positive ticks was
noted in two parks, WLP (17%) in urban and in BPP
(19%) in natural areas, and was reflecting the highest
percentage of positive adult ticks (24 and 25%,
respectively) (Table 2).
Thus, Borreliella spp. and/or B. miyamotoi prevalence
was significantly higher in parks (subtype 2, more
changed habitat) in comparison to forests (subtype 1, less
transformed forest) (Subtype of area × presence/absence
of Borreliella spp. and/or B. miyamotoi: χ2 = 7.6, df = 1,
P = 0.006) (Table 2).
Species of spirochaetes detected in the study
Species typing was performed on the basis of sequencing
of flagellin gene fragment (~600 bp product); 230 of 338
positive PCR samples were sequenced. Alignment and
BLAST-NCBI analyses revealed presence of six Borreliella
species: B. afzelii, B. burgdorferi, B. garinii, B. lusitaniae,
B. spielmani and B. valaisiana (Table 3). Additionally, five
B. miyamotoi sequences were obtained. Borreliella afzelii
was the dominant species (131/230; 57%) (Table 3), the
second most frequent was B. garinii, followed by B.
burgdorferi (Table 3). Other species, like B. valaisiana, B.
lusitaniae, B. spielmani and B. miyamotoi were relatively
rare (< 5%) (Table 3). We grouped these as ‘rare’ in further
analysis. Borrelia miyamotoi was identified only in
samples from WKF (2/648) in urban and BNW (3/186) in
natural areas, while B. spielmani was found only in one
tick from WBF (0.4%).
Interestingly, the distribution of species (frequency)
differed between natural and urban areas (Type of area ×
species: χ2 = 67.6, df = 6, P < 0.001) (Table 3). Borreliella
afzelii was present in over 2/3 of positive ticks from
urbanized and in almost 1/3 of positive ticks from natural
areas (Table 3). Also B. burgdorferi was slightly more
common in urban/suburban sites than in natural sites
(Table 3). However, B. garinii was more common in
natural sites, being detected in almost half of the positive
samples (Table 3). ‘Rare’ species were more frequent at
natural sites near Białowieża town (15.6 vs 5.8% at urban
sites). Three most common Borreliella species (B. afzelii,
B. burgdorferi or B. garinii) represented the great
majority of positive samples in both natural and urban
areas: 84.4% (65/77) and 93.5% (143/153), respectively
(χ2 = 0.3, df = 1, P = 0.595). Distribution of species
differed also between parks and forests (Subtype of area ×
species: χ2 = 16.6, df = 6, P = 0.011). In parks, B. afzelii
was identified in almost 3/4 of positive ticks, while
frequency of B. burgdorferi and B. garinii was much lower
and only few B. valaisiana infections were detected. In
forests in both types of areas, all seven species of
spirochaetes were present, B. afzelii was present in a half of
samples and B. garinii was present in 25% of positive
ticks (Table 3).
Among all sequences obtained there were some
unclear sequences with ambiguous nucleotides, for which
further analysis, in some cases, revealed co-infection of
two species: B. afzelii and B. garinii, as well as B.
valaisiana and B. lusitaniae, B. burgdorferi and B. miyamotoi.
However these additional data were excluded from
further phylogenetic and frequency analyses. One case of
infection with B. miyamotoi was confirmed by
sequencing of 424 bp product of flagellin gene fragment with
use of B. miyamotoi-specific primers (excluded from
sequence analysis). There were four more B.
miyamotoipositive samples detected, however in most cases
sequencing was inconclusive. There were a few discordant
results after sequencing the same samples with different
primers, e.g. two samples typed by BLAST as B.
miyamotoi by analysis of 424 bp product of primers specific
for B. miyamotoi, further typed by BLAST as B. afzelii
and B. burgdorferi in another sample with use of
~600 bp product of general Borreliella spp. and B.
miyamotoi primers. For that reason, positive samples detected
by B. miyamotoi-specific primers were excluded from
distribution or heterogeneity analysis, although overall
prevalence of B. miyamotoi was estimated 0.33% (10/
2993; 95% CI: 0.16–0.61%).
Sequence analysis and phylogeny
All chromatograms were checked manually for
sequence quality. Each sequence containing ambiguous
nucleotides was resolved in comparison to the
reference sequence of greatest homology (from GenBank
BLAST analysis). Following chromatogram reading,
the ambiguous nucleotide sites were assigned either
in accordance or alternatively to a reference sequence.
The alternative sequences were subjected to the
secondary BLAST analysis. If the most similar
sequence in GenBank was the same species as the
reference sequence, the secondary sequence was saved
and subjected to further heterogeneity and
phylogenetic analyses, while the sample was qualified as
multistrained. If the secondary sequence was most similar
to different species, it was excluded from further
analyses, while the sample was qualified as co-infected
with two species. The 230 samples species-typed by
sequence BLAST were involved in analyses of
frequency, while the 264 sequences resolved from that
230 samples were used in heterogeneity analysis by
Jaccard Index of similarity and were clustered for
phylogenetic analysis. Subsequently, a 547 bp
consensus alignment was analysed and identical sequences
were clustered for further phylogenetic analysis.
Overall, 38 unique variants of flaB sequence were
obtained: five variants of B. afzelii, six variants of B.
burgdorferi, 18 variants of B. garinii, one variant of B.
lusitaniae, five variants of B. valaisiana, one variant
of B spielmani and two variants of B. miyamotoi
(Table 5). Comparison of the distribution of flaB
sequences among natural and urban areas revealed that
flaB sequences of all species, except B. lusitaniae
(JI = 1), differed between types of areas, although
were most similar for B. afzelii (JI = 0.6, Table 4).
There was minor diversity in B. afzelii flaB sequences
(544 bp). Among B. afzelii sequences (n = 143) derived
from 131 positive samples, overall five B. afzelii variants
with similarity levels of 99.4–99.8% (541–543/544
nucleotides) were recognised (Table 5). Our variants were
either identical to, or most similar with sequences from
Germany, Czech Republic and Poland (Table 5,
Additional file 5: Figure S1). Beside two variants, our B.
afzelii variants were present in samples from both natural
and urban areas (Table 5).
Sequences (n = 29) of B. burgdorferi (544 or 547 bp)
were quite diverse. Six variants were recognized among
29 sequenced samples, the similarity level of variants
was 97.6–99.8% (534–543/544 or 547). Our B. burgdorferi
variants displayed the highest similarity with sequences
from the USA, Switzerland, Germany, Poland, Russia or
Turkey (Table 5, Fig. 2, Additional file 5: Figure S1). The
first variant, Bb_V1 (n = 1) displayed highest similarity
(only 98% of 547 bp, 3 gaps) with strain B31 from I.
scapularis from the USA (CP009656). Five of seven of our
B. burgdorferi variants were reported exclusively in urban
sites, mostly in WKF (Table 5). A higher number of B.
burgdorferi flaB variants was detected in urban areas
Borreliella garinii was the most heterogenic species.
Eighteen variants of flaB sequence (544 bp) were
recognized among 69 B. garinii sequences obtained from 49
sequenced samples. Our B. garinii variants displayed the
highest similarity with sequences from Czech Republic,
Poland and Russia, as well as from Turkey, mostly from
I. ricinus ticks and Apodemus spp. mice (Table 5,
Additional file 5: Figure S1). One variant (Bg_vEc from
WBF) was present exclusively in urban areas (Table 5).
The other 17 B. garinii variants were present in natural
areas; five of these were recorded also in urban areas
(Table 5). The number of flaB variants of B. garinii was
higher in natural areas (Table 4).
The only variant of B. lusitaniae (n = 4) was identical
with previously obtained sequences from I. ricinus from
Poland (KF422804, DQ016623, HM345914) and was
present in both urban (WKF, n = 3) and natural (BSW,
Table 4 Comparison of heterogeneity of Borreliella species in
the two types of areas studied in Poland (2013–2015)
Sum of variants Natural Urban N + U Jaccard index
B. burgdorferi 6
B. miyamotoia 2
Ba + Bb + Bg 29
Abbreviation: N + U both in natural and urban
aData not sufficient for heterogeneity comparison
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n = 1) areas, so distribution was identical in both types
of areas (Table 4).
The single B. spielmani sequence obtained from urban
WBF was identical with sequences from I. ricinus from
France (KF422808) and red fox (Vulpes vulpes) from
Among B. valaisiana sequences (n = 12) from 11 positive
samples, five variants were identified (Table 5). Our B.
valaisiana sequences displayed the highest similarity with
sequences from Poland, Russia and Turkey (Table 5,
Additional file 5: Figure S1). Variant Bv_V1 was present
either in urban or natural areas. All other variants were
detected exclusively in natural areas. Thus, a higher
number of B. valaisiana flaB variants was detected in natural
areas (Table 4).
Molecular phylogenetic analysis supported typing of
Borreliella species with BLAST in all cases except one B.
burgdorferi sequence (Additional file 5: Figure S1). Most
of our sequences localised on single-species
(monophyletic) branches, together with reference sequences of the
same species as typed by BLAST. The B. miyamotoi
clade rooted the Borreliella tree in this case. However,
one sequence (547 bp) typed as B. burgdorferi Bb_V1
(from I. ricinus male, WKF, Warsaw) built a unique
branch with a close relation to B. burgdorferi group
(Additional file 5: Figure S1), forming a polyphyletic B.
burgdorferi branch. Our variant Bb_V1 on “B.
burgdorferi and relative species” phylogenetic tree clustered with
novel European species, B. finlandensis (contig
ABJZ02000005 and sequence KU672551), however, as a
sister group (Fig. 3). The Bb_V1 sequence differed from
all known Borreliella spp. sequences by ACG insertion.
In reference to B. finlandensis SV1 contig 143,008–
144,018 (ABJZ02000005), there were some changes in
positions: 349 (G:A), 445 (T:C), 485 (T:G), 496 (G:A),
508 (A:G), 661 (C:T), an ACG-insertion in position 669–
671, 688 (G:A), 712 (C:T), resulting in substitution in
amino-acid sequence in position 162 (S:A) and additional
glutamine (Q) amino-acid after site 222. The atypical
insertion was confirmed by additional 2-repeats of sequencing
in both directions with consensus sequence of 677 bp in
length (MF150046). Analysis of an additional molecular
marker ospA confirmed 100% homology of our sequence
(MF150082) to ‘B. finlandensis Subtype 1j1 OspA partial
gene’ (KM069331). On the phylogenetic tree based on the
ospA gene fragment, our Bb_V1 sequence localised on a
branch together with Borrelia sp. SV1 CP001524 plasmid
(B. cf. ‘finlandensis’) and ‘B. finlandensis Subtype 1j1 OspA
partial gene’ (KM069331) (Additional file 6: Figure S2).
Among B. miyamotoi flaB sequences obtained in the
study, two variants were detected from five positive ticks
with direct sequencing of PCR products (5/10 positive
samples): Bm_V1 and Bm_V2. Four of the B. miyamotoi
sequences obtained were identical (Bm_V1; KT948321–3)
with 100% identity with sequences from I. ricinus from
Poland (KX646199, FJ18804). However, one sequence
from WKF (Bm_V2; KT948324) differed by the one
nucleotide (position 751; T:G) (99.8% similar to Bm_V1),
changing the amino-acid sequence in position 249 (S:A)
(ref. AY604981). On the B. miyamotoi phylogenetic tree
based on flagellin gene fragment, our sequences clustered
on Polish-origin branch of sequences, forming a separate
clade (Fig. 4).
The main finding of our study is the discovery of similar
risk of contracting tick bite and borreliosis (estimated on
the basis of almost identical prevalence of spirochaetes
in ticks) from two areas with opposite levels of human
impact (anthropopressure). Although we compared tick
abundance, prevalence and species composition of
spirochaetes in I. ricinus ticks from distant geographically
areas of Warsaw forests and park versus semi-natural
forest/park sites from the vicinity of BPN, known
worldwide as primeval forest habitat, we found only minor
differences in tick abundance (in general, 60% higher in
natural areas, but very similar in late summer-autumn
period), no significant differences in the percentage of
infected ticks and identical number of bacteria species
(six). Despite the number of genetic variants identified
within Borreliella species, the observed genetic
differences were very minor, with the exception of one B.
burgdorferi variant (Bb_V1), which may actually
constitute a new species. Interestingly, although all
spirochaetes species were found in both natural and urban
sites, the analysis of common variants confirmed that
only about 1/3 of B. garinii and B. burgdorferi variants
were present in both areas. The majority of variants
(60%) were associated with certain type of area, urban or
Abundance of ticks
Despite annual fluctuations, we observed well established
tick population in urban areas, as was found in natural
endemic areas. However, we observed different trends in
4year tick abundance for these two types of areas. In urban
areas the abundance oscillates on a similar level in
following years. Interestingly, tick population in natural areas
seemed to grow since 2013, while it was quite stable in
Warsaw agglomeration. Nevertheless, mean tick
abundance is over 50% higher in natural areas. Tick abundance
was two times higher in natural forests compared to urban
forests, which resembles findings of other studies [
Additionally, we have registered different tick abundance
between sites, which is with concordance with other
studies, underlining that tick density depends on local
properties of their habitat [
26, 46, 48
According to our data, total tick abundance is also
generally two times higher in forests in comparison to
fenced parks, despite the level of human impact in the
area (natural or urban), which is similar to results of
another study [
], though there is no difference in adults
abundance, rather only in nymph abundance. Lower tick
abundance in parks, in comparison to forests, could be
explained by maintenance practice (direct
anthropopressure), mowing and isolation from relatively large hosts
(such as ruminants) through fencing [
restrains the forming of optimal vegetation structure
(providing optimal microclimate) for ticks, while fencing
limits access of large mammals, hosts for adult females,
affecting tick reproduction [
We also observed independent effect of Season on tick
abundance in two types of areas: natural and urban.
Generally, higher tick densities were detected in the
spring-early summer season, similarly to that found in
other studies [
]. What is interesting is that even
though tick abundance was much higher in spring-early
summer season in natural areas, compared to urban areas
(26.9 ± 1.9 vs 12.2 ± 1.2 ticks/100 m2) (Table 1), the mean
tick abundance in late summer-autumn season was very
similar in either natural or urban areas (6.1 ± 2.2 vs
7.9 ± 1.4 ticks/100 m2) (Table 1). We suspect this might
be connected with host availability in spring-summer
season. We assume that in natural areas the increased host
availability allows more ticks to find their hosts before
summer diapause, permitting the continuation of their
life-cycle, resulting in similar abundance in late
summerautumn season in natural and urban areas; this concept
requires further investigation.
Prevalence of spirochaetes
We discovered almost identical prevalence (11.3%) of
Borreliella spp. and/or B. miyamotoi in ticks from
both types of habitats. In our study, prevalence in
adults and nymphs varied from 8.4% in WBF to
18.7% in BPP, which is with agreement with results
from other European and Polish studies which
showed that these varied locally, from 4% to over
22, 29, 44, 47, 55–60
]. We have recorded
significantly higher prevalence of infection with spirochaetes in
I. ricinus ticks in second season, as opposed to recent
study in the United Kingdom , but in agreement with
the findings in the Netherlands [
]. A possible
explanation of this phenomenon is a cumulative effect of
transstadial transmission on prevalence of infection in I. ricinus
during a year. There is very little possibility of
transmission of these bacteria to the next generation (transovarial
], thus together with lower tick
abundance in summer-autumn, each year this cumulative effect
is probably compensated for. Despite the lower abundance
of ticks in the second season compared to spring-early
summer, the risk of acquiring borreliosis may be still high
due to this higher prevalence of infection.
Interestingly, neither the year nor the type of area
has effect on the prevalence of infection with
spirochaetes in our study. This risk parameter in urbanized
areas was identical to endemic areas of NE Poland. It
seemed that level of human impact on the area does
not contribute to spirochaetes prevalence in I. ricinus
ticks. Similar results were obtained in the USA [
The limitation of our study is pooling of the
nymphs and use of MIR for estimation of prevalence,
also for the overall prevalence in ticks. It could have
resulted in lower prevalence values through ignoring
possibility of more than one nymph in each pool
being infected. To test this, we calculated also NIP as
a measure of direct risk of infection, which assumes
that more than one specimen in a pool could be
infected in comparison to MIR values. However,
differences between MIR and NIP were not significant.
The NIP parameter was identical for both types of
areas as well, meaning that the risk of acquiring the
disease in case of tick bite in Warsaw urban parks
and forests is equal to risk of borreliosis in endemic,
low transformed forests of NE Poland.
Our findings suggest however, that in parks there
might be considerably higher prevalence of Borreliella
spp. and/or B. miyamotoi than in forests (18 vs 10%,
respectively). Still, numerous European studies have shown
this varies locally [
]. With almost identical prevalence
of infection in ticks, and despite lower tick abundance in
urban areas in spring-summer, the risk of acquiring
borreliosis seems to be similar in both types of areas, as
previously suggested [
]. Although there is 60% greater
chance of tick encounter in natural forests, there are
many more visitors in urban areas [
populations were shown to be well established in the city and
the abundance of ticks is generally similar in urban and
The overall lack of significant differences in prevalence
of Borreliella spp. and/or B. miyamotoi in I. ricinus ticks
from low and high-transformed areas is the opposite of
our previous findings for tick-borne Rickettsiales. For
Rickettsiales, a higher prevalence in urban habitats was
explained by dilution effect [
]. However, for Borreliella
spp. the dilution effect was recently criticized [
Our findings support the lack of a dilution effect for
spirochaetes prevalence and on general risk of disease
appearance , yet further study of a potential reservoir of
these bacteria in both types of areas is needed.
Diversity of spirochaetes
Spirochaetes species richness in those two types of
areas was also similar. Despite one species, B.
spielmani, detected only once, all detected Borreliella spp.
and B. miyamotoi were present in both natural and
urban areas (six species). The diversity within species
was minor. We found significant differences in the
distribution of the species among sequenced samples.
The dominant species in the study, B. afzelii, was also
dominant in urban community (almost 70% of
analyzed samples), while the second most frequent in
urban areas was B. burgdorferi. On the other hand, in
the bacteria community from natural areas, B. garinii
was dominant (~50%), B. afzelii constituted only 30%
and B. burgdorferi was less common than B.
valaisiana (~4% vs ~10%, respectively). Also the diversity of
flaB marker was greater for B. garinii and B.
valaisiana in natural areas but for B. burgdorferi in urban
areas. Both these phenomena could be explained by
different host availability in both types of areas. In
comparison to agglomeration-surrounded forests and
city park, park and forests in low-transformed areas
of NE Poland are inhabited/visited by the much
higher number of host species, particularly numerous
species of birds. There are 117 bird species registered
in the Białowieża area, of which 90 (84%) are typical
forest species [
]. On the other hand, in Warsaw, 42
bird species were recorded in parks and 70 in city
], and are probably less abundant. Due to
increased anthropopressure in cities, the contact of
avian hosts with ticks might be limited in comparison
to natural areas. Both B. garinii and B. valaisiana are
associated with birds [
], which also suggest the
role of avian hosts in distribution of spirochaetes in
natural areas, where these bacteria constitute 60% of
species in positive I. ricinus ticks. High diversity
among B. garinii sequences could also be explained
via high rates of migration of avian hosts [
urban areas dominated B. afzelii and B. burgdorferi,
species associated with rodent hosts, which are
probably the main tick hosts and enzootic reservoir of
many TBD pathogens [
]. Both larvae and nymphs
of I.ricinus preferably feed on rodents, so there is
high chance for them to get B. afzelii in highly
urbanized areas, where rodents likely constitute the
main tick hosts.
Strikingly, the ratio of B. afzelii in urban areas in our
study is almost identical with frequency of B. afzelii in
the Netherlands, highly urbanized and thus of high
human impact region of Europe (70% of Borreliella spp.
infections in I. ricinus ticks) [
]. However, also in Sweden
the frequency of B. afzelii was estimated on similar level
The presence of relapsing fever agent, B. miyamotoi,
was limited to only two sites: BNW in Białowieża and
WKF in Warsaw, and the the overall estimated
prevalence and frequency were low (0.33% and 2.2%,
respectively). As not all positive samples were sequenced, we
cannot provide prevalence of the certain species of
Borreliella genus or B. miyamotoi. Both estimated
prevalence, and the ratio value of B. miyamotoi, are however
similar to prevalence registered in other studies, varying
between 0.22–3.8% [
13, 22, 44, 47, 59, 71–82
]. A study
in Norway suggested that despite low prevalence of B.
miyamotoi, BMD should be considered as a possible
tick-borne disease [
]; we strongly agree with this
conclusion. On the basis of our B. miyamotoi sequences,
and other deposited in GenBank, we report also
presence of potentially specific Polish strain of B. miyamotoi.
Sequences of B. miyamotoi obtained in this study
clustered with other Polish sequences on the phylogenetic
tree, confirming that there is heterogeneity in flagellin
sequences of European lineage of these bacteria. To date,
B. miyamotoi is considered as conserved within 3
‘continental’ genotypes [
74, 83, 84
]. We also detected a single
unique variant of B. miyamotoi in WKF, which has no
relevance currently, but further investigation might show
whether it is a unique strain or aberration.
In several tick homogenates, evidence for co-existence
of two species (co-infection), not only of-genus Borreliella
but also between-genera (Borreliella sp. and B.
miyamotoi), was recognized. These results raise the question of
adequacy of standard PCR screening protocols and create
the need for simultaneous detection and typing of
different spirochaetes in ticks, e.g. via multiplex qPCR with
probes. How B. miyamotoi interacts with Borreliella spp.,
and if such coexistence affects transmission, remains
unknown and needs further scientific attention.
Finally, for the first time in Poland, we report the
presence of a novel Borreliella sp. in I. ricinus tick from
Warsaw Kabacki Forest. On the basis of phylogenetic
analysis of flaB and ospA gene fragments we classified it as a
new species related to ‘B. finlandensis’ (B. cf.
‘finlandensis’), as proposed by Casjens et al. [
]. Driven by unique
flaB sequence unique features and its distinctive position
in the phylogenetic tree, as well as close similarity of ospA
sequence and formation of a separate clade with B.
finlandensis Subtype 1j1 OspA partial gene (KM069331), we
conclude that B. finlandensis Subtype 1j1 may in fact not
belong to the ‘B. finlandensis’ species, but to potentially
new, related species we have detected in our study.
Further investigation is needed for confirmation whether
our isolate is a B. finlandensis or a new species.
There are no significant differences in Borreliella spp. and/
or B. miyamotoi prevalence between low transformed,
endemic areas of NE Poland and high human impact urban
areas. Despite twice higher tick abundance in natural areas
in spring, the mean abundance in both urban and natural
areas is not dramatically different, particularly in late
summer-autumn or in parks. Thus, the borreliosis risk
factors appear to be similar in urban and natural areas, in
cities and endemic forest areas. Analysis of Borreliella spp.
frequency suggests that in natural areas it is more likely to
develop neuroborreliosis, caused by B. garinii, while in
urban areas there may be an increased risk of skin
borreliosis, caused by B. afzelii. Although the prevalence of B.
miyamotoi in ticks is relatively low it might be
underestimated due to co-infections with Borreliella spp. in ticks
and due to lower detectability. The risk of developing
borreliosis or BMD seems to be similar in the city and in
endemic areas in case of tick-bite, but the overall risk requires
further investigation. Awareness of tick-borne
spirochaetoses should be increased, in concordance with ‘One
Additional file 1: Table S1. Ticks abundance in two subtypes of area:
forests and parks (mean ± SE). (DOCX 19 kb)
Additional file 2: Table S2. Ixodes ricinus nymph abundance in natural
and urban areas (mean ± SE). (DOCX 17 kb)
Additional file 3: Table S3. Abundance of I. ricinus females and males
in natural and urban areas (mean ± SE). (DOCX 21 kb)
Additional file 4: Table S4. Statistical table of ANOVA (GLM) analysis of
tick abundance and ML HILOGLINEAR analyses of spirochaetes
prevalence and distribution. (DOCX 17 kb)
Additional file 5: Figure S1. Molecular phylogenetic analysis of flaB
variants of sequences obtained in the study. The phylogenetic tree was
obtained with use of Maximum Likelihood method of tree construction
with Tamura-Nei + G evolutionary model chosen with accordance to
data by implemented model-test. The percentage of trees in which the
associated taxa clustered together is shown next to the branches. The
tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. The analysis involved 94 nucleotide sequences.
There were a total of 547 positions in the final dataset. Evolutionary
analyses were conducted in MEGA v. 6.06. The newly generated sequences
and their origin (type of area) are indicated with black symbols: triangle,
natural areas; upside down tringle, urban areas; diamond, both natural
and urban areas. (DOCX 1021 kb)
Additional file 6: Figure S2. Molecular phylogenetic analysis of Bb_V1
ospA fragment. The phylogenetic tree was obtained with use of
Maximum Likelihood method of tree construction with Tamura-Nei + G
evolutionary model chosen with accordance to data by implemented
model-test. The percentage of trees in which the associated taxa
clustered together is shown next to the branches. The tree is drawn to scale,
with branch lengths measured in the number of substitutions per site.
The analysis involved 19 nucleotide sequences. There were a total of 652
positions in the final dataset. Evolutionary analyses were conducted in
MEGA v. 6.06. The newly generated sequences are indicated with black
symbols. Underneath the main tree is visualized zoomed ‘B. finlandensis’
branch. (DOCX 475 kb)
The study was supported by Ministry of Science and Higher Education grant
NN404795240 and was supported by the Ministry of Science and Higher
Education through the Faculty of Biology, University of Warsaw intramural
grant DSM 501/86–112617. None of the funding sources were involved in
study design, data collection, data analysis, data interpretation or in writing
Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article and its additional files. Representative sequences are submitted in
the GenBank database under the accession numbers MF150046–MF150082
The study was designed and performed by MK. TS co-performed the field
study. RWF participated in molecular analyses. ES was the author of the
conception of the study. GK participated in collecting the material. AB
supervised statistical analyses, MK and AB drafted the manuscript. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
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