Abstract
ABSTRACT: Ixodes ricinus (Linnaeus 1758) and Ixodes persulcatus (Schulze 1930) ticks are involved in the transmission of a wide variety of pathogens with considerable impact on human and animal health. The co-distribution zone of these two tick species is situated in the Baltic countries, which provides a special setting for the population studies. In the present study, genetic variability of I. ricinus ticks collected in allopatric and sympatric locations in the Baltic countries has been investigated using a sequence analysis of the mitochondrial DNA control region, 16S rRNA and cytb genes. There were 32 haplotypes (Hd: 0.8551) and 27 haplotypes (Hd:0.8213) of control region sequences from ticks in allopatric and sympatric zones detected, respectively. Out of 47 16S rRNA gene haplotypes, 32 haplotypes (Hd: 0.7213) were found in the allopatric zone and 27 (Hd:0.9572) in the sympatric zone. The Cytb gene was very conserved and monomorphic in ticks from the allopatric zone, whereas three unique haplotypes were observed in the sympatric zone. The higher number of unique haplotypes of the control region was detected in the allopatric zone. Median joining network and Fst analysis did not reveal a clear separation between ticks from the two zones. Journal of Vector Ecology 41 (2): 244-253. 2016.
INTRODUCTION
The two closely related tick species, Ixodes ricinus (Linnaeus 1758) and Ixodes persulcatus (Schulze 1930) , play a major role in the transmission of tick-borne diseases, in particular Lyme borreliosis, tick-borne encephalitis, anaplasmosis, babesiosis, tularemia, and rickettsiosis in the Baltic countries. During the past two decades, the application of molecular markers in the study of Ixodid ticks has provided new essential information about their taxonomic relationships and population structures. Ticks have been studied at the individual, population, and species level. I. ricinus is the first tick species to have been used in studies of tick population genetics (Healy 1979a,b) .
INTRODUCTION
Two isozyme loci (a-GPDH and PGM) were analyzed and differences between allelic frequencies were found between males and females of I. ricinus for a-GPDH but not for PGM. Between spring and autumn, samples of PGM allele frequencies showed differences (Healy 1979b ). This indicated a temporal difference in mating phenology between two sympatric populations, which would limit the gene flow and lead to an increase in genetic differentiation. Healy (1979a) also showed that a-GPDH in females was positively related to walking activity, because heterozygous females displayed greater activity than homozygous females. The population structure of I. ricinus in Europe based on allozyme loci showed separate clusters of Western European (UK, Ireland, France, Netherlands, and Denmark) and Central European (Germany, Poland, Switzerland, and Italian Alps) tick populations. The other populations (Norway, Sweden, Spain, Slovakia, and Czech Republic) were separated from the main groups (Estrada-Peña et al. 1996) . Later, eighteen allozyme loci of I. ricinus were analyzed in five locations in Switzerland, of which only two appeared polymorphic. Based on these two loci, no evidence of geographic structuring was observed (Delaye et al. 1997) .
INTRODUCTION
Three major findings were based on the analysis of microsatellite markers in I. ricinus population studies. This species presents moderate to high genetic structure at a local scale and the host use and mating are not random (de Meeûs et al. 2002 , Kempf et al. 2009 , Araya-Anchetta et al. 2015 . A genetic analysis of microsatellites of I. ricinus ticks collected in Switzerland and Tunisia originally presented no differentiation at a local scale (de Meeûs et al. 2002) . However, microsatellite data in I. ricinus exhibited a deficit in heterozygotes that was not explained solely by the null alleles. Later it was discovered that population substructuring within the Swiss populations was obscured by the Wahlund effect (Kempf et al. 2010 ). This analysis also showed differences in host use between males and females. Assortative mating was observed in I. ricinus from two out of four locations in northern France (Kempf et al. 2009) . A non-random use of hosts was shown in I. ricinus ticks collected from birds, rodents, lizards, roe deer, and wild boar using microsatellite analysis (Kempf et al. 2011) .
INTRODUCTION
While many different methods have been used in the past, DNA markers have been shown to be most revealing in tick genetic studies. Mitochondrial DNA has a higher rate of base substitution than most nuclear genes, hence mtDNA sequences are a useful phylogenetic marker for clades that have diverged no more than a few million years ago (Curole and Kocher 1999) . Genetic differences were investigated across the entire range of I. ricinus by sequencing multiple genes from a small number of ticks per site (Casati et al. 2008 , Noureddine et al. 2011 ). The major genetic division between I. ricinus in Eurasia and North Africa was based on the analysis of six mtDNA genes and four nuclear genes (Noureddine et al. 2011) . Later, the African branch was proved to be a separate species (Estrada-Peña et al. 2014) . A new member of the I. ricinus-I. persulcatus complex, Ixodes inopinatus, was described in dry areas of the Mediterranean region in Spain, Portugal, Morocco, Algeria and Tunisia (Estrada-Peña et al. 2014 ). An additional variation within Europe has been detected using gene sequences from six mitochondrial genes (ATP6, COI, COII, COIII, CYTB, and 12S) that comprise a new multilocus sequence typing (MLST) system for ticks and showed differences between I. ricinus ticks in Latvia and the UK (Dinnis et al. 2014) . Lack of genetic variability within continental Europe was explained by Porretta et al. (2013) . During the last glacial phase, I. ricinus never experienced a prolonged allopatric divergence in separate glacial refugia but persisted with interconnected populations across southern and central Europe. The generalist behavior in host choice of I. ricinus would have played a major role in maintaining connections between its populations. Although most of the hosts persisted in separate refugia, from the point of view of I. ricinus, they represented a continuity of 'bridges' among populations.
INTRODUCTION
The sympatric zone of I. ricinus and I. persulcatus covers the southern and eastern parts of Estonia , the eastern part of Latvia (Bormane et al. 2004) , and a small part of northern Lithuania (Paulauskas et al. 2015) ( Figure 1 ). The processes that take place at the boundaries of tick distribution are most revealing, providing clues to the factors that control tick survival. For this reason, the zone of sympatry between I. ricinus and I. persulcatus is worth special attention (Bugmyrin et al. 2013) . Studies show that the sympatric zone in the northern periphery of the range of these two species has widened mainly because of the increased abundance of I. persulcatus (Bugmyrin et al. 2013) . Climate change was named as the most important factor for the increased numbers of I. persulcatus ticks in the Karelia and Archangelsk regions in Russia (Tokarevich et al. 2011 , Bugmyrin et al. 2013 , and I. ricinus in Sweden (Jaenson et al. 2012) and Norway (Jore et al. 2011 (Jore et al. , 2014 . Extensive collections of I. ricinus analyzed for pathogen detection have been amassed in Estonia since the 1980s. Although fluctuations in tick abundance were noted, no specific studies on changing abundance or distribution have been conducted. In Latvia, I. ricinus is considered largely absent from the eastern parts of the country where I. persulcatus is more common and no evidence of a recent spread is available. However, the landscape changes both in newly re-generated woodlands and in the existing forests in Latvia could continue affecting tick abundance and distribution in the future (Medlock et al. 2013) . In Lithuania, I. ricinus ticks have been investigated since 1987 but mainly for pathogen (B. burgdorferi s.l.) screening (Motiejūnas et al. 1994 , Paulauskas et al., 2008 , Žygutienė et al. 2008 . I. ricinus has been found in all parts of Lithuania, while I. persulcatus ticks were present just in the northern part of the country (Žygutienė 2009 (Žygutienė , Paulauskas et al. 2015 .
INTRODUCTION
According to Filippova (1999) , the sympatric zone of I. ricinus and I. persulcatus formed 8,000 -10,000 years ago and is sufficient for producing approximately 2,000 generations of these ticks. It was proposed that sympatry of vectors has an effect on the groups of pathogens biologically associated with them. I. persulcatus ticks in the zone of sympatry significantly enhance Borrelia circulation among the I. ricinus ticks (Alekseev 1993a , Alekseev et al. 1993b , and pathogens exchange tick vectors (Geller et al. 2012 ). Both tick species have an independent and original functional role in the focal ecosystem, which contributes to the stability of this ecosystem under conditions of sympatry. I. ricinus and I. persulcatus ticks not only share the same hosts and habitats, but they have no morphological barrier to mating (Filippova 2002 ) and can produce hybrids (Kovalev et al. 2015) . This interspecific competition between I. ricinus and I. persulcatus ticks may have consequences on the genetic diversity of I. ricinus ticks and the epidemiology of tick-borne diseases in the area.
INTRODUCTION
The aim of our study was to determine the intraspecific variability of the mtDNA (16S rRNA gene, Cytb gene and control region) sequences among I. ricinus ticks collected from the sympatric and allopatric zones of the Baltic countries.
MATERIALS AND METHODS

Tick collection and identification
I. ricinus ticks were collected in Lithuania, Latvia, and Estonia in 2008 and 2012 from vegetation (Table 1, Figure   Figure 1 . Green dots indicate tick sampling sites. Dashed area represents the sympatric zone of I. ricinus and I. persulcatus according to Bormane et al. 2004 , Paulauskas et al. 2015 . Grey area indicates I. ricinus allopatry zone. Table 1 . Tick sampling sites. 1). Ticks were collected from vegetation by means of flagging and stored in micro-centrifuge tubes filled with 70% ethanol and brought to the laboratory for DNA extraction. Ticks were identified using morphometric analysis according to Fillipova (1985) and molecular methods. Tick DNA was extracted using 2.5% ammonium hydroxide solution as described by Stanczak et al. (1999) and Paulauskas et al. (2008) . Oligonucleotide primers Ixri-F Ixri-R designed by A. Jenkins (A/S Telelab, Skien, Norway) were used in PCR reaction. These primers amplify 150 bp fragment of the 5.8 S rRNA gene, which is specific for I. ricinus (Fukunaga et al. 1996) . The identification of species was confirmed by mtDNA sequence analysis.
Tick collection and identification
PCR amplification of the control region, 16S rRNA, and cytb was performed in 25 μl reaction volume with 2 μl extracted DNA template, 1 μl of each primer (10 pmol/μl ) ( Table  2) , 2 μl dNTP, 1.5 μl 25 mM MgCl 2 , 0.17 μl AmpliTaqGold polymerase (Thermo Fisher Scientific, U.S.A.), 2.5 μl 10xPCR buffer, and 14.83 μl ddH 2 O. Amplifications were performed using the following program: one denaturation cycle at 95º C for 10 min, 35 cycles with 45 s at 95º C, 45 s at 50º C for cytb gene and the control region, 45º C for 16S rRNA gene, and 45 s at 72º C. The final step was at 72º C for 7 min in an Eppendorf Mastercycler Nexus (Germany). PCR amplifications were visualized with 1% agarose gel electrophoresis. PCR products were purified using ExoSAP-IT protein (Affymetrix, U.S.A.) following the manufacturer's instructions.
DNA sequencing and analysis
DNA sequencing was performed (Table 2) in a 3100-Avant™ Analyzer System. Sequence alignments were conducted with ClustalW Multiple Alignment using MEGA6 software (Tamura et al. 2011) were constructed using the Network 83.161.120.155 package. F ST genetic distances Analyses of Molecular Variance (AMOVAs) applying different population clustering criteria were performed, as indicated in the tables of the results section using Arlequin 3.5 (Excoffier and Lischer 2010) .
RESULTS

Control region sequences
A total of 172 control region sequences (Table 4 ) was analyzed (64 from Lithuania, 52 from Latvia, and 56 from Estonia) ( Table 3 ). There were 76 sequences from the allopatric and 96 from the sympatric zone. A total of 45 different haplotypes was identified in the data set (haplotype diversity, Hd: 0.8403). Sequences from the allopatric zone had 32 haplotypes (Hd: 0.8551) when sites with alignment gaps were considered. When the sites with gaps were considered as missing data and excluded from calculations, there were 22 haplotypes left (Hd: 0.8032). Sequences from the sympatric zone had 27 haplotypes (haplotype diversity, Hd: 0.8213) when the sites with alignment gaps were considered. After the sites with gaps had been excluded there were 24 haplotypes left (Hd: 0.8162) ( Table 3) .
Control region sequences
Median joining network was constructed in order to compare I. ricinus genetic variability between the ticks with and without coexistence with I. persulcatus ticks ( Figure  2 ). Eight haplotypes were observed in both sympatric and allopatric zones. The sympatric zone had 16 unique haplotypes, whereas the allopatric zone had 19 (Figure 4) . AMOVA analysis showed a 0.831% genetic variation between the ticks from sympatric and allopatric zones, while the variation within these zones was 99.183% (Table 5) . A very low and insignificant Fst value (F = 0.00817, p = 0.353) indicated no genetic differentiation and a high gene flow between the sympatric and allopatric zones.
16S rRNA gene sequences
In total, 110 16S rRNA gene sequences were analyzed (39 from Lithuania, 32 from Latvia, and 39 from Estonia) ( Table 4) . A total of 47 haplotypes were observed in the data set (Hd: 0.9560). As many as 54 sequences were from the allopatric zone and 53 were from the sympatric zone. Sequences of 16S rRNA from the allopatric zone had 32 haplotypes (Hd: 0.9574) and sequences from the sympatric zone had 27 haplotypes (Hd: 0.9572) when the sites with gaps were considered. Only 15 haplotypes (Hd: 0.7275) from the allopatric and ten (Hd: 0.7213) from the sympatric zone were observed when the sites with gaps were excluded. Median joining network showed that 14 haplotypes were observed in both zones (Figure 3 ), while 18 haplotypes were found unique for the allopatric zone and 16 haplotypes unique for the sympatric zone (Figure 4) . The most common haplotype in both zones was 16S1. AMOVA analysis showed negative insignificant Fst value (F = -0.01558, p = 0.611) (Table 6), which indicates genetic similarities in 16S rRNA sequences in allopatric and sympatric zones.
Cytb gene sequences
Thirty cytb gene sequences were analyzed (Table 4) . Cytb gene did not reveal any variation in ticks from the allopatric zone. All sequences were identical and belonged to CB1 haplotype. The analysis of the marker revealed four haplotypes (Hd: 0.3137) from the sympatric zone. However, 83.3% of the ticks belonged to the most common CB1 haplotype. Haplotypes CB2, CB3, and CB4 were found only in the sympatric zone (Figure 4 ). CB2 and CB3 differed from CB1 in one nucleotide, whereas CB4 differed in five nucleotides. The results show that cytb gene is the most conserved and has low differentiation in I. ricinus ticks.
Cytb gene sequences
AMOVA analysis of cytb gene sequences revealed low and insignificant differences between the allopatric and sympatric zones (Fst =0.05753, p =0.653) ( Table 7 ). The control region sequences (GenBank accession numbers KT070717 -KT070760) were the most variable with thirtyseven (9%) variable sites (Table 3) , nineteen (4.7%) of which were parsimony-informative (Pi). The most conserved was cytb gene (KT070761 -KT070764) that had six (2.7 %) variable sites and only one (0.5%) parsimony-informative site, whereas the other five were singletons. 16S rRNA gene sequences (KT070765 -KT070811) had twenty (5.46%) variable sites, twelve (3.3%) of which were parsimony-informative.
DISCUSSION
Our results of the analyzed mitochondrial sequences from 189 ticks collected in the Baltic countries show a larger number of unique haplotypes in the control region and 16S rRNA gene in the allopatric zone. However, the cytb gene was very conserved and monomorphic in this zone. As stated before, climate change was explained to have an impact on rapid range and abundance increase of I. ricinus tick species in Baltic countries (Medlock et al. 2013) . As explained by Noureddine et al. (2011) , the absence of the genetic structure among the European I. ricinus ticks may be the consequence of a recent rapid range expansion of this species, as evidenced by the observed high haplotype diversity contrasting with the low nucleotide diversity. The population expansion increases the retention of new mutations (Watterson 1984) and creates an excess of haplotypes that differ only in one or a few mutations (Slatkin and Hudson 1991, Rogers and Harpending 1992) . This could account for a greater haplotype diversity of cytb gene in the sympatric zone.
DISCUSSION
Haplotype diversity was higher in the allopatric zone than in the sympatric zone when the sites with gaps were included in the calculations, and the opposite result was observed when gaps were excluded (Table 3) . Other authors eliminate gaps from the analysis (Casati et al. 2008 , Dinnis et al. 2014 ), but our results show that they are important in the analysis.
DISCUSSION
Median joining network analysis of I. ricinus ticks did not reveal a clear separation between the two zones. Lack of genetic differences between these ticks can be explained by ecological factors. Habitat conditions of ticks are very strict and dependent on environmental conditions such as relative humidity, temperature, and vegetation. These factors result in discontinuous geographical distribution even though the ticks can colonize more than 300 different avian and mammalian wild species Humair 2002, Casati et al. 2008 ). Ticks have low mobility when separated from their hosts; consequently, the rates and patterns of the gene flow are determined by host movements during infestation. Highly mobile mammals and birds promote homogeneity among closely related populations (Casati et al. 2008 ). This shows that slight differences in temperature and ecology of the habitats do not affect the genetic variation in this species. No correlation between the various types of habitat and genetic variability of ticks was found in Serbia by Cakic et al. (2014) . It has been estimated that the maximal distance that an I. scapularis nymph can be dispersed is 425 km (Ogden et al. 2008) , based on the maximum length of time a nymph can remain attached to an avian host (i.e., five days; Ogden et al. 2005 ) and the mean distance a passerine can fly in that time. Different routes (e.g., flyways) used by passerines migrating northwards into the Baltic countries may have a strong influence on the spatial occurrence of different tick haplotypes brought from Europe. There are two dominant bird migratory routes in Lithuania (Patapavičius 1998 (Patapavičius , 2006 . In spring, the birds migrate from the southwest towards the northeast through Vilnius, Biržai, and Daugavpils. This route passes through inland area, 200 km from the Baltic Sea. In autumn, the birds migrate from northeast towards southwest through the Baltic Sea coastal area and can have influence on dispersing haplotypes brought from northern parts of the Baltic countries (Paulauskas et al. 2006) .
DISCUSSION
Despite a large number of ticks that might be transported by birds between the locations, few establish new populations. This may be due to the fact that the survival rate of each tick stage is poor, with 90% of a generation failing to reach the next developmental stage (Randolph et al. 2002) . Randolph et al. (2002) estimated that the survival of just ten adult ticks would on average require 1,000 larvae. The likelihood of a successful transmission of mtDNA haplotypes to a new region would also be halved as, in the majority of cases, only females are able to impart their mtDNA genetic material to the next generation (Breton et al. 2007 ). Bi-parental inheritance of mitochondria is rare in many species and unlikely to influence the result significantly (Dimauro and Davidzon 2005, Dinnis et al. 2014) . However, recent studies of I. ricinus and I. persulcatus hybrid ticks showed the presence of heteroplasmy (16%), i.e., they had mtDNA of both species, which has been confirmed by sequencing. It can be hypothesized that mtDNA might be paternally inherited in ticks (Kovalev et al. 2015) . Therefore, a male tick surviving to reproductive age would affect the mitochondrial genetic structure of the future population far less than would a female.
DISCUSSION
Low or even negative Fst values indicate high gene flow and suggest genetic exchange between the populations. Special living conditions of I. ricinus in the sympatric zone, sharing their habitats and hosts with closely related I. persulcatus ticks, and the ability to transmit new pathogens do not have a significant impact on the tick population structure and genetic differentiation from allopatric ticks.

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