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Geographic Uniformity of the Lyme Disease Spirochete (Borrelia burgdorferi) and Its Shared History With Tick Vector (Ixodes scapularis) in the Northeastern United States
Wei-Gang Qiu1,a, Daniel E. Dykhuizena, Michael S. Acostaa, and Benjamin J. Luftba Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794-5245
b Department of Medicine, Health Science Center, State University of New York, Stony Brook, New York 11794-8153
Corresponding author: Daniel E. Dykhuizen, State University of New York, Stony Brook, NY 11794-5245., dandyk{at}life.bio.sunysb.edu (E-mail)
Communicating editor: S. YOKOYAMA
| ABSTRACT |
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Over 80% of reported cases of Lyme disease in the United States occur in coastal regions of northeastern and mid-Atlantic states. The genetic structure of the Lyme disease spirochete (Borrelia burgdorferi) and its main tick vector (Ixodes scapularis) was studied concurrently and comparatively by sampling natural populations of I. scapularis ticks along the East Coast from 1996 to 1998. Borrelia is genetically highly diverse at the outer surface protein ospC. Since Borrelia is highly clonal, the ospC alleles can be used to define clones. A newly designed reverse line blotting (RLB) assay shows that up to 10 Borrelia clones can infect a single tick. The clone frequencies in Borrelia populations are the same across the Northeast. On the other hand, I. scapularis populations show strong regional divergence (among northeastern, mid-Atlantic, and southern states) as well as local differentiation. The high genetic diversity within Borrelia populations and the disparity in the genetic structure between Borrelia and its tick vector are likely consequences of strong balancing selection on local Borrelia clones. Demographically, both Borrelia and I. scapularis populations in the Northeast show the characteristics of a species that has recently expanded from a population bottleneck. Major geological and ecological events, such as the last glacial maximum (18,000 years ago) and the modern-day expansion of tick habitats, are likely causes of the observed "founder effects" for the two organisms in the Northeast. We therefore conclude that the genetic structure of B. burgdorferi has been intimately shaped by the natural history of its main vector, the northern lineage of I. scapularis ticks.
THE evolutionary history of a species is in essence a coevolutionary process of species interactions (![]()
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Lyme disease (also called Lyme borreliosis; ![]()
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The genome of B. burgdorferi has been sequenced (![]()
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0.91 Mb and at least 21 linear and circular plasmids with combined size of >0.5 Mb. B. burgdorferi is basically clonal, showing very little evidence for recombination or transfer of plasmids (![]()
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Being a vector-borne, obligate parasite, B. burgdorferi undergoes severe population bottlenecks during both the host-to-tick and tick-to-host transmissions (for reviews of the transmission cycle see ![]()
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| MATERIALS AND METHODS |
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Study sites, tick processing, and determination of Borrelia infection:
Blacklegged ticks (I. scapularis) were collected from vegetation by flagging during their host-seeking seasons (in the fall for adult ticks and early June for nymphs) during 19971998. The study area includes multiple sites in eastern Long Island, New York (Fig 1A) and 12 sites along the East Coast (Fig 1B). Samples of other tick species (I. Pacificus and Dermacentor andersoni ) were collected from western North America and used as outgroups in phylogenetic analysis. The sources of the ticks are listed in Table 1. In the laboratory, frozen ticks were bisected individually. Total DNA was extracted using Chelex as described previously (![]()
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Cold single-strand conformation polymorphism analysis of tick mitochondrial 16S rRNA genes:
Tick mitochondrial 16S ribosomal RNA genes were amplified using nested PCR. A 460-bp fragment was amplified for 15 cycles using primers 16S + 1 (primer sequences are listed in Table 2) and 16S - 1 in a 25-µl reaction mixture containing 5 µl of the tick DNA extract, 200 µM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.1 µM of each external primer, and 1 unit of recombinant Taq polymerase (Life Technologies, Rockville, MD). The mixture was first heated to 96° for 1 min and then amplified for 15 cycles at 94° for 1 min, 48° for 1 min, and 72° for 1.5 min in a PTC-100 thermal cycler (MJ Research, Watertown, MA). Two microliters of this first amplification was used as a template for the second round of PCR. In the second round, a 300-bp portion of the 460-bp fragment was amplified using the same forward primer 16S + 1 and a reverse primer 16S - 2 in a 50-µl mixture containing 200 µM each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.5 µM each primer, and 1.25 units of recombinant Taq polymerase. The amplification was run for 35 cycles at 94° for 1 min, 54° for 1 min, and 72° for 1 min. The final PCR products were electrophoresed on a 2% agarose gel (NuSieve:SeaKem 2:1; FMC BioProducts, Rockland, ME) to view the results. These same PCR primers have been used to amplify mitochondrial DNA from 36 species of hard- and soft-body ticks (![]()
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The PCR-amplified 300-bp fragment of 16S rDNA was subjected to cold single-strand conformation polymorphism (SSCP) analysis (![]()
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Unique alleles, as recognized by distinct SSCP mobility patterns, were reamplified from the first amplification for an additional 30 cycles using 2 µl of the product of the first PCR reaction as template and primers 16S + 1 and 16 - 1. The 460-bp PCR product was purified using the Prep-A-Gene kit (Bio-Rad, Richmond, CA) according to the protocol recommended by the manufacturer. Each unique allele was then cycle sequenced from both directions using the PCR primers on a ABI 373S DNA sequencer (Perkin-Elmer, Norwalk, CT). The sequences were assembled and edited using the software Sequencher (Gene Code, Ann Arbor, MI). A total of 25 haplotypes were identified and their GenBank accession nos. are AF309008, AF309009, AF309010, AF309011, AF309012, AF309013, AF309014, AF309015, AF309016, AF309017, AF309018, AF309019, AF309020, AF309021, AF309022, AF309023, AF309024, AF309025, AF309026, AF309027, AF309028, AF309029, AF309030, AF309031, AF309032.
Cold SSCP analysis of ospA variation:
The genetic diversity of Borrelia was analyzed at the ospA locus (a single-copy gene located on a linear plasmid, lp54; see ![]()
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Reverse line blotting assay of ospC variation:
We also measured the genetic diversity of Borrelia populations using the outer surface protein locus, ospC (a single-copy gene located on a circular plasmid, cp26; see ![]()
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10% different from each other while sequences within groups are
1% different from each other (![]()
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- RLB is more sensitive. The average number of ospC alleles in an infected tick was 1.5 using the SSCP (
WANG et al. 1999 ), compared to 2.6 using RLB assays (see RESULTS).
- Results of the RLB assay were generally less ambiguous since some ospC alleles show similar mobility patterns on a SSCP gel.
- Individual bands tend to smear on SSCP gels when there is a high multiplicity of types.
- The current RLB design identifies major groups but not sequence variation within a major group, while SSCP reveals DNA sequence change regardless of the number of bases changed. In other words, SSCP does not distinguish major groups from variation within these groups.
RLB has the following disadvantages: (1) Major groups, like group C, which are recombinants of other groups, can easily be misclassified (see below); and (2) unknown groups can escape detection (the universal probe can indicate a missing group only when that group is the only clone in a tick). Thus, the presence of some sequence groups like group C will sometimes have to be confirmed using other methods.
Design of ospC allele-specific probes:
Among the 21 major ospC groups of B. burgdorferi identified (![]()
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Fluorescein-labeled PCR amplification:
A portion of the ospC gene was PCR amplified directly from infected ticks using a pair of primers OC6 (+) and OC602 (-) (![]()
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Tailing of oligonucleotide probes with poly(dT):
Short stretches of oligonucleotides usually do not contain a sufficient number of thymine (T) residues to bind to a nylon membrane when activated by UV light (![]()
Membrane preparation:
The tailed oligonucleotide probes were fixed onto a 15 x 15-cm nylon membrane (Stratagene, La Jolla, CA), using a MiniSlot 30 manifold (Immunetics, Cambridge, MA). The probes (40 pmol or 10 µl from 50-µl tailing product) were diluted in 2 ml of TE buffer and evenly applied into individual slots (0.8 mm x 13 cm) created by the manifold. The wet membrane was immediately UV crosslinked (125 mJ), briefly rinsed in water, and stored at 4° in 2x SSC. Once prepared, the membrane can be reused (up to 10 times) by stripping the membrane between uses (![]()
Checkerboard hybridization assays:
Up to 1350 individual hybridization assays (30 probes x 45 samples) can be performed simultaneously using a checkerboard hybridization system (Checkerboard System 1; Immunetics; ![]()
Statistical methods:
Gene frequency estimation:
The frequencies of the SSCP alleles of ospA in a Borrelia population were directly estimated from the occurrence of individual mobility classes in an SSCP assay. In the checkerboard hybridization assay of ospC variation using RLB, a positive signal produced on the membrane indicated the presence of a major ospC group in the tick. Numbers of positive signals were used as absolute gene frequencies of individual alleles in a population. The direct counting method (for both SSCP alleles and ospC major sequence groups) tends to underestimate the frequent alleles and overestimate rare ones, biasing toward an evenness in allele frequency distribution. ![]()
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Tests of natural selection:
The Ewens-Watterson-Slatkin test of neutrality (![]()
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Phylogenetic inference:
The nucleotide sequences of all distinct haplotypes of the 460 bp of the tick mitochondrial 16S rRNA gene were aligned using CLUSTAL W (![]()
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Genetic structure and spatial statistics:
Genetic differentiation among local Borrelia populations was tested using the G-test of homogeneity of allele frequency distributions, using the biostatistical package BIOM (![]()
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Demographic history:
The program FLUCTUATE (![]()
) and population growth rate (g, exponential growth rate, with the unit of 1/µ individuals per generation).
| RESULTS |
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B. burgdorferi population structure:
Infection rate:
The infection rate as determined by PCR amplification of the Borrelia ospA gene was 2080% in the Northeast and 07% in North and South Carolina (Table 1). One inland tick population from Pennsylvania (PA) also had low Borrelia infection rate (1 out of 39 ticks or 2.6%). All these adult ticks were collected in the same season (November 1997) and the density of adult I. scapularis ticks was as high in the South as in the Northeast. These results are in agreement with the findings of other field studies (![]()
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Genetic diversity at ospA:
Four SSCP mobility classes (five sequence haplotypes, MC1a, MC1b, MC2, MC3, and MC4) of ospA were identified in previous surveys of eastern Long Island, New York (![]()
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We determined the ospA alleles of the Borrelia from 109 infected I. scapularis ticks collected from 10 locations along the East Coast. Only one new ospA SSCP mobility class, MC5, was found in this much-expanded biogeographic survey. The new mobility class was observed only once in one of the three infected adult I. scapularis ticks from Pea Island, North Carolina. DNA sequencing revealed that the mobility shift was caused by a single third-base synonymous substitution at the nucleotide position 465 as compared with the B31 type. The creation of MC5 is most parsimoniously interpreted as a recombination between MC1a and MC4, not as a new mutation.
Not only did all but one Borrelia in infected ticks belong to one of the four previously identified mobility classes, but the frequencies of the mobility classes were uniform from Massachusetts to Maryland for 1997 (MA, RI, NY1, NJ, MD1, and MD2; P = 0.1837, Table 3). This geographic uniformity was also found for the 1996 samples from Long Island, New York (LI-96) and from Connecticut (CT; P = 0.7170, Table 3). The frequencies of mobility classes are temporally stable as well in the northeast region from 1995 to 1997 (LI-95, LI-96, CT, and NY1; P = 0.207), with the pattern of MC2 and MC3 relatively common and that of MC1 and MC4 relatively rare (Table 3). However, the frequencies of the mobility classes collected from Long Island, New York during the fall of 1994 were different from the pattern found in the later years (LI-94 vs. LI-95 and LI-96; P < 0.001). This difference between 1994 and the other years was confirmed by analysis of the frequencies of major ospC groups (Table 4).
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Multiple infections of ospC clones:
A much higher level of DNA sequence diversity exists at ospC than in ospA. Fig 3 shows the chemiluminescence image of an RLB assay for a highly infected population (NY1, from Millbrook, New York). The controls were amplified from cloned ospC alleles and should hybridize with their respective probes only. The off-diagonal signals in the figure were therefore caused by cross-hybridization among alleles. We were able to remove the cross-hybridization of A, G, and J probes by using shorter oligonucleotides as probes (Table 2 shows the revised probes). Since the group C allele is a recombinant of multiple alleles (B, I, and E; see ![]()
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Most of the infected adult ticks from the Northeast were infected with multiple Borrelia clones (Fig 3). The degree of multiplicity (the average number of distinct ospC clones per infected tick) is particularly high in localities with high Borrelia prevalence. For example, ticks from Millbrook, New York (NY1) had a multiplicity of 3.1 clones per infected tick. Some ticks from that sample (ticks 2 and 32) were infected with at least 8 clones. On the other hand, all of the Borrelia-infected ticks from the southeast were infected with only a single clone (Table 4). Even in the Northeast, fewer nymphal ticks were infected with Borrelia and the multiplicity of infecting clones was lower in infected ticks. Infected nymphal ticks from Long Island were infected with about one-half the number of clones as the adults. To compare the results from an SSCP assay with RLB, the sample (SI-94) used in the original SSCP study (![]()
The five coastal (from Massachusetts to Maryland) samples (MA, NY1, SI-96, MD1, and MD2) were not significantly different in their frequency distribution of major ospC groups (samples with small numbers, such as RI, NY2, and NJ, were excluded in the test). Major group K, one of the four major ospC groups that cause disseminating Lyme disease in humans (![]()
Allele frequency distributions at both ospA and ospC in all the local Borrelia populations were significantly more than expected even from neutral distributions (results of Ewens-Watterson tests, not shown), suggesting balancing selection in the form of frequency-dependent selection. For examples of the actual distributions, see ![]()
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Linkage between ospA and ospC:
Because of the high degree of clonality of B. burgdorferi (![]()
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I. scapularis population structure:
Divergence of northern and southern populations:
Cold SSCP analysis of the I. scapularis mitochondrial 16S rRNA gene revealed 10 common haplotypes (named AJ) in tick samples collected from the northeastern United States. Four rare haplotypes, each observed only once, were also identified. SSCP mobility patterns of the tick mitochondrial 16S haplotypes found in the northern populations are shown in Fig 4A. In the Southeast, three haplotypes, M, N, and F, dominated most of the ticks in two samples from Beaufort, South Carolina. The F allele was found in both the Southeast and the Northeast. Haplotypes found in tick samples from mid-Atlantic sites (MD2 from southern Maryland; NC1 and NC2 from North Carolina) carried mostly common alleles (e.g., D and F) of the Northeast. Samples from the mid-Atlantic region, however, had a relatively large proportion of locally distinct haplotypes that were absent from either the northeastern or the South Carolina samples. SSCP mobility patterns of unique haplotypes from the North and South Carolina samples are shown in Fig 4B. A total of 31 distinct I. scapularis haplotypes were identified from this and two other studies (![]()
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Local populations of southern I. scapularis (e.g., NC2 and SC) are genetically much more heterogeneous than northern populations (Table 6). Inland I. scapularis populations may also be much more diverse than coastal populations, as indicated by comparing two North Carolina samples (NC1 to NC2, Table 6). Variation of DNA sequences and allele frequencies of these tick samples do not deviate from neutral expectations, as analyzed by Ewens-Watterson tests (results not shown) and TAJIMA's (1989) tests (Table 6).
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Minimum spanning tree (Fig 5) and mismatch distributions (Fig 6) clearly show differences in the genetic composition between the northern and southern I. scapularis populations. The minimum spanning tree revealed two major mitochondrial clades, in agreement with the findings of previous studies (![]()
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Temporal stability:
Exact tests of population differentiation (![]()
AMOVA:
The genetic composition of local tick populations shows enough local similarity that the populations can be subdivided into three localities (Table 7), as revealed by AMOVA (![]()
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The three-group structure is caused by the genetic distinctness of I. scapularis in the mid-Atlantic region. The M and N haplotypes, which predominated samples from Beaufort, South Carolina, were absent from the tick samples from southern Maryland (MD2) and North Carolina (NC1 and NC2). Ticks from Washington County, North Carolina (NC2) contain mostly typical northern haplotypes (A, D, E, and F) but a large proportion of haplotypes in this sample (12 of 35 ticks or 34%) are distinct and found only in this region (6 O type ticks, 3 K type, 1 L type, and 2 singletons, NC2-22 and NC2-29; Table 6). Haplotype O belongs to clade B while K, L, and NC2-22 and -29 are all clade A (Fig 5), suggesting that these haplotypes had local origins and were not immigrants from either the northern or the southern populations. Moreover, haplotypes found in this region tend to be phylogenetically intermediate between clades A and B. The genetic distinctness of mid-Atlantic I. scapularis (especially inland populations) implies local origin of these populations and limited gene flow between distant tick populations.
Coastal vs. inland populations in North and South Carolina:
The dissimilarity in the genetic composition of tick populations over a short distance is most striking between the two samples from North Carolina. The population NC1 was collected from a bird sanctuary on a coastal island (Pea Island National Wildlife Refuge, Cape Hatteras National Seashore, North Carolina) and NC2 from a nearby inland site
80 km away (Washington County, North Carolina). Whereas all but one tick from the coastal site were northern types, the 35 ticks from the inland site were a mixture of northern and southern types. The coastal population was very much like the northeastern populations, implying a possible role of migratory birds in bringing the northern ticks south during the fall. Since no southern haplotypes were found in this coastal site, the long-distance gene flow mediated by migratory birds may be unidirectional (i.e., from north to south only). The ongoing long distance transportation of ticks from north to south by birds was further supported by evidence from the South Carolina samples (SC 97 and 98), collected also from a coastal site (Spring Island, South Carolina). While the stable presence of the most common northern haplotype in these two samples (F, at
20%) may be the result of mitochondrial introgression, the rarer northern haplotypes (A and G) that were found in the sample from November 1998 could be new immigrants from the North. More sampling from coastal and inland sites will be needed to test this hypothesis.
High tick diversity in the New York-Connecticut region: Within the northern tick populations, the samples from New York and Connecticut showed higher levels of genetic diversity. The number of haplotypes in these populations ranged from six to eight (Table 6), while samples from Rhode Island, Massachusetts, New Jersey, Pennsylvania, and northern Maryland showed lower diversities with three to four haplotypes (Table 6). The higher levels of tick genetic diversity in the New York-Connecticut region imply that this region could be the source of the current geographic expansion of the northern ticks.
Comparisons between I. scapularis and B. burgdorferi:
Population structure:
Within the range of northern blacklegged ticks, local populations are differentiated. On the other hand, local populations of Borrelia are similar within this range. Noncorrespondence in the level of genetic differentiation among local geographic samples between the northern blacklegged ticks and Borrelia was shown by pairwise FST tests (Fig 7). As discussed below, we propose that balancing selection at ospC creates this nonconcordance. The balancing selection counters the effects of drift in B. burgdorferi, but, of course, not in the blacklegged tick. The other possibility for the geographic uniformity of Borrelia is that the uniformity is maintained by a high level of gene flow mediated by some long-ranging reservoir hosts, such as birds.
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Demographic history:
Maximum-likelihood tests of population growth (![]()
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| DISCUSSION |
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Contemporary patterns of biogeographic variation of a species are a result of both ongoing ecological and selective processes (such as gene flow and natural selection) and species histories (e.g., range expansion and contraction; ![]()
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Geographic uniformity of Borrelia in the Northeast:
This survey of local Borrelia populations along the East Coast showed that the clone frequencies of the Lyme disease bacteria in the northeastern and mid-Atlantic region of the country are uniform. Not only are the frequencies of ospA and ospC alleles the same in all localities (Table 3 and Table 4), but the linkage relationships between the alleles of these two loci are also the same across localities.
Is the geographic uniformity of B. burgdorferi in the Northeast caused more by gene flow or by selection? We think that selection is more important than gene flow in maintaining the constant allele frequencies across Borrelia populations for the following reasons:
- The gene-frequency uniformity across populations of Borrelia cannot be explained by gene flow via ticks since the tick populations show genetic differentiation (Fig 7). Movements of mammals and migratory birds infected with the spirochete can mediate gene flow among Borrelia (
SONENSHINE 1993 ). Long distance flow of Borrelia via migratory birds is used to explain the high genetic diversity of Borrelia in low-density southern populations. However, it is unlikely that the migration of infected small mammals or birds is at a high enough frequency to homogenize the spirochete populations.
- Despite the geographic uniformity in the frequency distribution of ospA and ospC alleles, the frequency distribution is not temporally stable. Borrelia infecting tick populations from the same location (Shelter Island, New York) collected in 1994 are different from those collected in 1996 as measured at both the ospA and ospC loci (Table 3 and Table 4). This sudden shift in allele frequencies of ospA between 1994 and 1996 happened both on Shelter Island and in Wildwood State Park on Long Island (
QIU et al. 1997 ), showing the change cannot be explained by migration, but must be explained by selection.
- The gene frequency distributions of Borrelia infecting nymphal ticks are significantly different from the distributions infecting the adults from the same cohort (
QIU et al. 1997 ). Since larval and nymphal ticks feed on different hosts, this suggests selection is maintaining the gene frequency distributions.
- The distribution is significantly different from that expected from neutrality, suggesting strong balancing selection. The balancing selection is evidenced by high local nucleotide sequence diversity at loci that are directly involved in establishing infection in mammalian hosts, such as ospC (
GILMORE and PIESMAN 2000 ;
SCHWAN and PIESMAN 2000 ). The effect of balancing selection is further displayed in the overdispersed allele frequency distribution at many nontargeted loci of selection (e.g., silent variations of ospA SSCP alleles). Thus, we conclude that strong balancing selection caused by the host immune response is the major force maintaining the geographic uniformity of Borrelia across the Northeast.
Divergence among North American Borrelia:
Compared with other Borrelia species found in North America (![]()
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It is possible to learn more about the population biology of B. burgdorferi by cross-species comparisons. ![]()
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The hypothesis of adaptive divergence between B. burgdorferi and B. bissettii was tested using McDonald-Kreitman tests (![]()
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Separate histories of the northern and southern I. scapularis populations:
Results of this study revealed substantial differences in genetic structure, evolutionary history, and epidemiological characters between the northern and southern blacklegged ticks.
First, it can be concluded that the species of I. scapularis is geographically structured across its range along the East Coast. Although the northern and southern blacklegged ticks are taxonomically now described as a single species (![]()
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0.2%. Ticks from the South consist of predominantly clade B haplotypes (mostly M and N) and the average within-population sequence diversity is much higher,
1.5% (Fig 5). Studies of hybridization between the northern and southern blacklegged ticks using nuclear loci have so far failed to detect genetic discontinuity (![]()
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Second, the patterns of the genetic composition of populations suggested that the northern and southern I. scapularis populations have separate and different evolutionary demographic histories. The northern tick population samples showed evidence of exponential increase in effective population size, such as star phylogenies (Fig 5) and unimodal distributions of pairwise nucleotide differences (Fig 6) among haplotypes (![]()
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Third, the B. burgdorferi infection rate in I. scapularis ticks is markedly different between ticks collected from the Northeast and those from the Southeast (Fig 3). The localization of Lyme disease in the range of the northern lineage of I. scapularis suggests that genetic (as well as ecological) factors involving the ticks may play a part in the concentration of the disease in the North. The low Borrelia infection rate in ticks from the South is generally attributed to the lack of an efficient enzootic cycle of immature ticks and white-footed mice in the South compared to the one in the Northeast (![]()
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Shared vicariant histories between B. burgdorferi and northern I. scapularis:
Both B. burgdorferi and its vector, the northern lineage of I. scapularis, exhibit characteristics of a young evolutionary lineage, such as a low level of neutral genetic polymorphism within populations. Furthermore, statistical inference of population history on the basis of genetic compositions of local population samples suggested that the parasite and its vector share a similar demographic history of recent population growth and geographic expansion (Fig 8). The similar demographic histories between Borrelia and I. scapularis in the North may have the same evolutionary causes.
It has long been observed that species diversity of many animals and plants decreases with higher latitude (![]()
18,000 years ago, may have played an important part in creating such latitudinal gradients in species diversity. Fragmentation of a formerly continuous species distribution caused by geological events such as tectonic movements and glaciation is known as the "vicariance hypothesis" in biogeography (![]()
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Conclusion:
This study showed that the population structure of B. burgdorferi, a vector-transmitted parasitic bacterial species, is strongly shaped by the evolutionary history of its vector, the northern lineage of I. scapularis. We saw high infection rates in the Northeast where the genetic variability of the tick is low. There is also strong evidence of recent expansion of both the tick and Borrelia populations in the regions that were covered by glacier 18,000 years ago, as opposed to the tick populations in the South where the populations were not similarly destroyed. However, the population genetics of the northern tick and Borrelia are different, presumably because of the frequency-dependent selection on the Borrelia clones by the mammalian host. We postulate that the epidemic of B. burgdorferi in the Northeast (not only in humans but also in ticks and other mammals) is due to the biotic and genetic deprivation, induced by geological events like glaciations and modern human activities like deforestation and reforestation (![]()
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| FOOTNOTES |
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1 Present address: Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850. ![]()
| ACKNOWLEDGMENTS |
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We thank Edward Bosler, Jason Campbell, Howard Ginsberg, Richard Ostfeld, Bill Golde, Daniel Taub, M. Bushmich, and, in particular, Rob Mactier for helping us to collect ticks. Much of the DNA sequencing was done in John Dunn's lab in the Brookhaven National Lab. The study benefited greatly from guidance and advice from Mike Bell, Dou








) were coestimated for a northern tick sample (LI2), a mid-Atlantic sample (NC2), a southern tick sample (SC, clade A haplotypes excluded), and Borrelia carried by the northern tick sample. The graphs are likelihood curves of population growth rate when the population size estimate is at its maximum-likelihood value. Estimates of population growth rate (g) for the northern tick sample and the Borrelia sample are both significantly greater than zero. Estimate of g for the mid-Atlantic and southern samples are not significantly different from zero. (The 95% confidence range of g is approximated by 2 log-likelihood units on each side of the maximum-likelihood estimates.)