Heredity 83 (1999) 378±386 Received 20 August 1998, accepted 7 July 1999

Mitochondrial DNA sequence variation in paci®cus (: )

DOUGLAS E. KAIN*, FELIX A. H. SPERLING , HOWELL V. DALY & ROBERT S. LANE Department of Environmental Science, Policy and Management, Division of Insect Biology, University of California at Berkeley, Berkeley, CA 94720, U.S.A.

The western black-legged , Ixodes paci®cus, is a primary vector of the , burgdorferi, that causes . We used variation in a 355-bp DNA portion of the mitochondrial cytochrome oxidase III gene to assess the population structure of the tick across its range from British Columbia to southern California and east to Utah. Ixodes paci®cus showed considerable haplotype diversity despite low nucleotide diversity. Maximum parsimony and isolation- by-distance analyses revealed little genetic structure except between a geographically isolated Utah locality and all other localities. Loss of mtDNA polymorphism in Utah is consistent with a post- Pleistocene founder event. The pattern of genetic di€erentiation in the continuous part of the range of Ixodes paci®cus reinforces recent recognition of the diculties involved in using genetic frequency data to infer gene ¯ow and migration.

Keywords: gene ¯ow, Ixodes paci®cus, Ixodidae, Lyme disease, mitochondrial DNA, population structure.

Introduction stages of I. paci®cus each feed once and then drop from the host to moult or lay eggs under leaf litter (Peavey & The western black-legged tick, Ixodes paci®cus, is one of Lane, 1996). E€ective gene ¯ow is likely to occur only the most medically important ticks in western North when there is sucient rainfall, ground cover and soil America. A primary vector of the Lyme disease humidity to allow completion of these critical life stages. spirochaete, , it may also transmit Ixodes paci®cus is distributed down the west coast of the rickettsial pathogen Ehrlichia equi (Lane et al., 1991; North America from British Columbia to northern Baja Richter et al., 1996). The genetic structure of popula- California, with disjunct populations occurring east to tions of I. paci®cus is potentially important in under- Utah (Fig. 1) (Dennis et al., 1998). Ixodes paci®cus standing the epidemiology and evolutionary dynamics varies geographically with respect to its host preferences of the diseases it vectors (Tabachnick & Black, 1995). (Arthur & Snow, 1968; Arnason, 1992), its internal Current gene ¯ow in I. paci®cus is probably the result transcribed spacers (ITS1 and ITS2) in nuclear ribo- of a complex interaction of factors such as host speci®- somal DNA sequences (Wesson et al., 1993), and its city, the type of host parasitized, and ecological require- allozymes (Kain et al., 1997). ments (Hilburn & Sattler, 1986). As with most ticks, Allozyme data provided an equivocal picture of the I. paci®cus is dependent on its hosts for long-range population structure and biogeography of I. paci®cus movement (Hilburn & Sattler, 1986). Gene ¯ow potential (Kain et al., 1997). Eleven of 12 loci sampled across the in I. paci®cus should be high, because it parasitizes mobile range of I. paci®cus, including Utah, exhibited little vertebrates and has a broad host range which includes 80 genetic di€erentiation. In contrast, the twelfth locus, species of reptiles, birds, and mammals (Lane et al., glucose-6-phosphate isomerase (GPI), showed a pattern 1991). However, ecological restrictions may counterbal- of genetic di€erentiation that suggested either an adap- ance this potential. The larval, nymphal and adult life tive cline or secondary contact along a broad zone in central and southern California. This zone demarcated a *Correspondence and current address: Department of Biology, Agnes northern group of populations (WVC, CNY, CHB; Scott College, Decatur, GA 30030, USA; E-mail: dkain@agnes- Fig. 1) from a southern group (MDO, SCI, MR, scott.edu UTAH). Kain et al. (1997) recognized that either a Current Address: Department of Biological Sciences, CW-405 Bio- logical Sciences Bldg., University of Alberta, Alberta, Canada T6G rapid range expansion or high rates of gene ¯ow could 2E9. explain the pattern of allozyme variation. However, they

378 Ó 1999 The Genetical Society of Great Britain. POPULATION STRUCTURE OF IXODES PACIFICUS 379 proposed that the most plausible explanation for the variation across the range of I. paci®cus. Theoretically, pattern of allozyme variation in I. paci®cus was that it mtDNA has a fourfold smaller e€ective population size had a high rate of gene ¯ow. than nuclear genes because of its uniparental inheritance The purpose of this study was to use a portion of the and haploid nature (Simon et al., 1994). Additionally, a mitochondrial DNA (mtDNA) cytochrome oxidase III higher mutation rate provides more lineages for genetic gene to survey the extent and pattern of population drift and gene ¯ow to sort (Simon et al., 1994). Mitochondrial DNA was used successfully to assess the phylogeography of the related tick , the vector of B. burgdorferi in the eastern United States (Norris et al., 1996). In the current study on I. paci®cus, populations were chosen to allow assessment of con- gruence of mtDNA variation with allozyme data, both with respect to the GPI locus and the lack of allozyme di€erentiation in isolated populations.

Materials and methods

Samples and DNA Collecting localities are shown in Fig. 1 and detailed in Table 1. Except for CHB (California, Alameda Co., Anthony Chabot Regional Park), localities correspond to a subset of those used by Kain et al. (1997). Host- seeking adult ticks were collected from vegetation with ¯annel tick-drags. Ticks were either stored in 95% ethanol or allowed partially to engorge on rabbits (to induce enzyme systems), then frozen at )70°C. Ethanol- preserved specimens were placed individually in 1.5 mL microcentrifuge tubes and centrifuged under vacuum for 15 min to remove excess alcohol. Ticks were then put in 0.5 mL microcentrifuge tubes, frozen in liquid nitrogen, and then dry-homogenized using a ¯amed 200 lL pipette tip as a pestle. Fifty lL of TE (0.100 M Tris- HCl, pH 8.3, 0.1 mmol EDTA) was added to the dry sample and the solution was boiled for 15 min. Frozen partially engorged ticks were processed using the Qiagen Fig. 1 Map of western North America showing range of Qiamp Tissue Kit according to the manufacturer's Ixodes paci®cus by stippled shading. Collecting localities are Tissue protocol. Homogenates and extracted DNA were indicated with dots, with code as in Table 1. stored at )20°C until further use.

Table 1 Collecting localities and locality codes for Ixodes paci®cus populations. (BC, British Columbia; OR, Oregon; CA, California; UT, Utah)

Code State/Prov. County Locality WVC BC N/A West Vancouver, Eagleridge Viewpoint. CNY OR Douglas Highway 5, Canyon Cr. Rd, approx. 5 km S Canyonville. CHB CA Alameda Anthony Chabot Regional Park, Willow Cr. Trail. MDO CA San Luis Obispo Montana de Oro State Park, Pecho Valley Rd., 3.8 km S park entrance. SCI CA Santa Barbara Santa Cruz Island, along road to Prisoner's Harbour. MR CA San Diego Highway 15, Mercy Road exit, along bike paths. UTAH UT Washington Dixie National Forest, NW of Silver Reef, along road to Oak Grove Campground.

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. 380 DOUGLAS E. KAIN ET AL.

We employed the polymerase chain reaction (PCR), Hartigan, 1996). For comparison, two FST analogues, using universal COIII oligonucleotide primers (modi®ed GCA (Cockerham & Weir, 1993) and NST (Lynch & from Simon et al., 1994) to obtain a partial COIII DNA Crease, 1990) were used to estimate gene ¯ow also. sequence. The resulting PCR product was cloned using a Gene ¯ow can be estimated from the equation h ˆ Nm puc18/SmaI vector and sequenced (GenBank accession for the continuous generation, island model of pop- no. AF082986). Next, PCR primers speci®c to I. paci®cus ulation structure, or h ˆ 2Nm for the discrete gener- were designed from the cloned sequence and used to ation model (Rannala & Hartigan, 1996). Under the amplify a 403-bp target (primers: IpA TTC ATA GAA continuous generation model of population structure, GAC TAT CAC C, IpB TTC AAA GCC AAA ATG the PMLE of h does not assume population equilib- ATG AG). From this, 355 bp of sequence was used to rium and incorporates both immigration and birth appraise geographical variation. PCR products were rates into its estimate based on the relationship sequenced directly using either biotinylated solid phase h ˆ immigration rate/birth rate (Rannala & Hartigan, manual sequencing (29 individuals) (Hultman et al., 1996). To estimate Nm under a discrete generation

1989) or automated DNA sequencing methods (51 model (Lynch & Crease, 1990), GCA and NST can be individuals) (ABI 377, Applied Biosystems, Perkin- used to calculate gene ¯ow for the haploid island Elmer, Inc., Foster City, CA). Sample sizes ranged model of population structure using the equation from 10 to 18 ticks per population. Nm ˆ 1/(2FST) ) 1/2, where GCA and NST ˆ FST. NST partitions nucleotide diversity within and between populations when multiple populations are compared Data analyses (Lynch & Crease, 1990). NST weights diversity esti- Sequences were aligned using either the ESEE sequence mates by haplotype genetic distances, whereas the editing program (Cabot & Beckenbach, 1989) or the other estimators assume equal genetic distance among Sequence Navigator DNA analysis software (Applied haplotypes (Lynch & Crease, 1990). For the compu- Biosystems Division). Character state changes and tation of h, we used equations 16 and 17 in Rannala nucleotide composition were calculated with PAUP 3.1 & Hartigan (1996). FSTAT (Goudet, 1995) and HAPLO2 (Swo€ord, 1993). The amino acid sequence was deter- (Lynch & Crease, 1990) software programs were used mined using MacClade 3.0.3 (Maddison & Maddison, to calculate GCA and NST, respectively. 1992). Estimates of nucleotide diversity were calculated An isolation-by-distance analysis (IBD) was perform- using HAPLO2 software (Lynch & Crease, 1990) and ed to examine further the in¯uence of gene ¯ow on the estimates of haplotype frequency were computed using partitioning of genetic variation in I. paci®cus (Slatkin &

ARLEQUIN 1.1 software (Schneider et al., 1997). Maddison, 1990). The log10 of pairwise Nm values were Relationships among unique haplotypes were assessed regressed against log10 of pairwise distance values by maximum parsimony (MP) using PAUP 3.1 with a (Slatkin & Maddison, 1990). heuristic search algorithm and the random stepwise addition option treating all characters as unordered Results multistate (Swo€ord, 1993). Ixodes scapularis (Gene- Bank accession no. AF083466) was used for outgroup In total, 80 I. paci®cus and one I. scapularis haplo- comparison. To obtain a single unrooted parsimony types were sequenced. Within I. paci®cus, 36 nucleo- network, a 50% majority rule consensus tree was tide sites out of 355 bp varied among all haplotypes computed. (Fig. 2) and 2% was the maximum di€erence between The null hypothesis of panmixia was tested using an exact test of the di€erentiation of haplotypes among c populations, using ARLEQUIN 1.1 (Schneider et al., Fig. 2 Unique haplotypes, variable nucleotide positions, rela- 1997). The exact test of population di€erentiation of tive haplotype frequencies, sample sizes, and nucleotide and haplotypes tests the hypothesis that the observed distri- haplotype diversity estimates within Ixodes paci®cus. Numbers bution of frequencies is less likely than the distribution in top row of left side of ®gure indicate nucleotide positions expected under panmixia (Schneider et al., 1997). Prob- relative to a cloned sequence (GenBank accession no. abilities were estimated by permutation analysis using AF082986). Numbers in the body of the right side of ®gure indicate relative haplotype frequencies within each population. 1000 randomly permuted r ´ k contingency tables Sample sizes and nucleotide diversity estimates (‹2 SE) are (r ˆ populations and k ˆ di€erent haplotypes) of haplo- arrayed along bottom of the right-hand side of ®gure. HT, type frequencies. haplotype code; N, sample size per population and total Gene ¯ow was calculated using three estimators. number of that haplotype. Locality codes as in Table 1. Note: The pseudo-maximum likelihood estimator (PMLE) was haplotype frequencies may not equal unity because of round- used to compute the scale parameter h (Rannala & ing errors.

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. POPULATION STRUCTURE OF IXODES PACIFICUS 381

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. 382 DOUGLAS E. KAIN ET AL. any pair of haplotypes. Thirty variable sites were at Table 2 P-values for the pair-wise exact test of the random the third codon position, ®ve were at ®rst codon distribution of haplotype frequencies to test the null positions, and one was at a second codon position. hypothesis of panmixia in Ixodes paci®cus. Locality codes The transition to transversion ratio (1.05:1) was as in Table 1 relatively low compared to insects (Simon et al., WVC CNY CHB MDO SCI MR 1994). Out of a total of four amino acid replacements, three were produced by ®rst codon substitutions CNY 0.139 resulting in conserved amino acid replacements [pos. CHB 0.069 0.792 239, Val/Leu, pos. 356, Ileu/Leu (both nonpolar); MDO 0.011 0.071 0.385 pos. 209, Asn/Tyr (polar neutral)] (Fig. 2). One SCI 0.002 0.010 0.037 0.034 MR 0.030 0.791 0.847 0.614 0.108 second codon position substitution resulted in a UTAH <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 nonconserved (polar neutral to nonpolar) amino acid replacement (pos. 84, Ser/Phe). This pos. 84 substitu- tion was found in one tick from the Oregon signi®cantly di€erentiated from all other populations. In population, in all ticks from the Utah population, contrast, no apparent geographical pattern to the and in the outgroup I. scapularis (Genbank accession di€erentiation was found in the continuous portion of no. AF083466). the range. Average nucleotide diversity within populations The PMLE provided an estimate of h ˆ 20.72. Under ranged from 0.0005 (UTAH) to 0.0086 (MR) and the the continuous generation model, Nm ˆ 20.72 mean within-population nucleotide diversity was 0.0057 (SE ˆ 9.67) and, under the discrete generation model,

(Fig. 2). Average among-population nucleotide diversity Nm ˆ 10.36. The NST value was 0.329 (SE ˆ 0.217) and was 0.0028 (Fig. 2). A total of 38 unique haplotypes was the resulting Nm ˆ 1.02. The GCA estimator provided a found within and among populations (Fig. 2). Haplo- value of 0.167 which gives a Nm ˆ 2.49. type diversity within populations ranged from 0.173 for The IBD results indicate that there is no relationship Utah to 0.905 for MR (Fig. 2). Overall haplotype between the magnitude of gene ¯ow and geographical diversity was 0.930. All populations except Utah shared distances (Fig. 3). The R2 value was 0.065 and was at least one haplotype with another population. Ticks nonsigni®cant (P ˆ 0.28) and the regression slope was from Utah only carried unique haplotypes (37 and 38). )0.76. Thirty-four haplotypes were unique to various localities Strict consensus MP analysis performed on 2000 (Fig. 2). equally parsimonious trees resulted in an almost com- The exact test of di€erentiation of haplotype frequen- pletely unresolved polytomous tree (not shown). A cy was signi®cant over all populations (P<<0.001). The parsimony network derived from the 50% majority rule pairwise di€erentiation test was signi®cant in 12 out of consensus tree is shown. An unrooted tree is presented 21 comparisons (Table 2). The UTAH population was (Fig. 4) because tree topology was not a€ected by

Fig. 3 Isolation by distance analysis for populations of Ixodes paci®cus; R2 ˆ 0.065, b ˆ )0.76, P ˆ 0.28.

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. POPULATION STRUCTURE OF IXODES PACIFICUS 383

Fig. 4 Unrooted parsimony network of 38 unique Ixodes paci®cus haplotypes derived from the 50% majority rule consensus tree of 2000 equally parsimo- nious trees derived from a heuristic search. Locality and haplotype codes as in Table 1 and Fig. 2, respectively, with number of individuals in parentheses. Circle sizes are approximately propor- tional to haplotype frequencies across the range. Short hatching indicates nu- cleotide substitutions and asterisks indi- cate nucleotide substitution that results in the nonconserved amino acid re- placement. Numbers at branches indi- cate consensus tree percentage support for that branch. removing I. scapularis as an outgroup. This was probably type diversity (0.173) in Utah ticks compared to other because of the large number of nucleotide substitutions populations (0.762) (Fig. 2). The Utah locality can be separating I. scapularis and I. paci®cus relative to the characterized as a montane island with sucient mois- small number of substitutions separating ticks within ture and ground cover to support I. paci®cus (Dennis I. paci®cus. With the exception of the Utah locality, the et al., 1998). It is ecologically isolated from the western tree revealed little geographical structure. Relationships portion of the range by a large expanse of arid high of many haplotypes were reconstructed as a basal desert habitat that is unsuitable to I. paci®cus (Dennis polytomy, whereas others clustered into weakly support- et al., 1998). Such a distributional pattern could result ed clades or clades that combined geographically distant from either Pleistocene range fragmentation or a more haplotypes. recent founding event (Hewitt, 1996). The loss of mtDNA polymorphism in the Utah population, coupled with the fact that haplotypes in this population were not Discussion unusually diverged (Fig. 4), suggests a post-Pleistocene founder event leading to a genetic bottleneck, rather Utah and Santa Cruz Island populations than Pleistocene vicariance (Hewitt, 1996). Ixodes The isolation of the Utah population is supported by the paci®cus has the potential for both natural and fact that the mtDNA of I. paci®cus from this population human-mediated long-range dispersal because it para- was distinguishable from that of all other tick popula- sitizes birds, large mobile mammals, livestock, and tions evaluated. Furthermore, there was reduced haplo- domestic pets (Lane et al., 1991).

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. 384 DOUGLAS E. KAIN ET AL.

Allozyme data did not distinguish the Utah population pattern where mtDNA in this species was divided into (Kain et al., 1997). Such a result could be explained by two distinct clades (Norris et al., 1996). Because third high rates of gene ¯ow, non-neutrality of alleles, or positions in COIII sequences are generally faster-evolv- retention of ancestral polymorphisms (Avise, 1994). ing than the faster bases in 12S and 16S sequences These alternative hypotheses are dicult to choose (Simon et al., 1994), it is reasonable to expect that the between. Our mtDNA data suggest that glucose-6-phos- mtDNA region sequenced in this study should be phate isomerase alleles may be in¯uenced by selection (see informative in I. paci®cus. below). Because gene ¯ow models assume demographic In fact, mtDNA data agreed with the lack of a equilibrium, we cannot distinguish gene ¯ow from reten- discernable pattern in 11 out of 12 allozyme loci (Kain tion of polymorphisms if the population is not in et al., 1997), and it is likely that this pattern character- equilibrium (Whitlock & McCauley, 1999). If either a izes a signi®cant part of the genetic structure of founder event, range fragmentation, or restriction of gene I. paci®cus. Because mtDNA showed no pattern sug- ¯ow have occurred, allozymes, as markers of gene ¯ow, gesting secondary contact, it is most plausible that the are not as sensitive as mtDNA (Avise, 1994). pattern in the twelfth allozyme locus, GPI, is caused by In contrast to the distinctness of the Utah population, selection on that locus. there was no evidence that ticks from Santa Cruz Island As for sampling biases, we note that we sampled ticks were di€erentiated from mainland populations. Allo- over a relatively broad area at each locality. Even if we zyme data, likewise, show no di€erences between Santa had unduly concentrated our sampling e€orts, we Cruz Island and mainland ticks (Kain et al., 1997). We should have found high homogeneity within samples suggest that this is most probably because of the known rather than the many rare alleles, which we actually high incidence of recent anthropogenic e€ects, particu- observed. It is conceivable, however, that our sample larly as movement of pets and livestock between the sizes were insucient to detect rare, but shared, haplo- mainland and island (Miller, 1985). types, which would lead to the appearance of many unshared haplotypes. Inadequacy of gene ¯ow models remains a reason- Western portion of range able factor in the apparent lack of pattern presented by No discernible geographical patterns of di€erentiation our data. Gene ¯ow estimates in this study varied in mtDNA were exhibited by I. paci®cus within the 20-fold and ranged from moderate, Nm ˆ 1.02 (NST)to western portion of its range, and high haplotype very high, Nm ˆ 20.72 (PMLE, continuous generation diversity contrasted with low nucleotide diversity. model). An average estimate generated from allozyme Although estimates of gene ¯ow were moderate to high, data gives a Nm ˆ 5.70 (Kain et al., 1997). On the the data do not comfortably ®t this scenario, because whole, it is dicult to determine which estimator is there were so many unique haplotypes restricted to most appropriate to this study (Whitlock & McCauley, single localities and no signi®cant isolation-by-distance 1999). Indirect measures of gene ¯ow and migration e€ect (Fig. 3). Likewise, recent, rapid range expansion that are extrapolated from gene frequency data are seems unlikely in the presence of such a large number of variants of FST, a standardized measure of genetic unique haplotypes. Another explanation is unusually variance among populations. The assumptions under- rapid local evolutionary rates, perhaps following an lying these extrapolations include (i) low rates of earlier rapid range expansion that spread the haplotypes mutation, (ii) a stepping stone pattern of migration, (1, 2, 15) that are now widely distributed. The failure to (iii) spatial and temporal demographic stability, settle unambiguously on a single hypothesis may be (iv) nonoverlapping generations (for some estimators), because of a number of factors, including unsuitability and (v) equilibrium between gene ¯ow and genetic drift of mtDNA as a gene target, sampling biases, or gene (Whitlock & McCauley, 1999). We consider these ¯ow models that are biased or whose assumptions are assumptions in succession below. violated. Gene ¯ow models assume equilibrium between As a violation of the ®rst assumption, high rates of the e€ective movement of genes into a population and mutation could produce a large number of unique genetic drift (Whitlock & McCauley, 1999). If such haplotypes in the face of moderate to high levels of gene equilibrium exists, then geographical genetic structure ¯ow (Schneider, 1996). Gene ¯ow models assume will result even in the presence of high rates of gene ¯ow relatively low rates of mutation, which may apply to (Whitlock & McCauley, 1999). nuclear genes, but which may not apply to mtDNA The general informativeness of mtDNA for this level genes (Whitlock & McCauley, 1999). A high rate of of analysis was demonstrated in a survey of the closely mutation in the COIII gene in I. paci®cus may be related I. scapularis (Norris et al., 1996). Here 12S and evidenced by the unusually low transition to transver- 16S mtDNA sequences exhibited a broad geographical sion ratio (1.05).

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. POPULATION STRUCTURE OF IXODES PACIFICUS 385

For the second assumption, that there is a stepping the range. This probably reveals failures in current gene stone pattern of migration, the isolation-by-distance ¯ow models to re¯ect adequately the complexity of model employed here uses a one- or two-dimensional evolutionary processes underlying the geographical stepping stone model. If spatial structure of gene ¯ow di€erentiation of mtDNA haplotypes in I. paci®cus. deviates from this model, then an isolation-by-distance Because many systematic analyses are begun with little e€ect may not be detected (Whitlock & McCauley, or no ecological or genetic data available on the 1999). Likewise, if the stepping stone model applies on a organism being investigated, it will be crucial to contin- local level, but not at larger distances, then an isolation- ue to develop realistic gene ¯ow models that can be by-distance e€ect would appear to be absent (Whitlock applied in empirical studies such as ours. & McCauley, 1999). The third assumption of gene ¯ow models is that there Acknowledgements is spatial and demographic stability. Deviations from this model have not been extensively explored, although We thank the following people for their invaluable there are some indications that there could be signi®cant assistance in collecting I. paci®cus: D. Kindree (West levels of gene ¯ow with the presence of many unique Vancouver), C. S. Arnason (Simon Fraser University), haplotypes and the lack of an isolation-by-distance R. A. Gresbrink (Oregon State Health Division, e€ect. NuÈ rnberger & Harrison (1995) in a study on retired), S. G. Bennett and J. P. Webb, Jr. (Orange mtDNA in gyrinid water beetles, found that demo- County Vector Control District), K. Padgett (University graphic instability appeared to have led to high levels of California at Berkeley), K. MacBarron (San Diego of haplotype heterogeneity in the presence of high rates County Department of Environmental Health), L. of gene ¯ow. These beetles were localized in a series of Laughrin (University of California Natural Reserve isolated ponds with high extinction rates followed by System, Santa Cruz Island Reserve), J. R. Clover founder events (NuÈ rnberger & Harrison, 1995). Their (California Department of Health Services), P. Coy study concluded that signi®cant inadequacies remain in (California Department of Transportation). We thank the ability of current population genetic models to M. Lynch and B. Rannala for their assistance with data analyse the relationship between population structure analyses. This project was partially funded by Sigma Xi, and gene ¯ow. and the Walker Fund of UC Berkeley to DEK, a Hatch For the fourth assumption, three of four estimators Grant to FAHS, and by grant AI22501 from the used in this study assume discrete generations. However, National Institutes of Health and California Agricultur- there is some evidence that I. paci®cus has overlapping al Experiment Station Project 5719-AH to RSL. generations (Peavey & Lane, 1996) and therefore the fourth, and highest, estimate of migration rate References (Nm ˆ 20.72) might be most accurate. Finally, if there is violation of the ®fth assumption of ARNASON, C. S. 1992. Biology of the Western Black-legged Tick, equilibrium between gene ¯ow and genetic drift, then Ixodes paci®cus, (Cooley and Kohls, 1943): A Potential estimates of gene ¯ow could be in¯ated because of the Vector of Lyme Disease in South Coastal British Columbia. lack of such equilibrium. Recent and rapid range M.S. Thesis, Simon Fraser University, B.C., Canada. , D. R.AND SNOW , K. R. 1968. Ixodes paci®cus Cooley expansion usually results in a pattern of little population ARTHUR and Kohls, 1943: its life-history and occurrence. Parasitol- genetic structure (Slatkin & Hudson, 1991), but under ogy, 58, 893±906. certain conditions can produce patterns of high within- AVISE, J. C. 1994. Molecular Markers, Natural History and population haplotype diversity within populations, and Evolution. Chapman & Hall, New York. high estimates of gene ¯ow (Patton et al., 1996). The CABOT, E. L.AND BECKENBACH , A. T. 1989. Simultaneous editing original gene pool would have to be highly polymorphic, of multiple nucleic acid and protein sequences with ESEE. with colonists expanding into new territory and carrying Comp. Appl. Biosci., 5, 233±234. a biased subsample of haplotypes that included both COCKERHAM, C. C.AND WEIR , B. 1993. Estimation of gene ¯ow rare, localized and more common alleles. The presence from F-statistics. Evolution, 47, 855±863. of a few ancestral haplotypes within most populations DENNIS, D. T., NEKOMOTO, T. S., VICTOR, J. C., PAUL, W. S.AND would in¯ate gene ¯ow estimates even if current rates PIESMAN, J. 1998. Reported distribution of Ixodes scapularis and Ixodes paci®cus (Acari: Ixodidae) in the United States. were low (Patton et al., 1996). J. Med. Entomol., 35, 629±638. In conclusion, we were able to ®nd mtDNA haplotype GOUDET, J. 1995. FSTAT, Version 1.2: a computer program to di€erentiation between a population of Utah ticks and calculate F-statistics. J. Hered., 86, 485±486. all populations of I. paci®cus where allozyme data were HEWITT, G. M. 1996. Some genetic consequences of ice ages, and not able to do so. We found no di€erentiation of their role in divergence and speciation. Biol. J. Linn. Soc., mtDNA haplotypes within the continuous portion of 58, 247±276.

Ó The Genetical Society of Great Britain, Heredity, 83, 378±386. 386 DOUGLAS E. KAIN ET AL.

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