Population Genetics of the Chewing Louse, Thomomydoecus Minor

Molecular Ecology (2015) 24, 4129–4144 doi: 10.1111/mec.13306

Host behaviour drives parasite genetics at multiple geographic scales: population genetics of the chewing louse, Thomomydoecus minor

SHEREE E. HARPER, THERESA A. SPRADLING, JAMES W. DEMASTES and COURTNEY S. CALHOUN Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614-0421, USA

Abstract Pocket gophers and their symbiotic chewing lice form a host–parasite assemblage known for a high degree of cophylogeny, thought to be driven by life history parameters of both host and parasite that make host switching difficult. However, little work to date has focused on determining whether these life histories actually impact louse populations at the very fine scale of louse infrapopulations (individuals on a single host) at the same or at nearby host localities. We used microsatellite and mtDNA sequence data to make comparisons of chewing-louse (Thomomydoecus minor) population subdivision over time and over geographic space where there are different potential amounts of host interaction surrounding a zone of contact between two hybridizing pocket-gopher subspecies. We found that chewing lice had high levels of population isolation consistent with a paucity of horizontal transmission even at the very fine geographic scale of a single alfalfa field. We also found marked genetic discontinuity in louse populations corresponding with host subspecies and little, if any, admixture in the louse genetic groups even though the lice are closely related. The correlation of louse infrapopulation differentiation with host interaction at multiple scales, including across a discontinuity in pocket-gopher habitat, suggests that host behaviour is the primary driver of parasite genetics. This observation makes sense in light of the life histories of both chewing lice and pocket gophers and provides a powerful explanation for the well-documented pattern of parallel cladogenesis in pocket gophers and chewing lice.

Keywords: cophylogeny, host, inbreeding, parasite population genetics Received 21 April 2015; revision received 12 June 2015; accepted 3 July 2015

microevolutionary host–parasite processes can drive Introduction macroevolutionary cophylogeny (Nadler et al. 1990; One of the most intimate associations in nature is that Huyse et al. 2005; Du Toit et al. 2013; Liu et al. 2013; of parasites with their hosts. Cophylogeny represents Koop et al. 2014). an especially intimate host–parasite interaction that has Pocket gophers (Rodentia: Geomyidae) and their taken place over long periods of evolutionary time as chewing lice (Phthiraptera: Trichodectidae) have been reflected by the similar evolutionary histories of both the subjects of considerable study with respect to lineages (Futuyma & Slatkin 1983). The study of para- cophylogeny. Parallel cladogenesis between these site population genetics is important for what it can groups is extensive, taking place at multiple phyloge- reveal on multiple scales (Nadler 1995; Criscione et al. netic levels ranging from the fine-scale comparisons 2005), including the perspective it can provide on how of conspecific hosts and their lice (Demastes et al. 2012) to more distantly related congeneric species of Correspondence: Theresa A. Spradling, Fax: 319-273-7125; hosts and their lice (Light & Hafner 2007) to the even E-mail: [email protected] more distantly related genera of hosts and their lice

(Hafner et al. 1994; Demastes et al. 2003 and references The few published data available on the population therein). The pattern of cophylogeny observed in each genetics of chewing lice of pocket gophers suggest of these cases is not perfect, but it is strong enough that louse dispersal from host to host is restricted and to signal a long history of intimate association, and it that inbreeding in some cases may be intense. Nessner generally exceeds the degree of phylogenetic et al. (2014) described a suite of microsatellite loci for congruence seen in other lice and their hosts (Clayton Geomydoecus ewingi, chewing lice of the pocket gopher, et al. 2004). Limited opportunity for dispersal by the Geomys breviceps, in Texas. Because the intent of that parasite seems to be the strongest correlate of study was to describe microsatellite loci, population- cophylogeny for the chewing lice of birds and level parameters were only reported for a small number mammals (Clayton et al. 2004), and the biology of of lice from a small number of hosts, and a more both pocket gophers and their chewing lice is thought thorough investigation of population genetics is war- to work together to effectively limit parasite dispersal ranted. Earlier studies of the population genetics of (Hafner & Nadler 1988). chewing lice from pocket gophers were based on allo- Pocket gophers are fossorial, occurring in patchily zyme data, which are typically less variable than more distributed populations, with genetically distinct recent microsatellite data (e.g. Sanchez et al. 1996; Irvin groups being largely parapatric (Daly & Patton 1990). et al. 1998; De Innocentiis et al. 2001). Only two of 11 Pocket gophers also are asocial animals except during allozyme loci examined in the pocket-gopher chewing brief mating encounters (Hall 1981). These factors are louse, Thomomydoecus minor, showed any polymor- expected to work in concert to limit parasite transmis- phism, and two of five infrapopulations were sion between pocket-gopher species, populations and monomorphic for all loci (Nadler & Hafner 1989). individuals. Chewing lice of pocket gophers are Allozyme genetic variation was similarly limited for wingless, eyeless insects that hold fast to the host’s another pocket-gopher chewing louse, Geomydoecus actu- hair and feed on skin detritus (Marshall 1981). Thus, osi, yet enough genetic variation existed in G. actuosi to the very specializations that make chewing lice well show significant subdivision of infrapopulations at a suited for a subterranean existence on a solitary host locality and to show that parasite FST between localities also should greatly reduce their ability to disperse. was similar to, but slightly smaller than, host FST When this poor dispersal ability is coupled with the between the same localities, providing the strongest solitary nature of pocket gophers, the probability of genetic evidence to date that louse gene flow is limited colonizing a new host individual is low. However, by gopher gene flow (Nadler et al. 1990). while pocket-gopher and chewing-louse life histories We aimed to expand current knowledge of chewing- are reasonably well understood, the effect of these life louse population genetics using highly variable history parameters on the genetics of the parasite at microsatellite data and mtDNA sequences. We chose a the finest scale, within and among populations and unique setting for the study, one that allowed multiple infrapopulations (i.e. all of the individuals of the comparisons of chewing-louse population subdivision same species on a single host, Esch et al. 1975), has over multiple layers of host reproductive activity. We yet to be fully investigated, and mode of transmission worked at a zone of contact between two pocket-gopher (i.e. from one infrapopulation to another) remains subspecies, Thomomys bottae connectens and T. b. opulen- poorly understood. Transmission may be considered tus, described by Smith et al. (1983). The region of horizontal if it involves movement between unrelated contact occurs at a narrowing of the Rio Grande Valley hosts or vertical if it involves transfer from mother to that we will refer to as the San Acacia constriction offspring, and parasite population genetic data are (Fig. 1), where pocket-gopher habitat is limited and useful for distinguishing the two transmission pocket gophers are patchily distributed and rare (Smith patterns (Criscione 2008). The hypothesis of strictly et al. 1983; J. W. Demastes, personal observation). To maternal transmission has been falsified for two the north and to the south of this zone, pocket gophers populations of chewing lice of pocket gophers are far more abundant in the wider, lusher and (today) (Demastes et al. 1998; Patton et al. 1984), but it more irrigated regions of the valley (Smith et al. 1983), remains unclear whether transmission is predomi- where their high density makes them a nuisance to nantly along genealogical lines with limited horizontal alfalfa farmers. The zone of contact between these two transmission or completely independent of genealogy pocket-gopher subspecies coincides with a marked shift (Demastes et al. 1998). Understanding mode of in vegetation along the Rio Grande Valley (Smith et al. transmission and the role of varying degrees of host 1983). These two subspecies of pocket gophers show a interaction on parasite population genetics is a remarkably low genetic similarity (69% based on key component of a more robust understanding of allozyme data), and their karyotypes, which have the host–parasite cophylogeny. same number of chromosomes (2N = 76), differ in

New Mexico zone, G. aurei has crossed over the host contact zone Rio Grande Las Nutrias and is experiencing an ongoing expansion of its range Albuquerque as it colonizes the southern host subspecies and Study site e displaces the usual louse species (Hafner et al. 1998 d n and J. W. Demastes, unpublished data). The ongoing a r G Northern Host range expansion of G. aurei past the zone of host o Ri Subspecies hybridization led us to question what might be hap- La Joya pening with another louse, Thomomydoecus minor, that 1992, 2011 also inhabits pocket gophers on both sides of the San Acacia constriction. Thomomydoecus and Geomydoecus San Acacia lice are well-differentiated genera morphologically (Hellenthal & Price 1984; Page et al. 1995) and geneti- host hybrid zone cally (Nadler & Hafner 1989; Hafner et al. 1994), and San Acacia they are frequent cohabitants that appear to partition 1990, 1991 resources on individual hosts (Reed et al. 2000). Our specific aims in this study were threefold. (i) We examined T. minor populations on opposite sides Southern Host Lemitar of the host contact zone to determine whether the Subspecies different host subspecies on opposite sides of the constriction may interact infrequently enough to create any genetic subdivision within T. minor. Given that G. aurei has recently crossed this boundary (Hafner 0 5 mi Socorro et al. 1998), we initially hypothesized a relatively low 1990 0 5 km level of population subdivision within T. minor at this geographic/host boundary. This aim involved also Fig. 1 Map of New Mexico (inset) showing study site along testing the current distribution of host pocket gophers the Rio Grande. Expanded view shows Thomomydoecus minor to determine whether the two host subspecies remain sample sites (squares), collection dates, and approximate in the same geographic regions today that they did region of suitable host habitat (grey shading). Designations of when studied by Smith et al. (1983) and Demastes northern host subspecies (open-square localities) and southern host subspecies (shaded-square localities) are expected based et al. (1998). (ii) We examined louse infrapopulations on Smith et al. (1983). at the same locality (i.e. in the same or a neighbouring alfalfa field) and at different localities approximately 16–24 km apart, but on the same side of the host chromosomal morphology by as many as 17 pairs of contact zone, to determine the degree of louse chromosomes that are biarmed in one subspecies and infrapopulation subdivision in regions where host uniarmed in the other (Smith et al. 1983). While these contact is relatively more likely. Given prior evidence levels of genetic divergence might suggest extremely that chewing-louse transmission at this site is not limited introgression would be possible, Smith et al. strictly maternal and that there are even mixed (1983) provided genetic evidence of hybridization communities of G. aurei and G. centralis on the same between these two highly differentiated forms where host (Demastes et al. 1998; Hafner et al. 1998), we they meet. Nevertheless, they concluded that, even in postulated that louse population subdivision could be the face of ongoing hybridization, ‘extensive introgres- weak and horizontal transmission frequent at this sion is not possible, even over time, since populations scale. (iii) Owing to available historical samples from through the zone [of contact] are at continual low our region of interest, we were able to compare louse density, have patchy distributions, and are subjected to population genetics and the genetic consequences of strong temporal perturbations due to periodic flooding’ multiple founder events and seasonal bottlenecks that (Smith et al. 1983, p. 15). are a routine part of the louse life cycle (Nadler et al. While chewing lice of pocket gophers are normally 1990) over a 19-year time span at the same locality. highly host specific, Hafner et al. (1998) described an Given a 40-day generation time estimated for a con- unusual case of recent host colonization at this particu- gener (G. oregonus; Rust 1974), this 19-year time span lar pocket-gopher contact zone. While the northern correlates with a 173-generation time span. We also pocket-gopher subspecies normally hosts the louse used molecular methods to test for inbreeding, which Geomydoecus aurei, and the southern pocket-gopher would be predicted to occur in isolated, bottlenecked subspecies normally hosts G. centralis, at this contact populations.

Methods Louse population sampling and DNA isolation Chewing lice were collected from 15 pocket-gopher Host genetics individuals at five locations, two north and three south To verify host subspecies for the lice examined herein of the San Acacia constriction (Fig. 1; Table 1). Lice and to assess whether the host contact zone is still from each host individual were placed in labelled Nunc located along the Rio Grande Valley near the San Acacia CryoTube vials (Nalge Nunc International, Denmark) constriction between the towns of La Joya and San Aca- and stored on dry ice or in liquid nitrogen until return ° cia, New Mexico, 15 pocket gophers were examined to the laboratory, where they were stored at 80 C. from five localities, two north and three south of the San Genomic DNA was extracted from individual lice = Acacia constriction. Eight of the specimens were col- (n 275) using the QIAamp DNA Micro Kit (Qiagen, lected in 2011, and the other seven were collected from Valencia, CA, USA). Manufacturer’s recommendations 1990 to 1992 (Fig. 1; Table 1). The New Mexico Depart- were followed with the following exceptions: prior to ment of Game and Fish (NMDGF) approved collection DNA extraction, individual louse bodies were placed of specimens, and procedures followed all guidelines set on a freezer block under the dissecting microscope and by the University of Northern Iowa Institutional Animal punctured six times using a #2 insect pin. Carrier RNA Care and Use Committee and the American Society of was added to AL buffer before the addition of ethanol. Mammalogists (Sikes & Gannon 2011). DNA extraction, Incubation before elution was increased to 5 min, and l PCR, DNA sequencing and sequence analysis followed DNA from each louse was eluted in 30 LH2O. Follow- standard methods, yielding sequences from the mito- ing DNA extraction, cleared louse bodies were mounted chondrial cytochrome c oxidase I (COI) gene and three on microscope slides for preservation as vouchers. nuclear genes, interphotoreceptor retinoid-binding protein (IRBP), b-fibrinogen (b-fib) and recombination activating gene 1 (Rag1; Supplementary Methods). Thomomydoecus mitochondrial DNA sequencing and population analysis A 710-base pair (bp) fragment of the mitochondrial Table 1 Collection localities, infrapopulation sample sizes and cytochrome oxidase subunit I gene (COI) was amplified collection dates for 275 individual lice from 15 pocket gophers (Table 1) using published universal insect primers LCO1490 and Louse Side Locality (in HCO2198 (Folmer et al. 1994) in reactions with 1.0 lLof Host sample of Socorro Collection DNA (approximately 0.47–2.0 ngl), 2.5 lM of each specimen size constriction Co., NM) year primer (10 lm), and 1X HotStart GoTaq Green Master 751 20 N 1.4 mi S, 0.8 mi 2011 Mix (Promega, Madison, WI) in a final volume of W Las Nutrias 20.0 lL. Thermal cycles for all samples included an 752 6 N 1.4 mi S, 0.8 mi 2011 initial denaturation at 95 °C for 2 min, followed by 40 W Las Nutrias cycles of denaturation at 94 °C for 45 s, annealing at 753 6 N 1.4 mi S, 0.8 mi 2011 45 °C for 45 s, and elongation at 72 °C for 45 s, with a W Las Nutrias final extension at 72 °C for 10 min. PCR products were 759 19 N 0.9 mi S, 0.1 mi 2011 W La Joya purified using ExoSAP-IT (USB, Cleveland, OH) and 760 15 N 0.9 mi S, 0.1 mi 2011 were sent to the Iowa State University DNA Facility for W La Joya sequencing on an Applied Biosystems 3730xl DNA 761 20 N 0.9 mi S, 0.1 mi 2011 Analyzer. Sequences were screened for error and edited W La Joya manually using GENEIOUS PRO (version 5.4.6; Biomatters 434 20 N 1 mi S La Joya 1992 Ltd), yielding a 583-bp fragment of sequence common 435 19 N 1 mi S La Joya 1992 to all individuals. A COI sequence for T. genowaysi, the 436 25 N 1 mi S La Joya 1992 437 23 N 1 mi S La Joya 1992 chewing louse of a Mexican pocket gopher, was used 3243 19 S 0.7 mi S, 0.2 mi 1990 for outgroup analysis (kindly provided by Verity E San Acacia Mathis). 1428 20 S San Acacia 1991 Uncorrected sequence divergence values and the 756 19 S 1.1 mi S, 0.75 mi 2011 most appropriate molecular substitution model were E Lemitar assessed using MEGA 6.06 (Tamura et al. 2013). Bayesian 757 20 S 1.1 mi S, 0.75 mi 2011 analysis was performed with the selected model using E Lemitar 3203 24 S Socorro 1990 the MRBAYES (Ronquist et al. 2012) plugin for GENEIOUS R8 (http://www.geneious.com; Kearse et al. 2012). Chain

© 2015 John Wiley & Sons Ltd HOST BEHAVIOUR DRIVES PARASITE GENETICS 4133 length was set to 1 500 000 with 4 heated chains, a 0.1 software CONVERT (version 1.31; Glaubitz 2004) was used heated-chain temperature and a subsampling frequency to reformat all data files for use in additional genetic of 300. Burn-in was set to 100 000 using unconstrained analysis programs. Locus quality was evaluated using branch lengths for priors. Output was evaluated to several measures, including reamplification and tests assess the quality of runs using three criteria recom- for null alleles (Data S1, Supporting information). mended in the MRBAYES documentation (http://mrbayes.sourceforge.net/manual.php). TCS 1.21 software Population analysis of Thomomydoecus microsatellites (Clement et al. 2000) was used as an additional means to visualize relative frequencies of haplotypes and the Standard AMOVA comparisons using the number of number of mutational steps between them. Population different microsatellite alleles (FST based) were made in Φ subdivision ( ST) values were calculated and standard ARLEQUIN (version 3.5.1.2; Excoffier & Lischer 2010).

AMOVA (analysis of molecular variance) comparisons Population subdivision values (FST) were calculated using pairwise differences in DNA sequences were using permutations calculated by the program for made in ARLEQUIN (version 3.5.1.2; Excoffier & Lischer assessing whether differences from 0 were significant 2010). (FSTAT version 2.9.3.2; Goudet 1995, 2001). STRUCTURE (version 2.3.4; Pritchard et al. 2000; Falush et al. 2003; Hubisz et al. 2009) was used to implement a Thomomydoecus microsatellite development and Bayesian algorithm to identify genetically homogenous microsatellite genotyping clusters of individuals. Population admixture and corre- To discover microsatellite loci in the genome of T. lated allele frequencies were assumed. Burn-in period minor, a pool of 160 T. minor lice was formed from the was set at 3 9 105 followed by 3 9 106 MCMC repeti- lice of five gophers from a single collection site (1.4 mi tions for most analyses to reach stable estimates of S, 0.8 mi W Las Nutrias, New Mexico). DNA was parameters. Five runs were evaluated for each cluster extracted and sent to the Evolutionary Genetics Core value, which ranged 1–5; quality of each run was Facility at Cornell University for microsatellite discov- assessed by examining variation in log-likelihood scores ery; detailed methods of microsatellite development and alpha. Infrapopulation identity was used as a prior and primer choice are provided in Supporting Informa- for the LOCPRIOR model to enhance clustering when tion (Supplementary Methods). genotypic signal is weak (Hubisz et al. 2009). Assign- For all 275 louse individuals included in the mtDNA ment of louse individuals to populations was made by analysis, seven microsatellite loci were amplified using host origin. For analysis of the 10 louse infrapopulations microsatellite primers developed for T. minor (Table S1, north of the constriction, settings described above were Supporting information) using the dye-labelled, nested, used, but burn-in period was set at 10 000 followed by 3-primer amplification technique (Schuelke 2000). All 100 000 MCMC repetitions. For a similar analysis of the reactions contained 0.5 lL DNA (approximately five louse infrapopulations from south of the constric- 0.23–1.0 ng), 0.4 lL each louse primer (1 lM to 10 lM as tion, burn-in was set at 100 000 followed by 100 000 described by Schuelke 2000), 0.4 lL designated MCMC repetitions and Alphapropsd was adjusted fluorophore-labelled M13 primer and 1X GoTaq Clear upward in some runs to reach stationarity. STRUCTURE HotStart Master Mix (Promega, Madison, WI, USA) in a HARVESTER (web version 0.6.93; Earl & von Holdt 2012) 10.0 lL reaction. Thermal cycles were as follows for all was used to implement the Evanno et al. (2005) method samples: 1 cycle of denaturation for 2 min at 95 °C, 10 to evaluate the appropriate number of clusters (K) for cycles of denaturation at 94 °C for 40 s, annealing at each analysis by examining the mean log-likelihood 58 °C for 40 s, and elongation at 72 °C for 40 s, scores and the ΔK for each cluster value. followed by 30 cycles of denaturation at 94 °C for 40 s, Because Structure analysis may sometimes group annealing at 53 °C for 40 s, and elongation at 72 °C for individuals in too few clusters, resulting in an inaccu- 40 s and a final extension at 72 °C for 15 min. rate assessment of the value of K (Kalinowski 2010), an Representative samples from each infrapopulation, alternative population clustering approach was including all negative PCR controls, were screened for performed in POPTREE2 (Takezaki et al. 2010). For all 15 amplification and contamination on 1.2% agarose gels. louse infrapopulations studied, pairwise DA values (Nei Cleanly amplified products were sent to the Iowa State et al. 1983) were calculated and neighbour-joining (NJ; University DNA Facility for analysis on an Applied Saitou & Nei 1987) trees were built based on genetic Biosystems 3730 DNA Analyzer. Output fsa files were distances. These analyses were performed for all 7 loci, scored, each by two workers, using GENEMARKER and for each locus individually to determine whether software (version 1.90; SoftGenetics, State College, PA), each locus seemed to be informative at a population coupled with visual inspection and editing. The level and whether a common ‘signal’ would be derived

© 2015 John Wiley & Sons Ltd 4134 S. E. HARPER ET AL. from each locus. These analyses were repeated for only between them (1035-bp sequence data). One haplotype northern infrapopulations (including all infrapopula- group was found exclusively in gophers expected to be tions and including only the infrapopulations with sam- T. b. connectens based on their collection north of the ple sizes >6), and they were repeated for only southern San Acacia constriction, and the other was found exclu- infrapopulations. sively in gophers expected to be T. b. opulentus based To examine any potential asymmetry in geneﬂow on their collection south of it (Fig. S1, Supporting infor- rates among infrapopulations, and to provide a method- mation). Nuclear genes provided a very similar set of ologically different approach to examining population relationships with some mixing of alleles consistent connectivity, recent migration rates were calculated with limited hybridization (4 of 16 pocket gophers each using BayesAss 3.0 following the guidelines in its docu- were heterozygous for one of the alleles that is typical mentation for optimizing the results (Wilson & Rannala of the other subspecies in the 3 loci examined; Supple- 2003; Rannala 2012). Ten iterations were performed mentary Results, Figs S2–S4, Supporting information). with different starting seeds to compare results. Trace ﬁles were evaluated using Tracer 1.6 (Rambaut et al. Characterization of newly discovered Thomomydoecus 2014) to assess whether burn-in and run length settings microsatellite loci were appropriate and to choose the runs with the best effective sample size (ESS) scores. The two small Seven tetranucleotide repeat microsatellite loci infrapopulations from Las Nutrias were excluded from (Table S1, Supporting information) proved informative the analysis to improve results, because the method and reliable for downstream population genetics tends to work poorly when sample size is small comparisons as they showed few genotyping errors, no (Meirmans 2014); 30 million iterations were required for evidence of genotypic disequilibrium, little evidence of these runs. null alleles, and observed and expected heterozygosity

Allelic richness and inbreeding coefficient (FIS) values values that were in close agreement (Supplementary were calculated in FSTAT (version 2.9.3.2; Goudet 1995, Results, Supporting Information). > 2001); tests for statistical significance of FIS values 0 were performed using permutations calculated by the Thomomydoecus population genetics at the host hybrid program. Global tests of Hardy–Weinberg heterozygote zone deficiency were assessed by infrapopulation in GENEPOP (version 1.2; Raymond & Rousset 1995; Rousset 2008). Of the 583 bp of COI sequenced for all T. minor individ- Markov chain parameters were set to 1000 dememoriza- uals, there were 24 variable nucleotide positions, 21 of tion steps and 100 batches with 1000 iterations per which corresponded to the third position of codons, batch. ARLEQUIN (version 3.5.1.2; Excoffier & Lischer two of which corresponded to first positions of codons 2010) was used to calculate observed and expected and one of which corresponded to a second position of heterozygosity (HO and HE) for individual loci in each a codon. A simple HKY model was determined to be of the 15 infrapopulations. the most appropriate for phylogenetic analysis by both In all statistical comparisons involving multiple tests BIC and AICc criteria. Bayesian analysis resulted in a of the same hypothesis, the Benjamini–Yekutieli (B–Y; tree rooted between two groups of haplotypes (Fig. S5, Benjamini & Hochberg 1995) modified false-discovery Supporting information). These haplotype groups were rate method for correction of the critical p-value was reflected clearly in TCS analysis, with two common used as advocated by Narum (2006) for providing a haplotypes, each of which has several rare haplotypes more appropriate correction to experiment-wise type I within a few mutational steps of it (Fig. 2A). The two error rate for population genetic data than does the major mitochondrial haplotype groups were concordant more common Bonferroni correction. These false-discov- with geography and with host subspecies, with one ery rate corrections were necessary for tests of linkage haplotype group found exclusively in lice collected on disequilibrium, deviation between HO and HE, global gophers north of the San Acacia constriction and the tests of heterozygote deficiency, FST and FIS. other found exclusively in lice south of it. The most common T. minor haplotypes from opposite sides of the constriction differed by 2.1% uncorrected sequence Results divergence compared with 0–0.3% sequence divergence among haplotypes north of the constriction and 0–0.2% Distribution of host subspecies sequence divergence among haplotypes south of the Mitochondrial DNA sequences from pocket gophers constriction. Thomomydoecus minor haplotypes differed sorted neatly in two haplotype groups that displayed from the outgroup, T. genowaysi, by 5.0–5.7% uncor- an average 8.0% uncorrected sequence divergence rected sequence divergence.

© 2015 John Wiley & Sons Ltd HOST BEHAVIOUR DRIVES PARASITE GENETICS 4135 0.2 0.0 0.4 0.6 0.8 1.0 AB north of the constriction vs. south of it, with no Las apparent asymmetry in direction of migration Nutrias (Tables 2 and S3). A neighbour-joining tree built from DA values based on all seven microsatellite loci grouped infrapopulations in the same two clusters indicated by other Northern analyses (Fig. S6, Supporting information), with an

average DA between northern and southern infrapopulations of 0.20 (Table 2). When each microsatellite

La locus was analysed separately using DA values to Joya build neighbour-joining trees for the 13 largest infrapopulations, six of the seven loci clustered infrapopulations strictly concordant with collection locality north vs. south of the San Acacia constriction. The single locus that did not cluster infrapopulations in this manner (locus 3495) also placed infrapopula- San tions primarily in a north vs. south grouping, with Acacia the exception of clustering one southern infrapopulation from near San Acacia with the northern infrapopulations. Therefore, all loci seemed Lemitar to be informative regarding population structure at Southern this level and were consistent in indicating a single overarching pattern of population structure. Socorro Tests of louse movement within and among localities Fig. 2 (A) TCS analysis of Thomomydoecus minor mtDNA sequences shows two divergent mtDNA lineages. Circle sizes Within localities, AMOVAs of both mtDNA and approximate haplotype frequency, and empty circles represent microsatellites indicated that a significant component inferred haplotypes not sampled. Each line segment represents of overall genetic variation could be partitioned among one mutational step. Light grey indicates haplotypes seen in contemporaneous infrapopulations (Table 2). These louse infrapopulations north of the San Acacia constriction, infrapopulations from the same locality also showed and dark grey indicates louse haplotypes found south of there. moderate to high levels of population subdivision as (B) Structure analysis of Thomomydoecus minor microsatel- measured by F and Φ (Table 2). Migration lite data (K = 2) sorts louse infrapopulations (separated by ST ST white bars) in a manner consistent with mtDNA and with estimates from one infrapopulation to another within a geography. locality were low, showing a maximum of about twice as many migrants between infrapopulations at the same locality as between infrapopulations separated by AMOVA indicated that the variation in mitochondrial geographic distances of 16–24 km (Tables 2 and S3). Φ DNA explained by differences between lice on opposite Most ST values between contemporaneous infrapopu- sides of the San Acacia Constriction is large and lations at the same locality, although large, were not significant (94% of variation, P < 0.001), with average significantly different from 0 after correction for Φ ST values between northern and southern infrapopula- multiple tests, but pairwise FST values based on tions being 0.82 (Table 2). microsatellite data were significantly different from Structure analysis of nuclear microsatellite data zero for 8 of 12 (67%) pairwise comparisons among indicated two clusters (K = 2) of lice that corre- northern infrapopulations and one of two (50%) sponded to geographic distribution of lice north of comparisons among southern infrapopulations after the San Acacia constriction vs. south of it (Fig. 2B). correction for multiple tests (Table S2, Supporting Population subdivision estimated from microsatellite Information). Structure analysis did not indicate data was high between northern and southern genetic partitioning of louse individuals by infrapopu- = infrapopulations (FST 0.43; Tables 2 and S2). A large lation within a locality. proportion of microsatellite genetic variation in lice Population subdivision between lice from different was explained by this relationship (AMOVA, 40%, localities, but collected at approximately the same time, < Φ P 0.001; Table 2). Bayesian analysis of recent migra- was high as indicated by both mtDNA-based ST tion rates indicated restricted migration between lice values and microsatellite-based FST (Tables 2 and S2).

Table 2 Measures of population structure as indicated by mitochondrial and microsatellite data

mtDNA sequences Microsatellites

Average Average Migration Φ † † †,‡ † AMOVA* ST AMOVA* FST Estimate DA

Northern infrapopulations vs. 94% 0.82 40% 0.43 Individuals 0.20 southern infrapopulations P = 0.001 P < 0.0001 in N from S: Avg. 1.3% (1.0–1.6%) Individuals in S from N: Avg. 1.3% (1.0–1.3%) § Between localities in the north 25.2% 0.40 3.15% 0.09 Avg. 2.5% 0.05 P = 0.11 P = 0.108 (1.5–5.8%) ¶ Among infrapopulations at a locality 10.6% 0.15 5.8% 0.06 Avg. 3.5% 0.03 P = 0.003 P < 0.0001 (1.3–6.9%) k Between localities in the south 14.9% 0.12 3.5% 0.09 Avg. 3.3% 0.04 P = 0.660 P = 0.662 (2.0–4.6%) Among infrapopulations at a locality** 23.3% 0.12 3.5% 0.03 Avg. 6.5% 0.02 P = 0.010 P = 0.011 (4.1–10.4%) †† Old infrapopulations vs. new at La Joya 1.5% 0.13 0.89% 0.04 Avg. 3.7% 0.03 P = 0.304 P = 0.818 (2.1–5.2%)

*AMOVAs are reported as % of total genetic variation explained by a given comparison and the associated probability value (P); sig- niﬁcant P values are indicated in bold. † Values averaged from pairwise comparisons. ‡ Recent migration rate estimates as a percent of infrapopulation estimated to be immigrants from a comparison infrapopulation, averaged over all relevant pairwise comparisons (the two small infrapopulations from Las Nutrias were omitted to improve estimation). § Only northern infrapopulations included, noncontemporaries (old La Joya infrapopulations) excluded. ¶ Only northern infrapopulations included, grouped by locality and by date, so comparisons are only between contemporaries at a locality. k Only southern infrapopulations included, grouped by locality, noncontemporaries (Lemitar infrapopulations) excluded. **Only southern infrapopulations included, grouped by locality and by date, so comparisons are only between contemporaries at a locality. †† Only La Joya infrapopulations included, grouped by year.

As would be expected under an isolation-by-dis- Structure analysis, they did not cluster with the other tance model, most analyses indicated greater genetic infrapopulation from the same locality, however. subdivision between localities than within a locality, Similarly, a neighbour-joining tree based on pairwise although the difference was not always a pronounced DA values for the 8 largest infrapopulations north of one, and the trend was even reversed for one the San Acacia constriction clustered infrapopulations Φ comparison ( ST among southern infrapopulations; from La Joya tightly with a distinct separation between Table 2). Furthermore, as would be expected under an them and the Las Nutrias infrapopulation. Inclusion of isolation-by-distance model, Bayesian estimates of the two smaller infrapopulations from Las Nutrias migration indicated greater proportions of migrants disrupted this pattern, with one of the small Las between infrapopulations at the same locality than Nutrias infrapopulations (752, n = 6) being more similar between infrapopulations at neighbouring localities on genetically to La Joya infrapopulations than to the same side of the constriction (Tables 2 and S3, other Las Nutrias infrapopulations (Fig. S7, Supporting Supporting information). information). AMOVAs of lice from different localities north of the Although among-locality differences were not San Acacia constriction indicated that between-locality significant in AMOVA of southern infrapopulations, genetic variation was not significant for either mtDNA and although Structure analysis of microsatellite data or microsatellites (Table 2). Structure analysis of the 8 for southern infrapopulations did not reflect any largest northern infrapopulations (eliminating infrapop- population structure reflecting between-locality differ- < ulations with 15 individuals) indicated two clus- ences, neighbour-joining trees based on DA values did ters (K = 2) that corresponded with locality. When the cluster southern infrapopulations by locality (Fig. S8, two small northern infrapopulations were included in Supporting information).

Infrapopulation characteristics data consistent with collection date, with the single exception of a weak cluster of older infrapopulations Most infrapopulations showed at least 2 alleles for each south of the San Acacia constriction (Fig. S8, Supporting locus (Table S4A, Supporting Information). Allelic information). When the noncontemporaneous infrapop- richness was similar for all infrapopulations, but ulations at La Joya (i.e. 2011 samples and 1991 samples) slightly higher in northern infrapopulations (averaging were treated falsely as contemporaries in Bayesian 2.02) than in southern infrapopulations (averaging 1.78; analysis, migration estimates ranged 2–5% (average Table S4B, Supporting Information). 3.7%, Tables 2 and S3, Supporting Information) among In northern infrapopulations, observed and expected noncontemporaries while true contemporary infrapopu- heterozygosity values averaged over seven loci were lations at this locality yielded similar estimates (1–7%, similar in each of the 10 infrapopulations (Table S4C, average 3.5%, Table 2). Supporting Information), and global tests of Hardy– Weinberg heterozygote deficiency indicated a deficit of heterozygotes in only a single northern infrapopulation Discussion (436 La Joya) after correction for multiple tests While marking parasites and recapturing them to allow (P = 0.010) Inbreeding coefficients (F ) were positive in IS a direct assessment of movement from host to host can six of ten northern infrapopulations and ranged from be particularly informative (e.g. Zohdy et al. 2012), in 0.05 to 0.21, with an average F of 0.01 (Table S4D, IS many parasite systems such as ours, a molecular Supporting Information). No infrapopulation showed approach, albeit indirect, represents the most feasible an F value that was significantly >0 after BY correction IS method for assessing parasite transfer. In the pocket-go- for multiple tests. pher and chewing-louse system, factors that make Most southern infrapopulations showed observed direct assessment of louse transfer under natural and expected heterozygosity values that were in close conditions much less tractable than assessment via agreement (Table S4C, Supporting Information), and molecular methods include, but are not limited to (i) global tests of Hardy–Weinberg heterozygote deficiency the short (approximately 40 day) generation time of lice indicated only a single infrapopulation (757 from (Rust 1974), which makes possible recapture time short; Lemitar) that showed significant heterozygote deficit (ii) the large proportion of the chewing-louse popula- after correction for multiple tests (P = 0.0028). Four of tion that consists of nymphs that will moult quickly; seven loci in this infrapopulation showed fewer (iii) the normal activity patterns of chewing lice, which heterozygotes than expected, particularly for the Allie would require louse anesthetization prior to marking locus, which showed 0 observed heterozygotes vs. an and return to the host; and (iv) the fossorial, asocial expected 5.2 heterozygotes. These five infrapopulations lifestyle of pocket gophers, which makes host mark–re- showed great variation in their positive F values, IS capture work challenging (but see Daly & Patton 1990) which ranged from 0.02 to 0.31, with an average of 0.09. and the possibility of host contact unlikely over a short Only one infrapopulation showed an F value that was IS time span. Fortunately, as demonstrated here, molecular significantly >0 (Table S4D, Supporting Information; data on parasite populations are informative of both P < 0.0014). host interactions and parasite interactions on multiple spatial scales, even including when hosts are near neighbours. The effect of time Genetic differences among infrapopulations collected Host genetics from the same locality (La Joya) 19 years (173 generations) apart in time did not compose a significant Our genetic sampling of pocket gophers confirms that component of the overall genetic variation among those there still are two genetically divergent lineages of hosts individuals in AMOVAs of mtDNA or microsatellites that are separated in geographic space at a region near (Table 2). Likewise, although moderate population the San Acacia constriction, as there was in earlier subdivision was observed in mtDNA and microsatellite sampling (Smith et al. 1983; Demastes et al. 1998). data for these across-time infrapopulation comparisons Importantly, for our purposes, our pocket-gopher data Φ as evidenced by ST, FST and DA, each of these values confirm that the host subspecies for each of our was comparable to values observed between contempo- chewing-louse samples is actually the host subspecies raneous infrapopulations from the same locality that would have been predicted from geography. In (Table 2). Neither Structure analysis nor a neighbour- addition, our genetic data provide confirmation of the joining tree based on DA (Fig. S7, Supporting informa- observations of Smith et al. (1983) by demonstrating tion) indicated any genetic structure in microsatellite that these two subspecies of pocket gophers are quite

© 2015 John Wiley & Sons Ltd 4138 S. E. HARPER ET AL. different genetically, on the order of differentiation that are distributed in a manner concordant with host expected between reproductively isolated species. The genetics. Lice from northern hosts are clearly 8.0% mtDNA COI-gene sequence divergence that we distinguishable from lice from southern hosts based on Φ observed between these pocket-gopher subspecies is mtDNA analyses (TCS, AMOVA and ST) and based substantial and near the mean sequence divergence on nuclear DNA analyses (Structure, AMOVA, FST, observed between 11 pairs of sister species of rodents differentiation in DA values and low Bayesian estimates for the mitochondrial cytochrome b gene (9.3% uncor- of migration). Therefore, a strong genetic discontinuity rected sequence divergence; Bradley & Baker 2001). exists in these lice from north and south of the San Karyotypic dissimilarity and genic dissimilarity for Acacia constriction. Considering these parasites as these pocket gophers also were near what would be independent measures of host gene ﬂow (Hafner et al. considered typical species-level differences (Smith et al. 2003) serves to highlight the limitations to host 1983). hybridization in the vicinity of the San Acacia constric- Our results further corroborate those of Smith et al. tion as there is no apparent ‘leakage’ of lice belonging (1983) in demonstrating potential hybridization between to the northern genetic group onto the southern host these two pocket-gopher lineages as all three strongly subspecies, or vice versa. It is possible that further differentiated nuclear loci sampled here were heterozy- sampling nearer the middle of the host contact zone gous in at least one individual pocket gopher in a would reveal some T. minor infrapopulations that con- manner consistent with hybridization between northern sist of both genetic types occurring on the same host or and southern subspecies. None of the individuals we large numbers of louse individuals that show obvious examined was heterozygous for more than one locus, admixture of northern and southern alleles. However, suggesting that there were no F1 hybrids in our just a few kilometres away from the contact zone, any analysis, however. Although ‘southern’ alleles in north- effects of hybridization between T. minor genetic groups ern pocket gophers may be present because they were are not readily apparent. common to the ancestor of both subspecies, rather than If molecular substitution rates in lice and pocket from hybridization, data from more extensive gophers were the same, genetic divergence estimates geographic sampling of Smith et al. (1983) indicate a would suggest a more recent divergence in the lice at smooth, narrow cline of allele frequencies in otherwise the San Acacia constriction than in their hosts, because strongly diagnostic loci, a pattern consistent with T. minor populations at this contact zone show only ¼ genetic introgression in the hosts beyond F1 hybrids. the genetic divergence of the hosts (COI sequence Based on these data, it seems likely that pocket-gopher divergence = 2.1% between northern and southern lice hybridization occurs occasionally near the San Acacia and 8.0% between their respective hosts). However, in constriction as suggested by Smith et al. (1983). light of data indicating that chewing lice typically have The fact that maternally inherited mtDNA shows a a 1.5- to 4-fold higher overall rate of COI substitution clean break in pocket-gopher haplotypes corresponding than their pocket-gopher hosts (studies reviewed by to the region of the San Acacia constriction (data from Light & Hafner 2007), the relative timing of louse this study and Demastes et al. 1998) could be evidence divergence appears even more recent relative to the that hybridization events at this zone are primarily the divergence of their hosts. This discrepancy in timing result of male dispersal rather than female dispersal. renders a cophylogeny scenario extremely improbable Despite a skewed sex ratio that favours females in for this pair of hosts and their parasites. Therefore, Thomomys bottae, Daly & Patton (1990) estimated that a either these T. minor genetic groups are experiencing single reproductively successful male may contribute as secondary contact at the San Acacia constriction, having much to the gene pool as 3–15 females owing to the arrived there from two source populations at a time highly variable reproductive success of male Thomomys independent of their hosts, or one of the genetic groups bottae. A better analysis of the roles of males vs. females of T. minor represents a long-established parasite popu- in hybridization at this site must await the sampling of lation at this zone and the other represents a relatively greater numbers of individuals from a wider span of recent invasion from one of the hosts in the region to geographic space. the other, with subsequent genetic divergence in relative isolation at this site. Wider geographic sampling of T. minor could help address the relative likelihoods of Thomomydoecus population genetics at the host hybrid these possibilities. zone Given that a pattern of cospeciation does not explain Although T. minor is recognized as a single species genetic patterns in T. minor relative to their hosts, some based on morphology, both mitochondrial DNA and other host-driven or parasite-driven aspect of life his- nuclear DNA indicate two genetically divergent groups tory is implicated. Sequence divergence values between

© 2015 John Wiley & Sons Ltd HOST BEHAVIOUR DRIVES PARASITE GENETICS 4139 northern and southern T. minor are lower than that 2.1% sequence divergence between northern and south- typically seen between sister species of insects for the ern T. minor, so the G. aurei host switch seems compara- COI gene (Hebert et al. 2003), so hybridization between tively much more recent. In fact, rate of range northern and southern lice would seem likely if they expansion for G. aurei suggests that the beginning of were to come in contact. In addition, if these northern the event may be closely tied to a major flood in 1929 and southern T. minor were to come in contact, it is and that the G. aurei invasion may be driven by com- likely that they would be capable of surviving on petitive superiority of the northern louse (Hafner et al. opposite host types, given that even more differentiated 1998). Our data from the geographic distribution of lice have proven capable of host switching (Reed & T. minor genetic groups serve to corroborate the hypoth- Hafner 1997). Finally, given the genetic similarity of esis of G. aurei competitive superiority by showing that northern and southern T. minor, strong natural selection there is not any sort of asymmetry in host gene flow against louse hybridization or selection favouring that would promote movement of different louse phenotypes for different hosts or for G. aurei onto a southern host subspecies. different ecogeographic regions seems unlikely to yield These two species of chewing lice, T. minor and such a crisp pattern of genetic structure within the G. aurei, have responded differently at the host hybrid species. Therefore, given the strong mitochondrial and zone in the face of infrequent host hybridization: nuclear genetic structure observed in louse populations T. minor populations have remained isolated on oppo- from opposite sides of the host contact zone, northern site sides of the host contact zone, while G. aurei and southern T. minor must rarely admix as a populations have recently crossed over this host contact consequence of host behaviour rather than as a conse- zone onto a new host subspecies. There are several quence of louse mating patterns, unsuitability of hosts, intriguing possibilities that could drive these different or natural selection. responses, including differences in the life history or Smith et al. (1983) considered the zone of low pocket- prevalence of the louse species themselves, topics that gopher population density where habitat is sparsest in have not yet been sufficiently investigated. Importantly, the vicinity of the San Acacia constriction to be the in the absence of any competitive advantage, for main impediment to greater genetic introgression of the northern T. minor to survive on the southern host hosts. As described later by Barton & Hewitt (1985), subspecies, a large number of lice would need to be local gradients in population density form effective transferred for a ‘selectively neutral’ rise in frequency traps for hybrid zones, holding them in place over time. to be established. However, with competitive superior- Any reduction in fitness of the hybrids would be ity of northern G. aurei over its southern congener, a expected to further fix the host contact zone in single establishment of lice on the southern host geographic space (Barton & Hewitt 1985), and the subspecies may have been all that was required to degree of genetic differentiation (especially chromoso- allow the invasion documented by Hafner et al. (1998). mal differentiation; Smith et al. 1983) between these hosts would suggest hybridization would be selected Tests of louse movement within and among localities against. Therefore, host-driven factors controlling geographic distribution, rather than parasite biology, Genetic subdivision among T. minor infrapopulations at likely serve as the main impediments to greater mixing localities sampled at the same time only 16–24 km apart of T. minor genetic groups. was strong enough that it was detectable with only Evidence that limited host density and limited host seven loci in Structure and neighbour-joining analyses interaction at the constriction are sufficient to maintain of DA. Only one of the smallest infrapopulations genetic structure in T. minor is particularly interesting sampled (n = 6) could not be sorted by locality using given that these same forces have not been sufficient to these analyses, which is not surprising given its small prevent G. aurei, the other louse that inhabits these sample size. With hosts having an estimated dispersal same host individuals, from breaking through the host distance of about 400 m per year at this site (Hafner contact zone and expanding its range southward to a et al. 1998), among-locality genetic differences in chew- point about halfway between San Acacia and Lemitar ing lice may not be surprising at distances such as by 1996 (towns shown in Fig. 1). The species continues these. to progress farther south at a rate of about 700 m per While mother-to-offspring transmission would seem year, displacing its congener, G. centralis, from its to provide the easiest colonization route for chewing natural host in the process (Hafner et al. 1998 and J. W. lice of pocket gophers, brief host-mating encounters Demastes, unpublished data). Sequence divergence in also provide the opportunity for horizontal louse trans- G. aurei from opposite sides of the constriction is negli- mission (Demastes et al. 1998). Although horizontal gible (T. A. Spradling, unpublished data), compared to transmission of chewing lice among pocket-gopher

hosts is expected to be rare given the short encounters (Nadler 1995). For T. minor, while FIS was high and between these otherwise asocial hosts, horizontal significant in two infrapopulations examined here, transmission has been demonstrated for G. aurei in the the majority of infrapopulations showed little or no same geographic region encompassed by this study inbreeding as measured by FIS. Nessner et al. (2014) (Demastes et al. 1998). Nevertheless, the lice studied found that 24–35% of the microsatellite loci they devel- herein, T. minor, show population structure that oped for G. ewingi, another chewing louse of pocket indicates that louse movement from one host to another gophers, departed from Hardy–Weinberg expectations is quite limited as evidenced by both nuclear and mito- with substantial homozygote excess and high FIS values. Φ chondrial data analyses including AMOVA, FST, ST However, because microsatellite loci are prone to null and DA. Bayesian estimates of migration further alleles, and because sample sizes were small in keeping suggest that louse infrapopulations at the same locality with their purpose of primer development, it is not operate as independent units, as migration values clear whether inbreeding will prove typical of that below about 10% can be taken as evidence of demo- species of chewing louse when a greater number of graphically independent populations (Hastings 1993; infrapopulations are examined or whether many of the Faubet et al. 2007), and all but one comparison of loci examined exhibit null alleles. Null alleles were infrapopulations at the same locality fell below this regarded by Martinu et al. (2015) as common in value. Moreover, restriction to gene flow among microsatellite loci they characterized for a chewing infrapopulations within a locality approached the level louse of mocking birds and a sucking louse of Eurasian observed among infrapopulations from different locali- field mice, although they also recognized the potential ties in nearly every measure. Therefore, all genetic impact of population subdivision on deviations from evidence points to predominantly vertical transmission Hardy–Weinberg expectations. Data from T. minor, for T. minor, even in alfalfa fields where pocket-gopher which appear to be relatively free of null alleles, density is high. suggest that inbreeding levels vary widely by louse Another chewing louse, Geomydoecus actuosi, that infrapopulation, perhaps because initial host coloniza- inhabits Colorado pocket gophers showed allozyme- tion is a relatively unpredictable process involving based FST values for infrapopulations from a locality small numbers of parasites in some cases and larger – = ranging 0.04 0.16 (unweighted average FST 0.09; numbers in others. Such variability in inbreeding level

Nadler et al. 1990). Average microsatellite-based FST among parasite infrapopulations was observed in tape- values for T. minor infrapopulations collected at worms and schistosomes, which also showed high approximately the same time and at the same locality levels of inbreeding in some infrapopulations and little were slightly smaller (0.03–0.06), but average observed or none in others (Stefka et al. 2009; Van den Broeck = heterozygosity for microsatellite loci (HO 0.34) was et al. 2014). substantially higher than for allozyme data for the Nadler et al. (1990) suggested that founder events at = same species (HO 0.01; Nadler & Hafner 1989), mak- initial host colonization and the seasonal population ing the microsatellite loci preferable for analyses of bottlenecks in louse populations observed by Rust population subdivision and inbreeding. Therefore, (1974) both can serve to decrease genetic diversity in microsatellite data from T. minor more robustly sup- chewing lice as compared to other insect species. Based port earlier evidence that pocket-gopher hosts act as on the presumed prevalence of population bottlenecks islands of habitat, isolated from other such islands in chewing lice, we expected to find significant genetic even in the same or a neighbouring alfalfa field. Gala- structure over time resulting from genetic drift. Despite pagos hawks recently were shown to create similar 19 years and an estimated 173 chewing-louse genera- genetic structure in their chewing lice (Koop et al. tions between collection periods at La Joya, no measure 2014). of population subdivision recognized time as an important component of the overall genetic variation at this locality for either mitochondrial data (AMOVA and Infrapopulation characteristics and genetic stability Φ ) or microsatellite data (AMOVA, Structure, F , D over time ST ST A and Bayesian estimates of migration). Therefore, Price (1980) recognized parasite infrapopulations as whatever series of founder events and seasonal bottle- being bound to resources that are patchily distributed necks may have occurred over the past 173 generations, in their environment in both time and space. While genetic drift did not alter population genetics in a isolation of infrapopulations could lead to inbreeding, measurable way over the time span considered. Perhaps numerous aspects of both the parasite life cycle and effective population size is high enough in these lice to that of its host will control the genetic make-up of render genetic drift no more important over time than parasite populations, rendering generalizations tenuous it is over space at the scales we examined. Few other

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Fig. S2 Pocket-gopher (host) genetics based on Bayesian and values generated based on seven microsatellite loci for 10 neighbour-joining analysis of nuclear b-ﬁbrinogen sequences. infrapopulations occurring north of the San Acacia constriction. Fig. S3 Pocket-gopher (host) genetics based on Bayesian and neighbour-joining analysis of nuclear IRBP sequences. Fig. S8 Chewing-louse infrapopulation relationships based on

a mid-point-rooted neighbour-joining tree built from DA val- Fig. S4 Pocket-gopher (host) genetics based on Bayesian and ues generated based on seven microsatellite loci for ﬁve neighbour-joining analysis of nuclear Rag1 sequences. infrapopulations occurring on south of the San Acacia constriction. Fig. S5 Chewing-louse genetics based on Bayesian analysis (HKY85 model) of mitochondrial COI sequences from 275 Table S1 Microsatellite loci, primer sequences, genetic diver- Thomomydoecus minor and one outgroup. sity measures, and measures of locus reliability. Fig. S6 Chewing-louse infrapopulation relationships based on Table S2 FST values for chewing-louse infrapopulations. a mid-point-rooted neighbour-joining tree built from DA values generated based on seven microsatellite loci for 15 Table S3 Migration rate estimates. infrapopulations occurring on both sides of the San Acacia constriction. Table S4 Microsatellite analyses for individual infrapopulations including total number of alleles sampled, allelic rich- Fig. S7 Chewing-louse infrapopulation relationships based on ness, observed (Ho) and expected (He) heterozygosity and a mid-point-rooted neighbour-joining tree built from DA the inbreeding coefﬁcient (FIS).