Supporting Information
Supplementary Methods
Host genetics
To verify that the contact zone between host subspecies is still located along the Rio
Grande Valley near the San Acacia constriction between the towns of La Joya and San Acacia,
New Mexico, 15 pocket gophers were examined from five locations, two north and three south
of the San Acacia Constriction (Fig. 1; Table S1). Eight of the specimens were collected in 2011
and the other 7 were collected from 1990-1992. The New Mexico Department of Game and
Fish (NMDGF) approved collection of specimens, and procedures followed all guidelines set by
the University of Northern Iowa Institutional Animal Care and Use Committee and the American
Society of Mammalogists (Sikes and Gannon 2011).
DNA was extracted from pocket gopher tissues using the DNeasy Tissue Kit (Qiagen,
Valencia, California) according to manufacturer specifications. The mitochondrial COI gene was
amplified in a polymerase chain reaction (PCR) using primers CO1-5285F and CO1-6929R
(Spradling et al. 2004) with Promega Hotstart Mastermix Mix (Promega, Madison, WI) according
to manufacturer specifications with 40 thermal-cycles at an annealing temperature of 45C. PCR
products were treated with ExoSAP-IT (USB, Cleveland, OH) and were sent to Iowa State
University DNA Facility for sequencing with primer CO1-5285F on an Applied Biosystems
3730xl DNA Analyzer. Three nuclear loci were amplified and sequenced similarly. A portion of
the first exon of the Interphotoreceptor Retinoid Binding Protein (IRBP) gene was amplified
primers 119A (Jansa and Voss 2000) and 1297D (Jansa and Voss 2000, Stanhope et al. 1992)
as described in Hafner et al. 2014. Both primers were used for sequencing. Amplification of a
portion of the Recombination Activating Gene 1 (Rag1) was performed using primers Rag1-S70
and Rag1- S73 (Steppan et al. 2004) at an annealing temperature of 54C. A portion of the βfibrinogen gene was amplified using primers FIB-B17L (Prychitko and Moore, 1997) and R2
(Spradling et al. 2004) with an annealing temperature of 50C. Both primers were used for
sequencing for all nuclear genes to better assess potential heterozygous sites. Sequences
were screened for error and edited manually for heterozygosity using Geneious Pro (version
5.4.6, Biomatters Ltd),
Heterozygous sequences in the three nuclear genes were duplicated and treated as two
different alleles, each with one of the two alternative nucleotides. Because cloning to determine
allele sequences for heterozygotes was beyond the needs of this analysis, where an individual
was heterozygous at multiple nucleotide positions for a nuclear gene, existing homozygous
genotypes of other individuals were used to guide nucleotide composition for each of the
reconstructed alleles.
Appropriate substitution models and uncorrected sequence divergence values were
assessed for the four genes using Mega 6.06 and Bayesian Information Criterion scores
(Tamura et al. 2013). Bayesian analysis was performed with the selected model using the
MrBayes (Ronquist et al. 2012) plugin for Geneious R8 (http://www.geneious.com, Kearse et al.
2012). Chain length was set to 1,500,000 with 4 heated chains, a 0.1 heated-chain temperature,
and a sub-sampling frequency of 300. Burn-in was set to 100,000 using unconstrained branch
lengths for priors. Output was evaluated to assess the quality of runs using three criteria
recommended in the MrBayes documentation (http://mrbayes.sourceforge.net/manual.php).
Neighbor-joining trees also were built using similar molecular substitution models for
comparison as the neighbor-joining trees depicted zero-branch lengths in a more intuitive
manner.
Louse microsatellite primer development
A DNeasy Blood & Tissue Kit (Qiagen, Valencia, California) was used to extract DNA.
From the pool of 160 lice. Manufacturer’s recommendations were followed with the following
exceptions: lice were used directly from the ultra-cold freezer and use of liquid nitrogen was
eliminated, after 4 hours of incubation in ATL buffer, an additional 20 µl Proteinase K was added
with additional crushing performed before continued incubation overnight at 56ºC, final elution in
AE Buffer was decreased from 200 µl to 50 µl, and incubation time was increased to 5 minutes.
At Cornell University, pooled louse DNA was treated with the restriction enzyme Hinc III
and then ligated to a double stranded SNX linker. The ligation procedure was modified to
generate Pme I sites if linkers ligated to themselves. Digested, ligated fragments were enriched
for microsatellites by hybridization to 3' biotinylated di-, tri-, and tetra-nucleotide repeat probes.
PCR amplified products were ligated to 1.0 µl of a Titanium Rapid Library MID adapter (10 µm
adapter stock), and small fragments were removed with Ampure beads. Libraries were
submitted to the Sequencing and Genotyping Facility at Cornell Life Sciences Core Laboratory
Center for FAM-quantification and Titanium 454 sequencing.
Single sequence reads and contigs from analysis of 454 sequencing data were analyzed
at University of Northern Iowa, and loci were chosen for amplification if they had a tetrameric
repeat structure with a minimum of five repeats. Loci chosen based on multiple sequence reads
were given "names"; loci chosen based on single sequence reads were numbered based on
sequencing read number. Primerselect software (Lasergene Core Suite package, DNAStar,
Madison, WI) was used to identify suitable primers to amplify each locus. A long M13 tag (5’CGAGTTTTCCCAGTCACGAC-3’) was added to the 5’ end of all locus-specific forward primers
to allow concurrent amplification with a fluorescent primer (Schuelke 2000). A short tag (5'GTTTCTT-3') was added to all locus-specific reverse primers to promote adenylation and
reduce stutter (Brownstein et al. 1996). Fluorescent tags (6-FAM, HEX, NED) were added to the
5’ end of universal M13 primers (5’-CGAGTTTTCCCAGTCACGAC-3’) to allow three-primer
amplification of PCR products and subsequent multiplex genotyping (Schuelke 2000).
Individual amplification of 7 target microsatellite loci in 275 chewing louse individuals
was performed as described in the manuscript “Host behavior drives parasite genetics at
multiple geographic scales: Population genetics of the chewing louse, Thomomydoecus minor”.
In order to check the accuracy of genotyping assignments of individual lice and to estimate
locus-specific genotyping error rates, 15 randomly chosen DNA samples out of an initial 157
samples (10%) were chosen for re-screening. At least two lice per host individual were included
in this re-assessment in which new dye-labeled, 3-primer PCR was performed and amplified
products were submitted to Iowa State University for analysis alongside a subset of previous
PCR products.
Locus quality was further evaluated by examining null allele frequencies for each of the
15 infrapopulations examined as estimated using the EM algorithm of Dempster et al. (1977) via
the FreeNA program (Chapuis and Estoup 2007). Tests for genotypic disequilibrium between
each pair of loci in each population were performed using Genepop 4.2 web version (Rousset
2008, http://genepop.curtin.edu.au/) using the likelihood-ratio test statistic to assess
significance. Observed heterozygosity (HO) and expected heterozygosity (HE) were calculated
in Arlequin (version 3.5.1.2, Excoffier and Lischer 2010) for the source population of
microsatellite sequences using three infrapopulations combined from Las Nutrias, NM.
Supplementary Results
Host genetics
Mitochondrial COI sequences from pocket gophers collected on opposite sides of the
San Acacia constriction, where host subspecies previously came into contact (Smith et al. 1983;
Table S1), sorted neatly in two different haplotype groups that displayed an average 8.0 %
uncorrected sequence divergence between them (1,035 bp sequence data). One haplotype
group was found exclusively in gophers collected north of the San Acacia constriction and the
other was found exclusively in gophers collected south of it (Fig. S1).
The 499 bp portion of -fib sequenced (461 bp of intron 7 and 38 bp of exon 8) had 8
variable nucleotide sites, 6 of which appeared heterozygous in one or more individuals.
Bayesian and neighbor-joining analysis of putative allele sequences created two clusters.
Pocket gopher alleles from individuals collected north of the San Acacia constriction formed one
genetic cluster, and individuals from south of the contact zone formed the other, with two
exceptions (Fig. S2). One allele from a southern gopher individual grouped with otherwise
northern alleles and one allele from one northern individual grouped with otherwise southern
alleles.
The 874 bp region of IRBP sequenced included 15 nucleotide positions that were
variable; eight of these positions were variable only due to autapomorphies. Of the seven
potentially phylogenetically informative positions, three were diagnostic for all pocket gophers
collected south of the San Acacia constriction. The other four positions were heterozygous in
many of the pocket gophers collected north of the San Acacia constriction. Bayesian and
neighbor-joining analysis of IRBP putative alleles yielded several genetic clusters, one
consisting solely of alleles from individuals collected south of the San Acacia constriction and a
larger and more genetically diverse cluster consisting of alleles from all northern individuals plus
a single allele from a southern individual (Fig. S3).
The 735 bp region of Rag1 sequenced included seven variable nucleotide positions, six
of which were potentially phylogenetically informative. All pocket gophers collected north of the
constriction had identical nucleotide sequences except for one unique nucleotide position
observed in two individuals, and all northern individuals differed from all southern individuals by
anywhere from one to four variable nucleotide positions. Bayesian and neighbor-joining analysis
of Rag1 alleles indicated two genetic clusters that were consistent with geographic distribution,
with the exception of a single southern allele that showed greatest resemblance to otherwise
Northern alleles (Fig. S4).
Taken together, mtDNA and all 3 nuclear genes show clear distinctions between
haplotypes or alleles carried by pocket gophers from north of the constriction vs. south of it, yet
there also were some exceptions to this pattern. Typically northern nuclear alleles were found in
the southern localities of Lemitar (2 of 6 nuclear alleles, each from different individuals, sampled
in 2011) and San Acacia (1 of 6 nuclear alleles sampled in 1990-1991), and typically southern
alleles were found in the northern locality of Las Nutrias (1 of 9 nuclear alleles sampled in
2011).
Quality of louse microsatellite loci
For these 7 loci, accuracy testing showed no genotyping errors in which allele size was
incorrectly identified in individuals after rescreening. Genotyping errors did occur for some of
these loci when an individual louse originally amplified as a heterozygote and later amplified as
a homozygote, or vice versa, but this non-repeatability was limited, yielding an allelic error rate
as high as 7% for a single locus, but near 0% for other loci (Table S1). Among the 7 loci chosen
for downstream analyses, there were no pairs of loci that showed evidence of genotypic
disequilibrium across all populations after correction for multiple tests, and there were no loci
that showed evidence of null alleles in any of the infrapopulations (Table S1). Observed and
expected heterozygosity values were not significantly different for these loci at the source
population from which the primer information was derived, and null alleles were estimated to be
rare (0-.01-0.03 averaged over all 15 infrapopulations; Table S1).
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Fig. S1. Pocket gopher host genetics based on Bayesian (HKY85 model) and neighborjoining analysis (Tamura-Nei model) of mitochondrial COI sequences yielded the same
tree structure. Neighbor-joining analysis is shown here for its more intuitive
representation of zero branch lengths. Gophers collected north of the contact zone are
shown in blue and names are designated with “N”; gophers collected south of the zone
are shown in red and names are designated with “S”. Individuals are identified with
nearest town and collector number.
Fig. S2. Pocket gopher host genetics based on Bayesian and neighbor-joining analysis
(JC69 model) of nuclear -fibrinogen sequences with heterozygous sequences split into
putative alleles based on other observed homozygous sequences. Both analyses
yielded the same tree structure, but neighbor-joining analysis is shown here for its more
intuitive representation of zero branch lengths. Gophers collected north of the contact
zone are shown in blue and names are designated with “N”; gophers collected south of
the zone are shown in red and names are designated with “S”. Individuals are identified
with nearest town and collector number.
Fig. S3. Pocket gopher host genetics based on Bayesian and neighbor-joining analysis
(HKY85 model) of nuclear IRBP sequences with heterozygous sequences split into
putative alleles based on other observed homozygous sequences. Both analyses
yielded the same tree structure, but neighbor-joining analysis is shown here for its more
intuitive representation of zero branch lengths. Gophers collected north of the contact
zone are shown in blue and names are designated with “N”; gophers collected south of
the zone are shown in red and names are designated with “S”. Individuals are identified
with nearest town and collector number.
Fig. S4. Pocket gopher host genetics based on Bayesian and neighbor-joining analysis
(JC69 model) of nuclear Rag1 sequences with heterozygous sequences split into
putative alleles based on other observed homozygous sequences. Both analyses
yielded the same tree structure, but neighbor-joining analysis is shown here for its more
intuitive representation of zero branch lengths. Gophers collected north of the contact
zone are shown in blue and names are designated with “N”; gophers collected south of
the zone are shown in red and names are designated with “S”. Individuals are identified
with nearest town and collector number.
Fig. S5. Chewing louse genetics based on Bayesian analysis (HKY85 model) of
mitochondrial COI sequences from 275 T. minor and one outgroup. Host number is
given first followed by louse individual number. Shortest branch lengths (e.g. between
louse 436.09 and louse 436.23 from the same host) represent identical sequences. Lice
collected on northern gophers are shown in blue (top clade); Lice collected on southern
gophers are shown in red (bottom clade). For clarity, only representative specimen
numbers are listed.
Fig. S6. Chewing louse infrapopulation relationships based on a midpoint-rooted
neighbor-joining tree built from DA values generated based on 7 microsatellite loci for
15 infrapopulations occurring on both sides of the San Acacia constriction (northern
infrapopulations are indicated by blue branches and southern infrapopulations by red
branches). Infrapopulations two major clusters consistent with geography (and host
subspecies). Clustering pattern within each group analyzed in isolation (Figs. S6 and
S7) differs slightly from the larger analysis shown here.
Fig. S7. Chewing louse infrapopulation relationships based on a midpoint-rooted
neighbor-joining tree built from DA values generated based on 7 microsatellite loci for
10 infrapopulations occurring north of the San Acacia constriction. Infrapopulations do
not show clustering strictly consistent with locality or time.
Fig. S8. Chewing louse infrapopulation relationships based on a midpoint-rooted
neighbor-joining tree built from DA values generated based on 7 microsatellite loci for 5
infrapopulations occurring on south of the San Acacia constriction. Infrapopulations
show clustering consistent with locality and, more weakly, with time.
Table S1. Microsatellite loci, primer sequences, genetic diversity measures, and measures of locus reliability.
Locus
name
3495
Primer sequences (5’  3’); primer tags are underlined,
species-specific sequences are not underlined
CGAGTTTTCCCAGTCACGACAATGAGTAAGTACGATCCAGCAC
GTTTCTTAGTGAGTTAAATGCTAGGCTGATG
1569
CGAGTTTTCCCAGTCACGACTTTAACAAAGAGGAATCGGATGC
GTTTCTTCATACTCCCTCACGATTCTGTCC
1451
CGAGTTTTCCCAGTCACGACCTCATGGTGATGGTCTTTGTCTC
GTTTCTTGTGTTCGAGAAGCTGTATCATCC
Jan
CGAGTTTTCCCAGTCACGACAATTGTGCTATGTCATCACTTGG
GTTTCTTCTTCAAACTTGCATTTACCTCTTC
Allie
CGAGTTTTCCCAGTCACGACACCACATACACTATCAGCAATCC
GTTTCTTTTAATTCAAGGACCAGTGGAGGG
Belle
CGAGTTTTCCCAGTCACGACATCCCTCTTTCTGTCGTTGGAAG
GTTTCTTTGCGGTTCGAAATTCTCCAAG
851
CGAGTTTTCCCAGTCACGACTTAATTCACACTCAATCCAGGCG
GTTTCTTGTGTAGGCGTCCCACAAATTTG
ano comparisons of Ho and He were significantly different (p = 0.17-1.0)
brepresents 2 miscalled alleles in 15 diploid genotypes
crepresents 1 miscalled allele in 15 diploid genotypes
# alleles,
and size of
smallest
allele (base
pairs)
HO and HE
at the Las
Nutrias,
NM,
localitya
Estimated null
allele frequency
averaged over 15
infrapopulations
3
190 bp
2
198 bp
3
337 bp
2
198 bp
4
211 bp
4
215 bp
3
436 bp
0.53—0.59
0.02
Estimated
allelic error
rate based on
sample reamplification
and rescoring
0.00
0.16—0.20
0.03
0.07b
0.41—0.45
0.01
0.00
0.53—0.63
0.03
0.03c
0.28—0.37
0.02
0.00
0.25—0.26
0.02
0.00
0.41—0.40
0.03
0.03c
Table S2. FST values calculated in FSTAT (version 2.9.3.2; Goudet 1995, 2001).
FST (from FSTAT); highlighted values are significant at p < 0.009 (BY correction value) based on results of 2100 permutations
LN751 LN752 LN753 LJ759 LJ760 LJ761 LJ434 LJ435 LJ436 LJ437 SA1428 SA3243 LE756 LE757
LN752 0.1055
LN753 0.0708 0.0857
LJ759 0.0503 0.0924 0.0920
LJ760 0.0934 0.0963 0.1175 0.0624
LJ761 0.0906 0.1287 0.0869 0.0302 0.0031
LJ434 0.0365 0.0381 0.0568 0.0110-0.0068 0.0078
LJ435 0.1203 0.1303 0.0532 0.0952 0.0408 0.0255 0.0534
LJ436 0.0663 0.0232 0.0742 0.0822 0.0418 0.0800 0.0195 0.0891
LJ437 0.0977 0.1289 0.1451 0.0337 0.0420 0.0094 0.0329 0.0686 0.0990
SA1428 0.4045 0.3567 0.3561 0.4823 0.3391 0.4126 0.3507 0.3263 0.2998 0.4527
SA3243 0.3940 0.3516 0.3586 0.4726 0.3130 0.3947 0.3261 0.3229 0.2526 0.4535 0.0629
LE756 0.5483 0.5737 0.5198 0.6238 0.4933 0.5498 0.5029 0.4558 0.4391 0.5915 0.1165 0.1871
LE757 0.4636 0.4637 0.4024 0.5415 0.4082 0.4734 0.4224 0.3827 0.3654 0.5174 0.0945 0.1645 0.0033
SO3203 0.4644 0.4568 0.4202 0.5442 0.4105 0.4747 0.4242 0.3949 0.3596 0.5079 0.0567 0.1353 0.0627 0.0429
Locality abbreviations:

LN = Las Nutrias, LJ = La Joya (759-761 = 2011; 434-437 = 1992), SA = San Acacia, LE = Lemitar, and SO = Socorro

Red values = FST comparisons between 1992 and 2011 at the same locality

Bold, underlined, italics values = FST comparisons between contemporaneous infrapopulations at the same locality
Table S3. Migration rate estimates from BayesAss 3.0 (Wilson and Rannala 2003) for migration into southern
infrapopulations from northern infrapopulations (below diagaonal) and into northern infrapopulations from southern
infrapopulations (above diagonal). Shading indicates comparisons of northern and southern infrapopulations (blue and
yellow shading), within locality (peach or lilac shading), between localities (orange and violet shading). Un-shaded values
were not considered as these comparisons involve both a spatial and a temporal component.
Las Nutrias
LaJoya new
LaJoya new
LaJoya new
old LaJoya
old LaJoya
old LaJoya
old LaJoya
San Acacia
San Acacia
Lemitar
Lemitar
Socorro
m[0][0]:
m[1][0]:
m[2][0]:
m[3][0]:
m[4][0]:
m[5][0]:
m[6][0]:
m[7][0]:
m[8][0]:
m[9][0]:
m[10][0]
m[11][0]
m[12][0]
0.7256
0.0187
0.0167
0.0155
0.018
0.0161
0.0142
0.0143
0.083
0.0111
0.0107
0.011
0.01
m[0][1]:
m[1][1]:
m[2][1]:
m[3][1]:
m[4][1]:
m[5][1]:
m[6][1]:
m[7][1]:
m[8][1]:
m[9][1]:
m[10][1]:
m[11][1]:
m[12][1]:
0.0583
0.6964
0.031
0.0572
0.0475
0.043
0.032
0.0443
0.0111
0.0111
0.0109
0.0105
0.0096
m[0][2]:
m[1][2]:
m[2][2]:
m[3][2]:
m[4][2]:
m[5][2]:
m[6][2]:
m[7][2]:
m[8][2]:
m[9][2]:
m[10][2]:
m[11][2]:
m[12][2]:
0.022
0.0416
0.6941
0.0439
0.0466
0.0291
0.034
0.0473
0.0127
0.0122
0.0115
0.0113
0.0118
in south from north
0.01322
in north from south
0.01325
within locality north
0.03484
within locality south
0.06498
between locality north
0.02522
between locality south
0.03263
over time at La Joya
0.03731
m[0][3]:
m[1][3]:
m[2][3]:
m[3][3]:
m[4][3]:
m[5][3]:
m[6][3]:
m[7][3]:
m[8][3]:
m[9][3]:
m[10][3]:
m[11][3]:
m[12][3]:
0.0201
0.0318
0.0553
0.6924
0.0381
0.0495
0.0303
0.0525
0.0119
0.0118
0.012
0.0108
0.0121
m[0][4]:
m[1][4]:
m[2][4]:
m[3][4]:
m[4][4]:
m[5][4]:
m[6][4]:
m[7][4]:
m[8][4]:
m[9][4]:
m[10][4]:
m[11][4]:
m[12][4]:
0.0328
0.0741
0.0382
0.0454
0.6959
0.0318
0.0693
0.0354
0.013
0.0123
0.0112
0.0112
0.0113
m[0][5]:
m[1][5]:
m[2][5]:
m[3][5]:
m[4][5]:
m[5][5]:
m[6][5]:
m[7][5]:
m[8][5]:
m[9][5]:
m[10][5]:
m[11][5]:
m[12][5]:
0.0131
0.0182
0.0254
0.0209
0.0188
0.6941
0.0134
0.0181
0.0111
0.0115
0.0113
0.0115
0.0099
m[0][6]:
m[1][6]:
m[2][6]:
m[3][6]:
m[4][6]:
m[5][6]:
m[6][6]:
m[7][6]:
m[8][6]:
m[9][6]:
m[10][6]:
m[11][6]:
m[12][6]:
0.0232
0.0244
0.0314
0.021
0.0247
0.0265
0.7127
0.0229
0.0126
0.0133
0.0133
0.0125
0.0125
m[0][7]:
m[1][7]:
m[2][7]:
m[3][7]:
m[4][7]:
m[5][7]:
m[6][7]:
m[7][7]:
m[8][7]:
m[9][7]:
m[10][7]:
m[11][7]:
m[12][7]:
0.0405
0.0388
0.0253
0.0382
0.0297
0.0471
0.0286
0.7123
0.0106
0.0108
0.0118
0.0104
0.0095
m[0][8]:
m[1][8]:
m[2][8]:
m[3][8]:
m[4][8]:
m[5][8]:
m[6][8]:
m[7][8]:
m[8][8]:
m[9][8]:
m[10][8]:
m[11][8]:
m[12][8]:
0.0144
0.0119
0.016
0.015
0.0165
0.0153
0.0125
0.0111
0.6957
0.041
0.0224
0.0161
0.0196
m[0][9]:
m[1][9]:
m[2][9]:
m[3][9]:
m[4][9]:
m[5][9]:
m[6][9]:
m[7][9]:
m[8][9]:
m[9][9]:
m[10][9]:
m[11][9]:
m[12][9]:
0.0141
0.0123
0.0204
0.0173
0.0156
0.0142
0.0197
0.0103
0.0636
0.7604
0.0269
0.0213
0.0362
m[0][10]:
m[1][10]:
m[2][10]:
m[3][10]:
m[4][10]:
m[5][10]:
m[6][10]:
m[7][10]:
m[8][10]:
m[9][10]:
m[10][10]:
m[11][10]:
m[12][10]:
0.0106
0.0105
0.0168
0.0108
0.0187
0.0108
0.0108
0.0097
0.0698
0.0434
0.784
0.104
0.1132
m[0][11]:
m[1][11]:
m[2][11]:
m[3][11]:
m[4][11]:
m[5][11]:
m[6][11]:
m[7][11]:
m[8][11]:
m[9][11]:
m[10][11]:
m[11][11]:
m[12][11]:
0.0113
0.0107
0.0163
0.0114
0.0167
0.0117
0.012
0.0102
0.0307
0.0324
0.0513
0.7446
0.0334
m[0][12]:
m[1][12]:
m[2][12]:
m[3][12]:
m[4][12]:
m[5][12]:
m[6][12]:
m[7][12]:
m[8][12]:
m[9][12]:
m[10][12]:
m[11][12]:
m[12][12]:
0.014
0.0105
0.0132
0.0112
0.0131
0.0108
0.0103
0.0114
0.0461
0.0286
0.0228
0.0248
0.711
Table S4. Microsatellite analyses for individual infrapopulations. For each infrapopulation, collecting locality relative to
the San Acacia constriction (N = north, S = south), collector number, and approximate locality is given. A) Total number
of alleles sampled, B) Allelic richness and average allelic richness, C) Observed (Ho) and expected (He) heterozygosity,
and D) Inbreeding coefficient (FIS).