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Increased differentiation and reduced gene flow in sex
chromosomes relative to autosomes between lineages
of the brown creeper Certhia americana
Joseph D. Manthey and Garth M. Spellman
J. D. Manthey (jdmanthey@gmail.com), Biodiversity Inst. and Dept of Ecology and Evolutionary Biology, Univ. of Kansas, Lawrence,
KS 66045, USA. – JDM and G. M. Spellman, Center for the Conservation of Biological Resources, Dept of Biology, Black Hills State Univ.,
Spearfish, SD 57799, USA.
e properties of sex chromosomes, including patterns of inheritance, reduced levels of recombination, and hemizygosity
in one of the sexes may result in the faster fixation of new mutations via drift and natural selection. Due to these patterns
and processes, the two rules of speciation to describe the genetics of postzygotic isolation, Haldane’s rule and the
large-X effect, both explicitly include quicker evolution on sex chromosomes relative to autosomes. Because sex-linked
mutations may be the first to become fixed in the speciation process, and appear to be due to stronger genetic drift (in
birds), we may identify pronounced genetic differentiation in sex chromosomes in taxa experiencing recent speciation
and diverging mainly via genetic drift. Here, we use nine sex-linked and 21 autosomal genetic markers to investigate
differential divergence and introgression between marker types in Certhia americana. We identified increased levels
of genetic differentiation and reduced levels of gene flow on sex chromosomes relative to autosomes. is pattern is
similar to those observed in other recently-divergent avian species, providing another case study of the earlier role of sex
chromosomes in divergence, relative to autosomes. Additionally, we identify three markers that may be under selection
between Certhia americana lineages.
Birds, snakes, butterflies, and some fishes and lizards exhibit
female heterogamety and Z–W sex determination. Certain
properties of sex chromosomes, including patterns of inheri-
tance, reduced levels of recombination, and hemizygosity in
one of the sexes (Vicoso and Charlesworth 2006) can result
in the faster fixation of new mutations via drift and natural
selection. ese properties have led to the explicit inclusion
of sex chromosomes in the two rules of speciation used to
characterize the genetics of postzygotic isolation: Haldane’s
rule and the large-X effect (Coyne and Orr 1989).
Haldane’s rule states that hybrid incompatibility is more
pronounced in the heterogametic sex due to expression of
hemizygous (Z-linked) alleles, whether recessive or domi-
nant (Haldane 1922). ree main hypotheses have been pro-
posed to explain Haldane’s rule, including the faster-male
hypothesis, dominance, and the fast-Z. e faster-male
hypothesis suggests evolution on sex chromosomes is quick-
ened due to sexual selection on males, which would reduce
the effective population size of Z-chromosomes (NEZ) rela-
tive to the effective population size of autosomes (NEA),
thereby increasing the effects of genetic drift (Wu and Davis
1993); the faster-male hypothesis could be exacerbated fur-
ther by skew in operational sex ratio, leading to larger vari-
ance in differences between NEZ and NEA. e dominance
hypothesis of Haldane’s rule states that mutations involved
in reduced hybrid fitness are on average recessive; therefore
the negative effects of these mutations is more pronounced if
they are located on sex chromosomes, where they would be
expressed in the heterogametic sex (Turelli and Orr 1995).
Finally, the fast-Z effect suggests that sex chromosomes may
exhibit faster rates of adaptive change compared to auto-
somes (Charlesworth et al. 1987), although these effects
may be caused in part by the faster-male or dominance
hypotheses, as well as vary in effect between species due to
differences in effective population size (Mank et al. 2010a).
e second rule of speciation is the large-X, or in the case of
birds, the large-Z effect, which states that mutations on the
Z chromosome have a relatively large effect on hybrid fitness
compared to similar mutations on autosomes (Charlesworth
et al. 1987, Coyne and Orr 1989).
Because the two rules of speciation explicitly tie sex
chromosomes to speciation, the Z chromosome often may
be responsible for the development of pre- (e.g. cellular
incompatibilities) or postzygotic (e.g. male plumage char-
acteristics; Mank et al. 2010b) reproductive isolation in
recently diverged species. Several recent studies of contact
zones between bird species (Sæther et al. 2007, Carling and
Brumfield 2009, Storchova et al. 2010, Backström and
Väli 2011, Elgvin et al. 2011) have shown reduced levels
of introgression between species in Z-linked markers
Journal of Avian Biology 44: 001–008, 2013
doi: 10.1111/j.1600-048X.2013.00233.x
© 2013 e Authors. Journal of Avian Biology © 2013 Nordic Society Oikos
Subject Editor: Staffan Bensch. Accepted 3 September 2013
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compared to autosomal markers, supporting the strong role
of sex-linked mutations in speciation. Additionally, a study
by Mank et al. (2010b) investigated the fast-Z effect using
the genome drafts of Taeniopygia guttata and Gallus gallus.
ey found a lack of positive selection, and hypothesized
that the fast-Z may be predominantly due to increased
effects of genetic drift, potentially from sexual selection in
males reducing NEZ (i.e. partially from the faster-male
hypothesis).
Because sex-linked mutations may be the first to become
fixed in the evolution of reproductive isolation, and appear
to be due to increased efficacy of genetic drift in birds (Mank
et al. 2010b), Z-linked markers may be relatively strongly
differentiated in taxa exhibiting recent speciation and diverg-
ing mainly under genetic drift. e brown creeper Certhia
americana, a widespread North American avian taxon, is cur-
rently considered a single species (AOU 1983); however,
recent phylogeographic studies (Manthey et al. 2011a, b)
suggest C. americana is two species. Mitochondrial and
nuclear DNA identified a basal split between northern and
southern populations at approximately 32°N latitude. is
split occurred recently (~ 1.50 million yr ago), with diver-
gence between populations caused by isolation (genetic drift)
and lack of gene flow (Manthey et al. 2011a, b).
Here, using nine sex-linked and 21 autosomal markers,
we measure divergence and fixation between Certhia lin-
eages and determine if genetic markers are evolving under
neutral processes or via selection. Although we cannot test
specific processes described above relating to fitness and
hybrid incompatibilities, we aim to examine the relative
effects of sex chromosomes on differentiation between
Certhia lineages. Using these data, we examine the follow-
ing hypotheses: (H01) Differentiation between lineages will
be greater in sex-linked markers than autosomal markers;
(H02) Sex-linked markers will exhibit relatively less gene
flow between lineages.
Methods
Sampling and laboratory procedures
Tissue samples of 16 brown creeper individuals were obtained
from two populations, representing the basal lineages identi-
fied in phylogeographic studies (Manthey et al. 2011a, b).
ese samples were from LaPlata County, CO, USA (n 8)
and the state of Jalisco in Mexico (n 8). One sample of the
Eurasian treecreeper Certhia familiaris was used as an out-
group taxon. We attempted to minimize any effects of sam-
ple size on estimates of polymorphism and divergence by
sampling mainly males (only one female included in this
study). Because one female was included, all analyses
included 31 alleles for Z-linked markers and 32 alleles for
autosomal markers.
Total genomic DNA was previously extracted for earlier
studies. e sequences of 21 autosomal loci were obtained
from Manthey et al. (2011b), which included anonymous
loci (developed in that study) and introns (developed by
Backström et al. 2008). An additional nine Z-linked introns
were obtained using polymerase chain reaction (PCR) ampli-
fication with previously designed primers (Backström et al.
2006, 2010, Kimball et al. 2009). PCR amplification of all
sequences was carried out in 15 ml reactions and included an
initial denaturation period of 10 min at 95°C, with 40
subsequent cycles of 95°C for 30 s, TA for 45 s, and 72°C for
1 min; annealing temperature varied with loci and samples
from 55–61°C. PCR products were purified and sequenced
using 10 ml ABI BigDye sequencing reactions. Sequencing
reactions were purified using a standard ethanol precipita-
tion clean-up followed by sequencing on an ABI 3130
Genetic Analyzer.
Phased and trimmed haplotype sequences were taken
directly from Manthey et al. (2011b) for the autosomal
sequences, which were previously checked (and trimmed if
necessary) for recombination. Newly sequenced loci were
processed using the following methodology. Sequences were
automatically aligned and manually checked and edited
using Sequencher 4.8 (GeneCodes). On introns, any partial
exon sequence was removed prior to analyses. Haplotypes
were inferred using PHASE (Stephens et al. 2001, Stephens
and Donelly 2003), with an output threshold of 0.7, as
implemented in DnaSP (Librado and Rozas 2009). Using
RDP3, and seven inclusive tests in the program (Smith 1992,
Padidam et al. 1999, Gibbs et al. 2000, Martin and Rybicki
2000, Posada and Crandall 2001, Martin et al. 2005, Heath
et al. 2006, Boni et al. 2007), we checked for recombination.
Using these tests, recombination was not detected in any of
the Z-linked loci.
Levels of variation, neutrality and selection
Measures of genetic diversity (polymorphic sites and nucle-
otide diversity (p)) and neutrality statistics (Tajima’s D;
(Tajima 1989) and Fu and Li’s D* (Fu and Li 1993)) were
estimated using DnaSP ver. 5 (Librado and Rozas 2009).
Significance of neutrality indices was inferred via 1000
bootstrap replicates implemented directly in DnaSP. Taji-
ma’s D uses the number of segregating sites and nucleotide
diversity in a genetic marker to identify an excess or lack of
polymorphisms, signifying population size changes or
selection. Similarly, Fu and Li’s D* investigates polymor-
phism frequencies, although focusing on singletons, to
identify purifying selection or recent selective sweeps.
To identify loci under selection, we used two tests: 1)
the Hudson–Kreitman–Aguade (HKA) test (Hudson et al.
1987, implemented online at: http://genfaculty.rutgers.
edu/hey/software#HKA ), and 2) a Bayesian method,
implemented in BAYESFST (Beaumont and Balding
2004). HKA compares ratios of polymorphisms to diver-
gence levels to identify signals (or lack thereof) of selection
in an entire dataset. We compared the two lineages of
C. americana, performing the test using 10 000 coalescent
simulations for three datasets: 1) all loci together,
2) Z-linked loci, and 3) autosomal loci. BAYESFST identi-
fies individual loci that exhibit a signal of selection by
implementing an MCMC to estimate locus, population,
and population-by-locus interaction effects. Using default
settings, we ran five iterations of BAYESFST to ensure
replicative results, again with three datasets (as with
the HKA test). We applied a 5% significance level to the
tests, which corresponds to an approximate transformed
p-value of 2.94; low outliers suggest loci evolving under
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balancing selection while high outliers suggest loci evolving
under diversifying selection.
Investigation of population structure and gene flow
We investigated population structure and genetic-structure
variation between autosomal and sex-linked markers using
several methods: 1) private, shared and fixed polymor-
phisms, 2) summary statistics, including the fixation index
(FST; Hudson et al. 1992) and relative node depth (RND;
Feder et al. 2005), and 3) a measure of genetic differentia-
tion and population sorting that uses phylogenies, the gene-
alogical sorting index (GSI; Cummings et al. 2008). e
RND is measured as the average pairwise divergence (Dxy;
Nei 1987) between lineages divided by Dxy between all
C. americana samples and the outgroup (C. familiaris).
e RND was used to identify ‘fast’ evolution between
C. americana lineages relative to the outgroup. e GSI is a
normalized statistic that quantifies exclusive ancestry of
populations in gene trees. Input for calculating the
GSI are gene trees for each locus. erefore, TOPALi ver.
2.5 (Milne et al. 2009), using a PhyML analysis (Guindon
and Gascuel 2003), was used to construct maximum likeli-
hood phylogenies for each locus. GSI values range from 0
(complete mixing of populations in the gene tree) to 1
(monophyly) with significance assessed by comparing
observed values to values obtained from 10 000 permuta-
tions of the individuals assigned to each gene trees’ tips.
Because the GSI is a standardized statistic, the value of GSI
for all loci can be combined to produce an ensemble GSI,
which measures the structure of populations in an entire
dataset.
To investigate gene flow between lineages we used a
coalescent method implemented in the Isolation with Migra-
tion (IMa) software (Hey and Nielsen 2007) using separate
datasets for autosomal and Z-linked loci. Trial runs were
used to identify appropriate priors for the IMa model;
following trial runs, we ran IMa for a burn-in period of
500 000 steps followed by 100 million iterations ( 100
effective sample size for each parameter). We performed
three runs of IMa with identical priors and different starting
seeds to assess convergence. Additionally, we used log-likeli-
hood ratio tests of nested models implemented in IMa to
determine if models identifying no or asymmetrical migra-
tion explained the data as well as the full model. Nested
models were ranked using Akaike’s information criterion
(AIC; Carstens et al. 2009).
Results
In total, 3271 base pairs from nine Z-linked loci were
sequenced for 16 brown creeper samples and supplemented
with 5063 base pairs from 21 autosomal loci from Manthey
et al. (2011b). One sample from the data matrix, from the
locus MUSK, could not be sequenced. For all Z-linked loci,
except BRM15, one sample of Certhia familiaris was
sequenced. Few (3/9) Z-linked loci shared any haplotypes
between populations, while the majority (15/21) of auto-
somal loci shared haplotypes between populations. DNA
sequences obtained in this study were deposited in GenBank
under accession numbers KF570392 – KF570652. Sequences
shorter than 200 base pairs are provided in the Supplemen-
tary material Appendix 1 because GenBank currently does
not accept sequences of this length.
Genetic diversity, neutrality and selection
Estimates of genetic diversity are shown in Table 1 and
Fig. 1. In general, the southern population exhibited greater
genetic diversity. In the northern population (southern
population), nucleotide diversity ranges from 0–0.67%
(0.03–0.88%) and 0–1.57% (0–1.99%) in Z-linked and
autosomal loci, respectively. Variance in nucleotide diversity
was significantly higher in autosomal loci for the northern
population (Levene’s test; p 0.028) but not significantly
different between marker types in the southern population
(p 0.235). None of the loci deviated from neutrality
using both Tajima’s D and Fu and Li’s D* (Table 1).
e HKA test did not reject evolution under the neutral
model for the Z-linked (p 0.595), the autosomal
(p 0.969) or the combined (p 0.973) datasets. BAYES-
FST results identified seven outlier loci (Fig. 2) with the full
dataset (all loci). ree loci (MUSK, Ca34, and Ca65) are
suggestive of disruptive selection and four (MADH2, 00895,
13093, and Ca9) of balancing selection. BAYESFST results
for the autosomal and Z-linked datasets identified the same
outliers (results not shown) with the exception of MUSK,
which was not an outlier in the dataset with only Z-linked
markers.
Genetic structure and gene flow
Z-linked markers exhibit a smaller level of shared polymor-
phisms between populations, while having more than
double the amount of fixed polymorphisms (Fig. 1, Table 1).
e mean FST for Z-linked loci is higher (Mann–Whitney
U-test; p 0.014) than autosomal loci (Table 1, Fig. 3).
Polymorphisms
Autosomal Loci
Z-linked loci
North
South
Shared
Fixed
0 0.008 0.016
Proportion of base pairs polymorphic
Figure 1. Rates of polymorphisms in autosomal and Z-linked
markers, represented as proportion of total number of base pairs
that are polymorphic. Private polymorphisms (North and South),
polymorphisms shared between lineages (Shared) and polymor-
phisms fixed between lineages (Fixed).
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Discussion
In this study, we identified increased levels of genetic dif-
ferentiation between lineages of C. americana on sex chro-
mosomes relative to autosomes (Fig. 1, Table 1), supporting
H01 (differentiation between lineages will be greater in
sex-linked markers than autosomal markers). e observed
levels of fixed differences on sex chromosomes (2.05 the
autosomal level) are higher than what would be expected
under neutral evolution (~ 1.33). e ratio drops, but is still
higher (1.56 the autosomal level) than what would be
expected, when loci putatively evolving under selection are
removed. ese deviations from strict neutral evolution
could be due to sexual selection in males, causing deviations
in NEZ (Wu and Davis 1993), specific population size effects
in birds (Mank et al. 2010a), or higher variance in male
reproductive success (Barker et al. 2008), leading to a
larger signal of faster-Z differentiation between lineages.
Alternatively, the levels observed here might be within the
expected variance of fixed differences given the number of
base pairs sampled.
Additionally, we find reduced gene flow between
lineages on sex chromosomes (Table 2), supporting H02
(sex-linked markers will exhibit relatively less gene flow
between lineages). ese estimates are limited in inferring
between pre-divergence genetic structure and post-divergence
gene flow (Becquet and Przeworkski 2009); however, either
interpretation (i.e. high pre-divergence structure or low post-
divergence gene flow on sex chromosomes) lends weight to
the hypothesis of the importance of sex chromosomes in the
speciation process in Certhia americana. Sampling individu-
als from a single population for each lineage introduces a
potential bias in parameter estimation of gene flow (includ-
ing potential biases in effective population size estimation);
however, this sampling regime limits the effects of genetic
structure within each lineage in inducing further biases
in IMa, as both the northern and southern lineages of
C. americana have strong population structure (Wahlund
1928, Manthey et al. 2011a, b).
Autosomal FST comparisons have a greater range (0.000–
0.941) than Z-linked FST comparisons (0.4438–0.9636),
but do not have a significantly greater variance (p 0.238).
Interestingly, in Z-linked loci, nucleotide diversity of the
southern population was negatively correlated with FST sta-
tistics (r –0.770, p 0.015) while northern nucleotide
diversity was not (p 0.585). In autosomal loci, FST com-
parisons are not correlated with nucleotide diversity in the
northern (p 0.071) or the southern lineage (p 0.089),
but nucleotide diversity between lineages is correlated
(p 0.001). While these properties may be biologically
meaningful (e.g. different patterns of genetic diversity rela-
tionships with FST in Z-linked markers between lineages),
genetic diversity patterns between populations can limit
upper bounds of FST estimates (Jakobsson et al. 2013) which
may have led to the observed pattern.
Estimates of RND and Dxy (Table 1, Fig. 3) were, on
average, higher in Z-linked loci, although it was not statisti-
cally significant (Mann–Whitney U-test; Dxy p-value 0.330;
RND p-value 0.647). Ensemble GSI statistics were
approximately the same for each population (Table 1). GSI
statistics were significant for a majority of loci in both the
southern (8/9 Z-linked and 15/20 autosomal) and the north-
ern (8/9 Z-linked and 9/20 autosomal) populations. Many
of the loci show nested monophyly of one population (5/9
Z-linked and 7/21 autosomal) with the other paraphyletic
(one population’s GSI 1.00 and the other’s GSI 0.88).
Gene flow estimates from coalescent-based analyses
(2Nm), as implemented in IMa, were lower in Z-linked
loci than autosomal loci (Table 2). Analyses of nested mod-
els identified models with reduced number of parameters
that could explain the data as well as the full model (Sup-
pementary material Appendix 1, Table A1, A2). However,
when using the AIC, the full model was best for the auto-
somal dataset and a model with zero migration north to
south was best for the Z-linked model. Although we
detected non-zero gene flow in coalescent-based estimates,
it is not substantial enough (2Nm 2) to counteract the
effects of genetic drift (Wright 1931, Slatkin 1987).
0
0.1
0.2
0.3
0.4
0.5
0.6
–5 –3 –1 1357
Transformed p-value; logit(2|P-0.5|)
Z-linked
Autosomal
FST
Ca34
Ca65
MUSK*
MADH2
00895
Ca9
13093
Figure 2. Results from the BAYESFST analysis of all loci. e estimates of FST plotted against empirical p-values for each locus. e
vertical bar shows the 0.05 significance level (~ 2.94 transformed p-value) used for identifying outlier loci. In analyses with autosomal or
Z-linked specific markers, results were the same as the full dataset with the exception of MUSK (denoted with an *) not being recognized
as an outlier.
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0.0 0.2 0.4 0.6 0.8 1.0
PTCH6
24105
IQGAP2
PPWD1
GPBP1
BRM15
A
LDOB3
MADH2
MUSK
13093
00895
02108
22528
04550
Ca4
Ca8
Ca9
Ca10
Ca13
Ca14
Ca15
Ca21
Ca29
Ca34
Ca47
Ca50
Ca51
Ca57
Ca58
Ca65
FST
Figure 3. FST values between lineages for each marker. e top nine markers are Z-linked and the bottom 21 are autosomal.
Lines through values indicate averages for each dataset separately.
Patterns of reduced gene flow between lineages and
increased genetic differentiation between lineages in sex
chromosomes are similar to patterns observed in other
closely related avian species, including Ficedula flycatchers
(Sætre et al. 2003, Borge et al. 2005), Luscinia nightin-
gales (Storchova et al. 2010), Passer sparrows (Elgvin et al.
2011) and Passerina buntings (Carling and Brumfield
2008, 2009). Alternatively, other avian species (often with
low overall genetic differentiation) have shown greater
intraspecific Z-linked gene flow (Dallimer et al. 2002, Li
and Merila 2010). ese patterns may suggest that
increased levels of differentiation on Z-linked markers and
reduced introgression only occur following significant
divergence in allopatry (as opposed to within species
between localities). On average, RND indicates the north-
ern and southern C. americana lineages are nearly as diver-
gent as C. americana is to its sister-species (C. familiaris),
providing further support to previous literature (Manthey
et al. 2011a, b) suggesting significant divergence in allopa-
try between C. americana lineages.
Higher levels of differentiation and reduced levels of
gene flow on sex chromosomes also could be attributed to
female biased dispersal. Multiple lines of evidence suggest
biased dispersal is not the cause of this pattern. While no
studies have investigated natal philopatry in C. americana,
studies in C. familiaris show patterns of site tenacity follow-
ing establishment of territories (Cramp and Perrins 1993,
Peach et al. 1995) while young birds will disperse at least one
kilometer (Suorsa et al. 2003). ough Certhia species may
have short natal dispersal distances, they are likely inconse-
quential; research on C. americana song in California dem-
onstrates the formation of local dialects in populations less
than 50 km apart (Baptista and Krebs 2000). Finally, coales-
cent estimates of gene flow (all estimates of 2Nm 2)
between the northern and southern clades of C. americana
(this study and Manthey et al. 2011b) indicate gene
flow, including male- or female-biased gene flow, was incon-
sequential in the evolutionary history of these lineages.
In general, the overall dataset did not deviate from
neutrality (HKA test, neutrality statistics; Table 1), however,
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Table 1. Per locus statistics of polymorphism neutrality, and divergence. Statistics include: base pairs of marker (BP), nucleotide
diversity ( 100) in the north (p N) and south (p S), number of polymorphisms in the northern (N) and southern (S) lineages, number of
shared polymorphisms (PS), number of fixed polymorphisms (PF), Tajima’s D (TD), Fu and Li’s D (FLD), FST, relative node depth (RND), and
genealogical sorting index of the southern (GSI S) and northern lineage (GSI N). Significance (p 0.05) of neutrality indices and sample-
size corrected significance (p 0.05) of GSI indicated by an asterisk (*).
Locus BP p N p S N S PSPFTD N TD S FLD N FLD S FST RND GSI S GSI N
MADH2 (Z) 482 0.667 0.535 10 10 5 0 0.24 20.62 0.53 20.73 0.4438 0.636 0.88*0.43*
PTCH6 (Z) 540 0.235 0.101 3 1 0 3 1.08 1.50 1.04 0.70 0.8120 0.743 1.00*0.88*
ALDOB3 (Z) 137 0 0.875 0 3 0 0 – 0.83 – 1.06 0.5079 0.759 0.03 0.76*
MUSK (Z) 349 0.108 0.044 3 1 0 7 21.70 21.15 22.20 21.37 0.9636 1.614 0.88*1.00*
GPBP1 (Z) 212 0.248 0.288 1 2 0 2 1.47 20.02 0.69 20.48 0.8468 1.883 0.88*1.00*
PPWD1 (Z) 423 0.238 0.515 6 7 0 0 20.58 20.18 0.91 20.75 0.4516 0.628 0.88*1.00*
IQGAP2 (Z) 395 0.095 0.034 3 1 0 3 21.70 21.16 22.20 21.43 0.9384 1.008 0.88*1.00*
BRM15 (Z) 290 0 0.795 0 8 0 1 – 20.23 – 0.33 0.5061 – 0.88*1.00*
24105 (Z) 443 0.102 0.194 2 4 0 0 20.65 20.97 20.50 20.60 0.5128 0.175 0.67*0.25
13093 (3) 322 1.571 1.991 16 14 11 0 20.06 1.28 0.41 1.23 0.0570 1.311 0.10 0.33
00895 (28) 218 0.665 0.887 3 7 3 0 1.75 20.29 1.04 20.44 0.0626 1.375 0.33 0.33
02108 (15) 194 1.349 0.851 6 4 2 0 1.53 1.15 1.27 1.14 0.1466 0.527 0.45*0.33
22528 (2) 265 0.579 0.333 5 3 0 0 0.06 20.07 0.46 1.04 0.1944 0.480 0.45*0.38
04550 (15) 89 0.539 0.627 1 1 1 0 1.03 1.53 0.69 0.69 0.0083 0.841 0.45*0.33
Ca4 (1A) 266 0.219 0 2 0 0 0 20.08 –20.50 – 0.2222 0.316 0.77*0.03
Ca8 (21) 172 – – – – – – – – – – – 0.000 – –
Ca9 (20) 274 1.183 1.128 9 10 7 0 0.72 0.10 0.91 20.35 0.0137 0.988 0.52*0.18
Ca10 (4A) 258 0.266 0.217 3 2 0 2 20.71 20.19 20.04 0.91 0.8125 0.906 0.88*1.00*
Ca13 (6) 266 0.595 0.746 4 9 1 0 0.98 20.99 1.14 21.01 0.4425 0.826 0.59*0.88*
Ca14 (3) 264 0.325 0.533 3 5 1 2 20.15 20.22 20.04 21.05 0.6832 1.300 1.00*0.88*
Ca15 (2) 227 0.737 0.671 5 6 2 0 0.19 20.68 0.46 20.05 0.1680 0.527 0.38 0.38
Ca21 (1) 216 0 0.847 0 5 0 2 – 0.19 – 0.46 0.6141 0.230 0.88*1.00*
Ca29 (1A) 189 0.401 0.366 2 2 1 0 0.66 0.91 0.91 0.91 0.0626 0.217 0.45*0.18
Ca34 (8) 222 0.180 0 1 0 0 1 0.65 – 0.69 – 0.8857 – 1.00*0.88*
Ca47 (24) 286 0.398 0.702 3 8 3 0 0.74 20.61 1.04 20.23 0.1126 0.807 0.14 0.33
Ca50 (12) 282 0 0.165 0 1 0 0 – 1.03 – 0.69 0.2666 0.086 0.88*1.00*
Ca51 (19) 290 0 0.129 0 3 0 0 – 21.70 –22.20 0.0000 2.031 0.00 0.27
Ca57 (1) 256 0.358 0.140 2 2 0 3 1.33 21.04 0.91 20.50 0.8477 0.912 0.88*1.00*
Ca58 (5) 246 0.262 0.458 2 4 0 0 0.84 20.25 0.72 20.63 0.4426 0.413 0.68*0.59*
Ca65 (3) 261 0.048 0.048 1 1 0 2 21.16 21.16 21.45 21.45 0.9411 2.000 1.00*0.88*
Z-linked 3271 0.188 0.376 28 37 5 16 0.6648 0.931 0.77*0.81*
Autosomal 5063 0.461 0.516 68 87 32 12 0.3326 0.804 0.58*0.55*
Overall 8334 0.379 0.474 96 124 37 28 0.4323 0.841 0.64*0.63*
Table 2. Coalescent-based (from IMa) estimates of gene flow (2Nm)
from north to south (southward) and south to north (northward),
with associated 95% confidence intervals. Three replicate runs were
performed for sex-linked (Z) and autosomal (A) loci with different
starting seeds.
Southward
Southward
95% CI Northward
Northward
95% CI
Z1 0.036 0.004–0.750 0.036 0.005–0.446
Z2 0.035 0.004–0.753 0.038 0.006–0.447
Z3 0.037 0.004–0.748 0.037 0.006–0.446
A1 0.230 0.022–1.221 0.385 0.114–1.452
A2 0.230 0.021–1.221 0.386 0.115–1.456
A3 0.229 0.021–1.219 0.383 0.115–1.461
in BayesFST analyses, three markers were identified as
potentially evolving under disruptive or directional selec-
tion (Fig. 2). Of these markers, more mapped to autosomes
than sex chromosomes, suggesting no clear trend in pat-
terns of selection based on marker type. ese results could
be caused by differential levels of gene flow between mark-
ers or marker-types, potentially causing positive results in
the BayesFST test; these caveats suggest we should inter-
pret these results with caution, as positive selection, or
genetic drift with reduced gene flow, may be driving the
observed patterns of sequence divergence in these loci.
When differentiation is investigated in concert with RND
(Table 1), the majority of Z-linked and autosomal markers
is not diverging between lineages faster than expected com-
pared to outgroup data, although two of the outliers in the
BAYESFST analysis (Ca65 and MUSK) had FST values
0.9 and RND values 1.6 (in Ca34 the outgroup
could not be amplified). ese combined patterns suggest
Ca65 and MUSK are good candidates for further investiga-
tion of selection.
Two lines of evidence suggest the selection patterns in the
data are driven by directional selection in the southern
lineage rather than disruptive selection between lineages.
First, we identified a negative correlation between differen-
tiation and genetic diversity in the southern lineage and no
relationship between differentiation and genetic diversity in
the northern lineage. Under strict neutral evolution, it is
expected that interspecific differentiation will be positively
correlated with intraspecific genetic diversity (Kimura 1983,
Li and Graur 2006). Second, in sex-linked markers, seven
loci show patterns of nested monophyly based on the GSI
(Table 1). Six of these indicate the southern lineage is nested
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Acknowledgements – We would like to thank the state, provincial,
and federal agencies and wildlife officers for their cooperation
and help obtaining permits that contributed to this research.
is project was funded in part by the Nathaniel R. Whitney Jr
Memorial Research Grant from the South Dakota Ornithologists’
Union and the National Science Foundation (NSF DEB
0814841).
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Supplementary material (Appendix JAV-00233 at www.
oikosoffice.lu.se/appendix ). Appendix 1.
