Contrasting Patterns of Introgression at X-Linked Loci Across the Hybrid Zone Between Subspecies of the European Rabbit (Oryctolagus cuniculus)

Abstract

Hybrid zones provide an excellent opportunity for studying the consequences of genetic changes between closely related taxa. Here we investigate patterns of genetic variability and gene flow at four X-linked loci within and between the two subspecies of European rabbit (Oryctolagus cuniculus cuniculus and O. c. algirus). Two of these genes are located near the centromere and two are located near the telomeres. We observed a deep split in the genealogy of each gene with the root located along the deepest branch in each case, consistent with the evolution of these subspecies in allopatry. The two centromeric loci showed low levels of variability, high levels of linkage disequilibrium, and little introgression between subspecies. In contrast, the two telomeric loci showed high levels of variability, low levels of linkage disequilibrium, and considerable introgression between subspecies. These data are consistent with suppression of recombination near the centromere of the rabbit X chromosome. These observations support a view of speciation where genomic incompatibilities at different loci in the genome create localized differences in levels of gene flow between nascent species.

Full text

Geraldes et al. 2005
1
Contrasting patterns of introgression at X-linked loci across the hybrid zone between subspecies of the
European rabbit (Oryctolagus cuniculus)
A
RMANDO GERALDES
*,†,‡
NUNO FERRAND
*,†
M
ICHAEL W. NACHMAN
‡
*
CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão,
4485-661 Vairão, Portugal
†
Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, 4099-
002 Porto, Portugal
‡
Department of Ecology and Evolutionary Biology, University of Arizona,
Tucson, AZ, 85721
Sequence data from this article have been deposited with the GenBank Data Libraries under accession nos. DQ306315-
DQ306490
Genetics: Published Articles Ahead of Print, published on April 2, 2006 as 10.1534/genetics.105.054106

Geraldes et al. 2005
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Running Head: Introgression at X-linked loci
Key words: X chromosome, Introgression, Nucleotide variability, Linkage disequilibrium, Speciation
Corresponding Author:
Armando Geraldes
Department of Ecology and Evolutionary Biology, Biosciences West Building, The University of
Arizona, P.O. Box 210088, Tucson, AZ 85721
Phone: (520) 626-4747 Fax: (520) 621-9190
Email: geraldes@email.arizona.edu

Geraldes et al. 2005
3
Hybrid zones provide an excellent opportunity for studying the consequences of genetic
changes between closely related taxa. Here we investigate patterns of genetic variability and gene
flow at four X-linked loci within and between the two subspecies of European rabbit (Oryctolagus
cuniculus cuniculus and O. c. algirus). Two of these genes are located near the centromere and two
are located near the telomeres. We observed a deep split in the genealogy of each gene with the root
located along the deepest branch in each case, consistent with the evolution of these subspecies in
allopatry. The two centromeric loci showed low levels of variability, high levels of linkage
disequilibrium and little introgression between subspecies. In contrast, the two telomeric loci showed
high levels of variability, low levels of linkage disequilibrium, and considerable introgression between
subspecies. These data are consistent with suppression of recombination near the centromere of the
rabbit X chromosome. These observations support a view of speciation where genomic
incompatibilities at different loci in the genome create localized differences in levels of gene flow
between nascent species.

Geraldes et al. 2005
4
A key problem in evolutionary genetics concerns the origin of reproductive isolation between
incipient species. Two important conclusions come from previous studies of the genetics of
reproductive isolation. First, barriers to gene flow often derive from incompatibilities between allelic
variants at two or more loci, i.e. epistasis (B
ATESON 1909; DOBZHANSKY 1936; MULLER 1940, 1942).
Empirical support for epistasis comes from a large body of work in Drosophila, beginning with
D
OBZHANSKY (1936). More recently, specific genes underlying reproductive isolation have been
identified, and all involve epistatic interactions (M
ALITSCHEK et al. 1995; TING et al. 1998; BARBASH
et al. 2003; PRESGRAVES et al. 2003). Second, loci contributing to reproductive isolation tend to be
overrepresented on the X chromosome in groups in which males are heterogametic (C
OYNE and ORR
1989). Evidence for the “large X effect” comes from mapping studies of hybrid sterility and hybrid
inviability (e.g. D
OBZHANSKY 1936; GRULA and TAYLOR 1980; TRUE et al. 1996; PRESGRAVES 2003;
PRESGRAVES et al. 2003; TAO et al. 2003). Moreover, Haldane’s (1922) rule (the sterility or
inviability of heterogametic hybrids) seems to be due largely to incompatibilities involving recessive
X-linked mutations (T
URELLI and ORR 1995, 2000). Finally, in a number of cases where sister species
hybridize in nature, X-linked loci introgress less than autosomal loci (e.g. H
AGEN 1990; SPERLING and
S
PENCE 1991; TUCKER et al. 1992).
The genetic basis of reproductive isolation has been studied both with laboratory crosses and in
natural hybrid zones, and both approaches have advantages and disadvantages. For example,
laboratory crosses make it possible to control the genetic background as well as the environment, and
they are repeatable. Hybrid zones offer the advantage of many generations of recombination, making
fine-scale mapping more feasible. In hybrid zones, it is possible to identify genes contributing to
isolation simply from patterns of gene flow without prior knowledge of the phenotype. Hybrid zones

Geraldes et al. 2005
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also allow us to study species that cannot be crossed in the laboratory. Finally, hybrid zones provide a
picture of the fitness of hybrid genotypes under natural conditions.
The European rabbit (Oryctolagus cuniculus) provides an opportunity to study the genetic basis
of reproductive isolation between recently evolved taxa. This species consists of two subspecies, O. c.
algirus in the southwestern portion of the Iberian Peninsula and O. c. cuniculus in the northeast of the
Iberian Peninsula and France. These two groups diverged in allopatry during the early Pleistocene and
have subsequently come into secondary contact in central Iberia, forming a contact zone that runs in a
NW-SE direction (Figure 1) (B
RANCO et al. 2000; BRANCO et al. 2002). The two subspecies are well
differentiated with respect to mtDNA (B
RANCO et al. 2000), the Y chromosome (GERALDES et al.
2005), and some allozyme loci (F
ERRAND and BRANCO 2006).
Motivated by the large X-effect documented in other species, here we focus on four X-linked
loci to understand the nature of reproductive isolation in rabbits. Two of these loci are near the
centromere and two are near the telomeres. We address three main questions. First, what are the
levels and patterns of genetic variation at genes on the rabbit X chromosome? Second, are patterns of
variation and introgression heterogeneous among loci, and if so, do the differences correlate with the
physical location of genes on the X chromosome? Third, are the data compatible with a model of
divergence without gene flow? We surveyed nucleotide variability at four X-linked loci, in a sample
of 43 male rabbits representing both subspecies and the area of contact. All four loci showed two
divergent lineages. Despite this deep divergence, there is still evidence of gene flow between
subspecies. Patterns of gene flow and nucleotide variability were heterogeneous among loci, being
low at the centromeric loci and high at the telomeric loci. We hypothesize that the centromeric region
of the X chromosome of the European rabbit may be involved in reproductive isolation between these
two subspecies.

Geraldes et al. 2005
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MATERIALS AND METHODS
Samples: Forty-three male European rabbits were sampled (Table 1). The samples were
divided into three groups: 20 from the northeast region of the Iberian Peninsula and from France,
corresponding to Oryctolagus cuniculus cuniculus (NE), 14 from the southwest region of the Iberian
Peninsula corresponding to O. c. algirus (SW) and nine from the contact zone (CZ) as defined by
mtDNA variation (B
RANCO et al. 2000). The geographic locations of the populations sampled are
shown in Figure 1, and collecting localities are given in Table 1. Additionally, one male Lepus
granatensis was used as an outgroup.
PCR amplification and sequencing: Genomic DNA was extracted either from blood, muscle,
or liver following S
AMBROOK and RUSSEL (2001). Introns of four X-linked loci were PCR amplified;
two are located near the centromere and two are near the telomeres (Figure 2). Amplification and
sequencing primers are listed in Supplementary Table 1. For each locus, two pairs of amplification
primers were designed. The first was based either on published rabbit sequences (Phka2) (D
AVIDSON
et al. 1992) or on conserved exonic regions between human, mouse and rat. Nested primers were then
designed specifically for the rabbit based on the first sequences obtained. Amplifications were carried
out in 50 µL volumes using Platinum Taq High Fidelity DNA Polymerase (Invitrogen, CA, USA)
following manufacturer recommendations. Cycling temperatures were as follows: an initial
denaturation step at 94º for 1min and 20 sec followed by 35 cycles of 94º for 20 sec, annealing for 20
sec and extension at 68º for 4 min. Annealing temperatures for each PCR are specified in
Supplementary Table 1. PCR products were purified using the QIAquick PCR Purification Kit
(Qiagen, CA, USA) prior to sequencing. Sequencing was carried out using an ABI 3700 automated

Geraldes et al. 2005
7
sequencer. All sequences have been deposited in GenBank under accession numbers DQ306315-
DQ306490.
Data Analyses: Sequences were inspected and concatenated using the computer program
Sequencher (Gene Codes, MI, US) and then aligned manually using the BioEdit software (H
ALL
1999). By sequencing the X chromosome in males we were able to recover haplotypes directly. The
analyses below were based on single nucleotide polymorphisms in introns only.
Basic population genetic parameters, including the number of segregating sites, number of
haplotypes, levels of nucleotide diversity,
(NEI and LI 1979), and the proportion of segregating sites,
θ (W
ATTERSON 1975), were estimated using the program DnaSP 4.00 (ROZAS et al. 2003) for the
entire data set and also for the NE, CZ and SW groups (Figure 1). Phylogenetic relationships among
alleles were estimated using the Median Joining (MJ) algorithm (B
ANDELT et al. 1999) as
implemented in Network v4.1.0.8 (http://www.fluxus-technology.com/).
We estimated divergence three ways. First, divergence between all O. cuniculus alleles and
Lepus granatensis was calculated as the average pairwise distance per nucleotide site, D
xy
(Nei 1987),
and as the number of net nucleotide substitutions per site, D
a
(NEI 1987). D
a
is defined as D
xy
- 0.5 (D
x
+ D
y
), where D
xy
is the average pairwise distance between groups and D
x
and D
y
are the average
pairwise distances within groups. Second, D
xy
and D
a
were calculated between the NE and SW groups
of O. cuniculus. Finally, to estimate the divergence time of the two subspecies of O. cuniculus,
maximum likelihood (ML) net nucleotide distances between L. granatensis and O. cuniculus, and
between the two main lineages found in O. cuniculus (see Results), were calculated using PAUP v 4.0
(S
WOFFORD, 2002). Divergence time between subspecies of O. cuniculus was calculated assuming a
divergence time between L. granatensis and O. cuniculus of 11.8 million years (MY) (M
ATTHEE et al.
2004).

Geraldes et al. 2005
8
The population recombination parameter, R (R= 3Nc for X-linked loci, where c is the
recombination rate per generation and N is the population size) between adjacent sites (H
UDSON
1987), the minimum number of recombination events, Rm (H
UDSON and KAPLAN 1985), and the
number of pairs of sites showing four gametic types were calculated using DnaSP 4.00 (R
OZAS et al.
2003). Another estimator of the population recombination parameter, γ (H
EY and WAKELEY 1997),
was calculated using the software SITES. While Hudson’s R is based on the variance of the number
of base pair differences between DNA sequences, γ is a maximum likelihood estimator developed
using a coalescent model for a sample of four DNA sequences with recombination. Linkage
Disequilibrium (LD) between pairs of polymorphic sites present at a frequency of at least 10% was
calculated within and between all loci, using the statistics D’ (L
EWONTIN 1964) and r
2
(HILL and
R
OBERTSON 1968) as implemented in DnaSP 4.00 (ROZAS et al. 2003).
Tajima’s D (T
AJIMA 1989) and Fu and Li’s D (FU and LI 1993) were calculated to test for
deviations from a neutral equilibrium frequency distribution using DnaSP 4.00 (R
OZAS et al. 2003).
Ratios of polymorphism within O. cuniculus to divergence between O. cuniculus and L. granatensis
were compared with the expectations under a neutral model using the Hudson-Kreitman-Aguadé
(HKA) test (H
UDSON et al. 1987). We performed one four-locus test and six pairwise comparisons
between loci using the HKA software (H
EY and KLIMAN 1993).
At each of the four loci we detected a deep split in the genealogy (see Results). We asked if
the observed pattern of nucleotide polymorphism is compatible with a single panmitic population, as
opposed to some form of population subdivision. If two populations have evolved in allopatry, the
basal branch of a gene genealogy may be longer than in a panmitic population. Furthermore,
mutations arising in an isolated subpopulation are unable to recombine with mutations in a different
subpopulation, resulting in higher levels of LD. W
ALL (2000) suggested two measures based on LD

Geraldes et al. 2005
9
that could be powerful indicators of population subdivision. The first, l
b
, is the number of congruent
sites, defined as the number of mutations that, on a pairwise basis, result in only two haplotypes. The
second, g
d
, is the maximum physical distance between congruent sites. Coalescent simulations of
panmixia were performed with the computer program ms (Hudson 2002). For each locus 50,000
genealogies of 43 individuals were simulated conditioned on the estimated values of θ and γ.
Additionally, for each locus, coalescent simulations were performed using two different values of the
population recombination parameter (3Nc=0.0015 and 3Nc=0.015, per site) chosen to reflect the range
of recombination rates known for other mammals (e.g. D
IETRICH et al. 1994; KONG et al. 2002;
J
ENSEN-SEAMAN et al. 2004). A computer program (GARRIGAN et al. 2005) was used to calculate l
b
and g
d
from the simulated datasets, and the distribution of the two statistics for each set of conditions
were plotted against each other. The probability of obtaining the observed values of l
b
and g
d
was
calculated as the proportion of simulated genealogies for which the values of l
d
and g
d
were greater
than the observed values.
Fst and Nm were calculated using the method of H
UDSON et al. (1992a) implemented in DnaSP
4.00 (R
OZAS et al. 2003). Genetic differentiation was also calculated using the test statistic Ks*
(H
UDSON et al. 1992b), and significance was assessed by performing 1000 permutations. To test for
significant population structure among populations and among groups of populations, analyses of
molecular variance AMOVA (E
XCOFFIER et al. 1992) between the SW and NE groups were performed
using ARLEQUIN (S
CHNEIDER et al. 2000).
One simple model of divergence is an isolation model in which two populations become
separated with no subsequent gene exchange. The HKA model (H
UDSON et al. 1987) takes this form
and further assumes that the ancestral species has a population size that is the average of the two
descendent species. More recent models relax this assumption. For example, W
AKELEY and HEY

Geraldes et al. 2005
10
(1997) proposed a model that is similar to the HKA model but includes an additional parameter, θ
A
,
the population mutation parameter for the ancestral species. While the HKA test only uses the number
of polymorphic sites and divergence, this model also incorporates the total number of polymorphic
positions in the two groups (S), the number of polymorphisms exclusive to one group (Sx
NE
and
Sx
SW
), the number of shared polymorphisms (Ss) and the number of fixed differences (Sf). We tested
the fit of our data to these two models in two different ways. First we performed pairwise
comparisons between all loci, and second we performed tests with all four loci together. The fit of our
data to the W
AKELEY and HEY model of divergence without gene flow was tested using the program
WH (WANG et al. 1997).
These models assume that there has been no gene flow between the two populations since the
initial split. In many cases this is an unrealistic assumption. H
EY and NIELSEN (2004) developed a
model of population divergence that allows for genetic drift (increasing population divergence) and
gene flow (retarding it) to act together, which they call the Isolation with Migration model. The
computer program IM is an implementation of the Markov Chain Monte Carlo method for the
analyses of genetic data under this model. We used IM to estimate the effective population size of O.
c. cuniculus and of O. c. algirus and to estimate migration rates for each locus between subspecies in
each direction. IM assumes that there is no recombination within loci. For Phka2 and Hprt1 we used
the largest region showing no evidence of recombination, following W
ON and HEY (2005). For Phka2
a portion of 687 bp containing 23 polymorphic sites was used, and for Hprt1 a region of 501 bp with
20 polymorphic sites was used. For Smcx all 20 NE and 14 SW individuals were used since the data
are free of recombination. For Msn we removed three recombinant individuals (Vau1, Rsl4 and Rsl10)
from the NE group. We assigned wide prior distributions of the parameters based on preliminary trial
runs. We ran the program under Metropolis Coupled Monte Carlo Markov Chains (MC3), using 10
chains with linear heating. We used a burn-in period of 1,000,000 steps and recorded results every 40

Geraldes et al. 2005
11
steps. To test that the chains were mixing well we ran the program with different random seed
numbers and the results were similar. We ran the program for 25,625,601 steps after the burn-in
period and recorded the results of 625,014 steps.
RESULTS
Levels and patterns of variation: We observed considerable variation at all four genes.
Polymorphic sites for each gene are shown in Figure 3, and summaries of variation are given in Table
2. The number of polymorphic sites varied from 56 at Msn to 151 at Phka2, but the number of
haplotypes observed at each gene was much more constant (from 23 at Smcx to 29 at Phka2). Levels
of polymorphism were high, both in the total sample and within each subspecies. However, nucleotide
diversity (π) at the two centromeric loci was considerably lower (0.52% at Smcx and 0.55% at Msn)
than at the two telomeric loci (0.70% at Phka2 and 1.26% at Hprt1). This contrast was even more
striking in the proportion of segregating sites, where θ at the centromeric loci was roughly half the
value seen at the telomeric loci.
We assessed the amount of LD in our sample in several ways, and all were consistent in
revealing more recombination (less LD) at the telomeric loci than at the centromeric loci (Table 2),
consistent with suppression of recombination near the centromere. The number of pairs of sites
showing all four gametic types was zero at Smcx, 77 at Msn, 390 at Phka2, and 180 at Hprt1. Rm, the
minimum number of recombination events in the history of the sample (H
UDSON and KAPLAN 1985)
was zero at Smcx, intermediate at Msn and Hprt1 (7 and 6 respectively) and highest at Phka2 (17).
Similarly, R (H
UDSON 1987) between adjacent sites was low at the two centromeric loci (Smcx and
Msn) and much higher at the telomeric loci (Phka2 and Hprt1). The values for γ (H
EY and WAKELEY

Geraldes et al. 2005
12
1997), a maximum likelihood estimator of the population recombination parameter, are concordant
with R in showing more recombination at the telomeric loci than at the centromeric loci. Thus,
although there are small differences among estimators, levels of recombination are higher at the
telomeric loci than at the centromeric loci. We assessed the significance of LD through pairwise
comparisons of polymorphic sites present at a frequency of at least 10% using a Fisher’s Exact Test.
From the 8515 pairwise comparisons performed, 2497 were significant in a two-tailed test, and 980
remained significant after a Bonferroni correction for multiple tests. Of these, 197 were between
polymorphic sites at Smcx, 219 between sites at Msn, 93 between sites at Phka2, and 130 between sites
at Hprt1. Interlocus LD was only detected between Smcx and Msn where there were 341 pairs of sites
that showed significant LD.
The distribution of allele frequencies as measured by Tajima’s D and Fu and Li’s D generally
conformed to expectations under a neutral model of molecular evolution (Table 2). For example, in
the total sample, Tajima’s D was either positive (Msn and Hprt1), very close to zero (Smcx) or
negative (Phka2), but not significantly different from zero. When the population groups were
analyzed separately, Tajima’s D was negative (except for Hprt1 in the NE and SW groups), but not
significantly so (P > 0.05 for all tests). We also tested a neutral model of molecular evolution by
comparing ratios of polymorphism within O. cuniculus (in the total sample) to divergence between O.
cuniculus and Lepus granatensis, using the HKA test. We performed one four-locus comparison as
well as six pairwise comparisons between loci, and none of these were significant (P > 0.05 for each).
Divergence and gene flow between subspecies: Fst estimates between O. c. cuniculus and O.
c. algirus are shown in Table 3. Fst was very high at the centromeric loci, Smcx (0.680) and Msn
(0.829), and one order of magnitude lower at the telomeric loci, Phka2 (0.027) and Hprt1 (0.022).
The two subspecies were significantly differentiated, using the Ks* test statistic, at all loci but Hprt1
(Table 3). Similarly, the AMOVA analyses revealed that at the two centromeric loci, Smcx and Msn,

Geraldes et al. 2005
13
most of the genetic variation is partitioned among the two subspecies (64% and 84% respectively)
while at the telomeric loci, Phka2 and Hprt1, most of the observed variation was partitioned among
populations within each subspecies (93% at both loci), and only a marginal proportion (2%) of the
variation was segregating between subspecies.
This differentiation can also be seen in the phylogeny of alleles for each gene (Figure 4). At
each locus there were two divergent groups of haplotypes, and in each case the root fell along the deep
branch separating these two groups. In this analysis, only Smcx was free of homoplasy. The locus
with the most homoplasy was Phka2. This homoplasy may be due to recombination or recurrent
mutation. Evidence for recurrent mutation comes from the observation that at Hprt1 three different
positions have three nucleotides segregating (Figure 3d). Other evidence of recurrent mutation is the
fact that the amount of homoplasy is slightly reduced if CpG sites, which are known to be
hypermutable, are excluded. For example, for Phka2, the consistency index (CI) increased from 0.778
to 0.803 when CpG sites were removed. However, much of the homoplasy is probably due to
recombination, as evidenced by the fact that the CI was 1.0 for Smcx, 0.889 at Msn, but was 0.778 at
and 0.717 at Phka2 and Hprt1, respectively. Moreover, at Phka2 and Hprt1 one and 16 individuals,
respectively, were identified as recombinants between the two divergent lineages based on their
position on the haplotype network and by visual inspection of the table of polymorphism (Figure 3 a
and d).
The degree of introgression between subspecies can also be seen by the concordance (or lack
thereof) between geography and phylogeny. For the two centromeric loci (Smcx and Msn) there was
good concordance between phylogeny and geography, i.e. the two major lineages correspond well
with each subspecies (Figure 4). At Msn we did not detect any introgressed haplotypes and at Smcx
we only observed three NE individuals with haplotypes from the lineage that is otherwise restricted to
the SW and CZ groups. At the two telomeric genes (Phka2 and Hprt1), in contrast, there seems to be

Geraldes et al. 2005
14
little or no concordance between phylogeny and geography. At all four genes, individuals from the
CZ group are scattered throughout the haplotype networks.
The proportion of congruent sites, l
b
, is greater at the two centromeric loci (representing 30%
and 32% of all polymorphic sites at Smcx and at Msn respectively), than at the telomeric loci (11% at
Phka2 and 16% at Hprt1). Similarly, the maximum distance between congruent sites, g
d
, is greater at
Smcx (85% of the total locus length) and at Msn (95%) than at Phka2 (65%) and Hprt1 (16%). We
calculated the probability of observing these values of l
b
and g
d
using coalescent simulations of 50,000
genealogies of 43 individuals evolving neutrally under panmixia with mutation (θ) and recombination
(γ) parameters estimated from the data. Results are shown in Table 4. Under these conditions, the null
model was rejected for Msn (P=0.00214). This test is quite conservative using γ estimated from the
data since population subdivision will increase LD and thus underestimate the true value of
recombination. Therefore, we also conducted simulations with a population size of 10
5
and per site
recombination rates of 0.5X10
-8
and 5X10
-8
, reflecting the range of recombination rates seen in other
mammals (e.g. D
IETRICH et al. 1994; KONG et al. 2002; JENSEN-SEAMAN et al. 2004). At the two
centromeric loci, Msn and Smcx, the null model was rejected using either value of recombination. For
Phka2 the null hypothesis was rejected only with the higher recombination rate, and Hprt1 was
marginally significant (P=0.066) only for the higher recombination rate (Table 4).
Another way of looking at divergence is to quantify the amount of shared and fixed variation
between the two groups (Table 5). The number of shared polymorphisms was low at the centromeric
loci (16% and 6% of all polymorphisms at Smcx and Msn respectively), and high at the telomeric loci
(42% and 64% at Phka2 and Hprt1 respectively). Only Msn showed fixed differences between the
two groups. These patterns of variation suggest that there has been gene flow between O. c. cuniculus
and O. c. algirus at some, but not all, loci. To further test this, we performed an HKA test between

Geraldes et al. 2005
15
NE and SW population groups. A multilocus test between all four loci failed to reject the null model,
but in one pairwise comparison (between Phka2 and Msn) the model was rejected (P=0.035). This
result seems to be mainly driven by the fact that divergence at Msn was much higher than expected
(observed D=26.80, expected D=10.68). The isolation without migration model (W
AKELEY and HEY,
1997) shares most of the assumptions with the HKA model, but estimates the ancestral population size
instead of assuming that it is the average of the population size of the extant populations. A four locus
test using this model also failed to reject the null hypothesis. We also tested the fit of the data using
all pairwise comparisons to Msn. Only the comparisons to Msn were performed because in the other
comparisons there are no fixed differences and the program is unable to simulate the distribution of the
expected values. The comparison between Smcx and Msn failed to reject the null model while the
other two comparisons did reject the null model (Phka2/Msn P
( 2)
=0.032 and P
(WH)
=0.030; Msn/Hprt
P
( 2)
=0.024 and P
(WH)
=0.009).
We used IM (H
EY and NIELSEN 2004) to obtain Maximum Likelihood Estimates (MLE) of the
effective population size for each subspecies. We also estimated migration rates for each locus in each
direction. The average estimate of the effective population size was approximately 882,000 for O. c.
algirus and 422,000 for O. c. cuniculus (Table 6). The probability distribution of the ancestral
population parameter was flat (not shown), as expected if the ancestral population existed long ago
(W
ON and HEY 2005). Similarly the probability distribution of t, the time since divergence, was flat,
but non zero (not shown). This suggests that the two subspecies were isolated in the past, but this
analysis does not provide a reliable estimate of the time of isolation. Gene flow at the telomeric loci
was higher from NE to SW than from SW to NE. For the centromeric loci, introgression of Msn is
quite low in both directions, while Smcx shows some unidirectional introgression from SW to NE.
Thus it seems that levels and patterns of gene flow are very different between centromeric and
telomeric loci.

Geraldes et al. 2005
16
We estimated divergence time between the two subspecies of O. cuniculus using a phylogenetic
approach. Assuming a divergence time of 11.8 MY (M
ATTHEE et al. 2004) between O. cuniculus and
L. granatensis, divergence time between O. c. cuniculus and O. c. algirus was estimated to be on the
order of 2-5 MY ago (Table 7).
DISCUSSION
We documented genetic variation at four X-linked loci in natural populations of the European
rabbit, O. cuniculus. At each locus, we observed a deep split in the phylogeny with the root lying
along the long internal branch. This pattern is consistent with the evolution of each subspecies in
allopatry and subsequent secondary contact. Despite this broad similarity among loci, we detected
heterogeneity among loci in terms of levels of nucleotide polymorphism, recombination, and
introgression between the two subspecies. This heterogeneity corresponds well with the physical
location of the loci on the rabbit X chromosome. The two centromeric loci had lower levels of
nucleotide polymorphism, higher levels of LD and reduced introgression in comparison to the two
telomeric loci. Although we do not have direct estimates of the frequency of crossing-over in rabbits,
these observations are consistent with suppression of recombination near the centromere, as has been
observed in other species (e.g. K
ONG et al. 2002).
Levels and patterns of variation: Across the entire sample (i.e. including both subspecies),
the average heterozygosity among all loci (π=0.76%) was high and roughly one order of magnitude
higher than heterozygosity at X-linked loci in humans (π= 0.081%; H
AMMER et al. 2004) and mice
(π=0.078%; N
ACHMAN 1997). Clearly, this high level of nucleotide variability reflects not only
nucleotide polymorphism within each subspecies but also the divergence between subspecies. One

Geraldes et al. 2005
17
gene (Msn) showed no introgression between subspecies. Levels of nucleotide polymorphism at this
gene were 0.14% for O. c. cuniculus and 0.26% for O. c. algirus, closer to values observed in humans
(H
AMMER et al. 2004) and mice (NACHMAN 1997).
We also observed variation in levels of polymorphism among loci. Interestingly, the two
centromeric loci had lower levels of π and θ than observed at the two telomeric loci, both for the entire
dataset and for each subspecies considered separately. Within each subspecies, this difference may be
explained by different levels of introgression. In other words, Smcx and Msn may be less variable
within each subspecies because they contain relatively few introgressed haplotypes, compared to
Phka2 and Hprt1. However, we also observe less variation at Smcx and Msn in the total sample. This
may be due in part to lower mutation rates at these genes. For example, divergence between
Oryctolagus and Lepus is lower at Smcx (Dxy=1.74%) and Msn (Dxy=4.08%) than at Phka2
(Dxy=6.06%) or Hprt1 (Dxy=4.43%) (Table 2). If recombination is suppressed near the centromere,
these differences in mutation rate may reflect an association between mutation and recombination (e.g.
H
ELLMANN et al. 2003). It is also possible that reduced variation at Smcx and Msn may be due partly
to the effect of either positive or negative selection at linked sites (M
AYNARD-SMITH and HAIGH 1974;
C
HARLESWORTH et al. 1993).
Indirect evidence that recombination is suppressed near the centromere comes from our
observation of increased LD at Smcx and Msn compared to Phka2 and Hprt1. Patterns of LD are
affected by many factors, including selection, mutation, recombination, and changes in population size
(e.g. A
RDLIE et al. 2002). However, in humans, there is good evidence that levels of LD are inversely
correlated with recombination rate over much of the genome (e.g. R
EICH et al. 2001; MCVEAN et al.
2004; M
YERS et al. 2005). Moreover, in many organisms, recombination is suppressed near the
centromeres, particularly in metacentric chromosomes (e.g. K
ONG et al. 2002). Thus, our observation

Geraldes et al. 2005
18
of increased LD at Smcx and Msn relative to Phka2 and Hprt1 is consistent with, but not proof of,
reduced recombination near the rabbit X centromere.
Divergence and gene flow between subspecies: RFLP surveys of mtDNA polymorphism in
the Iberian Peninsula and France have shown that O. cuniculus is composed of two deeply divergent
mtDNA lineages that are thought to have diverged about 2 MYA (B
IJU-DUVAL et al. 1992; BRANCO et
al. 2000). A survey of nucleotide variability at Sry also found evidence for the existence of two
divergent lineages in the Y chromosome (G
ERALDES et al. 2005). These two lineages are associated
with O. c. algirus and O. c. cuniculus (B
RANCO et al. 2000), and are thought to have evolved in
allopatry. Our X chromosome data confirm the existence of two divergent evolutionary units in O.
cuniculus, and we show that in general the data reject the evolution of the two lineages under
panmixia. The divergence time estimated from these loci is in good agreement with divergence time
estimated from mitochondrial genes, and places the origin of these two subspecies at the
Pliocene/Pleistocene boundary. We observed high levels of population differentiation at the two
centromeric loci, but not at the telomeric loci. At the centromeric loci the two divergent lineages
correspond well with the described subspecies and are broadly concordant with the patterns of
differentiation seen at the Y chromosome and at the mtDNA. The same was not observed at the two
telomeric loci, where geography and phylogeny are largely decoupled.
If two populations evolve in allopatry for a sufficiently long time and then come into secondary
contact with little or no gene flow, a high percentage of fixed differences and a small number of
shared polymorphisms are expected. In our data this is seen only at Msn where 36% of all
polymorphisms correspond to fixed differences between groups, and 6% correspond to shared
polymorphisms. At all other loci, there are no fixed differences between subspecies and the
percentage of shared polymorphisms varies from 16% at Smcx (centromeric) to 64% at Hprt1
(telomeric). This heterogeneity among loci is also reflected in the rejection of an isolation without

Geraldes et al. 2005
19
gene flow model using the HKA test between Phka2 and Msn and the rejection of the null model using
the WH test between Phka2 and Msn, and between Hprt1 and Msn.
It is noteworthy that the patterns of reduced introgression seen at Smcx and Msn, which may
experience reduced recombination, are similar to the patterns seen previously at the mtDNA (B
RANCO
et al. 2000) and the Y chromosome (Geraldes, unpublished data), genomic regions with no
recombination. In contrast, a survey of 14 allozyme loci revealed higher, but variable, levels of
introgression (φ
ct
between subspecies ranged from 0 to 0.46), comparable to the patterns observed at X
chromosome loci. Differences among loci in levels of introgression have also been documented in
other organisms. For example, genomic regions with suppressed recombination as a result of
chromosomal rearrangements introgress less than co-linear regions in comparisons between
Drosophila pseudoobscura and D. persimilis (N
OOR et al. 2001; MACHADO et al. 2002) and between
hybridizing sunflowers of the genus Helianthus (R
IESEBERG et al. 1999). Based on such observations,
N
OOR et al. (2001) and RIESEBERG (2001) have argued that chromosomal rearrangements may
promote speciation, not through underdominance directly as in traditional models (e.g. W
HITE 1978),
but by suppressing recombination and thereby extending the effects of isolation genes to linked sites.
Our finding of low levels of introgression in an area of high LD near the X chromosome centromere of
the rabbit is consistent with similar observations in fruit flies and sunflowers. Similarly, in Anopheles
mosquitoes, two (out of three) areas of reduced introgression map to centromeres (T
URNER et al.
2005).
Our observations also have some interesting parallels with studies of hybridization in the house
mice, Mus musculus and M. domesticus. In the house mouse hybrid zone in Western Europe, the Y
chromosome shows reduced introgression (V
ANLERBERGHE et al. 1986; TUCKER et al. 1992; DOD et
al. 1993), and the X chromosome shows lower levels of introgression than do the autosomes (T
UCKER
et al. 1992; D
OD et al. 1993; MUNCLINGER et al. 2002), although there is also considerable variability

Geraldes et al. 2005
20
in levels of introgression among loci on the X chromosome (P
AYSEUR et al. 2004). In a similar
fashion, we observe some X linked loci with much reduced introgression in rabbits, providing further
support for the importance of the X chromosome in reproductive isolation. Interestingly, the
differences among loci in migration estimates (Table 6), may provide some clues to the nature of
incompatibilities underlying reproductive isolation. In particular we note that estimates of the number
of migrants from NE to SW for the centromeric loci are slightly lower than in the opposite direction.
This is in agreement with the expected asymmetric behavior of young Dobzhansky-Muller interactions
(O
RR 1995) and suggests that incompatibilities may derive from interactions between the cuniculus X
chromosome and an algirus genetic background.
One ultimate goal of speciation studies is to determine the identity of genes involved in
reproductive isolation between nascent species. With the completion of the sequence of the rabbit
genome expected in the next few years, it may soon be possible to identify candidate genes for
reproductive isolation in this species. The results presented here suggest that some of these genes may
lie near the centromere of the X chromosome.

Geraldes et al. 2005
21
ACKNOWLEDGEMENTS
This work was supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/4621/2001
PhD grant to AG and Research Project POCTI/BSE/40280/2001, and by a National Science
Foundation grant to MWN. We thank R. Villafuerte for help in collecting rabbit samples, J. Good and
C. Pinho for valuable discussions, D. Garrigan, T. Salcedo and M. Dean for help with ms simulations,
and J. Hey for help with IM. We would also like to thank two anonymous reviewers for suggestions on
a previous version of this manuscript.

Geraldes et al. 2005
22
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Table 1 - Individuals sampled and their geographic locations
Population Sample Size Population no.
a
Group Individual ID
Versailles 1 1 NE Ver1827
Vaulx-en-Velin 1 2 NE Vau1
Carlucet 1 3 NE Cau19
Perpignan 1 4 NE Pep18
Zaragoza 2 5 NE Zrg16, Zrg20
Castelló 2 6 NE Rsl4, Rsl10
Benavente 1 7 NE Bnv3
Zamora 2 8 NE Zam1, Zam20
La Rioja 2 9 NE Lrj3, Lrj6
Madrid 1 10 NE Mdr7
Alicante 3 11 NE Alic1, Alt107, Alt120
Cartagena 1 12 NE Cat12
Cuenca 2 13 NE Cue1, Cue3
Galicia 1 14 CZ Gal25c3
Bragança 2 15 CZ Bra1, Bra13
Toledo 3 16 CZ Tol25, Tol50, Tol64
Ciudad Real 2 17 CZ Cre1, Vdm12
Las Amoladeras 1 18 CZ Amo2
Córdoba 3 19 SW Luc4, Luc9, Luc17
Sevilla 3 20 SW Pfr1, Pfr5, Pfr7
Doñana 1 21 SW Don6
Vila Real 3 22 SW Vrl1, Vrl4, Vrl7
Idanha-a-nova 1 23 SW Id85
Elvas 2 24 SW Elv3, Elv6

Geraldes et al. 2005
32
Vila Viçosa 1 25 SW VV1_1/94
a
Population numbers from Figure 1.

Geraldes et al. 2005
33
Table 2 - Levels of polymorphism, allele frequency spectrum tests of neutrality, divergence and recombination
Polymorphism
Frequency Spectrum Tests of
Neutrality
Divergence (%)
Recombination
Between NE and SW
Between O. cuniculus
and L. granatensis
n L
a
K
b
S
c
π (%) θ (%) Tajima's D Fu and Li's D Da
d
Dxy
e
Da
f
Dxy
g
γ
h
R
i
Rm
j
4gt
k
All 43 3168 29 151 0.699 1.102
-1.337 -0.995
0.008 0.659
5.719 6.059
0.0051 0.0025 17 390
NE 20 3168 14 105 0.574 0.934 -1.584 -2.106 0.0019 0.0018 10 95
CZ 9 3168 9 86 0.924 0.999 -0.385 0.121 0.0052 0.0028 3 25
Phka2
SW 14 3168 10 95 0.708 0.945 -1.109 -1.109 0.0074 0.0032 10 137
All 43 2709 23 60 0.517 0.512 0.035 -1.499 0.531 0.782 1.475 1.735 0 0.0012 0 0
NE 20 2709 13 44 0.341 0.458 -1.021 -0.647
0 0 0 0
CZ 9 2709 6 29 0.390 0.394 -0.054 0.667 0 0.0003 0 0
Smcx
SW 14 2709 8 21 0.160 0.244 -1.438 -2.172 0 0.0020 0 0
All 43 2825 25 56 0.553 0.458 0.733 -0.849 0.786 0.949 3.803 4.083 0.0023 0.0011 7 77
NE 20 2825 8 14 0.079 0.140 -1.591 -1.390
0.0003 0.0002 1 3
CZ 9 2825 8 36 0.462 0.469 -0.073 0.423 0 0.0005 0 0
Msn
SW 14 2825 11 23 0.246 0.256 -0.161 -0.649 0.0016 0.0117 2 7
All 43 1473 28 68 1.256 1.115 0.454 -0.381 0.027 1.256 3.848 4.428 0.0088 0.0086 6 180
NE 20 1473 13 47 1.129 0.899 1.025 0.978
0.0071 0.0034 2 58
CZ 9 1473 8 49 1.228 1.249 -0.087 0.134 0.0094 0.0109 4 104
Hprt1
SW 14 1473 12 50 1.328 1.132
0.760 0.642
0.0079 0.0120 4 111
a
Length of the sequence in bp.
b
Number of haplotypes.

Geraldes et al. 2005
34
c
Number of polymorphic sites.
d
Net nucleotide divergence per site (NEI 1987) between NE and SW (CZ was excluded from this analysis).
e
Average pairwise nucleotide substitutions per site (NEI 1987) between NE and SW (CZ was excluded from this
analysis).
f
Net nucleotide divergence (NEI 1987) between the haplotype found in Lepus granatensis and all O. cuniculus
sequences.
g
Average pairwise nucleotide substitutions per site (NEI 1987) between the haplotype found in Lepus granatensis
and all O. cuniculus sequences.
h
Maximum likelihood estimate of the population recombination parameter between adjacent sites (HEY and
W
AKELEY 1997).
i
Hudson’s (1987) estimator of the population recombination parameter between adjacent sites.
j
Minimum number of recombination events in the history of the sample (HUDSON and KAPLAN 1985).
k
Number of the pairs of sites that show all four gametic types.

Geraldes et al. 2005
35
Table 3 - Genetic differentiation between NE and SW groups at four X-linked loci
Fst
a
Nm
b
φ
ct
c
φ
st
d
φ
sc
e
Da(%)
f
Phka2 0.0266* 12.22 0.02 0.07 0.05 0.008
Smcx 0.6796*** 0.16 0.64 0.80 0.43 0.531
Msn 0.8286*** 0.07 0.84 0.86 0.11 0.786
Hprt1 0.0218 14.99 0.02 0.07 0.05 0.027
a
Fst was calculated using the method proposed by HUDSON, SLATKIN and MADDISON (1992).
Statistical significance for the estimation of Fst between the two groups was obtained with
the Kst* statistic (H
UDSON et al. 1992) (*P<0.05; **P<0.01; ***P<0.005).
b
Nm was calculated according to WRIGHT’s (1951) island model of population structure,
using the expression (Fst=1/(1+3Nm)) for X-linked loci.
c
φ
ct
is the fixation index for the amount of variation segregating between NE and SW groups,
calculated using the AMOVA framework (E
XCOFIER et al. 1992). For the NE group the
thirteen populations studied were pooled into seven subgroups (NE1 - populations 1, 2, 3 and
4; NE2 - population 5; NE3 - population 6; NE4 - populations 7 and 8; NE5- populations 9
and 10; NE6 - populations 11 and 12; and NE7 - population 13). For the SW group, the seven
populations studied were pooled into four subgroups (SW1 - population 19; SW2 -
populations 20 and 21; SW3 - population 22; and SW4 - populations 23, 24 and 25).

Geraldes et al. 2005
36
d
φ
st
is the fixation index for the amount of variation segregating within each subgroup,
calculated using the AMOVA framework (E
XCOFIER et al. 1992).
e
φ
sc
is the fixation index for the amount of variation segregating among subgroups within
each group, calculated using the AMOVA framework (E
XCOFIER et al. 1992).
f
Da is the net nucleotide distance per base pair between populations (NEI 1987).

Geraldes et al. 2005
37
Table 4- Probabilities of observing the number of congruent sites, l
b
, and maximum distance between
congruent sites, g
d
, under a single panmitic population.
γ estimated from data
a
γ =0.0015
b
γ =0.015
c
l
b
g
d
l
b
and
gd
l
b
g
d
l
b
and
gd
l
b
g
d
l
b
and
gd
Phka 2 0.35364 0.96288 0.34002 0.76470 0.99352 0.76296 0.04484 0.8919 0.04032
Smcx 0.39566 0.79952 0.37298 0.09558 0.33292 0.04248 0.00010 0.10748 0.00004
Msn 0.03470 0.03466 0.00214 0.06722 0.04908 0.00638 0.00006 0.01082 0.00000
Hprt 1 0.17324 0.99916 0.17302
0.62354 0.99992 0.62354
0.06626 0.99742 0.06626
Probabilities were calculated as the proportion of simulated genealogies with values of l
b
, g
d
, or both,
equal or greater than those observed in our data.
a
For Phka2 γ=0.0051 per site, for Smcx γ=0 per site, for Msn γ=0.0023 per site and for Hprt1 γ=0.0088
per site.
b
γ=3Nc, where N=1X10
5
and c=0.5X10
-8
per site.
c
γ=3Nc, where N=1X10
5
and c=5X10
-8
per site Table 4 - Shared and fixed variation between NE and SW
groups at 4 X-linked loci.

Geraldes et al. 2005
38
Table 5- Shared and fixed variation between NE and SW groups at four X-linked loci
a
S
b
Sx
NE
c
Sx
SW
d
Ss
e
Sf
f
Phka2 140 45 (64.6) 35 (56.5) 60 (41.1) 0 (10.9)
Smcx 56 35 (16.9) 12 (14.7) 9 (10.7) 0 (2.8)
Msn 53 11 (15.2) 20 (13.3) 3 (9.7) 19 (2.6)
Hprt1 59 9 (20.3) 15 (17.7) 38 (12.9) 0 (3.4)
a
The expected values under the population parameters estimated with software WH
(W
AKELEY and HEY 1997) are shown between parentheses
b
S- Number of polymorphic positions.
c
Sx
NE
- number of exclusive polymorphisms in the NE group.
d
Sx
SW
- number of exclusive polymorphisms in the SW group.
e
Ss- Number of shared polymorphisms.
f
Sf- number of fixed differences.

Geraldes et al. 2005
39
Table 6 - Maximum-Likelihood Estimates (MLE) and the 90% Highest Posterior Density (HPD) Intervals
a
of
demographic parameters
Ne
SW
Ne
ΝΕ
Nm from NE to SW
Nm from SW to NE
Phka2 Smcx Msn Hprt1
Phka2 Smcx Msn Hprt1
MLE 882,675 422,149 10.215 0.008* 0.008* 3.470 0.281 0.404 0.012 0.951
HPD 520,833-1,737,939 202,851-685,307 2.600-16.092 0.008-6.480 0.008-0.411 0.733-14.695
0.004-7.696
+
0.004-3.569 0.004-0.358 0.004-6.687
*The estimated value of Nm is at the lower limit of resolution; i. e. 0.008 corresponds to the first bin of the parameter space
surveyed for the migration parameter from SW to NE.
+
The actual interval was larger than this and could not be estimated reliably, because the likelihood surface was relatively
flat.
a
The 90% HPD intervals contain 90% of the probability density for each estimate.

Geraldes et al. 2005
40
Table 7- Uncorrected and corrected
a
net nucleotide (Da)
b
divergences between O. cuniculus and
L. granatensis and between subspecies of O. cuniculus, and estimates of divergence time (MY)
between subspecies of O. cuniculus.
O. cuniculus/L. granatensis
O. c. cuniculus/O. c. algirus
Uncorrected
Corrected
Uncorrected
Corrected
Da (%)
Da (%)
Da (%)
Divergence Time
(MY)
Da (%)
Divergence Time
(MY)
Phka2 5.719
8.483
c
1.153 2.38
1.263
c
1.76
Smcx 1.475
1.529
d
0.735 5.88
0.750
d
5.79
Msn 3.803
4.634
e
0.711 2.21
0.819
e
2.08
Hprt1 3.848
5.313
f
1.633 5.01
1.911
f
4.24
a
Appropriate models of nucleotide substitution to correct for multiple hits were selected for each
gene using MODELTEST 3.06 (P
OSADA and CRANDALL 1998) with the Akaike Information
Criterion (P
OSADA and BUCKLEY, 2004).
b
Pairwise distances (Dxy) per site were calculated using PAUP v 4.0 (SWOFFORD, 2002) with
locus specific estimated models of substitution. Net nucleotide divergences (Da) per site were
calculated as Dxy - 0.5 (Dx +Dy). Recombinant haplotypes were excluded from this analysis.
c
TAMURA-NEI model (1993) with proportion of invariable sites of 0.6578 and estimated
α parameter describing the gamma distribution of 1.0022.
d
TAMURA-NEI model (1993) with proportion of invariable sites of 0.5536.
e
Transversion Model with estimated α parameter describing the gamma distribution of 0.2204.
f
TAMURA-NEI model (1993) with proportion of invariable sites of 0.8524.

Geraldes et al. 2005
41
Figure legends
Figure 1 - Populations of European rabbit sampled and their geographic locations. Dark grey area
indicates SW populations, light grey indicates CZ populations and white indicates NE populations.
The name and number of samples from each population, and population names are specified in Table
1.
Figure 2 - Chromosomal location of the four X-linked loci used in the present study. Modified from
C
HANTRY-DARMON et al. 2003 and HAYES et al. 2002.
Figure 3- Polymorphisms for the 4 X-linked loci. a) Phka2, b) Smcx, c) Msn and d) Hprt1. The
nucleotide found in the Lepus granatensis sample was used to infer the ancestral state for each
position. Numbers in the columns indicate the position of the polymorphism in the alignment. Each
row represents one individual. A dot (.) represents the ancestral state, and letters (A, C, G, and T)
represent the derived nucleotide. The name of the haplotype defined by each sequence is shown in
parentheses. Positions polymorphic for bases other than the one found in Lepus granatensis are
indicated with an asterisk (*) and positions segregating for three different bases in O. cuniculus are
indicated with the symbol (#). Whenever a polymorphic site in O. cuniculus corresponded to a
deletion in L. granatensis we treated the most frequent base in our sample of O. cuniculus as the
ancestral state.
Figure 4- Median-Joining haplotype networks representing the phylogenetic relationships between all
the alleles found in the European rabbit. a) Phka2, b) Smcx, c) Msn and d) Hprt1. The size of the
circles is proportional to the frequency of each haplotype. The population group of the individuals
that are represented in each haplotype is denoted by black (SW), grey (CZ) and white (NE). The point
in the network from which the outgroup sequence of Lepus granatensis stems is represented by an
arrow. Haplotype IDs correspond to Figure 3.

Geraldes et al. 2005
42
Figure 1
14
1
2
3
5
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
25
24

Geraldes et al. 2005
43
Figure 2
S
m
cx
Msn
P
hk
a2
13
11
12
21.1
23.3
25
15
23.1
21.5
21.3
H
pr
t1

Geraldes et al. 2005
44
Figure 3 a)
1111111111111111111111111111111111111111112222222222222222222222222222222222222222222222222222222233333
11112222234444445556667777777888889999999990001111222333444455556666677788888888999990001111111122222333333444444555555556666677777788889999900011
1277902250117880155683663997778999235770333567781172355244568222403481227234123478881334869901266899011480245893477888122244691467724707811450277902704
617999086419833801724706336367307968246723491143076682967887~824965333127848462059023614722038570681880239538264191704928957937663592047959072707934732
* * *
L. granatensis ATCAGGCTGTGCAAGTGCATTCAGAGAATGCGGTAGGCTGTCCAGCGGGGTACAAGCGTCCTCCGGGGGCGACGAGGTTCCGCGACTGTGCGTTAAGGGGGCGGCGCTCGGGACTACCCGTGCAGAAGCCCACAGCTCAAGCGGCGGCGGG
Ver1827 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Vau1 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Cau19 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Pep18 (P2) ....C.....A........C.....A..C.....TA.....TGG.......G.C..T...T........T..T..AA.......T.......C.......C.......G........T..CA.C.G............GGA.......CA.
Zrg16 (P3) ..T.CA....A.......G..T...A..CA....TA.....TGG.....A.G.C....G.T.......AT..T..AA.......T...C...C.......C.AA................C..C.G..............A.......C..
Zrg20 (P4) ....CA....A.......G..T...A..CA....TA.....TGG.T.A.A.G.C....G.T.......AT..T..AA.......T.......C.G.....C..A................C.TC.G.............GA.......C..
Rsl4 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Rsl10 (P5) .C..C.....A..........T...A..CA....TA.....TGG...A.A.G.C.A..G.T...A...AT..T..AA.......T....A..C.G.....C..A................C.TC.G.....C..A....G........C..
Bnv3 (P6) ..T.CA....A....C.....T...A..CA....TA.....TGG...A.A.G.C....G.T.......AT..T..AA.....T.T.......CC......C..A..........A.....C..C.............T..A.......CA.
Zam1 (P7) .......C..................GGC.....C...G.....A...A.....G................TTAG..........TC........GA..AC...............G.T.C..T.G....T........GA....A..C..
Zam20 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Lrj3 (P8) ...GC.....A..............A..C...A.TA.....TGG.....A.G.C......T........T..TA.AA.......T.......C....A..C......C.......G....C..C.G.............GA..A...TC..
Lrj6 (P9) ..T.C.....AT...C.....T...A..CA....TA.....TGG...A.A.GGC....G.T.T.....AT..T..AA.......T.......CC......C..A................C..C...............GA.......C..
Mdr7 (P10) ...GC.....A..............A..C.....TA.....TGG.....A.G.C......T........T..TA.AA.......T.......C....AA.C......C.......G....C..C.G......T......GA..A...TC..
A
lt1 (P11) ..T.CAT...A..........T...A..CA..A.TA...ACTGG...A.ACG.C....G.T.......AT..T..AA.......T...C...C.......CGAA..T.............C..C.G.............GA.......C..
A
lt107 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
A
lt120 (P12) GC..C.....A..G...........A..C.....TA.....TGG.......G.C..T...T........T..T..AA....A..T.......C.......C.......G........T..CA.C.G............GGA.......CA.
Cat12 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Cue1 (P13) .C..C....CA.G........T...A..CA....TA.....TGG...A.A.G.C....G.T.......AT..T..AA.......T...C...C.......T..AT.T.............C..C.G.............GA...T...C.A
Cue3 (P14) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T..A.A..C....A..C......C...A...G....C..C.G...T.........GA..A....C..
Gal25C3 (P15) .......C..................GGC.TT..C...G.....A...A.....G....T.......A...TTAG..........TC........GA...C............T..G.T.C..T.G.............GA.......C..
Bra1 (P16) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C....G.T.......AT..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Bra13 (P1) ....C.....A.....C........A..C.....TA.....TGG.....A.G.C......T........T..T..AA.......T.......C....A..C......C.......G....C..CAG.............GA..A....C..
Tol25 (P6) ..T.CA....A....C.....T...A..CA....TA.....TGG...A.A.G.C....G.T.......AT..T..AA.....T.T.......CC......C..A..........A.....C..C.............T..A.......CA.
Tol50 (P17) ....C.....A...AC.....T...A..CA....TA.....TGG...A.A.G.C....G.T.......AT..T..AA......AT.......CC......C..A.A...........T.AC..C.G.............GA.......C..
Tol64 (P18) .......C..................GGC.....C...G.....A...A.....G................TTAG..........TC........GA..AC...............G.T.C..T.G....T........GA.......C..
Cre1 (P13) .C..C....CA.G........T...A..CA....TA.....TGG...A.A.G.C....G.T.......AT..T..AA.......T...C...C.......T..AT.T.............C..C.G.............GA...T...C.A
Vdm12 (P19) ..T.CA....A....C.....T...A..C.....TAA....TGG...A.A.G.C....G.T.T.....AT..T..AA.......T.......CC......C..A................C..C.........C.....GA.......C..
A
mo2 (P20) ..T.CA....A..........T...A..CA..A.TA...ACTGG...A.ACG.C....G.T.......AT..T..AA...T...T...C...C.......C.AA..T.............C..C.G.............GA.......C..
Luc4 (P21) ....C.....A.........C....A........TA.....TGG.....A.G.C......TC......AT..T..AA.......T.......C.......C........A..T.......C..C.G.............GA.A.....C..
Luc9 (P22) .C..C.....A......T....TA.A..C.....C..T...TGG.......G.C.................T.AG..........TC.............C.....T...A.....G.T.C..T.G.T..T........G.T......T..
Luc17 (P23) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T.......C....A..C......C.......G....C..C.G.............GA..A....C..
Pfr1 (P24) ....CA....A....C.....T...A..CA....TA.....TGG..AA.A.G.C....G.T.......AT..T..AA.......T.......C.......C..............G....CA.C.G.........T..GGAT......C..
Pfr5 (P25) .C..C.....A..........T...A..CA....T......TGG...A.A...C.A.AG.T..T.A...T..TA.AA.C.....T.......C.......C...................C..C.GT.T.......C..GA.......C..
Pfr7 (P26) ..T.CA....AT...C.....T...A..CA....TA.....TGG...A.A.G.C....G.T.T.....AT..T..AA.......T.......CC......C..A................C..C....T..........GA.......C..
Don6 (P25) .C..C.....A..........T...A..CA....T......TGG...A.A...C.A.AG.T..T.A...T..TA.AA.C.....T.......C.......C...................C..C.GT.T.......C..GA.......C..
Vrl1 (P27) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T.....T.C....A..C......C.......G....C..C.G.............GA..A....C..
Vrl4 (P27) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T.....T.C....A..C......C.......G....C..C.G.............GA..A....C..
Vrl7 (P18) .......C..................GGC.....C...G.....A...A.....G................TTAG..........TC........GA..AC...............G.T.C..T.G....T........GA.......C..
Id85 (P28) .C..C.....A..............A..C....GTA.....TGG.....A.G.C......T.......AT..TA..A..T....T.......C....A..C..............G....C..C.G.............GA..A....C..
Elv3 (P27) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T.....T.C....A..C......C.......G....C..C.G.............GA..A....C..
Elv6 (P27) ....C.....A..............A..C.....TAA....TGG.....A.G.C......T........T..TA.AA.......T.....T.C....A..C......C.......G....C..C.G.............GA..A....C..
VV1_1/94 (P29) ....C...A.A.............GA..C.....C..T...TGG.........C......T.....A..TA.T...AC......T......AC.......C...........T..........C.G.............GA.....A.C..

Geraldes et al. 2005
45
Figure 3 b)
111111111111111111111111111122222222222
12222444456666669999000000111122333344455566678901224455666
872578023630357992679044478111123478945806747992656074517577
791227657304237563981314743136867116226267558886109309012445
*
L. granatensis GTAGTGAGGAAGTGGTATCTAGGCCCGCGAGAGAGGCGCGGTACAGCAGACCGCCTTTCA
Ver1827 (S1) ...A..G.A...C..G.C.CG.AT..A.......C..C.A.CG.....A...........
Vau1 (S2) ...A..G.A...C..G.C.CG.AT..A.......C......CG.....A...........
Cau19 (S3) ...A..G.A...C..G.C.CG.AT..A.......C..C...CG.....A...........
Pep18 (S2) ...A..G.A...C..G.C.CG.AT..A.......C......CG.....A...........
Zrg16 (S4) ...A..G.....C..G.C.CG.AT..A.C.....C......................G..
Zrg20 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
Rsl4 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Rsl10 (S7) ...A..GA....C..G.C.CG.AT..A.......C.........................
Bnv3 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Zam1 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Zam20 (S8) ...A..G..G..C..G.C.CG.AT..A.......C.........................
Lrj3 (S9) ...A..G.....C..G.CTCG.AT..A.......C...............G.........
Lrj6 (S10) ...A..GA....C..G.C.CG.AT..A..TA...C...A.....................
Mdr7 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Alic1 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Alt107 (S2) ...A..G.A...C..G.C.CG.AT..A.......C......CG.....A...........
Alt120 (S11) ...A..G.....C..G.C.CGAAT..A.......C.........................
Cat12 (S3) ...A..G.A...C..G.C.CG.AT..A.......C..C...CG.....A...........
Cue1 (S12) ..C..AG....TCT.A........T..T....T.............AG.G....TCC...
Cue3 (S13) ..C..A.....TCTAA...........T....T..........T.A...G....TC....
Gal25c3 (S14) A.C..A.....TCT.A...........T....T............A...G....TC....
Bra1 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Bra13 (S6) ...A..G.....C..G.C.CG.AT..A.......C.........................
Tol25 (S15) ..C..A.....T.T.AG..........T....T............A...G....TC....
Tol50 (S16) ..C..A.....TCTAA...........T....T............A...G....TC....
ToL64 (S17) ..C..AG....TCT.A...........T....T...........C..G.G....TCC...
Cre1 (S15) ..C..A.....T.T.AG..........T....T............A...G....TC....
Vdm12 (S15) ..C..A.....T.T.AG..........T....T............A...G....TC....
Amo2 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
Luc4 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
Luc9 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
Luc17 (S18) ..C..AG....TCT.A...........T....T............A...G....TC....
Pfr1 (S18) ..C..AG....TCT.A...........T....T............A...G....TC....
Pfr5 (S16) ..C..A.....TCTAA...........T....T............A...G....TC....
Pfr7 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
Don6 (S19) ..C..A.....TCT.A...........T...GT...T........A...G.G..TC....
Vrl1 (S20) ..C..A.....TCTAA...........T....T............A...G...TTC....
Vrl4 (S16) ..C..A.....TCTAA...........T....T............A...G....TC....
Vrl7 (S21) .AC..AG....TCT.A...........T....T.......A....A...G..A.TC....
ID85 (S18) ..C..AG....TCT.A...........T....T............A...G....TC....
Elv3 (S22) ..C..A....GTCT.A...........T...GTT...........A...G.G..TC..AG
Elv6 (S5) ..C..A.....TCT.A...........T...GT............A...G.G..TC....
V
V
1
_
1/94 (S23) ..C.AAG....TCT.A........TT.T....T..A..........AG.G....TCC...

Geraldes et al. 2005
46
Figure 3 c)
11111111111111111111111122222222222222
112344566677889900012234445566777778889912333455567788
67228067002568463456714872693956566778990933344505770400
42461247745510585032319375099132613195582964905593460557
L. granatensis TCCGCACCCGGCAGTTCCGGCTCTTGCTAGTCGCATTTTTACTAGGACGGTACGGC
Ver1827 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Vau1 (M2) C...G..T.A.TTA...T.A..T...TC.AC...GC.C.C....CT....C..A..
Cau19 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Pep18 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Zrg16 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Zrg20 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Rsl4 (M3) C...G..T.A...A...T....T....C..C..TGC.C.C..C.CT....C..A.A
Rsl10 (M4) C...G....A...A...T....T....C..C..TGC.C.C..C.CT....C..A.A
Bnv3 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Zam1 (M5) C...G....A...A...T....T....C..C....C.C.C....CT.T..C..A..
Zam20 (M6) C...G..T.A...A...T....T...TC.AC...GC.CCC....CT....C..A..
Lrj3 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Lrj6 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Mdr7 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Alic1 (M7) C...G..TGA...A...T....T....C..C...GC.C.C....CT....C..A..
Alt107 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Alt120 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Cat12 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Cue1 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Cue3 (M8) C...G..T.AC..A...T....T...TC.AC...GC.C.C....CT....C..A..
Gal25c3 (M9) .T.A.G.........CA....G........C...G.C.....CC.....T......
Bra1 (M1) C...G..T.A...A...T....T...TC.AC...GC.C.C....CT....C..A..
Bra13 (M10) .T.A.G.........CA....G......G.C...G.C.....CC.....T......
Tol25 (M11) .T.A.G.........CA....G........C...G.C.....CC............
Tol50 (M12) .....G.........CA....G.A......C...G.C.....C.............
Tol64 (M9) .T.A.G.........CA....G........C...G.C.....CC.....T......
Cre1 (M13) .....G.....T...CA..A.G.........G..G.C...................
Vdm12 (M14) .....G.........CA....G........C...G......T..............
Amo2 (M15) C.T.G..T.A...A...T....T....C..C..TGC.C.C....CT....C..A..
Luc4 (M16) .....G.....T...CA..A.G..C......G..G.C...................
Luc9 (M16) .....G.....T...CA..A.G..C......G..G.C...................
Luc17 (M12) .....G.........CA....G.A......C...G.C.....C.............
Pfr1 (M17) .....GG....T...CA..A.G.........G..G.C...................
Pfr5 (M18) ...A.G.........CA....G........C.A.G.C...T.C.....C.......
Pfr7 (M19) .....G.........CA....G...A....C...G.C.........G....G..A.
Don6 (M20) .....G........ACA....G........C...G.C.....C.............
Vrl1 (M21) .T.A.G.........CA....G........C...G.C.....CC..G..T......
Vrl4 (M22) .....G.....T...CA.AA.G.........G..G.C.........G.....T...
Vrl7 (M21) .T.A.G.........CA....G........C...G.C.....CC..G..T......
Id85 (M23) .....G.....T...CA..A.G.........G..G.C...............T...
Elv3 (M21) .T.A.G.........CA....G........C...G.C.....CC..G..T......
Elv6 (M24) .....G.........CA....G........C...G.C.....C...G.........
VV1
_
1/94 (M25) .T.A.G.........CA...TG........C...G.C.....CC..G..T......

Geraldes et al. 2005
47
Figure 3 d)
111111111111111
11122222233334444556777777777777888888888888899999000012222333344
15734703488904490224374001122334458000133345889902355146712557445601
75321245157570474459160592837281907145818901190618112406290589160621
# * # #
L. granatensis GAACTCGGATAGCCTTCATCCCTCCAGGCGCTTGGAACCGAGCCCTGACGGTTCAAACAAGGAAAGGG
Ver1827 (H1) ......A.........T.G....AA......CCAT..A.A..TA....T.....GG....A...G...
Vau1 (H1) ......A.........T.G....AA......CCAT..A.A..TA....T.....GG....A...G...
Cau19 (H2) ......A.........T......AA......CCAT..A.A..TA....T.....GG....A...G...
Pep18 (H3) ........G...............A......CCAT..A.A..TA...GT......G..GT.....A..
Zrg16 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Zrg20 (H5) ........G...............A......CCAT..A.A..TA...GTA.....G..GT.....A..
Rsl4 (H6) ........G...............A..A..TCCAT..A.A..TA...GT......G..GT.....A..
Rsl10 (H6) ........G...............A..A..TCCAT..A.A..TA...GT......G..GT.....A..
Bnv3 (H7) ........G...............A......CCAT..A.A..TA...GT......G..GT...G.A..
Zam1 (H8) ......A...G.....T..T......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Zam20 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Lrj3 (H9) .......A.C.A...........AA......CCAT..A.A..TA...GT....TGG....A...G...
Lrj6 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Mdr7 (H10) ........G.............C.A......CCAT..A.A..TA...GT......G..GT.....A..
Alic1 (H11) .......A.C..A..GT......AA....A.CCAT..A.A..TA...GT.....G.....A...G...
Alt107 (H12) .......A.C.............AA....A.CCAT..A.A..TA...GT.....G..T..A...G...
Alt120 (H1) ......A.........T.G....AA......CCAT..A.A..TA....T.....GG....A...G...
Cat12 (H13) .......A.C..............A......CCAT..A.A..TA...GT......G..GT.....A..
Cue1 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Cue3 (H9) .......A.C.A...........AA......CCAT..A.A..TA...GT....TGG....A...G...
Gal25c3 (H14) .......A.C.............AA......CCAT..A.A..TA...GTA....G.....A...G...
Bra1 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Bra13 (H15) ...T..A......T..TTG.......A.T..CG.T....T....TC.G...GG.GG....A...G...
Tol25 (H16) ......A.........T.G..T...GA.T...G.T...T.....TCAG...GG.GG..GT.....A..
Tol50 (H17) ........G...............A......CCAT..A.A...A...GT......G..GT.A...A..
Tol64 (H18) ........GC......T.G.....A......CCAT..A.AG.TA...GT......G..GT.....A..
Cre1 (H19) ......A.........T.G.T....GA.T...G.T.........TC.G...GG.GG..GT.....A..
Vdm12 (H19) ......A.........T.G.T....GA.T...G.T.........TC.G...GG.GG..GT.....A..
Amo2 (H20) ....C.....G.....T.G.......A.T...G.TC........TC.G...GG.GG..GT.....A.A
Luc4 (H21) A.C.............T.G.....A.A.T..CG.T....T.A..TC.G...GG.GG..GT.....A..
Luc9 (H22) ........G..............AA....A.CCAT..A.A..TA...GT.....G.....A...G...
Luc17 (H4) ......A...G.....T.GT......A.T..CG..C........TC.G..TGG.GG..GT.....AAA
Pfr1 (H23) .G......G...............A......CCAT..A.A..TA...GT......G..GT..T..A..
Pfr5 (H5) ........G...............A......CCAT..A.A..TA...GTA.....G..GT.....A..
Pfr7 (H24) ...T..A......T..TTG.......A.T..CG.T.T..T....TC.G...GG.GG....A...G...
Don6 (H25) .....T.A.C.A............A......CCAT..A.A..TA...GT.....GGG...A...G...
Vrl1 (H14) .......A.C.............AA....C.CCAT..A.A..TA...GTA....G.....A...G...
Vrl4 (H15) ...T..A......T..TTG.......A.T..CG.T....T....TC.G...GG.GG....A...G...
Vrl7 (H14) .......A.C.............AA....C.CCAT..A.A..TA...GTA....G.....A...G...
Id85 (H26) .............T..TTG.......A.T..CG.T....T....TC.G...GG.GG..GT.....A..
Elv3 (H27) ........G.....A.........A......CCAT..A.A..TA...GT......G..GT.....A..
Elv6 (H28) ...T.........T..TTG.......A.T..CG.T.T..T....AC.G...GG.GG....A...G...
VV1
_
1/94 (H4) ......A...G.....T.GT......A.TCGCC..GCA.CGAG...CGGC.GG.GG..GT.....
A
A
A

Geraldes et al. 2005
48
Figure 4 a)
Lepus granatensisLepus granatensis

Geraldes et al. 2005
49
Figure 4 b)
Lepus
granat ensis
Lepus
granat ensis

Geraldes et al. 2005
50
Figure 4 c)
Lepus granatensisLepus granatensis

Geraldes et al. 2005
51
Figure 4 d)
Lepus granatensisLepus granatensis

Geraldes et al. 2005
52
Supplementary Table 1- X-chromosome loci details, PCR and Sequencing Primers
a
Primer no. Primer name Type
b
Primer sequence (5'-3') Temp.
c
1 Phka2e2f Pcr CCTGGGTGCGAGACAACATCTACAG
2 Phka2e4r Pcr CCAGGAACAGGAGAAAGAGGGAGGT
66º
3 NPhka2e2f NPcr CATGGCCTACCGCAAGAATG
4 NPhka2e4r NPcr GCAGGTGGCAGTGTTGTACTT
62º
5 P730r S TTTCGAGGCTCCCTACTTCA
6 P640f S GATTCCCACCAAAGCAAAGA
7 P1900f S ACCTCCTCCCCTCAACCTTA
8 P2015r S TGTGGAAAACACAACCTGGA
9 P2630f S TAGAGGGTCACAGTCTCGGG
10 SPhka2_e2f2
S TCAGAAGCCAAGATTCCCAC
11 Sphka2_e3r S CCTCATCATGCACTGGAGAA
12 Sphka2_e3f S GAAGCTGATGCGAGGTCTTC
13 Sphka2_e3r2 S GCACATTACTTCCCGTGTCC
14 Sphkf S ACGAACACCTCACCTGCTTAT
15 Slehf S GCCATCCTCCACTGCACTC
16 SP2919r S ATCAGTGTGTTCTTTGCCCC
17 SP851f S TGTCAGGTCTAGGAAGAGT
18 LP2000r S CTCCTGCACCAGCCCGCC
19 SP1410r S TCACTGGGAGATGGCCACGT
20 Smcxe2f Pcr GGACTGGCAGCCACCCTTTGCTGTG
21 Smcxe4r Pcr CACAAGGTTGGCTCCAGACTGGTACA
64º
22 NSmcxe2f NPcr/S
GTAACAGGGTTGGGAAAACGGATACC
23 Nsmcxe4r NPcr/S
TGTTTTGCCTGGTGGATAGTTGAGG
62º
24 SSmcxf2 S GGGAAGTGAGAGTGAGTGCC
25 SS1580r S TTCACTTCCTCATGCACCAG

Geraldes et al. 2005
53
26 Ssmcxr2 S TCTCCAAATGAACCGAAACC
27 Msne4f2 Pcr CCCAGCGCCTGTTCTTTCTGCAAG
28 Msne7r3 Pcr/S CCTGATTTCACTCCAAGGGAAGCC
62º
29 M105f NPcr GGTCTGTGCCTCTCCATTAGGG
30 M3226r NPcr/S
TGGTTAGTTGCCAACACTGAGGG
62º
31 M847r S ACCCAGATCTACTACACTGT
32 M769f S TAGGGACAGTAAGCTGTGC
33 M1513f S ACAGAGATGATGCTGGCACT
34 M2099f S TATCTGAAGATTGCTCAAGA
35 M3300r S AATAGAATAGAGTAAGAGT
36 Msne4f3 S TCAACAAGGAAGTGCACAAGTGTGG
37 Msne5f1 S CACAAACTCAACAAGGACCAGTGGG
38 Msne6r1 S ATTCTGCTCATAGATGTTGAGACCC
39 Smsnf S AGTGAACTACTTCAGCATTA
40 Smsnr S AGTGCCAGCATCATCTCTGT
41 Hprt11e2f Pcr GCGATGATGAACCAGGTTATGACC
42 Hprt11e3r Pcr TAGCTCTTCAGTCTGATAAAATCTACAG
64º
43 NpHprt1_f NPcr/S
ATTACGTCGAGGACTTGGAAAGG
44 NpHprt1_r NPcr/S
AGCAGGTCAGCAAAGAACTTATAGCC
68º
45 Hprt1r2 S GGATTCTAGTCCCGGTTGCT
46 Sh654r S TCCAATGAATGTTAGAACTA
47 Sh520f S CTCTCCTCAGCTTCCTGTA
a
Primers 1 through 19 were used for the region between exons two and four of Phka2
[Phosphorylase kinase alpha 2 (liver)] located on O. cuniculus Xp15 (C
HANTRY-DARMON et al.
2003). Primers 20-26 were used for the region between exons two and four of Smcx [Smcy
homolog, X-linked (mouse)] located on O. cuniculus Xp11 (C
HANTRY-DARMON et al. 2003).

Geraldes et al. 2005
54
Primers 27 through 40 were used in the study of the region between exons six and nine of Msn
(Moesin) located on O. cuniculus Xq12prox (C
HANTRY-DARMON et al. 2003). For this gene the
number of exons in the three species compared is not the same. The primers used for the
amplifications are in exons six and nine of humans, which correspond to exons three and six in
the house mouse and exons four and seven in the rat. Finally, primers 41 through 47 were used
for the region between exons two and three of Hprt1 [Hypoxanthine phosphoribosyltransferase 1
(Lesch-Nyhan syndrome)] located on O. cuniculus Xq23 (H
AYES et al. 2002).
b
Purpose for which the primers were used: PCR- primers used for PCR, NPCR- primers used for
Nested PCR and S- primers used for most sequencing reactions, SA- alternative sequencing
primers used to sequence individuals in which the S primers did not work.
c
Annealing temperature used in the respective PCR or Nested PCR reactions.