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Hybrid speciation through sorting of parentalincompatibilities in Italian sparrows
JO S. HERMANSEN,* FREDRIK HAAS,* 1 CASSANDRA N. TRIER,*1 RICHARD I. BAILEY,*
ALEXANDER J . NEDERBRAGT,* ALFONSO MARZAL† and GLENN-PETER SÆ TRE*
*Department of Biosciences, Centre for Ecological and Evolutionary Synthesis, University of Oslo, PO Box 1066, Blindern,
N-0316 Oslo, Norway, †Department of Zoology, University of Extremadura, ES-06071 Badajoz, Spain
Abstract
Speciation by hybridization is emerging as a significant contributor to biological
diversification. Yet, little is known about the relative contributions of (i) evolutionary
novelty and (ii) sorting of pre-existing parental incompatibilities to the build-up of
reproductive isolation under this mode of speciation. Few studies have addressed
empirically whether hybrid animal taxa are intrinsically isolated from their parents,
and no study has so far investigated by which of the two aforementioned routes intrin-
sic barriers evolve. Here, we show that sorting of pre-existing parental incompatibili-
ties contributes to intrinsic isolation of a hybrid animal taxon. Using a genomic cline
framework, we demonstrate that the sex-linked and mitonuclear incompatibilities iso-
lating the homoploid hybrid Italian sparrow at its two geographically separated
hybrid–parent boundaries represent a subset of those contributing to reproductive iso-
lation between its parent species, house and Spanish sparrows. Should such a sorting
mechanism prove to be pervasive, the circumstances promoting homoploid hybrid spe-
ciation may be broader than currently thought, and indeed, there may be many cryptic
hybrid taxa separated from their parent species by sorted, inherited incompatibilities.
Keywords: birds, hybridization, introgression, Passer, reproductive barriers, speciation
Received 28 May 2014; revision received 29 August 2014; accepted 1 September 2014
Introduction
Natural hybridization is increasingly recognized as an
important contributor to biological diversification
(Abbott et al. 2013; Seehausen et al. 2014). Existing taxa
exchange adaptations through introgression (Heliconius
Genome Consortium 2012) and new species form—not
only in the face of gene flow (Nosil 2012), but also as a
direct consequence of interbreeding (Rieseberg 1997;
Mallet 2007; Rieseberg & Willis 2007; Mavarez & Lin-
ares 2008; Abbott & Rieseberg 2012; Abbott et al. 2013).
The latter process, known as hybrid speciation, encom-
passes the origin of genetically admixed taxa that are
reproductively isolated from their parents. The develop-
ment of reproductive barriers plays a central role in
hybrid speciation, yet the nature and development of
such barriers remain poorly understood (Rieseberg
1997; Mallet 2007; Abbott et al. 2013; Schumer et al.
2014). An important task in hybrid speciation research
is therefore to unveil the evolution of the barriers that
separate hybrid taxa from their parent species.
Both pre- and postzygotic barriers have been shown
to act between hybrid taxa and their parents (Rieseberg
1997; Mallet 2007; Mavarez & Linares 2008; Abbott et al.
2013). A recent survey of the topic suggested that eco-
logical divergence and geographic isolation are likely to
be of greater importance for the establishment of hybrid
taxa than are intrinsic postzygotic barriers, especially in
animals (Abbott & Rieseberg 2012). The evolution of
intrinsic reproductive isolation between hybrids and
parents is poorly studied and understood, however
(Buerkle et al. 2000; Coyne & Orr 2004; Jiggins et al.
2008; Schumer et al. 2014). The only direct empirical evi-
dence comes from the Helianthus sunflower system,
Correspondence: Glenn-Peter Sætre, Fax: +47 22854001;
E-mail: [email protected] authors contributed equally to this work.
© 2014 John Wiley & Sons Ltd
Molecular Ecology (2014) 23, 5831–5842 doi: 10.1111/mec.12910
where intrinsic isolation seems to result from a mixture
of sorting of pre-existing chromosomal structural differ-
ences and structural rearrangements induced by recom-
bination (Rieseberg et al. 1995, 2003; Lai et al. 2005). To
what extent intrinsic barriers separating hybrids from
their parents develop through sorting of pre-existing
parental incompatibilities or through de-novo epistatic
interactions in the hybrid genomes therefore remains an
open question. This question can, however–as sug-
gested by Rieseberg (1997)–be addressed by comparing
the locations of genomic regions contributing to intrin-
sic reproductive barriers between the parent species
with those isolating the hybrid species from their par-
ents. If the genomic regions contributing to reproduc-
tive isolation in a hybrid taxon are a subset of those
acting between its parents, then the sorting hypothesis
would be supported.
Here, we apply the approach suggested by Rieseberg
(1997) to investigate the evolution of intrinsic barriers
during homoploid hybrid speciation in Passer sparrows.
In this species complex, interbreeding between house
P. domesticus and Spanish sparrows P. hispaniolensis has
resulted in a taxon of hybrid origin, the Italian sparrow
P. italiae, which genetically and phenotypically is a mix-
ture of its parent species (Meise 1936; Elgvin et al. 2011;
Hermansen et al. 2011; Trier et al. 2014). The Italian spar-
row is a small seed-eating bird occupying a human-
commensal niche similar to that of the house sparrow
(Summers-Smith 1988). It is distributed over the entire
Italian Peninsula and meets the house sparrow in a nar-
row hybrid zone in the Alps. It lives sympatrically with
the Spanish sparrow in restricted areas of mainland Italy
(Summers-Smith 1988; Hermansen et al. 2011) and
exchanges migrants with Spanish sparrows from Sardi-
nia (Trier et al. 2014). The Italian sparrow’s parent spe-
cies are themselves broadly sympatric across large parts
of the Spanish sparrow range, maintaining phenotypic
distinctness in all but a few locations (Summers-Smith
1988). This demonstrates the existence of reproductive
barriers typically isolating house and Spanish sparrows.
Previous work has shown that the Italian sparrow
has developed reproductive barriers against both parent
species (Hermansen et al. 2011; Trier et al. 2014). Evi-
dence suggests that intrinsic barriers consist mainly of
sex-linked and mitonuclear incompatibilities (Trier et al.
2014). A set of seven candidate reproductive isolation
(RI) loci have been identified, three of which exhibit
steep clines at the Italian-house sparrow boundary and
four which exhibit steep clines at the Italian–Spanish
sparrow boundary. Among these candidate RI loci, sex
chromosome (Z)-linkage is overrepresented, and the
patterns of variation suggest a role for mitonuclear con-
flict in isolating Italian and Spanish sparrows (Trier
et al. 2014).
Here, we take the next step in the study of reproduc-
tive isolation in the Passer system and investigate
whether these intrinsic barriers have formed through
sorting of pre-existing incompatibilities or through
de-novo epistatic interactions in the hybrid genome. We
accomplish this by replicating the hybrid–parents study
of Trier et al. (2014) in an area of parental species symp-
atry and hybridization on the Iberian Peninsula.
Materials and methods
Focal population
Our focal population is situated on the Iberian Penin-
sula, close to Badajoz, Extremadura, Spain (38.64866N,
�7.215453E, Fig. 1). This is an area where house and
Spanish sparrows occur sympatrically, including breed-
ing in the same stork nests. As in our previous hybrid–
parents study (Trier et al. 2014), only male individuals,
which are diploid for the Z chromosome, were analysed
to avoid issues related to haplodiploidy of the Z chro-
mosome. One hundred and sixty-three males (pheno-
typic house sparrows: N = 76, phenotypic Spanish
sparrows: N = 87) were caught using mist nets, and a
blood sample (20–50 lL) for use in genetic analyses was
taken from each individual by puncturing a brachial
vein. DNA was extracted from blood samples stored in
standard buffer using Qiagen DNeasy 96 Blood and Tis-
sue Kits (Qiagen N.V., Venlo, the Netherlands) accord-
ing to the manufacturer’s instructions with the minor
adjustment of adding 100 lL of blood/buffer in the ini-
tial step. Authorization to catch birds and take blood
samples was obtained from the appropriate authorities,
and all birds were released upon finished sampling.
Reference populations
As reference house sparrows, we used previously pub-
lished data from two allopatric populations; Oslo, Nor-
way (N = 58) and Hradec Kr�alov�e, Czech Republic
(N = 27) (Fig. 1; Trier et al. 2014). Population genetic
analyses have shown that the house sparrow exhibits
very little geographic structuring genetically (Sætre
et al. 2012). This is likely a result of a recent, massive
range and population expansion associated with the
sparrows’ recent adaptation to human commensalism in
connection with the rise and spread of agriculture
(Sætre et al. 2012). House sparrows from Norway and
Czech Republic are therefore suitable allopatric refer-
ence populations for use in comparative analyses with
the sympatric area on the Iberian Peninsula.
As reference Spanish sparrows, we used a population
on the Gargano Peninsula in east-central Italy (N = 52)
(Fig. 1). This reference population was chosen based on
© 2014 John Wiley & Sons Ltd
5832 J . S . HERMANSEN ET AL.
an assessment of hybrid indices from our available col-
lection of samples. This assessment indicated that these
birds, albeit locally sympatric with Italian sparrows, are
genetically pure Spanish sparrows and hence should
give good estimates of pure Spanish sparrow allele fre-
quencies. In contrast, allopatric Spanish sparrows from
Sardinia show evidence of some gene exchange with
mainland Italian sparrows, through episodes of migra-
tion (Trier et al. 2014; see also Fig. 2A).
Hybrid–parent comparison
To obtain a direct comparison with hybrid–parent
boundaries, we reanalysed the data set utilized by Trier
et al. (2014) using the exact same marker set and paren-
tal reference populations as used when analysing the
focal sympatric parental population. The hybrid–parents
data set consists of 57 populations of Italian sparrows,
Italian-house hybrids and parapatric house sparrows
from the Italian peninsula and the Alps (N = 385), as
well as parapatric Spanish sparrows from Sardinia
(N = 48).
Marker set
We utilized 77 species-informative SNP markers (Table
S1, Supporting information) from 75 genes developed
through 454 transcriptome sequencing of cDNA from
heart, liver and brain tissues (described in Trier et al.
2014). To obtain species-informative SNPs from 454
transcriptome data of six house and six Spanish spar-
rows lacking reference genomes, we mapped against
the most closely related reference genome available,
that of the zebra finch Taeniopygia guttata (Warren et al.
2010). First, we mapped all cDNA reads from the house
sparrows against the zebra finch transcriptome and
used the consensus called on the mapped reads as ref-
erence for mapping all cDNA reads from both house
and Spanish sparrows. Then, we singled out the bases
where all reads from one species exhibited a different
House sparrowPasser domesticus
Italian sparrowPasser italiae
Spanish sparrowPasser hispaniolensis
Oslo
Sardinia
Gargano
Badajoz (3)
Hradec Králove (2)
(1)
(4)
(5)
Fig. 1 Geographic distribution of the studied Passer taxa. Grey indicates the distribution of the house sparrow, orange indicates the
distribution of the Italian sparrow, and red indicates the distribution of the Spanish sparrow. Hatched areas indicate regions where
the house sparrow and Spanish sparrow (red-grey) or Italian sparrow and Spanish sparrow (red-orange) distribution overlap. Black
dots refer to populations presented in Fig. 2A. Bird drawings indicate species-specific male plumage characteristics of the house
sparrow, Italian sparrow and Spanish sparrow.
© 2014 John Wiley & Sons Ltd
HYBRID SPECIATION BY SORTING OF INCOMPATIBILITIES 5833
base than that of the other species, that is putatively spe-
cies-diagnostic SNPs. These steps were repeated a
second time starting with the cDNA reads from the
Spanish sparrows (for detailed description of the
species-diagnostic SNP detection method, see
https://github.com/lexnederbragt/454_transcriptome_snps).
Based on the species-diagnostic SNPs from the 12 sam-
ples, multiplex sets of PCR primers were designed and
all genotyped at each of 77 SNP loci (with the exception
of five loci where genotyping failed for 44 of the Span-
ish sparrow reference individuals, see Table S1, Sup-
porting information) using the Sequenom MassARRAY
platform at CIGENE, Norwegian University of Life Sci-
ences, �As, Norway. Genes were annotated by blasting
against the zebra finch and chicken Gallus gallus (Inter-
national Chicken Genome Sequencing Consortium 2004)
genomes. Two exceptions were SNPs within CHD1Z
and ND2 genes, which were genotyped using existing
primers (see Elgvin et al. 2011). The CHD1Z SNP in this
set is located within an intron.
We note that the six Spanish sparrows used for
transcriptome sequencing and SNP detection were
sampled from the same sympatric locality on the
Iberian Peninsula as the focal sparrows in this study,
whereas the six house sparrows used for transcriptome
sequencing and SNP detection were sampled in Oslo,
Norway. Thus, all else being equal, one might expect to
observe more introgression into house sparrows than
Spanish sparrows in the area of parental sympatry, as
the house sparrows in the parental sympatric popula-
tion were not among the populations used for identifi-
cation of species-informative markers. This potential
problem should not, however, adversely affect the com-
parison of locus-specific introgression between hybrid–
parents and parent–parent systems, that is the compara-
tive genomic clines analyses (see below).
Comparative investigation of introgression patterns
We used Bayesian admixture analysis as implemented
in STRUCTURE (Pritchard et al. 2000; Falush et al. 2003)
to compare the genetic makeup of individuals in the
focal sympatric area in Spain (house: N = 76, Spanish:
N = 87) to that of other populations of house and Span-
ish sparrows and hence to assess patterns of introgres-
sion. As representative allopatric house sparrow
(A)
(B) (C)
Fig. 2 Genetic composition of house and Spanish sparrows in parental sympatry. (A) Comparison of house and Spanish sparrow
genetic makeup using STRUCTURE. Each bar represents one individual, and the coloration of a bar represents the relative house
(white) and Spanish (black) genomic contribution, that is its admixture proportion. Numbers in lower left corners indicate sampling
localities shown in Fig. 1 except 6, which indicates F1 house X Spanish sparrow hybrids from aviary crosses. (B) BGC generated
Bayesian estimates of hybrid index (HI) for each individual in the focal sympatric population near Badajoz, Spain. HI estimates are
presented in ascending order, and 0 denotes pure house sparrow, whereas 1 denotes pure Spanish sparrow. The point estimates are
based on the median from the posterior distribution, and black lines indicate 95% credibility intervals. Colour of dots indicates the
species origin of the mitochondrial DNA of each individual; blue denoting house sparrow and red denoting Spanish sparrow origin.
(C) Frequency distribution of HI in parental sympatry.
© 2014 John Wiley & Sons Ltd
5834 J . S . HERMANSEN ET AL.
populations, birds from Oslo, Norway (N = 58) and
Hradec Kr�alov�e, Czech Republic (N = 27) were used.
As representative Spanish sparrow populations, birds
from the Gargano Peninsula, Italy (N = 52) and Sardi-
nia, Italy (N = 48) were used. Known F1 aviary hybrids
between house (Oslo, Norway) and Spanish (Badajoz,
Spain) sparrows (N = 6) were also included to verify
that STRUCTURE assigned admixed individuals cor-
rectly. In STRUCTURE, the admixture model was used
and we assumed two groups (K = 2) with correlated
allele frequencies and ran 1 000 000 iterations after a
burn-in of 500 000 iterations.
Detection of SNPs associated with reproductiveisolation
To test for loci potentially associated with reproductive
isolation, we applied a genomic cline approach (Payseur
2010; Gompert & Buerkle 2011; Fitzpatrick 2013). This
approach takes advantage of the fact that introgression
can vary across the genome due to recombination in
admixed individuals. Genomic cline analysis quantifies
locus-specific introgression relative to a genome-wide
average as estimated by hybrid index or average gen-
ome-wide ancestry. We used genomic cline analysis to
identify loci exhibiting restricted introgression relative
to the genome-wide average, as such loci are candidates
for being associated with reproductive isolation. The
basic assumption is that alleles at loci responsible for
barriers to gene flow and neutral loci linked to such
selected loci will exhibit reduced introgression into the
foreign genomic background, due to selection against
unfit admixed individuals. This will result in steep and
possibly shifted genomic clines. By contrast, gene flow
at unlinked, neutral loci is not expected to be influenced
by such barriers. Neutral loci therefore exhibit shal-
lower clines that do not change more sharply with
changing hybrid index than a neutral expectation.
We used genomic cline analysis as implemented in
the BGC software (Gompert & Buerkle 2012). This
approach allows for the use of markers that are not
fixed in each parent species. BGC uses Markov Chain
Monte Carlo (MCMC) methods to obtain Bayesian esti-
mates of two parameters that describe the bias and rate
of locus-specific introgression into foreign genomic
background, based on ancestry (Gompert & Buerkle
2011, 2012). For a given locus, cline parameter a speci-
fies the probability of ancestry from one of the parental
species where an increase in probability produces a
positive parameter value and a decrease, a negative
parameter value. Cline parameter b, on the other hand,
specifies the rate of transition from low to high
probability of ancestry from the same parental species
as a function of hybrid index, with an increased rate
yielding a positive parameter value and a decreased
rate, a negative parameter value (Gompert & Buerkle
2012). a is therefore analogous to cline centre in geo-
graphic cline analysis, whereas b is analogous to cline
steepness. Locus-specific introgression differs from the
genome-wide average when credibility intervals for the
posterior probability distributions of a and b do not
overlap with zero (Gompert & Buerkle 2011, 2012).
As parental reference populations in the BGC analy-
ses, and hence to indicate parental allele frequencies,
we used house sparrows from Oslo, Norway (N = 58)
and Hradec Kr�alov�e, Czech Republic (N = 27), and
Spanish sparrows from the Gargano Peninsula, Italy
(N = 52). For the BGC analyses in parental sympatry,
all individuals from the sympatric parental population
were pooled into one admixed test population. Simi-
larly, for the reanalysis of the hybrid–parents data set,
all individuals from the Alps, the Italian peninsula
(excluding Spanish sparrows from the south-east Italian
sympatric zone), Sicily and Sardinia were pooled into
one admixed test population.
Genotypes at the mitochondrial locus ND2 were
coded as diploid homozygotes as BGC failed to run
with a haploid marker included. Ten independent runs
of 100 000 iterations each were run with the first 25 000
iterations discarded as burn-in, MCMC samples thinned
by recording every fifth value, while the rest of the
BGC settings were as default. We verified that the runs
gave qualitatively similar results, and for each system,
we present the results from the run with the best fit,
that is the run with the lowest mean negative log-likeli-
hood. SNPs were identified as significantly deviating
from genome-wide expectations when the 95% credibil-
ity intervals of the cline parameters a and b did not
cross zero. We also estimated the quantiles of the cline
parameters in the genome-wide distribution of intro-
gression, namely qa and qb. These quantiles represent
the position of each SNP on the posterior probability
distribution of the genome-wide average for each of aand b, respectively.The genomic cline model implemented in BGC
assumes no drift and that all loci have the same effec-
tive population size. As Z-linked loci have a lower
effective population size than autosomal markers (3/4),
this could lead to an overrepresentation of Z linkage of
steep clines simply as an effect of stronger drift at these
loci. To test for this, we also analysed the Z-linked and
autosomal loci in parental sympatry separately using
the same procedure as when analysing the full data set.
To compare the patterns of introgression between the
parent species in sympatry on the Iberian peninsula
with the introgression patterns between the Italian spar-
row and its parent species in Italy, we investigated
which markers exhibited significant excess ancestry
© 2014 John Wiley & Sons Ltd
HYBRID SPECIATION BY SORTING OF INCOMPATIBILITIES 5835
(significant a parameter) and/or steep clines (signifi-
cant, positive b parameter) in either or both systems.
We also estimated Pearson’s product–moment correla-
tion coefficients among systems for both cline parame-
ters (a and b) for the autosomal markers, for the
Z-linked markers, for the candidate intraspecific incom-
patibilities in the Italian sparrow and for the putative
reproductive isolation markers (see below). The correla-
tions were performed on point estimates based on the
median of the posterior distribution for a and b. Finally,we plotted the quantiles of the cline parameters in the
genome-wide distribution of introgression, qa and qb,to visualize where in the genome-wide distribution of
locus-specific introgression, the markers were situated.
Results
Patterns of introgression in parental sympatry
Our Bayesian admixture analysis revealed a pattern of
asymmetric introgression in the area of parental sympa-
try. The majority of individuals with house sparrow
phenotype showed signs of introgression when com-
pared to allopatric house sparrow populations (Fig. 2A).
Spanish sparrows, on the other hand, did not exhibit
signs of extensive introgression. They showed less intro-
gression than the insular Spanish sparrow population
on the Mediterranean island of Sardinia previously
shown to undergo gene exchange with Italian sparrow
migrants from mainland Italy (Fig. 2A; Trier et al. 2014).
With the aforementioned SNP ascertainment bias caveat
in mind, however, this apparent asymmetry in intro-
gression must be interpreted cautiously as it may to
some degree reflect an artefact of the SNP detection
strategy used in this study. Nonetheless, the relative
difference in introgression between allopatric and sym-
patric house sparrow populations as evident in Fig. 2A
is a signal of significant ongoing introgression in symp-
atry. Moreover, the distribution of hybrid indices esti-
mated using the Bayesian algorithm implemented in
BGC provided an informative basis for investigating
locus-specific introgression using genomic clines analy-
sis (Fig. 2B, C).
Genomic clines analysis in parental sympatry
Ten SNPs exhibited significant excess house sparrow
ancestry (negative a), whereas eight SNPs exhibited
excess Spanish sparrow ancestry (positive a) in parental
sympatry (Fig. S1, Supporting information). This indi-
cates that locus-specific introgression does not show the
same overall bias towards introgression into house
sparrows as previously described for admixture propor-
tion results from STRUCTURE. Furthermore, 19 SNPs
exhibited significantly steep clines (positive b), indicat-ing reduced introgression and hence a potential associa-
tion with reproductive isolation, whereas 10 SNPs
exhibited significantly shallow clines (negative b) (Fig.
S1, Supporting information). Of the 19 loci exhibiting
steeper clines than expected given the marker set aver-
age, 15 were Z-linked: a significant overrepresentation
of sex-linkage compared to the genome-wide expecta-
tion from other passerine birds (two-tailed binomial
test: null probability based on flycatcher genome (Elle-
gren et al. 2012) = 0.066, successes = 15, trials = 19,
P = 5.90 9 10�15). This remains a significant overrepre-
sentation of sex-linkage also when compared to the
proportion of sex-linked markers in our marker set
(Two-tailed binomial test: null probability based on pro-
portion of sex-linked marker in marker set = 0.273, suc-
cesses = 15, trials = 19, P = 4.10 9 10�6). One could
argue that this overrepresentation of Z-linked loci may
result from stronger drift due to lower effective popula-
tion size of Z-linked loci compared to autosomal loci,
and we cannot exclude the possibility that differences
in effective population size between Z and autosomes
may contribute to the observed pattern. However, our
independent analyses of Z-linked and autosomal loci
gave qualitatively the same results for the b parameter
as when all markers were analysed together (Z-linked
loci: N = 21, r = 0.964, P = 2.15 9 10�12, autosomal loci:
N = 55, r = 0954, P = 2.20.0 9 10�16, Fig. S2, Supporting
information). This indicates that there is a real overrep-
resentation of Z linkage among the candidate RI loci.
Genomic clines reanalysis of hybrid–parent data set
In our reanalysis of the hybrid–parents data set using a
different, larger, parental Spanish reference set than
previously, 26 SNPs exhibited significant excess house
sparrow ancestry, whereas 29 SNPs exhibited excess
Spanish sparrow ancestry. Furthermore, 13 SNPs exhib-
ited significantly steep clines, whereas 17 SNPs exhib-
ited significantly shallow clines (Fig. S3, Supporting
information). All Z-linked loci exhibiting steep clines at
the hybrid–parent range boundaries in Trier et al. (2014)
also exhibited significantly steep and shifted genomic
clines when reanalysing the hybrid–parent data set
(Figs 3 and 4; Fig. S3, Supporting information). That is,
the Z-linked candidate reproductive isolation loci
remained unchanged: CETN3 and CHD1Z at the Ital-
ian/house boundary and HSDL2, MCCC2 and GTF2H2
at the Italian/Spanish boundary. Autosomal candidate
marker RPS4 did not exhibit a significantly steep cline
in the reanalysis, however, but was the marker exhibit-
ing the strongest excess Spanish sparrow ancestry
(Figs 3 and 4; Fig. S3, Table S2, Supporting informa-
tion). In our previous study of hybrid–parent barriers,
© 2014 John Wiley & Sons Ltd
5836 J . S . HERMANSEN ET AL.
(A) (B)
(C) (D)
Fig. 3 Comparison of locus-specific patterns of introgression between parental sympatry and hybrid–parent range boundaries. Dis-
played are cline estimates for the 77 markers used in our BGC analyses. Each symbol denotes the point estimate of the given parame-
ter based on the median of the posterior distribution. White symbols denote autosomal loci, black symbols denote Z-linked loci, grey
symbols denote putative internal incompatibilities in the Italian sparrow, red symbols denote the putative nuclear reproductive isola-
tion genes situated at hybrid–parent range boundaries, and the yellow symbol denotes mitochondrial marker ND2. The shape of the
symbol denotes the significance level in each comparison based on 95% credibility intervals of the posterior distribution. Squares
denote comparisons where the parameter estimates are significant in both parental sympatry and in the hybrid–parents analysis, tri-
angles denote comparisons where the parameter estimates are significant only in parental sympatry, diamonds denote comparisons
where estimates are significant only in the hybrid–parents analysis, circles denote comparisons where the parameter estimates do not
deviate from neutral expectations in any of the two systems, and finally, crosses denote where the estimates are significant in both
systems but in opposite direction. Numbers refer to the different putative RI markers as specified in panel A. In all panels, parame-
ters from Italy (hybrid–parents) are plotted against parameters from Spain (parental sympatry) (A) a (bias) parameter: positive and
negative values indicate bias in favour of Spanish sparrow and house sparrow alleles, respectively. Note different scale on axes. (B)
b (rate) parameter: positive values indicate restricted introgression, and negative values indicate elevated introgression. Note differ-
ent scale on axes. (C) qa parameter: values closer to 0 and 1 indicate increasing evidence for excess house or Spanish sparrows ances-
try, respectively. (D) qb parameter: values closer to 0 and 1 indicate increasing evidence for shallow and steep clines, respectively.
© 2014 John Wiley & Sons Ltd
HYBRID SPECIATION BY SORTING OF INCOMPATIBILITIES 5837
FST outlier analysis revealed RPS4 to be the strongest
candidate for being under directional selection in the
Italian-house hybrid zone in the Alps (Trier et al. 2014).
Moreover, analysis of spatial genetic variation using
GENELAND (Guillot et al. 2005a,b) showed that RPS4
exhibited a sharp shift from house to Spanish sparrow
genotypes in the Alps hybrid zone (Trier et al. 2014).
We thus retain RPS4 as a candidate RI locus between
Italian and Spanish sparrows when plotting the results
from the comparative genomic cline analysis as well as
in our correlation comparison of candidate hybrid–
parent RI loci. Furthermore, in our reanalysis, six loci
exhibited steep clines not situated at either hybrid–par-
ent boundary, compared with 15 such loci reported by
Trier et al. (2014). These six markers were, however, a
subset of the 15 loci that Trier et al. (2014) interpreted
as candidates for being unpurged intraspecific incom-
patibilities within the Italian sparrow.
Hybrid–parents intrinsic barriers represent a subset ofthe barriers isolating the parent species
All Z-linked candidate hybrid–parent RI loci (CETN3,
CHD1Z, HSDL2, MCCC2 and GTF2H2) were among the
19 loci exhibiting significantly steep clines in parental
sympatry (Figs 3 and 4; Figs S1 and S3, Supporting
information). Indeed, there was strong concordance
between the two systems in patterns of locus-specific
introgression for the candidate nuclear RI loci (Figs 3
and 4; Table 1, Supporting information, correlation
among systems for the a parameter for the nuclear
putative RI markers: N = 6, r = 0.986, P = 2.77 9 10�4;
correlation among systems for the b parameter for the
nuclear putative RI markers b: N = 6, r = 0.942,
P = 4.94 9 10�3).
Of the six candidate intraspecific incompatibility loci
in the Italian sparrow, three exhibited restricted intro-
gression also in parental sympatry (Fig. 3; Figs S1 and S3,
Table S2, Supporting information). Furthermore, more
Fig. 4 Comparison of genomic clines for loci associated with
hybrid–parent reproductive isolation in Italian sparrows. Geno-
mic clines for the hybrid–parents analysis in Italy (left panel)
and parental sympatry in Spain. Markers PIK3C3 (Z-linked)
and DYRK4 (autosomal) are shown as ‘control loci’ that do not
deviate from neutral expectations between hybrid and parent.
Significant excess house sparrow ancestry is indicated by H,
significant excess Spanish sparrow ancestry is indicated by S,
significantly steep clines are indicated by +, and significantly
narrow clines are indicated by �. For estimates not differing
from neutral expectations, this is indicated by ns. Red clines
denote loci with significant excess Spanish sparrow ancestry,
and blue clines denote loci with significant excess house spar-
row ancestry. Black clines are not significantly shifted in either
direction.
© 2014 John Wiley & Sons Ltd
5838 J . S . HERMANSEN ET AL.
loci appeared to be involved in reproductive isolation in
parental sympatry than between hybrid and either parent
as ten loci exhibited steep clines only in parental sympa-
try (Fig. 3; Figs S1 and S3, Supporting information).
In parental sympatry, mitochondrial marker ND2
exhibited the same steep cline and excess house spar-
row ancestry as in Italian sparrows (Figs 3 and 4; Figs
S1 and S3, Supporting information). Similarly, Z-linked
nuclear-encoded mitochondrial markers HSDL2 and
MCCC2 also exhibited the same steep clines and excess
house sparrow ancestry in parental sympatry as at the
Italian-Spanish boundary (Figs 3 and 4; Figs S1 and S3;
Table S2, Supporting information).
Discussion
In this study, we investigated the evolution of intrinsic
reproductive isolation in a hybrid species system. We
compared loci exhibiting restricted introgression at the
range boundaries between the hybrid Italian sparrow
and its parent species, house and Spanish sparrows, to
loci exhibiting restricted introgression in an area where
the parent species co-occur and hybridize. The rationale
behind our approach is that if the hybrid–parent barri-
ers have arisen through sorting of pre-existing parental
incompatibilities, the same loci should also act as barri-
ers between the parents when they hybridize in sympa-
try. If the hybrid–parent barriers have arisen through
de-novo interactions in the hybrid genome, no such con-
cordance is expected (Rieseberg 1997).
Hybrid–parent intrinsic barriers represent a subset ofthose isolating the parent species
Consistent with the main prediction from the sorting
hypothesis, all five Z-linked candidate RI loci between
the Italian sparrow and either parent species were
among the 19 loci exhibiting restricted introgression in
parental sympatry. Moreover, these five loci all exhib-
ited similar patterns of excess ancestry in parental
sympatry as in the hybrid taxon, indicating that the
sorting of these incompatibilities in the Italian sparrow
was driven by a deterministic process. Autosomal mar-
ker RPS4 did not exhibit a significantly steep cline in
parental sympatry, however, suggesting that this
locus may be involved in a novel barrier that has devel-
oped between the hybrid Italian sparrow and the house
sparrow.
Our comparative analysis further revealed a set of
ten autosomal loci to exhibit restricted introgression
only in parental sympatry. This supports the prediction
from the sorting hypothesis that hybrid–parent RI genes
should represent a subset of parent–parent RI genes. It
further suggests that the association of these loci with
reproductive isolation is caused by epistasis through
linkage disequilibrium, which has been broken down
by recombination in the hybrid taxon. In sum, our
results support the hypothesis that the previously
reported intrinsic barriers between the Italian sparrow
and its parents have arisen through the sorting of pre-
existing parental incompatibilities. Allelic mosaicism of
hybrid species genomes has previously been demon-
strated in Helianthus sunflowers (Rieseberg et al. 1995,
2003) as well as in Lycaeides (Gompert et al. 2006; Nice
et al. 2013) and Papilio butterflies (Kunte et al. 2011) and
cichlids (Keller et al. 2013), but this is the first time
mosaicism of parental intrinsic incompatibility alleles is
demonstrated.
Not all narrow clines in the hybrid Italian sparrow
are located at hybrid–parent range boundaries, indicat-
ing a possible presence of unpurged intraspecific
incompatibilities (Trier et al. 2014). Such incompatibili-
ties may become coupled with extrinsic barriers and
this can set the stage for further diversification of the
hybrid taxon (Bierne et al. 2011). Findings consistent
with isolation-by-adaptation have previously been
reported from the Passer system, so such a coupling
mechanism may be at work (Eroukhmanoff et al. 2013).
In our comparative genomic cline analysis, three of the
six candidate intraspecific incompatibilities exhibited
restricted introgression also in parental sympatry. This
suggests that a subset of the candidate intraspecific
incompatibilities in the Italian sparrow may represent
unpurged parental incompatibilities, albeit further care-
ful investigation is needed to unveil their potential role
in further diversification.
The set of markers associated with restricted intro-
gression in parental sympatry exhibited a strong over-
representation of Z chromosome linkage. This finding
must, however, be interpreted with caution. We cannot
fully control for the lower effective population size of
Z-linked markers in our analyses, and hence this
Table 1 Pearson’s product–moment correlations between cline
parameters in different marker groups. The correlations were
performed on point estimates based on the median of the pos-
terior distribution for a and b
Marker group N
a-Parameter b-Parameter
r P r P
RI (nuclear) 6 0.986 2.77 9 10�4 0.942 4.94 9 10�3
II 6 0.617 0.192 0.030 0.956
Z-linked 21 0.558 8.56 9 10�3 0.240 0.295
Autosomal 55 0.426 1.17 9 10�3 0.485 1.77 9 10�4
RI refers to candidate reproductive isolation loci and II refers
to candidate unpurged intraspecific incompatibilities within
the Italian sparrow.
© 2014 John Wiley & Sons Ltd
HYBRID SPECIATION BY SORTING OF INCOMPATIBILITIES 5839
finding may to some degree be influenced by elevated
drift at Z-linked loci. However, the finding is consistent
with a growing number of studies demonstrating that
sex chromosome linkage plays a disproportionate role
in reproductive barriers between species with chromo-
somal sex determination systems (Qvarnstr€om & Bailey
2009), including in the Passer sparrows (Elgvin et al.
2011; Trier et al. 2014). The disproportionate role for sex
chromosomes in reproductive isolation is thought to
result from the higher mutation rate in the male germ
line, combined with exposure of recessive alleles to
selection in the heterogametic sex, an overrepresenta-
tion of genes with sexual function on the sex chromo-
somes, high average linkage between the sex-linked
genes involved in reproductive isolation, and stronger
drift due to a lower effective population size of sex-
linked markers (Charlesworth et al. 1987; Coyne & Orr
2004; Qvarnstr€om & Bailey 2009; Mank et al. 2010; Sætre
& Sæther 2010; Ellegren et al. 2012).
Mother’s curse influences isolation both between theparents and between hybrid and parent
‘Mother’s curse’ is the phenomenon by which selection
in males has no direct effect on mitochondrial fitness
due to the maternal inheritance of mitochondria,
favouring female (irrespective of male) interests, conse-
quently creating a selective sieve allowing for the build-
up of male-detrimental mutations (Frank & Hurst 1996;
Gemmel et al. 2004). This in turn selects for suppressor
alleles that restore male fitness. In female-heterogametic
taxa (ZZ/ZW), Z-linked genes spend two-thirds of their
time in the male lineage, and suppressor alleles are
therefore expected to be disproportionately situated on
the Z chromosome (Rice 1984; Trier et al. 2014).
Mismatches between mitochondrial alleles and nuclear
suppressor alleles lead to detrimental effects in hybrids
whose parents differ in mitonuclear system (Frank &
Hurst 1996; Gemmel et al. 2004).
In parental sympatry, we found both mitochondrial
marker ND2 and Z-linked nuclear-encoded mitochon-
drial markers HSDL2 and MCCC2 to exhibit the same
steep clines and shifts in favour of the house sparrow
allele as in Italian sparrows at the Italian–Spanish
boundary (Trier et al. 2014). The finding of strong house
sparrow excess ancestry for mitochondrial DNA also in
parental sympatry suggests that the fixation of house
rather than Spanish sparrow mitochondrial DNA in the
Italian sparrow was driven by deterministic rather than
stochastic processes. The mechanism favouring house
sparrow mitochondria remains unknown, but the candi-
dacy of HSDL2 and MCCC2 as Z-linked mother’s curse
suppressors is strengthened as these genes exhibit
among the steepest clines both at the Italian-Spanish
sparrow boundary and in parental sympatry, as well as
exhibiting significant shifts in the same direction as
mtDNA in both systems.
Conclusions
Our findings suggest that intrinsic barriers isolating the
Italian sparrow from its parent species have mainly
developed through the sorting of pre-existing sex-linked
parental incompatibilities and that isolation in both
cases is driven in part by mitonuclear conflict involving
the Z chromosome. This represents the first evidence
that the sorting process contributes to the persistence of
a hybrid animal taxon. It remains to be further investi-
gated whether and to what extent novel evolution in
the hybrid Italian sparrow also contributes to reproduc-
tive isolation against its parent species, but autosomal
marker RPS4 suggests that de-novo epistatic interactions
may also play a role in this system. Should a sorting
mechanism similar to the one described here prove to
be pervasive, the circumstances promoting homoploid
hybrid speciation may be broader than currently sus-
pected, and indeed, there may be many cryptic hybrid
taxa separated at two boundaries by sorted, inherited
incompatibilities, as in the Italian sparrow.
Acknowledgements
We thank M. H. Tu, M. Moan, P. Munclinger, M. F. G. Rojas,
S. A. Sæther as well as numerous field assistants for help with
acquiring the data, B. Dogan for help with laboratory work
and N. H. Barton, A. Runemark and anonymous referees for
helpful comments on a previous draft of the manuscript. This
work was supported by The Research Council of Norway,
Molecular Life Science (MLS), University of Oslo, and Centre
for Ecological and Evolutionary Synthesis (CEES), University
of Oslo.
References
Abbott RJ, Rieseberg LH (2012) Hybrid speciation. In: eLS. John
Wiley & Sons, Ltd, Chichester. DOI: 10.1002/9780470015902.
a0001753.pub2.
Abbott R, Albach D, Ansell S et al. (2013) Hybridization and
speciation. Journal of Evolutionary Biology, 26, 229–246.Bierne N, Welch J, Loire E, Bonhomme F, David P (2011)
The coupling hypothesis: why genome scans may fail to
map local adaptation genes. Molecular Ecology, 20, 2044–
2072.
Buerkle CA, Morris RJ, Asmussen MA, Rieseberg LH (2000)
The likelihood of homoploid hybrid speciation. Heredity, 84,
441–451.Charlesworth B, Coyne JA, Barton NH (1987) The relative rates
of evolution of sex chromosomes and autosomes. The Ameri-
can Naturalist, 130, 113–146.
Coyne JA, Orr HA (2004) Speciation, 1st edn. Sinauer Associ-
ates, Sunderland.
© 2014 John Wiley & Sons Ltd
5840 J . S . HERMANSEN ET AL.
Elgvin TO, Hermansen JS, Fijarczyk A et al. (2011) Hybrid spe-
ciation in sparrows II: a role for sex chromosomes? Molecular
Ecology, 20, 3823–3837.
Ellegren H, Smeds L, Burri R et al. (2012) The genomic land-
scape of species divergence in Ficedula flycatchers. Nature,
491, 756–760.Eroukhmanoff F, Hermansen JS, Bailey RI, Sæther SA, Sætre
G-P (2013) Local adaptation within a hybrid species. Hered-
ity, 111, 286–292.
Falush D, Stephens M, Pritchard JK (2003) Inference of
population structure using multilocus genotype data: linked
loci and correlated allele frequencies. Genetics, 164, 1567–1587.Fitzpatrick BM (2013) Alternative forms for genomic clines.
Ecology and Evolution, 3, 1951–1966.Frank SA, Hurst LD (1996) Mitochondria and male disease.
Nature, 383, 224.
Gemmel NJ, Metcalf VJ, Allendorf FW (2004) Mother’s curse:
the effect of mtDNA on individual fitness and population
viability. Trends in Ecology and Evolution, 19, 238–244.
Gompert Z, Buerkle CA (2011) Bayesian estimation of genomic
clines. Molecular Ecology, 20, 2111–2127.
Gompert Z, Buerkle CA (2012) bgc: software for Bayesian esti-
mation of genomic clines. Molecular Ecology Resources, 12,
1168–1176.Gompert Z, Fordyce JA, Forister ML, Shapiro AM, Nice CC
(2006) Homoploid hybrid speciation in an extreme habitat.
Science, 314, 1923–1925.
Guillot G, Estoup A, Mortier F, Cosson JF (2005a) A spatial sta-
tistical model for landscape genetics. Genetics, 170, 1261–1280.
Guillot G, Mortier F, Estoup A (2005b) GENELAND: a com-
puter package for landscape genetics. Molecular Ecology
Notes, 5, 712–715.Heliconius Genome Consortium (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among spe-
cies. Nature, 487, 94–98.
Hermansen JS, Sæther SA, Elgvin TO et al. (2011) Hybrid
speciation in sparrows I: phenotypic intermediacy, genetic
admixture and barriers to gene flow. Molecular Ecology, 20,
3812–3822.
International Chicken Genome Sequencing Consortium (2004)
Sequence and comparative analysis of the chicken genome
provide unique perspectives on vertebrate evolution. Nature,
432, 695–716.
Jiggins CD, Salazar C, Linares M, Mavarez J (2008) Hybrid trait
speciation and Heliconius butterflies. Philosophical Transactions
of the Royal Society B: Biological Sciences, 363, 3047–3054.Keller I, Wagner CE, Greuter L et al. (2013) Population genomic
signatures of divergent adaptation, gene flow and hybrid
speciation in the rapid radiation of Lake Victoria cichlid
fishes. Molecular Ecology, 22, 2848–2863.Kunte K, Shea C, Aardema ML et al. (2011) Sex chromosome
mosaicism and hybrid speciation among Tiger Swallowtail
butterflies. PLoS Genetics, 7, e1002274.
Lai Z, Nakazato T, Salmaso M et al. (2005) Extensive chromoso-
mal repatterning and the evolution of sterility barriers in
hybrid sunflower species. Genetics, 171, 291–303.Mallet J (2007) Hybrid speciation. Nature, 446, 279–283.
Mank J, Nam K, Ellegren H (2010) Faster Z-evolution is pre-
dominantly due to genetic drift. Molecular Biology and Evolu-
tion, 27, 661–670.
Mavarez J, Linares M (2008) Homoploid hybrid speciation in
animals. Molecular Ecology, 17, 4181–4185.Meise W (1936) Zur Systematik und Verbreitungsgeschichte
der Haus- und Weidensperlinge, Passer domesticus (L.) und
hispaniolensis (T.). Journal f€ur Ornithologie, 84, 631–672.
Nice CC, Gompert Z, Fordyce CA et al. (2013) Hybrid specia-
tion and independent evolution in lineages of alpine butter-
flies. Evolution, 67, 1055–1068.Nosil P (2012) Ecological Speciation, 1st edn. Oxford University
Press, Oxford.
Payseur BA (2010) Using differential introgression in hybrid
zones to identify genomic regions involved in speciation.
Molecular Ecology Resources, 10, 806–820.
Pritchard JK, Stephens M, Donnelly P (2000) Inference of popu-
lation structure using multilocus genotype data. Genetics,
155, 945–959.Qvarnstr€om A, Bailey RI (2009) Speciation through evolution
of sex-linked genes. Heredity, 102, 4–15.Rice WR (1984) Sex chromosomes and the evolution of sexual
dimorphism. Evolution, 38, 735–742.Rieseberg LH (1997) Hybrid origins of plant species.
Annual Review of Ecology and Systematics, 28, 359–389.Rieseberg LH, Willis JH (2007) Plant speciation. Science, 317,
910–914.Rieseberg LH, Van Fossen C, Desrochers AM (1995) Hybrid
speciation accompanied by genomic reorganization in wild
sunflowers. Nature, 375, 313–316.
Rieseberg LH, Raymond O, Rosenthal DM et al. (2003) Major
ecological transitions in wild sunflowers facilitated by
hybridization. Science, 301, 1211–1216.
Sætre G-P, Sæther SA (2010) Ecology and genetics of speciation
in Ficedula flycatchers. Molecular Ecology, 19, 1091–1106.
Sætre G-P, Riyahi S, Aliabadian M et al. (2012) Single origin of
human commensalism in the house sparrow. Journal of Evolu-
tionary Biology, 25, 788–796.Schumer M, Rosenthal GG, Andolfatto P (2014) How com-
mon is homoploid hybrid speciation? Evolution, 68, 1553–1560.
Seehausen O, Butlin RK, Keller I et al. (2014) Genomics and the
origin of species. Nature Reviews Genetics, 15, 176–192.
Summers-Smith JD (1988) The Sparrows: A Study of the Genus
Passer, 1st edn. T & AD Poyser, Calton.
Trier CN, Hermansen JS, Sætre G-P, Bailey RI (2014) Evidence
for mito-nuclear and sex-linked reproductive barriers
between the hybrid Italian sparrow and its parent species.
PLoS Genetics, 10, e1004075.
Warren WC, Clayton DF, Ellegren H et al. (2010) The genome
of a songbird. Nature, 464, 757–762.
The study was conceived and designed by J.S.H. and
G.P.S. Fieldwork was carried out by J.S.H., F.H., C.N.T.,
A.M. and G.P.S. Bioinformatics work was carried out
by A.J.N. The data were analysed by J.S.H. with contri-
butions from C.N.T., R.I.B. and F.H. The study was
written by J.S.H. with contributions from R.I.B., G.P.S.
and the other authors.
© 2014 John Wiley & Sons Ltd
HYBRID SPECIATION BY SORTING OF INCOMPATIBILITIES 5841
Data accessibility
Genotype data for all individuals used in the presented
analyses, estimated cline parameters for all markers,
hybrid indices, BGC input files and code used for run-
ning BGC are available through Dryad (doi:10.5061/
dryad.v6f4d).
Supporting information
Additional supporting information may be found in the online ver-
sion of this article.
Fig. S1 Individual Bayesian genomic clines for all 77 markers
in parental sympatry analysis.
Fig. S2 Comparison of estimates of genomic cline steepness in
parental sympatry for Z-linked and autosomal loci analysed
separately and together with all markers.
Fig. S3 Individual Bayesian genomic clines for all 77 markers
in hybrid–parents analysis.
Table S1 Details of SNPs used in analyses.
Table S2 Estimates of a-parameter (‘cline shift’) and b-parame-
ter (‘cline steepness’) in hybrid–parents and parental sympatry
analyses for candidate RI markers and putative unpurged
intraspecific incompatibilities in the Italian sparrow.
© 2014 John Wiley & Sons Ltd
5842 J . S . HERMANSEN ET AL.