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Hybrid speciation through sorting of parental incompatibilities in Italian sparrows JO S. HERMANSEN,* FREDRIK HAAS,* 1 CASSANDRA N. TRIER,* 1 RICHARD I. BAILEY,* ALEXANDER J. NEDERBRAGT,* ALFONSO MARZAL and GLENN-PETER SÆTRE* *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 hybridparent 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 formnot 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: g.p.satre@ibv.uio.no 1 These authors contributed equally to this work. © 2014 John Wiley & Sons Ltd Molecular Ecology (2014) 23, 5831–5842 doi: 10.1111/mec.12910
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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: g.p.satre@ibv.uio.no1These 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 andSpanish 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 Spanishsparrows: N = 87) were caught using mist nets, and ablood sample (20–50 lL) for use in genetic analyses wastaken 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 geneticanalyses 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) andHradec 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 hybridsbetween house (Oslo, Norway) and Spanish (Badajoz,

    Spain) sparrows (N = 6) were also included to verifythat STRUCTURE assigned admixed individuals cor-

    rectly. In STRUCTURE, the admixture model was used

    and we assumed two groups (K = 2) with correlatedallele 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 clinesteepness. Locus-specific introgression differs from the

    genome-wide average when credibility intervals for the

    posterior probability distributions of a and b do notoverlap 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), andSpanish 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 notcross zero. We also estimated the quantiles of the cline

    parameters in the genome-wide distribution of intro-

    gression, namely qa and qb. These quantiles representthe 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 theZ-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 exhibitedexcess Spanish sparrow ancestry (positive a) in parentalsympatry (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 couldargue 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 parameteras 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, Supportinginformation). 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 andnegative 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 nuclearputative RI markers: N = 6, r = 0.986, P = 2.77 9 10�4;correlation among systems for the b parameter for thenuclear 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 significantlynarrow clines are indicated by �. For estimates not differingfrom 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 0.295Autosomal 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.

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    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.

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    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.