This is a pre-acceptance preprint of: Irwin DE, Rubtsov AS, Panov EN. Mitochondrial introgression andreplacement between yellowhammers (Emberiza citrinella) and pine buntings (E. leucocephalos; Aves,
Passeriformes). Biological Journal of the Linnean Society, in press (accepted 4 April 2009). For thedefinitive version, please see the Biological Journal of the Linnean Society.

Mitochondrial introgression and replacement between yellowhammers(Emberiza citrinella) and pine buntings (E. leucocephalos; Aves,
Passeriformes).
DARREN E. IRWIN1*, ALEXANDER S. RUBTSOV1,2, AND EUGENE N. PANOV3
1

Biodiversity Research Centre, and Department of Zoology, University of British
Columbia, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada
2State Darwin Museum, Moscow 117292, Russia
3
Severtsov Institute of Ecology and Evolution, Moscow 117071, Russia*

Corresponding author: E-mail: irwin@zoology.ubc.ca

In studies of phylogeography and taxonomy, strong emphasis is usually placed on the
study of mitochondrial DNA (mtDNA). Here we present a remarkable case in whichhighly phenotypically divergent species have almost no divergence in mtDNA.
Yellowhammers (Emberiza citrinella Linnaeus) and pine buntings (E. leucocephalos S.
G. Gmelin) differ noticeably in appearance and song but hybridize in some areas ofcontact. They share a variety of closely related mtDNA haplotypes, with little
divergence in frequencies, indicating a mitochondrial divergence time sometime
during or after the last major glacial period. In contrast, nuclear DNA (AFLP
markers and CHD1Z gene sequences) differs more strongly between the species, andthese differences can be used to identify intermediate genetic signatures of hybrids.
The combined amount of mitochondrial diversity within yellowhammers and pinebuntings is very low compared to other Emberiza species pairs, whereas the level ofvariation at the nuclear gene CHD1Z is comparable to that within other species pairs.
While it is difficult to completely reject the possibility that the two species split
extremely recently and experienced rapid nuclear and phenotypic differentiation, we
argue that the evidence better supports another possibility: the two species are older
and mtDNA has recently introgressed between them, most likely due to a selective
sweep. Mismatches between mitochondrial and nuclear phylogeographic patterns
may occur more commonly than often thought, and could have important
implications for the fields of phylogeography and taxonomy.

ADDITIONAL KEYWORDS: AFLP – CHD1Z – hybridization – introgression –mtDNA – phylogeography – pine bunting – Siberia – speciation – yellowhammer.

1

INTRODUCTION
The study of variation in mitochondrial DNA (mtDNA) has played a central role instudies of taxonomy and phylogeography (Avise, 2004; Ballard & Whitlock, 2004;
Zink & Barrowclough, 2008). Mitochondrial DNA is unusual in that, in most taxa
(such as birds), it is inherited solely through the maternal line. As a result, mtDNA
generally has a lower effective population size than nuclear DNA, causing mtDNA
within a single population to have a lower expected coalescence time than mostnuclear genes, assuming that no selection is involved. This characteristic potentiallymakes mtDNA more useful than a single nuclear gene in inferring phylogeographic
history (Avise, 2004; Zink & Barrowclough, 2008).
Despite these advantages, the sole use of mtDNA in inferring history has
recently come under criticism (Irwin, 2002; Ballard & Whitlock, 2004; Edwards etal., 2005; Rubinoff & Holland, 2005; Bazin, Glémin & Galtier, 2006), due primarily
to the fact that the entire mitochondrial genome is inherited as a single unit and may
capture only a portion of phylogeographic history. Because of stochastic effects, sexbiased gene flow, lack of recombination between distinct mitochondrial lineages, anddirect selection on mitochondrial variation (Dowling et al., 2008), patterns ofgeographic variation in mtDNA might be highly discordant with patterns of variation
throughout the nuclear genome. In response to these criticisms, Zink &Barrowclough (2008) have defended the primary use of mtDNA, emphasizing its
greater ability to capture patterns of population division due to its lower effective
population size and arguing that the use of mtDNA leads to accurate conclusions
regarding taxonomic relationships the great majority of the time. Zink (2004) evenargued that mtDNA is of greater importance than phenotypic traits and nuclear DNA
in determining subspecies boundaries. Here, we explore these issues by studying
patterns of genetic differentiation in mitochondrial and nuclear DNA between two
phenotypically distinct but hybridizing taxa that have been considered distinct
species since their scientific description in the late 1700’s.
The debate regarding the importance of mtDNA as an indicator of population
history has important implications for the study of speciation, in which genetic
patterns are compared between relatively young taxa. Past variation in climate andhabitat distribution, for instance during the Pleistocene glaciations, has frequently
divided species ranges into geographically separated areas, and then allowed thoseareas to expand and come into contact, producing contact zones between divergent
groups. Such contact zones enable an examination of interactions between closely
related taxa and an assessment of whether they are reproductively isolated. One
region where such contact zones are common is central Siberia (Haffer, 1989;
Rogacheva, 1992; Newton, 2003; Irwin & Irwin, 2005), where many pairs of related
western and eastern forms meet. Siberia was treeless (although not highly glaciated)
for long periods during Pleistocene glaciations (Frenzel, 1968; Adams & Faure,1997), hence any forest-dependent species found there now must have expandedfrom other regions. In birds, patterns of migratory behavior (Irwin & Irwin, 2005)
and molecular biogeography (Irwin, Bensch & Price, 2001; Irwin et al., 2005)
suggest that many species expanded into Siberia from a forested refugium in central
Asia (i.e. the region west and northwest of the Tibetan Plateau and Taklamakan

2Desert), while others expanded from a refugium in eastern or northeastern China (ornearby regions such as the Korean Peninsula; Nazarenko, 1990). Some species had
related forms in both of these regions.
Thus central Siberia is a meeting place of distinct avian faunas, making it aprime location for the study of how related forms interact when they come into
secondary contact. Yet little research has focused on the genetic relationships
between western and eastern relatives in central Siberia. In perhaps the only previous
such study, genetic variation indicated that western and eastern forms of greenish
warblers (Phylloscopus trochiloides viridanus and P. [t.] plumbeitarsus) started
diverging long before the last major period of Pleistocene glaciation and then moved
into central Siberia from different directions, one from central Asia and the otherfrom eastern China (Irwin et al., 2001, 2005).
Here, we investigate interactions between the yellowhammer (Emberizacitrinella Linnaeus 1758) and the pine bunting (E. leucocephalos S. G. Gmelin
1771), two sister taxa in the Emberizinae family (Alström et al. 2007). Both species
breed across a wide region of western and central Siberia, with yellowhammers
extending westward to western Europe and pine buntings extending eastward to theRussian Far East (Fig. 1). The two species differ noticeably in plumage patterns, but
a variety of phenotypic intermediates across the sympatric area suggest that theyhybridize extensively (Panov 1989; Panov, Rubtsov & Monzikov, 2003; Panov,
Rubtsov & Mordkovich, 2007). The two forms are similar morphometrically but
apparently differ to some degree in habitat preference, with yellowhammers being
more likely to inhabit shrubby habitat along forest edges and in mountain steppe
vegetation, and pine buntings being more likely to inhabit open forests whereconiferous trees predominate (Ravkin, 1973; Panov et al., 2003).
This study was motivated by the desire to use yellowhammers and pinebuntings as a model system for the study of speciation between western and eastern
Palearctic forms. Our primary goal was to use patterns of genetic differentiation to(1) reconstruct the time of population splitting between yellowhammers and pinebuntings and (2) clarify patterns of current introgression due to hybridization. We
hypothesized that genetic variation would indicate that the two forms started
diverging sometime before the last of a long series of major Pleistocene glaciations;this last glacial period began roughly 110,000 years ago (Adams & Faure, 1997). We
intended to use mtDNA as the primary genetic marker in this effort, as is often donein the field of phylogeography (Avise, 2004; Zink & Barrowclough, 2008). Our
surprising initial results of virtually no mitochondrial divergence between the species(see Results) prompted us to investigate patterns of variation in the nuclear genome
as well, using AFLP analysis and the sequencing of a sex-linked gene (CHD1Z) on
the Z-chromosome. Given the large phenotypic differences between yellowhammers
and pine buntings, we hypothesized that the two groups are genetically divergent in
nuclear DNA even though they are so similar in mtDNA. We also hoped to use
nuclear DNA markers that differed between yellowhammers and pine buntings to test
whether phenotypically intermediate individuals (i.e., apparent hybrids) haveintermediate patterns of genetic variation. Our results reveal a variety of nuclear
markers that differ between the taxa and are useful in identifying hybrids. More
importantly, our findings indicate rapid mitochondrial introgression and fixation, a3phenomenon that is often not considered in the fields of phylogeography andtaxonomy.

MATERIALS AND METHODSS AMPLING
We obtained blood and/or tissue (muscle or liver) samples from field-caught birds
and from museum specimens. In total we examined 156 yellowhammers, 87 pinebuntings, and 20 phenotypic hybrids (Table S1). Each individual from which a sample
was obtained (both live birds and museum specimens) was photographed anddescribed phenotypically according to the standard scoring system used by Panov etal. (2003). We also obtained samples of eight other Emberiza species in order toestimate the genetic relationships of the yellowhammer/pine bunting complex to otherspecies within the genus. Sample sizes and localities are given in Fig. 1 and Table S1.
From birds captured in the field, we took about 50 µl of blood and diluted it in
500 µl of “Queen’s Lysis Buffer” (Seutin, White & Boag, 1991). Blood samples
were stored in a cool place during field work and frozen just after returning from thefield. Tissue samples from the museum collections were stored in 96% ethanol. DNA
was extracted using a standard phenol-chloroform protocol.

DNA SEQUENCE ANALYSISIn order to test whether yellowhammers and pine buntings differ in mtDNA wesequenced a subset of samples of each species from allopatric populations:yellowhammers from the Baltic Sea, Kursk and Krasnodar regions (41 samples), and pinebuntings from Chita and Sakhalin regions (33 samples; see Table S1 and Fig. 1 forlocations). We amplified 1032 base pairs (bp) of the ND2 gene using the primers
L5215: 5’-TATCGGGCCCATACCCCGAAAAT-3’ and H1064:
5’-CTTTGAAGGCCTTCGGTTTA-3’ (Drovetski et al., 2004). PCR amplification was
conducted using 1 µM each of primers L5215 and H1064, 1x PCR buffer and 1.5 U Taq
DNA polymerase (New England Biolabs), 2.5 mM MgCl2 (Invitrogen), and 0.25 mM
dNTP-mix (New England Biolabs) in 25 µl total volume. PCR included an initialdenaturing step of 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30
sec, and 72°C for 1 min, followed by a final elongation step at 72°C for 10 min. To
compare levels of ND2 variation in the yellowhammer/pine bunting species complex withthat throughout the Emberiza genus, we sequenced the same ND2 fragment from eight
other species: E. aureola, E. calandra, E. cioides, E. cirlus, E. hortulana, E.
spodocephala, E. stewarti, and E. tristrami (Table S1).
We are reasonably confident that our ND2 sequences are from the mitochondrial
genome rather than from a nuclear pseudogene (Sorenson & Quinn, 1998), for several
reasons. First, we used a combination of tissue samples and blood samples, and the twosources of DNA gave compatible results (pseudogenes are more likely to be amplified
from blood than from tissue). Second, our resulting ND2 phylogeny of 10 Emberizaspecies (see Results) is highly consistent with the cytochrome b phylogeny presented by4

Alström et al. (2007), differing only slightly in nodes that received low bootstrap support
in the cytochrome b phylogeny (e.g. the placement of E. cirlus). Third, our ND2phylogeny had much greater depth than our CHD1Z phylogeny, consistent with the
expectation that mitochondrial sequences evolve more quickly than nuclear sequences(Sorenson & Quinn, 1998).
We sequenced 612 bp of an intron of a sex-linked gene, CHD1Z on the Zchromosome, (Fridolfsson & Ellegren, 1999), from 173 samples representing bothparental species and their hybrids. PCR amplification was performed using 1 µM each ofprimers 2550F (5’-GTTACTGATTCGTCTACGAGA-3’) and 2718R(5’-ATTGAAATGATCCAGTGCTTG-3’; Fridolfsson & Ellegren, 1999) and other PCR
reagents as described above for ND2, in 25 µl total volume. PCR included an initialdenaturing step of 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 50°C for 30
sec, and 72°C for 40 sec, and a final elongation step at 72°C for 10 min. To compare
CHD1Z variation within the yellowhammer/pine bunting species complex with thatthroughout the genus, we sequenced the same CHD1Z fragment from four other species:
E. calandra, E. hortulana, E. cirlus, and E. stewarti (Table S1). Purification andsequencing of PCR products for both the ND2 gene and the CHD1Z intron were
performed by Macrogen (Seoul, South Korea).
Sequences were aligned and edited using BIOEDIT (Hall, 1999). Both avian sex
chromosomes (Z and W) have a copy of the CHD1Z gene, but the intron used here differsin length between these chromosomes (Fridolfsson & Ellegren, 1999). For sequencing weused the samples obtained from the homogametic sex (males) only, to avoid sequencing
complications from the presence of the W-chromosome copy. To reconstruct haplotypes
from unphased genotypes we used the program FASTPHASE under default settings(Scheet & Stephens, 2006).
For both ND2 and CHD1Z, we produced minimum spanning haplotype networks
with the assistance of the programs TCS (Clement, Posada & Crandall, 2000) and
ARLEQUIN 3.11 (Excoffier, Laval & Schneider, 2005), which produced identical results.
ARLEQUIN was also used to calculate ΦST, defined as the fraction of variance in pairwise
sequence differences (i.e. the number of mismatches) that is explained by the difference
between two groups (Excoffier, Laval & Schneider, 2006; this ΦST takes into accountboth genetic distance between and frequency of haplotypes, and is therefore notequivalent to some formulations of FST that use only haplotype frequency).
To estimate genetic distances and phylogenetic trees among Emberiza species
based on ND2 and CHD1Z, we used the program TREE-PUZZLE (Schmidt et al., 2002),
which uses a maximum likelihood approach. We used an HKY-Γ model of sequence
evolution (following Price, 2008, pp. 38-40), eight gamma-distributed rate categories,
and accurate parameter estimation using neighbor-joining trees. A likelihood ratio test, as
implemented in TREE-PUZZLE, revealed that for both the ND2 and the CHD1Z trees theassumption of a constant molecular clock does not significantly decrease the likelihood of
the tree; hence we use the clock-like trees for illustration.
While there is support for a reasonably constant molecular clock for mtDNA in
birds (Weir & Schluter, 2008), we did not assume the same clock would apply to
CHD1Z. To calculate a range of reasonable estimates for the CHD1Z molecular clockthat was independent of the yellowhammer/pine bunting data, we compared ND2

5

distances with CHD1Z distances for the other species pairs that were in both datasets (E.
calandra, E. hortulana, E. cirlus, and E. stewarti).

DEMOGRAPHIC MODELING BASED ON MTDNA
Many studies of phylogeography, historical demography, and conservation genetics rely
heavily on inference from mtDNA-based demographic modeling, using a variety ofmethods. We used such methods to explore the range of historical scenarios that areconsistent with the mitochondrial data under certain assumptions. We note that our goal
was not to infer the precise history of the two species; rather, we wished to determine
whether the historical scenarios inferred using mtDNA were consistent with variation in
nuclear DNA and phenotypic traits.
To test whether the ND2 data were consistent with neutral evolution underconstant population size, we used ARLEQUIN to perform three tests of selective neutrality,each tested for significance using 10,000 simulations: Tajima’s D test (Tajima, 1989),
Fu’s FS test (Fu, 1997), and Ewens-Watterson-Slatkin’s exact test (Slatkin 1996).
To estimate the time of divergence and demographic history of allopatricyellowhammers and allopatric pine buntings based on mitochondrial ND2 data and anassumption of neutrality, we used the program IMA (Hey, 2007; Hey & Nielsen, 2007),
which used a Bayesian Markov chain Monte Carlo approach to fit the ND2 sequence data
to an “Isolation with Migration” model (Nielsen & Wakeley, 2001). This model assumes
that two populations (in this case, allopatric yellowhammers and allopatric pine buntings)
split some time ago from a single population, and that there has been some constant rateof gene exchange since the populations split. Given an estimated mutation rate, IMA
attempts to simultaneously estimate six parameters: the effective population sizes of bothcurrent populations and the ancestral population, the time since splitting of populations,and the migration rates of each population to the other. The model assumes selective
neutrality and no recombination within a locus.
We ran IMA under two scenarios, using ND2 sequence data from allopatricyellowhammers and allopatric pine buntings. The purpose of these model runs was not toprecisely determine the true history of the two species, as the model assumptions are too
restrictive and simple for that purpose. Rather, our goal was to estimate the range of
population splitting times that are consistent with the mitochondrial ND2 data, in thehopes that this information would help us to determine whether mitochondrial patterns
were consistent with patterns in nuclear DNA. First, we ran IMA to estimate all six
parameters simultaneously. Second, we ran the model with the maximum migration ratein each direction set to zero, effectively making the model into a two-island equilibrium
model. Several preliminary runs were conducted under each scenario to determine
proper upper bounds on parameters as well as run times necessary to achieve
convergence and have sufficient sampling, as recommend by Hey (2007). Each model
was run with a burn period of 107 steps followed by a recording period of 108 steps. Eachscenario was run twice using different random number seeds, to check repeatability of
results. In the runs that included post-split migration, we used IMA to record the meantime of migration events (Won & Hey, 2005).
Conversion of the IMA parameters t and θA to divergence time in years andeffective population size requires an estimate of generation time and mutation rate (Hey6

2007). We used an estimated generation time of 1.7 years (the average time for sevenpasserine species; Kondo et al., 2008; after Sæther et al., 2005; note that using a different
estimate of generation time would affect estimates of population size but would not affectthe resulting estimate of time in years since population expansion). We based ourestimate of the ND2 mutation rate on the well-supported mitochondrial molecular clock
estimate of 1% mutation per million years along a single lineage (2% between lineages;
Lovette, 2004; Weir, 2006; Weir & Schluter, 2008). This rate corresponds to 1.03 * 10 -5
mutations per entire ND2 sequence (1032 bp) per year, or an estimate for µ of 1.75 * 10 -5
per generation. To account for uncertainty in the rate of mutation, we also calculatedparameter values for both 0.5% and 1.5% per million year mutation rates, which seemed
appropriate given the apparent variation in calibrated rates reported by Weir & Schluter(2008).

AFLP ANALYSIS
Whereas mitochondrial DNA is inherited matrilineally as a single unit, AFLP
(amplified fragment length polymorphism) markers are spread throughout the nuclear
genome and represent a variety of genealogical histories. To generate AFLP markers weused the protocol provided by LI-COR Biosciences (2003), based on the method
developed by Vos et al. (1995). We used the restriction enzymes EcoRI and MseI todigest genomic DNA, and then synthetic oligonucleotides (“adaptors”) were ligated to thefragments. We performed two rounds of PCR using primers corresponding to, in the firstround, the adaptor plus 1 arbitrary base pair, and in the second round, the adaptor plus 3
arbitrary base pairs. Fluorescein-labeled primers were used in the second round of PCR(the “selective amplification”). The products were separated in 6.5% KBPlus gels andvisualized using a LI-COR 4300 DNA Analyzer.
We generated two sets of AFLP data for subsequent analysis; each allowed
particular questions to be addressed most effectively:
AFLP dataset 1: genetic differentiation between taxa
Here, our goal was to obtain an unbiased estimate of genetic differentiation between
allopatric yellowhammers and allopatric pine buntings. We scored all AFLP bands,
regardless of their patterns of variation, from 5 primer combinations. This was done for asubset of samples from allopatric yellowhammers (n = 13) and allopatric pine buntings(n=15). We did not include bands that appeared in 3 or fewer individuals. We
summarized variation in the resulting presence/absence matrix using principal
components analysis, using R (R Development Core Team, 2006).
We used AFLP-SURV (Vekemans, 2002) to calculate FST, the proportion of total
variance in allele frequencies that is explained by differences between the two species.
Estimation of allele frequencies was done using a Bayesian approach (with non-uniform
prior distributions of allele frequencies) assuming Hardy-Weinberg equilibrium withineach species. This approach is necessary to avoid biases in the estimation of allele
frequencies from dominant markers (Lynch & Milligan, 1994; Zhivotovsky, 1999).
Locus-specific FST's were generated using these allele frequencies, using the equation

7

( p1 " p) 2 + ( p2 " p) 2FST =,2 p(1" p)!

where px is the frequency of the allele for band presence in population x, and p is themean of p1 and p2.
Some previous AFLP studies of population differentiation have reported analternative FST statistic that is based on AFLP band frequencies rather than allele
frequencies. This statistic is based simply on two phenotypes, these!being presence orabsence of each AFLP band. Because a number of other studies report this statistic (e.g.
Svensson et al., 2004; Bensch & Åkesson, 2005; Helbig et al., 2005; Irwin et al., 2005;
Parchman, Benkman & Britch, 2006; Toews & Irwin, 2008), for purposes of comparison
we also calculated it for our data, using ARLEQUIN 3.11 (Excoffier et al., 2005).
AFLP dataset 2: selected markers for distinguishing taxa and hybrids
Here, our goal was to select only the most useful markers in distinguishing the taxa andpresumably in identifying hybrids as well. We examined AFLP profiles from 10 primer
combinations and determined AFLP markers that differed in presence/absence
frequencies between 26 allopatric yellowhammers (group 1 below) and 15 allopatric pinebuntings (group 7 below) according to the following two criteria: we selected all markers
that were (1) at least 1.5 times more common in one of the groups than in the other, and(2) present in at least 50% of individuals in the group with the highest frequency of thatmarker. These criteria ensured that selected markers were picked objectively and had alarge difference in frequency between the two groups.
This procedure resulted in 20 selected markers, which were then also scored inindividuals from within or close to the contact zone. In total, this analysis included 65
samples divided into 7 phenotypic groups (for detailed descriptions of each group see
Panov et al., 2003): 1) allopatric yellowhammers (from Baltic Sea, Kursk, Moscow, andOrenburg): n = 26; 2) phenotypic yellowhammers from the contact zone: n = 4; 3) yellow
hybrids: n = 8; 4) white hybrids: n = 7; 5) pine buntings with slight hybrid phenotypes: n= 2; 6) pine buntings in or near the contact zone: n = 3; 7) allopatric pine buntings (fromEast Transbaikalia and Sakhalin): n = 15. We again summarized variation in the resulting
presence/absence matrix using principal components analysis, using R (R Development
Core Team, 2006). We also used STRUCTURE 2.2 (Pritchard, Stephens & Donnelly, 2000;
Falush, Stephens & Pritchard, 2007) to calculate assignment probabilities of individuals
to two genetic clusters based on their AFLP signatures.

RESULTS
MITOCHONDRIAL DNA
Allopatric yellowhammers and pine buntings differ remarkably little in mtDNA (Fig. 2a),with many haplotypes shared between the two species. ΦST is only 0.025, which does notdiffer significantly from zero (P = 0.073). Out of 1032 basepairs (bp) sequenced, theaverage pairwise difference between species, after correction for within-species8polymorphism, was only 0.056 basepairs (uncorrected pairwise differences between
species: 2.231 bp; within yellowhammers: 2.285 bp; within pine buntings: 2.064 bp).
This amounts to a percentage divergence of 0.0054 %, an extraordinarily low amount forsuch phenotypically divergent taxa.
A variety of analyses indicate that mtDNA of both yellowhammers and pinebuntings have experienced a selective sweep and/or population growth. Tajima's test of
selective neutrality (Tajima, 1989) was significant for yellowhammers (D = -1.49, P =0.049) and pine buntings (D = -1.81, P = 0.018) as well as all samples combined (D = 2.06, P = 0.004), where significance indicates rejection of the neutral model. Fu's (1997)
test also rejected neutrality in each case (yellowhammer: FS = -9.87, P < 0.00001; pinebunting: FS = -9.16, P = 0.00010; all samples combined: FS = -24.37, P < 0.00001).
Finally, Ewens-Watterson-Slatkin’s exact test (Slatkin 1996) also indicated a selective
sweep and/or population growth (yellowhammer: P = 0.0182; pine bunting: P = 0.0007;
all samples combined: P < 0.00001).
The IMA analyses provided fairly precise estimates of the time of population
splitting of yellowhammer and pine bunting mtDNA. All four runs (two allowed
migration after the split and two did not) produced similar posterior probability
distributions for the time of the split (Fig. S1), with the peak time being 32,000 years ago(95% confidence interval: 20,000 – 48,000). The analysis also produced a narrow rangeof estimates of the ancestral effective population size (Fig. S1), which was relatively
small, the peak estimate being 110,000 (95% confidence interval: 47,000 – 290,000).
Incorporating uncertainty in the rate of mutation ranging from 0.5% to 1.5% per million
years along a lineage, these 95% confidence intervals expand to 14,000 to 97,000 years
ago for the time of splitting and the ancestral effective population size of 31,000 to
580,000. The IMA runs were not able to produce stable distributions for the estimates ofpresent population sizes nor migration rates after the time of the split (Fig. S1).
The most divergent mitochondrial haplotypes within the yellowhammer/pinebunting complex differ by only 8 substitutions, or 0.8 % sequence divergence. Thismaximal amount of sequence divergence within the yellowhammer/pine bunting complex
is very low compared to that between other species of Emberiza buntings (Table 1; Fig.
3a; to represent yellowhammers and pine buntings in the phylogeny, we used the mostdivergent sequences, denoted with asterisks, in Fig. 2a). The genetic distance between themost divergent haplotypes in the pine bunting/yellowhammer complex is 16 times lessthan between the pine bunting and another closely related species, the chestnut-breasted
bunting (E. stewarti). These results closely parallel the patterns seen at the mitochondrial
cytochrome b gene, as presented by Alström et al. (2007).

CHD1Z SEQUENCES
Sequences from 173 samples revealed five single nucleotide polymorphisms and one
insertion/deletion (‘indel’) within 613 bp of the CHD1Z intron. Positions of thesepolymorphisms in the sequence, and the alternative characters at those positions, are as
follows: 132:A/G, 160:T/O, 241:T/C, 379:G/A, 495:T/C, 501:T/A, where ATGC
represent mononucleotides and O represents a deletion. The program FASTPHASE (Scheet& Stephens, 2006) revealed that the genotypes most likely consist of 11 haplotypes, fiveof which are quite rare (Fig. 2b). Common haplotypes are shared between species,9indicating that CHD1Z would be of little use in characterizing the genetic origin ofindividuals in the hybrid zone. We therefore focused our analysis on allopatricyellowhammers (75 samples) and pine buntings (36 samples), addressing the question of
how genetically divergent the two groups are.
ΦST between the allopatric groups based on CHD1Z is 0.176, which issignificantly different than zero (P < 10-5) and much larger than ΦST based on ND2(0.025, see above). This is a surprising result, since under neutral theory estimates of ΦST
based on mitochondrial sequences are expected to exceed estimates of ΦST based on
nuclear genes (Zink & Barrowclough, 2008).
The haplotype network shows multiple possible pathways by which haplotypes
might have arisen, an apparent pattern of reticulate evolution suggesting a role forrecombination in generating new haplotypes (Fig. 2b). Explaining the pattern without
recombination would require postulating that several cases of point mutations arose
multiple times, which seems quite unlikely. The two most highly diverged of thecommon haplotypes differ substantially in frequency between the species, with GTTGTT
more common in yellowhammers (chi-squared contingency test: χ2 = 10.7, P = 0.002)
and AOCGCA more common in pine buntings (χ2 = 26.0, P < 10-6; Fig. 2b). Taken
together, a possible explanation for these patterns is that GTTGTT is an ancestral
yellowhammer allele, whereas AOCGCA is an ancestral pine bunting allele. All of theother haplotypes, with the sole exception of the low-frequency haplotype AOCACA,
could have arisen through hybridization and recombination without any novel point
mutation.
For the purpose of estimating the CHD1Z relationships of the yellowhammer andpine bunting with four other bunting species, we took the common haplotypes in the two
species (GTTGTT for yellowhammer and AOCGCA for pine bunting) as representing
these species respectively. In contrast with the mtDNA result, CHD1Z shows a genetic
distance between the yellowhammer and pine bunting comparable with other closely
related species, E. cirlus and E. stewarti (Table 1, Fig. 3b). A comparison of the CHD1Z
distances and ND2 distances for the other species pairs that were in both datasets (E.
calandra, E. hortulana, E. cirlus, and E. stewarti), and assuming an ND2 molecular clockof 2% per million years, results in CHD1Z clock estimates ranging from 0.1% (E.
hortulana - E. stewarti) to 0.2% (E. calandra - E. stewarti) per million years. Applying
this range of estimates to the divergence between the two most common haplotypes leads
to an estimated time of divergence of these two haplotypes of roughly 3 to 6 million years
ago.

AFLP ANALYSIS
Genomic differentiation between taxa
AFLP analysis of allopatric yellowhammers and allopatric pine buntings reveals amoderately strong signal of divergence in their nuclear genomes. Our analysis of 5 AFLP
primer pairs (AFLP dataset 1) revealed 367 AFLP markers in 13 allopatricyellowhammers and 15 allopatric pine buntings. Of these, 63 markers (17.2%) werevariable. A principal components analysis (Fig. 4) reveals that the two taxa are clearly
separated along the first principal component (PC1), which explains 11.4% of thevariance in the dataset. This difference in PC1 is highly significant (t-test: t26 = 13.57, P <1010-12). FST, defined as the fraction of variance in AFLP signatures that is explained by thedifference between two groups, is 0.078 based on estimated allele frequencies (using
AFLP-SURV) and 0.140 based on band frequencies (using ARLEQUIN), both of which aresignificantly different from zero (P < 10-5 and P < 10-4, respectively). While the overallsignal of differentiation between the species is clear, it is due to a relatively small numberof markers (Fig. 5), a pattern that is generally expected for multilocus genetic data
(Whitlock, 2008).
AFLP analysis of hybridization
By examining bands produced by 10 AFLP primer pairs combinations, we identified 20
markers that showed strong frequency differences between 26 allopatric yellowhammersand 15 allopatric pine buntings (Table 2; see Materials and Methods). A principal
components analysis of these 20 markers on all phenotypic groups (AFLP dataset 2)
reveals that phenotypic hybrids tend to have intermediate AFLP signatures (Fig. 6), andthe STRUCTURE analysis produced similar results (Figure S2). Many of the phenotypic
hybrids are generally more similar to allopatric yellowhammers than to allopatric pinebuntings.

DISCUSSIONGENETIC DIFFERENTIATION BETWEEN THE SPECIESYellowhammers and pine buntings differ noticeably in plumage, song, and geographic
distributions (Panov et al., 2003, 2007; Rubtsov, 2007), suggesting a long period ofallopatric divergence. Thus we expected to find sizeable genetic divergence between
them. Surprisingly, patterns of mtDNA variation were extraordinarily similar between thespecies, with many shared haplotypes, a nonsignificant ΦST of 0.025, and a correctedsequence divergence of only 0.00054%. The estimated mitochondrial divergence date
between species based on both the isolation model and the isolation with migration model
is only roughly 30,000 years ago, an extraordinarily short time compared to mostestimated divergence times between avian sister species, the bulk of which are between
one and six million years ago (Weir & Schluter, 2007). A variety of analyses (Tajima's D,
Fu’s FS, mismatch analyses, and IMA results) all indicate that mtDNA has undergone
substantial population growth and/or selective sweeps, and that the pattern inyellowhammers and pine buntings are remarkably similar.
In contrast to mtDNA, nuclear markers do show moderately strong divergence
between yellowhammers and pine buntings. Significant divergence was observed at thenuclear CHD1Z intron in frequencies of alleles, although alleles were shared between thespecies. In AFLP markers there is a clear signal of divergence, although it is relatively
modest compared to amounts of AFLP divergence between other avian species pairs. Our
FST estimate based on AFLP band frequencies is 0.14, whereas this measurement ofdivergence is 0.18 between greater and lesser spotted eagles (Aquila clanga and A.
pomarina; Helbig et al., 2005), 0.38 between white-winged crossbills and Hispaniolan
crossbills (Loxia leucoptera and L. megaplaga; Parchman et al., 2006), 0.4 between two
reproductively isolated taxa of greenish warbler (Phylloscopus trochiloides viridanus and11

P. [t.] plumbeitarsus; Irwin et al., 2005), and 0.42 between two cryptic species of winterwrens (Troglodytes troglodytes and T. [t.] pacificus; Toews & Irwin, 2008). In three ofthese other cases, divergence in mtDNA has been assessed and is quite substantial(eagles: 1.75 % in cytochrome b; warblers: 5 % in control region; wrens: 6.2 % in ND2).
These findings lead to two strong conclusions. First, the two species do differ
genetically, suggesting that the two species are evolutionarily significant units that haveexperienced some period of relatively independent evolution. Second, this genetic
difference can be clearly seen only in roughly 10% of the genome (Fig. 5). These results
demonstrate that estimates of genetic divergence can differ dramatically between
different molecular markers, and that a small but important subset of the genome candiffer markedly even when most of the genome does not. It is possible that the few AFLP
markers that show high divergence between the species are closely linked to genes thatare under divergent selection in the two species.
These patterns can be explained in two ways. First, if we were to assume that the
mitochondrial relationships are representative of true history, we would have to conclude
that yellowhammers and pine buntings shared a common ancestor roughly 30,000 years
ago (95% confidence intervals: 14,000 to 97,000 years). Under this scenario, thedifferentiation in phenotypes and in some AFLP markers would have occurred extremely
rapidly, most likely due to strong selection. This would qualify as one of the fastest
known cases of bird speciation, which in most cases takes more than a million years(Price, 2008). This scenario is extremely difficult to reconcile with the noticeable
divergence in plumage color, songs, ecology, and nuclear DNA.
Second, a much more parsiminoious explanation of these patterns is recent
introgression of mtDNA between divergent forms. Recent hybridization might haveintroduced mtDNA from one species into the other, and that mitochondrial clade might
have then become fixed in both species. This process can in theory occur rather easily,
even when there is fairly strong selection against hybrids (Takahata & Slatkin, 1984).
The smaller effective population size of mtDNA compared to nuclear DNA makes
mtDNA particularly susceptible to fixation of foreign haplotypes (Funk & Omland,2003). A selective advantage of one type of mitochondria over another could have
hastened this process (Grant, Spies & Canino, 2006; Dowling et al., 2008). This processof introgression, drift, and possible selection would also apply to nuclear genes. Over along period of hybridization, much of the genome could have become homogenized
between the species, whereas parts that were strongly selected in different directions
continued to diverge between the species. We suggest that this second scenario, of
introgression between two highly distinctive species, is easier to reconcile with theobserved patterns.
The estimated time at which this mitochondrial introgression occurred (14,000 to
97,000 years ago) is difficult to reconcile with paleoclimatological history, as this span oftime occurred during the last major glacial period, which began roughly 110,000 years
ago and ended roughly 12,000 years ago (Adams & Faure, 1997). If yellowhammers andpine buntings were confined to separate refugia (e.g. the former in Europe or central
Asia, the latter in southeast Asia), it might seem unlikely that hybridization would haveoccurred during this major glacial period. However, there is evidence that the period from55,000 to 25,000 years ago was relatively mild, with evidence for some wooded
vegetation across Siberia (Adams & Faure, 1997). Hence it is possible that12

yellowhammers and pine buntings did hybridize during that period, which would beconsistent with the estimates from IMA. This is the first study we of aware of thatsuggests evidence of such genetic contact between western and eastern Palearctic refugial
forms during that more mild phase of the last glacial period. An important caveat is that
the IMA analysis assumes selective neutrality; it is possible that introgression of mtDNA
may have occurred much more recently through the actions of selection.
CHD1Z patterns are remarkably supportive of this introgression scenario rather
than a scenario of recent speciation. The CHD1Z haplotype network strongly suggests a
recombinant origin of many haplotypes, as the alternative explanation of so many
identical mutations occurring multiple times is implausible. The two most common
haplotypes (GTTGTT, the most common haplotype in yellowhammers; and AOCGCA,
the most common haplotype in pine buntings) are also the most divergent of all
haplotypes (except for one rare haplotype, AOCACA), suggesting that they represent the
haplotypes ancestral in the two species. These two haplotypes are roughly as divergent as
we might expect sister species of Emberiza to be, given the genetic distances throughout
the Emberiza phylogeny (e.g., the GTTGTT and AOCGCA haplotypes are as divergent
as the GTTGTT haplotype is from E. stewarti). All of the other haplotypes, with the sole
exception of AOCACA, are simple combinations of these major haplotypes. If mutation
rates were high, we would expect to see many haplotypes that differ from a common one
by a single mutation, a pattern that is not seen. Overall, the patterns are suggestive ofrecombination between highly divergent forms of CHD1Z. These forms likely correspondto distinct yellowhammer and pine bunting forms of CHD1Z before hybridization,
recombination, and introgression led to the current mixed pattern. The estimated time ofdivergence between these haplotypes, three to six million years ago, provides one very
rough maximum estimate for when the two species started diverging. It should be notedthat the Z chromosome may play an especially large role in speciation in femaleheterogametic groups such as birds, and may be less subject to genetic mixing between
incipient species compared to other parts of the genome (Qvarnström & Bailey, 2009).

MOLECULAR IDENTIFICATION OF HYBRIDIZATION
Our analysis strongly supports the hypothesis that hybrids can be identified based on
appearance, as individuals with apparent hybrid phenotypes usually had intermediate
AFLP signatures. This result confirms the utility of screening large numbers of AFLP
markers between two allopatric samples, and then using a subset of markers that are mostdivergent in frequency to test whether there is hybridization in sympatry and to compare
the genetic signatures of birds in the hybrid zone to those in allopatry. It would require
much further analysis as well as larger sample sizes to accurately assign birds in thecontact zone to hybrid categories, such as F1, F2, backcross, etc.; this goal was beyond
the scope of this paper.
Most phenotypic hybrids are more similar to allopatric yellowhammers than they
are to allopatric pine buntings. This pattern suggests that gene flow from the contact zone
has affected allopatric European populations of yellowhammers to a greater extent thanallopatric populations of pine buntings. Perhaps backcrosses with yellowhammers havehigher fitness than backcrosses with pine buntings, or perhaps yellowhammers havelarger dispersal distances, leading to greater gene flow across their range. Whatever the13

cause of this pattern, it is in accordance with the distribution of various color phenotypes
within the yellowhammer breeding range. Populations of yellowhammers in the easternpart of their range tend to have some chestnut coloration on the throat; this and otherplumage traits have lead some authors to treat them as a distinct subspecies (Emberizacitrinella erythrogenys; Cramp & Perrins, 1994; Byers, Curson & Olsson, 1995),
although this variation is subtle and varies clinally from the traits of the European
Emberiza citrinella citrinella. The suggestion of Panov et al. (2003) thatE. c. erythrogenys could be a product of ancient hybridization between yellowhammers
and pine buntings appears to be consistent with our molecular data. These results point to
the possibility of hybridization being a creative force, rearranging gene combinations tocreate novel phenotypes that are relatively successful over time (Arnold, 1997).

MITOCHONDRIAL INTROGRESSION
There is growing recognition that introgression of mtDNA between species might bequite common (Good et al., 2008), as is suggested by the high rate of species paraphyly
and polyphyly in interspecific mitochondrial gene trees (Funk & Omland, 2003). Cases ofpartial introgression of heterospecific mtDNA has been observed in a variety of species(e.g., Rohwer, Bermingham & Wood, 2001; Weckstein et al., 2001; Good et al., 2003,
2008; Deffontaine et al., 2005; Melo-Ferreira et al., 2005; Plötner et al., 2008), but thecase of the yellowhammer and pine bunting is unusual in the magnitude of phenotypic
differentiation between the species and the extent of mitochondrial blending, with thetype of one species apparently completely replacing that of the other. Such complete
replacement is remarkable between species that differ so noticeably in appearance. Casesof complete replacement might be much more common than presently thought, since they
are difficult to detect. When mtDNA has only partially introgressed, it is detectablebecause members of one species have two very divergent forms of mtDNA, one of which
is similar to the other species (e.g Plötner et al., 2008). When complete replacement has
occurred, there are no surviving examples of the extinct haplotype group to reveal thepresence of introgression; the remaining pattern is simply one of mtDNA similarity
between the two species, which could be mistakenly interpreted as recent population
splitting.
There is growing recognition that mitochondrial DNA could often be underselection, challenging the assumption of neutrality that is common to many analyticalmethods used in studies of phylogeography, speciation, and conservation genetics(Ballard & Whitlock, 2004; Bazin et al., 2006; Dowling et al, 2008). Selection can occur
in many ways, including local adaptation of different mtDNA variants to different
environmental conditions, coevolution between mitochondrial and nuclear genes(Dowling et al., 2008), and selective sweeps of universally favored mitochondrial
mutations. We suggest that a selective sweep is the likely explanation for the
mitochondrial introgression between yellowhammers and pine buntings, as it seems
implausible that a complete replacement of one species' mtDNA by another occurred bydrift alone. A possible scenario consistent with the data is the following: a favorable
mutation arose in mtDNA of one of the species and rose in frequency due to selection. A
short time later, hybridization introduced this variant to the other species. The favorable
variant then continued to grow in frequency within both species, and hybridization

14

continued to transfer variants of this favorable mtDNA between the species. Eventually,
all other variants of mtDNA vanished from both populations. Plötner et al. (2008)
suggested that a variant of mtDNA that arose in one species of water frog has spread toanother species because it is selectively advantageous to both species in the more
northerly parts of their ranges. Parallel shifts in climate or other environmental variables
may be quite common for sister species; during such shifts, the mitochondria in one of
the species may become better adapted to the new conditions and, assisted byhybridization, sweep to fixation in both species.
These findings highlight the challenges inherent in the use of molecular variation
to reconstruct biogeographic history and identify evolutionarily significant units. Becauseof both shared ancestral polymorphism and introgressive hybridization, two groups thathave experienced much independent evolution can be similar in most of their genome
while still differing in those parts that are under divergent selection. These differences
can be maintained even when two groups hybridize extensively, given strong enoughselection for alternative alleles in the two groups (Barton & Hewitt, 1985, 1989; Wu,
2001). Hybridization is being increasingly recognized for its potentially creative role inevolution, as differential introgression can lead to novel gene combinations (Arnold,
1997; Mallet, 2005; Price, 2008). In the case of yellowhammers and pine buntings,
mtDNA appears to have introgressed between two phenotypically distinct forms thatdiffer in parts of the nuclear genome. Thus mtDNA, as well as many parts of the nuclear
genome, would provide a misleading picture of the history of the species complex.
Our findings should be noted in light of the traditional emphasis on the use of
mtDNA in taxonomy at the species and subspecies level. For example, Zink (2004)
argued that phenotypically-defined subspecies should not be considered evolutionarily
significant units if they do not correspond to distinct mitochondrial clades. Suchreasoning, when applied to our study group, would lead to the mistaken conclusion thatyellowhammers and pine buntings are a single genetically undifferentiated group.
Plumage patterns (Panov et al., 2003, 2007), song (Rubtsov, 2007), AFLP, and CHD1Z
lead to a different conclusion. Introgression is just one of the many reasons that patternsof relationships in mtDNA may not accurately represent relationships in nuclear DNA(Irwin, 2002; Ballard & Whitlock, 2004; Chan & Levin, 2005; Edwards et al., 2005;
Jennings & Edwards, 2005; Shaffer & Thomson, 2007). We predict that distinct
phenotypic groupings will increasingly be supported by multilocus nuclear-DNA surveys
based on AFLP (e.g. Bensch, Åkesson & Irwin, 2002), single nucleotide polymorphisms(SNPs; e.g. Shaffer & Thompson, 2007), or multiple gene sequences (e.g. Jennings &Edwards, 2005), even when mtDNA does not differ between groups.

ACKNOWLEDGEMENTS
We are grateful to Trevor Price for assisting with the development of this project, as wellas much helpful discussion. We thank Mikhail Markovetz for collecting blood samples
from the Baltic Sea region, and Andrei Lissovskii (Zoological Museum of Moscow
University) and Sergei Drovetskii (University of Alaska) for assistance with fieldsampling and advice regarding molecular analysis. We are grateful to the following
individuals and the museums they represent for providing tissue samples for this work:15

Sharon Birks (Burke Museum, University of Washington), Robert Zink (Bell Museum,
University of Minnesota), David Willard (Field Museum of Natural History, Chicago),
Per Alström (Swedish Museum of Natural History, Stockholm), and Jon Fjeldså
(Zoological Museum, University of Copenhagen). Sincere thanks are extended to David
Toews, Alan Brelsford and Jason Weir (University of the British Columbia, Vancouver)
for technical support and advice regarding the molecular analysis, and to Alan Brelsford,
Carol Irwin, Jessica Irwin, Johan Lindell, and anonymous referees for comments on the
manuscript. For financial support we thank the U.S. Civilian Research and Development
Foundation (grant № RUB1-2630-MO-04), the Natural Sciences and Engineering
Research Council of Canada, the Canadian Foundation for Innovation, and the British
Columbia Knowledge Development Fund.

REFERENCES
Adams JM, Faure H, eds. 1997. Review and atlas of palaeovegetation: preliminary land
ecosystem maps of the world since the Last Glacial Maximum. Oak Ridge
National Laboratory, TN, USA. Accessed online (7 August, 2008):
http://www.esd.ornl.gov/projects/qen/adams1.html
Alström P, Olsson U, Lei F, Wang H-t, Gao W, Sundberg P. 2007. Phylogeny andclassification of the Old World Emberizini (Aves, Passeriformes). MolecularPhylogenetics and Evolution 47: 960-973.
Arnold ML. 1997. Natural hybridization and evolution. Oxford: Oxford University
Press.
Avise JC. 2004. Molecular markers, natural history, and evolution. Sunderland,
Massachusetts: Sinauer Associates.
Ballard JWO, Whitlock MC. 2004. The incomplete natural history of mitochondria.
Molecular Ecology 13: 729-744.
Barton NH, Hewitt GM. 1985. Analysis of hybrid zones. Annual Review of Ecology and
Systematics 16: 113-148.
Barton NH, Hewitt GM. 1989. Adaptation, speciation and hybrid zones. Nature 341:
497-503.
Bazin E, Glémin S, Galtier N. 2006. Population size does not influence mitochondrial
genetic diversity in animals. Science 312: 570-572.
Bensch S, Åkesson M. 2005. Ten years of AFLP in ecology and evolution: why so few
animals? Molecular Ecology 14: 2899-2914.
Bensch S, Åkesson S, Irwin DE. 2002. The use of AFLP to find an informative SNP:
genetic differences across a migratory divide in willow warblers. Molecular
Ecology 11: 2359-2366.
Byers C, Curson J, Olsson U. 1995. Sparrows and buntings: a guide to the sparrows
and buntings of North America and the world. New York: Houghton Mifflin.
Chan KMA, Levin SA. 2005. Leaky prezygotic isolation and porous genomes: rapid
introgression of maternally inherited DNA. Evolution 59: 720-729.
Clement M, Posada D, Crandall K. 2000. TCS: a computer program to estimate genegenealogies. Molecular Ecology 9: 1657-1660.

16

Cramp S, Perrins CM, eds. 1994. The birds of the western Palearctic. Vol. 9. Oxford:
Oxford University Press.
Deffontaine V, Libois R, Kotlik P, Sommer R, Nieberding C, Paradis E, Searle JB,
Michaux JR. 2005. Beyond the Mediterranean peninsulas: evidence of central
European glacial refugia for a temperate forest mammal species, the bank vole
(Clethrionomys glareolus). Molecular Ecology 14: 1727-1739.
Drovetski SV, Zink RM, Fadeev IV, Nesterov EV, Koblik EA, Red’kin YA, Rohwer
S. 2004. Mitochondrial phylogeny of Locustella and related genera. Journal of
Avian Biology 35: 105-110.
Dowling DK, Friburg U, Lindell J. 2008. Evolutionary implications of non-neutral
mitochondrial genetic variation. Trends in Ecology and Evolution 23: 546-554.
Edwards SV, Kingan SB, Calkins JD, Balakrishnan CN, Jennings WB, Swanson
WJ, Sorensen MD. 2005. Speciation in birds: Genes, geography, and sexual
selection. Proceedings of the National Academy of Sciences of the U. S. A. 102:
6550-6557.
Excoffier L, Laval G, Schneider S. 2005. ARLEQUIN ver. 3.0: An integrated software
package for population genetics data analysis. Evolutionary Bioinformatics
Online 1: 47-50.
Excoffier L, Laval G, Schneider S. 2006. ARLEQUIN ver 3.1 user manual. URL:
http://cmpg.unibe.ch/software/arlequin3
Falush D, Stephens M, Pritchard JK. 2007. Inference of population structure using
multilocus genotype data: dominant markers and null alleles. Molecular Ecology
Notes 7: 574-578.
Frenzel B. 1968. The Pleistocene vegetation of northern Eurasia. Science 161: 637-649.
Fridolfsson A-K, Ellegren H. 1999. A simple and universal method for molecularsexing of non-ratite birds. Journal of Avian Biology 30: 116-121.
Fu Y-X. 1997. Statistical tests of neutrality of mutations against population growth,
hitchhiking and background selection. Genetics 147: 915-925.
Funk DJ, Omland KE. 2003. Species-level paraphyly and polyphyly: frequency, causes,
and consequences, with insights from animal mitochondrial DNA. Annual Review
of Ecology, Evolution, and Systematics 34: 397-423.
Good JM, Demboski JR, Nagorsen DW, Sullivan J. 2003. Phylogeography andintrogressive hybridization: chipmunks (genus Tamias) in the northern Rocky
Mountains. Evolution 57: 1900-1916.
Good JM, Hird S, Reid N, Demboski JR, Steppan SJ, Martin-Nims TR, Sullivan J.
2008. Ancient hybridization and mitochondrial capture between two species ofchipmunks. Molecular Ecology 17: 1313-1327.
Grant WS, Spies IB, Canino MF. 2006. Biogeographic evidence for selection on
mitochondrial DNA in North Pacific walleye pollock Theragra chalcogramma.
Journal of Heredity 97: 571-580.
Haffer J. 1989. Parapatrische Vogelarten der paläarktischen Region. Journal fürOrnithologie 130: 475-512.
Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:
95-98.

17

Helbig AJ, Seibold I, Kocum A, Liebers D, Irwin J, Bergmanis U, Meyburg BU,
Scheller W, Stubbe M, Bensch S. 2005. Genetic differentiation andhybridization between greater and lesser spotted eagles (Accipitriformes: Aquilaclanga, A. pomarina). Journal of Ornithology 146: 226-234.
Hey J. 2007. Using the IMA program. Distributed by the author:
http://lifesci.rutgers.edu/~heylab/heylabsoftware.htm
Hey J, Nielsen R. 2007. Integration with the Felsenstein equation for improved Markov
chain Monte Carlo methods in population genetics. Proceedings of the National
Academy of Sciences of the U. S. A. 104: 2785-2790.
Irwin DE. 2002. Phylogeographic breaks without geographic barriers to gene flow.
Evolution 56: 2383-2394.
Irwin DE, Bensch S, Irwin JH, Price TD. 2005. Speciation by distance in a ring
species. Science 307: 414-416.
Irwin DE, Bensch S, Price TD. 2001. Speciation in a ring. Nature 409: 333-337.
Irwin DE, Irwin JH. 2005. Siberian migratory divides: the role of seasonal migration in
speciation. In: Greenberg R, Marra PP, eds. Birds of two worlds: the ecology andevolution of migratory birds. Baltimore, Maryland: Johns Hopkins University
Press, 27-40.
Jennings WB, Edwards SV. 2005. Speciational history of Australian grass finches(Poephila) inferred from thirty gene trees. Evolution 59: 2033-2047.
Kondo B, Peters JL, Rosensteel BB, Omland KE. 2008. Coalescent analyses ofmultiple loci support a new route to speciation in birds. Evolution 62: 1182-1190.
LI-COR Biosciences. 2003. Applications manual: Model 4300 DNA Analyzer. Lincoln,
Nebraska: LI-COR Biosciences.
Lovette IJ. 2004. Mitochondrial dating and mixed-support for the “2% rule” in birds.
Auk 121: 1-6.
Lynch M, Milligan B. 1994. Analysis of population genetic structure with RAPD
markers. Molecular Ecology 3: 91-99.
Mallet J. 2005. Hybridization as an invasion of the genome. Trends in Ecology and
Evolution 20: 229-237.
Melo-Ferreira J, Boursot P, Suchentrunk F, Ferrand N, Alves PC. 2005. Invasion
from the cold past: extensive introgression of mountain hare (Lepus timidus)
mitochondrial DNA into three other hare species in northern Iberia. Molecular
Ecology 14: 2459-2464.
Nazarenko AA. 1990. [Avifaunal interchange between south and north Asia at theeastern periphery of the continent: the last glacial-interglacial cycle.] Zhurnal
Obshchei Biologii 51: 89-106 (in Russian).
Newton I. 2003. The speciation and biogeography of birds. London: Academic Press.
Nielsen R, Wakeley J. 2001. Distinguishing migration from isolation. A Markov chain
Monte Carlo approach. Genetics 158: 885-896.
Panov EN. 1989. [Hybridization and ethological isolation in birds.] Moscow: Nauka (inRussian).
Panov EN, Rubtsov AS, Monzikov DG. 2003. Hybridization between yellowhammer
and pine bunting in Russia. Dutch Birding 25: 17-31.

18

Panov EN, Rubtsov AS, Mordkovich MV. 2007. [New data on interrelationships of two
bunting species (Emberiza citrinella, E. leucocephala) interbreeding in zone of
their ranges overlap.] Zoologicheskiĭ Zhurnal 86: 1362-1378 (in Russian).
Parchman TL, Benkman CW, Britch SC. 2006. Patterns of genetic variation in theadaptive radiation of New World crossbills (Aves: Loxia). Molecular Ecology 15:
1873-1887.
Plötner J, Uzzell T, Beerli P, Spolsky C, Ohst T, Litvinchuk SN, Guex G-D, Reyer
H-U, Hotz H. 2008. Widespread unidirectional transfer of mitochondrial DNA: acase in western Palaearctic water frogs. Journal of Evolutionary Biology 21: 668681.
Price, T. 2008. Speciation in birds. Greenwood Village, Colorado: Roberts andCompany.
Pritchard JK, Stephens M, Donnelly P. 2000. Inference of population structure using
multilocus genotype data. Genetics 155: 945-959.
Qvarnström A, Bailey RI. 2009. Speciation through evolution of sex-linked genes.
Heredity 102: 4-15.
R Development Core Team. 2006. R: a language and environment for statisticalcomputing. Vienna, Austria: R Foundation for Statistical Computing. Available
from URL: http://www.R-project.org.
Ravkin YS. 1973. [The birds of north-eastern Altai.] Novosibirsk (in Russian).
Rogacheva H. 1992. The birds of central Siberia. Husum, Germany: Husum Druck- undVerlagsgesellschaft.
Rohwer S, Bermingham E, Wood C. 2001. Plumage and mitochondrial DNA haplotype
variation across a moving hybrid zone. Evolution 55: 405-422.
Rubinoff D, Holland BS. 2005. Between two extremes: mitochondrial DNA is neither
the panacea nor the nemesis of phylogenetic and taxonomic inference. Systematic
Biology 54: 952-961.
Rubtsov AS. 2007. [Variation in songs of yellowhammer and pine bunting (Emberizacitrinella, E. leucocephala) as an evidence for the population structure dynamics
and evolutionary history of species.] Zoologicheskiĭ. Zhurnal 86: 863-876 (inRussian).
Sæther B-E, Lande R, Engen S, Weimerskirch H, Lillegård M, Altwegg R, Becker
PH, Bregnballe T, Brommer JE, McCleery RH, Merilä J, Nyholm E, Rendell
W, Robertson RR, Tryjanowski P, Visser ME. 2005. Generation time andtemporal scaling of bird population dynamics. Nature 436: 99-102.
Scheet P, Stephens M. 2006. A fast and flexible statistical model for large-scale
population genotype data: Applications to inferring missing genotypes andhaplotypic phase. American Journal Human Genetics 78: 629-644.
Schmidt HA, Strimmer K, Vingron M, von Haeseler A. 2002. TREE-PUZZLE:
maximum likelihood phylogenetic analysis using quartets and parallel computing.
Bioinformatics 18: 502-504.
Seutin G, White BN, Boag PT. 1991. Preservation of avian blood and tissue samples for
DNA analyses. Canadian Journal of Zoology 69: 82-90.
Shaffer HB, Thomson RC. 2007. Delimiting species in recent radiations. Systematic
Biology 56: 896-906.
Slatkin M. 1996. A correction to the exact test based on the Ewens sampling distribution.

19

Genetical Research 68: 259-260.
Sorenson MD, Quinn TW. 1998. Numts: a challenge for avian systematics andpopulation biology. Auk 115: 214-221.
Svensson EI, Kristoffersen L, Oskarsson K, Bensch S. 2004. Molecular population
divergence and sexual selection on morphology in the banded demoiselle(Calopteryx splendens). Heredity 93: 423-433.
Tajima F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA
polymorphism. Genetics 123: 585-595.
Takahata N, Slatkin M. 1984. Mitochondrial gene flow. Proceedings of the National
Academy of Sciences of the U. S. A. 81: 1764-1767.
Toews DPL, Irwin DE. 2008. Cryptic speciation in a Holarctic passerine revealed bygenetic and bioacoustic analyses. Molecular Ecology 17: 2691-2705.
Vekemans X. 2002. AFLP-SURV version 1.0. Distributed by the author. Laboratoire deGénétique et Ecologie Végétale, Université Libre de Bruxelles, Belgium.
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J,
Peleman J, Kuiper M, Zabeau M. 1995. AFLP: A new technique for DNA
fingerprinting. Nucleic Acids Research 23: 4405-4414.
Weckstein JD, Zink RM, Blackwell-Rago RC, Nelson DA. 2001. Anomalous variationin mitochondrial genomes of White-crowned (Zonotrichia leucophrys) andGolden-crowned (Z. atricapilla) Sparrows: pseudogenes, hybridization, orincomplete lineage sorting? The Auk 118: 231-236.
Weir JT. 2006. Divergent patterns of species accumulation in lowland and highland
neotropical birds. Evolution 60: 842-855.
Weir JT, Schluter D. 2007. The latitudinal gradient in recent speciation and extinction
rates in birds and mammals. Science 315: 1928-1933.
Weir JT, Schluter D. 2008. Calibrating the avian molecular clock. Molecular Ecology
17: 2321-2328.
Whitlock MC. 2008. Evolutionary inference from QST. Molecular Ecology 17: 18851896.
Won, YJ, Hey J. 2005. Divergence population genetics of chimpanzees. Molecular
Biology and Evolution 22: 297-307.
Wu, C-I. 2001. The genic view of the process of speciation. Journal of Evolutionary
Biology 14: 851-865.
Zhivotovsky LA. 1999. Estimating population structure in diploids with multilocus
dominant DNA markers. Molecular Ecology 8: 907-913.
Zink RM. 2004. The role of subspecies in obscuring avian biological diversity andmisleading conservation policy. Proceedings of the Royal Society of London B
271: 561-564.
Zink RM, Barrowclough GF. 2008. Mitochondrial DNA under siege in avian
phylogeography. Molecular Ecology 17: 2107-2121.

20

Table 1. Pairwise genetic distances (see Materials and Methods for details) between the
10 species of Emberiza genus for mtDNA (ND2 gene, below the diagonal) and Zchromosome (CHD1Z intron, above the diagonal). For yellowhammers (E.
citrinella) and pine buntings (E. leucocephalos), haplotypes indicated withasterisks in Fig. 2 were used to calculate genetic distances.
1. E. calandra
2. E. tristrami
3. E. aureola
4. E. spodocephala
5. E. cioides
6. E. hortulana
7. E. cirlus
8. E. stewarti
9. E. citrinella
10. E. leucocephalos

1
0.394
0.419
0.463
0.249
0.313
0.346
0.265
0.307
0.307

2

3

4

5

0.140
0.126
0.404
0.505
0.524
0.466
0.490
0.494

0.117
0.456
0.545
0.510
0.471
0.562
0.566

0.466
0.545
0.545
0.490
0.558
0.563

0.234
0.302
0.243
0.275
0.282

6
0.023

7
0.031

8
0.028

9
0.032

10
0.028

0.308
0.213
0.243
0.239

0.016
0.179
0.216
0.222

0.012
0.010
0.084
0.082

0.015
0.010
0.010
0.005

0.012
0.010
0.007
0.007
-

Table 2. Identities of the 20 informative AFLP fragments that differ in frequency (seeMaterials and Methods for criteria) between allopatric yellowhammers and pine buntings.
EcoRI primer1
MseI primer2
Approximate(NNN-3’)(NNN-3’)fragment length
AAC
CTT
112
AAC
CTT
135
ACA
CTA
231
ACA
CTA
406
ACC
CAA
65
ACC
CAA
223
ACC
CAA
323
ACC
CAT
468
ACT
CAA
64
ACT
CAA
225
ACT
CAA
226
ACT
CAA
236
ACT
CAA
251
ACT
CAT
176
ACT
CAT
182
ACT
CAT
279
ACT
CAT
303
AGC
CTT
245
AGG
CTA
187
AGG
CTA
552
1
EcoRI primer: 5’-GACTGCGTACCAATTCNNN-3’
2
MseI primer: 5’-GATGAGTCCTGAGTAANNN-3’.

Frequency inyellowhammers
0.19
0.62
0.08
0.27
0.31
0.38
0.00
0.19
0.46
0.31
0.27
0.23
0.23
0.38
0.15
0.31
0.31
0.58
0.69
0.15

Frequency in pinebuntings
0.73
0.07
0.53
0.53
0.53
0.67
1.00
0.80
1.00
0.60
0.80
0.60
0.67
1.00
0.60
0.53
0.60
0.20
0.13
0.67

21

Figure 1. Geographic distribution of yellowhammers (Emberiza citrinella; solid lines)
and pine buntings (E. leucocephalos; dashed lines), which hybridize extensively intheir area of overlap in western and central Siberia. Sampling sites are indicated bysmall circles (one sample) or large circles (multiple samples, with numbers
indicating sample sizes). Phenotypic yellowhammers are indicated by light grey
circles, phenotypic hybrids by dark grey, and phenotypic pine buntings by black.

22

Figure 2. Minimum spanning network showing the relationships among (a) ND2
haplotypes and (b) CHD1Z haplotypes of allopatric yellowhammers (light gray) and pinebuntings (dark gray). In (a), circle area is proportional to the number of samples with that
haplotype, with the smallest size corresponding to a single sample, and small dots
representing missing haplotypes. Asterisks indicate the two most distantly related
haplotypes in the two species; these haplotypes were used in the interspecific phylogeny(Fig. 3a) and to calculate maximum genetic distance (Table 1). In (b), the circle area is
proportional to the number of haplotypes of that sequence (with a single individual
represented by GTCGTT). The light and dark areas of the circles represent the relative
frequencies of particular haplotypes in yellowhammer and pine buntings respectively.
Missing haplotypes are indicated by small dots. Most haplotypes could have been
produced by recombination of the two most common haplotypes (GTTGTT, which ismore common in yellowhammers; and AOCGCA, which is more common in pinebuntings). These two most common haplotypes (marked with asterisks) were used in thephylogenetic reconstruction in Figure 3b.

23

Figure 3. Phylogenetic trees of Emberiza species based on (a) ND2 and (b) CHD1Z.
Numbers to the left of nodes represent quartet puzzling support values, with numbers
above 90 indicating very strong confidence in the clade joined at that node. Scale bars
show expected rates of nucleotide divergence between two lineages, under the HKY + Γ
substitution model (see Materials and Methods). The grey areas between E. citrinella andE. leucocephalos denote haplotype sharing between the two species, presumably due toongoing gene flow. For these two species, highly divergent haplotypes were used torepresent the two species in the phylogeny (indicated with asterisks in figure 2; see
Results).

24

Figure 4. Variation in AFLP signatures (based on AFLP dataset 1) among allopatricyellowhammers (open circles) and pine buntings (filled diamonds), illustrated using
principal components analysis. All variable markers (63 total, not chosen based on theirpattern of variation but simply included if they were variable) from 5 primer
combinations were used (these were only determined for allopatric samples; seeMaterials and Methods). PC1 explains 11.4% of the variation, while PC2 explains 8.7%.
There is no overlap between the two species in their AFLP signatures, and the differencein PC1 is highly significant (t-test: t26 = 13.57, P < 10-12).

Figure 5. Distribution of FST values between allopatric yellowhammers and allopatricpine buntings for 63 variable AFLP markers from 5 primer combinations (AFLP dataset1). FST was calculated based on allele frequencies estimated using AFLP-SURV(Vekemans 2002). Most markers differ little in frequency between the species, but asmall percentage show a strong difference (7 out of 63, or 11%, show an FST larger than
0.1).
25

Figure 6. Genetic variation among yellowhammers, pine buntings, and phenotypic
hybrids. The primary axis of variation (PC1) in a principal components analysis of 20
informative AFLP markers (AFLP dataset 2) is shown for seven phenotypic categories(Panov et al. 2003), with each diamond representing a single individual. Group numbers
along the horizontal axis indicate 1) allopatric yellowhammers, 2) yellowhammers fromthe contact zone, 3) yellow hybrids, 4) white hybrids, 5) pine buntings with slight hybrid
phenotypes, 6) pine buntings in or near the contact zone, and 7) allopatric pine buntings.
PC1 explains 20.1 % of the variance, and varies significantly among phenotypic groups(ANOVA: F = 261.64, df = 1 and 63, P < 10-15). AFLP variation clearly distinguishes thetwo species (t-test between allopatric groups: t39 = -19.56, P < 10-15), and hybrids have arange of intermediate values.

26