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Extensive introgressive hybridization within the northern oriole group (Genus Icterus) revealed by three-species isolation with migration analysis


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Until recently, studies of divergence and gene flow among closely-related taxa were generally limited to pairs of sister taxa. However, organisms frequently exchange genes with other non-sister taxa. The "northern oriole" group within genus Icterus exemplifies this problem. This group involves the extensively studied hybrid zone between Baltimore oriole (Icterus galbula) and Bullock's oriole (I. bullockii), an alleged hybrid zone between I. bullockii and black-backed oriole (I. abeillei), and likely mtDNA introgression between I. galbula and I. abeillei. Here, we examine the divergence population genetics of the entire northern oriole group using a multipopulation Isolation-with-Migration (IM) model. In accordance with Haldane's rule, nuclear loci introgress extensively beyond the I. galbula-I. bullockii hybrid zone, while mtDNA does not. We found no evidence of introgression between I. bullockii and I. abeillei or between I. galbula and I. abeillei when all three species were analyzed together in a three-population model. However, traditional pairwise analysis suggested some nuclear introgression from I. abeillei into I. galbula, probably reflecting genetic contributions from I. bullockii unaccounted for in a two-population model. Thus, only by including all members of this group in the analysis was it possible to rigorously estimate the level of gene flow among these three closely related species.
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Extensive introgressive hybridization within the northern
oriole group (Genus Icterus) revealed by three-species
isolation with migration analysis
Frode Jacobsen & Kevin E. Omland
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA
Allele sharing, coalescent, Icterus, IMa2,
incomplete lineage sorting, introgression,
multipopulation, northern orioles.
Frode Jacobsen, Department of Biological
Sciences, University of Maryland Baltimore
County, 1000 Hilltop Circle, Baltimore, MD
21250, USA. Tel: +1 410 455 1704;
Fax: +1 410 455 3875; E-mail:
Funding Information
This work was funded by National Science
Foundation grants to K.E.O. (DEB-0347083
and DEB-1119506), and Maryland
Ornithological Society Avian Research Grants
and an American Ornithologist’s Union
Research Grant to F.J.
Received: 20 June 2012; Revised: 30 July
2012; Accepted: 31 July 2012
doi: 10.1002/ece3.365
Until recently, studies of divergence and gene flow among closely-related taxa
were generally limited to pairs of sister taxa. However, organisms frequently
exchange genes with other non-sister taxa. The “northern oriole” group within
genus Icterus exemplifies this problem. This group involves the extensively stud-
ied hybrid zone between Baltimore oriole (Icterus galbula) and Bullock’s oriole
(I. bullockii), an alleged hybrid zone between I. bullockii and black-backed oriole
(I. abeillei), and likely mtDNA introgression between I. galbula and I. abeillei.
Here, we examine the divergence population genetics of the entire northern
oriole group using a multipopulation Isolation-with-Migration (IM) model. In
accordance with Haldane’s rule, nuclear loci introgress extensively beyond the
I. galbulaI. bullockii hybrid zone, while mtDNA does not. We found no
evidence of introgression between I. bullockii and I. abeillei or between I. galbula
and I. abeillei when all three species were analyzed together in a three-population
model. However, traditional pairwise analysis suggested some nuclear introgres-
sion from I. abeillei into I. galbula, probably reflecting genetic contributions
from I. bullockii unaccounted for in a two-population model. Thus, only by
including all members of this group in the analysis was it possible to rigorously
estimate the level of gene flow among these three closely related species.
The study of phylogeography and population divergence
is challenged by the difficulty of distinguishing between
shared retained ancestral polymorphism and introgressed
alleles (Nielsen and Wakeley 2001). The much larger
effective population size of nuclear DNA relative to
mtDNA slows the rate of lineage sorting and the speed at
which reciprocal monophyly is achieved at the nuclear
level (Avise 2004). Slow and stochastic lineage sorting
thus causes a pattern of allele sharing among species that
can seem similar to patterns caused by gene flow through
introgressive hybridization, even between long-divergent
lineages (Hudson and Turelli 2003). Multiple unlinked
loci and coalescent-based “divergence population genet-
ics” methods are needed to distinguish between these two
processes (e.g., Kliman et al. 2000; Machado et al. 2002).
The Isolation-with-Migration (IM) model (Hey and
Nielsen 2004, 2007) provides a powerful tool for the study
of population divergence and for testing different specia-
tion models and demographic scenarios (Hey 2006; Peters
et al. 2007; Kondo et al. 2008; Pinho and Hey 2010).
Shared alleles allow multilocus coalescent approaches such
as IM to reconstruct the divergence history of closely
related species (Nielsen and Wakeley 2001; Hey and Niel-
sen 2004, 2007). The original IM model was designed for
two-population studies between sister taxa that have not
exchanged genes with other taxa since they split (Hey and
Nielsen 2004, 2007). However, organisms frequently
exchange genes with other non-sister taxa (Arnold 2006;
Nosil 2008; Petit and Excoffier 2009). Genetic contribu-
tions from other source populations not included in the
analysis (i.e., “ghost” populations) could thus severely bias
or invalidate inference about the history of closely related
ª2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited and is not used for commercial purposes.
taxa (Beerli 2004; Slatkin 2005; Strasburg and Rieseberg
2010). Thus, until recently, IM analyses were limited to
pairs of taxa that share more recent common ancestry and
gene flow with each other. However, this limitation was
removed with the release of IMa2, allowing the simulta-
neous consideration of up to ten populations granted a
population tree (i.e., phylogeny) is available for the taxa
involved (IMa2: Hey 2010b).
In this study, we used IMa2 to analyze the three species
in the “northern oriole” group within the New World
orioles (Jacobsen and Omland 2011; Fig. 1). This group
includes the well-known hybrid zone between the eastern
Baltimore oriole (Icterus galbula) and Bullock’s oriole
(I. bullockii) (Sutton 1938, 1968; Sibley and Short 1964;
Rising 1969, 1970, 1983, 1996; Corbin et al. 1979). I. galbula
breeds across the eastern United States south to Louisiana,
and across most of Canada from Alberta in the west to
Nova Scotia in the east (Fig. 2). The breeding range of
I. bullockii extends across western North America from
British Columbia, Canada in the north to Durango,
Mexico in the south. Both I. galbula and I. bullockii are
long-distance migrants and mainly overwinter in southern
Mexico and Central America (Rising et al. 1998, 1999).
Their winter ranges overlap with the range of the third
member of the group, I. abeillei, a Mexican endemic lim-
ited to the central Mexican plateau (Jaramillo and Burke
1999) and traditionally considered a close relative and
subspecies within I. bullockii due to their overall pheno-
typic similarity (Jaramillo and Burke 1999).
The parapatric breeding ranges of I. galbula and I. bull-
ockii overlap along a long and narrow transect (150 km
in Kansas, Allen 2002) that stretches over 1800 km from
southern Alberta to central Texas (Rising 1983). The two
orioles interbreed extensively in this contact zone, which
falls within the “Rocky Mountain-Great Plains” suture
zone (Remington et al. 1968), a hotspot of avian hybrid
zones between eastern and western species pairs (e.g.,
Carling and Brumfield 2008; Flockhart and Wiebe 2009;
Mettler and Spellman 2009). Documentation of viable
hybrid offspring within this contact zone (Sutton 1938;
Sibley and Short 1964; Sutton 1968; Rising 1970) led to
the lumping of I. galbula and I. bullockii (including
I. abeillei) into a single species, the northern oriole (AOU
1973). Later studies found that the I.galbulaI. bullockii
hybrid zone was stable (Rohwer and Manning 1990; Roh-
wer and Johnson 1992; Rising 1996; Allen 2002), and full
species status was restored for all three members of the
group (AOU 1995). Hybridization has never been firmly
documented between I. abeillei and the other two mem-
bers of the group, but there is some evidence suggesting
hybridization with I. bullockii where their ranges abut in
northern Durango, Mexico (Miller 1906; but see Rising
1973). We are unaware of any documented instances of
hybridization between members of the northern oriole
group and other oriole species.
Despite the extensive research on the northern oriole
group, we have limited knowledge about the extent of
nuclear gene flow within this species complex. Allen
(2002) examined clinal variation of phenotypic and
molecular markers across the I. galbulaI. bullockii hybrid
zone in Kansas, and found that neither male plumage
Figure 1. Adult males of the three species in the northern oriole
group; (a) Baltimore oriole Icterus galbula (photo: Frode Jacobsen), (b)
Bullock’s oriole I. bullockii (photo: Frode Jacobsen), and (c) black-
backed oriole I. abeillei (photo: Jonathan Hiley)
2©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
traits nor mtDNA haplotypes introgressed beyond the
zone of contact. These findings were recently corrobo-
rated by Carling et al. (2011), who estimated the
mitochondrial cline to be 328 km wide. In contrast, of
152 neutral AFLP markers examined by Allen (2002),
there were no fixed differences between the two species,
and only ten AFLP bands differed more than 40% in
frequency across the hybrid zone. Allen (2002) interpreted
this apparent lack of differentiation in AFLP markers
between I. galbula and I. bullockii as evidence of unham-
pered nuclear introgression since the two species came
into secondary contact. However, such extensive allele
sharing among closely related species could also result
from the slow sorting of retained ancestral polymorphism
(Moore 1995; Hudson and Turelli 2003).
The goals of this study were to (1) estimate levels of
introgression among all three species in the northern oriole
group while accounting for their recent shared ancestry
using a three-population IMa2 model and (2) examine
the effects of genetic exchange with unsampled taxa in
two-population analyses.
Material and Methods
Taxonomic sampling
We sought a broad representation from throughout the
ranges of all three species whilst avoiding known areas of
sympatry (see Appendix1 for sampling locations). Our
sampling scheme provided a total of 21 to 24 individuals
of each of the three species, I. abeillei,I. bullockii and
I. galbula. In addition, a single individual of western
meadowlark (Sturnella neglecta) was included as an out-
group taxon.
Multilocus nuclear data
This study included sequence data from eight intron-
spanning loci that map to six autosomes and the Z sex
chromosome in the zebra finch (Taeniopygia guttata)
genome (see Table 1 for details). The two Z-linked loci
are well separated on the zebra finch Z chromosome
(>7 Mb apart) and were therefore treated as independent
¨m et al. 2006, 2010). All eight loci were ampli-
fied and sequenced following procedures described in
previous phylogenetic studies on Icterus (Jacobsen et al.
2010; Jacobsen and Omland 2011). Resulting DNA chro-
matograms were aligned and edited in SEQUENCHER
4.7 (Gene Codes Corp., Ann Arbor, MI). Edited sequences
for all loci were imported into MEGA5 (Tamura et al.
2011) and aligned using MUSCLE (Edgar 2004) with final
manual adjustments. The haplotypes of individuals het-
erozygous at more than one position at a given locus
were determined using the software PHASE v.2.1.1
(Stephens et al. 2001; Stephens and Scheet 2005), with
the following parameter settings: burn-in =1000, number
of iterations =10,000, and thinning interval =1. Each
analysis was repeated 10 times using different random
starting seed and the run that received the highest
log-likelihood was used to infer the haplotypes. To avoid
systematic bias in downstream coalescent analyses, all
haplotypes resolved at a probability of 0.5 or higher were
Breeding distribution northern orioles
Baltimore oriole
(I. galbula)
Bullock’s oriole
(I. bullockii)
Black-backed oriole
(I. abeillei)
Figure 2. Breeding distributions of the three northern oriole species: Icterus abeillei (light gray), I. bullockii (dark gray), and I. galbula (black).
Hatched areas indicate range overlap between I. bullockii and I. galbula. Digital distribution maps for each species were downloaded from
NatureServe (Ridgely et al. 2007).
©2012 The Authors. Published by Blackwell Publishing Ltd. 3
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
included in the study (Garrick et al. 2010). An ambigu-
ously aligned single-nucleotide repeat (poly-A) region
within locus FGB4 was excluded from the alignment prior
to analyses. Linkage analysis of the two Z-linked loci
(ALDOB5 and BRM15) using HAPLOVIEW v.4.2 (Barrett
et al. 2005) revealed no evidence of LD among polymor-
phic sites (results not shown). We tested for intralocus
recombination using the difference in sums of squares
(DSS) test in TOPALi v.2.5 (Milne et al. 2004), with 500
bootstrap replicates to determine statistical significance. A
significant DSS peak was detected at locus b-ACT2, and
only the largest non-recombinant sequence block (558 bp)
was retained for downstream analysis. Accession numbers
for sequences obtained from previous studies on this group
are listed in Appendix 2. All new sequences collected in
this study have been deposited in GenBank under accession
numbers JX403068-JX403342 and JX412950-JX412955.
Three common measures of nucleotide polymorphism
were calculated using ARLEQUIN v.3.11 (Excoffier and
Lischer 2010): number of haplotypes (h), haplotype
diversity (Hd), and the average number of nucleotide dif-
ferences per site between two sequences (p). Haplotype
networks illustrating the relationships among inferred
alleles were constructed for each locus using the median-
joining algorithm in NETWORK v. (Bandelt et al.
1999, The networks
were edited using NETWORK PUBLISHER v.
Tests for selection were conducted using a multilocus
HKA test (Hudson et al. 1987) implemented in the HKA
program available at heylab/
HeylabSoftware.htm. We chose western meadowlark
(Sturnella neglecta) as outgroup for this test, the most
distant relative within the family Icteridae available for
comparison across the eight loci examined in this study.
Kimura 2P distances between the three northern oriole
species and S. neglecta were calculated using DNASP
v. and Rozas 2009). Statistical significance
of the HKA test was determined through 10,000 coales-
cent simulations based on the number of sampled alleles
and observed levels of polymorphism and divergence
across loci. These simulations also allowed us to conduct
a multilocus test of Tajima’s Dstatistic (Tajima 1989).
Fu’s (1997) Fs and Ramos-Onsins and Rozas (2002)R
neutrality statistics were also calculated using DnaSP.
Confidence intervals and statistical significance of test
values were calculated using 10,000 random permutations
and coalescent simulations.
Multilocus coalescent analyses
We examined patterns of gene flow among all three
members of the northern oriole group using the Bayesian
IM model implemented in the program IMa2 (Hey
2010b), which assumes random mating, constant popula-
tion sizes, selective neutrality, free recombination among
loci and no recombination within loci (Hey and Nielsen
2004, 2007; Hey 2010b).
By default, all model parameters estimated by IMa2 are
scaled to the population mutation rate. The parameter
estimates were converted into demographic units (i.e.,
effective population sizes in number of individuals and
divergence times in years) using a neutral mutation rate
of 1.35*10
substitutions per site per year for autosomal
loci (Ellegren 2007) and 1.45*10
substitutions per site
per year for Z-linked loci (Axelsson et al. 2004; Ellegren
2007). To reflect the different modes of inheritance, auto-
somal (1.0) and Z-linked (0.75) loci were assigned respec-
tive inheritance scalars in the IMa2 input file. Population
sizes were converted into effective number of individuals
Table 1. Locus information. Chromosome: intron location determined based on BLAST searches of zebra finch reference genome assembly, with
start coordinates for Z-linked loci provided in parentheses; Length: size (in base pairs) of largest non-recombinant alignment segment; N: number
of non-recombinant alleles included in the study.
Chromosome Length
Primer sourceI. abeillei I. bullockii I. galbula
b-ACT2 22 558 24 26 24 (Waltari and Edwards 2002)
a-ENO8 21 252 46 42 46 (Kondo et al. 2008)
FGB4 4 575 24 30 28 (Barker 2004)
GAPDH11 1 327 36 30 30 (Primmer et al. 2002)
RDP2 12 298 36 28 26 (Waltari and Edwards 2002)
TGFb53 575 30 32 28 (Bures
ˇet al. 2002)
ALDOB5 Z (400,071) 253 36 35 24 (Kondo et al. 2008)
BRM15 Z (7,528,458) 362 40 35 36 (Borge et al. 2005)
Full locus information is as follows: Beta-actin gene, intron 2 (b-ACT2); alpha-enolase gene, intron 8 (a-ENO8); fibrinogen B beta polypeptide
gene, intron 4 (FGB4); glyceraldehyde-3-phosphate dehydrogenase gene, intron 11 (GAPDH11); rhodopsin gene, intron 2 (RDP2); and transform-
ing growth factor beta-2, intron 5 (TGFb5); aldolase-B fructose-biphosphate intron 5 (ALDOB5); Brahma protein gene, intron 15 (BRM15).
4©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
using a generation time of 1.7 years, the average age at
reproduction documented for multiple passerine lineages
(Sæther et al. 2005). The actual generation time in orioles
may well be slightly longer, as most oriole species exhibit
delayed plumage maturation, which could delay the onset
of reproduction (for males in particular) (e.g., Flood
1984; Rohwer and Manning 1990; Butcher 1991; Richard-
son and Burke 1999). Effective population sizes reported
in this study are therefore likely conservative estimates of
the actual effective population sizes. The HKY mutation
model was specified for all loci.
Three-population IMa2 analysis
First, we estimated levels of gene flow among all three
species within the northern oriole group simultaneously
in a three-population IMa2 analysis. We conducted sepa-
rate analyses with two alternative population trees: (1) the
nuclear species tree inferred by our recent species tree
analysis of Icterus (Jacobsen and Omland 2011): (I. galbula,
(I. abeillei,I. bullockii)) and (2) the mtDNA gene tree
inferred by Omland et al. (1999): (I. bullockii,(I. abeillei,
I. galbula)).
Short preliminary runs were conducted to determine
appropriate upper bounds of the demographic parame-
ters. Subsequently, two identical long runs of 35*10
MCMC steps with a burnin period of 1*10
steps were
started with different starting seeds and run until
minimum effective sample sizes (ESS) of 200 were
achieved. We assessed convergence by monitoring the
mixing properties of the MCMC during each run and by
ensuring that similar parameter estimates were obtained
from the two independent runs. Acceptable chain mixing
(i.e., absence of long-term trends in plots of L[P] and
tover the course of each run) and low autocorrelations
(<0.1) were achieved by running 100 chains under a
geometric heating scheme (with heating parameters set to
a=0.975 and b =0.75). Population-specific parameter
priors were provided in a separate file. We used exponen-
tial priors on migration rates (optionj7), and determined
appropriate prior distribution means based on the peak
posterior parameter estimates from preliminary runs.
Two-population IMa2 analyses
To assess the effects of violating the crucial assumption of
no allelic contributions from other source (i.e., “ghost”)
populations, we also conducted three separate pairwise
IMa2 analyses of the three northern oriole species. Run
settings were kept unchanged, except for slight adjustment
of parameter priors where posterior density distributions
exceeded the upper bounds determined by preliminary
runs. Each MCMC (M) mode analysis was replicated
using different random seeds to ensure convergence onto
similar posterior distributions, using the same criteria for
assessing convergence as described above. Finally, we con-
ducted nested model testing by running IMa2 in “Load
genealogies” (L) mode to specifically compare the fit of
reduced models that do not allow for post divergence
gene flow to the full IMa2 model (i.e., including all six
parameters Θ
, t). This was performed
by sampling 2.5*10
genealogies evenly from the two
MCMC runs performed on each population pair and
compared the log-likelihood scores of all possible nested
models provided in a separate file in the IMa2 distribu-
tion package.
Polymorphism and divergence
The three species within the northern oriole group were
polymorphic at all loci used in this study, except for
I. abeillei that was monomorphic at ALDOB5. Sample
sizes and data for individual loci are summarized in
Table 1. The three measures of sequence polymorphism
were all lower in I. abeillei than in I. bullockii and I. galbula
(Table 2), but not significantly so (single factor ANOVA:
Table 2. Locus-specific estimates of number of haplotypes (h), haplotype diversity (Hd), and Jukes & Cantor nucleotide diversity (p).
I. abeillei I. bullockii I. galbula
Locus h Hd phHd phHd p
b-ACT2 5 0.656 0.00147 4 0.397 0.00094 4 0.239 0.00060
a-ENO8 7 0.283 0.00136 9 0.490 0.00224 9 0.481 0.00234
FGB4 10 0.786 0.00300 20 0.956 0.00741 15 0.942 0.00603
GAPDH11 8 0.830 0.00472 8 0.823 0.00492 14 0.883 0.00590
RDP2 7 0.711 0.00385 7 0.804 0.00438 3 0.578 0.00216
TGFb53 0.393 0.00087 6 0.585 0.00213 14 0.923 0.00430
ALDOB5 1 0 0 4 0.395 0.00217 3 0.507 0.00372
BRM15 3 0.145 0.00068 4 0.217 0.00062 2 0.413 0.00114
©2012 The Authors. Published by Blackwell Publishing Ltd. 5
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
h: F
=0.656, P=0.529; Hd: F
=0.585, P=0.566;
=0.943, P=0.405; respectively). The differences
in mean genetic diversity between I. abeillei and the two
other members of the group are mainly driven by the lack
of sequence variation at the sex-linked locus ALDOB5
(Table 2).
The haplotype networks revealed extensive allele shar-
ing and few fixed differences among the three northern
oriole species (Fig. 3). Some loci were characterized by a
distinct star-shaped topology (e.g., a-ENO8,ALDOB5 and
BRM15), with a common central (and presumed
ancestral) haplotype surrounded by several rare, derived
haplotypes one or two mutational steps away from the
ancestral haplotype (Fig. 3b,g,h).
The three species showed varying degrees of genetic dif-
ferentiation across loci, but generally exhibited extensive
haplotype sharing across loci (Table 3, also see Fig. 3).
Strongest differentiation was observed between the highly
disjunct I. abeillei and I. galbula, with highly significant
values at six of the eight loci examined. The two
widely hybridizing I. bullockii and I. galbula were strongly
differentiated at only half (4/8) of the loci examined. The
(a) β-ACT2 (b) α-ENO8
(c) FGB4
(d) GAPDH11
(e) RDP2 (f) TGFβ5
(g) ALDOB5
(h) BRM15
I. abeillei
I. bullockii
I. galbula
Figure 3. Haplotype networks of eight nuclear intron loci (6 autosomal +2 Z-linked) revealing extensive haplotype sharing among the three
northern orioles: Icterus abeillei (white), I. bullockii (gray), and I. galbula (black). Each circle corresponds to a unique haplotype and the size of
each circle reflect the relative total sample size of each haplotype. Small open circles in (c) indicate unsampled/extinct haplotypes, and
perpendicular bars along lines connecting haplotypes indicate additional mutations distinguishing neighboring haplotypes.
6©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
two sister species, I. abeillei and I. bullockii, were only
weakly differentiated and only two loci (FGB4 and
ALDO5) remained significant after Bonferroni corrections
(Table 3).
The multilocus HKA test indicated no significant
departures from neutrality in our dataset. The multilocus
Tajima’s Dtest was significant only for I. bullockii,a
result that is mainly driven by significantly more negative
than expected Dvalues at loci a-ENO8 and BRM15
(Table 4). Fs values were negative at the majority of loci,
indicative of recent population growth, but this statistic
deviated only significantly from neutral expectations at
less than half the loci following Bonferroni corrections
(Table 4). The R
test was only significant at a single
locus a-ENO8 (Table 4).
Three-population IMa2 analysis
The inclusion of all three members of this species com-
plex in a single IMa2 analysis revealed some interesting
patterns of gene introgression (Fig. 4). The two compet-
ing population trees (nDNA vs. mtDNA) yielded similar
parameter estimates (with one important exception dis-
cussed below). We therefore only report the results based
on the nuclear population tree (I. galbula,(I. abeillei,
I. bullockii)).
First, we found clear evidence of substantial introgression
across the hybrid zone between I. galbula and I. bullockii,
I. galbula >I. bullockii
=3.388, 95% HPD =0.4112.30,
vs. 2Nm
I. bullockii >I. galbula
=2.125, 95% HPD =0.193
6.504, Fig. 4a). Interestingly, the gene flow appears to be
slightly skewed in the direction of I. bullockii. In contrast,
we did not detect any gene flow between the sister species
I. bullockii (Fig. 4b). The gene flow estimates virtually
peaked at zero (2Nm=0.001 in both directions) and the
95% HPD intervals ranged from 0 to 1.148 and 0 to 0.495
in the direction of I. bullockii and I. abeillei, respectively.
Similarly, the three-population analysis did not detect any
gene flow between the highly allopatric I. galbula and
I. abeillei (Fig. 4c), with gene flow estimates peaking near
zero and 95% HPD intervals ranging from 0 to 0.631 and
0 to 0.719 in the direction of I. galbula and I. abeillei,
respectively. There was no evidence of historical gene flow
between the I. galbula lineage and the ancestral I. abeillei/
I. bullockii lineage.
IMa2 provided very concise estimates of the timing of
divergence events within the group. The analysis based on
the nuclear species tree suggested that the initial diver-
gence between the I. galbula lineage and the ancestor of
I. abeillei and I. bullockii appears to have occurred
approximately 350,000 years ago (95% HPD =215,838
568,125), whereas I. abeillei and I. bullockii split much
more recently approximately 95,000 years ago (41,865
159,087, Fig. 5a). In contrast, the analysis based on the
mtDNA population tree (I. bullockii,(I. abeillei,I. galbula))
resulted in perfectly overlapping estimates of the two
splitting events around 215250,000 years ago, with nearly
perfectly overlapping HPD intervals (Fig. 5b). Effective
Table 3. Population differentiation among three northern oriole pop-
ulations, Icterus abeillei,I. bullockii, and I. galbula, estimated based
on pairwise genetic distances between populations. Significant F
values are indicated at the 0.05 >P>0.01 (*) and P<0.01 (**) level.
Bolded values are significant at the Bonferroni-adjusted a-level of
Locus I. abeilleiI. bullockii I. abeilleiI. galbula
I. bullockiiI.
b-ACT2 0.151** 0.372** 0.096*
a-ENO8 0.010 0.019*0.019*
FGB4 0.102** 0.383** 0.173**
GAPDH11 0 0.064*0.052*
RDP2 0.069*0.289** 0.177**
TGFb50.065*0.400** 0.289**
ALDOB5 0.131** 0.241** 0.017
BRM15 0.018 0.186** 0.186**
Table 4. Locus-specific tests for deviations from selective neutrality and constant population sizes in Icterus abeillei (a), I. bullockii (b), and I. gal-
bula (g). Significance of observed values is indicated at the 0.05 >P>0.01 (*) and P<0.01 (**) level. Tests were inapplicable for I. abeillei at
ALDOB5 due to lack of sequence variation. Bolded values remained significant after Bonferroni corrections for multiple testing (adjusted a-level of
Tajima’s DFu’s F
Ramos-Onsins & Rozas R
Locus a b g a b g a b g
b-ACT2 0.048 0.821 1.884** 1.406 1.227 1.398 0.14 0.108 0.118
a-ENO8 1.725*1.97** 1.708*6.561** 7.748** 7.315** 0.048 0.043** 0.05*
FGB4 1.412 0.24 0.711 4.64** 11.431** 6.177** 0.085 0.118 0.152
GAPDH11 0.712 0.152 0.993 1.889 2.089 8.774** 0.154 0.131 0.078
RDP2 0.136 0.019 0.474 1.978 1.962 0.464 0.114 0.126 0.16
TGFb50.024 0.047 0.144 0.058 0.973 7.268** 0.125 0.13 0.128
ALDOB5 0.559 0.434 0.88 1.258 0.099 0.157
BRM15 1.423 1.559*1.047 1.112 -2.963** 1.369 0.061 0.08 0.206
©2012 The Authors. Published by Blackwell Publishing Ltd. 7
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
population sizes (N
) were approximately 398,000 black-
backed orioles, 557,000 Baltimore orioles, and 921,000
Bullock’s orioles (Fig. 6).
Two-population IMa2 analyses
A summary of the three separate two-population analyses
is presented in Table 3. Estimates of gene flow were
mostly consistent between the pairwise two-population
analyses and the three-population analysis. However, the
two-population analysis of I. abeillei and I. galbula sug-
gested a small but significant amount of introgression
from I. abeillei into I. galbula (2Nm
I. abeillei > I. galbula
0.477 (Fig. 4f). Although the 95% HPD interval included
zero (see Table 5), nested model testing conducted in
IMa2 indicated that a reduced model with zero migration
was a significantly worse fit to the data than the full
model allowing for migration (results not shown).
Furthermore, the two-population analysis of I. galbula
and I. bullockii indicated lower but equal rates of intro-
gression in both directions (2Nm
I. bullockii >I. galbula
1.331 and 2Nm
I. galbula >I. bullocki
=1.275, see Fig. 4d).
The divergence time estimates from the pairwise two-
population analyses were comparable to those produced
by the three-population IMa2 analysis, except that both
divergence events (I. galbulaI. abeillei/I. bullockii split,
and I. abeilleiI. bullockii split) were estimated to have
occurred roughly 70,000 years earlier than that indicated
by the three-population analysis (see previous section,
Fig. 5). In agreement with the nuclear species tree of this
group (Jacobsen and Omland 2011), the most recent
divergence in this group between I. abeillei and I. bullockii
was estimated at approximately 163,000 years ago, well
within the 95% HPD interval of the three-population
estimate (see previous section). Similarly, the older split
between the I. galbula lineage and the sister taxa I. abeillei
Introgression rates
I. bullockii I. galbula
I. galbula
I. galbula
I. galbula
I. galbula
I. galbula
12 14 16 18 20
0 5 10 15 20
I. bullockii I. galbula
I. galbula
I. bullockii I. abeillei
I. abeillei
I. bullockii I. abeillei
I. bullockii
I. bullockii
I. bullockii I. abeillei
I. abeillei
I. abeillei
I. abeillei
I. abeillei
I. bullockii
Figure 4. Posterior probability plots of bi-directional migration rates (given in effective number of gene copies - 2Nm) estimated between Icterus
bullockii and I. galbula (a,d), between I. abeillei and I. bullockii (b,e), and between I. abeillei and I. galbula (c,f) based on a combined three-
population analysis and three separate pairwise analyses in IMa2.
8©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
and I. bullockii were estimated at 411,000 and 424,000
years ago, respectively.
Effective population size (N
) estimates from the pair-
wise two-population analyses were also roughly similar to
those estimated by the three-population analysis. The
peak estimates were slightly higher in the two-population
analyses, but the 95% HPD intervals overlap greatly with
the estimates from the three-population analysis. The
most striking deviations in population size estimates arose
when the non-sister species, I. galbula and I. abeillei, were
analyzed separately, which resulted in overestimation of
I. galbula
and a slight underestimation of N
I. abeillei
(Table 5). Similarly, N
I. bullockii
was substantially smaller
when paired with the non-sister I. galbula than when cor-
rectly paired with its sister species I. abeillei (Table 5,
Fig. 6).
The recent development of a multispecies coalescent frame-
work for the study of divergence and gene flow among
three or more taxa simultaneously in the program IMa2
(Hey 2010b) has proven useful for understanding the
population dynamics of complex taxonomic groups con-
sisting of several recently diverged species (e.g., Hey 2010a;
Illera et al. 2011; Schoville et al. 2011; So et al. 2011; Li
et al. 2012). In this study, we were able to estimate levels of
nuclear introgression between allopatric populations of the
three species in the northern oriole group while also
accounting for their recent shared ancestry.
The hybrid zone between the non-sister species,
I. galbula and I. bullockii, represents perhaps one of the
most blatant case examples for the need of a multipopula-
tion framework to disentangle the demographic history
of larger groups of taxa. The reproductive barriers that
so effectively prevent mtDNA and phenotypes of both
I. galbula and I. bullockii from introgressing beyond the
stable hybrid zone appear to be “leaky”, allowing near-
0 500 000 1 000 000 1 500 000 2 000 000
Eective population sizes
I. abeillei
I. bullockii
I. galbula
Figure 6. Posterior probability plot of effective population sizes (N
of extant populations of I. abeillei,I. bullockii, and I. galbula. Peak
estimates of N
are very similar for all three populations, with highly
overlapping confidence intervals (95% HPDs).
0 100 000 200 000 300 000 400 000 500 000 600 000 700 000 800 000
I. abeillei - I. bullockii split
~ 95 000 years ago
I. galbula - ancestor I. abeillei/I. bullockii split
~ 350 000 years ago
Divergence time estimates
Figure 5. Posterior probability plot of divergence (splitting) times
within the northern oriole group. According to IMa2, the initial split
between Icterus galbula and the ancestor of I. abeillei and I. bullockii
occurred roughly 350,000 years ago, whereas the more recent split
between I. abeillei and I. bullockii occurred approximately 95,000
years ago.
Table 5. Summary of pairwise two-population IMa2 analyses of
black-backed oriole (Icterus abeillei), Bullock’s oriole (I. bullockii), and
Baltimore oriole (I. galbula). Given are highest posterior parameter
estimate (HiPt) and lower (HPD95Lo) and upper (HPD95Hi) bounds of
95%HPD intervals for the effective population size of population 1
), population 2 (N
), and ancestral population (N
), splitting time
(t), and population migration rate from population 2 into population
1 (2Nm1>2) and from population 1 into population 2 (2Nm2>1)
forward in time. Population sizes are given in 1000 individuals and
time since divergence in 1000 years.
I. abeillei (1) vs. I. galbula (2)
HiPt 328 951 169 411 0.001 0.477
HPD95Lo 200 532 66 216 0 0
HPD95Hi 527 1,937 332 750 0.351 1.486
I. abeillei (1) vs. I. bullockii (2)
HiPt 517 1,198 225 163 0.005 0.025
HPD95Lo 252 528 118 79 0 0
HPD95Hi 1,184 4,732 373 299 2.404 7.461
I. bullockii (1) vs. I. galbula (2)
HiPt 595 688 163 424 1.275 1.331
HPD95Lo 350 397 62 245 0 0.069
HPD95Hi 1,005 1,215 313 749 3.206 4.25
©2012 The Authors. Published by Blackwell Publishing Ltd. 9
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
neutral nuclear alleles to introgress and successfully
spread in a heterospecific genetic background without
erasing the diagnostic species differences. Such a substan-
tial rate of introgression evident in populations several
hundred miles away from the nearest known areas of
sympatry has major implications for our understanding
of the population dynamics of all three species within the
group. Limited mtDNA introgression (Allen 2002; Carling
et al. 2011) but extensive nDNA introgression (Allen
2002; this study) follows the predictions of Haldane’s rule
(Haldane 1922), wherein reduced female hybrid fitness
prevents maternally inherited mtDNA, but not bi-paren-
tally inherited nDNA from introgressing (Brookfield 1993;
Turelli and Orr 1995; Orr 1997). Sex-biased (i.e., male
driven) gene flow due to female heterogamety and the
effects of Haldane’s rule have increasingly been docu-
mented in a wide range of avian groups (e.g., Borge et al.
2005; Carling and Brumfield 2008; Carling et al. 2010;
´et al. 2010; Backstro
¨m and Va
¨li 2011).
A question of interest to researchers studying divergence
and gene flow is to determine the timing of migration
events between diverging populations. If possible, knowing
when gene flow occurred in the speciation process would
be of great importance to distinguish between an allopatric
speciation model with recent secondary contact following
range expansion (gene flow late) and a parapatric specia-
tion model where divergence occurred in the presence of
gene flow with subsequent decrease, then cessation of gene
flow once reproductive barriers evolved. However, it is
now clear that IMa2 and similar methods are unable to
distinguish between these different modes of speciation
(Sousa et al. 2011; Strasburg and Rieseberg 2011). We
therefore cannot infer with confidence whether the cur-
rently hybridizing I. galbula and I. bullockii diverged in
allopatry and only recently came back into secondary con-
tact following postglacial range expansion, or if they
diverged in parapatry while still exchanging genes
throughout the early stages of divergence. Nonetheless, the
fact that these two orioles are nearly 5% divergent in
mtDNA (Omland et al. 1999) indicates that they have
been evolving independently for an extended period of
time, consistent with allopatric speciation followed by one
or more episodes of gene flow during the last glacial cycles
of the Pleistocene epoch. Pliocene or Pleistocene diver-
gence followed by postglacial expansion has been proposed
to explain the current distribution of many parapatric spe-
cies found in North America and Eurasia (Klicka and Zink
1999; Hewitt 2004; Johnson and Cicero 2004). A recent
exchange of nuclear variation through introgressive
hybridization during secondary contact in the late Pleisto-
cene would explain the wide gap between the mtDNA and
nDNA divergence time estimates for these two species,
even though the TMRCA for a single locus (e.g., mtDNA)
will necessarily always predate the divergence between two
species (Edwards and Beerli 2000).
In contrast, the IMa2 analyses revealed no evidence of
introgression between I. abeillei and I. bullockii. The
region of parapatry in Durango, Mexico where hybridiza-
tion between these two species is alleged to have taken
place was unfortunately not represented in this study. It
is therefore possible that some hybridization could occur
within this region, but restricted enough to prevent wide-
spread introgression into the core ranges of either species.
Thus, the ubiquitous nuclear haplotype sharing observed
between these sister species most likely represents ances-
tral polymorphism retained in both descendent lineages
since their recent split in the late Pleistocene (Hudson
and Turelli 2003)
The low rate of unidirectional introgression from I. ab-
eillei into I. galbula that was indicated by the two-popula-
tion IMa2 analysis is somewhat puzzling, given that no
evidence of gene flow between these two species was
revealed in the three-population analysis. Previous studies
on these two species using IM based on mtDNA and two
nuclear introns also found no evidence of gene flow
between these two mitochondrial sister taxa (Kondo et al.
2004, 2008). The fact that they are reciprocally monophy-
letic at the mtDNA level separated by a single fixed sub-
stitution also strongly suggests that there is no recent or
ongoing mitochondrial gene flow between these highly
allopatric species (Kondo et al. 2008).
Rather, we suspect that this signature of gene flow is a
spurious outcome arising when violating the assumption
of the IM model that no genetic exchange has occurred
between the two focal populations and ghost populations
(Beerli 2004; Slatkin 2005). As I. abeillei shares a more
recent evolutionary history with I. bullockii than with
I. galbula (Jacobsen et al. 2010; Jacobsen and Omland
2011), retained ancestral polymorphisms shared between
I. abeillei and I. bullockii could introgress into I. galbula
via I. bullockii over the extensive hybrid zone. This study
revealed just how easily neutral nuclear alleles can cross
this species boundary and freely introgress across the
entire range of the other species. By excluding I. bullockii
from the analysis, such shared ancestral alleles could mis-
takenly be inferred by IMa2 to have introgressed directly
from I. abeillei into I. galbula (Fig. 7). Furthermore, the
many alleles introgressing from I. bullockii into I. galbula
have probably contributed to the high nucleotide diversity
in I. galbula, causing IMa2 to overestimate the current
effective population size of I. galbula in analyses that do
not account for I. bullockii.
Thus, exclusion from the analysis of other taxa that
have exchanged genes with either of the two focal taxa
through shared ancestry or gene flow can bias estimation
of population sizes and gene flow in IM, and the severity
10 ©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
of the bias depends on how large the genetic contribution
from the third taxon has been (Strasburg and Rieseberg
Our study reveals the complexity of evolutionary
processes (recent divergence, slow sorting of ancestral var-
iation, and introgressive hybridization) that have contrib-
uted to produce the observed pattern of rampant allele
sharing among the three species within the northern ori-
ole group. Furthermore, our findings highlight the impor-
tance of accounting for all potentially interbreeding taxa
when interpreting the divergence history of a group of
taxa, and the extent to which gene flow may retard the
rate of divergence at neutral loci. Thus, this study pro-
vides an interesting perspective on the broad-scale
impacts of introgressive hybridization, which has impor-
tant implications for everything from understanding
adaptive evolution to inferring species trees. The ease with
which genome-scale datasets can now be obtained will
soon enable researchers to study even more complex
clades with four or more taxa that may interbreed.
We thank the curatorial staff at ANSP, LSUMNS, MZFC,
BMNH, FMNH, UWBM, and H. L. Gibbs for providing
the tissue samples used in this study. We especially thank
A. G. Navarro-Sigu
¨enza, B. E. Herna
˜os, and M.
Honey for help in collecting in Mexico. We further thank
D. Kenny for laboratory assistance, and J. L. Peters for
assistance with coalescent analyses. This work was funded
by National Science Foundation grants to K. E. O. (DEB-
0347083 and DEB-1119506), and Maryland Ornithological
Society Avian Research Grants and an American Orni-
thologist’s Union Research Grant to F. J.
Conflict of Interest
None declared.
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14 ©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
Appendix 1. Taxonomic sampling
Species Museum
ID Sex Collection Locality
Icterus abeillei MZFC 2339 M Mexico, Morelos, Carretera
Icterus abeillei MZFC 5381 M Mexico, Guerrero, Apaxtla
Icterus abeillei MZFC 3468 M Mexico, Guanajuato, San Pedro
Icterus abeillei MZFC KEO27 F Mexico: Guanajuato, Salvatierra
Icterus abeillei MZFC KEO28 M Mexico: Guanajuato, Salvatierra
Icterus abeillei MZFC KEO29 M Mexico: Guanajuato, Salvatierra
Icterus abeillei MZFC 1087 M Mexico, Guanajuato, Yuriria
Icterus abeillei MZFC 5441 M Mexico, Guanajuto, Yuriria
Icterus abeillei MZFC KEO30 M Mexico: Guanajuato, Yuriria
Icterus abeillei MZFC KEO31 M Mexico: Guanajuato, Yuriria
Icterus abeillei MZFC 3467 M Mexico, Jalisco, Laguna de Chapala
Icterus abeillei MZFC 7966 M Mexico: Jalisco
Icterus abeillei MZFC 10733 M Mexico: Jalisco
Icterus abeillei MZFC 11483 M Mexico: Jalisco
Icterus abeillei MZFC KEO32 M Mexico: Jalisco, Chapala
Icterus abeillei MZFC KEO33 M Mexico: Jalisco, Chapala
Icterus abeillei MZFC KEO34 M Mexico: Jalisco, Chapala
Icterus abeillei MZFC KEO35 F Mexico: Jalisco, Chapala
Icterus abeillei MZFC KEO37 F Mexico: Jalisco, Chapala
Icterus abeillei MZFC KEO45 M Mexico: Michoaca
´n, Brisen
Icterus abeillei MZFC KEO46 M Mexico: Michoaca
´n, Brisen
Icterus abeillei MZFC KEO48 F Mexico: Michoaca
´n, Brisen
Icterus abeillei MZFC KEO49 M Mexico: Michoaca
´n, Brisen
Icterus bullockii MVZ 176515 M USA: NM, Mora Co.
Icterus bullockii LSUMNS 3874 F USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3875 F USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3876 M USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3877 F USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3878 M USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3879 M USA: CA, Inyo Co.
Icterus bullockii LSUMNS 3880 M USA: CA, Inyo Co.
Icterus bullockii FMNH 330029 M USA: CA, San Bernardino Co.
Icterus bullockii FMNH 341933 M USA: CA, Imperial Co.
Icterus bullockii FMNH 341934 M USA: CA, Monterrey Co.
Icterus bullockii FMNH 341937 M USA: CA, Monterrey Co.
Icterus bullockii FMNH 341938 M USA: CA, Monterrey Co.
Icterus bullockii UWBM 55942 M USA: WA, Grant Co.
Icterus bullockii UWBM 55951 F USA: WA, Douglas Co.
Icterus bullockii UWBM 55975 M USA: WA, Douglas Co.
Icterus bullockii UWBM 55976 F USA: WA, Douglas Co.
Icterus bullockii UWBM 55978 F USA: WA, Douglas Co.
Icterus bullockii UWBM 59055 F USA: WA, Asotin Co.
Icterus bullockii UWBM 59056 M USA: WA, Asotin Co.
Icterus bullockii UWBM 59058 M USA: WA, Asotin Co.
Icterus galbula LSUMNS 131146 M USA: LA, Cameron Co.
Icterus galbula ANSP 10126 M USA: PA, Bucks Co.
Icterus galbula ANSP 10134 M USA: PA, Bucks Co.
Icterus galbula ANSP 10148 F USA: PA, Bucks Co.
Icterus galbula USFWS 17860 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17861 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17862 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17864 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17866 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17867 M USA: MD, Baltimore Co.
Icterus galbula USFWS 17869 M USA: MD, Baltimore Co.
©2012 The Authors. Published by Blackwell Publishing Ltd. 15
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
Appendix 1. (Continued).
Species Museum
ID Sex Collection Locality
Icterus galbula USFWS 17870 M USA: MD, Baltimore Co.
Icterus galbula UWBM 67201 M USA: MD, Prince Georges Co.
Icterus galbula BMNH 42547 M USA: MN, Becker Co.
Icterus galbula BMNH X7630 M USA: MN, Hennepin Co.
Icterus galbula BMNH X7763 F USA: MN, Chisago Co.
Icterus galbula BMNH X7799 M USA: MN, Chisago Co.
Icterus galbula FMNH 350604 M USA: IL, Cook Co.
Icterus galbula FMNH 395866 F USA: IL, Cook Co.
Icterus galbula HLG LG03 M Canada: Manitoba
Icterus galbula HLG LG04 M Canada: Manitoba
Icterus galbula HLG LG05 M Canada: Manitoba
Icterus galbula HLG LG06 F Canada: Manitoba
Sturnella neglecta NMNH 586115
Museums are abbreviated as follows: ANSP =Academy of Natural Sciences, Philadelphia; BMNH =J. F. Bell Museum of Natural History, University
of Minnesota; FMNH =Field Museum of Natural History; HLG =H. Lisle Gibbs, blood samples; LSUMNS =Louisiana State University, Museum of
Natural Science; MVZ =Museum of Vertebrate Zoology, University of California, Berkeley; MZFC =Museo de Zoologı
´a, Facultad de Ciencias, Uni-
versidad Nacional Auto
´noma de Me
´xico; NMNH =National Museum of Natural History; UWBM =University of Washington, Burke Museum and
USFWS =United States Fish and Wildlife Service band numbers.
San Pedro is the short form of San Pedro de Los Naranjos, and Brisen
˜as is the short form of Brisen
˜as de Matamoros.
16 ©2012 The Authors. Published by Blackwell Publishing Ltd.
Divergence Genetics of Northern Orioles F. Jacobsen & K. E. Omland
Appendix 2. GenBank accession numbers for DNA sequences obtained from previous studies on Icterus (Kondo et al. 2008; Jacobsen et al.
2010; Jacobsen and Omland 2011).
Species GenBank Accession Nos.
I. abeillei DQ898412-DQ898433, DQ898358-DQ898385, GU972843, GU972875, JN169143-JN169144, JN169230-JN169239,
JN169317-JN169326, JN169404-JN169413, JN169491-JN169500, JN169578-JN169587, JN169665-JN169674
I. bullockii JN169159-JN169170, JN169246-JN169257, JN169333-JN169344, JN169422-JN169431, JN169507-JN169518, JN169594-JN169605,
I. galbula DQ898316-DQ898331, DQ898344-DQ898347, DQ898356-DQ898357, DQ898386-DQ898392, DQ898407, GU972855, GU972887,
JN169191- JN169196, JN169274-JN169283, JN169361-JN169370, JN169448-JN169457, JN169535-JN169544,
JN169622-JN169631, JN169709- JN169718
S. neglecta GU972873, GU972905
F. Jacobsen & K. E. Omland Divergence Genetics of Northern Orioles
©2012 The Authors. Published by Blackwell Publishing Ltd. 17
... Evidence supporting Haldane's rule derives from several sex determination systems (Laurie 1997;Presgraves and Orr 1998;Brothers and Delph 2010), including the avian ZZ/ZW system: heterogametic female birds (ZW) may exhibit lower fitness than homogametic males (ZZ; Tegelström and Gelter 1990;Carling and Brumfield 2008). Reduced introgression of maternally inherited loci further suggests that hybrid female birds suffer greater fitness disadvantages (Carling and Zuckerberg 2011;Jacobsen and Omland 2012;Gowen et al. 2014). ...
... The chickadee hybrid zone follows Haldane's rule, with increasingly male hybrid offspring associated with parents with an increasingly hybrid pair GI. Haldane's rule is typically illustrated by genetic evidence such as reduced cline widths or increased differentiation of sex chromosome and mitochondrial loci (Jacobsen and Omland 2012;Gowen et al. 2014). Haldane's rule specifically states, "when in the F 1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous sex" (Haldane 1922, p. 7). ...
Hybridization often occurs at the parapatric range interface between closely related species, but fitness outcomes vary: hybrid offspring exhibit diverse rates of viability and reproduction compared with their parental species. The mobile hybrid zone between two chickadee congeners (Poecile atricapillus × Poecile carolinensis) has been well studied behaviorally and genetically, but the viability of hybrids and the underlying mechanisms contributing to hybrid fitness have remained unclear. To better characterize the fitness costs of hybridization in this system, we analyzed 21 years of data from four sites, including more than 1,400 breeding attempts by the two species, to show that rates of hatching success changed substantially as the zone of hybridization moved across the landscape. Admixture-associated declines in hatching success correlated with reduced proportions of heterogametic (female) offspring, as predicted by Haldane's rule. Our data support an underlying mechanism implicating genetic admixture of the homogametic (male) parent as the primary determinant of offspring sex ratio, via incompatibilities on the hemizygous Z chromosome. Our long-term study is the first to directly measure changes in fitness costs as a vertebrate hybrid zone moves, and it shows that changes in these costs are a way to track the distribution of a hybrid zone across the landscape.
... Sutton (1938) was the first to describe hybrid orioles, from specimens collected in western Oklahoma, although he was unable to confirm that intermediate-plumaged birds were indeed hybrids and instead described them as "oddly plumaged" orioles. In subsequent decades the Baltimore and Bullock's orioles and their hybrids were studied extensively (Sibley and Short 1964, Sutton 1968, Rising 1969, 1970, 1973, 1983a, 1996Corbin and Sibley 1977, Corbin et al. 1979, Jacobsen and Omland 2012. These diverse investigations better defined the patterns and consequences of hybridization in these orioles, and featured in the more general discussions of the role J. Walsh, S. M. Billerman, V. G. Rohwer, et al. ...
... Their breeding ranges overlap across a narrow region of the Great Plains of North America, stretching from northern Texas in the United States to southern Saskatchewan and Alberta in Canada (Rising 1983a(Rising , 1983b. Where their ranges overlap, the 2 species often interbreed and produce phenotypically intermediate hybrids (Sibley and Short 1964, Corbin and Sibley 1977, Rising 1983a, 1983b, 1996Carling et al. 2011, Jacobsen andOmland 2012). ...
Hybrid zones are powerful natural settings for investigating how birds diversify into distinct species. Here we present the first genomic-scale exploration of the Baltimore (Icterus galbula) and Bullock’s (I. bullockii) oriole hybrid zone, which is notable for its long history of study and for its prominence in debates about avian species concepts and species limits. We used a reduced-representation sequencing approach to generate a panel of 3,067 genetic markers for 297 orioles sampled along the Platte River, a natural west-to-east transect across the hybrid zone. We then explored patterns of hybridization and introgression by comparing variation in genomic and plumage traits. We found that hybridization remains prevalent in this area, with nearly all orioles within the hybrid zone showing some degree of genomic mixing, and 41% assigned as recent-generation (F1/F2) hybrids. The center and width of the genomic and plumage gradients are concordant and coincident, supporting our finding that classically scored plumage traits are an accurate predictor of pure vs. hybrid genotypes. We find additional support for previous suggestions that the center of this hybrid zone has moved westward since it was first intensively sampled in the 1950s, but that this westward movement had slowed or ceased by the 1970s. Considered in concert, these results support previous inferences that some form of ongoing selection is counteracting the potential homogenization of these orioles via hybridization, thereby supporting their continued taxonomic separation as distinct species.
... At the same time, the incidence of an admixed individual among a panel of 38 birds suggests that hybridisation between C. glaucicomans and C. hainanus is probably not exceptional, although their very different songs would seem to act as a reproductive isolating barrier (Gwee et al., 2019;Zhang et al., 2016). Previous research has shown that hybrid zones are possible between non-sister species (Jacobsen & Omland, 2012). The genomic hybrid individual we identified was sampled in Guangdong, outside of the breeding range of C. glaucicomans, but was sampled on 20 April 2014, during the spring migration that would see many individuals from further north pass through this area. ...
Full-text available
Morphology has been a leading taxonomic guiding light to systematists for the last couple of hundred years. However, the genetic and – more recently – genomic revolution have produced numerous demonstrations of erroneous classifications that were based on labile morphological traits. We used thousands of genome‐wide markers to shed light on evolutionary dynamics in a confusing and taxonomically obscure group of Asian Cyornis flycatchers. Using genomic data, we corroborated recent findings based on three mitochrondrial and five nuclear genes that the two taxa hainanus and klossi which were previously treated as separate species (Cyornis hainanus and Cyornis rubeculoides klossi, respectively) are genomically homogeneous and form a single species, C. hainanus. We also uncovered a novel case of interbreeding between C. hainanus and a non‐sister species, C. glaucicomans, illustrating these flycatchers' ability to hybridise in marginal situations even after substantial times of divergence. Our study illustrates how genome‐wide loci can shed light on complicated taxonomic problems, resulting in a better integration of phenotypic and genotypic data.
... We detected secondary gene flow between stricklandii from Sabah and the eastern Javan clade ( fig. 1), even though they are distantly related in the complex, adding to previous insights that secondary gene flow does not necessarily have to be restricted to sister lineages (Dasmahapatra et al. 2012;Jacobsen and Omland 2012;Edelman et al. 2019;Gwee et al. 2020). ...
Full-text available
Quantifying the magnitude of the global extinction crisis is important but remains challenging, as many extinction events pass unnoticed owing to our limited taxonomic knowledge of the world’s organisms. The increasing rarity of many taxa renders comprehensive sampling difficult, further compounding the problem. Vertebrate lineages such as birds, which are thought to be taxonomically well understood, are therefore used as indicator groups for mapping and quantifying global extinction. To test whether extinction patterns are adequately gauged in well-studied groups, we implemented ancient-DNA protocols and retrieved whole genomes from the historic DNA of museum specimens in a widely known songbird radiation of shamas (genus Copsychus) that is assumed to be of least conservation concern. We uncovered cryptic diversity and an unexpected degree of hidden extinction and terminal endangerment. Our analyses reveal that >40% of the phylogenetic diversity of this radiation is already either extinct in the wild or nearly so, including the two genomically most distinct members of this group (omissus and nigricauda), which have so far flown under the conservation radar as they have previously been considered subspecies. Comparing the genomes of modern samples with those from roughly a century ago, we also found a significant decrease in genetic diversity and a concomitant increase in homozygosity affecting various taxa, including small-island endemics that are extinct in the wild as well as subspecies that remain widespread across the continental scale. Our application of modern genomic approaches demonstrates elevated levels of allelic and taxonomic diversity loss in a songbird clade that has not been listed as globally threatened, highlighting the importance of ongoing reassessments of extinction incidence even across well-studied animal groups. Key words: extinction, introgression, white-rumped shama, conservation.
... When non-sister species hybridized in the past, an introgressed gene might yield a topology that depicts a faulty sister relation between these species. This has been observed in several bird genera, such as Manacus (Brumfield et al. 2008) and Icterus (Jacobsen and Omland 2012). In addition, if the introgression event happened far back in time, it can lead to misleading branching arrangements at deep phylogenetic nodes. ...
What is a species? This seemingly simple question has occupied the minds of numerous biologists and philosophers, resulting in the formulation of many species concepts. From a theoretical point of view, the species problem has been resolved by equating species with independently evolving lineages (i.e. the evolutionary species concept or the general lineage concept). However, the practical issues with describing and delineating species remain. The origin of species is a gradual process that typically requires thousands to millions of years, creating a grey zone of species delimitation in which taxonomy is often controversial. To account for this, an integrative taxonomy has been proposed in which different taxonomic concepts and methods are integrated in the delimitation of species. In this chapter, I argue that genomics provides another line of evidence in this pluralistic approach to species classification. Indeed, genomic data can be combined with classical species criteria, such as diagnosability, phylogeny and reproductive isolation. First, genomic data can provide an extra diagnostic feature in species delimitation. Compared to ‘old-school’ genetic markers, the use of genome-wide markers leads to a significant rise in statistical power. Second, phylogenomic analyses can resolve the evolutionary relationships within rapidly diverging or hybridizing groups of species while taking into account gene tree discordance. Third, genomic data can be used to pinpoint the genetic basis of reproductive isolation and provide a detailed description of the speciation process. All in all, the genomic era will supply avian taxonomists with a new tool box that can be applied to old concepts, leading to better informed decisions in cataloguing biodiversity.
... They found evidence for unidirectional gene flow from Reed Warbler (A. scirpaceus) to Marsh Warbler (A. palustris) and from Reed Warbler to Blyth's Reed Warbler (A. dumetorum). Similarly, a study on three Icterus orioles applied IM-models to describe gene flow patterns (Jacobsen and Omland 2012). They concluded that "only by including all members of this group in the analysis was it possible to rigorously estimate the level of gene flow among these three closely related species. ...
Full-text available
p>Hybridization is not always limited to two species; often multiple species are interbreeding. In birds, there are numerous examples of species that hybridize with multiple other species. The advent of genomic data provides the opportunity to investigate the ecological and evolutionary consequences of multispecies hybridization. The interactions between several hybridizing species can be depicted as a network in which the interacting species are connected by edges. Such hybrid networks can be used to identify 'hub-species' that interbreed with multiple other species. Avian examples of such 'hub-species' are Common Pheasant (Phasianus colchicus), Mallard (Anas platyrhynchos) and European Herring Gull (Larus argentatus). These networks might lead to the formulation of hypotheses, such as which connections are most likely conducive to interspecific gene flow (i.e. introgression). Hybridization does not necessarily result in introgression. Numerous statistical tests are available to infer interspecific gene flow from genetic data and the majority of these tests can be applied in a multispecies setting. Specifically, model-based approaches and phylogenetic networks are promising in the detection and characterization of multispecies introgression. It remains to be determined how common multispecies introgression in birds is and how often this process fuels adaptive changes. Moreover, the impact of multispecies hybridization on the build-up of reproductive isolation and the architecture of genomic landscapes remains elusive. For example, introgression between certain species might contribute to increased divergence and reproductive isolation between those species and other related species. In the end, a multispecies perspective on hybridization in combination with network approaches will lead to important insights into the history of life on this planet.</p
... markers that are fixed or highly differentiated between two parental species) are predicted to be under divergent selection, exhibiting reduced introgression (Yuri et al. 2009). Lastly, differential introgression of sex-linked and mitochondrial markers relative to autosomal loci is expected, a pattern often attributed to Haldane's rule and observed in a number of avian systems (Saetre et al. 2003, Carling and Brumfield 2008, Jacobsen and Omland 2012. This approach might help reveal the evolutionary history of the young Amazilia species complex, where detecting divergence in a few genes under selection may be critical to establish proper species limits. ...
Pleistocene climate cycles have been recognized to be a major driver of postglacial northward range expansion of North American bird populations. During glacial maxima, allopatric lineages that were reproductively isolated might have come into secondary contact with one another during expansion periods and the genetic signatures of past hybridization as a result of secondary contact events should produce detectable hybrid zones. The white-chested hummingbirds, Amazilia violiceps and A. viridifrons, constitute a species complex showing phenotypic similarity across its range. One exception is the subspecies found in the Central Depression of Chiapas (A. viridifrons villadai), which shares some plumage traits with the endemic but allopatric green-fronted populations in Oaxaca. Phylogenetic relationships, taxonomy and species limits among violiceps, viridifrons and villadai have been controversial for decades. We assessed genetic structure of populations and introgression in this species complex by analysing 95 individuals at ten nuclear microsatellites and morphology. Bayesian analysis yielded four clusters. However, only two clusters generally match previously described mtDNA haplogroups, one parental taxon in the south (villadai) and a cluster with two admixed taxa (viridifrons and violiceps) that cannot be attributed to any pure parental population. High genetic admixture was recorded in the violiceps/viridifrons range, probably as a consequence of a postglacial northern expansion of violiceps. Signs of admixture and gene flow between violiceps/viridifrons and villadai were low. Historical and contemporary migration rates and Approximate Bayesian computations support a scenario of divergence with gene flow: a Pleistocene basal split separating A. violiceps and the other two clades are derived from a second split (villadai and viridifrons) or from a merger of violiceps and villadai into viridifrons due to gene flow.
The Baltimore–Bullock’s oriole hybrid zone is one of the best-studied avian hybrid zones in North America, yet our understanding of the causes of selection against hybrids remains poor. We examine if endohelminth parasites may cause selection against hybrid orioles but found no evidence for this hypothesis. Of the 139 male orioles we examined, 43 individuals contained endohelminth parasites from at least 1 of these groups: Cestoda, Acanthocephala, or Nematoda. Across the hybrid zone, Baltimore Orioles (Icterus galbula) and Bullock’s Orioles (I. bullockii) differed in their parasite communities, such that Baltimore Orioles frequently contained both Acanthocephala and Cestoda parasites whereas Bullock’s Orioles primarily contained Cestoda parasites. Despite these differences in parasite communities between parental species, the frequency of hybrid orioles with parasites was similar to parentals, suggesting that hybrids were as susceptible to endohelminth parasites as parentals. Using a subset of 99 adult male orioles, we explored how parasites may be associated with the expression of orange carotenoid-based plumage in hybrids and parentals. Associations between carotenoid-based plumage color and parasites were most strongly expressed in Bullock’s Orioles, but patterns were subtle and counterintuitive because individuals with parasites often had more enhanced color measures compared to individuals without parasites. Taken together, these data suggest that endohelminth parasites impose little fitness costs to male orioles on the breeding grounds and likely do not cause selection against hybrids.
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According to Haldane’s Rule, the heterogametic sex will show the greatest fitness reduction in a hybrid cross. In birds, where sex is determined by a ZW-system, female hybrids are expected to experience lower fitness compared to male hybrids. This pattern has indeed been observed in several bird groups, but it is unknown whether the generality of Haldane’s Rule also extends to the molecular level. First, given the lower fitness of female hybrids, we can expect maternally inherited loci (i.e. mitochondrial and W-linked loci) to show lower introgression rates than biparentally inherited loci (i.e. autosomal loci) in females. Second, the faster evolution of Z-linked loci compared to autosomal loci and the hemizygosity of the Z-chromosome in females might speed up the accumulation of incompatible alleles on this sex chromosome, resulting in lower introgression rates for Z-linked loci than for autosomal loci. I tested these expectations by conducting a literature review which focused on studies that directly quantified introgression rates for autosomal, sex-linked and mitochondrial loci. Although most studies reported introgression rates in line with Haldane’s Rule, it remains important to validate these genetic patterns with estimates of hybrid fitness and supporting field observations to rule out alternative explanations. Genomic data provide exciting opportunities to obtain a more fine-grained picture of introgression rates across the genome, which can consequently be linked to ecological and behavioral observations, potentially leading to novel insights into the genetic mechanisms underpinning Haldane’s Rule.
Disentangling historical, ecological, and abiotic drivers of diversity among closely related species can benefit from morphological diversity being placed in a phylogenetic context. It can also be aided when the species are variously in allopatry, parapatry, and sympatry. We studied a clade of Australian thornbills (Passeriformes: Acanthizidae: Acanthiza) comprising the Brown Thornbill (A. pusilla), Inland Thornbill (A. apicalis), Mountain Thornbill (A. katherina), and Tasmanian Thornbill (A. ewingii) whose distributions and ecology facilitate this approach. We first clarified phylogenetic relationships among them and then detected a low level of gene flow in parapatry between a non‐sister pair (Brown, Inland). Further work could partition relative roles of past and current hybridization. We identify likely cases of ecologically driven divergent selection and one of convergent evolution. Divergent selection was likely key to divergence of Inland Thornbills from the Brown–Mountain sister pair. In contrast, convergence in plumage between the non‐sister Brown and Inland Thornbills has been driven by their mesic forest habitats on opposite sides of the Australian continent. Finally, morphological distinctiveness of Tasmanian populations of Brown Thornbills could reflect character displacement in sympatry with the ecologically similar Tasmanian Thornbills. Collectively, the combined morphological, genetic, and ecological evidence points to diverse evolutionary processes operating across this closely related group of birds.
The neutral theory of molecular evolution predicts that regions of the genome that evolve at high rates, as revealed by interspecific DNA sequence comparisons, will also exhibit high levels of polymorphism within species. We present here a conservative statistical test of this prediction based on a constant-rate neutral model. The test requires data from an interspecific comparison of at least two regions of the genome and data on levels of intraspecific polymorphism in the same regions from at least one species. The model is rejected for data from the region encompassing the Adh locus and the 5′ flanking sequence of Drosophila melanogaster and Drosophila sechellia. The data depart from the model in a direction that is consistent with the presence of balanced polymorphism in the coding region.
An accurately resolved gene tree may not be congruent with the species tree because of lineage sorting of ancestral polymorphisms. DNA sequences from the mitochondrially encoded genes (mtDNA) are attractive sources of characters for estimating the phylogenies of recently evolved taxa because mtDNA evolves rapidly, but its utility is limited because the mitochondrial genes are inherited as a single linkage group (haplotype) and provide only one independent estimate of the species tree. In contrast, a set of nuclear genes can be selected from distinct chromosomes, such that each gene tree provides an independent estimate of the species tree. Another aspect of the gene-tree versus species-tree problem, however, favors the use of mtDNA for inferring species trees. For a three-species segment of a phylogeny, the branching order of a gene tree will correspond to that of the species tree if coalescence of the alleles or haplotypes occurred in the internode between the first and second bifurcation. From neutral theory, it is apparent that the probability of coalescence increases as effective population size decreases. Because the mitochondrial genome is maternally inherited and effectively haploid, its effective population size is one-fourth that of a nuclear-autosomal gene. Thus, the mitochondrial-haplotype tree has a substantially higher probability of accurately tracking a short internode than does a nuclear-autosomal-gene tree. When an internode is sufficiently long that the probability that the mitochondrial-haplotype tree will be congruent with the species tree is 0.95, the probability that a nuclear-autosomalgene tree will be congruent is only 0.62. If each of k independently sampled nuclear-gene trees has a probability of congruence with the species tree of 0.62, then a sample of 16 such trees would be required to be as confident of the inference based on the mitochondrial-haplotype tree. A survey of mtDNA-haplotype diversity in 34 species of birds indicates that coalescence is generally very recent, which suggests that coalescence times are typically much shorter than internodal branch lengths of the species tree, and that sorting of mtDNA lineages is not likely to confound the species tree. Hybridization resulting in transfer of mtDNA haplotypes among branches could also result in a haplotype tree that is incongruent with the species tree; if undetected, this could confound the species tree. However, hybridization is usually easy to detect and should be incorporated in the historical narrative of the group, because reticulation, as well as cladistic events, contributed to the evolution of the group.
Specimens of Northern ("Baltimore" and "Bullock's") orioles collected at six sites in western Kansas in 1976 and 1978 are compared with specimens collected at the same sites in the mid-1960's. No changes in the location or size of the step-cline ("hybrid zone") between the two taxa are indicated by comparisons of the plumage features of the male specimens; the distribution of phenotypes of birds from sites where both Baltimore and Bullock's orioles occur is not bimodal, and, therefore, there is no indication of selection against intermediate birds. Female Bullock's-like orioles, however, are found farther east along the Cimarron River in southwestern Kansas than they were in the 1960's. The correlations between the amount of precipitation at a locality in western Kansas and the phenotype of oriole that occurs there are high (about 0.90). This suggests that precipitation per se is a factor that determines the distributional limits of these birds relative to each other. There are no recent changes, however, in the average climatic conditions in western Kansas. Perhaps the orioles present at a site in any given season are a reflection of the conditions there the previous season or two. The analyses of size variation among samples collected in the 1970's show an east-west cline that is more-or-less congruent with that found for plumage features; at any given site, the variability in size is positively correlated with the variability in plumage. Where a diversity of oriole phenotypes occurs, there is no tendency for females to associate with males of the "same type" (i.e. Bullock's with Bullock's or vice versa); this suggests that assortative mating does not occur.