Erschienen in: Science ; 344 (2014), 6190. - S. 1410-1414

The genomic landscape underlying
phenotypic integrity in the face ofgene flow in crows
J . W . Poelstra,'*N. Vijay,'* C. M. Bossu,'* H. Lantz, z,s B. Ryll,4 I . M'tiller,.r, 6 V. Baglione,7
P. U rmeberg, 8 M. Wikelski, 5 ' 6 M. G . Grabherr,3 J. B. W . Wolf't
The importance, extent, and mode of interspecific gene flow for the evolution of species
has long been debated. Characterization of genomic differentiation in a classic example ofhybridization between all-black carrion crows and gray-coated hooded crows identified
genome-wide introgression extending far beyond the morphological hybrid zone. Gene
expression divergence was concentrated in pigmentation genes expressed in grayversus black feather follicles. Only a small number of narrow genomic islands exhibited
resistance to gene flow. One prominent genomic region (<2 megabases) harbored 81
of all 82 fixed differences (of 8.4 million single-nucleotide polymorphisms in total)
linking genes involved in pigmentation and in visual perception- a genomic signal reflecting
color-mediated prezygotic isolation. Thus, localized genomic selection can cause marked
heterogeneity in introgression landscapes while maintaining phenotypic divergence.
enomic studies increasingly appreciate theimportance of interspecific gene flow inthe context of species diversification (1, 2),
including that of hominids (3). Yet our understanding of the forces generating het
erogeneity in differentiation across the genome
and their relationship to phenotypic evolution
is limited (3 5). The European hybrid zone be
tween an black carrion crows [Cbrvus (carune)
corone] and gray coated hooded crows [C (carune)
rornix] (6) provides ideal conditions for studying
the evolutionazy consequences of introgression
in a phase of early species divergence. The geo
graphical distribution pattern of these species
suggests a population history shaped by glaci

C

1

Science for Ufe Laboratory and Department of Evolutionary
Biology, Evolutionary Biology Centre, Uppsala Univers~y.
75236 Uppsala. Sweden. 2Bioinformatics Infrastructure for
LWe Sciences, Uppsala University. 75124 Uppsala. Sweden.
3 Science for Ufe Laboratory and Department of Medical
Biochemistry and Microbiology, Uppsala University. 75123
Uppsala, Sweden. 40epartment of Organismal Biology,
Evolutionary Bioi<V, Centre. Uppsala University. 75236
Uppsala, Sweden. Max Planck Institute for Ornithology,
78315 Radolfzell. Germany. 6Department of Biology.
University of Konstanz, 78464 Konstanz. Germany.
7
Departamento de Ciencias Agro Forestales. Campus La
Yutera, Universidad de Valladolid. 34004 Palencia, Spain.
8
Science for LWe Laboratory and Department of Biochemistry
and Biophysics, Stockholm University. 17121 Solna. Sweden.
• These authors contributed equaUy to this work. j{:orrespondlng
author. E man: pchen.wolf@ebc.uu.se

ation cycles during the Pleistocene, when periodsof isolation in distinct southern refugia alter
nated with periods of range expansion and sec
ondary contact (7). The near absence of neutral
genetic differentiation across the hybrid zone (S)strongly contrasts with abrupt spatial segregation of plumage coloration (Fig. 1), which has
remained remarkably stable over the past cen
tury despite successful backcrossing of hybrids( 6). Evidence for color assortative mating (9)and the independent recurrence of pied piu
m age phenotypes in the otherwise all black genus
Carous has prompted the hypothesis that thiscolor polymorphism is promoting speciation(fig. Sl).
We a<>sembled a high quality reference genomeof one hooded crow male sequenced to 152x
coverage (table Sl). The final assembly consisted
ofl298 scaffolds with an N50 size of 16.4Mb (10)and a cumulative length of 1.04 Gb, including
35 2% gaps; 95% of the a<>sembly was contained
within the 100 largest scaffolds (size range: 1 to50 Mb), which approached chromosome size
(crow karyotype: 2n = 80; size range of zebra
finch dlromosomes: 1 to 156Mb) (fig. S2, A andB, and table S2). Evidence based annotation of the
genome with mRNA sequencing data (table S3)
identified a set of 20,794 protein coding genes.
We subsequently resequenced 60 genomes of
unrelated individuals from two populations eachof carrion and hooded crows (Fig. 1) to a mean

sequence coverage of 12.2x (range: 7.1x to 28.6 x,
table S4); 8.44 million single nucleotide polymorphisms (SNPs) segregated across all four
populations, 5.27 million of which were shared
between carrion and hooded crows. Several lines
of evidence suggest substantial genome wide geneflow across the hybrid :zone. The major axes ofgenetic variation (accounting for ll5% of the total variance) coincided with the presumed direc
tion ofspatial expansion out of Spain, yet German
carrion crows clustered more closely with bothhooded crow populations [Fig. 1; for pairwise,fixation index (F~rr) estimates see Fig. 2A, fig. S3,
and table S5; see fig. S4 for mitochondrial DNA
divergence]. Extensive gene 'flow between hooded
crows and the German carrion crow population
was further supported by ABBA BABA tests (fig.
S5 and table S6), admixture analyses (fig. S6),
·and coalescence based parameter estimates of
an isolation with migration model (fig. S7). Con·sistent with a scenario of admixture upon sec·ondary rontact, an populations but Spain showedsignatures of expansion and had higher nucleo
tide diversity than that of the presumably refugial
Spanish population (table S5~
The low genome wide differentiation acro;s the
hybrid zone was also reflected in gene express
ion divergence as measured by mRNA sequenc
ing (RNA seq) from 19 individuals and five tissues
under rontrolled ronditions. The overall proportion of differentially expressed genes between
carricn and hooded a'O'iYS was low, between 0.00%
and 0.41% across tissues [Fig. 3B; see (B); false
disco\el')' rate <0.05). The main difference in gene
expression patterns corresponded to phenotypic
divergence in plumage coloration. Differentiallyexpressed genes predominated in growing fea
ther follicles from the torso, where carrion crows
produce black feathers and hooded crows grayfeathers (Figs. 1 and 3A; X2 df 4 = 169.34, p <0.001), with an overrepresentation of genes in
the melanogenesis pigmentation pathway (Fig.
.3, B and C; n = 21, X:dJ 4 = 147.16, P < 0.001; seetable fn for Gene Ontology terms). Nineteen of
20 differentially expressed melanogenesis related
genes were underexpressed in the gray hooded
crow torso (table SS). The pathway wide patternof reduced expression could not be attributed to·differences in melanocyte density (Fig. 4), but.rather to upstream regulatory changes inducing
broad scale down regulation in follicles produc
ing gray feathers (Fig. 3C).
We surveyed the landscape of genomic diver·gence with a window based approach based on
population genetic summary statistics derived
for 22,072 nonoverlapping 50 kb windows (Fig.
2A and fig. SS) and a hypothesis free clustering

1 41 0

Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-260687

algorithm reoonstructing local genomic phylog
enies (so called "cacti") (10) (Fig. 2C). Phylogen
etic clustering segmented the genome into two
major topologies (class I and class II cacti here
after; fig. 89), with only 0.28% of the genome
following a topology that clearly separated car
rion and hooded crows. Both methods identified
one L95 Mb genomic region exhibiting extreme
genetic differentiation between carricn and hooded
crows on scaffolds 78 and 60, which, on the basisof synteny with other birds, are located adja
cently on chromosome 18 [Fig. 2, A and B, andfig. S8A; for a detailed exploration of divergencepeaks, see (10) and fig. SS]. This region, harboring 81 of an 82 fixed sites between carrion andhooded crows, oontained 40 annotated protein
coding genes, had strongly reduced levels of nu
cleotide diversity, and showed increased linkage
disequilibrium (LD). Disrupted patterns of LD,
taxon specific dropout of paired end read map
ping (10) (fig. 810), and the presence of two local
F!rr peaks connected by a saddle (11) suggest thepossibility of an inversion in this region.
In the center of the right local F!IT peak (Fig.
2B), a region about 4.1 kb upstream of the PRKCA
gene exhibited longer haplotype blocks (iES statistic) and showed evidence for recent, hooded
crow specific, positive selection: It was enriched
for fixed hooded crow specific derived variants(22 of 28) and had significantly reduced values ofFu andU'sD statistic(P<O.fiS) in hooded crows.
Furthermore, this region contains a tandem ar
ray of voltage dependent calcium channel subunits,
including those encoded by CACNG5, CACNG4,
and CACNGI. The CACNG gene fumily codes fortransmembrane regulators of AMPA receptors,
which influence the microphthalmia associatedtranscription factor gene MITF, a central regu
latory element of the melanogenesis pathway (12)(Fig. 3C). At least 11 of the 21 melanogenesis
genes underexpressed in gray hooded crow fea
ther follicles are directly regulated by MI1F (e.g.,
TYR, TYRPl, SLOJSA2, RAB38, EDNR.B2) or interactwith.M17Fin feedbackloq>S (MCJR,HPGES)(Fig. 3C and table 88). Moreover, RAS GRFJ, acentral mediator between calcium concentrationand downstream gene expression (13), was sig
nificantly underexpressed in the gray feather fol
licles of hooded crows (Fig. 3C and table 88). Thistentatively links gene expression, color pheno
type, and the signature of divergent selection in
the divergence peak.
Two additional strongly differentially expressedmelanogenesis genes were located in divergent
genomic regions: NDP (Norrie disease pseudo
glioma) and HPGDS (hematopoietic prosta
glandin D synthase), which also act dosely
upstream of MI1F (14) (Fig. 3C and fig. 811).
.HPGDS was among the most strongly underexpressed genes in t he hooded crow torso and

Fig . 1. The crfNi s ystem. Illustrations of carrion
crow (left) and hooded crow (right). We sampledgene expression data of regrowing feathers from thehead and torso, which differ in coloration. The map
below shows the European distribution of the carrion
crow (dark gray area) and hooded crow (light grayarea): the hybrid zone is shown as a black solid line.
Sampling locations of this study in Spain (Sp) .
Germany (Ge). Poland (Po). and Sweden (Sw) areindicated by filled circles (black. carrion crow; gray.
hooded crow). Superimposed on the geographicalmap are the major axes of genetic variation (principal
components PCl and PC2) derived from wholegenome resequencing data from the four populat ions (black and gray dots. respectively). [Drawings
courtesy of Dan Zetterstrom]also showed an extreme reduction in variance,
consistent with selection (Ansari Bradley test, P: 0.0039). A multigenic architecture of the color
trait is consistent with the segregation patternof different crow color phenotypes in the hy
brid zone (fig. Sl2), indicating that a number ofgenes likely contribute to color divergence.
Under the left local F!rr peak, fixed differences
enriched for carrion crow derived variants wereclustered in a narrowly defined area (Fig. 2B). An.exception included a hooded derived fixed vari
ant in the regulatory region of the gene RGS9
(regulator of G protein signaling 9), which affects
visual perception in vertebrates (15), was highly
expressed in eye (higher than in any other tissue·by a factor of 5), and showed lower expression in:hooded crows across tissues (fig. Sll). Not only is
RGS9 involved in phototransduction, but differ.ent splice forms (also detected in crows; fig. Sll)regulate dopamine and opioid signaling in the'brain (15). This potential pleiotropy is of interest
because the main differen ces between carrion
and hooded crows are coloration, oolor ass:>rtative
mate choice (9), and hormone mediated social
dominance behavior (16). These diverse biolog
ical functions may also be explained by the SC£24
gene family (17): SLC24A2 andSLC24A3, involved'in vision and hormonal regulation, respectively,
were associated with class I cacti in crows (Fig.
2C), and SLC24A4, involved in pigmentation,

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Fig . 2. The genomi c landscape of divergence. (A) Pairwise genetic differentiation (Fsr) in 50-kb sliding windows across the genome between carrion
crows and hooded crows (top panel). between carrion crow populations(second panel). between hooded crow populations (third panel), and between
carrion crows and hooded crows from across the hybrid zone (bottom panel).
Alternating colors denote the different chromosomes; and the dotted horizontal lines mark the 99th-percentile Fsr estimates of autosomes and the
Z chromosome. respectively. Red arrows indicate taxon-specific peaks ofconsecutive elevated Fsr windows; black arrows highlight outlier peaks thatare specific to comparisons with the refugial Spanish population. (B) The
largest and most extreme Fsr peak was located on scaffolds 78 and 60.

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Plotted within the peak are 81 of 82 total fixed variants (black bars in top
panel. ancestral alleles: gray bars. derived hooded crow- specific alleles). (C)Localized phylogenetic patterns within the genome ("cacti").T he majority of
the genome exhibits neutral genetic divergence classified as class 11 cacti(divergent Spanish population or complete admixture).Two cacti display clear
separation of carrion crows (black: Spain. solid circles; Germany, open
circles) and hooded crows (gray: Sweden. solid circles. Poland. open circles)(class I cacti; fig. S9). Four exemplar gene models associated with themelanogenesis pathway and associated with visual perception are depicted(green blocks and lines represent exons and introns . respectively) along withthe regions assigned to class I cacti (blue).

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Fig. 3. The functional genomic basis of pi ~.m age col or differences. (A) Photograph of the developmental stage of feather f ollicles used for gene expression
quantification. (B) Percentage of all expressed genes (IM'lite) and melanogenesis genes (striped) inferred to be differentially expressed. (C) Schematic
overview of t he melanogenesis pathway. with genes underexpressed in skinfrom torso in hooded crows shown in blue. genes overexpressed shown in red
Fig. 4. Characterization of feather melanocytes.
Immunohistochemistry on feather follicles of carrion
crows (upper panel) and hooded crows (lower panel)for one of the melanogenic enzymes (a-Trpl , cyan)differentially expressed in our RNA-seq data confirms differential melanogenesis pathway activity(left column; view on to barbed ridges of wholemount feather bulges. yellow arrows) but shows nostriking differences in melanocyte density (rightcolumn; detail of sectioned f eather follicle. magenta
square). Matched samples from carrion crows andhooded crows were stained in parallel and imaged
under identical conditions to ensure comparability.
br. barbed ridge; p, pulp. Scale bar. 50 J!m. [Drawing
courtesy of Kristina Fraune; f eather photo from
Wikimedia Commons]pro1e1n acuva110n
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MELA N O SOME

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compound(color scale indicates relative change). Genes either not supported by RNA-seq
data or expressed at FPKM (fragments per kilobase of exon per million
fragments mapped) levels below 1.0 in both hooded and carrion crows areshown as gray boxes. Pathway relationships are shown for transcriptional
activation (black double arrows), protein activation (e.g.. by phosphorylation)(black single arrows). and transport (gray single arrow. dotted line).

brcarrion crowhooded crow1413

was strongly underexpressed in hooded crows(Fig. 3C).
Our results show that the pronounced pheno
typic differences between hooded and canion
crows are maintained in the face of substantial
gene ftow by variation in less than 1% of the
genome. The crow system thus constitutes anintriguing test case for the appropriate level oftaxonomic delineation (18) and the interpretation of local peaks of divergenre (often called"speciation islands'') (19). Gene flow slightly reduced the width of the main divergence peakand eroded linkage disequilibrium at the peakmargins (fig. SlOB) but slightly increased thepeak amplitude (10). Hence, divergent selection
acting on small genomic regions did not appear
to expand local differentiation or seed genome
wide reproductive isolation (19), although it did
maintain phenotypic differentiation in the faceof gene flow. Our findings further highlight therole of inversions in evolution (20, 21), poten
tially linking key genes involved in amatingtrait
and a tentative visual preference locus. This es
tablishes an interesting parallel to studies inwhich single genes (22), "supergenes" (4), or large
scale inversions (23) have been implicated indriving morphological change relevant to matechoice, but contrasts with studies in which manyeoologically relevant loci (21) or structural genomic
features dominated the genomic landscape of divergence (24). A key feature that distinguishes

1 414

the crow system is the apparent lack of ecologicalselection on the maintenance of separate pheno
types. Instead, the data presented here are oon
sistent with the idea that assortative mating andsexual selection can exclusively cause phenotypicand genotypic differentiation (25).

21 F. C. Jones et al, Nature 484. 55 61 (2012).
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23. H. Thaneycroft. EvoiJUCtt 29. 611 621 (1975).
24. S. Renaut et al.. NaL Corrmun. 4. 1827 (2013).
25. C. A. Mclem. D. Stuart Fox. Bioi. lilev. Camb. ~ilos. Soc.
10.11Wbrv.l2083 (2014).
ACKNOWLEDG M ENTS

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We thank W. Fledler. K. Temnow. L. Vossen, M. OOptner. T. Part.
K. H. Siebenrock. and the other statf d the Max Planck Institutefor Ornithology for help with field work and animal husbandry.
R. Burn assisted in population geneijc malyses. Supported by
VolkswagenStittung grant 1/83 496, Swedish Research Counci
grant 6212010 5553, the Wallenberg AdvalCed Bianformatics
Infrastructure (funded by the Knut and Afice Wallenberg

Foundation), Ca~ Tryggers foundation grant CTSI1517. CTSl2543.

m Euroj:em Research Cruncil grant ERCStG 336536 (J.B.W.W.):

the Max Planck Society (M.W.. I.M.): and SciUfelab Uppsala(M.G.G .. H.l.). C. !R:hyrhynchos samples were provided bycourtesy of the American Museum of Natural History, New York:fblish hooded crows by A. Kruszewicz and M. Gajownik.
Warsaw Zoo. We thank the UPPMAX Next Generation Sequencing
Cluster m SJorage (UPPNEX) prqect. funded by the Knut m
Alice Wallerberg Foundation m the Swedish NatiCttal lrtrastructure
for Computing, for access to computational resources. All
sequencing data m the genome assembly a-e available in the
NCBI short read archive under project PRJNA192205.