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We have a limited understanding of the genetic and molecular basis of evolutionary changes in the size and proportion of limbs. We studied wing and pectoral skeleton reduction leading to flightlessness in the Galapagos cormorant ( Phalacrocorax harrisi). We sequenced and de novo assembled the genomes of four cormorant species and applied a predictive and comparative genomics approach to find candidate variants that may have contributed to the evolution of flightlessness. These analyses and cross-species experiments in Caenorhabditis elegans and in chondrogenic cell lines implicated variants in genes necessary for transcriptional regulation and function of the primary cilium. Cilia are essential for Hedgehog signaling, and humans affected by skeletal ciliopathies suffer from premature bone growth arrest, mirroring skeletal features associated with loss of flight.
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A genetic signature of the
evolution of loss of flight in
the Galapagos cormorant
Alejandro Burga,*Weiguang Wang, Eyal Ben-David, Paul C. Wolf, Andrew M. Ramey,
Claudio Verdugo, Karen Lyons, Patricia G. Parker, Leonid Kruglyak*
INTRODUCTION: Changes in the size and pro-
portion of limbs and other structures have
played a key role in the evolution of species.
One common class of limb modification is re-
current wing reduction and loss of flight in
birds. Indeed, Darwin used the occurrence of
flightless birds as an argument in favor of his
theory of natural selection. Loss of flight has
evolved repeatedly and is found among 26 fam-
ilies of birds in 17 different orders. Despite the
frequency of these modifications, we have a
limited understanding of their underpinnings
at the genetic and molecular levels.
RATIONALE: To better understand the evolu-
tion of changes in limb size, we studied a classic
case of recent loss of flight in the Galapagos
cormorant (Phalacrocorax harrisi). Cormorants
are large water birds that live in coastal areas
or near lakes, and P. harrisi istheonlyflight-
less cormorant among approximately 40 extant
species. The entire population is distributed
along the coastlines of Isabela and Fernandina
islands in the Galapagos archipelago. P. harrisi
has a pair of short wings, which are smaller
than those of any other cormorant. The extreme
reduction of the wings and pectoral skeleton
observed in P. harrisi is an attractive model
for studying the evolution of loss of flight be-
cause it occurred very recently; phylogenetic evi-
dence suggests that P. harrisi diverged from its
flighted relatives within the past 2 million years.
We developed a comparative and predictive ge-
nomics approach that uses the genome se-
quences of P. harrisi and its flighted relatives
to find candidate genetic variants that likely
contributed to the evolution of loss of flight.
RESULTS: We sequenced and de novo assem-
bled the whole genomes of P. harrisi and three
closely related flighted cormorant species. We
identified thousands of coding variants exclu-
sive to P. harrisi andclassifiedthemaccording
to their probability of altering protein function
based on conservation. Variants most likely to
alter protein function were significantly enriched
in genes mutated in human skeletal ciliopathies,
including Ofd1,Evc,Wdr34,andIft122.Wecar-
ried out experiments in Caenorhabditis elegans
to confirm that a missense variant present in
the Galapagos cormorant IFT122 protein is
sufficient to affect ciliary function. The pri-
mary cilium is essential for Hedgehog (Hh)
signaling in vertebrates, and individuals af-
fected by ciliopathies have small limbs and
ribcages, mirroring the phenotype of P. harrisi.
We also identified a 4amino acid deletion in
served transcription factor
that has been expe rim ent -
ally shown to regulate limb
growth in chicken. The four
missing amino acids are
perfectly conserved in all
birds and mammals se-
quenced to date. We tested the consequences
of this deletion in a chondrogenic cell line and
showed that it impairs the ability of CUX1 to
transcriptionally up-regulate cilia-related genes
(some of which contain function-altering var-
iants in P. harrisi) and to promote chondrogenic
differentiation. Finally, we show that positive
selection may have played a role in the fixation
of the variants associated with loss of flight in
P. harrisi.
CONCLUSION: Our results indicate that the
combined effect of variants in genes necessary
for the correct transcriptional regulation and
function of the primary cilium likely contrib-
uted to the evolution of highly reduced wings
and other skeletal adaptations associated with
loss of flight in P. harrisi. Our approach may
be generally useful for identification of var-
iants underlying evolutionary novelty from ge-
nomes of closely related species.
Burga et al., Science 356 , 921 (2017) 2 June 2017 1of 1
The list of author affiliations is available in the full article online.
*Corresponding author. Email:
(A.B.); (L.K.)
Cite this article as A. Burga et al., Science 356, eaal3345
(2017). DOI: 10.1126/science.aal3345
Comparative and predictive genomics of loss of flight. Comparison of the genomes of four closely related cormorant species allowed us
to predict function-altering variants exclusively affecting the Galapagos cormorant and to test their functional consequences. Our results
implicate ciliary dysfunction as a likely contributor to the evolution of loss of flight.
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A genetic signature of the
evolution of loss of flight in
the Galapagos cormorant
Alejandro Burga,
*Weiguang Wang,
Eyal Ben-David,
Paul C. Wolf,
Andrew M. Ramey,
Claudio Verdugo,
Karen Lyons,
Patricia G. Parker,
Leonid Kruglyak
We have a limited understanding of the genetic and molecular basis of evolutionary
changes in the size and proportion of limbs. We studied wing and pectoral skeleton
reduction leading to flightlessness in the Galapagos cormorant (Phalacrocorax harrisi).
We sequenced and de novo assembled the genomes of four cormorant species and applied
a predictive and comparative genomics approach to find candidate variants that may
have contributed to the evolution of flightlessness. These analyses and cross-species
experiments in Caenorhabditis elegans and in chondrogenic cell lines implicated variants
in genes necessary for transcriptional regulation and function of the primary cilium.
Cilia are essential for Hedgehog signaling, and humans affected by skeletal ciliopathies
suffer from premature bone growth arrest, mirroring skeletal features associated with
loss of flight.
The evolution of loss of flight is one the most
recurrent limb modifications encountered
in nature (1). Indeed, Darwin used the occur-
rence of flightless birds as an argument in
favor of his theory of natural selection (2).
He proposed that loss of flight could evolve as a
result of selection in favor of larger bodies and
relaxed selection due to the absence of predators.
Loss of flight has evolved repeatedly and is found
among 26 families of birds in 17 different orders
(1). Moreover, recentstudies strongly suggest that
the ratites (ostriches, emus, rheas, cassowaries,
and kiwis), long thought to derive from a single
flightless ancestor, may constitute a polyphyletic
group characterized by multiple independent in-
stances of loss of flight and convergent evolution
(35). However, despite the ubiquity and evolu-
tionary importance of loss of flight (6), the under-
lying genetic and molecular mechanisms remain
The Galapagos cormorant (Phalacrocorax harrisi)
mately 40 extant species (7).Theentirepopulation
is distributed along the coastlines of Isabela
and Fernandina islands in the Galapagos archi-
pelago. P. harrisi has a pair of short wings, which
are smaller than those of any other cormorant
(Fig. 1A)a deviation from the allometric relation-
ship between wing length and body mass (7). The
radius and ulna are disproportionately small rela-
tive to the humerus, but no digits have been fused
or lost, unlike in some ratites (8). In addition, the
Galapagos cormorant differs from its flighted rela-
tives in a delay in the onset of several develop-
mental landmarks after hatching (9), shortened
remiges (flight feathers), underdeveloped pectoral
muscles, a long and narrow skull and pelvis, a dis-
proportionately long tibiotarsus, a factor of 1.6 in-
crease in body mass, and a highly reduced keel (7).
The keel is an extension of the sternum that runs
along its midline and provides an attachment sur-
face for the flight muscles, the largest muscles in
birds. Flightless taxa, such as ratites and Cretaceous
Hesperornis, have evolved flat sternums in which
the keel has been largely reduced or lost (10).
In contrast to ratites and penguins, which be-
came flightless more than 50 million years ago
(Ma) (5,11), the Galapagos cormorant and its flighted
relatives are estimated to share a common ancestor
at ~2 Ma (12). This recent and extreme modification
of wing size and pectoral skeleton makes P. harrisi
an attractive model for studying loss of flight.
High-quality genome sequences of four
cormorant species
To identify variants associated with loss of flight,
we sequenced and de novo assembled the 1.2-Gb
genomes of the Galapagos cormorant (Galapagos
Islands, Ecuador) and three flighted cormorant spe-
cies: the double-crested cormorant (Phalacrocorax
auritus; Minnesota, USA), the neotropical cormo-
rant (Phalacrocorax brasilianus; Valdivia, Chile),
and the pelagic cormorant (Phalacrocorax pelagicus;
Alaska, USA). P. auritus and P. brasilianus are the
closest relatives of P. harrisi (1214), and P. pelagicus
Genomes were assembled from a combination of
short insert and mate-pair Illumina libraries with
SOAPdenovo2 (15) (table S1). Among these four
genomes, the Galapagos cormorantsassembly
had the longest contig and scaffold N50 metrics
(contig N50, 103 kb; scaffold N50, 4.6 Mb; table
S1B). We evaluated the completeness of the cor-
ber of uniquely annotated proteins in each assembly
andbyusingtheCEGMApipeline(16,17). Overall,
we found agreement between these two indepen-
dent metrics in a data set including the four cor-
morant genomes and 17 recently published bird
genomes (r
table S2).
Commonly used metrics of assembly quality,
such as contig and scaffold N50, were very poor
predictors of the total number of proteins present
in each assembly (r
0.06, P= 0.81; fig. S1 and table S2). Three of the
four cormorant genomes (P. harrisi,P. auritus,
and P. pelagicus) obtained the highest CEGMA
scores and numbers of uniquely annotated genes
among all bird genomes (red triangles, Fig. 1B).
The following CEGMA scores, a means to estimate
genome completeness, were obtained for the cor-
morants: P. harrisi,90.3%;P. auritus,91.3%;
P. brasilianus,72.6%;P. pelagicus,87.1%.Incon-
trast, Sanger and PacBio genomes had lower scores
for other birds: Gallus gallus (Sanger assembly)
(18), 80.7%; Taeniopygia guttata (Sanger assembly)
(19), 71.4%; and Melopsittacus undulatus (PacBio
assembly) (20), 79.0%. Thus, our cormorant ge-
nomes perform even better than genomes assem-
bled from Sanger sequences and PacBio long
reads (complete statistics in table S2).
Phylogeny and genetic diversity
We reconstructed the cormorant phylogeny using
a Bayesian framework (17) and confirmed the phy-
logenetic relationship among the four sequenced
species (Fig. 1C). Moreover, our results indicate
that P. harrisi last shared a common ancestor with
P. auritus and P. brasilianus at ~2.37 Ma, in
agreement with an estimate from mitochondrial
DNA (12) (Fig. 1C). Española, the oldest extant
island in the Galapagos archipelago, emerged no
earlier than 4 Ma, and proto-Galapagos islands
existed at 9 Ma or earlier (21). Our results are con-
sistent with the view that P. harrisi lost the abil-
ity to fly while inhabiting the archipelago.
We calculated the proportion of single-nucleotide
polymorphism (SNP) heterozygous sites for each
sequenced individual to estimate the levels of in-
traspecific genetic diversity (Fig. 1D). P. harrisi
showed the lowest proportion of heterozygous
SNPs among the sequenced cormorants (0.00685%;
Fig. 1D). The heterozygosity of P. harrisi is even
lower than that of the crested ibis, Nipponia nippon,
a highly endangered bird with a small effective
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 1of8
Department of Human Genetics, Department of Biological
Chemistry, and Howard Hughes Medical Institute, University
of California, Los Angeles, CA, USA.
Departments of
Molecular, Cell and Developmental Biology and Orthopaedic
Surgery, University of California and Orthopaedic Institute for
Children, Los Angeles, CA, USA.
Wildlife Services, U.S.
Department of Agriculture, Roseburg, OR, USA.
U.S. Geological
Survey Alaska Science Center, Anchorage, AK, USA.
Instituto de Patología Animal, Facultad de Ciencias
Veterinarias, Universidad Austral de Chile, Valdivia, Chile.
Department of Biology and Whitney Harris World Ecology
Center, University of Missouri, St. Louis, MO, USA.
Institute, Saint Louis Zoo, St. Louis, MO, USA.
*Corresponding author. Email: (A.B.); (L.K.)
on June 13, 2017 from
population size and a known recent population
bottleneck (22) (0.0172%; Fig. 1D). The low level
of heterozygosity found intheGalapagoscormo-
rant is most likely due to its small population
size (~1500 individuals) and multiple popula-
tion bottlenecks (23).
Discovery and characterization of
function-altering variants in P. harrisi
To investigate the genetics of flightlessness evo-
lution, we developed a comparative and predic-
tive genomics approach (24,25) that uses the
genome sequences of P. harrisi and its flighted
relatives to identify genetic variants that likely
contributed to the evolution of loss of flight. Both
coding and cis-regulatory variants have been im-
plicated in the evolution of morphological traits
(26,27). However, determining the impact of
regulatory variants is not straightforward. To iden-
tify the contribution of regulatory variants to the
evolutionoflossofflightinP. harrisi,wesearched
for ultraconserved noncoding sequences showing
accelerated molecular evolution (2830). We iden-
tified 11 ultraconserved noncoding regions in tetra-
pods that show accelerated evolution in P. harrisi
but not in the other cormorants [false discovery
rate (FDR) < 5%]. One of these regions was lo-
cated in an intron of the gene FTO (fig. S2), which
has been associated with obesity in humans (31);
however, none of these regions overlapped with
experimentally validated or putative mouse limb
enhancers (17,32,33)(tableS7).
We thus focused on characterizing coding var-
iants because we are better able to predict their
molecular consequences. For our variant discovery
approach to be comprehensive, it was imperative
to interrogate most of the Galapagos cormorants
genes. To increase our power to do so, we annotated
genes using homology-based and transcriptome-
based gene annotations. The latter were derived
with mRNA expression data from the developing
wing of a double-crested cormorant embryo (fig.
S3C) (17). We then predicted all missense, dele-
tion, and insertion variants in ortholog pairs be-
tween P. harrisi and each of its three flighted
relatives (fig. S4) (17).
We used PROVEAN (34), a phylogeny-corrected
variant effect predictor, to evaluate the impact on
predictions have been validated in experimental
evolution studies that mimic the process of grad-
ual accumulation of mutations in nature (35). A
PROVEAN score is calculated for each variant;
the more negative the score, the more likely a
given variant is to alter protein function. We ex-
amined the distribution of PROVEAN scores ob-
tained when comparing 12,442 ortholog pairs
between P. harrisi and P. auritus (Fig. 2A). Of
these, 4959 pairs (40%) did not contain coding
variants; the remaining 7483 pairs contained a
total of 23,402 coding variants: 22,643 single
amino acid substitutions, 456 deletions, and 303
insertions (Fig. 2B). Most variants were predicted
to be neutral (the distribution is centered around
zero). As expected, deletions and insertions were
enriched in the tails of the distribution (Fig. 2B).
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 2of8
Fig. 1. The Galapagos cormorant, a model for studying flightlessness evolution. (A) The
average wing length of an adult Galapagos cormorant male is 19 cm (3.6 kg body mass), whereas the
wing length of its closest relative, the double-crested cormorant, is 31.5 cm (2.2 kg body mass).
[Illustration by Katie Bertsche from specimens 134079 and 151575, Museum of Vertebrate Zoology at
Berkeley] (B) The CEGMA score is a good predictor of genome completeness from a gene-centric
perspective. The blue line is the linear regression model (r
). Genomes
reported in this study are red triangles; other published avian genomes are black circles (table S2).
(C) Bayesian phylogram reconstructed with fourfold degenerate sites from whole genome
sequences. The orange bar illustrates the time span between the approximate origin of the proto-
Galapagos archipelago (9 Ma) and the origin of the oldest extant island, San Cristobal (4 Ma). Nodes
represent median divergence ages. Blue bars indicate the 95% highest posterior density interval.
(D) Heterozygosity levels inferred from whole genome sequences. Birds are not drawn to scale.
Table 1. Function-altering variants in P. harrisi are enriched for genes that cause skeletal
ciliopathies in humans. Sanger-validated examples of function-altering variants (PROVEAN score
<5) in P. harrisi. Cilia/Hh-related genes were found on the basis of functional enrichment for
human syndromes. PCP (planar cell polarity) genes were selected according to literature evidence
linking cilia and PCP. These variants are fixed in the population.
Gene Pathway Variant PROVEAN score Human syndrome
Ofd1 Cilia/Hh R325C
Orofaciodigital and Joubert
................................... ................................................................ ............................................................... ....................................................
Talpid3 Cilia/Hh D759V 7.805 Joubert and Jeune
................................... ................................................................ ............................................................... ....................................................
Evc Cilia/Hh T341I 5.546 Ellisvan Creveld
................................... ................................................................ ............................................................... ....................................................
Dync2h1 Cilia/Hh P2733S 7.431 Short-rib thoracic dysplasia
................................... ................................................................ ............................................................... ....................................................
Ift122 Cilia/Hh Q691L 5.491 Cranioectodermal dysplasia
................................... ................................................................ ............................................................... ....................................................
Wdr34 Cilia/Hh P188R 6.337 Short-rib thoracic dysplasia
................................... ................................................................ ............................................................... ....................................................
Kif7 Cilia/Hh R833W 6.827 Joubert and acrocallosal
................................... ................................................................ ............................................................... ....................................................
Gli2 Hh P1086T 5.117 Culler-Jones
................................... ................................................................ ............................................................... ....................................................
Fat1 PCP S1717L
................................... ................................................................ ............................................................... ....................................................
Dchs1 PCP G2063D 6.45 Van Maldergem
................................... ................................................................ ............................................................... ....................................................
Dvl1 PCP P103L 8.23 Robinow
................................... ................................................................ ............................................................... ....................................................
*Based on phenotypic similarity to mutant mouse model.
on June 13, 2017 from
Very similar numbers of variants and PROVEAN
score distributions were obtained for the other
homology-based (fig. S5) and transcriptome-based
annotations (fig. S3).
Enrichment for genes mutated in
skeletal ciliopathies
To identify proteins carrying function-altering
variants in the Galapagos cormorant, we applied
a stringent threshold to our four prediction data
sets: PROVEAN score less than 5, twice the thresh-
old for discovery of human disease variants (17,34)
(Fig. 2A). In our data set, variants with a PROVEAN
score less than 5 typically occur at residues that
have been perfectly conserved at least since mammals
and birds last shared a common ancestor (~30 0 Ma;
fig. S6). Consequently, changes in these residues
are likely to alter protein function or stability.
On the basis of theoretical and experimental
considerations (36), we hypothesized that flight-
lessness is likely to have a polygenic basis and
that the underlying variants would be enriched
in certain biological pathways. Consistent with this
hypothesis, gene enrichment analysis of function-
altering variants in the Galapagos cormorant
revealed that genes implicated in human develop-
mental disorders were significantly overrepresented
(17) (table S3A). Strikingly, 8 of the 19 significantly
enriched categories (Human Phenotype Ontology)
consisted of genes that affect limb development
when mutated, leading to disorders such as poly-
dactyly, syndactyly, and duplication of limb bones.
Control analyses showed no enrichment of these
categories in the flighted cormorants (17)(table
S3, B and C).
Many of the genes underlying the enrichment
for limb syndromes are those mutated in a family
of human disorders known as ciliopathies. For
instance, 17 of 25 genes (65%) in the duplication
of hand bonescategory and all 12 genes (100%)
in the preaxial hand polydactylycategory are
mutated in human ciliopathies (table S4). More-
over, ciliopathy-associated genes were present in
all of the enriched categories (table S4). Ciliopathies
comprise a phenotypically diverse group of rare
genetic disorders that result from defects in the
formation or function of cilia (37).
Cilia are hairlike microtubule-based structures
that are nucleated by the basal body (centriole and
associated proteins) and project from the surface
of cells. Primary cilia are essential for mediating
Hedgehog (Hh) signaling in vertebrates, serving
as antennae for morphogens during development
(38). We confirmed by Sanger sequencing the
presence of predicted function-altering variants
in Ofd1,Evc,Talpid3,Dync2h1,Ift122,Wdr34,and
Kif7, all of which are necessary for the assembly
or functioning of the primary cilium and are mu-
tated in human ciliopathies, particularly those
affecting the skeleton (Table 1). We also found
a likely function-altering variant in Gli2,atran-
scription factor necessary for Hh signaling (39)
(Table 1). Humans affected by diverse skeletal
ciliopathies have small limbs and ribcages (37),
suggesting a parallel with the main features of
the Galapagos cormorant: small wings and a flat-
tened sternum. However, the consequences of
ciliopathies in humans are often more severe and
pleiotropic, likely as a consequence of the over-
representation of loss-of-function alleles in patients
(40). Interestingly, although ciliopathies do not
exclusively affect the forelimb in humans, differ-
monly found among patients. For example, digital
the feet among oral-facial-digital syndrome type I
(40,41)andEllisvan Creveld (42) patients, which
suggests that forelimbs and hindlimbs differ in
their intrinsic sensitivity to ciliary dysfunction.
The Galapagos cormorant ortholog of human
OFD1 (mutated in oral-facial-digital syndrome 1)
contains three predicted function-altering var-
iants with a PROVEAN score less than 5: Arg
Cys (R325C), 6.913; Lys
Thr (K517T),
5.673;and Glu
Gly (E889G), 5.068 (fig. S6,
AandB).Ofd1 knockout mice display polydactyly
and shortened long bones (43). Also, a function-
altering missense variant [Gln
Leu (Q691L),
score 5.491] was found in IFT122, a component
the ciliary localization of Shh pathway regulators
(44). Null Ift122 mutants show severe limb and
skeletal phenotypes in mice (45), and mutations
in Ift122 have been associated with Sensenbrenner
syndrome in humans, which is characterized by
craniofacial, ectodermal, and skeletal abnormal-
ities, including limb shortening (46). Strikingly,
the mutated Gln in IFT122 is virtually invariant
among eukaryotes as different as green algae,
C. elegans,Drosophila, and vertebrates (fig. S6C).
To directly test whether the nonsynonymous sub-
stitution in IFT122 affects protein function, we
generated a C. elegans knock-in strain using CRISPR/
Cas9 homology-directed genome editing (Fig. 3A).
The edited strain carries the Galapagos cormorant
missense variant at the corresponding orthologous
position in daf-10 [Gln
Leu (Q862L)], the
ortholog of IFT122 in C. elegans (47).
In invertebrates, cilia do not mediate Shh sig-
naling but are necessary for detecting external sen-
sory inputs (48). The only ciliated cells in C. elegans
are sensory neurons, and mutations in cilia com-
ponents affect dispersal behavior, chemotaxis, and
dauer formation (49). We tested the bordering be-
havior of worms (accumulation of animals on the
thickest part of a bacterial lawn), which is known
to be mediated by ciliated neurons (50). We found
that daf-10(e1387) mutants carrying a premature
stop (Q892X) displayed an increase in bordering
behavior in a dispersal assay relative to the wild
type [73% for daf-10(e1387) versus 30% for N2; P=
0.019, ttest;Fig.3,B,C,andE].Twoindepen-
dently generated daf-10 Q862L knock-in lines
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 3of8
Fig. 2. Distribution of the effect of variants between P. auritus and P. harrisi.(A) We used PROVEAN to predict the effect on protein function of
23,402 variants contained in 12,442 orthologous pairs between P. auritus and P. harrisi; 4966 pairs contained no variants. The more negative the score,
the more likely the variant affects protein function. PROVEAN score thresholds used in this study are drawn as vertical dashed lines. Numbers of proteins
and variants found for each threshold are shown in the inset. (B) Density of PROVEAN scores for each class of variant.The same variants shown in (A) were
classified as single amino acid substitutions, deletions, and insertions. Numbers of variants in each class are indicated. a.u., arbitrary units.
on June 13, 2017 from
phenocopied the effect of the premature stop
allele (70% for line 1 and 69% for line 2 versus
30% for N2; P= 0.018 and 0.016, respectively;
Fig. 3, B, D, and E; see movie S1). Daf-10(e1387)
ciliary neurons fail to incorporate fluorescent dyes,
like many other loss-of-function mutants in cilia
components (49). In contrast, the daf-10 Q862L
knock-in worms incorporated the DiO dye in the
same manner as wild-type worms, which suggests
that this allele is hypomorphic (fig. S7). Overall,
these results indicate that the IFT122 Q691L
missense variant present in the Galapagos cormo-
rantcanaffectproteinfunctioninvivoinC. elegans.
The planar cell polarity (PCP) pathway exhib-
its a genetic link to cilia (38,5153). We found
function-altering variants in members of the PCP
pathway in P. harrisi: Fat1 atypical cadherin
(Fat1), Dachsous cadherin-related 1 (Dchs1), and
Disheveled-1 (Dvl1) (Table 1). The Galapagos cor-
morant FAT1 contains two function-altering var-
iants, Ser
Leu and Tyr
Cys (S1717L
and Y2462C; Table 1). The mutated Ser and Tyr
are conserved from zebrafish to humans (fig. S5D).
Fat1 knockout mice show very selective defects
in muscles of the upper body but not in posterior
muscles (54). In addition, Dvl1 is mutated in hu-
mans with Robinow syndrome, characterized by
limb shortening (55,56).
Sanger sequencing of 20 Galapagos cormorant
individuals from two different populations (Cabo
Hammond and Cañones Sur) (57)revealedonly
homozygous carriers for all of the variants in
Table 1, indicating that these variants are most
likely fixed in the Galapagos cormorant. In sum-
mary, we found an overrepresentation of pre-
dicted function-altering variants in genes that,
when mutated in humans and mice, cause skeletal
ciliopathies and bone growth defects.
CUX1 is mutated in P. harrisi
To identify the most likely function-altering var-
iants in P. harrisi,weappliedamorestringent
PROVEAN score threshold: 12.5 delta alignment
score, five times the threshold used for discovery
of human disease variants (34). This strategy nar-
rowed our search to 23 proteins (0.16% of anno-
tated proteins in P. harrisi)(tableS5).Wemanually
curated these 23 proteins and performed addi-
tional Sanger sequencing, reducing the list of pro-
teins with confirmed or putative variants to 12
(table S5) (17). These variants were exclusively
small deletions. Among these 12 proteins, two stood
out from their known role in development: LGALS-
3 and CUX1. LGALS-3 is affected by a 7amino acid
deletion in P. harrisi (PROVEAN score, 26.319).
LGALS-3 (Galectin-3) is localized at the base of
the primary cilium and is necessary for correct
ciliogenesis in mice (58),butithasnotbeenim-
plicated in human ciliopathies. Moreover, LGALS-
3physicallyinteractswith SUFU, an important
regulator of mammalian Hh signaling (59), and
knockout mice show pleiotropic defects in chon-
drocyte differentiation (60).
In addition, we found a 4amino acid deletion
(PROVEAN score, 15.704) in CUX1. CUX1 (cut-
like homeobox 1), also known as CDP, is a highly
conserved transcription factor with diverse roles
in development. CUX1 contains four DNA bind-
ing domains: three CUT domains (CR1 to CR3)
and one homeodomain (HD) (Fig. 4A) (61). The
full-length isoform, which contains four DNA bind-
ing domains (CR1-3HD), acts exclusively as a tran-
scriptional repressor and has rapid and unstable
DNA binding dynamics. In contrast, smaller iso-
forms such as CR2-3HD and CR3HD can act as
both repressors and activators of gene expression,
and show slow and stable DNA binding dynam-
ics in vitro (61).Althoughinsectandbirdwings
evolved independently, it is noteworthy that cut,
the Drosophila ortholog of Cux1, is necessary for
the proper development of wings and flight muscles
in flies (62). In chicken, Cux1 mRNA expression
in the limb at embryonic stage 23 is restricted to
the ectoderm bordering the apical ectodermal ridge
(AER) (63).TheAERisoneofthekeysignalingcen-
ters that drive limb development. At later stages,
Cux1 is expressed in the developing joints of both
chicken (64)andmouse(fig.S8)andisalsode-
tected in chondrocytes in developing bones of
mice (fig. S9). A function-altering variant in CUX1
is a strong candidate to contribute to loss of flight
in P. harrisi, because adenovirus-mediated over-
expression in the developing chicken wing of a
form of CUX1 missing the Cut2 DNA binding do-
main results in severe wing truncation (63,65).
These truncations most strongly affect distal skel-
etal elements (digits, radius, and ulna). As already
noted, in P. harrisi the radius and ulna are dis-
proportionately small relative to the humerus (7).
We Sanger-sequenced and confirmed the pre-
dicted Cux1 12base pair (bp) deletion in P. harrisi.
We also confirmed that this variant is fixed in the
population and absent in the other cormorant
species (fig. S10A). The 12-bp deletion in Cux1 re-
moves four amino acids, Ala-Gly-Ser-Gln (AGSQ),
immediately adjacent to the C-terminal end of
the homeodomain (Fig. 4B). We refer to this var-
iant as CUX1-D4aa. Alignment of CUX1 orthologs
from available vertebrate genomes revealed that
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 4of8
Fig. 3. The Galapagos cormorant variant IFT122 Q691L affects ciliary function in vivo. (A) The
daf-10 gene (IFT122 ortholog) was targeted with CRISPR/Cas9 homology-mediated repair in
C. elegans to introduce a nonsynonymous substitution present exclusively in the Galapagos cormorant
(IFT122 Q691L). The resulting edited knock-in strain contains the daf-10 Q862L substitution and
10 synonymous substitutions (17). Edited strains were sequenced with Sanger sequencing to
confirm genotypes. gRNA, guide RNA. (Bto D) Representative bordering behavior of N2 wild-type
worms (B), daf-10(e1387) containing a premature stop codon Q892X (C), and daf-10 Q862L knock-in
strain (D). (E) Quantification of bordering behavior in N2, daf-10(e1387), and two independently
generated knock-in daf-10 Q862L strains (n=3,ttest). *P< 0.05; error bars denote SE.
on June 13, 2017 from
the four missing residues are extremely con-
served among tetrapods (Fig. 4B). The deleted
Ser is phosphorylated in human cells (66), but the
consequences of this modification are unknown.
The Cux1 deletion does not include any of the
predicted residues responsible for DNA contact
and recognition (67), but given its close proximity
to the homeodomain, we decided to test whether
the DNA binding activity of CUX1 was affected.
We chose to express the CR3HD isoform because
Western blot analysis revealed that this was the
most abundant CUX1 isoform expressed in the
developing wing of mallard embryos (~50 kDa;
Fig. 4A). We performed electrophoretic mobility
shift assay (EMSA) with purified CR3HD CUX1-
Ancestral and CUX1-D4aa protein variants (fig.
S10B) as described (68) and found that DNA bind-
ing was not abolished in the deletion variant (fig.
S10C). CUX1 is able to both directly repress and
activate gene expression through its C-terminal
tail (69,70). We performed a luciferase reporter
assay (69,71) and found that both variants were
equally capable of repressing the expression of a
UAS/tk luciferase reporter (Fig. 4D). Thus, the
Galapagos cormorant CUX1-D4aa variant appears
repression activity in COS-7 cells.
CUX1 regulates the expression of cilia
and PCP genes
We hypothesized that the Cux1 deletion variant
is mechanistically related to the enrichment
of function-altering variants in ciliopathy-related
genes. This inference came from the fact that trans-
genic mice overexpressing the CR3HD-CUX1 iso-
form develop polycystic kidneys. Cilia in cystic
epithelial cells from these animals were twice
as long as the ones in control epithelial cells (72).
Furthermore, the CR2CR3HD-CUX1 isoform has
been shown to directly up-regulate the expression
of RPGRIPL1, also known as FTM, a component
of the cilia basal body that is involved in Shh
signaling and mutated in human ciliopathies (73).
Also, Cux1 knockout mice show deregulation of
SHH expression in hair follicles (74).
To test whether Cux1 could globally regulate
expression of cilia genes, we analyzed expression
array data from human-derived Hs578t cells stably
expressing a short hairpin RNA against Cux1,as
well as cells overexpressing the human CR2CR3HD-
CUX1 isoform (75). In concordance with the role
of Cux1 as a regulator of cell growth and pro-
liferation (76), genes significantly up- or down-
regulated in both conditions (P<0.05and>10%
change) were enriched for pathways such as cell
cycleand mitotic G
/S phases(P=3.99×10
and 0.016, respectively; table S6). We also found
enrichment for cilia-related categories such as
assembly of the primary ciliumand intraflagellar
transport(P= 0.00012 and 0.0057, respectively;
table S6). These results suggest that cilia-related
genes are enriched among Cux1 targets.
To further test whether Cux1 can regulate cil-
generated ATDC5 stable lines expressing N-
terminal His-tagged versions of CR3HD CUX1-
Ancestral and CUX1-D4aa variants. ATDC5 is a
well-characterized mouse chondrogenic cell line
that largely recapitulates in vitro the differentia-
tion landmarks of chondrocytes (77). We performed
quantitative reverse transcription polymerase chain
reaction (RT-qPCR) on a selected number of genes
containing predicted strong function-altering var-
iants in P. harrisi (Table 1) and showing detect-
able levels of expression in ATDC5 cells. In addition,
we measured the expression of Ptch1,thereceptor
the CUX1-Ancestral variant transcriptionally
up-regulated the expression of Ofd1 (factor of 1.7,
;Fig.4C)andFat1 (factor of 1.8, P=
0.029; Fig. 4C) and down-regulated the expres-
sion of Ift122 (factor of 0.77, P=0.0025;Fig.4C)
and Ptch1 (factor of 0.53, P=0.014;Fig.4C)relative
to the control line. In contrast, neither Dync2h1
nor Wdr34 expression levels were changed by
CUX1-Ancestral overexpression (Fig. 4C). These
results suggest that cilia- and Hh-related genes
Impaired transcriptional activity of the
Galapagos cormorant CUX1
The Galapagos cormorant CUX1 showed impaired
transcriptional activity relative to the ancestral var-
iant. Ofd1 was significantly up-regulated in CUX1-
D4aa cells relative to control cells [factor of 1.2, P=
0.021, analysis of variance (ANOVA) and Tukey
honest significant difference (HSD) test, Fig. 4C];
however, Ofd1 up-regulation was significantly
reduced in CUX1-D4aa cells relative to CUX1-
Ancestral cells (factor of 1.2 versus 1.7, P=6×10
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 5of8
Fig. 4. The Galapagos cormorant Cu x1 is a transcriptional activation hypomorph. (A) Western
blot showing the expression of CUX1 isoforms in the developing wing of a mallard embryo (22 days).
The most abundant band corresponds to the predicted size of the CR3HD CUX1 isoform. (B)Protein
alignment showing the deleted AGSQ residues in the Galapagos cormorant CUX1 and their high degree
of conservation among vertebrates. (C) Differential up-regulation of genes by CUX1-Ancestral and
CUX1-D4aa variants in ATDC5 cells. Pooled stable lines carrying CR3HD CUX1-Ancestral or CUX1-D4aa
variants were generated by lentiviral transduction and puromycin selection. Control cells were
transduced with an empty vector. Gene expression levels were measured by RT-qPCR (n= 5 biological
replicates, each comprising three technical replicates). (D) Luciferase-based assay to test the
repression activity of CUX1 C-terminal domain lacking CR3 and HD domains. GAL4 DNA binding
domain was fused to CUX1-Ancestral or CUX1-D4aa variants. Both constructs equally repressed a
promoter containing UAS binding sites in COS-7 cells (n= 3 biological replicates, each comprising three
technical replicates). In (C) and (D), error bars denote SE. *P<0.05,**P<0.01,***P< 0.001 (ANOVA
and Tukey HSD); black and red asterisks respectively denote significant difference of CUX1 variant
versus control and CUX1-Ancestral variant versus CUX1-D4aa variant.
on June 13, 2017 from
Fig. 4C). Similarly, although Fat1 was significantly
up-regulated in CUX1-Ancestral cells relative to
control cells (factor of 1.8, P=0.029),Fat1 ex-
pression levels in CUX1-D4aa cells were not sig-
nificantly different from control lines (factor of 1.2,
P= 0.57; Fig. 4C). The difference between Fat1
up-regulation in CUX1-D4aa and CUX1-D4aa cells
was not significant (factor of 1.8 versus 1.3, P=
0.17; Fig. 4C). In contrast, CUX1-D4aa cells signif-
icantly repressed both Ift122 (factor of 0.78, P=
0.0026) and Ptch1 (factor of 0.56, P=0.024),and
there were no significant differences between
CUX1-Ancestral and CUX1-D4aa cells (factor of
0.78 versus 0.78 for Ift122,P= 0.99; factor of 0.53
versus 0.56 for Ptch1,P= 0.96; Fig. 4C). These
results suggest that the 4amino acid deletion in
the Galapagos cormorant CUX1 affects its ability
to activate but not to repress gene expression;
they are also consistent with our luciferase reporter
assays, which showed no effect on repression (Fig.
4D). It is notable that both the transcriptional
activator (Cux1) and its target genes (Ofd1 and
Fat1) exhibit function-altering variants in the
Galapagos cormorant.
CR3HD-CUX1 promotes chondrogenesis
Chondrocytes are the main engine of bone growth.
The growth of skeletal elements depends on the
precise regulation of chondrocyte proliferation and
hypertrophy. Mutations that affect cilia result in
premature arrest of bone growth due to defects in
Indian Hedgehog (IHH) signaling in chondrocytes
(78). To test the role of CR3HD-CUX1 in chondro-
genesis, we differentiated control, CUX1-Ancestral,
and CUX1-D4aa ATDC5 cell lines and quantified
the expression of Ihh and Sox9, two well-established
markers of chondrocyte differentiation in vitro
and in vivo (78). Overexpression of both CR3HD
CUX1-Ancestral and CUX1-D4aa variants promoted
chondrogenic differentiation of ATDC5 cells after
7 and 12 days of differentiation (Fig. 5A). How-
ever, the CUX1-D4aa variant was not as efficient
as the ancestral variant, showing significant dif-
ferences from CUX1-Ancestral in Ihh expression
after 7 days of differentiation (~50% decrease, P=
and in Sox9 expression after 12 days (~15% de-
crease, P=1.6×10
; Fig. 5A). These results
suggest that the Galapagos cormorant CUX1 is
probably not as effective as the ortholog from
its flighted relatives in promoting chondrogenic
differentiation, and that mutations in Cux1 may
affect the dynamics of chondrogenesis. This obser-
vation is further supported by findings that CUX1
is expressed in the hypertrophic chondrocytes of
developing bones in mice, and that the bones of
Cux1 mutant mice are thin and flaky (79).
Possible evolutionary scenarios
Loss of flight has traditionally been attributed to
relaxed selection. In this scenario, the first cor-
morants that inhabited the Galapagos Islands
found a unique environment that lacked preda-
tors and provided food year-round, drastically
reducing the need to migrate. However, we found
no evidence for pseudogenization of developmen-
tal genes in P. harrisi (17) (tables S10 and S11). On
the other hand, loss of flight in the Galapagos
cormorant is thought to confer an advantage for
diving by decreasing buoyancy via shorter wings
and by indirectly allowing increased oxygen stor-
age via larger body size (80). This advantage could
make flightlessness a target of positive selection.
We evaluated whether any of our candidate
genes (Table 1) showed signatures of positive
estimating the ratio of nonsynonymous to syn-
onymous substitutions (w=dN/dS). This is a very
stringent test of selection because it assumes that
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 6of8
Fig. 5. CR3HD CUX1 promotes chondrogenesis. (A) ATDC5 control cells and cells carrying CR3HD
CUX1-Ancestral or CUX1-D4aa variants were differentiated into chondrocytes. Gene expression levels
were measured by RT-qPCR (n= 4 biological replicates, each comprising three technical replicates)
after 7 and 12 days. Error bars denote SE. *P<0.05,**P<0.01,***P< 0.001 (ANOVA and Tukey
HSD); black and red asterisks respectively denote significant difference of CUX1 variant versus control
and CUX1-Ancestral variant versus CUX1-D4aa variant. (B) Proposed mechanism for the reduction
of wing size in P. harrisi. Left: Normal functioning of IHH signaling pathway in vertebrates. CUX1
regulates the expression of cilia-related genes such as Ofd1 and promotes chondrogenesis. Right:
State of the IHH pathway in P. harrisi. Proteins in red have predicted function-altering variants in
P. harrisi. We propose that these variants would affect both cilia formation and functioning, leading
to a reduction in IHH pathway activity. As a result, the pool of proliferating chondrocytes would
decrease in wing bones and the number of hypertrophic chondrocytes would increase, resulting in
impaired bone growth.
on June 13, 2017 from
all sites in a protein are evolving under the same
selective pressure, a condition rarely met in highly
conserved regulatory genes (81). We found that 3
of 11 tested genes showed signs of positive selec-
tion (w>1)intheGalapagoscormorantlineage
compared to a background phylogeny of 35 taxa
(Ofd1 w=1.92,Evc w=1.93,Gli2 w=1.10;tableS8).
tically significant difference [w=1.10(Galapagos
branch) versus w= 0.11 (background branch), P=
0.0024; table S8]. In contrast, Gli2 showed no
sign of selection in the sister group of P. harrisi
(P. auritus and P. brasilianus)[w= 0.0001 (sister
branch) versus w= 0.11 (background branch), P=
0.46]. As a control, we also analyzed Gli3,the
partially redundant paralog of Gli2,whichalsomedi-
ates Hh signaling but has no predicted function-
altering variants in P. harrisi, and found no evidence
for positive selection [w=0.04(Galapagosbranch)
versus w= 0.15 (background branch), P=0.11;
table S8]. These results suggest that selection
toward flightlessness may be partially respon-
sible for the phenotype of P. harrisi.
The study of evolution of flightlessness in the
lessness could be a fast-evolving heterochronic con-
dition (10,82). Heterochrony, the relative change
in the rate or timing of developmental events
among species, is thought to be an important fac-
tor contributing to macroevolutionary change (83).
Yet virtually nothing is known about its genetic
and molecular mechanisms.
Diverse myological, osteological, and develop-
mental observations suggest that flightlessness
in the Galapagos cormorant is caused by the re-
tention into adulthood of juvenile characteristics
affecting pectoral and forelimb development (a
class of heterochrony known as paedomorpho-
sis) (7). Here, we propose a genetic and molecular
model that may explain this heterochronic con-
dition, where the perturbations of cilia/Ihh sig-
naling may be responsible for the reduction in
growth of both keel and wings in the Galapagos
cormorant (Fig. 5B). However, we cannot rule
out a role of Cux1 in the AER. Of special interest
is the gene Fat1,atargetofCux1 (Fig. 4D), which
contains two putative function-altering variants
(Table 1). Fat1
show selective defects in facial, pectoral, and
shoulder muscles but not in hindlimb muscles
(54). Thus, variants in Fat1 could explain the
underdeveloped pectoral muscle s of P. harrisi.
Although we have identified multiple variants
that likely contribute to the flightless phenotype
of P. harrisi,wecannotexcludethepossibility
that other genes and pathways may contribute
to the phenotype, nor the contribution of noncoding
regulatory variants (17). Further characterization
of the individual and joint contributions of the
variants found in this study will help us to recon-
struct the chain of events leading to flightless-
ness and to genetically dissect macroevolutionary
change. We hypothesize that mutations in cilia or
functionally related genes could be responsible
for limb and other skeletal heterochronic trans-
formations in birds and diverse organisms, in-
cluding humans.
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Supported by the Jane Coffin Childs Memorial Fund for Medical
Research (A.B.), the Howard Hughes Medical Institute (L.K.), the
U.S. Geological Survey through the Wildlife Program of the
Ecosystems Mission Area (A.M.R.), a Gruss-Lipper postdoctoral
fellowship from the EGL Charitable Foundation (E.B.-D.),
FONDECYT grant 11130305 (C.V.), and the National Institute of
Arthritis and Musculoskeletal and Skin Diseases (K.L.). Field
collection permits and export permits were by the Galapagos
National Park, facilitated by the Charles Darwin Foundation. Any
use of trade names is for descriptive purposes only and does not
imply endorsement by the U.S. government. We thank C. Koo and
M. Arbing at the UCLA-DOE Protein Expression Laboratory Core
Facility for protein purification; S. Feng at the Broad Stem Cell
Research Center High Throughput Sequencing Core for assistance;
C. Cicero for granting access to cormorant specimens (134079
and 151575) at the Museum of Vertebrate Zoology at Berkeley;
C. Valle (Universidad San Francisco de Quito) for advice; and
K. Garrett (Natural History Museum of Los Angeles), K. Burns
(San Diego Natural History Museum and San Diego State
University Museum of Biodiversity), and R. Duerr (International
Bird Rescue) for providing samples used in preliminary stages of
this study. Author contributions: A.B. and L.K. conceived the study;
A.B. coordinated the collection of samples, prepared libraries,
assembled and annotated genomes, and performed analyses
and experiments; E.B.-D. and A.B. performed the accelerated
evolution analysis; P.C.W., A.M.R., C.V., and P.G.P. provided DNA or
tissue samples; W.W. and A.B. carried out ATDC5 cell line
experiments supervised by K.L.; A.B. and L.K. wrote the
manuscript; and all authors discussed and agreed on the final
version of the manuscript. All sequencing data from this study is
available through the NCBI Sequence Read Archive under
Bioproject accession number PRJNA327123. Alignments used for
phylogenetic analysis and selection test are available at DRYAD
doi:10.5061/dryad.8m2t5. The authors declare no competing
financial interests.
Materials and Methods
Figs. S1 to S10
Tables S1 to S13
Movie S1
References (84109)
4 November 2016; accepted 13 April 2017
Burga et al., Science 356, eaal3345 (2017) 2 June 2017 8of8
on June 13, 2017 from
(6341), . [doi: 10.1126/science.aal3345]356Science
Parker and Leonid Kruglyak (June 1, 2017)
Andrew M. Ramey, Claudio Verdugo, Karen Lyons, Patricia G.
Alejandro Burga, Weiguang Wang, Eyal Ben-David, Paul C. Wolf,
Galapagos cormorant
A genetic signature of the evolution of loss of flight in the
Editor's Summary
, this issue p. eaal3345; see also p. 904Science
cormorant's loss of flight.
function of the genes may be responsible for skeletal changes associated with the Galapagos
involved in primary ciliogenesis. Functional analyses of these variants suggest that the impaired
flightless Galapagos cormorant (see the Perspective by Cooper). They identified variants in genes
. sequenced genomes from three cormorant species and compared them with that of theet alBurga
evolutionary history. However, the genetic changes that ground avian species are not well understood.
Although rare among existing birds, the loss of flight appears to have occurred multiple times in
Loss of flight in the Galapagos cormorant
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on June 13, 2017 from
... The flightless Galápagos cormorant (Phalacrocorax harrisi) has a short radius and ulna relative to its humerus (Bickley & Logan, 2014;Burga et al., 2017). Compared to flying cormorant species, the Galápagos cormorant has a deletion of four amino acids in the CUX1 coding sequence (Burga et al., 2017). ...
... The flightless Galápagos cormorant (Phalacrocorax harrisi) has a short radius and ulna relative to its humerus (Bickley & Logan, 2014;Burga et al., 2017). Compared to flying cormorant species, the Galápagos cormorant has a deletion of four amino acids in the CUX1 coding sequence (Burga et al., 2017). In experiments with mouse cell lines, the resultant protein was less effective in activating Ihh, a gene important for the proliferation and differentiation of cartilage cells (Burga et al., 2017;Kronenberg, 2003;Peckham et al., 2003). ...
... Compared to flying cormorant species, the Galápagos cormorant has a deletion of four amino acids in the CUX1 coding sequence (Burga et al., 2017). In experiments with mouse cell lines, the resultant protein was less effective in activating Ihh, a gene important for the proliferation and differentiation of cartilage cells (Burga et al., 2017;Kronenberg, 2003;Peckham et al., 2003). ...
Full-text available
Appendages have been reduced or lost hundreds of times during vertebrate evolution. This phenotypic convergence may be underlain by shared or different molecular mechanisms in distantly related vertebrate clades. To investigate, we reviewed the developmental and evolutionary literature of appendage reduction and loss in more than a dozen vertebrate genera from fish to mammals. We found that appendage reduction and loss was nearly always driven by modified gene expression as opposed to changes in coding sequences. Moreover, expression of the same genes was repeatedly modified across vertebrate taxa. However, the specific mechanisms by which expression was modified were rarely shared. The multiple routes to appendage reduction and loss suggest that adaptive loss of function phenotypes might arise routinely through changes in expression of key developmental genes. Appendages have been independently reduced or lost hundreds of times during vertebrate evolution. Despite that selection seems to routinely favor a reduced appendage, we found that the specific mechanisms to appendage reduction and loss are rarely shared, even when the same genes were involved.
... Furthermore, the differentially expressed genes (DEGs) in breast muscle also showed that changes of bone development related genes played a key role in the differences between the wild and domestic ducks. Many studies suggested that loss of ight in birds was always accompanied with limb modi cation and skeletal changes [18,19,32]. ...
... Morphological changes could contribute to loss of ight in birds. Compared to their ightless relatives, ight birds always have a lighter mass, higher wing area and longer bone length [18,32,36]. This change is consistent with the breeding goal of domesticated ducks as body weight is a major breeding goal in ducks. ...
... This unusual expression difference furtherly supports our hypothesis. We also checked the RNA-seq data for expression levels of the genes including DYRK1A, IFT122, CUX1 and ACOT7, which have previously been identi ed to be associated with ight in other birds [18,32,47,48]. ...
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Background Domestication alters lots of phenotypic, neurologic and physiologic traits between domestic animals and their wild ancestors. Domestic ducks were originated from mallards (Anas platyrhynchos) and some documents also showed that spot-billed ducks (Anas zonorhyncha) could also genetically contribute a small part to the domestication. Compared with the two ancestral species, domestic ducks generally present changes in body size and bone morphology, which is supposed to lead to loss of fight in domestic ducks. In the present study, we performed both genomic and transcriptomic analysis to identify candidate genes in order to elucidate the genetic mechanism underlying the phenotypic variation. Results Our results showed that genes associated with the skeleton systems were positively selected during domestication by Fst analysis between the wild and domestic ducks. We also found that many differentially expressed genes (DEGs) in the breast muscle between the wild and domestic ducks were enriched in the pathway for ossification. Among the genes, FGF14 and EIF2AK3 were also under strong selection by the genomic data, and they were both reported to be associated with limb morphology, bone development and flightlessness in some bird species. Conclusions Our study showed that the skeleton related genes were positively selected in the process of domestication, which could also cause the loss of flight in domestic ducks.
... Several recent studies have also employed whole-genome sequencing to understand the genomic basis of morphological changes associated with the evolution of flightlessness. By comparing whole genomes of the flightless Galapagos cormorant (Phalacrocorax harrisi) to three flighted cormorant species (Phalacrocorax auratus, Phalacrocorax brasilianus, Phalacrocorax pelagicus)-although examining only the coding portions of those genomes- Burga et al. (2017) discovered variants exclusive to Galapagos cormorants and enriched in genes that are also known to cause human skeletal disorders. Leveraging the repeated evolution of flightlessness in ratites and steamer ducks (Tachyeres spp.), Sackton et al. (2019) and Campagna et al. (2019), respectively, analyzed whole genomes to investigate the genomic basis and degree of genomic convergence underlying traits related to flightlessness. ...
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The increased capacity of DNA sequencing has significantly advanced our understanding of the phylogeny of birds and the proximate and ultimate mechanisms molding their genomic diversity. In less than a decade, the number of available avian reference genomes has increased to over 500—approximately 5% of bird diversity—placing birds in a privileged position to advance the fields of phylogenomics and comparative, functional, and population genomics. Whole-genome sequence data, as well as indels and rare genomic changes, are further resolving the avian tree of life. The accumulation of bird genomes, increasingly with long-read sequence data, greatly improves the resolution of genomic features such as germline-restricted chromosomes and the W chromosome, and is facilitating the comparative integration of genotypes and phenotypes. Community-based initiatives such as the Bird 10,000 Genomes Project and Vertebrate Genome Project are playing a fundamental role in amplifying and coalescing a vibrant international program in avian comparative genomics. Expected final online publication date for the Annual Review of Ecology, Evolution, and Systematics, Volume 52 is November 2021. Please see for revised estimates.
... This functional change in bone development may have led to the loss of flight in domestic ducks. Compared to their wild relatives, wild birds have a lighter mass, larger wing area, and greater bone length [41][42][43]. These differences are consistent with the major breeding goals. ...
Full-text available
Background Domestication alters several phenotypic, neurological, and physiological traits in domestic animals compared to those in their wild ancestors. Domestic ducks originated from mallards, and some studies have shown that spot-billed ducks may have also made minor genetic contributions to domestication. Compared with the two ancestral species, domestic ducks generally differ in body size and bone morphology. In this study, we performed both genomic and transcriptomic analyses to identify candidate genes for elucidating the genetic mechanisms underlying phenotypic variation. Methods In this study, the duck genome data from eight domestic breeds and two wild species were collected to study the genetic changes during domestication. And the transcriptome data of different tissues from wild ducks and seven domestic ducks were used to reveal the expression difference between wild and domestic ducks. Results Using fixation index (Fst) algorithm and transcriptome data, we found that the genes related to skeletal development had high Fst values in wild and domestic breeds, and the differentially expressed genes were mainly enriched in the ossification pathway. Our data strongly suggest that the skeletal systems of domestic ducks were changed to adapt to artificial selection for larger sizes. In addition, by combining the genome and transcriptome data, we found that some Fst candidate genes exhibited different expression patterns, and these genes were found to be involved in digestive, immune, and metabolic functions. Conclusions A wide range of phenotypic differences exists between domestic and wild ducks. Through both genome and transcriptome analyses, we found that genes related to the skeletal system in domestic ducks were strongly selected. Our findings provide new insight into duck domestication and selection effects during the domestication.
... Altered Hh signaling was suggested to play a role in the skeletal diversification of several species, including canids 73 , cichlids 74 and cormorants 75 . Hh signaling may represent a recurrent target of selection because its dosage-dependent effects allow fine-tuning of morphology. ...
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Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human–chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human–chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.
... Furthermore, enhancer evolution leading to decreased expression of the Hedgehog receptor Ptch1 may underly the evolution of bovine limbs (39). Alteration in genes encoding components of primary cilia, involved in transducing Hedgehog signals, may affect wing shape in flightless Galapagos cormorants (40). Our results indicate that another member of the Hedgehog pathway, Gli2, is regulated by a critical, evolved enhancer ( Figure 7). ...
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Human accelerated regions (HARs) are sequences that have evolved at an accelerated rate in the human lineage. Some HARs are developmental enhancers. We used a massively parallel reporter assay (MPRA) to identify HARs with enhancer activity in a mammalian testis cell line. A subset of HARs exhibited differential activity between the human and chimpanzee orthologs, representing candidates for underlying unique human male reproductive biology. We further characterized one of these candidate testis enhancers, 2xHAR.238. CRISPR/Cas9-mediated deletion in a testis cell line and mice revealed that 2xHAR.238 enhances expression of Gli2, encoding a Hedgehog pathway effector, in testis Leydig cells. 4C-seq revealed that 2xHAR.238 contacts the Gli2 promoter, consistent with enhancer function. In adult male mice, deletion of 2xHAR.238 disrupted mouse male-typical behavior and male interest in female odor. Combined, our work identifies a HAR that promotes the expression of Gli2 in Leydig cells and may have contributed to the evolution of human male reproductive biology.
1. A total of 772, 420-day-old Xingguo gray geese (XGG) were sequenced using a low-depth (~1×) whole-genome resequencing strategy to reveal the genetic mechanism of wing length-related traits by genome-wide association analysis (GWAS).2. The results showed that 119 SNPs had genome-wide significance for wing length in five regions of chromosome 4, of which the most significant locus (P=7.95E-11) was located upstream of RBM47 and explained 7.3% of phenotypic variation.3. A total of 219 SNPs located on chromosome 4 that were associated with 2-joint-wing length, of which four SNPs reached the genome-wide significant level. However, for the length of 1-joint-wing and primary feather, we did not detect any associated locus.4. Six promising candidate genes, RBM47, SLAIN2, GRXCR1, SLC10A4, APBB2 and NSUN7 on chromosome 4, may play an important role in the growth and development of feathers, muscles and bones.
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The understanding of how genetic information may be inherited through generations was established by Gregor Mendel in the 1860s when he developed the fundamental principles of inheritance. The science of genetics, however, began to flourish only during the mid-1940s when DNA was identified as the carrier of genetic information. The world has since then witnessed rapid development of genetic technologies, with the latest being genome-editing tools, which have revolutionized fields from medicine to agriculture. This review walks through the historical timeline of genetics research and deliberates how this discipline might furnish a sustainable future for humanity.
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Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical.
Studying the genetic signatures of evolutionary diversification in young lineages is among the most promising approaches for unveiling the processes behind speciation. Here, we focus on Oreotrochilus chimborazo, a high Andean species of hummingbird that might have experienced rapid diversification in the recent past. To understand the evolution of this species, we generated a dataset of ten microsatellite markers and complementary data on morphometrics, plumage variation and ecological niches. We applied a series of population and coalescent-based analyses to understand the population structure and differentiation within the species, in addition to the signatures of current and historical gene flow, the location of potential contact zones and the relationships among lineages. We found that O. chimborazo comprises three genetic groups: one corresponding to subspecies O. c. chimborazo, from Chimborazo volcano and surroundings, and two corresponding to the northern and southern ranges of subspecies O. c. jamesonii, found from the extreme south of Colombia to southern Ecuador. We inferred modest levels of both contemporary and historical gene flow and proposed the location of a contact zone between lineages. Also, our coalescent-based analyses supported a rapid split among these three lineages during the mid-to-late Holocene. We discuss our results in the light of past and present potential distributions of the species, in addition to evolutionary trends seen in other Andean hummingbirds.
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Birds are prolific colonists of islands, where they readily evolve distinct forms. Identifying predictable, directional patterns of evolutionary change in island birds, however, has proved challenging. The "island rule" predicts that island species evolve toward intermediate sizes, but its general applicability to birds is questionable. However, convergent evolution has clearly occurred in the island bird lineages that have undergone transitions to secondary flightlessness, a process involving drastic reduction of the flight muscles and enlargement of the hindlimbs. Here, we investigated whether volant island bird populations tend to change shape in a way that converges subtly on the flightless form. We found that island bird species have evolved smaller flight muscles than their continental relatives. Furthermore, in 366 populations of Caribbean and Pacific birds, smaller flight muscles and longer legs evolved in response to increasing insularity and, strikingly, the scarcity of avian and mammalian predators. On smaller islands with fewer predators, birds exhibited shifts in investment from forelimbs to hindlimbs that were qualitatively similar to anatomical rearrangements observed in flightless birds. These findings suggest that island bird populations tend to evolve on a trajectory toward flightlessness, even if most remain volant. This pattern was consistent across nine families and four orders that vary in lifestyle, foraging behavior, flight style, and body size. These predictable shifts in avian morphology may reduce the physical capacity for escape via flight and diminish the potential for small-island taxa to diversify via dispersal.
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Noncoding polymorphisms in the fat mass and obesity-associated (FTO) gene represent common alleles that are strongly associated with effects on food intake and adiposity in humans. Previous studies have suggested that the obesity-risk allele rs8050136 in the first intron of FTO alters a regulatory element recognized by the transcription factor CUX1, thereby leading to decreased expression of FTO and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Here, we evaluated the effects of rs8050136 and another potential CUX1 element in rs1421085 on expression of nearby genes in human induced pluripotent stem cell-derived (iPSC-derived) neurons. There were allele-dosage effects on FTO, RPGRIP1L, and AKT-interacting protein (AKTIP) expression, but expression of other vicinal genes, including IRX3, IRX5, and RBL2, which have been implicated in mediating functional effects, was not altered. In vivo manipulation of CUX1, Fto, and/or Rpgrip1l expression in mice affected adiposity in a manner that was consistent with CUX1 influence on adiposity via remote effects on Fto and Rpgrip1l expression. In support of a mechanism, mice hypomorphic for Rpgrip1l exhibited hyperphagic obesity, as the result of diminished leptin sensitivity in Leprb-expressing neurons. Together, the results of this study indicate that the effects of FTO-associated SNPs on energy homeostasis are due in part to the effects of these genetic variations on hypothalamic FTO, RPGRIP1L, and possibly other genes.
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Author The limb is a classic example of vertebrate homology and is represented by a large range of morphological structures such as fins, legs and wings. The evolution of these structures could be driven by alterations in gene regulatory elements that have critical roles during development. To identify elements that may contribute to bat wing development, we characterized sequences that are conserved between vertebrates, but changed significantly in the bat lineage. We then overlapped these sequences with predicted developing limb enhancers as determined by ChIP-seq, finding 166 bat accelerated sequences (BARs). Five BARs that were tested for enhancer activity in mice all drove expression in the limb. Testing the mouse orthologous sequence showed that three had differences in their limb enhancer activity as compared to the bat sequence. Of these, BAR116 was of particular interest as it is located near the HoxD locus, an essential gene complex required for proper spatiotemporal patterning of the developing limb. The bat BAR116 sequence drove robust forelimb expression but the mouse BAR116 sequence did not show enhancer activity. These experiments correspond to analyses of HoxD gene expressions in developing bat limbs, which had strong forelimb versus weak hindlimb expression for Hoxd10-11. Combined, our studies highlight specific genomic regions that could be important in shaping the morphological differences that led to the development of the bat wing.
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Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII's natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.
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The paleogeography of the Galápagos Archipelago is examined in light of a modern understanding of regional geology, eustatic sea level changes, and volcano subsidence. Newly determined ages and bathymetric data are used to assess the present biogeography and the Neogene paleogeography of the archipelago. The ages of emergence of the Galápagos Islands are consistent with the Nazca Plate moving 59 km/My to the east. Island age is found to be an important correlate of the number of native and unique endemic species on the islands, explaining additional variation beyond island size, which is the most important control. The age effect is most important shortly after emergence of an island. The islands' elevations are proportional to the square root of their age, leading them to sink below sea level. Holocene sea-level rise of 125 m over the past 20,000 years has led to the drowning of many islets and several major islands, and the fragmentation of larger islands into the currently isolated smaller islands. Thus, the geographic template for studies of organismal colonization and dispersal within the archipelago has changed drastically over recent geologic time. The integrated area of the Galápagos Islands was much greater during the Pleistocene than today, and although many more islets existed, some of the major islands were connected. Island fragmentation has led to diversification by vicariance as well as dispersal. Over the past five million years, at least seven major islands have existed within the archipelago, and the cumulative area of the islands has been at least 50% of that today.
The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses — the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here, we report that direct injection of in vitro-assembled Cas9/crRNA-tracrRNA ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-bases homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method of Arribere et al., 2014, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins. Copyright © 2015, The Genetics Society of America.