Robert H. S. Kraus - Avian Genomics in Ecology and Evolution - From The Lab Into The Wild-Springer International Publishing (2019)

Robert
H. S. Kraus Editor
Avian
Genomics in
Ecology and
Evolution
From the Lab into the Wild
Avian Genomics in Ecology and Evolution
Robert H. S. Kraus
Editor
Avian Genomics in
Ecology and Evolution
From the Lab into the Wild
Editor
Robert H. S. Kraus
Department of Migration
and Immuno-Ecology
Max Planck Institute for Ornithology
Radolfzell, Germany
Department of Biology
University of Konstanz
Konstanz, Germany
ISBN 978-3-030-16476-8 ISBN 978-3-030-16477-5 (eBook)

https://doi.org/10.1007/978-3-030-16477-5
# Springer Nature Switzerland AG 2019

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Contents
An Introduction to “Avian Genomics in Ecology and Evolution: From

the Lab into the Wild” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Robert H. S. Kraus
A Historical Perspective of Avian Genomics . . . . . . . . . . . . . . . . . . . . . . 7
Michael Wink
Avian Genomics in Animal Breeding and the End of the Model
Organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Alain Vignal and Lel Eory
Avian Chromosomal Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Joana Damas, Rebecca E. O’Connor, Darren K. Griffin,
and Denis M. Larkin
Repetitive DNA: The Dark Matter of Avian Genomics . . . . . . . . . . . . . . 93
Matthias H. Weissensteiner and Alexander Suh
Resolving the Avian Tree of Life from Top to Bottom: The Promise
and Potential Boundaries of the Phylogenomic Era . . . . . . . . . . . . . . . . 151
Edward L. Braun, Joel Cracraft, and Peter Houde
Avian Species Concepts in the Light of Genomics . . . . . . . . . . . . . . . . . . 211
Jente Ottenburghs
Population Genomics and Phylogeography . . . . . . . . . . . . . . . . . . . . . . . 237
Jente Ottenburghs, Philip Lavretsky, Jeffrey L. Peters, Takeshi Kawakami,
and Robert H. S. Kraus
Avian Population Studies in the Genomic Era . . . . . . . . . . . . . . . . . . . . 267
Arild Husby, S. Eryn McFarlane, and Anna Qvarnström
The Contribution of Genomics to Bird Conservation . . . . . . . . . . . . . . . 295
Loren Cassin-Sackett, Andreanna J. Welch, Madhvi X. Venkatraman,
Taylor E. Callicrate, and Robert C. Fleischer
Jurassic Park: What Did the Genomes of Dinosaurs Look Like? . . . . . . 331
Darren K. Griffin, Denis M. Larkin, and Rebecca E. O’Connor
v
An Introduction to “Avian Genomics
in Ecology and Evolution: From the Lab into
the Wild”
Robert H. S. Kraus
Abstract
Recently, the use of next-generation DNA sequencing developed from a technol-
ogy that only industry could use into a common tool in nonmodel biology. Not
long ago, ecologists and evolutionary biologists would rather shy away from
taking on a project in which whole-genome sequencing would be the major tool.
However, much sooner than anticipated, a new generation of students had been
trained in the use of bioinformatics tools and concepts and they would leverage
genomic technologies to their full potential—not only in medical sciences but
also in ecology and evolution. Tumbling prices on the sequencing
market allowed the large ornithological community to adopte this technology
early. Birds can be seen as a model group among many other organismal groups.
Substantial efforts of the community have built a foundation of avian genomics in
ecology and evolution. In this book, we summarize the major advances that now
constitute the first steps taken into making whole-genome analyses common-
place. In ten peer-reviewed chapters (in addition to this introduction) we provide
an overview of the use of genome technologies in avian biology research espe-
cially for an audience that might not currently be part of the “genomics revolu-
tion.” We thereby aim to mediate between early adopters of avian genomics and
interested professionals.
Keywords
Genome · Birds · Environment · Organismal biology · Non-model species ·
Sequencing · Phylogeny · Speciation · Conservation · Dinosaurs
R. H. S. Kraus (*)
Department of Migration and Immuno-Ecology, Max Planck Institute for Ornithology, Radolfzell,
Germany
Department of Biology, University of Konstanz, Konstanz, Germany
e-mail: rkraus@orn.mpg.de
# Springer Nature Switzerland AG 2019 1

R. H. S. Kraus (ed.), Avian Genomics in Ecology and Evolution,
https://doi.org/10.1007/978-3-030-16477-5_1
2 R. H. S. Kraus
1 Introduction
The recent rise of next-generation DNA sequencing technologies has transformed

biological and medical research (Shendure et al. 2017). In a single decade, not only
so-called second-generation sequencing technologies have become established but
also third-generation sequencers are now routinely used. While the former offered
orders of magnitude of increased throughput at the cost of accuracy and length of the
output DNA sequences, the latter revolutionized the field, resulting in very long
DNA sequences. Third-generation sequencing was advocated as the solution to the
“short read era” of second-generation sequencing (Munroe and Harris 2010), but it
took many more years to become stable, sufficiently cheap (it is still more expensive
than second-generation sequencing), and established (Lee et al. 2016). Most
recently, a relatively old idea of pulling DNA strands through genetically engineered
nanopores while measuring changes in electric potential (Ashkenasy et al. 2005;
Deamer and Akeson 2000) hit the market and now pushes the boundaries of what is
possible in terms of the lengths of a DNA strand that can be sequenced in one piece
(Ashton et al. 2015; Goodwin et al. 2015; Madoui et al. 2015). A profound impact on
the molecular sciences is expected, in particular for medical applications—from
cancer research to genetic disease, from personalized medicine to genome-wide
association studies via population re-sequencing. But what does all of this have to
do with birds?
Birds have been important for biological research disciplines throughout the
history of biology. They attract members of the general public like no other group
of animals; and birders—people with a personal interest in watching and enjoying
the life of birds—are perhaps the largest nonprofessional naturalist community. This
may well be due to an omnipresence of birds across biomes and habitats, and the ease
with which they can be observed everywhere: in cities, in the countryside, in forests,
in high mountain ranges, or on the coast. They are also the most speciose class
among terrestrial vertebrates, with around 10,500 extant species (varying depending
on the checklist of choice). The richness of forms triggered the curiosity of many
early naturalists who later emerged as leading personalities for biology as a whole.
For instance, the theory of evolution proposed by Darwin (1859) was greatly
inspired by his studies on the intriguing diversity of ground finches, later called
the “Darwin Finches,” on his 1826 travels to the Galápagos Islands (Lack 1947).
In addition, birds play an important role in medical and pharmaceutical research
(e.g., eggs used as bioreactors), and they are an important source of human food, i.e.,
meat and eggs (Petitte and Mozdziak 2007). Chickens, for example, are bred
industrially, with a global population exceeding that of humans by more than
two-fold. Therefore, among the first genomes of vertebrates to be fully sequenced
after the Human Genome project (International Human Genome Sequencing Con-
sortium 2001) was indeed the chicken (Hillier et al. 2004). Genomics in birds is a
major contributor to the general knowledge base of all biological fields (reviewed in
Jax et al. 2018; Kraus and Wink 2015).
Many avian studies adopted molecular methods very early on, even before the
onset of the first generation of DNA sequencing (Sanger 1981; Sanger et al. 1977)
An Introduction to “Avian Genomics in Ecology and Evolution: From the. . . 3
and polymerase chain reaction (PCR; Saiki et al. 1988, 1985). Michael Wink
reviews the early adoption of molecular methods, particularly in avian systematics,
from a historical perspective and looks into the origins of genomics in avian research
and where it might go (Wink 2019). Next-generation DNA sequencing has
proliferated in applications in ecology and evolution studies too, beyond the core
technological and medical applications, and several biological disciplines have been
transformed into big data sciences, with all the accompanying benefits but also costs
(Ekblom and Wolf 2014). Although the above-mentioned early avian model species
such as chicken or zebra finch have been studied intensively, many other species
have become models themselves. When the zebra finch became genome-sequenced
(Warren et al. 2010), it did not take long to announce that a new era of nonmodel
avian genomics had started (Balakrishnan et al. 2010). Obviously, this was not that
straightforward because not every type of knowledge can be transferred that easily
between species. Several large-scale bird sequencing projects were carried out over
the following years to provide reference genome assemblies for a broad taxonomic
range of birds (Zhang et al. 2014a). However, when comparing research groups with
a tradition in genomics with those groups emerging from the sequencing revolution,
the great value that established model species have when embarking on deciphering
genomes of nonmodel species can be observed. Without a detailed understanding of
the chicken genome and a comprehensive suite of wet and dry laboratory
technologies pioneered by the chicken genetics and genomics community, most of
today’s nonmodel genomics would not be possible. In this book, Vignal and Eöry
(2019) look into the question of whether or not advances in sequencing technologies
today indicate the advancing obsolescence of the classical bird model.
One of the earliest fields of genome-scale studies was to microscopically examine
chromosomes. Combined with genetics, this discipline is called cytogenetics and has
contributed greatly to our knowledge of genome organization across the avian tree of
life. Damas et al. (2019) explain the contributions of early and modern cytogenetic
research that today go hand in hand with DNA sequencing technologies. Closely
related to the large-scale organization of genomes at the chromosomal and the
sub-chromosomal levels, and highly relevant for questions regarding genome assem-
bly, are repetitive regions. In early work in the human genome consortia, one of the
major findings was that large portions of the vertebrate genome are not only
noncoding but also highly repetitive (International Human Genome Sequencing
Consortium 2001). Genome scientists have called these regions “genomic dark
matter” and Weissensteiner and Suh (2019) light the way for us with an extensive
overview of the known repeat structures in the genomes of birds and what their roles
might potentially be.
The outcome of evolution is reflected in the tree of life. Forms arise, change, and
vanish. The paleontological record clearly shows this diversity of forms across the
eons. Biologists have been fascinated for decades and centuries with deciphering
phylogenetic relationships and building trees of speciation histories. The first group
of animals to be sequenced for their whole genome and in a phylogenomic context
was indeed birds (Jarvis et al. 2014). One representative of every order of birds
underwent genome sequencing and was comparatively analyzed (Zhang et al.
4 R. H. S. Kraus
2014b, c). However, this was just the start of a new era of phylogenomics and
comparative genomics across all the bird species in the world (Zhang 2015); an
avian-specific project that serves as a blueprint for future studies in other groups of
organisms (Jarvis 2016). With genome-scale data sets, the field of phylogeny has
encountered significant challenges that need to be well balanced against the wealth
of information that accompanies whole-genome data (Delsuc et al. 2005). There are
not only technical hurdles such as computational complexities of big data but also
difficult choices such as which parts of the genome should be used for inference, and
in what way can the core questions be addressed using the large amount of avian
genome data that are available. In this book, Braun et al. (2019) cover many aspects
of why phylogenomics is difficult, and how problems can be overcome. Looking at
the branches of a phylogenetic tree reveals a problem of its own: what is a species?
Generations of biologists have had long-standing arguments on this question and the
latest genomic techniques have not solved the puzzle. In this book, we deal with the
issue in detail in a chapter on species concepts, with specific reference to avian
species (Ottenburghs 2019).
Processes at the level of the population have been routinely studied in ecology
and evolution for decades. An extensive body of literature has accumulated on how
geography and climates of the past have shaped the population structure of today.
These phylogeographic studies make use of population genetic theory and with the
advent of genome-scale data they deliver ever finer and more complex models and
predictions (Ottenburghs et al. 2019). Population studies on recent time scales, in
particular incorporating detailed knowledge of pedigree and other related informa-
tion within populations, also make quantitative predictions. Husby et al. (2019)
show how combined knowledge and technology of classical genetics and genomics
from animal breeding model species (e.g., the chicken) and genomic studies of wild
populations advance our understanding of trait evolution on short time scales (micro-
evolution). Understanding the background of avian phenotypes and the heredity of
traits enriches the tool box of conservation managers. In addition, there is an obvious
and omnipresent discussion on the role of genetic and genomic diversity in species’
abilities to cope with environmental change. Cassin-Sackett et al. (2019) look at this
and at the role that genomics can play versus more traditional genetic technology.
Our book closes with a special notion uniting the historical and paleontological
aspects of avian research with the modern approaches of cytogenetics and compara-
tive genomics. Both morphological and genomic evidence have clearly shown that
birds are in fact dinosaurs, and Griffin et al. (2019) take you on a journey through
Jurassic Park to appreciate this curious thought to its fullest potential.
Our team of authors thanks you, the reader, for taking the time to delve into the
breadth and depth of avian genomics. We appreciate that some parts may be too
detailed for a certain audience and others too vague. We aimed to strike a balance by
addressing a nongenomics audience but are aware that this can never work perfectly.
Our aim was to get as close to this idealistic and challenging goal as we possibly
could. The chapters of this book were peer reviewed by blinded independent experts
in their fields to make sure that no important details are missing and that no chapter
becomes too obfuscated with specialist terminology. The various aspects of
An Introduction to “Avian Genomics in Ecology and Evolution: From the. . . 5
genomics, whether technical or conceptual, require some time for familiarization.

We hope that our texts contribute to this process and provide starting points for
digging deeper where necessary and/or desired.
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A Historical Perspective of Avian Genomics
Michael Wink
Abstract
A traditional aim of avian taxonomists and systematists was to establish a reliable
phylogenetic framework, the avian tree of life. Until 50 years ago, the only way to
establish systematic relationships relied on the comparison of morphological
characters, which could be misleading because of convergent character evolution.
The first molecular approach used the electrophoretic separation of proteins (from
eggs). This was followed in the 1980s by DNA-DNA hybridisation. Both
methods provided some insight but did not show sufficient resolution. Better
results were obtained from nucleotide sequencing of marker genes, which started
in the 1990s. A real breakthrough came with next-generation sequencing (NGS),
which allowed sequencing a large portion of the avian genome. The review
illustrates and briefly discusses the achievements in the past and limitations of
the different methodological approaches.
Keywords
Systematics · Taxonomy · Classification · Morphology · Phylogenetics · Genome ·
DNA-DNA hybridisation · Sequencing · Next-generation sequencing
1 Ornithology: From Aristoteles to Linné
Today’s sciences are shaped mainly by Greek/Latin history that has strongly
influenced Western European scholars. I am aware that naturalists around the
world have studied birds and quite scientifically so. But, in this overview, I have
focussed mainly on Western science.
M. Wink (*)
Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Heidelberg, Germany
e-mail: wink@uni-heidelberg.de

https://doi.org/10.1007/978-3-030-16477-5_2
8 M. Wink
The Greek philosopher and naturalist Aristotle (384–322 BC) already recognised
140 bird species and described their morphology, anatomy, behaviour, distribution
and biology. The Roman naturalist Plinius (23–79 AC) produced a Historia
naturalis with 37 volumes and ordered birds according to their anatomy of legs
and feet. Aristotle and Plinius remained the authorities for more than 1000 years, and
most later, bird descriptions [e.g. from Albertus Magnus (1193–1280)] were copied
from their books. The Holy Roman Emperor Frederick II (1194–1250) was an
exception in so far, as he used his own observation for his book on falconry De
arte venandi cum avibus (Stresemann 1951).
The Renaissance (from the fifteenth century onwards) was important for the
development of natural sciences; it also brought progress for ornithology. The
invention of book printing by Johannes Gutenberg around 1450 facilitated the
rapid dissemination of novel findings and the publication of illustrated animal and
plant books. The following three authors were especially important for the progress
in ornithology: William Turner (1500–1568), Pierre Belon (1517–1564) and Conrad
Gessner (1516–1565). In the illustrated Historia animalium, Gessner described
180 bird species. His knowledge was derived from his own observations and of
those of other experts. After several reprints of Gessner’s popular work, John
Jonston (1603–1675) produced the Historiae naturalis de avibus libri VII in 1650
with excellent illustrations from Matthaeus Merian (Fig. 1).
Fig. 1 Illustrations from Historiae naturalis de avibus libri VII, in which species were ordered
according to similarity. As can be seen, shrikes and cuckoo were grouped with raptors because of their
bill morphology (a) and even bats were classified as birds because of their wings (b) (Photo M. Wink)
A Historical Perspective of Avian Genomics 9
Progress of ornithology also profited from the expeditions into foreign countries
and continents, which started around 1500. Explorers brought back live birds, which
were kept in aviaries (which already existed also many hundred years earlier), from
which predecessors of zoos were later derived.
Based on the early collections, naturalists started to elaborate on a taxonomy of
birds after 1600, which largely replaced the concepts of Aristotle and Plinius.
Influential bird specialists include Walter Charleton (1619–1707), Francis
Willughby (1635–1672) and especially John Ray (1628–1704). The Ornithologiae
libri tres of Ray can be regarded as the first modern ornithology handbook. Ray and
other naturalists based it on systematic observations (Stresemann 1951).
The “An historical review of bird taxidermy in Britain” cites René Antoine
Ferchault de Réaumur (1748) saying—the whole progress of ornithology was
being retarded because of preservation problems. The more detailed studies on
preservation came from the early 1770s (at least in Britain). With the development
of bird taxidermy (Schulze-Hagen et al. 2003), exotic bird skins were bought by
private collectors, which were later sold or donated to the public. The need to make
these collections widely available led to the opening of natural history museums,
which we have today.
The Swedish naturalist and medical doctor Carl Linnaeus (1707–1778)
introduced a binary nomenclature, which allowed the unequivocal description of
animal and plant species. For example, the European blackbird was named Turdus
merula; the first name corresponds to the genus and the second to the species. No
other animal will carry this name. Linné tried to introduce systematics in order to
arrange similar species into genera, orders and classes. Linné distinguished 6 bird
orders with 85 genera, which were defined by the morphology of beaks and feet. A
common phenomenon can be seen in unrelated organisms living under similar
constraints in that they evolve similar adaptations; they are called convergent traits.
As there are many convergent developments in these characters, some of the
systematic affiliations were wrong. The following six orders were recognised in
the tenth edition of Systema Naturae (1758):
1. Accipitres: raptors, owls, parrots, shrikes and waxwings

2. Picae: woodpeckers, hornbills, cuckoos, crows, hoopoes, birds of paradise and
creepers
3. Anseres: all water birds, pelicans, loons, grebes, gulls, terns and cormorants
4. Grallae: waders, flamingos, storks, herons, cranes, coots, bustards and ratites
5. Gallinae: wild fowl, guans, grouse and quails
6. Passeres: pigeons, thrushes, larks, humming birds, crossbills, wagtails, tits, swifts
and nightjars
The binary concept of Linné was promoted in Germany and Europe during the
eighteenth century by E. Pontoppidan, M. T. Brünnich, G. A. Scopoli, T. Pennant,
M. Tunstall, Statius Müller, P. S. Pallas, M. Brisson and J. F. Gmelin. In England the
binary system of Linné was accepted end of the eighteenth century, whereas in
France the zoologist G.-L. Buffon (1707–1788) developed his own system in his
Histoire Naturelle des Oiseaux (1770) (Stresemann 1951).
10 M. Wink
Avian taxonomy and systematics rapidly progressed during the eighteenth and
nineteenth century. As many explorers set out to travel and to explore Asia, Africa,
Australia and the Americas, many new bird species were found and described.
Scientifically curated bird collections were established in this time in Paris (1793),
London (1881), Frankfurt, Munich, Dresden and Halle, which allowed a better
understanding of systematics. This was a period when questions concerning the
status of species and subspecies were extensively discussed. Important ornithologists
of this period were P.S. Pallas (1741–1811), J.R. Forster (1728–1798), G. Forster
(1754–1794), F. Levaillant (1753–1824), C. Illiger (1775–1813), J. Kaup
(1803–1873), J.F. Naumann (1780–1857), C.L. Brehm (1787–1864), J.H. Blasius
(1809–1870), G. Hartlaub (1814–1900), J. Cabanis (1816–1906), A. Reichenow
(1847–1914), M. Fürbringer (1846–1920), H. Gadow (1855–1928) and
O. Kleinschmidt (1870–1954) in Germany; J. Latham (1740–1837), N. Vigors
(1785–1836) (knew already 3125 bird species), P.L. Sclater (1829–1913),
T.H. Huxley (1825–1895), R. Swinhoe (1836–1877), G.R. Gray (1808–1872),
E. Hartert (1859–1933), R.B. Sharpe (1847–1909) and W. von Rothschild
(1868–1937) in Great Britain; C.J. Temminck (1778–1858), H. Schlegel
(1804–1884), O. Finsch (1838–1917) and H. Boie (1784–1827) in the Netherlands;
J.B. de Lamarck (1744–1829) and G. Cuvier (1769–1832) in France; and J. Bartram
(1699–1777), C.W. Peale (1741–1827), A. Wilson (1766–1813), J.J. Audubon
(1785–1851), C.L. Bonaparte (1803–1857), J. Cassin (1813–1869), W. Swainson
(1789–1855), R. Ridgway (1850–1929) and J.A. Allen (1838–1921) in North
America (Stresemann 1951; Walters 2003).
Avian systematics saw many changes in the eighteenth and nineteenth century.
The discussion on the taxonomy of birds changed when Charles Darwin
(1809–1882) and A.R. Wallace (1823–1913) introduced the theory of evolution
through natural selection and the concept of phylogeny. This theory allowed an
understanding how adaptations in morphology, behaviour and other traits could
evolve via natural selection. In addition, the idea of phylogenetic trees and the
descent from commons ancestors was elaborated by C. Darwin. These were revolu-
tionary new concepts, since many people believed at that time that God had created
each species separately and that species do not change.
2 Towards a Natural System of Birds in the Twentieth

Century
Ornithologists after Darwin tried to establish a “natural system” of birds, which

would reflect a common phylogeny. More than 40 different systems have been
proposed during the last 200 years (Walters 2003). In the twentieth century, several
systems were introduced, which were used to arrange the order of bird families in
handbooks and field guides (e.g. Gadow 1892; Wetmore 1930–1960; Peters 1931–
1986; Stresemann 1927–1934; Mayr and Amadon 1951; Wolters 1982; Sibley and
Monroe 1990; Dickinson 2003; Clements 2007). A summary of some of these
classification systems can be found in Sibley and Ahlquist (1990).
Taxonomy and systematics were mainly based on morphological characters, such

as plumage colouration and shapes of beak, head and feet but also anatomical details.
In the twentieth century, other biological, biogeographical, ecological and biochem-
ical characters were additionally studied. The main concept of classifying species in
all these systems was overall similarity of characters. The more similar, the more
closely related two taxa were assumed to be. In most instances, avian taxonomists
usually agreed on the inclusion of a group of similar taxa into a common genus.
More difficult was sometimes the attribution to families and orders. Difficulties
could arise, because in many birds, plumage can change according to sex, age and
season. Only, when large collections became available, it was possible to place all
the different plumage forms into a single taxon. Furthermore, a number of bird
species can hybridise; thus hybrids might be wrongly classified in bird collections.
As mentioned before, many characters are adaptive which can undergo convergent
evolution. Thus, similar characters could evolve in unrelated groups. Placing them
together in a common assemblage would create artificial and polyphyletic groups
(groups, which contain members from other lineages). Taxonomy and classification
also depend on species concepts, which also changed over the last 200 years. Avian
species concepts and speciation processes will be discussed in detail in Ottenburghs
(2019) and Cassin-Sackett et al. (2019).
An important school of taxonomy, cladistics, was founded by Willi Hennig
(1913–1976) in the 1950s. He introduced plesiomorphic, apomorphic and
synapomorphic traits to define groups of organisms of common ancestry. Groups,
which included all descendants from a common ancestor, were called “monophy-
letic”. If taxonomists follow the rules of cladistics, they can distinguish between
monophyletic, paraphyletic and polyphyletic groups. Only monophyletic groups
should be the base of a natural system of classification. Whenever para- and
polyphyletic groupings are discovered, a taxonomic revision becomes necessary.
Especially, since the introduction of DNA analyses (which allowed the detection of
non-monophyletic assemblages), many revisions at the genus and family level had
become necessary (Wink 2011, 2014) (see Ottenburghs 2019; Cassin-Sackett et al.
2019).
3 Systematics and Phylogeny in the Age of DNA Analysis
In all areas of biology, including ornithology, a new era came with the study of DNA
and genetics. The structure DNA was elucidated in 1953 by James Watson and
Francis Crick. Progress in the field of genetics depended on new technologies: In
1978, DNA sequencing was established, 1985 polymerase chain reaction (PCR) and
after 2000 next-generation sequencing (NGS), which allows the parallel sequencing
of thousands of DNA sequences (see Weissensteiner and Suh 2019; Braun et al.
2019; Ottenburghs et al. 2019; Husby et al. 2019; Griffin et al. 2019).
DNA is present in the cells of all living organisms, from simple bacteria to Homo
sapiens. Before any cell division, DNA is copied, so that a daughter cell obtains an
almost identical copy of the mother cell. Since all cells originate from a mother cell
12 M. Wink
through cell division, all cells, which exist in all individuals on this planet today,
must have been derived from common ancestral cells. Thus, we have a continuous
lineage of cells and DNA back to the origin of life. DNA undergoes a few mutations
during the replication process or through mutagenic agents (chemicals, radiation)
and spontaneous chemical hydrolysis of nucleotide bases. These mutations are
preserved in specific positions in the genomes, if they are not corrected by repair
enzymes or not lethal for the individual. If these mutations occur in the germline and
are transferred to the next generation, they will (in general) continue further on in
subsequent generations and over time can become fixed in the population. As a
consequence, all individuals, which live today, carry a DNA with millions of
changes in their genomes which distinguish them from their ancestors and other
species. Consequently, millions of single nucleotide polymorphisms (SNPs),
insertions and deletions, as well as other larger-scale mutations, exist. The analyses
of these mutations enable phylogeneticists to reconstruct a common descent of all
organisms and to infer a reliable “tree of life”. Mutations usually occur at random
and at almost similar frequency and speed. Therefore, the concept of a molecular
clock can be used to date ancient divergence events (Thorpe 1982) (see Braun et al.
2019; Weissensteiner and Suh 2019; Griffin et al. 2019).
As in other animals, avian DNA is present in the nucleus of a cell arranged in
chromosomes. Most birds have more chromosomes than most mammals: For exam-
ple, the diploid genome of a heron consists of 68 chromosomes, while many other
bird taxa have 80 and more chromosomes. In general, 25% of these chromosomes
are categorised as large macrochromosomes (>40 Mbp), while the others are tiny
microchromosomes (<20 Mbp), which undergo a high degree of recombination (see
Damas et al. 2019). The sex chromosomes of birds are different from mammals in
the sense that the females are the heterogametic sex. Females are characterised by
WZ and males by ZZ gonosomes (as opposed to the mammalian XX and XY,
respectively) (see Damas et al. 2019). The bird genomes are also smaller than the
average mammalian genomes and usually contain over 1 billion nucleotides in the
haploid genomes and encode about 20,000 protein-coding genes, similar to
mammals. The content of repetitive DNA is lower in birds than in mammals
(Weissensteiner and Suh 2019; Kraus and Wink 2015; Zhang et al. 2014).
In addition to nuclear DNA, eukaryotic cells carry 100 to 1000 mitochondria
(organelles important for energy metabolism) which carry their own circular and
maternally inherited DNA (mtDNA). mtDNA is rather small and consists of
16,000–19,000 nucleotides, which encode for 13 proteins, 22 tRNA genes and
2 rRNA genes. Mitochondrial DNA is inherited maternally, as the oocyte has several
mitochondria and the spermatocyte only few. Upon fertilisation, only the maternal
mitochondria are maintained. Studying mtDNA alone, hybridisation can distort the
phylogenetic interpretation. As mtDNA is more variable than protein-coding nuclear
DNA, the sequencing and comparison of mtDNA have become an important tool to
study bird taxonomy and systematics (Avise 1987; Shields and Helm-Bychowski
1988; Funk and Omland 2003) (see Braun et al. 2019; Ottenburghs et al. 2019).
DNA can be recovered from any bird tissue but is also present in feathers or in
buccal swabs. Using PCR, marker genes (such as mitochondrial cytochrome b, COI,
ND2) can easily be amplified and sequenced using the chain termination method of
Sanger. A wider set of genes or even complete genomes and transcriptomes can be
sequenced simultaneously using NGS methodology (Kraus and Wink 2015), as will
be outlined in later chapters (see Weissensteiner and Suh 2019; Braun et al. 2019;
Ottenburghs et al. 2019; Husby et al. 2019; Griffin et al. 2019).
4 DNA-DNA Hybridisation
Already before DNA information could be read letter by letter using emerging
sequencing technologies, isolated DNA had its use in the study of bird systematics.
Charles Sibley was the first scientist to use DNA analysis for the study of bird
systematics. Before this, he had tried to use the analysis of proteins from eggs
(Sibley 1960; Sibley et al. 1974). However, the information gained from electropho-
retic profiles of proteins was too limited as to be of wider use in taxonomy. As a new
endeavour, he started his DNA work in 1975 when DNA sequencing was still in its
infancy. Instead of sequencing, Sibley used DNA-DNA hybridisation. The two
complementary strands of the DNA double helix can be separated, for example,
by heating. When the temperature is lowered again, then the complementary DNA
strands will find each other and form a double helix, the process of which is called
reassociation. Depending on the abundance of GC pairs, the thermal stability of
DNA can differ. GC-rich DNA melts at a higher temperature than GC-poor DNA.
The melting temperature can be reliably and accurately measured. Sibley used the
concept of DNA-DNA hybridisation, discovered by Doty et al. (1960) and Marmur
and Doty (1961). Briefly, as DNA molecules from different species differ in their GC
content, consequently their melting temperatures should also be different. The more
closely related two species are, the more similar their melting temperatures be. A
thorough description of DNA-DNA hybridisation is provided by Sibley and
Ahlquist (1990), with details on DNA isolation, preparation of single-copy tracer
DNA, hydroxyapatite (HAP) chromatography to generate single-copy DNA, radio-
active labelling and reassociation kinetics. The authors also provide details on data
processing, data corrections, controls and tree reconstructions by UPGMA clustering
(Sibley and Ahlquist 1990).
In an extraordinary study, Charles Sibley together with Jon Ahlquist analysed the
DNA melting curves of more than 1700 bird taxa. The results of their studies were
published in 1990 as the Phylogeny and Classification of Birds. Sibley used this
information to create a new avian systematics, which was published by Sibley and
Monroe in 1990 as the Distribution and Taxonomy of Birds of the World. The
authors used the following ordering system, starting with the class Aves; they
distinguished the categories subclass Neornithes (with the infraclasses Eoaves and
Neoaves) and parvclasses, orders (with suborders and infraorders), superfamilies and
families, tribes and genera.
14 M. Wink
5 Comparison of the Sibley Phylogeny with NGS Data (See

Braun et al. 2019)
The infraclass Eoaves is basal and includes the Struthioniformes and Tinamiformes.
This grouping was also recognised by more recent studies (Hackett et al. 2008; Jarvis
et al. 2014; Prum et al. 2015) which are based on partial or full-genome DNA
sequence analyses but termed “Palaeognathae” (Fig. 2). The rest of the birds are
recognised as Neognathae in modern phylogenies, which was termed Neoaves by
Sibley and Monroe (1990). However, the Neoaves of current phylogenies does not
include the Galloanserae, which were recognised as the parvclass Galloanserae (with
the orders Craciformes, Galliformes and Anseriformes) by Sibley and Monroe in
1990. In general, the general structure of the genomic avian trees of life (based on
genomic sequences; Hackett et al. 2008; Jarvis et al. 2014; Prum et al. 2015) is
similar but not identical to that of Sibley and Monroe (1990).
Sibley and Monroe (1990) recognised five more parvclasses: the parvclass
Turnicae (with the order Turniciformes), the parvclass Picae (with the order
Piciformes), the parvclass Coraciae (with the orders Galbuliformes, Bucerotiformes,
Upupiformes, Trogoniformes, Coraciiformes), the parvclass Coliae (with the order
Coliiformes) and the large parvclass Passerae (with the orders Cuculiformes,
Psittaciformes, Apodiformes, Trochiliformes, Musophagiformes, Strigiformes,
Columbiformes, Gruiformes, Ciconiiformes and Passeriformes). With regard to
DNA sequence (Hackett et al. 2008; Jarvis et al. 2014; Prum et al. 2015), strong
discrepancies have been discovered (see Braun et al. 2019). For example, the large
order Ciconiiformes consisted in Sibley’s systematic of the suborders Charadrii
[including sandgrouse (Pteroclidae), waders, gull and auks] and Ciconii (including
raptors, falcons, grebes, storks, herons, flamingos, penguins, loons and shearwaters).
In DNA sequence phylogenies, sandgrouse, raptors, falcons, grebes and flamingos
have very different phylogenetic positions (see Braun et al. 2019). I will restrict the
discussion to these few examples, which show that Sibley and Ahlquist (1990) and
Sibley and Monroe (1990) were correct in some groupings but also very wrong in
others. These and other discrepancies can be partly explained by the methodology of
DNA-DNA hybridisation, which does not provide sufficient resolution at the level of
some phylogenetic categories and was also prone to laboratory artefacts (e.g. how to
separate single-copy DNA from repetitive DNA). Sibley and Ahlquist (1990) were
aware of the limitations of the DNA-DNA hybridisation method, but, at that time, it
was the best method available.
6 The Era of DNA Sequence Analysis
The development of rapid DNA sequencing technologies was important for most
branches of biology. In the 1970s two sequencing methods were developed, the
chemical sequencing by Maxam and Gilbert and the enzymatic Sanger sequencing
(Sanger 1981; Saiki et al. 1985, 1988). At first, DNA fragments were separated by
gel electrophoresis on high-resolution gels. At that period, DNA sequencing was a
TINAMIFORMES Tinamidae
STRUTHIONIFORMES RHEIFORMES CASUARIIFORMES APTERYGIFORMES

PALAEOGNATHAE
ANSERIFORMES Anhimidae Anseranatidae Anatidae
GALLIIFORMES Megapodiidae Cracidae Numididae

GALLOANSERAE Phasianidae Odontophoridae
PODICIPEDIFORMES Podicipedidae
PHOENICOPTERIFORMES Phoenicopteridae
PHAETHONTIFORMES Phaethontidae
PTEROCLIDIFORMES Pteroclididae
MESITORNITHIFORMES Mesitornithidae
NEOGNATHAE
COLUMBRIFORMES Columbidae
“METAVES”
EURYPYGIFORMES Eurypygidae Rhynochetidae
Psophiidae Aramidae Gruidae

GRUIFORMES Sarothruridae Heliornithidae Rallidae
Podargidae Steatornithidae
CAPRIMULGIFORMES Nyctibiidae Caprimulgidae
APODIFORMES Aegothelidae Hemiprocnidae Trochilidae Apodidae
OTIDIFORMES Otididae
NEOAVES
CUCULIFORMES Cuculidae
MUSOPHAGIFORMES Musophagidae
GAVIIFORMES Gaviidae
SPHENISCIFORMES Spheniscidae
Diomedeidae Procellariidae
PROCELLARIFORMES Hydrobatidae Pelecanoldidae
CICONIIFORMES Ciconiidae
Scopidae Pelecanidae Ardeidae
“CORONAVES”
PELECANIFORMES Balaenicipitidae Threskiornithidae

Fregatidae Sulidae Anhingidae
SULIFORMES Phalacrocoracidae
Charadrii Lari Scolopaci

Burhinidae Chionidae Turnicidae Dromadidae
Ibidorhynchidae
Scolopacidae
Jacanidae
CHARADRIIFORMES Pluvianellidae
Haematopodidae Glareolidae Rostratulidae
Charadriidae Pluvianidae Pedionomidae
Recurvirostridae Stercorariidae Thinocoridae
Laridae Alcidae
ACCIPITRIFORMES Cathartidae Sagittariidae Pandionidae Accipitridae
STRIGIFORMES Tytonidae Strigidae
COLIIFORMES Coliidae
TROGONIFORMES Trogonidae
BUCEROTIFORMES Upupidae Phoeniculidae Bucerotidae Bucorvidae
Meropidae Coraciidae
CORACIIFORMES Brachypteraciidae Todidae “Piciformes”
Momotidae Alcedinidae
CARIAMIFORMES Cariamidae
FALCONIFORMES Falconidae
PSITTACIFORMES Psittacidae Cacatuidae Strigopidae
PASSERIFORMES Suboscines Oscines
Fig. 2 The “avian tree of life” after Hackett et al. (2008) based on nucleotide sequences of 19 nuclear
genes
challenge and time consuming. The next technological jump in the 1990s was the
development of capillary electrophoresis sequencers, for example, from ABI. Major
improvements were coming from upgrades to machines that have 4-8-24-48 up to
96 capillaries so that several sequences could be analysed at the same time. Large
16 M. Wink
sequencing core facilities utilised multiple machines, which allowed the sequencing
of the human genome in 2001. Next, there was a suture in DNA sequencing, when a
new generation of DNA sequencers (the pyrosequencer 454 from Roche) hit the
market with a complete new concept: parallel sequencing. Further developments of
next-generation sequencing (NGS) were sequencers from Illumina, SOLiD, Ion
Torrent, PacBio and Nanopore which enable sequencing of complete genomes and
transcriptomes (Metzker 2010; van Dijk et al. 2014; Krauss and Wink 2015; Jax
et al. 2018) (see also Weissensteiner and Suh 2019; Braun et al. 2019; Ottenburghs
et al. 2019; Husby et al. 2019; Griffin et al. 2019).
When DNA sequencing became more widely available, bird phylogenies were
reconstructed from an analysis of the nucleotide sequences of one or more marker
genes [in the beginning only mtDNA, later mtDNA and nuclear DNA (ncDNA)
were used] from each species. These phylogenies provided a much higher resolution
at the family and genus level but often failed to infer divergences in the far past
(Kraus and Wink 2015). A breakthrough was achieved by Hackett et al. in 2008 who
sequenced 19 nuclear genes of each of the major bird families using traditional
capillary electrophoresis sequencers. A simplified phylogeny is shown in Fig. 2.
Hackett et al. (2008) had already recognised important clades, which were confirmed
later by genome sequencing via NGS (Jarvis et al. 2014; Prum et al. 2015) (see
Braun et al. 2019; Weissensteiner and Suh 2019). These are the sister-pair relation-
ship of grebes and flamingos, a common ancestry of swifts and nightjars, the
separation of falcons from diurnal raptors, inclusion of New World vultures in the
raptor clade and a new clade combining falcons, parrots and passerine birds (Wink
2015).
In comparison to other animal groups, birds entered the era of genomics rather
late (Kraus and Wink 2015; Braun et al. 2019). The first sequenced avian genome
was that of the chicken (Gallus gallus) (Hillier et al. 2004), followed by the genome
of the zebra finch (Taeniopygia guttata) (Warren et al. 2010), and then the turkey
(Meleagris gallopavo) (Dalloul et al. 2010), the pied and collared flycatcher
(Ficedula hypoleuca, F. albicollis) (2012) and peregrine, saker falcon and mallard
(Falco peregrinus, F. cherrug, Anas platyrhynchos) (Huang et al. 2013) (Zhang
et al. 2014). Most early genomes were from agriculturally important species
(chicken, turkey, duck). These approaches were driven by breeding genomics or
veterinary/biomedical applications (see Vignal and Eöry 2019).
The above-mentioned genome data were instrumental for avian phylogenetics:
An important step forward came, when real genome data of representative taxa of the
avian tree of life were produced by NGS. The presently used Illumina technology
allows the rapid parallel sequencing of up to 250 million short sequences (50–200
nucleotides) in a single run; newer technologies aim for longer reads (Kraus and
Wink 2015).
A first phylogenomic avian tree of life was published in 2014 by the Avian
Phylogenetic Consortium (Jarvis et al. 2014), which was followed in 2015 by a more
detailed genome analysis of Prum et al. (2015) based on nucleotide sequences of
259 nuclear genes and a total of 394,000 sites, which covers 198 species in
122 families and 40 orders, providing the most up-to-date and comprehensive
avian phylogeny (details in Braun et al. 2019). The study of Prum et al. (2015) can be
regarded as an extension of Hackett et al. (2008). The advantage of the multigene
analysis of Prum et al. (2015) over Jarvis et al. (2014) is that they could include more
taxa and that all the genes sequenced could be unequivocally aligned. Jarvis et al.
(2014) used partial genome data, which suffer from difficulties to align homologous
DNA sequences and to avoid inclusion of chimeric sequence assemblies and
pseudogenes.
Sequencing technologies will develop further. From a technological point of
view, Illumina has won the battle for throughput, but third-generation sequencing
such as PacBio or Nanopore sequencing is beginning to reach the laboratory. These
new NGS technologies allow the generation of long sequence runs. The new era of
long-read sequencing coupled with near-chromosome scaffolding is bringing huge
improvements especially in gene completeness of assemblies (Gordon et al. 2016).
The longer the reads and the higher the quality of the sequences, the better the
phylogenies (see also Weissensteiner and Suh 2019; Braun et al. 2019; Ottenburghs
et al. 2019; Husby et al. 2019; Griffin et al. 2019).
As we presently recognise well over 10,300 bird species (some estimates assume
even more than 18,000 bird taxa; Barrowclough et al. 2016), it will certainly take
some time until we have the “avian tree of life”, which will reliably provide the
phylogenetic position and history for each of the avian species. Having a tree of life,
we will finally have the framework for systematics but also to study the evolution of
traits and adaptations in birds.
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346:1311–1320
Avian Genomics in Animal Breeding
and the End of the Model Organism
Alain Vignal and Lel Eory
Abstract
Avian genomics have long benefited from chicken being not only an important
agricultural species but also a model organism for various fields of research
including genetics, immunology, embryology and more recently vertebrate
genome organisation and function. Thanks to this, chicken was in 2004 amongst
the first vertebrates sequenced, and its genome annotation still benefits from a
large community of users. Hundreds of quantitative trait loci (QTLs) have been
mapped in chicken, and genome-wide information is now used in breeding
programmes in agriculture. The chicken genome sequence was soon followed
by the zebra finch sequencing, a model for the biology of learned vocalisation.
Shortly after, the advent of new sequencing technologies, reducing cost and
increasing speed of sequencing, allowed the turkey and duck genomes to follow.
Now, close to 50 bird genomes are published, and with the ever-decreasing cost
of sequencing and most recent genome assembly techniques, many more are
expected soon. In the new era of genomics technologies, will model organisms
remain essential?
Keywords
Chicken · Animal breeding · Model organism · Genomics · Avian · Genome ·
Sequence · Bird · Genomics
A. Vignal (*)
GenPhySE, Université de Toulouse, INRA, INPT, INP-ENVT, Castanet Tolosan, France
e-mail: alain.vignal@inra.fr
L. Eory
Royal (Dick) School of Veterinary Studies, The Roslin Institute, University of Edinburgh, Easter
Bush, Midlothian, UK
e-mail: lel.eory@roslin.ed.ac.uk

https://doi.org/10.1007/978-3-030-16477-5_3
22 A. Vignal and L. Eory
1 Introduction: Chicken as a Model Organism for Birds
The domestication of chicken is thought to have taken place between 6000 and
10,000 years ago in South East Asia (Tixier-Boichard et al. 2011) and possibly also
in China (Xiang et al. 2014, 2015). Nowadays, chickens are raised worldwide for
meat and egg production and are amongst the main sources of protein for human
nutrition. Domestic animal species are tame and readily available, making them an
easy source of study, with scientific observation using chicken as a model going as
far back Aristotle’s description of the embryo (Lennox 2017). Since then, chicken
has been a model organism for the study of many other biological questions and is
still essential in cancer, cardiovascular and embryology research (Burt 2007; Kain
et al. 2014). As a consequence, the species has become one of the major animal
species and certainly the most prominent bird studied in the genomics era (Schmutz
and Grimwood 2004).
Breeders have worked for long towards selecting desirable quantitative traits such
as growth, feed efficiency, carcass composition or the number and quality of eggs
laid. Meanwhile, interests have also focused on qualitative variations in traits such as
plumage, skin or eggshell colours, now the hallmark of many local breeds. All these
quantitative and qualitative genetic variations are conserved in commercial or
experimental populations and in fancy breeds and are now broadly studied both to
improve phenotype–genotype interactions and breeding practices and to understand
basic biology (Delany 2004; Tixier-Boichard 2007). Examples of chicken pheno-
typic variation are shown in Fig. 1. Concerning basic research, a number of
discoveries stemming from using chicken as a model organism include the discovery
of oncogenic viruses through the transmission of cancer by way of a cell-free filtrate
from a sick to a healthy chicken (Rous 1911) or the delineation of the thymic and
bursal lymphoid systems in 1965 (Cooper et al. 1965). But embryology remains the
main domain in which chicken is a prominent model organism, thanks to an easy
access to the embryos in the eggs and a precise description of its developmental
stages goes back to 1951 (Hamburger and Hamilton 1992). Paving the way towards
the era of whole genome sequencing, the construction of molecular genetic maps
started in the early 1990s (Bumstead and Palyga 1992; Levin et al. 1994), thus
establishing chicken as a reference for other studies in birds. Progress in molecular
genetics in the second part of the twentieth century has been extremely rapid, from
the identification of DNA as the physical support for genetic information (Avery
1944) to the first publication of the human genome (Lander et al. 2001; Venter et al.
2001). Since then, a lot more has been done to understand the function of genomes,
such as exemplified in the ENCODE project (ENCODE Project Consortium et al.
2012). Farm animals were amongst the very few vertebrates to follow the path of
human genomics, together with model organisms such as mouse, rat or zebrafish.
Amongst these, chicken was the first to have a whole genome sequence published as
soon as 2004, thanks to its importance also as a model organism, for being the most
studied representative of birds in addition to its economic importance.
The first draft vertebrate genome assemblies produced in the 2000s have always
been the fruit of coordinated efforts organised by large consortiums of research
Avian Genomics in Animal Breeding and the End of the Model Organism 23
Fig. 1 A sample of selected phenotypic variation in chicken
groups collaborating with one or several high-capacity sequencing centres (Mullikin

and McMurray 1999). In the case of the human genome project, sequencing centres
from many countries were involved, each of which having dozens of semiautomated
Sanger sequencing machines and all the associated laboratory equipment (Lander
et al. 2001). A totally different picture can be seen nowadays, with the so-called NGS
(next-generation sequencing) technologies allowing the production of very large
datasets at low cost (Shendure et al. 2017). As a consequence, other bird species than
chicken have had their genomes sequenced, either because of their interest in basic
research, such as the quail in embryology and the study of neural crest cell migration
(Le Douarin and Teillet 1974) or the zebra finch for the neurobiological basis of
vocal learning (Mello 2014), or due to their interest in human nutrition, such as the
common duck or the turkey (Dalloul et al. 2010; Huang et al. 2013). With increasing
sequencing capacity and decreasing costs, the number of bird species having their
genome sequenced is steadily increasing (Zhang et al. 2014b; Kraus and Wink
2015). Having a whole genome sequence assembly to use as a reference is now
considered a prerequisite for any work involving genomics, but how much will this
new era of cheap genome sequencing affect the position of chicken as prominent
bird model species?
To answer this question, we will explore the long and winding path towards the
present state of knowledge in chicken, starting with the observation of chromosomes
and the first molecular genetic maps and having now led to a deep understanding of
the genome through sequencing and annotation, both structural and functional. We
will also show a few examples of biological questions of interest to breeders and to
molecular ecology and evolution, whose study have benefited from this knowledge.
2 The Dawn of Chicken Genomics: From Maps to Genome

Sequence and Annotation
For technical descriptions of maps and genome assembly structure, we offer the
readers Fig. 2 and legend.
2.1 Genomics for Improving Breeding Practices
Interestingly, the first coordinated efforts towards genome-wide studies in chicken

were targeted at developing genetic maps for breeding perspectives. The intention
was to understand the mechanisms underlying the heritable portion of trait
variability, to discover genetic markers linked to traits of interest and thereafter to
use this information for genetic improvement through marker-assisted selection
(MAS). MAS is especially interesting for traits that are too complex or even
impossible to measure, such as resistance to disease. The most common approach
to find markers for MAS was to screen specifically designed second-generation “F2”
or backcross generation “BC” populations to detect quantitative trait loci (QTLs)
with markers uniformly distributed along the genome. To obtain such collections of
markers, the development of genetic maps was required, and these first maps later
appeared handy as a backbone for the whole genome sequence assembly. Indeed, by
solely relying on sequence data, many species having their genomes sequenced do
not have assemblies presented at the chromosome level but are instead in the form of
a collection of continuous genomic DNA sequences named contigs, themselves
assembled into discontinuous scaffolds (Fig. 2). These scaffolds are not assigned
to chromosomes and are not even ordered relatively to one another. For instance, in
the absence of dense genetic maps or other types of mapping information, the current
A: Chicken karyotype
B: maps, sequence and structural annotation

Genetic map
Radiation hybrid map
Cytogenetic map
BAC contig map
Sequence
A GTA
AT T GATA
T CCAT
ATAGAT
A NNNNNTA
T GACATG
A ATATACCTT
TTANNNNNNNNNNTGACAGTA
T AACCAT
ATATACCATG
A GT CTGACTA ATANNNTGCATA
T ACCGAT T CG
Contig
Scaffold
Genes
Alignement to genome: structural annotation
EST
T nscript sequencing (RNA-Seq)
Tra
Tissues Protein identification
Fig. 2 Chicken karyotype, genome maps and sequence assembly. (a) The chicken karyotype is
typical of bird genomes, having a few macrochromosomes and a larger number of
microchromosomes. The male is the heterogametic sex, having Z and W chromosomes.
Microchromosomes cause specific problems for mapping and sequencing, mainly due to their
common duck genome assembly is composed of 227,597 contigs assembled into

78,487 scaffolds. The longest contig and scaffold are 263 kb and 5.9 Mp long,
respectively (Huang et al. 2013), which is much smaller than the size of chromosome
arms, estimated to range between 10 Mb and more than 100 Mb (Pichugin et al.
2001). None of the chromosomal assignment positions of these contigs and scaffolds
are known, for lack of other substantial mapping information. In the case of chicken,
the genome assembly is presented as chromosome-level sequences, thanks to the
availability of a wealth of mapping data in the form of cytogenetic, genetic, radiation
hybrid (RH) and bacterial artificial chromosome (BAC) contig maps, most of which
produced in the pre-genome sequence era for helping in the identification of genes of
interest for the breeding industry. Chicken also benefits from extensive data pro-
duced on gene transcripts, essential for the annotation of the genome and the
construction of gene models (Yandell and Ence 2012).
2.2 The Definition of the Karyotype and Cytogenetic Maps
The definition of the karyotype, a description of the number and appearance of

chromosomes for a given species, is a prerequisite for having any mapping or
sequence data assigned properly to a chromosome location. Karyotypes are
established by cytogenetic observation of chromosomes when condensed at the
metaphase stage of cell division, and once the standard karyotype is defined by
classical cytogenetics, fluorescent in situ hybridisation (FISH) will allow the
Fig. 2 (continued) small size and high (G + C) content. Karyotype courtesy of V. Fillon
(GenPhySE, INRA Toulouse, France). (b) The chicken genome assembly is the result of coordi-
nated efforts of a consortium of collaborating laboratories. Whole genome sequencing strategies do
not allow the direct production of an ideally continuous sequence covering each chromosome arm.
The combination of sequence reads, together with short-distance read-pairing information, allows
the production of stretches of continuous sequence (contigs) and for the assembly of these contigs
into discontinuous blocks of sequence (scaffolds). Gaps in scaffold sequence are reported by
stretches of “N”, whose length corresponds to the estimated size of the gaps. Missing sequence
can be due to many factors including local sequence complexity and base composition. This
discontinuous nature of the sequence assembly has an important incidence on the detection of
genes, whose parts (exons, splice sites, 50 or 30 UTR, etc.) may be missing, introducing
approximations and errors in the genome annotation. According to various parameters including
depth of coverage and the sequencing technology used, a genome assembly may be composed of
thousands of scaffolds. For the assembly of the chicken genome scaffolds into chromosome-level
sequences, mapping information from genetic, physical and radiation hybrid maps was used. FISH
mapping allows the assignation of the sequence to the chromosomes. However, some sequence
scaffolds may not be mapped to chromosomes, in which case they are in a category called the
unknown sequence. In the chicken assembly, some of the unknown sequence may correspond to the
not yet identified microchromosomes. Genetic and radiation hybrid mapping is currently used to
group this unknown sequence into linkage groups. The more recent sequencing technologies, such
as PacBio, produce long reads and by consequence longer contigs. The mapping technology now
used most widely for assembling the scaffolds into chromosomes is optical mapping. This combi-
nation allows for higher quality genome assemblies at a much lower cost than previously
assignment of DNA fragments to chromosomal regions. Without this, genome

entities such as genetic markers, genes or genome sequence portions cannot have
chromosomal correspondence, let alone coordinates.
Classical cytogenetic studies were performed for hundreds of bird species,
revealing their very particular karyotype structure and suggesting relationships
between species by the observation of chromosome rearrangements (Christidis
1990). The chicken karyotype is typical of the vast majority of birds, with a few
large chromosomes called macrochromosomes, a higher number of small
microchromosomes and the Z and W gonosomes, the female being the heteroga-
metic sex (Schoffner and Kristan 1965). The microchromosomes are very difficult to
identify, due to their appearance as small spots on metaphase preparations, and
amongst the 38 pairs of autosomes composing the chicken karyotype, only the 8–10
largest ones can be identified by classical cytogenetic methods and are usually
referred to as the macrochromosomes (Ladjali et al. 1995). To identify each
microchromosome individually, FISH was used with large insert-containing clones,
usually BACs. Simultaneous hybridisation of groups of clones by two-colour
FISH allowed the identification of a first series of 16 microchromosomes (Fillon
et al. 1998), and the definition of the complete karyotype was done by using a
combination of large insert-containing clones and chromosome paints obtained
by microdissection (Masabanda et al. 2004). However, only large-insert clones
allow linking maps and the genome assembly to the karyotype. As a consequence,
microchromosomes still lacking a clone-based identification do not appear in the
assembly, although some of their sequence may be present in the unknown fraction
(see legend to Fig. 2) and in new unassigned genetic map linkage groups (Warren
et al. 2017).
2.3 Genetic Maps and the Initial Mapping of Phenotypic Traits
Genetic mapping is the oldest technique for ordering loci along chromosomes, as it
works by following the transmission in pedigrees of the different alleles segregating
at each locus. In chicken, these were at first very simple and directly observable
phenotypes such as shank colouration or feather shape (Bitgood 1993). Phenotypes
expressed by alleles whose underlying genes are at loci close together on a chromo-
some will tend to be transmitted together in successive generations, an indication of
genetic linkage, for instance, dominant white and frizzle plumage (Hutt 1933). The
distance between two loci, a function of the frequency of crossing-overs at meiosis,
is estimated statistically by calculating the frequency at which phenotypes are
transmitted together from parents to offspring. A set of loci linked together is a
linkage group, and in theory one linkage group per chromosome should be obtained.
However, if the density of markers is too low, some chromosomes may not be
represented at all, and two linkage groups belonging to the same chromosome may
appear as segregating independently. The numerous mutations affecting easily
observed phenotypes such as plumage colour, comb shape, feather shape, skin
colour or skeleton size, and considered as essential characteristics in the description
of breeds, were used to build genetic maps as early as 1933 (Hutt 1933). These were
followed by a collaborative effort to produce what was later called the classical
genetic map (Bitgood 1993). However, due to low marker density, these maps were
quite crude, with only a few chromosomes represented, each of them only partially,
and molecular markers were thereafter used to solve this problem.
Restriction fragment length polymorphisms (RFLP) were the first generation of
molecular or DNA-based genetic markers that could be used on a large scale and
were successfully used for the construction of a first genetic map in human (Donis-
Keller et al. 1987). The long-term goal was to map genetic diseases and possibly
identify the genes involved in their aetiology. Following this path, a similar approach
was thought possible for mapping QTL governing the expression of agronomic traits
in farm animals, including chicken (Soller and Beckmann 1986). The idea was that if
one or several genetic markers positioned close enough to the DNA polymorphism
underlying the phenotypic variation could be identified by linkage analysis, these
could thereafter be used to develop a genetic test for the selection of favourable traits.
Of special interest are traits that are difficult to measure and therefore not used in
classical selection, such as genetic resistance to diseases such as coccidiosis or
Marek’s disease, or to colonisation by Salmonella. The first molecular genetic map
in chicken composed of RFLP markers was published in 1992 (Bumstead and
Palyga 1992), but by that time microsatellites, a new generation of genetic markers,
were shown to be far easier to use by PCR and to have a much higher number of
alleles, allowing for higher mapping precision (Litt and Luty 1989; Weber and May
1989). High-density genetic maps are required, and a chicken consensus genetic map
containing 1889 markers was produced through a worldwide collaborative effort
(Groenen et al. 2000). This map proved very useful for the initial mapping of
Mendelian traits such as naked neck or polydactyly (Pitel et al. 2000) or QTL such
as meat quality (Nadaf et al. 2007) or resistance to salmonella (Mariani et al. 2001).
With the advent of technologies allowing for the genotyping of single nucleotide
polymorphism (SNP) in high numbers and at a low cost, the chicken genetic map
was greatly improved to a map containing 9268 markers (Groenen et al. 2009), and
now a chicken genotyping chip containing over 580,000 SNPs is available (Kranis
et al. 2013). With such high marker densities, the mapping of traits does not rely
anymore on linkage mapping in pedigreed populations but on genome-wide associ-
ation studies (GWAS) in populations. This approach, by taking the historical
crossing-overs having occurred in populations into account, allows for higher
precision in mapping, as, for instance, in Romé et al. (2015).
Although genetic maps were made in first instance for mapping QTL, they later
proved essential for assembling the chicken genome sequence. As a few small
microchromosomes are still absent from the genome sequence assembly, efforts
towards their inclusion involve different approaches including linking together
genomic sequence scaffolds currently non-assigned to chromosomes by means of
additional genetic markers and linkage groups, in addition to performing long-read
sequencing (Warren et al. 2017).
2.4 Physical Maps: A Step Towards Positional Cloning
MAS requires genetic markers positioned as close as possible to the DNA polymor-
phism underlying the phenotypic variation of interest, and ideally the causative
polymorphism itself should be used, whose identification could only be done by
the positional cloning approach. When using the first-generation microsatellite
genetic maps, QTL mapping had a precision in the order of several hundred
kilobases (kb), intervals typically containing dozens of genes. The only sequence
information then available was restricted to the few hundred base pairs (bp) that were
sequenced to design the PCR primers for the markers, and new sequence information
had to be developed in the mapping interval to develop additional markers. The best
option for this was then to use large insert-containing DNA clones identified as being
in the interval of interest. Several efforts were undertaken, first to construct BAC
libraries (Crooijmans et al. 2000; Romanov et al. 2003) and thereafter to order them
into a physical map (Ren et al. 2003; Wallis et al. 2004). As for the genetic map,
beyond their primary purpose for positional cloning, physical maps also later proved
essential as backbones for the whole genome assembly and later on for sequencing
regions missed by the shotgun approach. Such regions were chosen because they
contained badly assembled sequence, large gaps in the genome assembly or genes of
particular interest that were missed for various reasons such as high repeat content.
2.5 Expression Maps, Comparative Maps and Low Chromosomal

Rearrangement Rates in the Bird Lineages
The percentage of vertebrate genomes coding for proteins is very small. For instance
the annotated exons of all genes cover only 2.94% of the human genome and the
protein-coding exons as little as 1.2% (ENCODE Project Consortium et al. 2012).
With the limited sequencing capacities available in the 1990s, a shortcut to the vital
information on coding gene information was to sequence spliced transcripts from
messenger RNA extracted from diverse tissues. The first large-scale project for such
EST (expressed sequence tag) production sequenced 64 cDNA libraries from
21 adult and embryonic tissues (Boardman et al. 2002). The development of genetic
markers within these ESTs representing gene sequences allowed mapping them on
chromosomes. These EST/transcript maps provided candidate genes in QTL
intervals and allowed to perform the first comparison of bird and mammalian
genomes at the chromosome scale using paralogous genes as anchor points,
suggesting different dynamics of chromosome evolution between the mammalian
and avian lineages (Burt et al. 1999). The sequence of gene transcripts is also
essential for the annotation of the genome assembly, by detecting exons.
2.6 The First Chicken Genome Assembly
Chicken was amongst the first large vertebrate genomes sequenced (Hillier et al.
2004), very soon following the human (Lander et al. 2001; Venter et al. 2001),
mouse (Consortium et al. 2002) and fugu fish (Aparicio et al. 2002) ones. It is
contemporary to the rat genome sequence release (Gibbs et al. 2004). Takifugu
rubripes is a special case, as its genome was sequenced due to its very small size
(400 Mb) in order to have a representative vertebrate genome at low cost, and
chicken completed well this first broad picture of vertebrate genomes. The fact
that chicken was sequenced early was partly due to its importance as agricultural
and biological model and to the fact that genome maps and other resources were
available for aiding the assembly but also mainly due to its phylogenetic position.
Indeed, the annotation of the human genome, including the detection of coding
genes, relied on many approaches amongst which whole genome comparisons of
phylogenetically close and distant species for the detection of conserved sequences.
For this approach, having a bird whole genome sequence was essential in addition to
the few mammalian and fish genomes available or being sequenced in the early
2000s (Margulies 2003). The size of the current GRCg6a chicken assembly is
1065 Mb, which is much smaller than the typical mammalian genomes, whose
sizes are in the order of 2500–3000 Mb.
The chicken sequence revealed many interesting features, many of which pertain
to differences between macrochromosomes and microchromosomes. Indeed, gene
density and CpG content in the latter was found to be higher, as was already
suggested by former results (McQueen et al. 1996, 1998). Alignment of the genetic
map to the sequence showed higher recombination rates for microchromosomes
(Hillier et al. 2004; Groenen et al. 2009), and comparison to very partial genomic
sequences from another bird indicated higher rates of nucleotide divergence
(Axelsson et al. 2005). The very low rate of interchromosomal rearrangements in
the chicken lineage was confirmed by comparing the chicken genome with that of
human, mouse and rat (Bourque et al. 2005), supporting the fact that most bird
karyotypes are so similar. Other interesting features of vertebrate genomes could be
investigated by analysing the chicken genome. For instance, a comparison of the
human, mouse and chicken genome showed that some gene deserts have an unex-
pected stability, resisting chromosomal rearrangements and thus suggesting the
existence of distant regulatory elements that need to be physically linked to their
neighbouring genes (Ovcharenko 2005). However, despite its undeniable qualities,
this first assembly was incomplete, with 10 of the smallest microchromosomes
missing altogether, out of a total of 38 autosomes. The Z chromosome was poorly
assembled due to the half coverage obtained through sequencing a ZW female
individual, and only little of the W chromosome was present due to the same reason
and also to a high repeat content. The sequence of the Z and W chromosomes was
completed specifically by targeted sequencing of BAC and fosmid clones, based on
maps (Bellott et al. 2010, 2017). Progress towards sequencing the missing
microchromosomes has since been very slow, needing the construction of specific
radiation hybrid or genetic maps, and is a work still under progress (Douaud et al.
2008; Warren et al. 2017). At the time of writing, six microchromosomes (GGA29
and GGA34–38) are still missing in the GRCg6a assembly.
2.7 The Missing Microchromosomes and Towards a Complete

Genome
Obviously, one major reason for the absence of the smallest microchromosomes in
the genome assembly is their very small size, with an estimated size of the smallest
microchromosome of 3.4 0.26 Mb (Pichugin et al. 2001). This makes their
identification by cytogenetic analysis extremely difficult. However, other reasons
account for this fact. One of them is their extremely high (G + C) content, which was
first suspected by classical cytogenetic studies (Auer et al. 1987). Regions with
abnormally high (G + C) contents tend to resist cloning and sequencing, and each
time a new technology comes into use, one important point to consider is its ability to
improve the quality of the sequencing of (G + C)-rich regions. For instance, the
chicken protamine gene is an extreme example. It was sequenced in 1989 by
classical manual Sanger sequencing and shown to have a (G + C) content, as high
as 88% in the coding region (Oliva and Dixon 1989). Consequently, this sequence
could not be found in the genome assemblies and only appears in the latest Galgal5
and GRCg6a versions of the genome, which were improved by using the Pacific
Biosciences (PacBio) long-read sequencing technology having an improved toler-
ance for extreme (G + C) contents (Shendure et al. 2017). This improvement in
coverage of (G + C)-rich regions can be observed as sequencing technologies
progress. The (G + C) content of the smallest microchromosomes in the first genome
assembly reached 50% (Hillier et al. 2004), whereas in the Galgal5 assembly,
chromosomes 30 and 23 have (G + C) contents higher than 60% (Warren et al.
2017). Another reason for the difficulties in sequencing some microchromosomes
can also be due to other specificities. FISH mapping showed that some of them can
have a very high repeat content. Indeed, single cosmid or BAC clones sometimes
give a very strong painting signal on one or several chromosomes at a time (Fillon
et al. 1998). The repeat content in avian genomes is discussed by Weissensteiner
et al. (2019). Chromosome 16 is another specific case and is a well-known example
of a microchromosome presenting specific challenges for sequencing. It contains the
two major histocompatibility complexes MHC-B and MHC-Y (Fillon et al. 1996)
and for that reason has been studied extensively. However, these two important gene
complexes represent only a small part of the chromosome, the rest being occupied by
the 18S, 5.8S and 28S ribosomal RNA genes, repeated about 150 times, by PO41
repeats and by olfactory receptor and scavenger receptor gene families (Miller and
Taylor 2016). Finally, chromosome 31 contains the chicken Ig-like receptor (CHIR)
complex, a large gene family of more than 100 genes that have evolved by recent
duplications (Laun et al. 2006), therefore causing specific problems for assembly.
The specific comparison of sequences from the reference assembly to published
CHIR gene sequences obtained elsewhere (Laun et al. 2006) allowed their inclusion,
Table 1 Gallus gallus genome assembly statistics

Statistic Galgal2.1 Galgal4 Galgal5 GRCg6a
Nb. autosomes 30 30 32
Length of assembly (Mb) 1098 1046 1230 1065
Contig-N50 (kb) 46.345 279.750 2894.815 17655.422
Scaffold-N50 (Mb) 11.063 12.877 6.379 20.7
Scaffold-N75 (Mb) 4.432 5.752 1.611 12.7
Scaffold-N90 (Mb) 0.971 2.116 0.014 6.1
Scaffold-L50 26 23 47 12
and as a consequence the inclusion of chromosome 31, in Galgal5 (Warren et al.

2017).
2.8 Statistics on the Chicken Genome Assemblies
Statistics on the four major chicken genome assemblies are indicated in Table 1.
Galgal3, sometimes also referred to as Galgal2.1, was generated mainly by Sanger
sequencing, with the addition of some targeted sequencing of BAC and fosmid
clones. Galgal4 saw the addition of first-generation NGS data to the Sanger assembly
in the form of Roche 454 sequences. Galgal5 is a new assembly starting with 50 of
PacBio sequence, corrected by 36 Illumina paired-end reads; assembled with the
help of BAC-end sequences and of improved physical, genetic and RH maps; and
completed with finished BAC clones, chosen specifically to close gaps (Warren et al.
2017). This resulted in the addition of two microchromosomes and of 183 Mb of
sequence to the assembly, having thus expanded to a total length of 1.21 Gb.
Another major improvement is the continuity of the sequence, with an increase of
the contig N50 from 279 to 2894 kb, meaning that the number of gaps in the
sequence is much lower. The latest assembly, GRCg6a, is composed of 82
coverage of Pacific Biosciences reads and shows a dramatic increase of both contig
and scaffold N50, together with a shorter overall assembly length (Table 1).
Finally, since the release of Galgal5, chicken is included in the Genome Refer-
ence Consortium (GRC) (Church et al. 2011), together with the genomes of human,
mouse and zebrafish, which are the only other species included at the time of writing.
The GRC allows having community input and continued curation of reference
genome assemblies and provides the access to patches, which are accessioned
scaffold sequences that represent assembly updates. These patches add information
to the assembly without disrupting the chromosome coordinates, allowing for
early access to new information. Sequencing information providing alternate
representations of haplotypes for multi-allelic loci can also be included and will
especially be important for regions such as the MHC or the CHIR complexes.
Interestingly, long-read sequencing technology allows the production of phased
sequences and haplotyping (Korlach et al. 2017). The fact of being able to represent
sequence insertions, deletions and inversions in the genome reference will also
increase its usefulness as such events will be detected with more ease in sequenced
populations.
2.9 Understanding the Genome: The Annotation
Once a genome is sequenced, a lot still has to be done to make sense out of this raw
information. Eric Lander, one of the leaders of the human genome project in 2003,
summarised this quite clearly: “Genome: Bought the book; hard to read”. And he
also mentioned the fact that “The only problem is: there’s no index” (http://www.
improbable.com/airchives/paperair/volume9/v9i6/nano/nano_6.html). And at that
time, one of the main problems that had to be figured out was only to try and
estimate correctly the number of genes coding for proteins which are contained in the
genome, a problem still not entirely resolved (Pertea et al. 2018)! Since then, efforts
have been devoted towards understanding many other aspects of genome biology,
such as finding target sequences for regulatory elements, studying histone marks and
chromatin accessibility. Much of this work was done on a genome-wide basis within
the frame of the Encyclopedia of DNA Elements (ENCODE) project (ENCODE
Project Consortium et al. 2012). Structural annotation is the task of finding the
functional elements in the genome, whose implications in the biology of the organ-
ism will be further specified by functional annotation, either by direct experimental
evidence or by similarity to orthologous genes or sequences in other organisms. This
annotation concerns of course in first instance the genes coding for proteins but also
the non-coding genes, mainly microRNAs and long non-coding RNAs, and finally
all the regulatory elements such as promoters, enhancers or chromatin domains, all
of which having potential roles in the genetic part of phenotypic variation via DNA
polymorphism. The most obvious approach to finding transcribed genes is to align
messenger RNA sequences, or even better protein sequences if available in the case
of coding genes, on the genome assembly (Yandell and Ence 2012). Numerous
efforts since the first large-scale of EST production in chicken (Boardman et al.
2002) were made. The three main problems when producing transcribed sequence
data for structural annotation are (1) to ensure that a wide variety of tissues are
sampled, so as to obtain all possible tissue-specific genes, (2) to normalise or to
sequence at a high enough depth for detecting genes expressed at a low level and
(3) to produce long sequence reads so as to encompass complete genes and to detect
all possible alternative splicing events. Large efforts are still underway through
transcript sequencing, to improve the gene annotation in chicken (Zhang et al.
2017; Muret et al. 2017; Kuo et al. 2017). Much of the genetic variation underlying
quantitative traits is likely to be located in regulatory sequences, and epigenetic
mechanisms are also likely to play an important role in phenotypic expression. To go
further towards characterising the functional elements of the genome, an internation-
ally coordinated project, Functional Annotation of Animal Genomes (FAANG),
aims at going deeper into RNA sequencing, studying histone marks and chromatin
accessibility (Andersson et al. 2015). This project works in a similar fashion to the
ENCODE project but on several farm animal species. Finally, another approach
towards detecting functional elements in the genome is to detect constrained

sequences, which are conserved across evolution, suggesting their importance.
This approach was used in mammalian genome biology, first for the detection of
exons and for counting human genes (Roest Crollius et al. 2000) and later for
identifying other functionally constrained elements of the genome (Davydov et al.
2010). With the availability of numerous bird genomes spanning the avian phyloge-
netic tree, a similar approach can now be taken to detect elements conserved between
birds and mammals and also elements specific to birds.
3 The Breeder’s Perspective on the Genome
3.1 Marker-Assisted Selection, Genomic Selection and QTL

Identification
The primary idea behind studying the chicken genome from a breeder’s perspective
was to provide tools in the form of DNA markers, to be used for MAS and now more
generally for genomic selection (GS). Marker-assisted selection was the first
utilisation of molecular genetic markers envisaged right at the beginning of molecu-
lar genetics and was the reason for the development of the first genetic maps in the
early 1990s. At the time, little was known on the molecular makeup of genomes, and
genotyping capacity was limited. Genetic markers identified through linkage to
QTLs were to be used subsequently for selecting favourable alleles of practical
interest in the breeding populations. Now, except for very specific cases in which a
phenotype cannot be measured routinely, such as disease resistance, MAS has been
replaced in the breeding industry by GS, thanks to the high-density SNP chips
containing over 580,000 markers in chicken and to affordable genotyping methods.
For GS, all that is required in terms of biological information for the markers is that
they cover the genome in such a way that the quantitative trait locus (QTL) to be
selected for are in linkage disequilibrium with at least one of them (Goddard and
Hayes 2007). In GS, phenotypes still have to be measured, but not on such a large
scale as previously done. Briefly, the association of genotypes and phenotypes is
done in a training population, and for other individuals the phenotypes are predicted
by using only the genotyping information. Using predicted phenotypes also allows
to reduce the generation time by selecting for the best individuals at young age,
which can be especially interesting in the case of phenotypes measured late in life or
estimated through the measure of offspring, for instance, selecting roosters on the
basis of predicted egg production parameters of their offspring.
3.2 Identification of Genomic Events Causing Variation

in Ornamental and Colouration Genes
Since the domestication of animals, man has often spotted out unusual ornamental
traits, which he has selected either for pleasure or because they were associated with
production traits. In the recent centuries, these ornamental traits have often been kept
within specific breeds and are considered as important characteristics described in
the breed standards. These can be quite precise, with descriptions including, for
instance, the shape and colour of the body, the head, the tail, the legs, the crest, the
wattles, etc. The description of the plumage is also important, as feathers can be of
varying length and be striped or frizzled and as body coverage can vary, with some
breeds having naked heads and necks or varying amounts of feathers on the legs. The
colour of the eggshell can also sometimes be unusual, with dark brown or even blue
colours. This wealth of diversity, together with the availability of genetic markers
and the genome sequence, has allowed the identification of a number of mutations
having impact on a number of qualitative traits when the genetic determinism is
simple, usually monogenic.
The polydactyly phenotype is an autosomal dominant trait, characterised by the
presence of one or two extra toes on one or both feet of chicken. Similar to what was
found in human, mouse and cat, a mutation in a regulatory region in the intron 5 of
the lmbr1 gene on chromosome 2 was shown to cause ectopic expression in the
developing limb of Shh, located almost 0.5 Mb away, a major ligand in the
Hedgehog signalling pathway and having a key role in organogenesis (Dunn et al.
2011).
The pea-comb phenotype is a reduction in the size of the comb and the wattles.
The genetic determinism for this trait is a copy number variation in the intron 1 of the
Sox5 gene on chromosome 1, coding for a transcription factor playing a role in
chondrogenesis and causing a transient ectopic expression of this gene. This
duplicated sequence is not conserved across species, suggesting it does not have
an important function by itself, but may disturb the action of regulatory elements in
the region (Wright et al. 2009). A variation between 20- and 40-fold in the copy
number, instead of 2 copies in wild-type animals, could possibly explain the
observed variable expression of the pea-comb phenotype. Interestingly, the Sonic
Hedgehog signalling pathway is also involved in the expression of this phenotype
(Boije et al. 2012).
The rose-comb mutation is another phenotype involving comb morphology and is
usually associated with sperm motility defects. Interestingly, the rose-comb and
pea-comb genes show epistatic interactions, with the individuals carrying both
mutant alleles having the walnut-comb phenotype. The rose-comb phenotype was
explained in most individuals by a 7.4 Mb inversion on chromosome 7 and in some
other individuals by a recombination between a chromosome with the 7 Mb inver-
sion and a wild-type chromosome. For the 7.4 Mb inversion, the proximal
breakpoint is in the 50 UTR of FKBP7, 72 bp upstream of the start codon, this
position being also 42 bp from the 50 UTR and 150 bp from the start codon of
PLEKHAS3. The distal breakpoint is in intron 3 of CCDC108, a part of the sperm
flagellum, which could explain the sperm motility defects. Finally, it was shown that
it is the transient ectopic expression in mesenchymal cells of MNR2, located 3 kb
inside the distal breakpoint, which is responsible for the rose-comb phenotype. This
transient expression of MNR2 could be due to the change of genomic context for the
gene, following the inversion. Finally, transient ectopic expression of both SOX2 and
MNR2 in the same population of mesenchymal cells could explain the walnut
phenotype (Imsland et al. 2012).
A third major comb phenotype, duplex comb, is also explained by the ectopic
expression of a transcription factor, eomesodermin also known as T-box brain
protein 2 (Tbr2), but this time in the ectoderm. The mutation is a 20 kb tandem
duplication containing several conserved putative regulatory elements located
200 kb upstream of the eomesodermin gene (Dorshorst et al. 2015).
Another cranial phenotype is Crest, in which the chicken has a tuft of elongated
feathers on the top of its head. In this case, the exact mutation is not yet known, but it
was mapped very close to the Hoxc8 gene, showing ectopic expression in the cranial
skin during development, and is therefore very probably also a regulatory mutation
(Wang et al. 2012).
The naked neck phenotype, characterised by a lack of feathers on the head and
neck, is associated with a large insertion of sequence from chromosome 1, containing
the Wnt11 and Uvrag genes, approximately 260 kb downstream the GDF7/BMP12
genes on chromosome 3. This insertion is linked with an increased expression level
of GDF7/BMP12, possibly by a long-range action of Wnt11 enhancers (Mou et al.
2011).
The case of colouration genes is complex, and classical genetics has shown that
the numerous colouration phenotypes observed in chicken are due to complex
epistatic interactions. Chicken can be, for instance, white, silver, lavender, buff,
red, various shades of brown or black. And also, the distribution of the colours on the
body can vary, with specific colours on the breast, the head, the tail, the legs, etc.
Colours can also be distributed in speckles or even as stripes on the feathers, such as
in the barred phenotype. A few mutations have been identified, explaining part of
this diversity, some of which pointing to mechanisms conserved across vertebrates
(Gross et al. 2009). Mutations in the MCIR (Melanocortin 1-receptor) gene at the
extended black locus E alter the relative amounts of eumelanin (black–brown
pigment) and phaeomelanin (yellow–red pigment) in melanocytes. Some missense
mutations in the gene are associated with a constitutively active receptor causing a
dominant black phenotype, while one other a loss-of-function causing lighter colours
(Kerje et al. 2003). Mutations in the SLC45A2 gene whose product is important for
vesicle sorting in melanocytes cause the silver colour (missense mutations) or
sex-linked imperfect albinism (a frameshift and a premature stop codon)
(Gunnarsson et al. 2007). Phenotypic variation controlled by the dominant white
locus I is caused by mutations in the PMEL17 gene, coding for a melanocyte-specific
protein having a role in the development of eumelanosomes, pigment granules
containing eumelanin in melanocytes. A 9 bp insertion in exon 10 adds three
amino acids in the transmembrane region and causes the dominant white phenotype.
A five-amino acid deletion in the transmembrane region causes the Dun (whitish)
phenotype. Finally, a 9 bp insertion in exon 10 associated with a 12 bp deletion in
exon 6 causes the Smoky (greyish) phenotype (Kerje et al. 2004). A retroviral
insertion in intron 4 of the tyrosinase gene, essential for the synthesis of both
eumelanin and phaeomelanin, is associated with the recessive white phenotype at
the C locus (Chang et al. 2006), while a six-amino acid deletion in the gene causes
autosomal albinism (Tobita-Teramoto et al. 2000). The lavender phenotype is due to

a dilution of both the eumelanin and phaeomelanin pigments caused by a missense
mutation in the MLPH (melanophilin) gene (Vaez et al. 2008). The dark brown
phenotype at the DB locus is associated with an 8 kb deletion upstream the SOX10
gene, having a role in melanocyte biology. A variation in the level of SOX10
expression could have an effect in turn on the expression of key enzymes of pigment
synthesis, such as tyrosinase (Gunnarsson et al. 2011). Finally, the sex-linked
barring phenotype, characterised by striped feathers, has several allele morphs of
varying dilution. These are due to different non-coding and coding changes in the
ARF transcript of the CDKN2A tumour suppressor locus (Schwochow Thalmann
et al. 2017). Interestingly, for most of these loci, the same genes are involved in
similar phenotypes in other vertebrate species. For instance, MC1R is also involved
in black colouration in wolves (Anderson et al. 2009) or in fishes (Gross et al. 2009).
Colouration genes can act on other parts of the body. The blue egg phenotype is due
to an endogenous avian retroviral insertion (EAV-HP) close to the SLCO1B3 gene
responsible for its overexpression in the oviduct, probably a cause for the accumula-
tion of biliverdin in the eggshell gland; interestingly, two independent but similar
mutations are responsible for the phenotype in Chinese and South American chicken
(Wang et al. 2013; Wragg et al. 2013). The yellow skin is due to a cis-acting
mutation inhibiting the expression of BCDO2 (beta-carotene dioxygenase 2) in the
skin (Eriksson et al. 2008).
Other phenotypes having molecular mechanisms identified include the silky
feathers due to a cis-regulatory mutation of PDSS2 (Feng et al. 2014) and frizzle
feather to an alpha-keratin mutation (Ng et al. 2012).
As seen in these cases of simple monogenic phenotypic traits having a high
penetrance, the identification of the causal variant is possible, especially if it involves
a change in the coding sequence. In the case of regulatory mutations, this can be
much more challenging and is mostly possible if they are due to large events that are
easy to detect such as insertions, deletions or insertions or if there is a deep
understanding of the biology underlying the trait, stemming from basic research.
3.3 The Complex Case of Quantitative Trait Loci (QTLs)
Identifying the molecular mechanisms underlying QTLs is much more challenging

than for the monogenic traits. The initial QTL mapping strategies based on F2 or
backcross designs crossing chicken lines differing for the traits of interest allowed
the mapping of hundreds of QTLs (Abasht et al. 2006), and in a few instances,
causative genes could be identified, such as the BCMO1 gene as a player in meat
colour (Le Bihan-Duval et al. 2011). A review on the mapping of QTLs related to
growth, egg quality, behaviour, metabolism and egg resistance (Abasht et al. 2006)
gives an idea on the diversity of traits studied and also shows the co-localisation of
QTL from independent studies and therefore their confirmation. More data on QTLs
can be found at the ChickenQTLdb website: http://www.animalgenome.org/cgi-bin/
QTLdb/GG/index. However, the mapping intervals that could be defined with such
strategies were very large, usually several Mb containing dozens of genes due to the
limited number of recombinants observable in such designs. For instance, in a
backcross design, these will be equal to the number of backcross offspring multiplied
by the number of recombination events, which will range in chicken between 0.5 for
microchromosomes and 5 for macrochromosomes (Groenen et al. 2009). To refine
mapping precision, advanced intercross lines (AIL) can be made by crossing over
subsequent generations. For instance, an AIL between a layer and a broiler line
performed over 16 generations allowed to map a growth QTL with high precision on
chromosome 4, involving a variation of expression of the satiety signal receptor gene
CCKAR (Dunn et al. 2013). However, in most cases, this approach using specific
crosses and divergent populations did not allow identifying genes underlying QTLs
and thanks to the availability of high-density SNP chips, the more recent studies can
now be based on GWAS, allowing for higher precision in mapping. The additional
benefit is that the QTLs are detected directly in the populations of interest to breeders
(Romé et al. 2015), whereas one could not be certain that QTLs found in experimen-
tal crosses would also segregate in the populations of interest.
The challenge behind elucidating molecular genetic mechanisms underlying
QTLs is also expected to be much higher than for monogenic traits due to their
possible determinism, either due to a polygenic nature, to strong interactions with the
environment or to complex regulatory mechanisms involving non-coding sequences,
such as promoters, enhancers, regulatory RNA, etc. Indeed, while it is often possible
to correlate the expression level of genes with that of traits in transcriptomic studies,
the causes of these variations at the genome level, which are of interest to the breeder
for selection of traits, remain elusive. The GWAS approach will help refine the
location of causative mutations in the case of quantitative traits, but as seen for
complex traits in human, causative variants may be difficult to identify, and further
information on genome biology such as the definition of regulatory regions will
often be required. A typical example is the identification of a SNP having an allele
causing obesity in human. This identification was greatly facilitated by the existence
of a series of whole genome datasets on different epigenetic marks in numerous
tissues (Roadmap Epigenomics Consortium et al. 2015). Mining this dataset allowed
to restrict the region suggested by the GWAS to part of an intron of the FTO gene
having characteristic enhancer properties only in mesenchymal adipocyte
progenitors. This last information allowed for functional tests to be performed
using the correct tissue, demonstrating the effect of the FTO mutation on both the
IRX3 and IRX5 genes located downstream (Claussnitzer et al. 2015).
4 Exploiting Breeding Populations and Model Organisms

for Bird Biology
4.1 Domestication Syndrome
The domestication syndrome refers to a combination of traits that are often found
together in domesticated animals that are not found in their wild ancestors. These
include increased tameness, variations of coat colour, reduction of craniofacial
dimension, nonseasonal oestrus cycles, prolongation of juvenile behaviour and
reduction in brain size (Wilkins et al. 2014). Such traits can be observed in domestic
chicken, and progress on understanding the domestication syndrome in animals will
benefit from comparisons with the wild Gallus gallus ancestor. See, for instance, the
link between tameness and brain size in Agnvall et al. (2017). A recent hypothesis
suggests the domestication syndrome traits are related to altered neural crest cell
development (Wilkins et al. 2014) and chicken is again an interesting model thanks
to its importance in embryology (Théveneau and Mayor 2012). In domestic animals,
it is however not always easy to separate selection due to the early stages of
domestication, from subsequent selection on production traits (Loog et al. 2017).
Candidate domestication genes were identified through the comparison of modern
chicken breeds and the red jungle fowl Gallus gallus, the major wild relatives. The
two major genes currently discussed are the thyroid-stimulating hormone receptor
TSHR and the beta-carotene dioxygenase 2 BCDO2 (Rubin et al. 2010). TSHR plays
a role in the photoperiodic control of reproduction, and selection could be linked to
the fact that domesticated animals tend to lose any strict seasonal reproduction. In the
case of layer chicken, selection has in fact gone very far in that respect, towards the
production of a very high number of eggs all year round. The allele present at high
frequency in domestic chicken has since been directly associated with photoperiodic
response, reproduction, reduced aggressive behaviours and decreased fear of
humans (Karlsson et al. 2015, 2016). BCDO2 cleaves carotenoids in the skin and
when inactive causes the yellow skin colouration due to their accumulation. The
yellow skin allele is thought to come from the grey jungle fowl Gallus sonneratii, a
close relative to the red jungle fowl Gallus gallus, suggesting a hybrid origin of
domestic chicken (Eriksson et al. 2008). It was first suggested that the selection on
these two genes took place at the very beginning of chicken domestication
6000 years ago, but new evidence produced by sequencing ancient DNA from
chicken remains in archaeological sites points towards more recent events (Flink
et al. 2014; Loog et al. 2017). The selection on the TSHR gene is now thought to
have taken place during the Middle Ages, at a time when the proportion of chicken
remains in archaeological data was increasing presumably due to changes in diet
preferences in humans causing higher flock densities and increased egg production.
The introgression of the BCDO2 yellow skin allele can be attributed to recent gene
flow 100–150 years ago from Asia at the time of the creation of the modern chicken
breeds, and its prevalence varies according to breeds.
4.2 Epigenetic Mechanisms Specific to Birds: Absence

of Imprinting of Z Dosage Compensation
Epigenetic mechanisms affecting the expression of genes according to development

stage, tissue type or to the environment are increasingly studied. Having a bird
model organism is essential to study these mechanisms from an evolutionary
standpoint and to deepen our understanding of mechanisms specific to birds. There
are two main examples in which bird genomes have epigenetic mechanisms that are
quite different to those of mammals. The first is dosage compensation of the
gonosomes, which allows for similar expression levels of chromosome X genes in
mammalian in female (XX) or male (XY) cells. The second is imprinting, whereby
only the paternal or maternal copy of a gene is expressed.
In birds, contrariwise to mammals, the heterogametic sex is the female, having Z
and W gonosomes. As gene expression levels usually correlate with gene dose, the
expression level of dosage-sensitive genes must be corrected to have equivalent
values in the cells of homogametic and heterogametic individuals. Several
mechanisms are possible for this, and, for instance, in mammalian females, one of
the X chromosomes is inactivated, and except for a few genes escaping inactivation,
gene expression is only from the active chromosome (Augui et al. 2011). Analyses
of male/female ratios of chicken and zebra finch chromosome Z gene expression in
several tissues showed that dosage compensation in chicken is incomplete, with on
average higher expression levels, although not double as expected with no compen-
sation at all, in males than females (Itoh et al. 2007). The dosage compensation effect
varies along the chromosome, with a cluster of completely compensated genes close
to the male hypermethylated region on chromosome Zp (Melamed and Arnold
2007). Recent analysis of allele-specific expression showed that for the majority of
genes expressed in males, both alleles were expressed at similar levels, suggesting
that the overall reduction of expression is not due to the inactivation of one of the two
chromosomes, but to a different mechanism (Wang et al. 2017).
Genomic imprinting is the imbalance in parental gene expression observed for
some genes, in which only the alleles of maternal or paternal origin are expressed.
The mostly accepted theory explaining imprinting is that of the parental conflict in
which the genome of paternal origin expressed in an embryo will favour its growth at
the expense of the mother, whereas the genome of maternal origin will tend to
restrict the use of resources, allowing for the mother to remain fit for producing more
offspring in the future (Haig 2014). According to this theory, it is usually accepted
that imprinting should be restricted to organisms in which maternal resources
directly affect the embryo and to genes whose functions are related to its growth.
The development of the bird embryo takes place in an egg, and therefore no
imprinting should be found. However, it was also shown that a number of imprinted
genes are also expressed in the brain and can influence adult phenotypes related to
the level of investment in reproduction, for instance, maternal care. This will be
particularly true in cases where a mother’s genes of paternal or maternal origin may
be unequally related to other individuals in a group (Haig 2014). Whole genome
transcriptomic analyses were performed in chicken embryo (Fresard et al. 2014) and
brain (Wang et al. 2015), with the intent of detecting eventual parent-of-origin allele-
specific expression, but no sign of imprinted genes could be found.
4.3 Speciation
The interesting questions of the mechanisms of speciation can be addressed with

genomics (see also Ottenburghs 2019). Only a limited number of speciation genes
have been discovered to date (Mack and Nachman 2017), and PRDM9 is the only
one that was found in vertebrates (Mihola et al. 2009). Positive selection on PRDM9
is responsible for the rapid relocation of recombination hotspots across populations
and species (Stevison et al. 2016), thus provoking an arrest of meiotic prophase
(Davies et al. 2016). However, PRDM9 is not present in all vertebrates and more
specifically is absent from all birds investigated so far (Baker et al. 2017). In
accordance with this observation, recombination hotspots are stable in birds in at
least two finch species investigated (Singhal et al. 2015). For such a study, a high-
quality reference genome was required to avoid the appearance of false recombina-
tion hotspots due to inversions in the assembly.
5 Deep Understanding of the Genome
Several approaches are currently undertaken to deepen our knowledge of the biology
of genomes, including those of birds. The two main themes to identify functional
elements are based either on comparative analysis of multiple genomes (Lindblad-
Toh et al. 2011) or on biochemical assays to study defined regions of the genome
exhibiting a particular biochemical signature (ENCODE Project Consortium et al.
2012). With the emergence of novel molecular methods for genome editing (Zhang
et al. 2014a), it is now possible to prove the functional significance of the identified
functional elements, whether protein-coding or regulatory, which is crucial if the aim
is to establish the link between certain phenotypes and the underlying genotypes.
Although comparative genomics has been extensively used to define protein-
coding gene repertoire of genomes, for example, by mapping protein-coding genes
to the assembly (Jarvis et al. 2014; Yates et al. 2016; NCBI Resource Coordinators
2017), full genome comparisons of multiple species provide a way to understand the
fraction of a genome which evolves nonneutrally, either due to purifying or to
positive selection (Lindblad-Toh et al. 2011). These selective signatures often
correctly identify novel functional regions in genomes and so can be used to
distinguish causative from linked genetic variants that exist in populations. Evolu-
tion of protein-coding genes also leaves detectable traces in full genome multiple
sequence alignments, thus providing a way to predict so far unannotated protein-
coding genes (Lin et al. 2011). Nevertheless, apart from the above example,
signatures of selection rarely indicate the exact biological function of the genomic
segment, and so function should be experimentally tested.
Next-generation sequencing technologies, both short-read (e.g. Illumina) and

long-read (e.g. Pacific Biosciences), are now readily available coupled with various
biochemical assays (1) to characterise the complexity of transcriptome and its
protein-coding and regulatory components (for a transcriptomic review targeted at
non-specialist ornithologist readers, see Jax et al. (2018); (2) to define promoters,
enhancers and other regulatory elements; (3) to characterise chromatin state and
DNA–DNA interactions; and (4) to identify epigenetic modifications. Similar to the
efforts made by the ENCODE project (ENCODE Project Consortium et al. 2012),
such approaches are currently undertaken within the Functional Annotation of
Animal Genomes project (FAANG) (Andersson et al. 2015) to deepen our knowl-
edge of the chicken genome biology.
5.1 Phylogenomics Analyses
The first genome-wide comparative analysis of a bird was done when the chicken
genome got sequenced and compared to that of the human. As the last common
ancestor of mammals and birds lived approximately 310 million years ago, the
nucleotide sequence has changed to such an extent that only 2.5% of the chicken
genome could be aligned between the two species. Most of the conserved sequences
were attributed to protein-coding regions, covering 75% of all coding exons, or
promoter regions of genes, while the rest were shown to be clustered together in gene
poor regions and were linked to known or predicted conserved regulatory elements
(Hillier et al. 2004). Indeed, functional elements are frequently shared between
species and are under selective constraint due to the effect of negative selection,
which removes selectively disadvantageous alleles in populations. The proportion of
the genome under negative selection and the distribution of constrained sites there-
fore have some functional consequence and are important parameters of a genome
(Lander et al. 2001; Eory et al. 2010; Lindblad-Toh et al. 2011).
The resolution of constrained sites within a genome primarily depends on the
total divergence rate across the phylogeny of species compared. The power to detect
signatures of selection increases with the number of genomes sequenced and how
broad the sampling was from the given phylogeny. With optimal sampling it is
possible to increase the total divergence rates to such an extent that the effect of
selection can be estimated at single nucleotide resolution (Lindblad-Toh et al. 2011).
A map of selectively constrained sites in the human genome, based on a 29-way
mammalian genome comparison, showed that over 5% of the human genome is
under purifying selection and the study helped to detect candidate protein-coding
exons, stop codon read-through events, novel RNA structural variants and
constrained regulatory elements (Lindblad-Toh et al. 2011).
To better understand avian genome evolution via comparative genomics, there
was a need for the availability of multiple bird genome assemblies, broadly sampled
across the avian tree. These were achieved by the collaborative effort of the Avian
Phylogenetic Consortium (Jarvis et al. 2014), which sequenced and released 45 bird
genomes and refined the phylogeny of birds.
The work of the Consortium shed light on many features of bird genome
evolution.
One of the many questions was why do birds have relatively small genome sizes
of ~1 Gb, compared to reptiles and mammals which vary between 1 and 8 Gb (Zhang
et al. 2014c). It was found that the main factors to maintain the observed smaller
genome sizes of birds are first, the lower number of repeat elements (4–10%) relative
to mammals (50%); second, the twice as high rate of small deletion events relative to
reptiles and last, the high number (118) of lineage-specific large segmental deletion
events relative to reptiles (Zhang et al. 2014c), which also led to a loss of 7% of the
macrochromosomal genes which were found to be present in green anole.
Despite the overall loss in number of genes, the number of gene gains and losses
is relatively small in birds, and there are indications that gene turnover is lower in
birds than in mammals (Zhang et al. 2014c).
Many bird-specific morphological adaptations were traced back to changes at
molecular level, including adaptive evolution in genes or in regulatory regions.
While lower divergence rates frequently mark regions being under negative selec-
tion, accelerated rate of evolution can indicate adaptive changes in the genome. For
birds, adaptive changes were found in genes, which are crucial to enable the capacity
for flight. Some of these genes or regulatory elements take part in bone development
(Zhang et al. 2014c) others in the development and diversification of feathers (Zhang
et al. 2014c; Lowe et al. 2015) or increased metabolism (Zhang et al. 2014c).
As the sampling of birds included species representing the main lineages, it was
possible to make comparisons, for example, of gene evolution between vocal-learner
(hummingbirds, songbirds and parrots) and nonvocal-learner birds. Two hundred
and thirty-seven candidate genes were identified showing signs of adaptive evolution
across vocal-learners, many of which are expressed in the brain of vocal-learner
birds (Zhang et al. 2014c).
When considering purifying selection, the 45 additional bird genomes greatly
increased the resolution to detect selectively constrained regions in birds, and it was
estimated that 7.5% of the avian genome was highly conserved (Zhang et al. 2014c).
Nevertheless, when genome sizes are factored in, the per Mbp constraint contents are
rather different between mammals and birds. While the human genome is predicted
to contain around 160 constrained sites per 1 Mbp (Lindblad-Toh et al. 2011), the
prediction for birds is only 75 nt (Zhang et al. 2014c). The more than twofold
difference in constrained sites between birds and mammals raises interesting
questions about what factors are influencing the constraint content of a genome. It
is possible that the difference is caused by differences in the way constraint was
calculated (i.e. purely technical), but this may also happen due to a weaker selection
regime on bird genomes in general, or if birds actually maintain their physiology
with smaller number of constrained functional elements or have more unique
lineage-specific elements. Which of the above can explain the difference is remained
to be seen.
Ongoing negative selection on the protein-coding regions of genomes leaves
phylogenetic signatures which are detectable in multiple sequence alignments.
Mutations in protein-coding codons which would change the optimal amino acid
into an unfavourable one will be eliminated by purifying selection. The effect of

selection is less severe on mutations resulting in amino acids with similar physico-
chemical properties to the original amino acid. Also, substitutions at third nucleotide
of fourfold degenerate codons (codons where the third nucleotide can freely change
without changing the amino acid) evolve nearly neutrally and are not under strong
negative selection (Ohta 1995; Lin et al. 2011). These unique selection signatures
can be detected in multiple sequence alignment of protein-coding regions of a
genome, and the phylogenetic coding potential can be estimated at codon per
codon resolution by PhyloCSF (Lin et al. 2011). This is illustrated in Fig. 3 for
one of the exons of the TMTC2 gene. While the protein-coding exon has limited
number of substitutions and mainly results in synonymous or conservative amino
acid changes, the introns contain many more substitutions which are inconsistent
with protein-coding sequence evolution. The splice acceptor and splice donor sites
are also conserved. The phylogenetic coding potential can correctly identify protein-
coding exon and its boundaries along with the strand and the reading frame of the
exon. Due to the large number of bird assemblies in the alignment, it is now possible
to identify novel exons of genes or cases when the annotated ORF of a transcript may
not cover the real ORF (see Fig. 4). Using the chicken genome as reference in a
49 sauropsida alignment, the genome-wide coding potential predicts that there are
23% more protein-coding nucleotides in the chicken and other bird genomes relative
to what had been annotated in the Galgal4 assembly. This estimate suggests that the
number of protein-coding genes may be much larger than the ~15,000 predicted
before (Zhang et al. 2014c; Yates et al. 2016) and this was indeed found using
transcriptome datasets for chicken (Kuo et al. 2017).
Although protein-coding sequences make up a significant proportion of
constrained sites, greater than 80% of highly constrained elements were predicted
to be non-protein-coding (Zhang et al. 2014c), and although predicting functional
consequence may be possible (Lindblad-Toh et al. 2011), establishing the function
would require biochemical evidence.
Besides the fact that selective constraint is largely unaware of function, another
technical limitation with such studies is that multiple sequence alignments were
mainly reference species based. For example, the MULTIZ multiple sequence
alignments are built from pairwise alignments for all possible pairs between the
reference and target species. As a consequence, such alignments, while fully
representing the reference genome, miss out on genomic regions shared within a
certain clade, but not present in the reference genome. The full representation of a
species in an alignment therefore requires that the species in question becomes in
turn the reference, a situation largely benefiting model organisms (Blanchette et al.
2004). For example, vertebrate alignments in Ensembl use the human genome as
reference (Yates et al. 2016), but by doing so those homologous regions which are
unique for murids were not present in the alignment, making it impossible to study
lineage-specific genome evolution. The emergence of novel algorithms for creating
(Paten et al. 2011) and storing (Hickey et al. 2013) reference-free multiple sequence
alignments provides a better conceptual and technical framework for comparative
genomics and enables the storage and analysis of arbitrary subclades in phylogenies
Fig. 3 Phylogenetic coding potential in multiple sequence alignments of birds. (a) The transcript
model of the single transcript TMTC2 gene on the forward strand is represented in UCSC notation.
Boxes represent exons, with taller boxes representing parts of the open reading frame. Joining lines
represent introns, which are spliced out from the final transcript product before the sequence gets
translated into protein. (b) Zoomed-in region of exon 8 with flanking intronic regions. (c) Codon by
codon PhyloCSF signal on the positive strand for the three possible reading frames. Positive scores
along the multiple sequence alignment (d) indicate potential protein-coding region in the genome
on the positive strand in 0 reading frame. (d) The underlying 47-way bird alignment shows the
region of interest as represented by CodAlignView. The region of exon 8 shows remarkable protein-
coding conservation. Substitutions are rare within this region, and the large majority of these lead
either to synonymous or conservative amino acid changes. Splice acceptor and donor sites flanking
the exon are also fully conserved. Unlike the coding exon the flanking intronic regions have
accumulated multiple substitutions and deletions without any evidence that these changes would
preserve an amino acid coding function. Indeed many of these changes would result in stop codons
or frameshifts if the sequence would be protein-coding
Fig. 4 Novel exon and coding sequence extension of the BAZ1B gene using phylogenetic coding
potential. The positive PhyloCSF signal predicts multiple exons in the BAZ1B genic region on
chr19 in the galGal4 assembly. While many of these overlap with exons of the Ensembl BAZ1B
transcript model, there are 21 novel PhyloCSF + regions which indicate novel protein-coding
sequences. Three of these are located within the coding region identified by Ensembl and represent
novel protein-coding exons. The rest overlap with exons which are annotated by Ensembl as non-
protein-coding exons, so-called untranslated regions. An alternative transcript model from PacBio
long-read and RNA-seq-based models verifies the three “within coding” exons, while PhyloCSF
prediction on the full-length alternative transcript model suggests a much longer coding sequence
within the transcript with alternative translation start and end sites
with the reconstructed ancestral sequences without the loss of species- or clade-
specific data. It is now becoming possible to store, share and visualise large
comparative genomic datasets and mapping annotation across all species within a
given phylogeny, therefore representing each species with its full assembly and
annotation individually (Nguyen et al. 2014).
5.2 Improving Annotation and Gene Organisation Using

Long-Read Transcriptome Sequencing
Understanding the protein-coding, structural and regulatory components of the

transcriptome coded in the genome, along with transcript isoforms and alternative
splicing is one of the first steps in understanding the relationship between the
genotype and phenotype of individuals within populations. While some model
organisms benefited from large cDNA libraries, effectively providing full-length
transcript sequences, others got their genome annotation either based on comparative
methods or from relatively cheap, high-throughput, short-read sequencing data. This
latter became prevalent as a cost-effective way to provide species-specific datasets
for multiple tissues and developmental stages with a twofold benefit (Wang et al.
2009). First, it is possible to reconstruct the transcriptome from RNA-seq dataset,
while it also enables the quantification of gene expression and as such helps to detect
physiological changes between different conditions or protein–protein or other
regulatory interactions through the study of differential expression or the analysis of

gene co-expression networks. Despite its benefits, understanding the transcriptome
from short-read data is error prone. This is in part a consequence of the technology,
which relies on relatively short, 50–150 nt long sequenced fragments of the full
transcripts, but also because the sequencing shows compositional bias and the read
coverage is unequal both within and between transcripts (Wang et al. 2009). Due to
these problems, mapping of the reads is ambiguous (Engström et al. 2013), and
reconstruction of the transcript isoforms is only probabilistic, software dependent
and error prone (Steijger et al. 2013), while it is impossible to recover the exact
transcription start and end sites. It is especially difficult to sequence genes belonging
to large gene families or residing in repeat regions, as unique mapping of reads
becomes almost impossible within such regions. As a consequence RNA-seq-based
transcriptomes may comprise fewer genes than expected with lower number of
isoforms (Kuo et al. 2017). The emergence of long-read sequencing technologies,
especially from Pacific Biosciences (Roberts et al. 2013) and Oxford Nanopore
Technologies (Laver et al. 2015), provides solutions to the above problems by
enabling full-length transcript sequencing as a single read. This makes the identifi-
cation of correct isoform structure unambiguous, and coupling with 50 cap and polyA
selection enables reliable identification of transcription start and end sites. Also,
when normalisation is applied during the RNA library preparation, sequencing of
low-abundance transcripts becomes possible leading to an enriched transcriptome
annotation (Kuo et al. 2017).
As short- and long-read datasets become available, it is now possible to better
understand the transcriptome complexity of bird genomes (Jax et al. 2018). For
example, the number of annotated protein-coding and non-coding Ensembl genes
and transcripts has been increased for the chicken genome (Table 2; Yates et al.
2016), and recent estimates suggest that the chicken genome has similar number of
genes and transcripts than the mammalian genomes (Kuo et al. 2017).
Long-read and deep RNA-seq datasets along with improved genome assemblies
will certainly help resolve ambiguities in gene sets of birds and will clarify the
evolutionary history of gene families. For example, a recent analysis of bird
genomes suggested that 274 protein-coding genes, which are present in conserved
syntenic clusters in non-avian vertebrates and involved in important biological
Table 2 Gallus gallus transcriptome annotation statistics

Number of genes Galgal2.1 (Ensembl 67) Galgal4 (Ensembl 85) Galgal5 (Ensembl 89)
Protein-coding 16,736 15,508 18,346
Small non-coding 1102 1408 1705
Long non-coding 0 0 4643
Misc non-coding 0 150 144
Pseudogenes 96 42 43
Total number of 23,392 17,954 38,118
transcript isoforms
processes, had been lost during avian genome evolution (Lovell et al. 2014).
Nevertheless an analysis of de novo assembled RNA-seq transcriptomes found
evidence that 137 of the assumed missing genes exist in chicken and suggested
that the reason of these genes not previously been identified was due to the poor
coverage of GC-rich genomic regions where they are located (Bornelöv et al. 2017).
Long-read datasets may also be used to improve the annotation of closely related
species, as full-length transcript sequences of one species can be mapped onto the
genome of related species (Kuo et al. 2017), but with long-read sequencing getting
more affordable, there is a real possibility that species-specific, rich transcriptome
annotations will be made available for non-model species, effectively enabling each
species to become “its own reference”.
5.3 Inferring Function with “Assay-by-Sequence” Experiments
Next-generation sequencing technology has transformed genomics research and can

contribute to high-quality, richly annotated reference genomes. While many protein-
coding genes are conserved between species, alternative splicing and the resulting
diverse transcriptomes are significantly different between species (Barbosa-Morais
et al. 2012) and therefore cannot be interpreted from comparative data. Similarly, a
significant fraction of the non-coding RNA genes are highly diverse and not
sufficiently conserved between species and so require species-specific transcriptome
information (Ulitsky and Bartel 2013). Access to species-specific transcriptome data
may therefore be necessary to link genotype with observed phenotypic differences.
Although the gene annotation and information on the context-specific expression
of genes and transcripts are important in the above process, understanding the
regulation of these genes can also play a crucial role in linking the sequence to the
consequence. Assaying the DNA and protein interactions (e.g. with chromatin
immunoprecipitation followed by sequencing, i.e. ChIP-seq) or the lack of it
(Assay for Transposase-Accessible Chromatin, i.e. ATAC-seq) may provide the
missing functional information to understand the trait of interest. Identifying protein
and DNA interactions can detect active promoter regions and individual transcrip-
tion factor (TF) binding events in the genome as well as can identify active
enhancers or repressors which modulate the level of expression of particular genes
or clusters of genes (Park 2009). The associations of multiple ChIP marks at the
same genomic location may provide detailed information on the regulatory/tran-
scriptional status of genomic regions through the process called “segmentation”
(e.g. Ernst and Kellis 2012), for example, by classifying enhancers into active,
poised or weak enhancers. Accessible, protein-free DNA can also be assayed by,
e.g. ATAC-seq (Buenrostro et al. 2013) which in turn can help to locate transcription
factor binding sites (TFBS) and can define nucleosome positions.
Although TFs in general have highly conserved DNA-binding preferences, most
of the binding events were found to be species specific in vertebrates (Schmidt et al.
2010), while enhancer sequences, at least in mammals, appear to be more diverse,
arise from DNA exaptation and undergo quick functional turnover (Villar et al.
2015). The fact that regulatory conservation is less prevalent between species
impedes the identification of these elements through orthology and suggests that
detailed and reliable regulatory maps will only be obtained by doing assays at
individual species level as opposed to relying on the annotation of a reference
species. As large-scale biochemical assay-based functional annotation is still expen-
sive and resource intensive, detailed functional maps of birds are nevertheless likely
to first become available for reference species through the joint effort of research
communities similar to the efforts of the ENCODE (ENCODE Project Consortium
et al. 2012) and FAANG (Andersson et al. 2015) project consortia.
6 Do We Still Need a Reference Organism?
6.1 How Complete Is the Existing Chicken Reference?
Despite all the efforts dedicated to producing a complete chicken reference genome
and of all the progress made in sequencing technologies, progress still has to be
made. Notably, six of the smallest microchromosomes are still missing in the most
recent GRCg6a assembly released in 2018. This problem has been a concern since
the very beginning of chicken genomics in the early 1990s, and although a complete
molecular karyotype was described (Masabanda et al. 2004), the lack of large insert-
containing BAC clones for the smallest microchromosomes has always been a
problem for assigning any other sort of information including maps and genome
sequence to them. Specific strategies were dedicated to the sequencing of
microchromosomes. One was to develop radiation hybrid (RH) linkage groups
with markers developed from chicken mRNA and expressed sequence tag (EST)
sequences that were absent in the genome assembly or from non-localised chicken
genomic sequence having sequence similarity to synteny blocks in human obviously
missing in the chicken assembly. This strategy allowed the addition of chromosomes
25 (Douaud et al. 2008) and 33 (Morisson et al. 2007; Warren et al. 2017). Another
approach used was to develop SNP markers from non-localised sequence in the
assembly, to build new genetic linkage groups (Warren et al. 2017). However, for
both these approaches, chromosome assignment still requires large-insert BAC
clones for FISH mapping, which proved often impossible to find when screening
the current existing libraries. Also, the smallest microchromosomes being difficult to
identify on metaphase preparations, a solution is to study them in the form of
lampbrush chromosomes observed at the diplotene stage of meiosis (Galkina et al.
2017). Although the exact number is still under debate, a number of genes for which
there is transcriptome data available are clearly absent from the Galgal5 genome
assembly, this fact having important implications on the description of the gene
content in chicken and by extension in other bird species (Bornelöv et al. 2017).
Orthologs to many of these transcripts are grouped in the human genome, suggesting
that chromosomal segments are absent in blocks (Bornelöv et al. 2017). Specific
regions of macrochromosomes can also cause problems, such as exemplified by the
leptin gene, coding for a satiety hormone, a key regulator of energy balance in
mammals (Halaas et al. 1995). For many years, although the leptin receptor gene was
unambiguously found in the genome, the leptin gene itself could not, and its
existence was subject to debate for around 17 years (Pitel et al. 2010). The proof
of its existence was only recently published, and radiation hybrid mapping localised
it on chromosome 1 (Seroussi et al. 2017). This and many other examples show the
interest of having a very high-quality genome assembly scrutinised by a large
community of scientists. Work is still ongoing, and there is no doubt progress will
be done, as chicken is one of the very rare species being member of the GRC.
6.2 Other Birds: Specific Reference Genomes
Other bird species have been or will be sequenced for various reasons: other poultry
species such as turkey (Dalloul et al. 2010) or duck (Huang et al. 2013) due to their
interest in breeding, the zebra finch because of its interest as a model organism for
the biology of learned vocalisation (Warren et al. 2010) and more than 40 other
species for bird phylogenomics and evolution studies (Zhang et al. 2014b). Thanks
to lowering costs and improved technologies, the number of bird genomes
sequenced will increase rapidly. Bird species bred for human nutrition purposes
are mostly restrained to the gallo-anseriformes and are therefore close to chicken
from a phylogenetic point of view. Despite this, many have their genome sequenced
at varying levels of resolution. Turkey (Dalloul et al. 2010) and quail (Morris et al.
2019) have genomes assembled at the chromosome level thanks to using genetic
(turkey and quail) and physical (turkey) maps. On another hand, the duck genome
sequence cannot yet be presented as chromosomes, as only sequence data were used
for the assembly (Huang et al. 2013). For guinea fowl (Vignal et al. 2019), the
assignment to chromosomes is based on alignment to the chicken genome, taking
into account the known rearrangements observed by cytogenetic analyses. While
having genome sequence data in a random order just as contigs and scaffolds with no
chromosomal assignment can be sufficient for studying genes, their numbers, their
structure or their expression, precise location along the chromosomes will be needed
for QTL or GWAS analyses and for most genetic applications in breeding.
New sequence technologies produce longer reads, allowing for a much higher
genome sequence continuity, especially longer contigs that are essential for a correct
annotation (Yandell and Ence 2012). Complementary techniques now also allow
assembling the contigs at a chromosome level at a fraction of the cost of former
physical mapping strategies. Optical mapping such as implemented by Bionano
Genomics™ allows mapping by direct observation of DNA molecules fluorescently
labelled at specific short sequence motifs defined by restriction enzyme cutting sites
(Lam et al. 2012; Dong et al. 2013). Long-range DNA interactions detected by a
Hi-C (chromosome conformation capture) approach on chromatin reconstituted
in vitro with long DNA fragments and nucleosomes, such as implemented by the
Dovetail Genomics™. Chicago™ is another long-range mapping technique
allowing for chromosome-level assemblies (Putnam et al. 2016). Bird species benefit
widely from these technical advances (Korlach et al. 2017), and continuous
improvements of the quality of reference genomes are expected. Bird genome

sequences produced now have a much higher quality than the first-generation
chicken genome at a fraction of the cost. Results stemming from these possibilities
are emerging in genomics of wild species, such as exemplified by the detection of
signatures of selection in genes related to neuronal functions and cognition in the
great tit genome (Laine et al. 2016), to reproductive strategies in the ruff
(Lamichhaney et al. 2016) or to migration and climate change in the yellow warbler
(Bay et al. 2018).
6.3 Genomics in Ecology and Evolution: With or Without a Model

Organism
Ideally, the best model for studying the biology of a given species will be a selection
of individuals from the species itself, observed in the closest possible conditions to
the natural ones. This is obviously not possible in practice for many reasons
including biological complexity, experimental possibilities, right of access to
biological material or even ethics. Indeed, species used as model organisms were
chosen because they are easy to breed in experimental settings and because they
have specific characteristics allowing to answer a range of biological questions.
Amongst the most prominent species, some were chosen due to their phylogenetic
proximity to human, such as the mouse; others because they allow to work on very
specific questions in basic research, such as the yeast, the fruit fly or the nematode
worm. Species having been used for a long time have now the added benefit of
accumulated knowledge and material, such as experimental protocols or cell lines
and more recently high-quality whole genome sequences. A large consortium of
research groups worked on producing the first-generation chicken genome allowing
for rapid progress in bird genome biology. With reduced costs and improved
sequencing quality, many birds are now sequenced or will be in a short time, thus
challenging the status of chicken as prominent model organism. For instance, in
embryology, quail is emerging as a new model as it is smaller and has a shorter
generation time than chicken (Huss and Lansford 2017), but one major limitation for
genomics in this species was the absence of a reference genome, a problem which
has now been addressed with a first-generation assembly now available and
annotated (Morris et al. 2019). In this specific example, both chicken and quail are
used to answer potentially similar questions in embryology, but in other fields of
biology, different model species will be required, such as the zebra finch for the
biology of learned vocalisation (Warren et al. 2010). With the advent of cheap
genome sequencing, access to genomic data information from an ever-increasing
number of model organisms will be possible (Kraus and Wink 2015; de Magalhaes
2015).
Whole genome sequencing improves the resolution of phylogenetic trees (Jarvis
et al. 2014), but although in theory all species should be considered equally in such
analyses, differences in annotation levels complicate the task, and a uniform annota-
tion had to be made using chicken and zebra finch as references (Jarvis et al. 2014).
Given the difficulties in obtaining species-specific RNA datasets and other annota-
tion datasets based on biological samples for many bird species, the annotation of a
limited number of model species will remain essential, and one option would be to
choose such a minimal set of reference species. Moreover, when comparing gene
counts between bird species and between avian species and other vertebrates, care
must be taken about genes that will be missing for purely technical reasons, such as
the ones potentially present on the six microchromosomes still missing in chicken.
Although they are small and may contain only a limited number of coding genes,
some selection pressure has acted towards their conservation. Having a real idea of
the typical gene content in birds may therefore not be trivial, requiring more
improvements in the chicken assembly, and the fact that the exact number of
genes is still not exactly known even in the human genome (Pertea et al. 2018)
highlights the importance of having at least one bird as part of the GRC.
There have been cases in which the sequencing of a genome has allowed directly
to answer and identify a genome modification responsible for a phenotypic variation
without using a model organism. For instance, genome sequencing of the ruff
Philomachus pugnax, followed by low-coverage re-sequencing of males having
different genetically defined mating behaviour morphs, allowed the identification
of a 4.4 Mb causative inversion (Lamichhaney et al. 2016). However, would a
subtler mutation like a discrete SNP in a regulatory region have been identified
without any prior knowledge on epigenetic marks, such as currently sought for in
chicken by the FAANG consortium? Indeed, quite often, causative genes in birds
were discovered because they were candidate genes stemming from work in chicken.
For instance, in quail, the SLC45A2 gene was shown to cause colour variation as in
chicken (Gunnarsson et al. 2007), the MITF gene to cause the silver phenotype
(Minvielle et al. 2010) and the MLPH gene the lavender plumage colour (Bed’hom
et al. 2012). Making the matter even more complex, many traits involved in adapta-
tion and behaviour that are studied in ecology will be polygenic, subject to environ-
mental factors or be caused by subtle differences in regulatory regions of genes that
will be difficult to point out. For instance, a SNP in the 30 untranslated region of the
circadian clock period gene in drosophila affects the thermal plasticity in its midday
siesta by affecting the thermosensitive splicing of its intron 8 (Cao and Edery 2017).
This was discovered thanks to drosophila being a well-studied model organism and
emphasises once again the need for a high-level annotation of regulatory elements in
several species by ChIP-seq or by other means. This being only possible for a
handful of species, phylogenomics approach is expected to benefit a wider range,
especially if any species genome can be used as the reference. How long will it take
to go from lists of genes possibly associated to traits, such as seen in the great tit and
the yellow warbler (Laine et al. 2016; Bay et al. 2018) to their confirmation and the
elucidation of the underlying mechanism? And will this be possible without using a
model organism available in a laboratory setting? For instance, the implication of
BMP4 in morphological variation in Darwin’s finches was confirmed by laboratory
experiments on chicken embryos (Abzhanov et al. 2004). The latest gene editing
technologies now allow a wider range of species to be manipulated, but there again, a
choice of models will have to be made for practical reasons.
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0172389
Avian Chromosomal Evolution
Joana Damas, Rebecca E. O’Connor, Darren K. Griffin,

and Denis M. Larkin
Abstract
An outstanding feature of avian karyotypes is an extraordinary degree of apparent
similarity from one species to the next, with the majority of avian species
exhibiting 2n ¼ 74–86. Several exceptions to this rule include avian clades that
have a large degree of chromosomal fusion and fission. In this chapter we
describe patterns of avian chromosomal evolution, including likely associations
between karyotype evolution and phenotype. We also describe novel approaches
that will facilitate avian chromosome studies at molecular level to unravel the
mystery of the significance of this very distinctive genomic structure.
Keywords
Avian karyotype · Chromosome evolution · Sex chromosomes · Universal
probes · Macrochromosomes · Microchromosomes · Evolutionary breakpoint
regions · Rearrangements · Genomes · Chicken
Author contributed equally with all other contributors. Joana Damas, Rebecca E. O’Connor, Darren
K. Griffin and Denis M. Larkin
J. Damas · D. M. Larkin (*)

Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London,
London, UK
R. E. O’Connor · D. K. Griffin
School of Biosciences, University of Kent, Canterbury, UK
e-mail: r.o'connor@kent.ac.uk; D.K.Griffin@kent.ac.uk

https://doi.org/10.1007/978-3-030-16477-5_4
70 J. Damas et al.
1 Overall Genome Structure
The overall genome structure of birds has several unique features. First, birds
possess the smallest genomes among amniotes, averaging 1.35 Gbp (Giga base
pairs) and ranging from 0.9 Gbp in the black-chinned hummingbird [Archilochus
alexandri; (Gregory et al. 2009)] to 2.1 Gbp in the ostrich [Struthio camelus; (Scanes
2014)]. This reduction in size from that of their forebears has been hypothesised to
reflect an adaptation to the high metabolic requirements of powered flight (Gregory
2002; Hughes and Friedman 2008). This is, in part, supported by the fact that
flightless birds have larger genomes than flying birds and bats’ genomes are smaller
than their mammalian sister groups (Gregory 2005). This notion has however been
challenged in that comparative analyses also suggest that the evolution of compact
genomes may have occurred before the emergence of flight (Tiersch and Wachtel
1991; Organ et al. 2007). Regardless of when it occurred, the compactness of avian
genomes in extant species is characterised by a low fraction of repetitive DNA,
shorter genes and non-coding regions and by the extensive loss of gene family
members (Zhang et al. 2014).
Considering each of these in turn, the fraction of repetitive elements in avian
genomes, including transposable elements (TEs), varies from 4 to 22%. These are
very low values when compared to the 35–52% found in mammalian genomes
(Lander et al. 2001; Zhang et al. 2014). Indeed, 47 out of the 48 avian genomes
analysed by the Avian Phylogenomics Consortium had a fraction of TEs below 10%.
The exception was the downy woodpecker (Picoides pubescens) with 22% of its
genome comprising TEs resulting from an either species- or lineage-specific expan-
sion of LINE-CR1 (long interspersed elements; chicken repeat I) transposons (Zhang
et al. 2014). Interestingly, even though genomes sequenced with short next-
generation sequencing (NGS) technologies are known to have an underrepresenta-
tion of repetitive sequences, the data reported by Farré and collaborators (2016)
demonstrates that budgerigar (Melopsittacus undulatus), common cuckoo (Cuculus
canorus) and downy woodpecker genomes (assembled with NGS) had more
transposable elements detected in their assemblies than chicken (Gallus gallus),
zebra finch (Taeniopygia guttata) and turkey (Meleagris gallopavo) that were
sequenced with traditional sequencing techniques or a combination of NGS and
traditional techniques (Farré et al. 2016). The average total length of SINEs (short
interspersed elements) in birds is also much lower than that of other reptiles with a
total length of ~1.3 Mbp (Mega base pairs) compared to ~12.6 Mbp in the alligator
and ~34.9 Mbp in green sea turtle (Chelonia mydas), likely indicating that the avian
lineage underwent a reduction in the number of SINEs (Zhang et al. 2014). When
considering gene size, avian genomes have shorter genes (50% shorter than mam-
malian genes and 27% shorter than other reptilian genes), mainly due to a reduction
of intron sizes and intergenic regions. This compression, shared with bats, may result
from a reduced number of TEs within these regions (Zhang et al. 2013, 2014).
Moreover, bird genomes show an overall reduction in the number of gene family
members when compared to other vertebrates, and the loss of these paralogs seems
to correlate with segmental deletions associated with large-scale structural
Avian Chromosomal Evolution 71
rearrangements ancestral to the avian lineage (Hughes and Friedman 2008; Lovell
et al. 2014; Warren et al. 2016). While all the above features characterise avian
genomes, it is perhaps the organisational structure of the genome, i.e. the karyotype,
that is the most peculiar.
1.1 Karyotype Structure
The avian karyotype is near-unique in nature, comprising more chromosomes than

any other vertebrate (approx. 2n ¼ 80). Sizes of avian chromosomes vary from
200 Mbp to as low as 3.4 Mbp (Ellegren 2010), roughly divided into ~10 pairs of
macrochromosomes, comparable in size with mammalian chromosomes and com-
prising approximately 70% of the avian genome, and around 30 pairs of almost
evenly sized, morphologically indistinguishable microchromosomes (Christidis
1990; Masabanda et al. 2004; Griffin et al. 2007). The morphological similarity of
the microchromosomes makes them almost impossible to distinguish by classic
cytogenetic techniques such as karyotyping, as they appear as tiny “dots” using
light microscopy. Nonetheless, there have been attempts at generating (albeit partial)
karyotypes from around 1000 species [most (800+) reported in Christidis (1990)],
approximately 10% of all birds currently described.
Size is not the only feature distinguishing macro- and microchromosomes.
Microchromosomes are GC-rich, gene dense (while accounting for only 23% of
the genome but 48% of the genes) and exhibit higher nucleotide substitution rates
and recombination rates than macrochromosomes (Smith et al. 2000; Habermann
et al. 2001; Burt 2002; Axelsson et al. 2005). The presence of microchromosomes is
not unique to birds, as they share this genomic feature with turtles and lizards, but
not with crocodilians, the sister clade of birds (Olmo 2008). Nonetheless, birds have
smaller microchromosomes in greater numbers than any other vertebrate. Burt
(2002) proposed that the vertebrate ancestor genome contained microchromosomes
suggesting that the characteristic avian karyotype was established at an extraordinary
early stage of evolution, with the absence of microchromosomes in crocodiles
arising by fusion of micro- and macrochromosomes after the crocodilian-bird
lineages diverged (Ellegren 2010)—see later section on ancestral karyotypes.
The majority of avian chromosomes are acrocentric (centromere locates at the
chromosome end with almost undetectable p-arm) (Christidis 1990; Griffin et al.
2007), and most chicken centromeres contain long [>100 Kbp (Kilo base pairs)]
arrays of chromosome-specific simple repeats. However, chicken chromosome
5, 27, and Z centromeres are remarkably short (~30 Kbp) and lack the usual repeat
structure (Shang et al. 2010). Moreover, centromere repositioning and the formation
of neo centromeres have been observed (Zlotina et al. 2012). As in other vertebrates,
avian telomeres possess the canonical TTAGGG repeat motif, but they constitute
large repeat blocks that can be up to 4 Mbp in length (Delany et al. 2007; O’Hare and
Delany 2009).
72 J. Damas et al.
Fig. 1 Chromosome complement variation in vertebrates. Chromosome numbers were obtained

from Gregory (2017), Beçak et al. (1971), Benirschke et al. (1973, 1975)
1.2 Diploid Number
A surprising feature when comparing most avian karyotypes, despite the overall
large diploid number, is an apparent similarity in terms of their sizes and genomic
content between species. More than 60% of all avian karyotypes contain a diploid
number of 74–86 chromosomes (Christidis 1990; Griffin et al. 2007) (Fig. 1), and
chromosomal painting shows that interchromosomal rearrangements are extremely
rare during avian evolution, at least among the larger chromosomes. There are
however exceptions to this rule, with, among others, the stone curlew (Burhinus
oedicemus) having 2n ¼ 40 and the Southern go-away bird (Corythaixoides
concolor) having 2n ¼ 142 (Christidis 1990; Griffin et al. 2007) (Table 1). Intri-
guingly, deviations from the typical avian chromosome number are limited to few
avian groups, such as penguins, some birds of prey (i.e. falcons and eagles) and
parrots (de Oliveira et al. 2005; Griffin et al. 2007; Nanda et al. 2007; Nishida et al.
2008) (Table 1). It is apparent that a diploid number reduction could be achieved
(such as in falcons and parrots) from microchromosomal tandem fusions and/or the
fusion of micro- and macrochromosomes (de Oliveira et al. 2005; Griffin et al. 2007;
Nanda et al. 2007; Nishida et al. 2008; Nie et al. 2009; Damas et al. 2017). For
Table 1 Examples of avian karyotypes with extreme deviations from the typical avian 2n 80
Diploid
Order Species Common name number (2n) References
Charadriiformes Burhinus Stone curlew 40 Christidis
oedicnemus (1990)
Charadriiformes Esacus Beach thick 40 Christidis
magnirostris knee (1990)
Falconiformes Falco Merlin 40 Nishida et al.
columbarius (2008)
Falconiformes Falco peregrinus Peregrine falcon 50 Nishida et al.
(2008)
Psittaciformes Melopsittacus Budgerigar 58 Nanda et al.
undulatus (2007)
Piciformes Picoides Downy 92 Shields et al.
pubescens woodpecker (1982)
Sphenisciformes Pygoscelis Adélie penguin 96 Ledesma et al.
adeliae (2003)
Coraciiformes Ramphastos toco Toco toucan 106 Venturini et al.
(1986)
Coraciiformes Alcedo atthis River kingfisher 138 De Smet (1981)
Musophagiformes Corythaixoides Southern 142 Christidis
concolor go-away-bird (1990)
example, a detailed analysis of the atypical avian karyotype of the peregrine falcon
(Falco peregrinus) revealed that this species presents chromosomes comprising as
many as four fused ancestral chromosomes each (Damas et al. 2017).
This similarity in karyotypes of multiple species across the phylogenetic Class
distinguishes birds from other clades, e.g. mammals and lizards, where interchromo-
somal rearrangements are relatively common (Organ et al. 2008; Graphodatsky et al.
2011; Carvalho et al. 2015) and chromosome numbers are more variable (Fig. 1). For
instance, in mammals, chromosome number can vary enormously between species
of the same genus. An extreme example of this can be seen in the muntjacs where the
Chinese muntjac (Muntiacus reevesi) has a diploid number of 46, while the Indian
muntjac (Muntiacus muntjak) has a diploid number of 6 (7 in males) (Wurster and
Benirschke 1970), illustrating that in closely related mammals (~5 MY divergence
time), karyotypic variation can be very high.
1.3 Sex Chromosomes
Opposite to the situation in mammals, in birds, females are the heterogametic sex,
and males are homogametic. Females thus have one copy of the Z chromosome and
one chromosome W that mostly do not undergo genetic recombination, and males
have two copies of chromosome Z in each cell (Graves 2014). This is just one
example of sex chromosome heterogamety, which evolved independently many
74 J. Damas et al.
times as it can also be found in butterflies, fishes, non-avian reptiles, and amphibians
(Matsubara et al. 2006).
In Neognathae, the Z and W chromosomes are differentiated in terms of size with
the chromosome W being largely heterochromatic, gene poor and significantly
smaller than the Z. Ratites, however, have a W chromosome which is of a similar
size to the Z and is homologous in its entirety, except, in the case of emus, of a small
region near the centromere (Shetty et al. 1999). It can thus be inferred that the ZW
system was evident prior to the divergence of the Palaeognathae and Neognathae
lineages (Deakin and Ezaz 2014). Interestingly, Rutkowska and collaborators (2012)
demonstrated that unlike the mammalian Y chromosome, the avian W chromosome
is not subjected to gradual length reduction over evolutionary time. Indeed, closely
related avian species could have W chromosomes that differ significantly in size due
to variation in the length of non-coding regions (Rutkowska et al. 2012). In chickens,
the Z chromosome is 80 Mbp in size and contains around 1000 genes making it far
less gene dense than the autosomes. It also has 60% more interspersed repeats than
the autosomes making it different from the rest of the chicken genome (Bellott et al.
2010) but interestingly, similar (in terms of relative gene and repeat content) to the
mammalian chromosome X (Graves 2014). Although morphologically resembling
the XY system seen in mammals (with the exception of the ratites of which the Z and
W are indistinguishable from each other cytogenetically), the two systems exhibit no
homology (Nanda et al. 1999). In fact, the avian Z chromosome shares homology
with human autosomes 5, 9, and 18 (Bellott et al. 2010), and the human X
chromosome shares homology with a block of the q-arm (long arm) of chicken
chromosome (GGA) 1 and 20 Mbp of the p-arm (short arm) of chicken chromosome
4 (an independent microchromosome in most birds) (Ross et al. 2005). There also
appears to be no homology between the majority of non-avian reptile ZW and avian
ZW systems. The Japanese four-striped rat snake (Elaphe quadrivirgata), for exam-
ple, has a ZW system that shares homology with GGA2 not Z or W (Matsubara et al.
2006). The exception to this rule appears to be the gecko lizard system (Gekko
hokouensis) which exhibits homology with the chicken Z chromosome (Kawai et al.
2009). The ZW system is not a feature that is present among all reptiles. Both XY
and ZW sex chromosome systems are observed in lizards and turtles, with lizards
showing the largest degree of variability among reptiles (Ezaz et al. 2017). Some
reptiles such as crocodiles also exhibit temperature-dependent sex determination
(TSD) with the Gekkonidae lizard family being particularly unusual in exhibiting all
three types of sex determination (O’Meally et al. 2012).
Unlike in mammals, in chicken at least the sex-determining gene is not SRY
(sex-determining region Y). In fact, the homologous gene of SOX3 (SRY-like high
mobility group box-containing gene 3), on the mammalian chromosome X that
evolved into SRY, lies on chicken chromosome 4 among other genes that are
X-borne in mammals (Graves 2013, 2014). It has been suggested that the gene
DMRT1 (doublesex and mab-3 related transcription factor 1) found on the chicken Z
chromosome may be the key to sex determination (Smith et al. 2009). The system is
dosage-dependent meaning that male determination requires two copies of the gene.
DMRT1 has also been shown to be required for testis formation (Smith et al. 2009).
There is, however, still much debate as to what determines sex in birds. Possible
candidates also include W-specific genes that may determine ovarian function
(Graves 2014), among other theories. Improvements in the assembly of the Z
chromosome achieved using a bacterial artificial chromosome (BAC by BAC)
approach (Bellott et al. 2010) along with work underway to improve the assembly
of the W chromosome (Chen et al. 2012; Smeds et al. 2015; Tomaszkiewicz et al.
2017) will assist with resolving these questions.
2 An Absence of Inter-macrochromosomal Rearrangement
The generation of chromosome-specific DNA probes (paints) for chicken

macrochromosomes (chromosomes 1–9, Z, and W) not only facilitated the
characterisation of the chicken karyotype (Griffin et al. 1999; Habermann et al.
2001; Masabanda et al. 2004) but also led to a surge in avian comparative genomics
research [e.g. Nanda et al. (1999), Shetty et al. (1999), Raudsepp et al. (2002),
Shibusawa et al. (2002), Itoh and Arnold (2005), Griffin et al. (2007), Nishida et al.
(2008), Nanda et al. (2011), Kim et al. (2013)]. Avian cross-species chromosome
painting showed a high degree of synteny, even between distantly related avian
lineages such as chicken, emu (Dromaius novaehollandiae), and zebra finch that
diverged ~90 MYA (Shetty et al. 1999; Guttenbach et al. 2003; Derjusheva et al.
2004; Romanov et al. 2014). This conservation is also noticeable with non-avian
reptiles such as crocodiles and turtles, which diverged from a common ancestor with
birds ~230 MYA (Matsuda et al. 2005; Kasai et al. 2012; Pokorna et al. 2012).
The use of microchromosomal paints, however, has been relatively limited
(Griffin et al. 1999; Shetty et al. 1999; Hansmann et al. 2009; Nie et al. 2009)
largely due to the fact that paints are represented by “pools” of microchromosomes,
i.e. probes that recognise more than one chromosome pair rather than being assigned
to separate, entire chromosomes. Chromosome paints are initially generated by flow
cytometry of a suspension of chromosomes, and the inability to resolve smaller
chromosomes by this approach means that multiple chromosome pairs are isolated in
the flow karyotype (Lithgow et al. 2014). Still, the limited number of studies using
chicken microchromosome paints on other bird karyotypes showed that, in most
cases, synteny is also conserved among these chromosomes, with most chicken
microchromosomes represented as a single chromosome in other species (Deakin
and Ezaz 2014). Exceptions to this rule are found for the species with an atypical
chromosome number (e.g., falcons and parrots), where, as mentioned above, the
reduction in the number of chromosomes resulted from microchromosome tandem
fusions or the fusion of micro- and macrochromosomes (de Oliveira et al. 2005;
Griffin et al. 2007; Nanda et al. 2007; Nishida et al. 2008; Nie et al. 2009; Damas
et al. 2017).
Molecular cytogenetic techniques give valuable insights into the detection of
interchromosomal rearrangement (or lack of it) in avian chromosomes; however,
their relatively low resolution means that they are limited in their usefulness in
assessing if this stability extends to intrachromosomal rearrangement. Indeed, the
76 J. Damas et al.
comparison of high-resolution physical maps and genome sequences has begun to

establish that unlike interchromosomal events, intrachromosomal ones
(e.g. chromosomal inversions) were relatively frequent during avian evolution
(Volker et al. 2010; Skinner and Griffin 2012; Zhang et al. 2014) (Fig. 2). These
studies clearly demonstrated the importance of the availability of assembly data for
the detection of the full collection of events that shaped genomes through evolution.
The recent comparison of 21 sequenced avian genomes revealed that during evolu-
tion, bird chromosomes had an average intrachromosomal rearrangement rate of
~1.25 evolutionary breakpoint regions per MY (EBRs/MY) (Zhang et al. 2014),
which is higher than the ~0.35 EBRs/MY reported for mammals (Farré et al. 2011).
These evolutionary rates, coupled with the smaller genome sizes in birds compared
to mammals, show that despite their karyotypic stability, avian genomes have a
higher density of genome rearrangements than, for instance, mammals and suggest
that intrachromosomal rearrangements might be significant contributors to the phe-
notypic diversity presented by the members of this Class. Furthermore, rearrange-
ment rates are highly variable between avian lineages. For instance, the origin of
Neognathae was accompanied by an elevated rate of chromosomal rearrangements,
~2.87 EBRs/MY (Zhang et al. 2014). Interestingly, vocal learning species [i.e. zebra
finch, medium ground finch (Geospiza fortis), American crow (Corvus
brachyrhynchos), budgerigar, Anna’s hummingbird (Calypte anna)] show higher
rearrangement rates than their non-vocal learning relatives [i.e. golden-collared
manakin (Manacus vitellinus), peregrine falcon, chimney swift (Chaetura pelagica);
P ¼ 0.0499] and even higher than all the other non-vocal learning species
(P ¼ 0.004), which might relate with the larger radiations these clades experienced
relative to the other bird groups (Zhang et al. 2014). Nonetheless, due to the dearth of
chromosome-level assemblies for most of the studied species, these rearrangement
rates could still be underestimated, as evolutionary breakpoint regions (EBRs)
located between scaffolds would not be included in the analysis, or be inaccurate
as assembly errors could be misinterpreted as EBRs.
3 Genome Stability Models
Karyotype differences between species arise from DNA aberrations in germ cells
that were fixed during evolution. As with any other mutation, differences in chro-
mosomal rearrangement rates between lineages can result from either changes in
their rate of mutation or their rate of fixation (Burt 2001). The level of evolutionary
chromosome rearrangement observed in extant species is an outcome of “arm-
wrestling” between these two rates. The study of factors leading to changes in
mutation and fixation rates therefore might help us understand the variability of
rearrangement rates between avian clades and why most avian genomes do not
appear to undergo a great deal of interchromosomal rearrangement when compared
to, for example, mammals and non-avian reptiles but nonetheless undergo significant
intrachromosomal rearrangement (Griffin et al. 2007; Skinner and Griffin 2012).
Fig. 2 Chicken chromosome 1 and 17 representations on the Evolution Highway comparative

chromosome browser (http://eh-demo.ncsa.uiuc.edu/birds/). Blue and pink blocks define homolo-
gous synteny blocks, in “+” and “” orientation, respectively, compared to the reference chicken
chromosomes for six avian genomes assembled to chromosome levels. Numbers within blocks
depict corresponding chromosome numbers in each of the species
78 J. Damas et al.
3.1 Generation Time
Generation time and the repetitive DNA content of a species genome are believed to
be significant contributors to mutation rate variability. The shorter generation times
of birds, relatively to mammals, for instance, could imply a higher propensity to the
occurrence of genome rearrangements as a result of a higher number of undergone
meiosis and associated crossovers (Brown et al. 2018). As crossovers require the
production of double-strand breaks (DSBs) that could be misrepaired, chromosomes
would then have a higher chance to rearrange. In fact, generation time was previ-
ously correlated with the differences in rearrangement rates between human and
mouse (Burt et al. 1999), where the short-lived mice present rearrangement rates at
least twice as high as that of humans. The same reasoning could also explain, at least
partially, why the lineages leading to the short-generation-time songbirds (Nam et al.
2010) demonstrate some of the highest rearrangement rates on the avian phyloge-
netic tree. Interestingly, there are some limited data showing that the recombination
rates observed in birds [1.7–2.6 cM/Mbp; (Pigozzi 2016)] are higher than those of
eutherian mammals [0.2–1.8 cM/Mbp; (Segura et al. 2013)], which could also imply
higher mutation rates in avian genomes. Nonetheless, more mammalian and avian
genomes need to be included in the analysis, and a direct comparison of homologous
sites for mutation rates would need to be performed to prove this hypothesis.
3.2 Repetitive Sequences
Repetitive DNA sequences [e.g. segmental duplications (SDs), transposable

elements (TEs) and tandem repeats (TRs)] are often used as a template for
non-allelic homologous recombination (NAHR) and because of that are considered
important contributors to genome evolution in eukaryotes (Gregory 2005; Wessler
2006; Lynch 2007). In this respect, the low repetitive content of avian genomes
could represent fewer opportunities for avian genomes to change due to a shortage of
templates for NAHR (Burt 2002; Ellegren 2010) and would have an opposite effect
on the rate of rearrangements compared to generation time presented in the previous
section. Indeed, EBRs are associated with repetitive sequences in many animal
groups. Lineage-specific EBRs were previously found enriched for SDs, TRs, and
long terminal repeats (LTRs) in mammals (Murphy et al. 2005; Farré et al. 2011;
Groenen et al. 2012), yeasts (Chan and Kolodner 2011), and Drosophila (Puerma
et al. 2016), among others. In birds, lineage-specific EBRs are usually enriched in
LTRs (Skinner and Griffin 2012; Farré et al. 2016), and the genomes of songbirds
show both an expansion of LTR elements and higher rearrangement rates than other
avian clades (Zhang et al. 2014; Kapusta and Suh 2017).
The repeat-poor nature of avian genomes was previously hypothesised to con-
tribute to the maintenance of independent chromosomes after breakage because of a
lack of non-allelic homologous sequences to remerge broken DNA fragments into
new chromosomes (Burt 2002). This hypothesis could explain the maintenance of
many small chromosomes in the avian lineage and was used to explain the larger
diploid number of bird karyotypes, when compared to non-avian reptiles (Burt

2002). Nonetheless, many avian species with highly rearranged karyotypes show a
predominance of chromosomal fusions, as shown, for instance, for the peregrine
falcon (Damas et al. 2017), parrots (Nanda et al. 2007) and eagles (de Oliveira et al.
2005). The higher repeat content of, for instance, mammalian and amphibian
genomes, when compared to birds, aligns with a much higher frequency of chromo-
somal fusion endorsing the role of repetitive sequences for chromosome merging
(Voss et al. 2011; Uno et al. 2012). However, the same pattern is not clearly
observed within birds where higher levels of rearrangements in the genomes of
falcons, penguins, and parrots do not seem to correlate with higher repetitive
contents (Damas et al. 2017). On one hand, this lack of correlation between the
number of repetitive sequences and the number of EBRs could be concealed by an
underrepresentation of repetitive elements in NGS genome sequences. On the other
hand, it is also possible that as was observed in the highly rearranged gibbon genome
(Carbone et al. 2014), the formation of chromosomal rearrangements could be
associated with segmental duplications or unidentified clade-specific TE families
or even result from repair mechanisms that do not require homologous templates,
such as non-homologous end joining (Moore and Haber 1996).
3.3 Functional Constraints
Independently of the rate of mutation observed in a lineage, after a chromosomal

rearrangement occurs, it will only influence the evolution of species if it is fixed or
nearly fixed (polymorphism). The probability of fixation of a chromosome rear-
rangement can increase (a) simply by chance (i.e. genetic drift) in small or inbreed
populations, (b) if one of the chromosomal variants is transmitted at higher rate to
the descendants (i.e. meiotic drive), or (c) if the novel chromosome variant has
selectively advantageous implications (Burt 2001). Nonetheless, in large, randomly
mating populations, chromosome rearrangements have a higher probability of being
fixed if they have neutral, nearly neutral, or selectively advantageous implications
(Burt 2001). This way, the relative compactness of avian genomes might be one of
the main contributors to their stability, by reducing the chances of fixation of
chromosome rearrangements.
As mentioned in the opening section, genome compactness was previously
hypothesised to relate to the high metabolic demands of powered flight (Gregory
2002; Hughes and Friedman 2008). Indeed, this theory is supported both by the
smaller genome sizes of bats, when compared to other mammals (Gregory 2005),
and the genome size variation within the Class Aves. Flightless birds [e.g. ostrich
(Struthio camelus)] have the largest avian genomes, and hummingbirds that have the
highest metabolic rates among birds have the smallest (Wright et al. 2014). None-
theless, having compact genomes with higher gene and regulatory elements density,
together with a lower number of gene family members, can also have some
drawbacks. One of them would be a lower tolerance to rearrangement due to a
higher probability of a rearrangement having significant biological implications
80 J. Damas et al.
that would not be tolerated by selection. The fact that avian genomic regions
that have large number of collinear conserved DNA sequences [multispecies
(ms) homologous synteny blocks (HSBs)] were found significantly enriched for
conserved non-coding elements [CNEs; (Farré et al. 2016)] supports this hypothesis.
CNEs are non-coding sequences evolving slower than at neutral substitution rate,
many of which are known to be regulatory elements (e.g. sites for transcription
regulatory factors) (Woolfe et al. 2004; Babarinde and Saitou 2016). Thus, the
disruption of CNE-dense regions could have a substantial impact on gene regulation
pathways. As birds have both a higher fraction of their genomes within CNEs [~7%;
(Zhang et al. 2014)] compared to mammals [~4%; (Lindblad-Toh et al. 2011)] and
smaller genome sizes, genome rearrangements might have a higher likelihood of
causing significant (and/or deleterious) functional implications, which will affect
their chances of fixation. In support of this hypothesis, genomic regions harbouring
lineage-specific EBRs demonstrate a lower CNE density than their immediately
adjacent regions (Damas et al. 2017). Moreover, EBRs flanking intrachromosomal
rearrangements have a higher fraction of CNEs than those EBRs that flank chromo-
somal fusions and fissions (Damas et al. 2017). This observation agrees with the idea
that interchromosomal rearrangements might have more drastic effects on cis gene
regulation (Skinner and Griffin 2012; Romanov et al. 2014), and because of that,
their fixation would be further restricted. Indeed, interchromosomal changes were
rarely fixed in avian genomes and are limited to few avian lineages and very few
cases in most genomes (Griffin et al. 2007). In contrast, the fact that larger genomes,
with longer non-coding regions, lower gene and CNE density, and higher repetitive
content (e.g. those of mammals and non-avian reptiles) tend to have higher rates of
interchromosomal rearrangement further supports this hypothesis. It is also notewor-
thy that in avian genomes when interchromosomal change occurs, it tends to recur at
the same site. For instance, the fusion of ancestral chromosomes 4 and 10 appears to
have occurred independently during the evolution in chicken, greylag goose (Anser
anser), collared dove (Streptopelia decaocto) and other species (Fig. 3) (Griffin et al.
2007) suggesting that there are either a limited number of places in the avian genome
where ancestral chromosomes can merge without any deleterious effects for the
carrier of the fused chromosome or presence of homologous sequence templates
which make fusion of ancestral chromosomes 4 and 10 likely to re-occur.
4 Ancestral Karyotypes
The first reconstructions of ancestral genome structures were based on

low-resolution karyotype comparisons using chromosome painting. These studies
offered the first insights into the genome rearrangements that shaped extant genome
structures. As mentioned previously, avian cross-species chromosome painting
showed a high degree of synteny both between avian lineages (Shetty et al. 1999;
Guttenbach et al. 2003; Derjusheva et al. 2004; Romanov et al. 2014) and between
birds and non-avian reptiles (Kasai et al. 2012; Pokorna et al. 2012), making it
relatively easy to predict some key features of the ancestral avian karyotype using
Fig. 3 Seven-colour
chromosome painting
illustrating syntenies between
chicken (Gallus gallus) and
goose (Anser anser). Like
chicken, the goose also has a
“fused” chromosome
4. Colour code: chromosome
1 in cyan, 2 in yellow, 3 in
blue, 4 in green, 5 in magenta,
Z in orange and W in white
cytogenetic data. Using this approach, Griffin and collaborators (2007) reconstructed
an avian ancestral karyotype representing chicken chromosomes 1–9 plus Z (Griffin
et al. 2007). Only one difference between the avian ancestor and the chicken
karyotype was detected, where chicken chromosome 4 results from the fusion of
avian ancestor chromosomes 4 and 10 (Griffin et al. 2007). This karyotypic
organisation is maintained in most avian lineages studied so far. The karyotypes of
Passeriformes also only show one difference to the proposed avian ancestral karyo-
type where the ancestral chromosome 1 is split into two independent chromosomes.
Galliformes (e.g. chicken, quails, pheasants, and turkey) also underwent only a few
fusions or fissions, and most ancestral chromosomes were maintained intact (Griffin
et al. 2007). The exceptions to this pattern are the birds of prey, where multiple
rearrangements were identified (de Oliveira et al. 2005).
As reviewed above, the low resolution of cytogenetic methodologies only permits
the detection of interchromosomal rearrangements, making the FISH-based compar-
ative maps of limited use for reconstructing karyotypes of older ancestors
(e.g. eutherian and amniote ancestors). Sequenced genome comparisons can, how-
ever, not only expand the evolutionary depth of ancestral karyotype reconstructions
but also facilitate the detection of the full range of rearrangements that shaped
species genomes through evolution. Nonetheless, most algorithms developed to
perform the reconstruction of ancestral karyotypes based on genome sequence data
[e.g. InferCARs (Ma et al. 2006) or ANGES (Jones et al. 2012)] require
chromosome-level genome assemblies as inputs, which together with the shortage
of chromosome-level genome assemblies available for birds restricted the study of
chromosome evolution in this clade. Indeed, only recently the first sequence-based
avian ancestor genome structure was proposed (Romanov et al. 2014). Romanov and
colleagues used information from the alignments of chicken, turkey, duck (Anas
platyrhynchos), zebra finch, ostrich, and budgerigar genomes to reconstruct
82 J. Damas et al.
chromosome structure for each macrochromosome and several microchromosomes

(Romanov et al. 2014). They noted that the inter- and intrachromosomal changes
leading to the genome organisation of the six extant species’ genomes would be best
explained by a series of inversions and translocations with common breakpoint reuse
(Romanov et al. 2014). They also observed that chicken had the lowest number of
chromosomal rearrangements compared to the avian ancestor and budgerigar and
zebra finch had the highest. Moreover, microchromosomes seemed to represent
conserved blocks of synteny (Romanov et al. 2014). These results also suggest
that there are mechanisms in action to maintain the stability of the avian karyotype.
In later sections, we will learn more about the history of avian and ancestral
karyotypes when novel algorithms that are designed to work with fragmented
genomes (e.g. the DESCHRAMBLER (Kim et al. 2017) are applied to the currently
available genomes. We will also learn how new cost-effective sequencing and
scaffolding technologies (see below) will allow to generate scaffolds or contigs of
a chromosome (or chromosome arm) length (Korlach et al. 2017) and how these
assemblies are compared to infer evolutionary histories of individual avian
chromosomes.
5 Implications of Chromosome Rearrangements
Chromosome rearrangements are known to play a role in the generation of genetic

and phenotypic diversity (Chen et al. 2013). Genomic rearrangements can be
associated with altered gene expression levels and profiles (Harewood and Fraser
2014). Balanced rearrangements (i.e. inversions and reciprocal translocations) result
in alterations of nucleotide order without gain or loss of genetic material and might
affect gene expression by moving genes into new regulatory environment or switch
off genes completely if their coding sequence is destroyed by breakpoint. Contrast-
ingly, unbalanced rearrangements (i.e. duplications, deletions and unbalanced
translocations) can affect gene dosage through the gain or loss of genetic material.
Chromosomal rearrangements have already been demonstrated to affect phenotypes
in multiple species. For instance, yeasts grown in stress-inducing environments, such
as a glucose-limited setting, show many chromosomal rearrangements after few
generations, and strains containing chromosomal rearrangements are more resilient
to starvation (Dunham et al. 2002; Coyle and Kroll 2008). Pig (Sus scrofa) lineage-
specific EBRs were found enriched for genes related to the sensory perception of
taste, which might explain why pigs can eat food that is unpalatable for humans
(Groenen et al. 2012). In addition, rhesus macaque (Macaca mulatta) EBRs are
enriched for genes related to immune response (Ullastres et al. 2014), which were
proposed to be involved in lineage-specific adaptation. Birds also show similar
patterns. For instance, Farré and collaborators (2016) found that budgerigar
lineage-specific EBRs are enriched in genes involved in forebrain development,
which might relate to the different organisations of the “vocal brain nuclei” on this
species when compared to other vocal learning birds (Farré et al. 2016). They also
found that Anna’s hummingbird lineage-specific EBRs were enriched for genes on
the hexose metabolic process that might relate to this species’ ability to digest sugar
(Farré et al. 2016). Moreover, there are several examples of association between
phenotypes and rearrangement polymorphisms in birds. For instance, the lavender
plumage colour in Japanese quail (Coturnix japonica) was associated with a com-
plex mutation in the region of melanophilin (MLPH) gene which involved two
inversions and one deletion affecting the order of the MLPH and three neighbouring
genes (Bed’hom et al. 2012). Additionally, the behavioural morphs in ruffs
(Philomachus pugnax) are probably caused by a balanced inversion with one
breakpoint in the CENP-N gene that encodes centromere protein N (Kupper et al.
2016).
Chromosome rearrangements are also believed to accelerate genic differentiation
between populations and, therefore, facilitate speciation (Ayala and Coluzzi 2005).
Indeed, some avian species were found to possess segregating inversion
polymorphisms (Itoh et al. 2011). These events can lead to the restriction of
recombination, building genetic incompatibilities that might result in speciation.
Such an example is a large pericentric inversion (~100 Mbp) found in white-
throated sparrow (Zonotrichia albicollis) chromosome 2, which is associated with
behavioural and plumage variations and believed to be an example of the early
stages of sex chromosome evolution (Thomas et al. 2008; Davis et al. 2011; Zinzow-
Kramer et al. 2015).
6 New Tools for the Study of Chromosomal Evolution
While cytogenetic techniques provide valuable insights into the gross evolutionary
structure of chromosomes, their relatively low resolution and inability to efficiently
detect intrachromosomal evolutionary changes poses significant limitations to
the study of avian chromosome evolution. Indeed, as referred to previously,
intrachromosomal rearrangements (e.g. chromosomal inversions) were much more
frequent in avian evolution than interchromosomal changes, suggesting that com-
plete chromosome assemblies (i.e. where each chromosome is represented by one
single contiguous sequence) are urgently needed to reveal missing patterns of avian
genome evolution (Volker et al. 2010; Skinner and Griffin 2012; Zhang et al. 2014;
Damas et al. 2017) and map economically important phenotypes in poultry species
(Tuiskula-Haavisto et al. 2002; Richards 2003; Dodgson et al. 2011).
The generation of chromosome (or near chromosome)-level genome assemblies
from next-generation sequencing (NGS) data has recently been facilitated by
the introduction of novel technologies. Explanation of the specifics of each of
these technologies in detail is beyond the scope of this chapter. Suffice to say
however, they are all designed towards achieving the goal of chromosome-level
assemblies. These include long-read single molecule real-time sequencing (SMRT)
commercialised by Pacific Biosciences (Eid et al. 2009) and Oxford Nanopore
(Clarke et al. 2009) and the highly multiplexed short-read sequencing used by
10X Genomics (Zheng et al. 2016). Contigs or scaffolds can further be joined into
long (super)scaffolds using information on native chromatin conformation [Hi-C;
84 J. Damas et al.
(Lieberman-Aiden et al. 2009)], reconstituted chromatin conformation [Dovetail

“Chicago”; (Putnam et al. 2016)] or optical mapping [e.g. BioNano; (Mak et al.
2016)] technologies. While the long-read sequencing technologies theoretically
should provide one contig (non-gapped DNA sequence) per chromosome, the
quality and quantity of available DNA as well as the presence of long repeats
(e.g. centromeres) often limit the length of non-gapped sequences. Moreover,
novel scaffolding techniques usually show significant levels of disagreement
between them. Additionally, bioinformatic approaches have been developed to
significantly improve the contiguity of already existing fragmented NGS assemblies,
e.g. the Reference-Assisted Chromosome Assembly (RACA; Kim et al. 2013) and
Ragout (Kolmogorov et al. 2014, 2016). Both algorithms efficiently use comparative
information (i.e. alignment to phylogenetically related and distant species’ genomes)
to estimate the order of scaffolds (gapped DNA sequences) in a de novo sequenced
genome that lacks independent physical maps. Due to limitations of novel sequenc-
ing and mapping techniques, the real promise for making accurate and complete
chromosome-level genome assemblies nowadays lies in integrative approaches that
combine several complementary techniques in a single genome assembly project.
Among birds, this has been achieved for the ostrich genome which was assembled
by combining Illumina sequencing and optical mapping (Zhang et al. 2015). This
resulted in an assembly with N50 18 Mbp with 90% of the genome included in
75 superscaffolds (contiguous lengths of DNA sequence), which corresponds to
approximately 3 superscaffolds per ostrich chromosome. Despite these outstanding
results, these combined approaches can still not guarantee that each chromosome
was assembled into a single scaffold and these approaches are associated with
significant expenses not always possible for sequencing projects. A different
approach proposed by Damas and co-workers (2017) involved the combination of
a bioinformatic tool (RACA), wet lab verification by polymerase chain reaction
(PCR) and fluorescence in situ hybridisation (FISH) to generate, verify and physi-
cally assign predicted superscaffolds to chromosomes. An important advantage of
this approach was in the design and use of a panel of universal bacterial artificial
chromosome (BAC) probes that can be utilised with high efficiency (>90%) to map
superscaffolds to any avian genome, provided metaphase cell preparations are
available (Fig. 4). Making metaphases can however be technically challenging,
an actively growing population of cells is an essential prerequisite, and well-
developed skills in cytogenetics are necessary. This is a cost-efficient way to map
superscaffolds to chromosomes guaranteeing chromosome-level assembly for
macrochromosomes and most microchromosomes. Costs associated with this
method are almost completely limited to FISH mapping of 150–200 universal
BAC probes to metaphase chromosomes. Damas and co-workers made use of this
approach to order and orient scaffolds along the rock pigeon (Columba livia) and
peregrine falcon chromosomes (Fig. 4), generating assemblies that are comparable,
in continuity and reliability, to those achieved using traditional mapping techniques
[e.g. with the assistance of radiation hybrid maps built based on the presence of
closely located DNA sequences in the same DNA fragment after breaking DNA with
irradiation (Baxevanis and Ouellette 2004)]. In the near future (funding permitting),
Universal BACs
Zebra Finch
Scaffolds
Chicken
PCFs
CH261-123O22
CH261-50C15
CH261-40G6
GGA2
CH261-186J5
CH261-177K1
CH261-172N3
CH261-169N6
CH261-192C19
CH261-186C5 GGA28
CH261-72A10
CH261-69D20
GGA14
CH261-49P24
CH261-122H14
TGMCBa-205N19
CH261-4M5 GGA12
TGMCBa-342P15
CH261-95H20
TGMCBa-305E19
(a) (b) (c) (d)
Fig. 4 Chromosome-level assembly of the peregrine falcon chromosome 5. FISH mapping of

universal BAC clones (e.g. CH261-50C15, CH261-72A10 and CH261-4M5) (a) allows to build a
sparse cytogenetic map (b) comparatively anchored to long superscaffolds/predicted chromosome
fragments (PCFs) with the universal BAC clone sequence alignments resulting in ordering and
orienting of superscaffolds (and scaffolds) along the chromosome (c). Alignment of assembled
chromosome to complete chicken and zebra finch genomes (c) allows for the final verification of the
peregrine chromosome structure using independent zoo-FISH painting with chicken chromosome-
specific probes (d)
avian chromosome-level assemblies will be achieved through the generation of long

contigs with long-read technologies (e.g. using Oxford Nanopore and Pacific
Biosciences SMRT technologies), followed by further contig scaffolding using at
least one mapping technique (e.g. Hi-C and/or optical mapping) and the assignment
(and verification) of the scaffolds to chromosomes with the set of universal BAC
clones using FISH. Variations of this approach (e.g. use of RACA instead of more
expensive scaffolding techniques) will help to rapidly generate chromosome-level
assemblies.
86 J. Damas et al.
7 Concluding Remarks
The preponderance of a similar genome organisation (karyotype) in the vast majority

of birds suggests an associated functional significance, perhaps more so than in other
species. By contrast, other species’ genome evolution (e.g. in mammals) is
characterised by wholesale chromosome change facilitated by the presence of a
greater number of transposable elements (Skinner and Griffin 2012; Farré et al.
2016; Damas et al. 2017). Nonetheless, until we fully understand the forces that
shaped avian genomes, there remain many unanswered questions. For example, the
mechanisms behind the higher propensity of some avian lineages to successfully fix
interchromosomal evolutionary changes (e.g. in birds of prey, parrots and penguins)
are still a matter of future investigation. In addition, the origin and role of
microchromosomes are not fully understood. Answering these intriguing questions
will require a greater number of complete chromosome-level assemblies. While new
sequencing and scaffolding technologies that permit this level of assembly are under
development, additional tools that allow efficient, inexpensive assembly of avian
genomes are readily available (e.g. the universal BAC clone panel). With these
methods in hand, it is therefore reasonable to expect that the “mystery” of conserved
avian karyotypes will be unravelled in the very near future.
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Repetitive DNA: The Dark Matter of Avian
Genomics
Matthias H. Weissensteiner and Alexander Suh
Abstract
How much do we really know about bird genomes? Like other eukaryotic
genomes, the genomes of birds contain repetitive DNA—tandem repeats,
transposable elements, and endogenous viruses. Repetitive regions are notori-
ously difficult to assemble and often remain inaccessible as gaps within genome
assemblies, a situation which may be metaphorically referred to as genomic “dark
matter.” Here we review avian repetitive DNA from an integrated avian genomics
and cytogenetics perspective. While bird genomes are generally relatively repeat-
poor, some genomic regions consist almost entirely of repetitive elements. Parti-
cularly repeat-rich are centromeres, telomeres, and surrounding regions, as well
as the female-specific non-recombining W chromosome. Many of these regions
are entirely inaccessible with short-read sequencing but may be much better
resolved with long-read sequencing and other single-molecule technologies. We
further discuss how repetitive elements may have directly impacted bird speci-
ation through host–parasite arms races, meiotic drive, and changes in genome
structure. We conclude with a model for improving genome assemblies and
anticipate that the resolution of genomic “dark matter” will permit a deeper
understanding of bird genomes.
M. H. Weissensteiner
Department of Evolutionary Biology, Evolutionary Biology Centre (EBC), Uppsala University,
Uppsala, Sweden
Division of Evolutionary Biology, Faculty of Biology, Ludwig-Maximilian University of Munich,
Planegg-Martinsried, Germany
e-mail: matthias.weissensteiner@ebc.uu.se
A. Suh (*)
Department of Evolutionary Biology, Evolutionary Biology Centre (EBC), Uppsala University,
Uppsala, Sweden
e-mail: alexander.suh@ebc.uu.se

https://doi.org/10.1007/978-3-030-16477-5_5
94 M. H. Weissensteiner and A. Suh
Keywords
Bird evolution · Genome assembly · Cytogenetics · Repetitive element ·
Transposon · Virus · Centromere · Telomere · Sex chromosome · Speciation
1 What Is Genomic Dark Matter?
Genome assembly—a substantial prerequisite for genomics analyses—is nothing

else but a complex jigsaw puzzle. Although there is now a multitude of different
sequencing technologies, each with their own advantages and disadvantages
(reviewed by Goodwin et al. 2016), none of these are capable of sequencing entire
chromosomes, at least for bird genomes. As a result, sequencing data are fragments
of the genome which need to be pieced together into genome assemblies. We thus
emphasize that all genome assemblies are only models of the “true” genome, as the
assembly process is comparable to doing a puzzle game without ever knowing how
the outcome shall look like.
Nearly all avian genome assemblies currently available have been sequenced
using DNA sequencing technologies that produce short sequence fragments, in the
order of 50–500 bp (Kraus and Wink 2015), hereafter referred to as short-read
sequencing data (see Table S1 in Kapusta and Suh 2017). Short-read sequencing
is usually done by paired-end sequencing of DNA fragments of a specific size
(reviewed by Goodwin et al. 2016), resulting in genome assemblies which consist
of contiguous sequences (“contigs”) where all nucleotides have been determined and
linked contigs (“scaffolds”) which contain assembly gaps of undetermined “N”
nucleotides as placeholders (Yandell and Ence 2012). Sequencing reads thus corre-
spond to tens of millions of puzzle pieces in a 1 Gb bird genome. Additionally,
eukaryotic genomes usually contain various types of repetitive elements (Fig. 1),
ambiguous pieces which may fit in several places of the puzzle. Individual units of
repetitive DNA can either be dispersed throughout the genome (“interspersed
repeats,” IRs; Fig. 1a) or repeated adjacently to each other (“tandem repeats,”
TRs; Fig. 1b) and are difficult to assemble either if individual repeat units are highly
similar or if repeat units or repeat arrays are longer than individual reads (Chaisson
et al. 2015). Even if read pairs span individual IRs or short arrays of TRs, the central
part of their sequence may be undeterminable and thus constitute a scaffold gap (left
half of Fig. 1). In addition, repetitive regions with several IRs or long arrays of TR
are usually not spanned by read pairs and thereby lead to scaffold ends (right half of
Fig. 1) (Chaisson et al. 2015).
Altogether, it seems not too surprising that most avian genome assemblies consist
of thousands or even tens of thousands of sequences (scaffolds), instead of a number
equal to their known chromosome number (Table 1). This 1000-fold excess in
numbers of expected versus assembled sequences results from between-scaffold
gaps of unknown in size. Additionally, most avian genome assemblies contain
multiple millions of “N” (i.e., unknown) nucleotides (Table 1) that correspond to
Repetitive DNA: The Dark Matter of Avian Genomics 95
Fig. 1 Schematic illustration of how repetitive elements are responsible for “dark matter” in
genome assemblies. (a) Interspersed repeats (IRs; blue) such as transposable elements (TEs) and
endogenous viruses (EVEs) can lead to assembly gaps within scaffolds. Large IRs or IR-rich
regions can lead to gaps between scaffolds. (b) Tandem repeats (TRs; subscript numbers denote
units per repeat array) such as microsatellites (red) and satellites (orange) can either lead to
assembly gaps within scaffolds or to muted gaps, i.e., sequence contraction relative to the “true”
genome. Tandem repeats with large units or arranged as large arrays can lead to gaps between
scaffolds. Figure from Peona et al. (2018), used under CC BY license
within-scaffold gaps of approximately known size. To add yet another metaphor, it

may be appropriate to collectively refer to these undetermined (but nevertheless
existing!) DNA sequences as genomic “dark matter,” “analogous to the unexplained
dark matter comprising much of the mass in the universe” (Johnson et al. 2005). It is
worth noting that genomic “dark matter” is not unique to birds—in fact, even the
best-studied human Drosophila and mouse genome assemblies are still missing large
parts of their highly repetitive centromeres and telomeres (reviewed by Miga 2015;
Peona et al. 2018).
One may ask how much of the bird genome has so far been accessible to avian
genomics and how much DNA constitutes genomic “dark matter.” Considering
existing fluorometric genome size measurements (Gregory 2017), we estimated an
average of 18% genomic dark matter for those bird species where both cytogenetic
and genome assembly data are available (estimates range from 0.9 to 42.4%;
Table 1, Peona et al. 2018). Most of these genomes are based on short-read
sequencing, indicating that a significant portion of avian genomes remains inacces-
sible. The only exceptions are zebra finch and chicken where the expected and
assembled genome sizes are very similar (Table 1). The zebra finch and chicken
96
Table 1 Quantification of genomic “dark matter” in bird species where both cytogenetic and genome assembly data are available
Haploid Assembly Expected size Assembly size Missing “N” gaps % “dark
Name karyotypea scaffoldsb (Gb)a (Gb)b (Mb)c (Mb)d matter”e
Anas platyrhynchos (Pekin duck) 40 78,487 1.41 1.11 303.27 34.69 24.0
Balearica regulorum (gray- 40 125,353 1.49 1.14 347.70 5.31 23.7
crowned crane)
Calypte anna (Anna’s 37 122,597 1.41 1.11 293.70 38.67 23.6
hummingbird)
Cariama cristata (red-legged 54 139,827 1.47 1.15 321.30 5.69 22.3
seriema)
Columba livia (pigeon) 40 38,878 1.30 1.11 189.16 20.50 16.1
Corvus brachyrhynchos 40 33,296 1.22 1.10 127.33 40.18 13.7
(American crow)
Falco peregrinus (peregrine 25 21,224 1.42 1.17 244.05 18.54 18.5
falcon)
Gallus gallus (chicken) [version 39 23,474 1.25 1.23 18.31 11.76 2.4
galGal5]
Haliaeetus leucocephalus (bald 33 346,419 1.40 1.26 139.75 49.34 13.5
eagle)
Melopsittacus undulatus 29 25,212 1.30 1.12 183.38 30.75 16.5
(budgerigar)
Phoenicopterus ruber (American 40 144,901 1.22 1.14 77.75 6.52 6.9
flamingo)
Struthio camelus (ostrich) 40 32,461 2.06 1.23 835.14 40.39 42.4
M. H. Weissensteiner and A. Suh
Taeniopygia guttata (zebra finch) 40 35,422f 1.22 1.22 0.88 10.12 0.9
Tinamus guttatus (white-throated 40 176,848 1.21 1.06 152.58 22.72 14.5
tinamou)
Tyto alba (barn owl) 46 166,074 1.50 1.14 357.53 11.27 24.6
Table modified from Peona et al. (2018), used under CC BY license
a
Chromosome number and genome size estimates from Gregory (2017) and Christidis (1990). Genome size estimates were converted from C-values into billion
base pairs (Gb) assuming 1 pg ¼ 0.978 Gb (Doležel et al. 2003)
b
Assembly metrics from Table S1 of Kapusta and Suh (2017)
c
Assembly size subtracted from expected genome size
d
Sum of all “N” nucleotides present in the genome assembly
e
Percentage of the expected genome size either missing in the assembly or assembled as “N” nucleotides
f
Although the zebra finch assembly contains 64 chromosome-level scaffolds, one of these (“chrUn”) is a concatenation of 35,359 unanchored contigs separated
by “N” gaps
Repetitive DNA: The Dark Matter of Avian Genomics
97
genome assemblies were generated using Sanger (Warren et al. 2010) and long-read
sequencing (Warren et al. 2017), respectively, technologies with reads over 5-fold
and 100-fold longer than short-read sequencing. Nevertheless, even these assemblies
usually contain tens of thousands of scaffolds (Table 1, Peona et al. 2018),
suggesting that having significantly fewer and larger puzzle pieces helps with the
assembly of many, but not all repetitive regions of the genome.
With this in mind, in this chapter we discuss the current knowledge from avian
genomics about the diversity, distribution, and evolution of repetitive elements. We
further highlight which parts of avian genomes currently constitute genomic “dark
matter” through the discrepancy between what is known from cytogenetics and what
is so far lacking in genomic data. Ultimately, a comprehensive resolution of genomic
“dark matter” requires an integration of cytogenetics into genomics and will permit a
deep understanding of bird genomes.
2 Interspersed Repeats
Interspersed repeats are repetitive elements occurring in multiple nonadjacent

locations and with direct or indirect means to disperse throughout genomes. Such
“genomic parasites” are present in virtually all cellular genomes (Lee and Langley
2010) and comprise two major groups, transposable elements (TEs) (Kazazian
Jr. 2004) and endogenous viral elements (EVEs) (Katzourakis and Gifford 2010).
Class I TEs (retrotransposons; also including retroviruses) mobilize through
retrotransposition, a copy-and-paste mechanism involving reverse transcription of
the TE RNA sequence and insertion of the resulting TE complementary-DNA
(cDNA) into the genome (Fig. 2a) (reviewed by, e.g., Levin and Moran 2011). On
the other hand, class II TEs (i.e., DNA transposons) usually disperse through
transposition, a cut-and-paste mechanism where the TE DNA sequence is excised
and reinserted into the genome (Fig. 2b) (reviewed by, e.g., Levin and Moran 2011).
A third major mechanism is the occasional insertion of non-retroviral EVEs into the
genome via nonhomologous end joining (NHEJ) between virus DNA and the
genome during DNA repair (Fig. 2c) (reviewed by, e.g., Feschotte and Gilbert 2012).
Birds have a much lower overall diversity of major TE groups (“superfamilies”)
than other land vertebrates (see Fig. 1 of Kapusta and Suh 2017) and considerably
less EVEs (Cui et al. 2014). Nevertheless, recent years of avian genomics have
discovered a variety of peculiarities across the avian tree of life, such as TE de novo
emergences, horizontal transposon transfer, and germline integration of
non-retroviral EVEs (Fig. 3). In the following, we discuss these findings in detail
in the context of the different propagation modes of genomic parasites. These
genomic parasites are either “autonomous” if they encode for their own cis-
mobilizing enzymes or “nonautonomous” if they cannot mobilize on their own but
rely on trans-mobilization by the enzymatic machinery of autonomous elements.
Fig. 2 Schematic illustration of the three major mechanisms for the mobility of interspersed
repeats. (a) Copy-and-paste retrotransposition typical for retrotransposons and retroviruses. A
reverse transcriptase enzyme (gray) binds the retrotransposon RNA (blue line) and catalyzes reverse
transcription and complementary-DNA (cDNA) integration into a new target site (white square) via
staggered cuts, resulting in a target site duplication flanking the TE insertion. (b) Cut-and-paste
transposition typical for DNA transposons. A dimer of the transposase enzyme (gray) binds the
transposon DNA (blue box) and catalyzes excision and integration into a new target site (white
square) via staggered cuts, resulting in a target site duplication flanking the TE insertion. (c)
Nonhomologous end joining (NHEJ) leading to virus integration. Following viral infection, a
DNA double-strand break (DSB) is repaired by NHEJ through short sequence homologies between
the target site (white square) and the viral DNA. In contrast to transposition and retrotransposition,
an endogenous viral element (EVE) insertion resulting from NHEJ is not flanked by a target site
duplication
2.1 Class I Transposable Elements
2.1.1 LINE Retrotransposons

Long interspersed elements (LINEs) are retrotransposons which encode a reverse
transcriptase (RT) protein with an endonuclease (EN) domain and often an ORF1
protein of unknown function (Fig. 4) (Wicker et al. 2007; Kapitonov and Jurka
2008). LINEs mobilize via target-primed reverse transcription (TPRT) (Luan et al.
1993; Ichiyanagi and Okada 2008). The TPRT mechanism is initiated by
EN-catalyzed nicking of the target site in the genome, followed by reverse transcrip-
tion of the LINE mRNA into cDNA starting from its 30 -end (reviewed by Martin
Fig. 3 Distribution of interspersed repeats across the avian tree of life. Key events of TE evolution
(Kapusta and Suh 2017) such as horizontal transfers (HT) were mapped on a recent consensus
phylogeny of major avian lineages (Suh 2016) which accounts for phylogenetic uncertainty in
Fig. 4 Long interspersed elements (LINEs) of birds. Schematic organization of the CR1, RTE, and
R2 superfamilies. Full-length elements exhibit 50 and 30 untranslated regions (50 and 30 UTRs, blue
and red) and a coding region (gray) encoding an unknown protein (ORF1), an apurinic endonucle-
ase (APE) or an endonuclease (EN), and a reverse transcriptase (RT). The 30 UTR ends with a
characteristic microsatellite motif, and white squares are target site duplications (“v” indicates
variable lengths). Figure modified from Kapusta and Suh (2017), used with permission from John
Wiley and Sons
2006; Levin and Moran 2011). Premature termination of TPRT is common, and
LINE daughter copies are therefore frequently 50 -truncated, rendering most LINE
copies “dead on arrival” because they lack the sequence features required for their
own transcription and retrotransposition (Levin and Moran 2011).
Nearly all avian LINEs (~65,000–630,000 copies per genome; Zhang et al. 2014)
belong to the chicken repeat 1 (CR1) superfamily and together make up 39–88% of
all TE copies in available bird genome assemblies (Hillier et al. 2004; Warren et al.
2010; Zhang et al. 2014). This CR1 dominance is common for the genomes of most
land vertebrates (Shedlock et al. 2007; Suh et al. 2015a; Janes et al. 2010). Avian
CR1 copies have been grouped into at least 14 families (Hillier et al. 2004; Warren
et al. 2010), many of which were active over long parts of avian evolution (Matzke
et al. 2012; Kriegs et al. 2007; Suh et al. 2011b) and have been used as phylogenetic
markers for the avian tree of life (e.g., Suh et al. 2011b; Haddrath and Baker 2012;
Kaiser et al. 2007). Although some CR1 copies appear to be ancient (Vandergon and
Reitman 1994), the CR1 families present in bird genomes emerged from a single
ancestral CR1 lineage after the bird/crocodilian divergence (Suh et al. 2015a; Suh
2015). The vast majority of CR1 copies is 50 -truncated (Vandergon and Reitman
1994; Wicker et al. 2005; Hillier et al. 2004), but even the shortest copies usually
Fig. 3 (continued) genome-scale phylogenies (Jarvis et al. 2014; Prum et al. 2015; Suh et al.
2015b). The distribution table (right) contains TEs which are ubiquitous across bird genomes (CR1
LINEs and LTR retrotransposons or endogenous retroviruses, ERVs; Kapusta and Suh 2017) and
known non-retroviral EVEs (hepadnaviruses, circoviruses, parvoviruses, and bornaviruses; Cui
et al. 2014). Interspersed repeats are either present in all (“+”), some (“”), or none (“–”) of the
genomes sampled from the respective lineage. Left part of figure modified from Kapusta and Suh
(2017), used with permission from John Wiley and Sons
contain the typical hairpin structure and 8 bp microsatellite motifs of the 30 UTR
(Fig. 4) (Suh 2015). Whether these CR1 structures affect nearby genes remains
unknown (Kapusta and Suh 2017).
In addition to the ubiquitous CR1 elements, most bird genome assemblies contain
very low copy numbers of R2 elements (Kojima et al. 2016), a peculiar LINE
superfamily widely distributed across animals and which exhibits site-specific
retrotransposition into 28S rRNA genes (Kojima and Fujiwara 2005). Finally,
some unrelated bird lineages (songbirds, some parrots, hornbills, trogons,
hummingbirds, mesites, and tinamous; Fig. 3) contain thousands of copies of the
AviRTE element from the RTE superfamily as a result of repeated horizontal
transfer between these birds and parasitic nematodes (Suh et al. 2016). The RTE
superfamily appears to be notorious for horizontal transfer in general, given that
another RTE element (BovB) “jumped” repeatedly between the genomes of some
mammals, reptiles, and ticks (Kordiš and Gubenšek 1998; Walsh et al. 2013).
2.1.2 SINE Retrotransposons

Short interspersed elements (SINEs) are parasites of LINEs. As the name implies,
they are too short for having protein-coding capacity and thus rely on the LINE
enzymatic machinery. SINEs are part of the small RNA transcriptome as their heads
(50 ends) are derived from small RNA genes (usually from tRNAs) and their internal
promoters allow high transcription levels (Wicker et al. 2007; Kapitonov and Jurka
2008). SINE tails (30 ends) are usually highly similar to the 30 UTRs of LINEs they
parasitize, which allows frequent retrotransposition of SINE RNAs by “hijacking”
the LINE RT protein (Ohshima et al. 1996). As a result, SINEs by far outnumber
LINEs in the human genome (~1,600,000 SINEs vs. ~900,000 LINEs; Lander et al.
2001).
In contrast, bird genomes have relatively low SINE copy numbers of
~6000–17,000 (vs. ~65,000–630,000 LINEs; Zhang et al. 2014). Most of these
SINEs belong to the ancient families MIR, AmnSINE, and LFSINE which were
mobilized long before the last common ancestor of birds (reviewed by Kapusta and
Suh 2017) and are present across land vertebrates (Hirakawa et al. 2009; Bejerano
et al. 2006; Nishihara et al. 2006; Green et al. 2014). At least some of these ancient
SINEs might be involved in 3D genome folding as previously shown for mammals
(Schmidt et al. 2012; Wang et al. 2015) and suggested by the high sequence
conservation of hundreds of orthologous SINE copies across birds (Craig et al.
2018).
There are, however, SINEs with recent activity in some avian lineages due to
recurrent de novo emergence (Fig. 3). CR1-mobilized SINEs emerged twice within
passerines and independently in pelicans and trogons (Suh et al. 2017; Kapusta and
Suh 2017; Warren et al. 2010) and contained tRNA-derived SINE heads and SINE
tails with the typical structural features of CR1 30 UTRs (Fig. 5) (Suh 2015).
Furthermore, RTE-mobilized SINEs emerged three times in suboscine passerines
and independently in some parrots and hornbills (Suh et al. 2016). These
RTE-SINEs have a peculiar diversity of SINE heads (e.g., derived from tRNA,
5S-rRNA, or 28S-rRNA genes; Suh et al. 2016) and tails derived from the 50 and 30
Fig. 5 Short interspersed elements (SINEs) of birds. Schematic organization of the CR1-SINE and
RTE-SINE superfamilies. Full-length elements exhibit a SINE head derived from a small RNA
(sRNA; yellow; usually a tRNA) and a SINE tail derived from the LINE element responsible for
SINE mobilization. In CR1-SINEs, the SINE tail is homologous to the CR1 30 UTR (red), and in
RTE-SINEs, it is homologous to the RTE 50 and 30 UTRs (blue and red). Corresponding to its
mobilizing LINE, the SINE tail ends with a characteristic microsatellite motif, and white squares are
target site duplications (“v” indicates variable lengths). Figure modified from Kapusta and Suh
(2017), used with permission from John Wiley and Sons
UTRs of AviRTE LINEs (Fig. 5). This bipartite organization of SINE tails seems to
be typical for RTE-mobilized SINEs (Gogolevsky et al. 2008; Kojima 2018).
2.1.3 LTR Retrotransposons

In contrast to LINEs and SINEs, often collectively termed “non-LTR
retrotransposons,” LTR retrotransposons mobilize via replicative retrotransposition,
a mechanism mediated by “long terminal repeats” (reviewed by, e.g., Kazazian
Jr. 2004; Levin and Moran 2011). LTRs are thus “repeats within a repeat”
(Fig. 6). Importantly, retroviruses belong to the group of LTR retrotransposons
(Wicker et al. 2007). Full-length LTR retrotransposons encode several protein
domains, including a reverse transcriptase and a capsid protein (Fig. 6). During
replicative retrotransposition, the LTR retrotransposon mRNA is first reverse-
transcribed within a virus-like particle in the cytoplasm, and the cDNA is transported
back into the nucleus for insertion into the genome (Levin and Moran 2011). In
contrast to TPRT which leads to frequent 50 -truncation of daughter LINEs and
SINEs, replicative retrotransposition yields full-length daughter LTR
retrotransposons (Levin and Moran 2011). Recently inserted LTR retrotransposons
therefore contain two LTRs which are identical to each other and often cause intra-
element ectopic recombination (Devos et al. 2002). This process deletes one of the
two LTRs and the internal protein-coding regions, thus resulting in a solo-LTR
within the original target site duplication (Fig. 6b). Solo-LTRs are unable to
retrotranspose; however, they contain promoters and may thus influence the tran-
scription of nearby genes (Kovalskaya et al. 2006; Chuong et al. 2017).
LTR retrotransposons are present in genome assemblies from all major avian
lineages (Fig. 3). Birds generally contain only retrovirus-like LTR retrotransposons
(endogenous retroviruses, ERVs), more precisely from the superfamilies ERV1,
ERVK, and ERVL (reviewed by Kapusta and Suh 2017; Suh et al. 2018). These
superfamilies can be distinguished by the specific lengths of their target site
duplications (Fig. 6) (Kapitonov and Jurka 2008), allowing even the classification
of solo-LTRs which by far outnumber full-length elements in birds (Wicker et al.
2005). On the other hand, the virological classification of ERVs is based on their
Fig. 6 Long terminal repeat (LTR) retrotransposons of birds. (a) Schematic organization of the
endogenous retrovirus (ERV) superfamilies ERV1, ERV2 (ERVK), and ERV3 (ERVL). Full-
length elements exhibit a pair of LTRs (green) and a coding region (gray) encoding a capsid protein
(GAG), an aspartic proteinase (AP), a reverse transcriptase (RT), an RNase H (RH), an integrase
(INT), and an envelope protein (ENV). White squares are target site duplications (numbers indicate
specific lengths). Figure modified from Kapusta and Suh (2017), used with permission from John
Wiley and Sons. (b) Schematic illustration of nonallelic homologous recombination (NAHR)
between the LTRs of a full-length LTR retrotransposon. This process deletes the internal coding
region and one of the LTRs, leaving behind a solitary LTR (solo-LTR)
protein-coding genes and suggests that bird genomes contain mostly

alpharetroviruses and gammaretroviruses (Hayward et al. 2015) (but see (Cui et al.
2014) reporting mostly betaretroviruses and gammaretroviruses). Total copy num-
bers of LTR retrotransposons are ~24,000–120,000 copies in 48 sampled genome
assemblies (Zhang et al. 2014), which suggests that some avian lineages witnessed
more LTR retrotransposon activity than others. Accordingly, analyses of
orthologous solo-LTRs for their presence/absence in bird genomes indicate abun-
dant and diverse activity of LTR retrotransposons, especially during the neoavian
radiation (Fig. 3) (Suh et al. 2011b, 2015b). Nevertheless, the highest LTR
retrotransposon activity so far reported from birds occurred relatively recently in
the genomes of songbirds (reviewed by Kapusta and Suh 2017). A comparison of the
three available in-depth, manually curated repeat annotations for birds (chicken,
zebra finch, and collared flycatcher) (Warren et al. 2010; Hillier et al. 2004; Suh et al.
2018) suggests that LTR retrotransposons are not only more abundant in songbirds
than in chicken but also more diverse in terms of the presence of distinct LTR
retrotransposon families and subfamilies (Suh et al. 2018). Most of these LTR
subfamilies are lineage-specific, i.e., existing in either zebra finch (175 subfamilies
Fig. 7 DNA transposons of birds. Schematic organization of the mariner and hAT superfamilies.
Full-length elements exhibit a pair of terminal inverted repeats (TIRs; purple) and encode a DDE
transposase. Nonautonomous (NA) elements lack transposase-coding capacity. White squares are
target site duplications (letters indicate specific motifs and numbers indicate specific lengths).
Figure modified from Kapusta and Suh (2017), used with permission from John Wiley and Sons
from 17 LTR families) or collared flycatcher (33 subfamilies from 14 LTR families),
which implies frequent germline invasions of songbirds by a variety of retroviruses.
Strikingly, this songbird LTR diversification coincides with a recent replacement of
LINE activity by LTR activity, which occurred independently in the collared
flycatcher and zebra finch lineages (Suh et al. 2018). In-depth repeat annotations
of further songbird genomes are necessary to establish the timeframe and extent of
co-speciation between retroviruses and the species-rich songbirds.
2.2 Class II Transposable Elements
2.2.1 DNA Transposons

DNA transposons usually move from one genomic locus to another through a cut-
and-paste mechanism catalyzed by their own transposase proteins (Fig. 7). A dimer
of this transposase recognizes the terminal inverted repeats (TIRs), excises the DNA
transposon at its termini, and reinserts the transposon DNA elsewhere (Fig. 2;
reviewed by, e.g., Kidwell 2005; Feschotte and Pritham 2007; Levin and Moran
2011). Some DNA transposons lack coding capacity for a transposase and are thus
nonautonomous (Feschotte and Pritham 2007). The only structural hallmarks present
in all DNA transposons are TIRs and target site duplications (TSDs) with specific
lengths and motifs (Fig. 7) (Wicker et al. 2007).
Bird genomes generally contain very low amounts of DNA transposons. Most of
these are Charlie7 elements from the hAT superfamily and accumulated during early
bird evolution, before the divergence of chicken and zebra finch (Warren et al. 2010;
Kapusta and Suh 2017). Then again, the chicken genome contains over 16,000
copies of relatively recently inserted DNA transposons, namely, Galluhop from the
mariner superfamily and Charlie12 from the hAT superfamily (Wicker et al. 2005).
These appear to have been active before the divergence of chicken and turkey
(Kapusta and Suh 2017). Very few chicken DNA transposon copies are
transposase-encoding elements; most are short nonautonomous elements (Fig. 7)
(Wicker et al. 2005). Recent evidence further suggests the presence of Galluhop
transposons in other Galliformes and a horizontal transfer event to hornbills (Fig. 3)
(Bertocchi et al. 2017).
2.2.2 Other Transposons

There are two subclasses among class II DNA transposons which employ different
mobilization mechanisms than the aforementioned cut-and-paste DNA transposons
(Wicker et al. 2007). These subclasses are Helitrons which copy and paste by rolling-
circle replication (Kapitonov and Jurka 2007) and Mavericks (also known as
Polintons) which copy and paste by self-replication (Kapitonov and Jurka 2006;
Feschotte and Pritham 2005). Both mechanisms differ from class I retrotransposons
in that they do not involve an RNA intermediate. Although discovered much more
recently than cut-and-paste DNA transposons, Helitrons and Mavericks are widely
distributed across eukaryotes including various vertebrates (Feschotte and Pritham
2007; Thomas et al. 2010; Pritham et al. 2007). To our knowledge, none of these
peculiar DNA transposons have yet been reported from bird genomes.
2.3 Endogenous Viruses
Endogenous viral elements (EVEs) are genomic “fossils” of virus infections of the
germline and have since been integrated in the genome. Thus far, EVEs from a wide
range of virus groups have been unearthed (reviewed by, e.g., Feschotte and Gilbert
2012). The most common and widespread EVEs in birds are retroviruses (Cui et al.
2014) which are reverse-transcribing single-strand RNA (ssRNA) viruses
(Retroviridae) with obligate integration into the host genome through retro-
transposition (Feschotte and Gilbert 2012). Retroviruses can also be classified as
LTR retrotransposons (see Sect. 2.1.3). On the other hand, although some
non-retroviral EVEs may occasionally integrate into host genomes with the help of
TE-encoded enzymes, many insertions lack typical hallmarks of (retro)transposition
such as target site duplications (Fig. 2a) (Feschotte and Gilbert 2012). A plausible
explanation is that most of the non-retroviral EVEs integrated through nonhomo-
logous end joining (Fig. 2c), an error-prone pathway of the host genome to repair
DNA double-strand breaks (Feschotte and Gilbert 2012). The diversity of
non-retroviral EVEs identified in birds comprises ssDNA viruses (Circoviridae,
Parvoviridae), ssRNA viruses (Bornaviridae), and reverse-transcribing dsDNA
viruses (Hepadnaviridae) (Cui et al. 2014), all of which are present in low copy
numbers. The reasons for the avian scarcity of non-retroviral EVEs remain debated
(Kapusta and Suh 2017).
The EVEs of circoviruses, parvoviruses, and bornaviruses have very patchy

distributions across the avian tree of life (Fig. 3) as they each were detected in
only 3–10 of the sampled 48 bird genomes (Cui et al. 2014). In contrast, EVEs of
hepadnaviruses have been detected in all analyzed bird genomes to the exception of
chicken and turkey (Suh et al. 2013; Cui and Holmes 2012; Cui et al. 2014; Liu et al.
2012a; Gilbert and Feschotte 2010; Katzourakis and Gifford 2010). While it remains
unclear for most EVEs whether they are present in closely related species due to
either orthologous or independent germline infiltration, presence/absence analyses
of some hepadnaviral EVEs have provided direct evidence that birds have been
infected by hepadnaviruses for >70 My since at least the ancestor of Neoaves (Suh
et al. 2013). This duration of hepadnavirus–bird association is the longest so far
known from their amniote hosts (Suh et al. 2014).
3 Tandem Repeats
Tandem repeats are repetitive elements which are adjacent to each other and thereby
form repeat arrays. These can emerge and proliferate through a variety of
mechanisms (Fig. 8). Below we discuss different types of tandem repeats in ascend-
ing order of repetitive element size—ranging from microsatellites, minisatellites, and
satellites to gene families and copy number variations.
3.1 Microsatellites and Minisatellites
Microsatellites are short DNA motifs (<10 bp) which occur in arrays that typically
do not extend further than a few hundred base pairs (Ellegren 2004). They are
ubiquitous in eukaryote genomes and scattered along chromosomes in numerous
places. Predominantly nonfunctional and thus under little selective constraint
(exceptions include regulation of chromatin organization and gene expression, Li
et al. 2002), microsatellites are among the fastest evolving sequences with mutations
arising through errors in the DNA replication process. The so-called replication
slippage (Fig. 8a), which is increased with the length of the microsatellite array,
causes alleles mainly to differ by copy number rather than single-nucleotide changes
(Ellegren 2004; Schlötterer and Tautz 1992). The resulting high number of alleles
accounts for the extremely high variation in microsatellite loci, compared to the
genome-wide average, thus facilitating a wealth of genetic analyses. Among the
most important is the genotyping in paternity analysis, which allows the correct
assignment of biological parents to a given offspring. Building on that,
microsatellites have been used early on for genetic mapping in pedigree studies, to
assess genetic diversity and structure in population genetic studies (e.g., Crooijmans
et al. 1996; Hansson et al. 2000), and more recently for population monitoring based
on environmental samples and wildlife forensics (De Barba et al. 2016; Jan and
Fumagalli 2016).
Fig. 8 Mechanisms of tandem repeat array proliferation. (a) Replication slippage is based on the
inaccuracy of the DNA polymerase. When replicating a tandem repeat array, it sometimes jumps
back a few base pairs and repeatedly replicates some nucleotides, leading to a repeat expansion. If
the array forms a hairpin loop, the polymerase might replicate only the bases connecting the hairpin,
resulting in a repeat contraction. (b) Gene conversion occurs if a double-strand break during meiosis
is not resolved with a crossing-over but with the total replacement of one allele in the homologous
sequence, resulting in a homogenization of alleles. This can also happen via ectopic recombination
including paralogous sequences. (c) Rolling-circle replication has thus far only rarely been shown in
relation to satellite DNA dynamics. After hairpin loop formation, the sequence—often one repeat
unit—circularizes and acts as a template for DNA replication. (d) In unequal crossing-over, high
sequence identity—typically the case in tandem repeat arrays—leads to imprecise pairing of
homologous chromosomes. After resolution of the crossing-over, half of the meiotic products are
imbalanced, with either deleted or duplicated sequence
Although occurring in a lower density than in, for example, humans (Primmer
et al. 1997; Guizard et al. 2016), a plethora of avian microsatellite loci and the
corresponding PCR primers have been described for many different species and
taxonomic groups. Given that the applicability of a certain locus for cross-species
amplification is usually constrained by the divergence between two species (Prim-
mer et al. 2005), there are only a few “universal” loci which can be used for a wide
range of species. With the emergence of readily available high-throughput sequenc-
ing and its wealth of sequence data, microsatellites were thought to become obsolete
(Schlötterer 2004). However, in cases where cost-efficiency, number of individuals,

or DNA quality plays a role (e.g., wildlife monitoring, behavioral studies),
microsatellites are still the method of choice (but see Kraus et al. 2015). Addition-
ally, the discovery of suitable loci has become much easier since many genome
assemblies have become available. By aligning genome assemblies of target species
to each other, conserved loci can be found (Küpper et al. 2008; Dawson et al. 2010,
2013), thus providing a valuable complement to traditional microsatellite studies.
Additionally, high-throughput sequencing technologies enable the genome-wide
investigation of microsatellite evolution, an area of research which is, due to
technological and analytical constraints (Fig. 1), only in its early stage of develop-
ment (Gymrek 2017).
Minisatellites are tandem repeat arrays with unit sizes longer than 10 bp. They
evolve via similar mechanisms as microsatellites, but they occur far less frequently
and are presumably more relevant to gene function (López-Flores and Garrido-
Ramos 2012). Although there are a few examples of minisatellite loci characterized
in birds (e.g., Hanotte et al. 1991, 1992), the much higher abundance of microsatel-
lite loci has led to a quick replacement of minisatellites for the abovementioned
applications (López-Flores and Garrido-Ramos 2012).
3.2 Satellites
Satellites are generally defined as repetitive DNA which occurs in tandem repeat
arrays with repeat units between 200 and several thousand base pairs, although
differences in the size limit vary and also depend on the organism studied (López-
Flores and Garrido-Ramos 2012; George and Alani 2012). The term “satellite DNA”
originated in the description of a density gradient in DNA centrifugation analysis
and refers to one or several separate bands seen, like satellites orbiting a planet. The
reason for this lies in the fact that satellite repeat sequences often deviate from an
equilibrium base composition (i.e., A, C, T, and G in roughly equal numbers), such
that the resulting density also changes compared to the genome-wide average. If the
satellite repeat then occurs in a high copy number, a discrete band will be seen in the
density gradient (Plohl et al. 2012). Another common technique to detect satellite
DNA is to digest genomic DNA with a restriction enzyme and fractionate bands via
centrifugation in cesium chloride (Manuelidis 1977). To determine the smallest
repeat unit, multiple enzymes are used sequentially with a size standard on the gel,
combined with cloning and sequencing of the band (Madsen et al. 1992). Once the
sequence of a repeat is known, it can be used for fluorescence in situ hybridization to
determine its location along the genome and associate it with certain chromosomal
features, such as the centromere (e.g., Longmire et al. 1988).
Satellites are an abundant source of repetitive DNA and seem to play an important
role in maintaining genome integrity and organization as a main element of consti-
tutive heterochromatin (Grewal and Jia 2007; Peng and Karpen 2008; George and
Alani 2012). These regions of the chromosome, which are usually associated with
repetitive sequences, are densely packed through histone modifications throughout
the stages of mitosis and meiosis. Although mainly distributed in or near

centromeres and near telomeres of most chromosomes, satellites also occur in
heterochromatic fractions of sex chromosomes (López-Flores and Garrido-Ramos
2012) and, in shorter arrays, scattered along various chromosomes (Saksouk et al.
2015).
Satellite arrays can expand and contract via a range of different mechanisms
(Fig. 8b–d). In gene conversion, a double-strand break during meiosis is followed by
the invasion of one strand and ultimately resolved by the complete replacement of
one allele, thus homogenizing the tandem repeat array (Chen et al. 2007, Fig. 8b).
Rolling-circle replication facilitates a circularized stretch of DNA (e.g., one unit of a
satellite) which replicates and inserts itself into the tandem array (Rossi et al. 1990,
Fig. 8c). Finally, unequal crossing-over is another mechanism facilitating the expan-
sion and contraction of tandem repeat arrays. Hereby an imprecise pairing of
homologous sequences during meiosis leads to unbalanced chromosomes after
meiosis (Graur and Li 2000, Fig. 8d). Additionally, Bersani et al. (2015) have
shown that satellites can proliferate via RNA–DNA hybrid molecules, although
this seems to be restricted to mitotic cancer cells.
Despite avian genomes being comparatively repeat-poor (reviewed by Kapusta
and Suh 2016), cytogenetic data suggest that satellites make up a considerable
proportion of the total amount of DNA of avian genomes (e.g., a 190 bp satellite
accounts for up to 6.8% of the entire genome of some parrot species, Madsen et al.
1992). Avian satellites have been extensively studied using cytogenetic and molec-
ular genetic methods, and a variety of different satellite families have been described
(e.g., Kodama et al. 1987; Matzke et al. 1990, 1992; Wang et al. 2002). Strikingly
though, there are very few examples of comprehensive genome-wide avian satellite
studies. This might partly stem from the fact that satellites, or rather entire arrays of
satellites, are notoriously difficult to assemble in a linear fashion due to reasons
mentioned above (see Sect. 1). One exception is the comprehensive characterization
of repetitive DNA in the previous chicken genome assembly (Guizard et al. 2016). In
this study, 0.24% of the genome assembly could be attributed to satellite DNA arrays
with more than 50 copies and unit sizes ranging from 60 bp to 2 kb. Furthermore, a
14 kb satellite with a 1.4 kb subunit has been identified in crows via optical mapping,
long-read sequencing, and manual sequence assembly curation and shown to poten-
tially be a component of centromeric and subtelomeric heterochromatin
(Weissensteiner et al. 2017). This satellite forms large-scale tandem repeat arrays
(considerably longer than 100 kb) which were anchored into the crow genome
assembly by the combination of different approaches and were shown to influence
population genetic parameters. In regions adjacent to these long tandem repeat
arrays, genetic diversity is drastically reduced, which also leads to increased genetic
differentiation between hooded and carrion crow populations. This study illustrates
the necessity of considering the location of structural genomic features (such as the
presence of large tandem repeat arrays) for downstream genomic analyses, in order
to complement conclusions drawn from single-nucleotide polymorphism data.
3.3 Gene Families and Copy Number Variation
Gene families represent genetic elements which originated by a duplication event of

a gene and are arranged either in a tandem array or spread out as interspersed copies
(López-Flores and Garrido-Ramos 2012). Depending on the age of the duplication
event, these paralogs can be quite diverged and can both lose their function or adopt
new ones. The number of paralogs can range between less than 10 and several
hundred (López-Flores and Garrido-Ramos 2012). Among the most notable avian
examples is the beta (β) keratin multigene family. In chicken, 111 complete β-keratin
paralogs have been identified, spread out on six different chromosomes (Greenwold
and Sawyer 2010). The diversification in and within several subfamilies of this
multigene family via recombination, deletion, and duplication events has led to the
subsequent evolution of claws and beaks from the scale subfamily and further the
feather and feather-like subfamily. Furthermore, the first large-scale sequencing
efforts targeting 48 bird species spanning all major avian clades has revealed a
higher number of opsin genes than in mammalian genomes and likewise found a
relationship between avian tetrachromatic vision and the presence of two to three
conopsin genes (Zhang et al. 2014). Another example for how new technologies are
aiding the discovery and characterization of gene families in non-model organisms is
provided in Larsen et al. (2014). They used long-read sequencing to investigate the
vomeronasal gene receptors (a highly diverse gene family in mammals) and
concluded that previous assemblies have underestimated the diversity of this com-
plex region and that long-read sequencing will greatly improve characterization of
gene families in other non-model organisms.
Copy number variation describes the differences in number of a certain chromo-
somal segment (e.g., a gene) between any two individuals. CNVs have been
extensively studied in birds, particularly in the field of poultry genetics. The majority
of studies have used hybridization arrays to detect CNVs, but also SNP-chips and
whole-genome sequencing have been applied (Wang and Byers 2014). It seems that
CNVs are an abundant source of genetic variation in chicken, with a total of 3961
reported CNVs contained in 1876 CNV regions (Wang and Byers 2014). This
accounts for 8.3% and 9.6% of the chicken genome and the ordered assembly,
respectively. In a study involving 16 species of birds, Skinner et al. (2014) found
790 cross-species CNVs in 135 unique regions, which refutes the previous assump-
tion that CNVs affect smaller regions and occur less frequent in birds than in
mammals. They supported the notion that microchromosomes exhibit more CNVs
than macrochromosomes, a pattern that interestingly is still observed in derived
macrochromosomes consisting of fused ancestral microchromosomes. This suggests
that not size but rather sequence-related features of microchromosomes are respon-
sible for increased meiotic recombination, leading to a higher frequency of CNVs.
Furthermore, they showed that 70% of detected CNVs contain genes, implying
functional importance (Skinner et al. 2014).
CNVs have been shown to have direct consequences for the phenotype of the
carrier; striking examples in chicken are the silkie phenotype (hyperpigmentation of
skin and connective tissue), which is caused by an inverted duplication and junction
of genomic regions more than 400 kb apart (Dorshorst et al. 2011), and the pea comb
phenotype (reduced comb and wattles), which can be traced back to a massive
expansion of a duplicated intronic sequence of the SOX5 transcription factor (Wright
et al. 2009; Vignal and Eory 2019). Clearly, the evolution of copy number variation
is highly dynamic with a massive effect on the phenotype, likely much more so than
genetic variation in single nucleotides (Wang and Byers 2014).
4 Chromosomal Distribution of Genomic “Dark Matter”
Nearly all bird species possess karyotypes with several large macrochromosomes
and numerous small microchromosomes (reviewed by Kapusta and Suh 2017;
Organ et al. 2008; Burt 2002; Damas et al. 2019; Griffin et al. 2019). Irrespective
of their size, eukaryotic chromosomes are generally characterized by the presence of
a centromere and two telomeres, typically highly repetitive regions with important
functions (reviewed by Mekhail and Moazed 2010) and often tied to local suppres-
sion of recombination (reviewed by George and Alani 2012). This together with the
assumption that one meiotic crossover is obligate per chromosome explains that the
meiotic recombination landscape is highly heterogeneous in birds, with
low-recombination regions near putative centromeres and generally higher recombi-
nation rates on smaller chromosomes (Kawakami et al. 2014; Backström et al. 2010;
Stapley et al. 2010). As increased levels of meiotic recombination increase the
efficacy of selection in purging slightly deleterious mutations (Ellegren and Galtier
2016), it is not too surprising that microchromosomes are generally less repetitive
than macrochromosomes (Fig. 9). Microchromosomes also have high gene densities
(Ellegren 2010; Organ and Edwards 2011; Burt 2002), rendering mutations such as
expansions of repetitive elements effectively deleterious in many sequence contexts.
On the other hand, the Z and W sex chromosomes have high and very high repeat
densities, respectively (Fig. 9), which correspond to their levels of meiotic recombi-
nation. Except for the pseudoautosomal region (PAR), the Z chromosome
recombines only in males, and the W chromosome is non-recombining and
female-specific (Smeds et al. 2014). Additionally, many microchromosomes and
macrochromosomes contain highly repetitive regions with repeat densities of up to
~50% near centromere-associated assembly gaps (Fig. 9).
Most of these repeat-rich regions only became visible in the newest chicken
galGal5 assembly, the only available chromosome-level bird genome assembly
based on long-read sequencing (Warren et al. 2017). This extends the observation
from Sanger and short-read sequencing that avian genomes are repeat-poor and
evolutionarily stable, i.e., show very few chromosomal rearrangements (Ellegren
2010). Kapusta and Suh (2017) recently noted that such a compartmentalization of
avian genomes into repeat-poor/gene-rich/stable regions and repeat-rich/gene-poor/
instable regions (Fig. 9) is reminiscent of the “two-speed genome” model in plant-
pathogenic fungi (Raffaele and Kamoun 2012). In the following, we discuss those
genomic regions which are particularly enriched in repetitive elements and/or
constitute genomic “dark matter.” These are either parts of chromosomes such as
Fig. 9 The heterogeneous landscape of repetitive elements and assembly gaps in the third-
generation chicken genome. Selected chromosomes are shown from a figure from Kapusta and
Suh (2017), used with permission from John Wiley and Sons
centromeres and telomeres or entire chromosomes such as the female-specific W

chromosome, the supernumerary B chromosome, and the immune gene-rich chro-
mosome 16.
4.1 Distribution Within Chromosomes
4.1.1 Centromeres
Centromeres are structural chromosomal features which ensure the faithful segrega-
tion of chromosomes during meiosis and mitosis (Graur and Li 2000). They do so by
providing the substrate for spindle fiber attachment which is responsible for the
poleward migration of chromosomes, a process essential for proper cell division.
Fig. 10 Shifted centromere

positions between chicken and
Japanese quail. Fluorescent in
situ hybridization was used to
compare centromere positions
in giant lampbrush
chromosomes. In both
orthologous chromosomes
14 and 15, the position of the
centromere differs due to de
novo centromere formation
and/or accumulation of
heterochromatin. Figure from
Zlotina et al. (2012), used with
permission from Springer
Nature
Nonfunctional or surplus centromeres result in cellular failure or inviable gametes

(Thompson et al. 2010). Although a crucial and ubiquitous feature of chromosomes,
the centromere has no clear definition from a DNA point of view. The general rule is
that the region at which centromeric proteins attach during meiosis exhibits at least
some repetitive DNA sequences, but most studies have shown that functional
centromeres are rather epigenetically determined and can form at any given location
on a chromosome (Plohl et al. 2014).
In chicken, the units of centromere-associated tandem repeat arrays are
chromosome-specific, but on three chromosomes (5, 27 and Z) the centromere is
short (~30 kb) and organized in a non-tandem repetitive fashion (Shang et al. 2010).
Melters et al. (2013) have assessed the most abundant tandem repeats in 282 species
of plants and animals and argued that those likely constitute centromeric repeats.
Among the four species of birds investigated in that study (chicken, mallard, zebra
finch, and gray partridge), repeat unit lengths ranged from 42 to 191 bp and GC
content from 38 to 56%, suggesting a highly dynamic evolution of centromere DNA
and very few general rules regarding size or sequence composition. It seems likely
that heterochromatic tandem repeat arrays also play a role in the overall positioning
of centromeres. For example, in quail (Fig. 10), despite evident chromosomal
inversions compared to chicken, centromere positions are shifted due to the forma-
tion of new centromeres and subsequent accumulation of centromeric tandem
repeats (Zlotina et al. 2012).
It is important to note that there is currently a conspicuous gap between results
from classical cytogenetic studies (e.g., mentioned in the previous paragraph) and
Assembled sequence Repetitive anchored map (RAM)

SR scaffold_100
Carrion Hooded crow
LR contig_000233F
OM
OM
25 kb
Fig. 11 Anchoring large, presumably heterochromatic tandem repeat arrays in the hooded crow
genome assembly. Using optical mapping, large-scale tandem repeat arrays (“repetitive anchored
maps” or RAMs) can be localized in short-read and long-read genome assemblies. Vertical lines in
boxes denote recognition motifs of a nicking endonuclease which can be visualized as a specific
“barcode”-like pattern. Note that the tandem repeat array is entirely absent in the short-read
assembly (dark green) and only partly assembled in the long-read assembly (light green), whereas
the two optical map assemblies (blue) are capable of visualizing the tandem repeat array to a much
larger extent. This is because optical mapping uses much longer (>150 kb) single DNA molecules
as input, thus containing much more long-range information. Figure from Weissensteiner et al.
(2017), used under CC BY-NC 4.0 license
more recent whole-genome sequencing efforts. Although in principle present in

high-throughput sequencing data sets, centromeric repeats are usually not assembled
(cf. Fig. 1), and thus the positions of centromeres are not included in genome
assemblies of non-model organisms. This is not surprising given the highly repeti-
tive nature of these regions, but certainly not desirable, since centromeres play a
fundamental role in genome biology and their positions thus should be incorporated
in downstream analyses. A promising development is the emergence of new
technologies which facilitate the long-range information from single DNA
molecules, e.g., long-read sequencing (Eid et al. 2009) or nanochannel optical
mapping (Lam et al. 2012). Only recently, it has become possible to assemble the
linear sequence of one entire centromere in humans (Jain et al. 2018), a task which
has yet to be completed in birds. However, it is at least possible to anchor large
potentially centromeric tandem repeat arrays into genome assemblies through optical
mapping (Fig. 11) (Weissensteiner et al. 2017).
4.1.2 Telomeres
Like centromeres, telomeres represent crucial structural chromosomal features in
eukaryotic genomes. Their main purpose is to protect ends of chromosomes from
deleterious alterations via enzymes or to prevent breakage and fusion with other
chromosomes during replication in mitosis and meiosis (Blackburn et al. 2006).
While centromeres show diverse organizations with DNA sequences differing even
between chromosomes of the same species (Plohl et al. 2014), the general structure
of telomeres is extremely conserved among eukaryotes. The basic feature is a
microsatellite of six nucleotides “TTAGGG,” which is organized in a tandem repeat
array with different lengths and associated with specific proteins (TRFs, “telomere
repeat binding factors”) (Bilaud et al. 1997). A striking feature of the telomere is that
the length of the array is species-, individual-, chromosome-, and age-specific,
meaning that it is one of the most dynamic regions of the genome (Monaghan
2010). Due to the nature of DNA replication, the array gets shortened in each
replication cycle, leading to those differences in telomere array length depending
on age. Telomerase, a ribonucleoprotein which carries its own RNA molecule,
elongates shortened telomere arrays after replication via reverse transcription and
thus acts against this telomere shortening (Blackburn et al. 2006). Interestingly, the
rate of the shortening is variable even within a species and also depends on
environmental conditions. For example, Asghar et al. (2015) found that great reed
warblers chronically infected with avian malaria have on average shorter telomeres
and also produce offspring with shorter telomeres in early life. Thus, it seems that
differences in fitness depending on environmental conditions are mediated by
differential telomere degradation and elongation.
Overall, birds have a high variation in telomere array length, with the largest
individual arrays of any vertebrate found in chicken (up to 2 Mb, Delany et al. 2003;
Monaghan 2010). Also, the content of telomeric sequence proportional to the entire
genome is ten times larger in chicken compared to human. The latter might stem
from the fact that in birds there is abundant occurrence of “TTAGGG” repeats far
away from chromosome ends, and some (micro)chromosomes even show telomere
arrays larger than the rest of the chromosome (Nanda et al. 2002). Furthermore,
mega-telomere arrays are present on four chicken chromosomes (9, 16, 28, and W)
in which three of them account for an entire chromosome arm (Delany et al. 2007).
Despite the development of computational tools for estimating telomere array length
from high-throughput sequencing (Ding et al. 2014; Nersisyan and Arakelyan 2015),
to date there is no study on an avian system adopting this approach.
4.2 Distribution Between Chromosomes
4.2.1 W Chromosomes
Birds have Z and W sex chromosomes which evolved from an ancestral pair of
autosomes (Fridolfsson et al. 1998). The female-specific W chromosome is
non-recombining except for the pseudoautosomal region (PAR), which is identical
to the Z, and normally pairing and recombining during meiosis (Smeds et al. 2014).
Similar to the XY sex chromosomes of mammals, ZW sex chromosome differentia-
tion has been commonly explained via recombination suppression through
inversions (Charlesworth and Charlesworth 2000) and is generally followed by
degeneration of the non-recombining sex chromosome (W or Y) through accumula-
tion of repetitive elements (reviewed by, e.g., Charlesworth et al. 2005; Mank 2012;
Abbott et al. 2017). However, although the sex chromosomes of Neognathae
(Neoaves + Galloanserae; Fig. 3) and tinamous are visibly differentiated (“hetero-
morphic”) (Mank and Ellegren 2007), ratites such as ostrich and emu exhibit largely
undifferentiated (“homomorphic”) sex chromosomes (Vicoso et al. 2013; Zhou et al.
2014; Yazdi and Ellegren 2014). Accordingly, genomic approaches suggest that the
ZW-recombining PAR of ratites spans two thirds of the sex chromosome pair
(Vicoso et al. 2013; Zhou et al. 2014), in contrast to neognaths and tinamous
where the PAR is very small (except for the tropic bird, Zhou et al. 2014). For
example, the PAR is only 630 kb in collared flycatcher and contains only 22 protein-
coding genes (Smeds et al. 2014). To our knowledge, the smallest avian PAR is that
of the chicken as it contains no genes but only telomeric and subtelomeric repeats
(Bellott et al. 2017).
Why the sex chromosomes of most birds are highly differentiated and others have
largely undifferentiated sex chromosomes is a question yet to be answered (Wright
et al. 2016). What is known is that sex chromosome differentiation happened in a
stepwise manner throughout avian evolution, as suggested by the presence of three
or four evolutionary strata on the Z of neognaths and tinamous (Zhou et al. 2014;
Suh et al. 2011a; Smeds et al. 2015; Nam and Ellegren 2008) and two evolutionary
strata on the Z of ratites (Vicoso et al. 2013; Zhou et al. 2014). Consequently, sex
chromosome differentiation into multiple evolutionary strata happened indepen-
dently not only in neognaths and tinamous (Mank and Ellegren 2007) but also
within neognaths throughout the diversification of Neoaves and Galloanserae
(Stiglec et al. 2007; Zhou et al. 2014). This evidence from avian genomics extends
earlier cytogenetic observations that avian W chromosomes did not gradually
become smaller over evolutionary time but are rather highly variable in size even
over short timescales, probably due to repeat expansions or contractions (Rutkowska
et al. 2012). This is exemplified by W size extremes among closely related
songbirds. The Z chromosome is generally very stable in size across the avian tree
of life, and the ZW size ratio is on average 1.9 in birds, with a range from 0.8 in the
crimson finch Neochmia phaeton to 4.4 in the common tailorbird Orthotomus
sutorius (Rutkowska et al. 2012). Interestingly, Rutkowska et al. (2012) further
noted that birds with smaller genomes have smaller W chromosomes, which is in
line with the observation by Kapusta et al. (2017) that birds with smaller genomes
have more TE activity and higher genome shrinking at the same time (see Sect. 5.1).
We therefore suggest that a combination of different degrees of differentiation
(leading to W shrinkage) and different W repeat expansions (leading to W growth)
were at play during avian evolution. The exact mechanisms have yet to be
elucidated, but it is almost certain that repetitive elements played a significant role
in sex chromosome differentiation.
As mentioned above (see Sect. 4), the W chromosome is by far the most repetitive
chicken chromosome (Fig. 9) and thus notoriously difficult to assemble, although
some other chicken chromosomes remain largely or entirely unassembled (Kapusta
and Suh 2017; Warren et al. 2017). Analyses of the W repeat content are so far
limited to two bird species, namely, the long-read chromosome-level assembly of
chicken (Fig. 9) (Bellott et al. 2017) and the short-read scaffold-level assembly of
collared flycatcher (Smeds et al. 2015). In both cases, the assembled sequence
contains TE densities of over 50%, with an over eightfold enrichment in LTR
retrotransposons compared to the autosomes (cf. Fig. 9). The W chromosome can
thus be considered as a “refugium” for retrovirus-like elements (Kapusta and Suh
2017). To our knowledge, chicken and collared flycatcher are the only W assemblies
so far analyzed with in-depth repeat annotations, because such annotations are so far
restricted to chicken, zebra finch, and collared flycatcher (reviewed by Kapusta and
Suh 2017; Suh et al. 2018). Although assemblies of W-chromosomal sequence are
Fig. 12 The complex repetitive structure of the chicken W chromosome. The W contains seven
cytogenetically distinct regions (“chromomeres”), of which the three euchromatic chromomeres
(yellow and blue) are mostly assembled by now (cf. Fig. 9) (Bellott et al. 2017). Figure modified
from Bellott et al. (2017), used with permission from Springer Nature
available for 17 additional bird species (Zhou et al. 2014; Davis et al. 2010), none of
these have been annotated for lineage-specific repetitive elements, and repeat
densities would thus likely remain vastly underestimated, especially for LTRs
What about other types of repetitive elements on the W? In contrast to mammals
with many gene-containing regions on the Y chromosome which are either tandemly
repeated with several copies next to each other (“ampliconic”) or present as inverted
copies (“palindromes”) (Skaletsky et al. 2003; Soh et al. 2014; Bellott et al. 2014;
Hughes et al. 2010; Bachtrog 2013), the only known ampliconic gene on the avian
W chromosome is HINTW, with ~40 copies of a 5 kb repeat unit in chicken (Bellott
et al. 2017) and high between-copy sequence similarity (Backström et al. 2005).
Further signatures of gene conversion have been detected in a 22 kb palindrome on
the W chromosome of the white-throated sparrows (Davis et al. 2010). Together,
these ampliconic and palindromic structures permit gene conversion on an otherwise
non-recombining chromosome, which leads to decreased degeneration and increased
potential for adaptation of the genes involved (Betrán et al. 2012).
To conclude this section, we wondered: How much genomic “dark matter” is
there on the W? Most suitable for this thought experiment was the chicken W
chromosome of which ~7 Mb of euchromatic sequence have been recently assem-
bled (Fig. 12) (Bellott et al. 2017). We used RepeatMasker (Smit et al. 1996–2010)
to annotate this assembly and determined a total repeat density of ~73%. Notably,
14% of the assembled sequence are CR1 retrotransposons, and 57% are LTR
retrotransposons. Assuming a chicken ZW size ratio of 2.14 (Rutkowska et al.
2012) and a Z chromosome size of >82 Mb (Warren et al. 2017), the “true” W
size should be >38 Mb, which means that >31 Mb (or >82%) of the W are currently
constituting genomic “dark matter.” Bellott et al. (2017) suggested that large parts of
the chicken W chromosome are heterochromatic, each containing tandem arrays of
one of three specific satellite repeats, the 0.5 kb repeat SspI (Itoh and Mizuno 2002),
the 1.1 kb repeat XhoI, and the 1.2 kb repeat EcoRI (Saitoh and Mizuno 1992;
Solovei et al. 1998). We therefore extrapolated that the currently missing W
sequence consists entirely of these satellite repeat arrays and telomeric tandem repeat
arrays, which would yield a “true” total repeat density of ~95%. More precisely, the
chicken W chromosome would then consist of ~82% tandem repeats, ~10% LTRs,
~3% CR1s, and only ~5% non-repetitive sequence. What is the W chromosome,
really—a sex-specific chromosome or a refugium for repetitive elements?
4.2.2 B Chromosomes
Contrary to the widespread assumption that all cells or all individuals of a species
contain essentially the same set of chromosomes, it has been known for nearly a
century that organisms may have “supernumerary” B chromosomes in addition to
the “standard” A chromosomes (Randolph 1928). These B chromosomes contain
large amounts of repetitive DNA, are considered to persist as genomic parasites
through meiotic drive, and have been reported from over 500 animal species
(reviewed in (Camacho 2005; Burt and Trivers 2006). For eukaryotes, Camacho
(2005) estimated that approximately 15% of all karyotyped species exhibit B
chromosomes. In the classical definition, B chromosomes are present in only some
individuals of a species and vary in population frequency over time (Camacho
2005). There are, however, rare cases where B chromosomes are stably inherited
within species but consistently eliminated from somatic tissues. These are germline-
restricted chromosomes (GRCs) (reviewed in Wang and Davis 2014). Such a cell-
type-specific B chromosome “maintains its evolutionary viability by preserving its
vertical transmission, but can prevent the most harmful effects from manifesting in
the host’s soma” (Camacho 2005).
To date, B chromosomes have been cytogenetically studied in great detail in two
bird species from the Estrildidae family, zebra finch (Pigozzi and Solari 1998) and
Bengalese finch (del Priore and Pigozzi 2014). In both cases, the B chromosome is
maintained in the germline but eliminated from pre-somatic cells during embryo-
genesis. Notably, these GRCs are the only known cases within vertebrates apart from
those of hagfishes and lampreys, which shared a common ancestor with birds ~550
million years ago (Smith 2017). The GRCs of zebra finch and Bengalese finch are
stably inherited through the female germline where they are present in two euchro-
matic and recombining copies and are present in the male germline as a single
heterochromatic chromosome which is eliminated from spermatocytes during meio-
sis (del Priore and Pigozzi 2014; Itoh et al. 2009; Goday and Pigozzi 2010;
Schoenmakers et al. 2010; Pigozzi and Solari 2005). It has further been noted that
the zebra finch GRC is silenced in spermatocytes similar to the W chromosome in
oocytes (Schoenmakers et al. 2010). Overall, a complex model for GRC trans-
mission has emerged through cytogenetics, but some details remain unknown (see
Fig. 11 of Itoh et al. 2009).
In addition to their enigmatic mode of transmission, the GRCs of zebra finch and
Bengalese finch are notable for the fact that they are larger than the largest A
chromosome of the respective karyotype (del Priore and Pigozzi 2014; Pigozzi and
Solari 1998) (Fig. 13). Considering that chromosome 2 is the largest chromosome of
the zebra finch reference genome (Warren et al. 2010), this would suggest that these
two GRCs are each >156 Mb in size. Only an intergenic fragment of 18.8 kb of the
zebra finch GRC has so far been sequenced, suggesting that at least this part of the
GRC is derived from chromosome 1 and potentially amplified to high copy number
(Itoh et al. 2009), as well as the mRNA of a single GRC-linked gene (Biederman
et al. 2018). Although it may seem surprising that the current era of avian genomics
has so far neglected to sequence these GRCs which are equivalent in size to ~13% of
the somatic genome (Table 1) (but see recent genome and expression data in Kinsella
Fig. 13 The germline-restricted chromosome (GRC; arrowhead) of the zebra finch. (a) Partial
karyotype of the eight largest chromosome pairs in female somatic cells. (b) Partial karyotype of the
eight largest chromosome pairs in male somatic cells. (c) Partial karyotype of the eight largest
chromosome pairs and the single GRC in male germline cells. The scale bar is 10 μm. Figure from
Pigozzi and Solari (1998), used with permission from Springer Nature
et al. 2018), genomic data for B chromosomes are scarce in general. B chromosome
assemblies of vertebrates are, to our knowledge, limited to a single cichlid fish
species (Valente et al. 2014) and zebra finch (Kinsella et al. 2018). Finally, it is
well possible that GRCs are widespread among (song)birds but have been
overlooked because they are not as prominent in size as those of zebra finch and
Bengalese finch (Fig. 13) (Kinsella et al. 2018; Torgasheva et al. 2018). We therefore
conclude this section with the words of Smith (2017) on GRCs: “Even for the vast
majority of species with ‘sequenced’ genomes, we simply haven’t looked.”
4.2.3 Chromosome 16
Chromosome 16 seems to be one of the most elusive among the avian chromosomes,
continuously withstanding assembly efforts. Even in the newest chicken genome
assembly (galGal5; based on long-read sequencing), only a fraction of the entire
chromosome is assembled (Warren et al. 2017). Several chromosomal properties—
with repetitive DNA as a common feature—are the likely reason for that (Fig. 14).
Firstly, chromosome 16 bears a mega-array of telomeric repeats, occupying the
shorter arm of the chromosome (Delany et al. 2007). Then there are two domains
which harbor the major histocompatibility complexes MHC-B and MHC-Y on the
long chromosome arm. These are thought to be one of the genetically most diverse
regions in the avian genome due to extreme variability in copy number (Hess and
Edwards 2002). The MHC-B and MHC-Y regions are separated by an array of the
PO41 repeat, a 41-bp-long and (G + C)-rich sequence which also acts as a recombi-
nation hotspot, rendering the two MHC regions essentially unlinked (Solinhac et al.
2010). However, the biggest obstacle for sequencing efforts is perhaps the nucleolus
organizer region (NOR). This chromosomal region is responsible for the initiation of
the nucleolus, a structure in the cell nucleus responsible for the formation of
Fig. 14 A physical
representation of genes and
intergenic regions on chicken
chromosome 16. The MHC
(major histocompatibility
complex) is split into two
(MHC-B and MHC-Y)
regions. Separated by a large
array of GC-rich PO41
repeats, the two MHC regions
exhibit elevated
recombination between them.
Note also the large nucleolar
organizer region (NOR),
which occupies
approximately 5–7 Mb on the
q-arm and contains ~150
copies of ribosomal RNA
genes. Figure taken from
Miller and Taylor (2016),
used under CC BY license
ribosomes and thus of extreme functional importance (Graw 2015), consisting of

enormous tandem repeat arrays of ribosomal RNA gene copies. Individual copies are
between 11 and >50 kb in size, while copy numbers range from 279 to 268. Since
this translates to 5–7 Mb of repeated DNA with high sequence identity among repeat
copies (Delany and Krupkin 1999), it is therefore not surprising that assembly efforts
had limited success so far (Warren et al. 2017).
Intriguingly, most of the (known) genes on chromosome 16 seem to be tied to the
immune system, for example, coding for peptide antigen presentation (MHC-B) or
the CD1 genes, which encode lipid-antigen-binding molecules (Miller and Taylor
2016). Together with the presence of recombination hotspots increasing allelic
diversity, chromosome 16 seems to be an ideal substrate for the evolution of
immune-related genes due to its repetitive nature, and an improved assembly of
this chromosome would certainly benefit various aspects of avian genomics.
5 Evolutionary Implications of Genomic “Dark Matter”
5.1 Accumulation of Repetitive Elements
While tandem repeats have been largely inaccessible to avian comparative genomics
as they are mostly unresolved as genomic “dark matter” (see Sect. 3), in-depth
characterizations of assembled interspersed repeats have revealed lineage-specific
events of de novo emergences and horizontal transfers (see Sect. 2). In addition to a
patchy distribution of some interspersed repeats across the avian tree of life (Fig. 3)
and assembly issues with very young TEs aside (Fig. 1), bird genomes vary
considerably in their long-term TE accumulation and TE turnover via deletions
(Fig. 15) (Kapusta et al. 2017; Kapusta and Suh 2017). This may seem counterintui-
tive given that not only assembly sizes but also TE copy numbers and densities vary
only moderately in avian genomes (assembly sizes of 1.0–1.3 Gb; 130,000–350,000
TE copies or 4.1–9.8% TE densities, except for the downy woodpecker with a TE
density of 22.2%) (Kapusta and Suh 2017; Zhang et al. 2014) but were readily
explained by the “accordion model” of genome size maintenance in birds and
mammals (Kapusta et al. 2017). Accordingly, insertion rates and deletion rates
covary in birds, with lineages experiencing more TE accumulation also undergoing
a higher turnover of TEs via deletions (Fig. 15). The most extreme examples are
ostrich and penguins with very low rates of TE accumulation and deletion on the one
end of the spectrum and zebra finch, downy woodpecker, and Anna’s hummingbird
on the other end (Kapusta et al. 2017). Surprisingly, this implies that secondarily
flightless birds have larger genomes not due to increased TE accumulation but due to
reduced shrinking compared to flying birds. Likewise, the smaller genomes of flying
birds are probably the result of increased shrinking due to higher TE activity and thus
increased genomic instability (Kapusta and Suh 2017).
Below, we more specifically address the accumulation of repetitive elements in
the context of genetic diversity, phylogenetic markers, and large-scale
rearrangements.
5.1.1 Genetic Diversity

Like all types of mutations, changes in tandem repeat array length or insertions of
interspersed repeats will initially exist as polymorphisms until reaching fixation in a
population. Notably, genetic diversity in repetitive elements may have a larger
impact on phenotypic variation than that of single-nucleotide polymorphisms
(Huddleston and Eichler 2016). While genetic diversity has been quantified in
several avian species for single-nucleotide polymorphisms (e.g., Dutoit et al. 2017;
Vijay et al. 2017) and microsatellites (see Sect. 3), the study of population-level
variation in large tandem repeats and interspersed repeats is still in its infancy in
avian genomics. Virtually nothing is known about length variation in tandem repeat
arrays in birds, although methods exist to do so (Wei et al. 2014; Weissensteiner
et al. 2017). On the other hand, single cases of TE presence/absence polymorphisms
(TEVs; Fig. 16a) have so far been reported for chicken and grebes, respectively (Lee
et al. 2017; Suh et al. 2012). A very recent genome-scale study of TEVs across
200 flycatcher genomes reported over 10,000 TEVs segregating within and between
the four sampled flycatcher species and suggesting recent activity of at least eight
distinct families of retrovirus-like LTR retrotransposons (Suh et al. 2018). We
emphasize that these estimates of genetic diversity through TEVs are likely
underestimates, given that the flycatcher reference assembly and re-sequencing
data are all based on short-read sequencing and thus only allow for detection of
TEVs in the well-assembled, non-repetitive part of the genome (Fig. 16b).
Fig. 15 The temporal landscape of TE accumulation across the avian tree of life. Dated TE
landscapes are shown as stacked histograms plotted on the Jarvis et al. (2014) phylogeny. The
major groups of TEs are non-LTR retrotransposons (green; mostly LINEs), LTR retrotransposons
Fig. 16 Discovery of TE presence/absence polymorphisms from re-sequencing data. (a) Sche-

matic illustration of a situation where the “true” genome of a re-sequenced bird individual differs
from the reference genome in the presence of a TE insertion (red) in an orthologous target site
(white square). (b) Read mapping against the reference will result in properly mapped read pairs
(blue) and discordant read pairs (orange) where one of the reads has sequence similarity to a
TE. Figure from Suh et al. (2018), used under CC BY license
5.1.2 Phylogenetic Markers

Rare genomic changes (RGCs) are “complementary markers with enormous poten-
tial for molecular systematics” (Rokas and Holland 2000). The most widely used
type of RGCs in phylogenetics is presence/absence patterns of retrotransposon
insertions (cf. Fig. 16a) which have been shown to very rarely exhibit homoplasy
(reviewed by, e.g., Ray et al. 2006; Shedlock et al. 2004; Han et al. 2011). In contrast
to other types of RGCs, retrotransposon presence/absence patterns can be polarized
without outgroups because the retrotransposition mechanism yields an insertion
flanked by a target site duplication as the derived state, while an empty insertion
site constitutes the ancestral state (Fig. 2a). Concurrent with the availability of more
and more genome assemblies, studies have shifted from sampling several or dozens
of retrotransposon markers across species via polymerase chain reaction and Sanger
sequencing (e.g., Kaiser et al. 2007) to now sampling hundreds or thousands of
markers from available genome sequencing data (e.g., Suh et al. 2015b, Cloutier
et al. 2018).
Retrotransposon markers (sampling CR1s, LTRs, or SINEs) have been applied to
a wide range of birds ranging from Galliformes (Kaiser et al. 2007; Kriegs et al.
2007; Liu et al. 2012b) over Passeriformes (Treplin and Tiedemann 2007; Suh et al.
2017, 2018) to other groups of birds such as grebes, penguins, storks, and
woodpeckers (Suh et al. 2012; Watanabe et al. 2006; Kuramoto et al. 2015; Han
et al. 2011). These studies were largely congruent with sequence-based phylogenies.
Furthermore, retrotransposon markers have helped settle long-standing phylogenetic
disputes such as the paraphyly of ratites within Palaeognathae (Baker et al. 2014;
Haddrath and Baker 2012; Cloutier et al. 2018) and several critical branches within
the explosive radiation of Neoaves (Suh et al. 2011b, 2015b; Matzke et al. 2012),
Fig. 15 (continued) (purple), DNA transposons (blue), and other interspersed repeats (black;
mostly unclassified). Species names in gray are those with low-quality genome assemblies.
Figure modified from Kapusta and Suh (2017), used with permission from John Wiley and Sons
Fig. 17 Retrotransposon markers suggest that the root of the neoavian radiation remains unre-
solved (cf. Fig. 3). Comparison of a pruned neighbor network of retrotransposon markers (left) with
a neighbor network of a simulated hard polytomy, i.e., simultaneous speciation (right). Nine
reciprocally monophyletic taxa emerge from this potential hard polytomy (yellow), namely,
Aequornithes/Phaethontimorphae (Ae/Ph), Caprimulgiformes (Ca), Charadriiformes (Ch),
Columbimorphae (Co), Gruiformes (Gr), Opisthocomiformes (Op), Mirandornithes (Mi),
Otidimorphae (Ot), and Telluraves (Te). Figure modified from Suh (2016), used under CC BY
4.0 license
such as the sister group relationship between parrots and passerines. Notably, a
reanalysis of genome-level retrotransposon markers suggested that most of the
neoavian radiation is now reliably resolved (congruent with the genome-level
sequence phylogeny of Jarvis et al. 2014), except for the eight deepest speciation
events which remain unresolved (Fig. 17) (Suh 2016; but see Braun et al. 2019).
5.1.3 Large-Scale Rearrangements

Repetitive elements can mediate large-scale rearrangements such as inversions,
deletions, duplications, and translocations (Konkel and Batzer 2010; Devos et al.
2002). This happens through ectopic recombination between homologous sequences
(cf. Fig. 6b) and can thus be expected to be more likely to occur between repeat-rich
regions of bird genomes (cf. Fig. 9). Additionally, rapid amplification of specific TE
families can provide genome-wide interspersed substrates for ectopic recombination
and thus increased genomic instability (reviewed by, e.g., Kapusta et al. 2017). In
line with this, genomic studies of deep evolutionary timescales of avian chromosome
evolution have shown that breakpoints of large-scale rearrangements are biased
toward regions enriched in repetitive elements, especially TEs (Farré et al. 2016;
Skinner and Griffin 2012). Furthermore, high numbers of large-scale
rearrangements, in particular inversions, have been reported from the zebra finch
and other Estrildidae (Hooper and Price 2015; Romanov et al. 2014; Warren et al.
2010), which coincides with a higher rate of expansion and diversification of
retrovirus-like LTR retrotransposons than in other songbird lineages (Kapusta and

Suh 2017; Suh et al. 2018).
On recent evolutionary timescales, inversions are frequently invoked to explain
patterns of linkage disequilibrium in population genomics (e.g., zebra finches and
flycatchers, Knief et al. 2016; Ellegren et al. 2012). However, physical (i.e.,
assembly-based) evidence for intraspecific inversions is rare in avian genomics. To
our knowledge, the only cases where inversion polymorphisms have been assembled
or at least their breakpoints determined are the ruff (Küpper et al. 2016;
Lamichhaney et al. 2016) and the white-throated sparrow (Davis et al. 2011; Tuttle
et al. 2016). Both cases are intraspecific megabase-scale inversions with consider-
able phenotypic effects on plumage and behavior, and were thus subject to large
targeted efforts of sequencing and assembly. It is well possible that inversion
polymorphisms are far more common in birds but currently constitute genomic
“dark matter” because their repeat-rich breakpoints are difficult to assemble without
targeted efforts and with short-read sequencing data alone.
The same might apply to another type of large-scale rearrangements, the duplica-
tion of sequence segments or even entire chromosome arms. Duplications are also
known to potentially cause dramatic alterations of the phenotype and thus constitute
the genomic substrate for evolution (Ohno 1970). To our knowledge, the only well-
understood case of a duplication event in birds with direct phenotypic consequences
is the duplex comb locus in chicken. A 20 kb duplication 200 kb upstream of the
EOMES gene causes the V-shaped and buttercup comb phenotypes of certain
chicken breeds (Dorshorst et al. 2015).
5.2 Genomic Conflict and Speciation
5.2.1 Host–Parasite Arms Races

“From the perspective of genomic parasites, such as transposons and viruses,
genomes of cellular organisms are not simply strings of DNA but complex
microcosms of interactions between and among parasitic genes and host genes”
The genome is in an everlasting arms race with the parasitic genes hidden within.
Recent studies of human evolution highlight this importance, as host–virus arms
races caused nearly a third of all adaptive protein changes (Enard et al. 2016) and
were responsible for adaptive introgressions between Neanderthals and modern
humans (Enard and Petrov 2017). Furthermore, the significance of host–TE arms
races is becoming widely appreciated, with dozens of TE repressor mechanisms so
far known from model organisms (reviewed by Kapusta and Suh 2017; Goodier
2016). Accordingly, host defense can occur “before TE transcription (DNA methyl-
ation, histone modifications, DNA hypermutation), during TE transcription (pre-
mature termination), after TE transcription (RNA hypermutation, piRNA binding,
siRNA binding), and before TE transposition (inhibition of ribonucleoprotein
complexes)” (Kapusta and Suh 2017).
Fig. 18 Host–TE arms races may directly influence speciation. Illustration of the model of Rogers
(2015) where reduced hybrid fitness results from transposon derepression in some gametes follow-
ing meiotic recombination of incompatible TE repressor systems. Figure loosely drawn after Rogers
(2015)
To our knowledge, the evolutionary significance of arms races between birds and
transposons/viruses has been overlooked, since only three studies have investigated
such repression mechanisms in birds. Firstly, a study on DNA methylation of TEs in
great tits suggested that non-CpG methylation is responsible for silencing of most
TE copies (Derks et al. 2016), but it has been noted that an annotation of TEs specific
to the great tit lineage is so far lacking (Kapusta and Suh 2017). Secondly, a study on
APOBEC cytosine deaminases among 123 sampled vertebrates reported that birds
(especially zebra finch and medium ground finch) had the strongest signals of C-to-U
hypermutation of LTR retrotransposons, a mechanism leading to defective LTR
daughter copies (Knisbacher and Levanon 2015). Thirdly, two recent studies on
PIWI-interacting RNAs (piRNAs) in chicken suggested that the mRNAs of all
germline-transcribed TEs are targeted by specific piRNAs with complementarity to
prevent (retro)transposition (Chang et al. 2018) and reported a de novo piRNA birth
which controls avian leukosis virus infections in domestic but not wild chicken
(Sun et al. 2017).
It is plausible that the rapidity of host–TE/virus arms races directly influences
speciation by contributing to reproductive isolation between new species (Fig. 18).
Accordingly, reduced gene flow between two populations may lead to divergence of
their TE landscapes and TE repressor systems which, on secondary contact, may
result in reduced hybrid fitness or even hybrid sterility if the parental TE repressor
systems are incompatible (Rogers 2015; Erwin et al. 2015). Rogers (2015) suggested
a possible scenario leading to TE derepression in the gonads of F1 hybrids, which
would be the unlinking of a TE repressor system during meiotic recombination and
their placement in separate gametes (Fig. 18). To our knowledge, there are no
empirical examples yet for TE-induced reproductive isolation in birds. However,
in Drosophila the divergence of parental piRNA pathways has been linked to TE
derepression in F1 hybrids (Romero-Soriano et al. 2017; Kidwell et al. 1977;
Erwin et al. 2015). Based on these results, forward simulations of a new TE invading
a population suggested that TE repression via small RNAs may evolve de novo
within less than 200 generations (Kelleher et al. 2017). We therefore hypothesize
that host–TE/virus arms races played an important role in bird speciation. This may
be especially the case in songbirds as they were frequently invaded by novel
retrovirus-like LTR retrotransposons (Warren et al. 2010; Suh et al. 2018) (see
Sect. 2.1.3), a situation which likely led to rapid divergence of TE/virus repressor
systems between songbird populations and species.
5.2.2 Meiotic Drive

Selection for a higher number of offspring can act on many different levels.
Arguably the most basic level is at DNA sequence level, the very carrier of heritable
information. Often seen as a process affecting individuals of a population, it is
astounding that selection can also happen in a way that individual genes or
chromosomes compete for a spot in the next generation. This process, termed
“meiotic drive,” commonly exploits the asymmetry of female meiosis (Sandler and
Novitski 1957). Out of four homologous chromosomes, only one is transmitted to
the egg; the other three are banned to the polar bodies and do not contribute to the
next generation. While usually a stochastic process with an even distribution of all
four possible chromosomes, mechanisms can evolve that cause the chromosome
carrying the meiotic driver to be preferentially transmitted (Lindholm et al. 2016). A
well-known example of such an ultra-selfish genetic element is the knob system in
maize (Buckler IV et al. 1999). Blocks of heterochromatic tandem repeats found
distal from centromeres outcompete chromosomes without such elements. In the
case of a heterozygote, the carrier chromosome wins the race for being included in
the egg and thus gains a selective advantage. Sometimes the affinity for the centro-
meric motor protein may depend on the length of a certain type of satellite array
(Henikoff et al. 2001), demonstrating the importance of accurate assessment of
repetitive elements. Due to negative effects of centromere drive in male meiosis
(e.g., chromosomes failing to separate properly during cell division), which lead to
infertility or inviability, it is expected that other components influencing segregation,
such as protein binding affinity, evolve rapidly to restore segregation balance (Malik
and Henikoff 2001). Direct observations of centromere drive are extremely rare
(Lindholm et al. 2016), and thus far there are only two studies which reported
segregation distortion in birds; for chicken, Axelsson et al. (2010) report indirect
evidence for rapid evolution in kinetochore protein genes and determine two chro-
mosomal regions which exhibit unfair segregation in 197 chicken families. Knief
et al. (2015) found a prezygotic transmission distorter to be active in both males and
females of zebra finches, which suggests other mechanisms to disproportionately

increase a certain genetic element fitness. We assume that these cases are just the tip
of the iceberg of actual meiotic drives as their confirmation requires strong drivers or
very large sample sizes.
5.3 Meiotic Recombination and Gene Conversion
The meiotic recombination landscape, i.e., the distribution of crossovers on

chromosomes during meiosis, is usually highly heterogeneous in eukaryotes
(de Massy 2013). Different factors influence this heterogeneity, among the most
important are chromosome size, gene density, epigenetic marks, and structural
chromosomal features such as centromeres and telomeres.
The physical rupture of chromosomal DNA during meiosis (i.e., double-strand
breaks) leads to the exchange of homologous chromosomes through homologous
recombination (Graur and Li 2000). This causes a shuffling of allelic combinations
and is an important source of genetic variation (Ellegren and Galtier 2016). How-
ever, if a double-strand break occurs in a repetitive region, ectopic or nonallelic
homologous recombination (NAHR) might occur (George and Alani 2012). Homol-
ogous repetitive sequences, stemming from different chromosomal regions or even
different chromosomes, align to each other and can induce chromosomal
rearrangements via NAHR (cf. Fig. 6b). While sometimes neutral or even beneficial
for the carrier, mutations such as large chromosomal rearrangements or chromosome
number changes are mostly deleterious and lead to inviable or infertile offspring
(Lupski and Stankiewicz 2005). It is thus likely that mechanisms evolve which
prevent such mutations via suppression of recombination in these regions (George
and Alani 2012). This is usually achieved by heterochromatization, meaning that the
DNA molecule is being tightly wrapped around histone proteins so that otherwise
open chromatin is transcriptionally suppressed and also not accessible for double-
strand breaks (Grewal and Jia 2007). However, it seems paradoxical then that
tandem repeat arrays evolve rapidly showing frequent expansions and contractions.
A solution to this paradox is offered by the model of gene conversion. In this form of
nonreciprocal genetic exchange, a double-strand break in one homologous chromo-
some is followed by the invasion and subsequent insertion of an exact copy of the
sequence of the homologous chromosome (see also Sect. 3.2 and Fig. 8b) (Talbert
and Henikoff 2010). Via this mechanism, not only repeat expansions and
contractions can be explained, but also the emergence of higher-order repeat
structures and the homogenization of copies within and between tandem repeat
arrays.
Evidence for gene conversion in birds is limited to a few examples, mainly due to
the fact that a high number of unique closely spaced markers are needed to detect
it. Only with the advent of high-throughput sequencing technologies has this become
feasible (Backström et al. 2005; Weber et al. 2014; Mugal et al. 2015); however,
detection of gene conversion in highly homogenous tandem repeat arrays is yet
impossible due to the abovementioned requirement.
Direct evidence for the influence of chromosomal features on recombination is

also rare and mainly restricted to recombination suppression by large-scale
rearrangements such as inversions (e.g., white-throated sparrows, Huynh et al.
2011; ruffs, Küpper et al. 2016). However, in crows it has been shown that large-
scale tandem repeat arrays influence population genetic statistics (e.g., low genetic
diversity and low population recombination rate) of nearby genomic regions,
suggesting that these potentially are (peri)centromeric (Weissensteiner et al. 2017).
Finally, certain repetitive elements might also be enriched in regions of high
recombination rates. In flycatchers, for example, recombination rate analysis based
on patterns of linkage disequilibrium showed an association between recombination
hotspots and certain transposable elements (TEs). While the initiation of double-
strand breaks and thus recombination might be directly influenced by TE activity, it
seems that the shared requirement of open chromatin both for transcription and TE
activity is causing the association of certain TEs with high-recombination regions
(Kawakami et al. 2017).
5.4 Histone Modification and Transcription
5.4.1 Satellite Repeats

The transcription of DNA is in general restricted to open chromatin (euchromatin),
which is not condensed during interphase and thus accessible to the transcription
machinery (Grewal and Jia 2007). However, recently it has become evident that
some repetitive sequences residing in densely packed heterochromatin are also
transcribed (Trofimova and Krasikova 2016). Two hypotheses have been put for-
ward to resolve this: the “read-through” hypothesis states that some tandem repeats
are transcribed simply because they are located in between two protein-coding genes
and transcription continues into the adjacent noncoding region, failing to determine a
stop codon (Varley et al. 1980). Evidence for this mechanism has been found in
lampbrush chromosomes (highly enlarged chromosomes of growing oocytes) of
newts (Varley et al. 1980; Diaz et al. 1981). An alternative explanation for the
transcription of satellite DNA has been provided by Deryusheva et al. (2007). By
analyzing chicken and quail lampbrush chromosomes, they found open reading
frames (ORFs) for an endogenous retrovirus (ERV) directly adjacent to arrays of
two 41-bp-long tandem repeats (CNM and PO41). They postulate that transcription
is initiated at those LTR promoters and read through into the tandem repeat arrays.
Genomic regions with a high concentration of satellite DNA in the form of
large heterochromatic tandem repeat arrays show in general different histone modifi-
cations than euchromatin (Grewal and Jia 2007). To prevent an excess of double-
strand breaks in these often very fragile regions, histone proteins (on which the DNA
is coiled around) are methylated (as opposed to acetylated in euchromatic regions)
and result in densely packed chromatin (George and Alani 2012). Hetero-
chromatization is also a defining feature of sex chromosomes, in particular the
avian W chromosome (Stefos and Arrighi 1971) (see Sect. 4.2.1). Also in
germline-restricted chromosomes (see Sect. 4.2.2), methylation of certain histones
is the mechanism providing condensation and transcriptional silencing (Goday and

Pigozzi 2010). However, further research is needed to uncover the exact mechanisms
of heterochromatization and details on the evolutionary dynamics between large
tandem repeat arrays and histone modification in bird genomes.
5.4.2 Transposable Elements

With their ability to disperse copies of themselves throughout the genome,
transposable elements may insert into genomic contexts where they influence the
transcription of nearby genes. This can have significant phenotypic effects, as
recently shown for a textbook example of evolutionary biology, the industrial
melanism in peppered moths (van’t Hof et al. 2016). The copies of many TEs
contain their own transcriptional promoters or enhancers (e.g., located within
SINE heads, solo-LTRs, and LINE 50 UTRs; see Sect. 2) and through these
structures may influence the expression of nearby genes or even gene regulatory
networks (reviewed by Slotkin and Martienssen 2007; Chuong et al. 2017). In the
human genome, thousands of ancient TEs have been co-opted as transcriptional
regulators (Bejerano et al. 2006; Lowe et al. 2007). Some copies from these same TE
families are also evolutionarily conserved in birds (Craig et al. 2018), which
suggests that they have had a gene regulatory function at least since the common
ancestor of birds and mammals. However, the gene regulatory consequences of TE
families active during the diversification of birds (Fig. 15) are poorly understood
(Craig et al. 2018). To our knowledge, the only case of a phenotypic effect of a
recent TE insertion in birds is the blue eggshell color of some chicken breeds. This
phenotype is caused by a tissue-specific over-expression of the SLCO1B3 gene due
to a nearby insertion of a retrovirus-like LTR retrotransposon copy (Wang et al.
2013; Wragg et al. 2013).
In addition to such TE-induced increases in transcription, TE insertions may
instead lead to lower transcription levels of nearby genes (Fig. 19). This is due to
the role of DNA methylation as a mechanism to repress TE activity (see Sect. 5.2.1).
According to the “sloping shores” model of Grandi et al. (2015), an individual TE
insertion may decrease nearby gene expression because the hypermethylation of the
TE may change the methylation status of a nearby promoter (Fig. 19b). Similarly,
recent evidence suggests that new TE insertions in Drosophila can lead to a spread of
repressive histone modifications up to 20 kb away from the TE (Lee and Karpen
2017). However, the “sloping shores” model remains to be tested in birds, because
knowledge on the methylation status of avian TEs is so far limited to ancient and
medium-age TE insertions from great tit (Derks et al. 2016) (see Sect. 5.2.1). Recent
or polymorphic TE insertions might be particularly promising candidates for causing
differential methylation or differential gene expression within and between bird
species, and recently developed methods are now capable to determine the methyla-
tion status of individual TE insertions (Suzuki et al. 2015; Daron and Slotkin 2017).
In this context, it is plausible that TE insertions with large phenotypic effects
facilitate rapid adaptation (Stapley et al. 2015). We thus emphasize the importance
of determining not only the phenotypic effects of candidate TEs but also of
establishing whether or not they are under positive selection. In contrast to SNPs,
Fig. 19 DNA methylation of transposon insertions may affect nearby gene expression. (a)
Schematic scenario of a TE insertion event close to a transcriptional promoter of a nearby gene.
(b) Illustration of the “sloping shores” model of Grandi et al. (2015) where, following a TE insertion
event, DNA hypermethylation of the TE (blue dashed box) may extend to a nearby promoter
(orange dashed box) and lead to a lower transcription level of the affected gene. Alternatively, DNA
hypomethylation of the TE may lead to a higher transcription level of a nearby gene. Figure loosely
drawn after Grandi et al. (2015)
the selective forces acting on individual TE insertions have been largely ignored so
far. Recent methodological improvements (reviewed by Villanueva-Cañas et al.
2017), together with long-read sequencing, promise to reveal the evolutionary
significance of TE-induced regulatory variation in birds.
6 Limitations and Future Avenues to Analyze Genomic “Dark

Matter”
Rapid technological advances in the fields of genomics (Peona et al. 2018) and
cytogenetics (Damas et al. 2017) have made it possible to continuously add layers of
information to (avian) genomic studies which were previously hidden within geno-
mic “dark matter.” The four most important advances after the introduction of high-
throughput sequencing are long-read sequencing, linked-read sequencing, optical
mapping, and chromatin interaction mapping (Eid et al. 2009; Lieberman-Aiden
et al. 2009; Lam et al. 2012; Weisenfeld et al. 2017). They all share the utilization of
long-range information from single DNA molecules. Long-read sequencing
produces unbiased (i.e., without PCR-induced error) sequence reads with mean
read lengths of routinely up to 20 kb, and while the sequencing error rate is usually
quite high (~15%), the introduced error is random and can be overcome by increas-
ing the amounts of reads covering any single position in the genome (e.g., Bickhart
et al. 2017). Assemblies produced with this technology have resulted in unprece-
dented contiguity with over 100-fold increases of contig N50 and thus enormously
reducing the number of gaps in the assembly (Korlach et al. 2017; Weissensteiner
Fig. 20 A model for the resolution of genomic “dark matter” with current sequencing and genome
mapping technologies. Gray bars indicate genomic “dark matter,” mostly nested within interspersed
repeats (IRs) and tandem repeats (TRs). Hi-C, chromosome conformation capture; LRC, linked-
read “cloud”; OM, optical mapping. Figure from Peona et al. (2018), used under CC BY license
et al. 2017). More precisely and as illustrated in Fig. 20, long-read sequencing
provides read lengths on the >10 kb level (with individual reads now well beyond
100 kb, Jain et al. 2018), and we assume that this may resolve assembly gaps within
most interspersed repeats and at the boundaries of tandem repeat arrays. Linked-read
sequencing relies on microfluidic barcoding of long DNA molecules in individual
beads and subsequent sequencing using short-read platforms. By that, reads can be
assigned to individual molecules forming linked-read “clouds,” containing long-
range information used for the assembly process (Weisenfeld et al. 2017). Optical
mapping provides linkage information on the >100 kb level and may therefore at
least anchor tandem repeat arrays into genome assemblies (Weissensteiner et al.
2017). Chromosome conformation capture (Hi-C) provides linkage information on
the >1 Mb level and may therefore span large tandem repeat arrays including
centromeres, yielding scaffolds of the length of individual chromosomes (Bickhart
et al. 2017; Dudchenko et al. 2017).
At the moment, the focus lies on improving the long-range information in
genome assemblies (as measured in contig or scaffold N50) because it is easily
measurable. However, the main potential of resolving genomic “dark matter” lies in
the ability to characterize the full spectrum of genetic variation occurring in eukary-
otic genomes (Huddleston and Eichler 2016; Sedlazeck et al. 2018). Compared to
the number of single-nucleotide polymorphisms, the amount of genetic variation due
to structural changes (e.g., chromosomal rearrangements, tandem repeat dynamics,
retrotransposition) accounts for the majority of differences in the DNA sequence
between two human individuals (Chaisson et al. 2015). Yet, it is also unclear how
much of this uncharacterized variation has a direct relevance for the phenotype of an
organism. A prime avian example for massive functional genetic variation hidden in
obscure parts of the genome is the major histocompatibility complex (MHC) on
chromosome 16, which plays an important role in immune function in vertebrates.
This gene family is known to be notoriously difficult to assemble by itself, but
additionally most of chromosome 16 is so repetitive that the vast majority of it
remains inaccessible even in recent chicken genome assembly based on long-read
sequencing (Warren et al. 2017) (see Sect. 4.2.3). Thus, even functionally important
regions can reside within repetitive regions which are difficult to sequence and
assemble. Studies on the genetic mechanisms underlying speciation are also likely
to miss important information when looking for the targets of selection. Until
recently, the focus lied solely on annotated protein-coding genes (Ellegren et al.
2012; Poelstra et al. 2014), largely ignoring the role of undetected promoters and
regulatory elements. The latter might include repetitive DNA if they emerged
through co-option of transposable elements (Chuong et al. 2017). Furthermore, it
is important to keep in mind that, for example, genetic elements that distort meiotic
segregation (e.g., centromere drivers) may reside in repeat-rich heterochromatin in
or near centromeres and are thus difficult to detect and assemble.
Altogether, ornithologists now have a diverse toolkit to tackle the most challeng-
ing tasks of avian genomics (Fig. 20), and we hope that the importance of resolving
the “dark matter” of bird genomes will become more widely appreciated.
Acknowledgments We thank Anne-Marie Dion-Côté and Cormac Kinsella for helpful

discussions and Anne-Marie Dion-Côté, Cormac Kinsella, and Karen Miga for comments on earlier
versions of this manuscript. We are also grateful for comments from Robert Kraus and three
anonymous reviewers who further improved this manuscript. A.S. was supported by grants from
the Swedish Science Foundation (2016-05139) and the SciLifeLab Swedish Biodiversity Program
(2015-R14).
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Resolving the Avian Tree of Life from Top
to Bottom: The Promise and Potential
Boundaries of the Phylogenomic Era
Edward L. Braun, Joel Cracraft, and Peter Houde
Abstract
Reconstructing relationships among extant birds (Neornithes) has been one of the
most difficult problems in phylogenetics, and, despite intensive effort, the avian
tree of life remains (at least partially) unresolved. Thus far, the most difficult
problem is the relationship among the orders of Neoaves, the major clade that
includes the most (~95%) named bird species. This clade appears to have
undergone a rapid radiation near the end Cretaceous mass extinction (the K-Pg
boundary). On the other hand, if one embraces a “glass half full” view, the fact
that most orders in Neoaves can be placed into seven clades, recently designated
the “magnificent seven,” could be viewed as remarkable progress. We propose
that the dawning era of whole-genome phylogenetics will only resolve the
remaining relationships, if we improve data quality, exploit information from
other sources (i.e., rare genomic changes), and learn more about the functional
and evolutionary landscape of avian genomes. Of course, it is possible that the
remaining unresolved relationships are unresolvable regardless of the data avail-
able, but we suggest that the community should avoid this conclusion until more
data collection has been completed and improved analyses have been conducted.
Author contributed equally with all other contributors. Edward L. Braun, Joel Cracraft and Peter
Houde
E. L. Braun
Department of Biology and Genetics Institute, University of Florida, Gainesville, FL, USA
e-mail: ebraun68@ufl.edu
J. Cracraft
Department of Ornithology, American Museum of Natural History, New York, NY, USA
e-mail: jlc@amnh.org
P. Houde (*)
Department of Biology, New Mexico State University, Las Cruces, NM, USA
e-mail: phoude@nmsu.edu

https://doi.org/10.1007/978-3-030-16477-5_6
152 E. L. Braun et al.
We say this because there is ample evidence that estimates of avian phylogeny
based on large-scale datasets may be affected by well-characterized artifacts (e.g.,
long-branch attraction, heterotachy, and discordance among gene trees) and by
subtle “data-type effects” that reflect poor fit to empirical data for available
models of sequence evolution. Even if these analytical challenges can be
addressed, we need to integrate phylogenomic and fossil data. Finally, we also
emphasize that, regardless of the resolution (or lack thereof) for relationships
among major avian clades, we are only at the dawn of the phylogenomics of birds.
Large-scale molecular data remain unavailable for the vast majority of the
~10,000 named bird species, and those named bird species probably represent
an underestimate of the true number of distinct evolutionary lineages of birds
(whether or not those lineages are assigned the rank of species) by as much as
threefold. A true biodiversity genomics effort in birds is likely to reveal many
additional examples of cases where it is very difficult to resolve relationships; the
effort to resolve as many of those relationships as possible will represent a major
scientific achievement and provide lessons for phylogenomic studies in other
parts of the tree of life.
Keywords
Bird phylogeny · Phylogenetic estimation · Base composition · GC-content ·
Heterotachy · Rare genomic changes · Multispecies coalescent · Whole-genome
sequencing · Phylogenomics
1 Introduction
A renaissance within systematics began in the late 1980s with the introduction of the
polymerase chain reaction (PCR), which provided a simple method for directly
harvesting and sequencing DNA for comparative studies (Higuchi and Ochman
1989; Kocher et al. 1989; Saiki et al. 1988). This had an almost immediate effect
within systematic biology in general (Hillis and Moritz 1990; Miyamoto and
Cracraft 1991), as well as in ornithology in particular (Edwards et al. 1991; Edwards
and Wilson 1990; Helm-Bychowski and Cracraft 1993; Mindell 1997; also see
Sheldon and Bledsoe 1993 for a review describing the early history of molecular
systematics in birds). The human genome project began during the same period
(Cantor 1990; Watson 1990). This led to remarkable advances in DNA sequencing
technologies and methods for the storage and analysis of sequence data that continue
to have a profound impact on comparative biology, including the field of systematics
(for additional historical details, see Wink 2019). These technical achievements
resulted in DNA sequence comparative datasets of ever increasing sizes. Thus, the
next decade saw the widespread expansion of the use of DNA sequence data in avian
systematics, and knowledge about the avian tree of life deepened at all taxonomic
levels (reviewed by Cracraft et al. 2004). In particular, studies of avian relationships
Resolving the Avian Tree of Life from Top to Bottom: The Promise and. . . 153
were characterized by increased taxon sampling and the inclusion of multiple

mitochondrial regions and nuclear loci.
The idea of “phylogenomics” has existed for about two decades. Although the
original usage included the inference of gene function using evolutionary history
(Eisen 1998; Eisen et al. 1997), the term has largely become synonymous with
the use of large amounts of sequence data in phylogenetics (Delsuc et al. 2005).
And today, there are many thousands of citations alluding to “phylogenomics,” and
the majority of these imply the use of “genomic” or “large-scale” approaches to
estimate the tree of life. In some sense, the phylogenomic era of avian systematics
began with Hackett et al. (2008), in which the “Early Bird” consortium of
investigators employed 19 loci (~32 kb of sequence) from 169 species to construct
a phylogeny that included all major avian lineages (Table 1). It might be more
accurate to view the Early Bird effort as the beginning of a “proto-phylogenomic
era” of avian systematics because it represented a significant increase in scale of data
collection relative to previous work but it still relied on PCR for gene sampling.
However, the use of PCR sampling was not a major limitation, and, at the time,
Hackett et al. (2008) provided the broadest support for relationships within and
(to some degree) among avian orders.
Table 1 Recent large-scale estimates of avian phylogenya

Number of Branch
Study neornithine taxa Data typeb Analysisc lengthsd
Fain and Houde (2004) 149 one nuclear locus (nc) MP/ML/ n/a
BI
Ericson et al. (2006) 111 five nuclear loci (c/nc) BI time
Livezey and Zusi (2007a) 150 morphologye MP n/a
Hackett et al. (2008) 169 19 nuclear loci (nc) ML mol
Kimball et al. (2013) 77 50 nuclear loci (nc) ML mol
McCormack et al. (2013) 33 1541 nuclear loci (nc) BI mol
Jarvis et al. (2014) 48 12,020 nuclear loci ML time/
(nc/c) mol
Prum et al. (2015) 198 259 nuclear loci (c) BI time
Claramunt and Cracraft 48/230f up to 1156 nuclear BI time
(2015) loci (c)
Reddy et al. (2017) 235 54 nuclear loci (nc) ML mol
a
We define large-scale trees as those that include most or all of the orders, as defined by Cracraft
(2013), in Neoaves
b
Data types are reported as “c” for primarily coding and “nc” for primarily noncoding (introns,
UCEs, and untranslated regions)
c
BI Bayesian inference, ML maximum likelihood, MP maximum parsimony
d
Branch lengths available as estimates of absolute time or molecular change (substitutions per site).
“n/a” indicates that branch lengths are not available
e
Livezey and Zusi (2007b) provide a detailed description of this morphological data; Mayr (2008)
discussed issues with character scoring and the interpretations of avian morphological variation that
are more congruent with molecular phylogenies
f
The 48-taxon tree in Claramunt and Cracraft (2015) reflects an analysis of 1156 clocklike coding
regions. The 230-taxon tree reflects an analysis of two coding regions. Both analyses were
constrained to the Jarvis et al. (2014) backbone. Additional analyses using the Prum et al. (2015)
backbone are reported in their supplementary material
This achievement was soon surpassed by studies that adopted various next-
generation sequencing technologies (Glenn 2011). Arguably, the new and innova-
tive methods for sequence capture represent the most important technology for avian
phylogenomics at this time. Those methods greatly expanded the harvesting of
hundreds or thousands of loci across the genome (Table 2), truly launching the era
of avian phylogenomics. Currently, one of the more popular sequence capture
methods is the use of probes that hybridize to ultraconserved elements (UCEs),
which correspond largely to noncoding regions that are conserved across most or all
vertebrates (Bejerano et al. 2004). Many noncoding UCEs appear to be involved in
gene regulation (Dimitrieva and Bucher 2012), but their function is not related to
their use in phylogenetics. Instead, the conserved nature of the sequences allows
probes that hybridize to UCEs to be used for sequence capture in many different
vertebrates; most or all of the phylogenetic information in UCE datasets actually
reflects the less-conserved sequences that flank the conserved core of the UCE
(Crawford et al. 2012; Faircloth et al. 2012; McCormack et al. 2012, 2013).
Analyses of UCEs have begun to advance our understanding of avian systematics
Table 2 Published phylogenomic studies using the UCE sequence capture data
Study Focal order Number of species Number of samplesa
Smith et al. (2014) Passeriformes 5 36
Sun et al. (2014) Galliformes 15 —
Bryson et al. (2016) Passeriformes 30 —
Hosner et al. (2016) Galliformes 23 —
Hosner et al. (2015b) Galliformes 90 —
Manthey et al. (2016) Passeriformes 11 28
McCormack et al. (2016) Passeriformes 1 (3)b 27
Meiklejohn et al. (2016) Galliformes 18 —
Moyle et al. (2016) Passeriformes 106 —
Persons et al. (2016) Galliformes 11 —
Zarza et al. (2016) Passeriformes 3 26
Andersen et al. (2017) Coraciiformes 21 —
Bruxaux et al. (2017)c Columbiformes 6 21
Campillo et al. (2017) Passeriformes 17 —
Hosner et al. (2017) Galliformes 115 —
Wang et al. (2017) Galliformes 20 —
White et al. (2017) Caprimulgiformes 12 —
Andermann et al. (2018) Caprimulgiformes 2 9
Musher and Cracraft (2018) Passeriformes 29 62
Younger et al. (2018) Passeriformes 3 23
a
The total number of samples is listed if multiple individuals were sequenced for at least one of the
focal species; a dash indicates that only one sample per species was sequenced
b
McCormack et al. (2016) sequenced 27 Western scrub jays (Aphelocoma californica) from
3 lineages that could represent species
c
Bruxaux et al. (2017) used genome skimming followed by bioinformatic extraction of UCE loci
(and other loci) rather than sequence capture
at all levels, from the deepest branches (e.g., McCormack et al. 2013; Gilbert et al.
2018) to the tips of the tree (Table 2). UCEs even appear to be useful at
phylogeographic scales (Harvey et al. 2016; Smith et al. 2014). Lemmon et al.
(2012) developed a similar approach based on a distinct probe set (one focused
largely on coding exons) that they called anchored hybrid enrichment; like UCE
sequence capture, anchored hybrid enrichment is capable of harvesting vast
quantities of data, and it is also being applied in avian systematics (Prum et al. 2015).
At present, the only higher-level phylogenetic study of birds that has employed
whole genomes (more accurately, draft genome sequences) is that of Jarvis et al.
(2014). However, analyses of draft genome sequences are increasingly informing
avian evolutionary studies (Cornetti et al. 2015; Lamichhaney et al. 2015;
Nadachowska-Brzyska et al. 2015; Nater et al. 2015; Poelstra et al. 2014; Toews
et al. 2016a; Tuttle et al. 2016; Ottenburghs et al. 2017a; Stryjewski and Sorenson
2017; Tiley et al. 2018; for recent general reviews of avian evolutionary genomics,
see Joseph and Buchanan 2015; Kraus and Wink 2015; Toews et al. 2016b). Indeed,
there are currently major efforts in multiple labs to increase the number of avian
genome sequences (like the B10K project; Zhang et al. 2015), and within just a few
years, the amount of comparative data available for phylogenetic and evolutionary
studies in birds will expand exponentially. Yet, although the last decade has seen a
great improvement in our understanding of avian relationships, these large-scale data
have also revealed remarkable incongruence in avian relationships due to using
different genes, distinct data types (e.g., across exons, introns, and UCEs), and
various taxon and character sampling regimes (e.g., Jarvis et al. 2014; Hosner
et al. 2015b, 2016; Ottenburghs et al. 2016a; Reddy et al. 2017). Thus, far from
heralding the “end of incongruence,” as Gee (2003) asserted, phylogenomics has
actually revealed the complex nature of the phylogenetic signals in genomes (e.g.,
Jarvis et al. 2014). This raises several questions: Why do some nodes in
phylogenomic trees have limited support even when large amounts of data are
analyzed? What does it mean to use “whole genomes” in phylogenetics? What are
the limitations to “whole-genome” analysis? If we shift our focus toward avian
phylogenomics more specifically, there are several additional questions that emerge:
What are the most problematic relationships in the bird tree based on the data and
analyses that are currently available? And finally, what are likely to be the best ways
forward to resolve many of the very difficult problems that still exist across the avian
tree?
In this chapter we seek to address these questions by examining several funda-
mental issues relevant to the theory and practice of phylogenetics, using the evolu-
tion of birds as a “model system” to understand phylogenomic methods. We focus
exclusively on extant birds (Neornithes) and refer readers to recent reviews (Brusatte
et al. 2015; Wang and Zhou 2017) for discussions of Mesozoic birds. We also avoid
discussion of non-avian dinosaurs, although we would like to point interested
readers to the chapter on “dinosaur genomics” by Griffin et al. (2019). This chapter
begins by highlighting the progress that has been made (or has not been made) in
elucidating the avian tree of life, largely focusing on higher-level taxonomic
relationships. Throughout, we have used common names (unless their use is
unwieldy) in order to make this chapter more accessible to readers working outside
of avian systematics (the scientific names associated with those common names in
Table 3). Then we discuss the lessons of current genome-scale efforts to estimate the
bird tree, focusing on analytical challenges, such as the impact of “data-type effects”
(Reddy et al. 2017) and the computational challenges (e.g., the need to use more than
400 years of CPU time to analyze 48 bird genomes; see Jarvis et al. 2014). We place
this review of avian phylogenomics in a broader context, discussing the potential for
phylogenomics to have an impact on the fields of systematics, paleontology, geno-
mics, molecular biology, evolutionary developmental biology (“evo-devo”), and
biodiversity studies more generally. We also discuss the potential for those fields
to influence avian phylogenomics.
We have sometimes been deliberately provocative in this review, making it our
goal to summarize hypotheses embraced by many in the avian systematics commu-
nity and to play devil’s advocate regarding those hypotheses. We believe that this
will stimulate integrative studies to answer the many remaining questions in avian
phylogeny. Finally, we take a look forward and discuss the potential impact of
very high-quality (“platinum” or “reference” quality) genome assemblies generated
using third- and fourth-generation sequencing technologies (for recent reviews of
sequencing technologies, see Bleidorn 2016; Feng et al. 2015; Korlach et al. 2017).
We certainly expect platinum-quality genome assemblies to have a major impact
on phylogenomics, especially when those high-quality data are combined with
improved analytical methods. However, the phylogenomic data available at this
time (e.g., sequence capture and draft genome assemblies) have already enabled the
community to solve phylogenetic problems that have long been thought to be
intractable; we expect this trend to continue.
2 What We Do (And Do Not) Know About the Avian Tree
Systematic studies during the twenty-first century rapidly affirmed the

non-monophyly of many traditionally recognized orders, notably Gruiformes,
Ciconiiformes, Pelecaniformes, and Falconiformes. All of these figured prominently
in classifications for more than a century, and their ordinal names have been retained
in modern taxonomies (Table 3). However, the results of analyses using molecular
data required subsuming some traditional orders into more inclusive ones (e.g.,
Ciconiiformes within Pelecaniformes, Apodiformes within Caprimulgiformes) or
defining other orders more narrowly (e.g., Gruiformes and Falconiformes). Like-
wise, some largely abandoned names have been resurrected (e.g., Accipitriformes)
and some families to have been reassigned to ordinal rank (e.g., Mesitornithiformes
and Eurypygiformes). Efforts based on many genes, including some early
phylogenomic efforts, have also begun to resolve superordinal clades with some
degree of confidence (see below). In some cases, these studies have revealed
counterintuitive relationships, like the sister relationships of grebes and flamingos
as well as the placement of tropicbirds sister to Eurypygiformes (Sunbittern
and Kagu).
Table 3 Comparison of ordinal circumscriptions in commonly used avian taxonomies (“—” indicates ordinal name identical to H&Ma)
Taxa H&M Clements IOC HBW “Traditional”
Number of orders: 37 40 40 36 n/a
Number of species: 10,021 10,550 10,694 10,964 n/a
Palaeognathae
Ostrich Struthioniformes — — — —
Rheas Rheiformes — — Struthioniformes —
Emu and cassowaries Casuariiformes — — Struthioniformes —
Kiwis Apterygiformes — — Struthioniformes —
Elephant birds{ Aepyornithiformes
Moas{ Dinornithiformes
Tinamous Tinamiformes — — Struthioniformes —
Galloanseres
Waterfowl Anseriformes — — — —
Landfowl Galliformes — — — —
Neoaves
1. Telluraves (“core landbirds”)
1a. Australaves (originally “Australavis” in Ericson 2012)
Passerines Passeriformes — — — —
Parrots Psittaciformes — — — —
Falcons Falconiformes — — — —
Seriemas Cariamiformes
Resolving the Avian Tree of Life from Top to Bottom: The Promise and. . .
— — — —
1b. Afroaves (monophyly uncertain; Cavitaves sensu Yuri et al. 2013 italicized)
Woodpeckers and allies Piciformes — — — —
Puffbirds and jacamars Piciformes Galbuliformes — — —
Rollers and allies Coraciiformes — — — Coraciiformes
Hornbills and allies Bucerotiformes — — — Coraciiformes
Trogons Trogoniformes — — — —
157
(continued)
Table 3 (continued)
158
Taxa H&M Clements IOC HBW “Traditional”

Cuckoo roller Leptosomiformes — — — Coraciiformes
Mousebirds Coliiformes — — — —
Owls Strigiformes — — — —
Hawks and allies Accipitriformes — — — Falconiformes
New World vultures Accipitriformes Cathartiformes — Cathartiformes Falconiformes
2. Aequornithes (“core waterbirds”)
Penguins Sphenisciformes — — — —
Tubenoses Procellariiformes — — — —
Pelicans Pelecaniformes — — — See text
Cormorants and allies Pelecaniformes Suliformes Suliformes Suliformes —
Storks Pelecaniformes Ciconiiformes Ciconiiformes Ciconiiformes Ciconiiformes
Loons Gaviiformes — — — —
3. Phaethontimorphae
Sunbittern and Kagu Phaethontiformes — — — Pelecaniformes
Tropicbirds Eurypygiformes — — — Gruiformes
4. Otidimorphae
Cuckoos Cuculiformes — — — Cuculiformes
Bustards Otidiformes — — — Gruiformes
Turacos Musophagiformes Cuculiformes — — Cuculiformes
5. Caprimulgiformes (Strisores)
Hummingbirds Caprimulgiformes — Apodiformes — Apodiformes
Swifts and tree-swifts Caprimulgiformes — Apodiformes — Apodiformes
Owlet-nightjars Caprimulgiformes — Apodiformes — —
Frogmouths Caprimulgiformes — — — —
Nightjars Caprimulgiformes — — — —
Potoos Caprimulgiformes — — — —
E. L. Braun et al.
Oilbird Caprimulgiformes — — — —
6. Columbimorphae
Mesites Mesitornithiformes — — — Gruiformes
Sandgrouse Pterocliformes — — — Columbiformes
Doves Columbiformes — — — —
7. Phoenicopterimorphae (Mirandornithes)
Grebes Podicipediformes — — — —
Flamingos Phoenicopteriformes — — — —
“Orphan orders” (Cursorimorphae sensu Jarvis et al. 2014 italicized, but monophyly uncertain)
Hoatzin Opisthocomiformes — — — See text
Cranes, rails, and allies Gruiformes — — — —
Shorebirds Charadriiformes — — — —
a
H&M ¼ (Dickinson and Christidis 2014; Dickinson and Remsen Jr. 2013); Clements ¼ (Clements et al. 2017); IOC ¼ IOC World Bird List (Gill and Donsker
2017); HBW ¼ (del Hoyo et al. 2017)
{
extinct
Resolving the Avian Tree of Life from Top to Bottom: The Promise and. . .
159
Below we showcase what is currently known with reasonable certainty about the
relationships of the supraordinal lineages of birds (summarized as a consensus tree in
Fig. 1), highlighting the contributions of phylogenomics to our understanding of the
bird tree. At present, Jarvis et al. (2014) and Prum et al. (2015) are the largest-scale
estimates of the avian tree. Jarvis et al. (2014) presented many trees, but we largely
focus on the “total evidence nucleotide tree” (TENT), which was based on a
maximum likelihood (ML) analysis of a data matrix comprising intronic, exonic,
and UCE data. Prum et al. (2015) presented a single tree based on a Bayesian
analysis of a largely exonic dataset. They also provided an ML tree with a virtually
identical topology but much lower support; in this chapter, we focus on the ML
bootstrap support because it is more comparable to the support values associated
with Jarvis et al. (2014) trees. The tree in Fig. 1 is a strict consensus of the Jarvis et al.
(2014) TENT and the Prum et al. (2015) tree, modified to include information about
specific taxa that were not sampled by Jarvis et al. (2014). For those taxa that were
not included in Jarvis et al. (2014), we also considered Reddy et al. (2017), who
presented ML and Bayesian analyses of a data matrix dominated by noncoding
sequences with a sample of taxa similar to the Prum et al. (2015) study. We view the
consensus tree in Fig. 1 as the best corroborated hypothesis for the bird tree, but the
topology of the bird tree remains far from certain at this time. We emphasize this
uncertainty by calling attention to the key unsolved problems of higher-level
relationships.
2.1 Palaeognathae (“Ratites” and Tinamous)
One of the most surprising results from the first comprehensive avian phylogeny
based on multiple nuclear loci (Hackett et al. 2008) was the failure to support
monophyly of ratites (the large, flightless paleognaths such as the ostrich and
emu). Indeed, Hackett et al. (2008) had 100% bootstrap support for a node
contradicting ratite monophyly, placing ostriches sister to a clade comprising the
other ratites and the volant tinamous. Ratites had long been regarded as a textbook
exemplar (e.g., Bergstrom and Dugatkin 2012; Futuyma 2005; Stearns and Hoekstra
2005) of Gondwana vicariance following a single loss of flight in their common
ancestor (Cracraft 1973, 1974). The widespread occurrence of volant Paleogene
paleognaths (lithornithids; Houde 1986, 1988) certainly raised questions regarding
the prevailing paradigm that the distribution of ratites reflects a single loss of flight
and vicariance due to the breakup of Gondwana in the Cretaceous. Likewise,
analyses of complete mitochondrial genomes conducted shortly before Hackett
et al. (2008) reported, at best, equivocal support for ratite monophyly (Braun and
Kimball 2002; Slack et al. 2007) and analyses of some nuclear loci conflicted with
ratite monophyly (MYC in Cracraft et al. 2004; GH1 in Yuri et al. 2008; combined
CLTC and CLTCL1 in Chojnowski et al. 2008). However, an explicit hypothesis of
ratite non-monophyly based on broad sampling of the genome was not advanced
until Hackett et al. (2008). Ratite non-monophyly does not, in and of itself, falsify
the Gondwana biogeography hypothesis. However, it does raise the possibility
Ostrich Struthioniformes
Rheas Rheiformes
Palaeognathae
Emu & Cassowaries Casuariiformes
Kiwis Apterygiformes
Elephant birds Aepyornithiformes
Moas Dinornithiformes
Tinamous Tinamiformes
Waterfowl Anseriformes
Landfowl Galliformes
Galloanseres
Flamingos Phoenicopteriformes
Grebes Podicipediformes
Neoaves
7 (Columbea)
Doves Columbiformes
Sandgrouse Pterocliformes
6 Mesites Mesitornithiformes
Shorebirds Charadriiformes
Cranes, Rails Gruiformes Neoaves
Hoatzin Opisthocomiformes (Passerea)
Oilbird Caprimulgiformes
Potoos Caprimulgiformes
Nightjars Caprimulgiformes
5 Frogmouths Caprimulgiformes
Owlet-nightjars Caprimulgiformes
Swifts Caprimulgiformes
Hummingbirds Caprimulgiformes
Turacos Musophagiformes
#
Bustards Otidiformes
4 Cuckoos Cuculiformes
Tropicbirds Phaethontiformes
3 Sunbittern (& Kagu) Eurypygiformes
Loons Gaviiformes
Pelicans & allies Pelecaniformes
2 Tubenoses Procellariiformes
Penguins Sphenisciformes
New World vultures Accipitriformes
Eagles, Hawks Accipitriformes
Owls Strigiformes
Mousebirds Coliiformes
Cuckoo-roller Leptosomiformes
Trogons Trogoniformes
Hornbills & allies Bucerotiformes
1 Rollers & allies Coraciiformes
Woodpeckers & allies Piciformes
Seriemas Cariamiformes
Falcons Falconiformes
Parrots Psittaciformes
Passerines Passeriformes
Fig. 1 Consensus phylogenomic tree of birds. This backbone is a strict consensus of the Jarvis
et al. (2014) “total evidence nucleotide tree” (TENT) and the Prum et al. (2015) tree. The division
of Neoaves into Columbea and Passerea is based on Jarvis et al. (2014), although the division
is not presented in the consensus tree because it is not present in the Prum et al. (2015) tree.
The numbered clades correspond to the “magnificent seven” of Reddy et al. (2017): (1) “core”
landbirds (Telluraves); (2) “core” waterbirds (Aequornithes); (3) tropicbirds and Sunbittern
of dispersal by a volant ancestor followed by independent losses of flight. The

initial suggestion of ratite monophyly was followed by a detailed reanalysis of
the Early Bird data focused on determining whether the support for ratite
non-monophyly reflects misleading phylogenetic signal (Harshman et al. 2008);
that study did not reveal any sources of bias (for a detailed discussion of bias in
phylogenetic analyses, see below, in Sect. 3). Subsequent molecular studies, includ-
ing reanalyses of mitochondrial DNA (Phillips et al. 2010), the addition of nuclear
genes (Baker et al. 2014; Haddrath and Baker 2012; Smith et al. 2013), analyses of
transposable element (TE) insertions (Baker et al. 2014; Haddrath and Baker 2012),
and whole-genome analyses (Sackton et al. 2018), have also corroborated ratite
non-monophyly. However, those studies have shown conflicts regarding the other
relationships within paleognaths (we emphasize this by presenting the base of
Palaeognathae except ostriches as a polytomy in Fig. 1).
The recent results raise a profound question about our understanding of
paleognath evolution: how strongly corroborated is the new paradigm? It is impor-
tant to recognize that this new view of palaeognath history actually has three major
components: (1) ratites are not monophyletic; (2) ratites had a volant and vagile
ancestor that lost flight independently after dispersing; and (3) the morphological
features that unite ratites reflect convergence. The first of these is strongly
corroborated, although the phylogenomic study of Le Duc et al. (2015), which
included an analysis of 623 coding regions, supported ratite monophyly. However,
the Le Duc et al. (2015) results are unlikely to be accurate because their taxon
sampling was poor (the only paleognaths sampled were ostrich, a kiwi, and a
tinamou) and analyzed coding data, which is more likely to be misleading than
noncoding sequences (see below, in Sect. 3). It is possible to argue that the second
and third components of the current hypothesis are more equivocal. They are also the
more interesting components of the current hypothesis. A tree topology that nests the
volant tinamous within the flightless ratites does not provide definitive evidence for
multiple losses of flight; logically, it could reflect a single loss of flight followed by a
reversal to a volant state in tinamous. In fact, a skeptic might point out that a single
loss of flight followed by the reacquisition of flight in tinamous is actually the most
parsimonious optimization of the volant/flightless character state. Several authors
(e.g., Harshman et al. 2008; Smith et al. 2013) argued against that position by
pointing out that there are many examples of evolution from a volant to flightless
state (e.g., Wright et al. 2016) whereas evidence for evolution in the other direction
is absent. That argument provides evidence for multiple losses of flight within
paleognaths when the data are interpreted in a likelihood framework. The final
Fig. 1 (continued) (Phaethontimorphae); (4) cuckoos, bustards, and turacos (Otidimorphae);

(5) nightjars, swifts, hummingbirds, and allies (Caprimulgiformes); (6) doves, mesites, and sand-
grouse (Columbimorphae); and (7) flamingos and grebes (Phoenicopterimorphae). We indicate the
limited support for Otidimorphae using a hash (#). Reddy et al. (2017) called shorebirds, cranes, and
the Hoatzin the “orphan orders”; shorebirds and cranes form a clade (Cursorimorphae) in the Jarvis
et al. (2014) TENT but they represent independent lineages in the Prum et al. (2015) tree
component of the current hypothesis (that the other features that appear to unite
ratites arose by convergence) might appear to have been resolved by Johnston (2011),
who presented a morphological phylogeny that supports ratite non-monophyly and
places ostrich sister to all other paleognaths (like the molecular studies). However,
the vast majority of morphological phylogenies support ratite monophyly, including
recent studies (Bourdon et al. 2009; Worthy and Scofield 2012; and the uncon-
strained analyses in Worthy et al. 2017). This suggests a truly remarkable degree of
convergence if the current molecular hypothesis is correct.
Understanding the developmental basis for the loss of flight in different ratite
lineages could provide a direct way to examine the multiple loss of flight hypothesis.
Faux and Field (2017) found that tinamous retain the ancestral pattern of wing length
development (assuming the chicken character state is ancestral) whereas ostriches
and emus exhibit different patterns of wing development. However, Faux and Field
(2017) ultimately map three character states onto a four-taxon tree; thus, all possible
topologies are equally parsimonious (all trees require two character state changes to
explain the observed data). It will probably be necessary to understand the molecular
basis for the loss of flight in each lineage to resolve this issue definitively. Whole-
genome sequencing along with the identification of functional elements (using
approaches similar to Seki et al. 2017) is likely to facilitate the necessary evo-devo
studies. Sackton et al. (2018) used this approach, analyzing 14 paleognath genome
assemblies and identifying 63 noncoding elements that are likely to be transcrip-
tional enhancers that also exhibit an unusually high degree of sequence divergence
in ratites. They examined one of these “ratite-accelerated regions” experimentally,
finding that the chicken or tinamou sequences had enhancer activity in the developing
chick forelimb, whereas the orthologous rhea sequence did not. Similar experiments
focused on “ratite-accelerated regions” from other paleognath species should be very
informative (e.g., Cloutier et al. 2018). Examining the activity of “resurrected”
ancestors of these regions (i.e., sequences reflecting computational ancestral state
reconstructions) could be even more informative; the types of experiments could be
conducted by combining standard ancestral state reconstruction methods (e.g.,
Huelsenbeck and Bollback 2001) with approaches from synthetic biology (reviewed
by Hughes and Ellington 2017). These types of tools are likely to usher in a new era
for our understanding of these fascinating birds.
Studies focused on the phylogenetic position of extinct paleognaths (moas and
elephant birds) represent another exciting research area in that they have now moved
into the phylogenomic era (Baker et al. 2014; Grealy et al. 2017; Yonezawa et al.
2017). Surprisingly, these investigations have further corroborated earlier studies
that placed the Neotropical tinamous as sister to the extinct New Zealand moas
(Haddrath and Baker 2012; Phillips et al. 2010; Smith et al. 2013), on the one hand,
and the New Zealand kiwis sister to the extinct Malagasy elephant birds, on the other
(Mitchell et al. 2014). Estimates of divergence times for key taxa in these clades
(50–60 Mya for the kiwi and elephant bird; Mitchell et al. 2014; Yonezawa et al.
2017) have also been interpreted as precluding plausible avenues of overland
dispersal. The very young age for crown paleognaths inferred by Prum et al.
(2015) implies that those divergence times could be even more recent. Berv and
Field (2018) suggested the conflicts in divergence times reflect accelerated molecu-
lar evolution early in the Paleogene. They attributed this to a combination of the
observation that birds with smaller body size also tend to exhibit higher rates of
molecular evolution combined with the “Lilliput effect,” which is the tendency for
the lineages that survive mass extinctions to exhibit a marked decrease in body size
(Urbanek 1993). However, estimates of paleognath divergence time are probably too
recent for overland dispersal even if the Berv and Field (2018) hypothesis of an early
Paleogene rate acceleration is incorrect; if their hypothesized rate acceleration
is correct, it would provide additional evidence against overland dispersals. Those
late divergences among paleognaths have been viewed as additional evidence
corroborating the multiple loss of flight hypothesis (reviewed by Allentoft and
Rawlence 2012; for a dissenting argument, see Worthy and Scofield 2012, p. 88).
Regardless of the details, it seems clear that studies focused on both extant and
extinct paleognaths will continue to provide many interesting findings in the
genomic era.
2.2 Galloanseres (Landfowl and Waterfowl)
In sharp contrast to Palaeognathae and Neoaves (see below), in which there is

substantial uncertainty regarding many relationships, the picture for Galloanseres
is one of greater certainty. Monophyly of Galloanseres was initially controversial
(e.g., Ericson 1996), but that controversy was resolved prior to the phylogenomic era
(cf. Cracraft 2001). Relationships among the families were also established without
phylogenomic data, with Cox et al. (2007) resolving the last major question, the
positions of New World quail (Odontophoridae) and guineafowl (Numidae), using
only eight nuclear loci and three mitochondrial regions. Phylogenomic approaches
have been remarkably successful within galloanserine families; species-rich trees
with 100% bootstrap support at almost every node are now available, using sequence
capture data for Galliformes (summarized in Table 2) and more than 6.6 million base
pairs (Mbp) of coding sequence data for Anseriformes (Ottenburghs et al. 2016a).
There are certainly a few relationships that remain poorly supported, both in
Galliformes (Hosner et al. 2015b; Meiklejohn et al. 2016) and Anseriformes (e.g.,
Reddy et al. 2017 was unable to resolve the radiation of tribes within Anatidae with
confidence). Indeed, the poor resolution of anatid tribes might be viewed as overly
pessimistic based on Sun et al. (2017), although that study reflected analyses of
the mitochondrial genome which is ultimately a single gene tree that can differ from
the species tree (see below in Sect. 3). Regardless, problematic nodes within
Galloanseres are the exception and not the rule. Likewise, some taxa have not
been included in phylogenomic trees because they have only been sampled for a
limited amount of data (or because molecular data remain unavailable). However,
analyses of sparse supermatrices that combine legacy markers (sequences obtained
by PCR) with phylogenomic data have proven to be a successful strategy even in
those cases for which specific taxa are data limited (e.g., Hosner et al. 2016; Persons
et al. 2016). Overall, it is very likely that a species-level phylogenomic tree of
Galloanseres will be completed in the near future (at least for the level of species
named in current checklists).
One area in which our knowledge of the early evolution of Galloanseres is limited
is the estimation of time-calibrated trees; this is problematic because the oldest
neornithine fossils are putatively galloanserines. The oldest of these, Austinornis
lentus from the Cretaceous Austin chalk, can probably be dismissed. The fossil plac-
ing Austinornis within Galloanseres (as a stem galliform) is fragmentary, and Clarke
(2004) was only able to score it for 9 of 202 characters; the skeptical position that
Austinornis is a stem galloanserine (or even some other Cretaceous bird lineage) is
more appropriate than viewing it as evidence for the existence of crown Galloanseres
ca. 85 million years ago (Mya). Another putative Cretaceous galloanserine, Vegavis
iaai, has attracted substantial attention because Clarke et al. (2005) placed it within
Anseriformes with high (99%) bootstrap support. However, more recent analyses
place Vegavis outside crown Anseriformes (Agnolín et al. 2017; Lee et al. 2014;
O’Connor and Zhou 2013; Worthy et al. 2017). Indeed, Mayr et al. (2018) went
further and questioned whether Vegavis was even galloanserine. Placing Vegavis
within crown Anseriformes has a major impact on divergence time estimates for the
avian tree as a whole (e.g., Prum et al. 2015), so resolving its position with
confidence is critical. Ancient galloanserine fossils that are reliably placed within
crown Anseriformes do exist (e.g., Prebyornithidae; De Pietri et al. 2016), but they
are younger than Vegavis. For example, the oldest fossil placed within the
anseriform crown in the maximum parsimony (MP) and Bayesian analyses of
Worthy et al. (2017) was the Eocene Presbyornis pervetus. Kurochkin et al.
(2002) did place the Cretaceous Teviornis gobiensis in Presbyornithidae, but the
presbyornithid affinities of Teviornis are questionable (Clarke and Norell 2004).
Indeed, all of the putative upper Cretaceous fossils assigned to extant avian orders,
including a number of galloanserines (see Hope 2002), are quite fragmentary.
Fountaine et al. (2005) suggested the fragmentary nature of the Cretaceous
neornithine fossil record reflects a biological signal given the collecting efforts; if
so, it strongly suggests that the ancient calibrations that have been used for
Galloanseres in many clock studies are inappropriate. Even within the Paleogene,
a number of galloanserine fossils appear to have been placed incorrectly (Ksepka
2009; Wang et al. 2016). Finding better ways to incorporate fossil evidence is likely
to be more important for establishing divergence times for Galloanseres (and birds as
a whole) than the availability of genome-scale sequence data.
2.3 Neoaves (All Remaining Extant Birds)
Recent phylogenomics studies support the division of Neoaves into ten major
lineages, seven of which contain multiple orders. Reddy et al. (2017) called the
superordinal clades the “magnificent seven” and referred to the remaining three
lineages as the “orphan orders.” Interrelationships among these major lineages
remain poorly resolved [Fig. 1; also see Thomas (2015) for another comparison of
the Jarvis et al. (2014) and Prum et al. (2015) trees]. Moreover, differences across
recent studies are sensitive to the data type used for analyses (i.e., the estimates of
phylogeny differ depending on whether exons, introns, noncoding ultraconserved
elements, conserved non-exonic elements, or TE insertions were used for analyses;
Jarvis et al. 2014; Reddy et al. 2017). The observed conflict among analyses is so
striking that Suh (2016) suggested that the relationships among these taxa might
reflect a hard polytomy. We are skeptical of this extreme hypothesis; there has been
remarkable progress toward a satisfying resolution of Neoaves in the early part of the
phylogenomic era (i.e., the convincing evidence for the magnificent seven). We
believe this progress represents a good reason to expect that continued data collec-
tion and method development will ultimately resolve relationships among these
major groups. However, it is clear that branching did occur over a very short interval
of time early in the Paleocene, and this has made Neoaves one of the most difficult
problems in modern phylogenetics. Below we summarize the ongoing progress
made toward resolving the neoavian tree and highlight some of the remaining
open questions (although we emphasize that our discussion is not exhaustive).
The Base of the Neoavian Tree Identifying the basal split within the Neoaves has
long been a vexing problem, but one that is seemingly approaching resolution. Initial
large-scale analyses (Fain and Houde 2004; Ericson et al. 2006; Hackett et al. 2008)
identified a large but relatively poorly supported cluster called Metaves (we indicate
the “metavian” in Fig. 2 with an asterisk). Metaves comprised Caprimulgiformes
(as currently defined to include nightjars, swifts, hummingbirds, and allies) along
with doves, mesites, sandgrouse, flamingos, grebes, the Kagu and Sunbittern
(Eurypygiformes), tropicbirds, and (in some analyses) the Hoatzin. Those studies
placed Metaves sister to all other neoavian taxa, the latter being designated
Coronaves by Fain and Houde (2004). Kimball et al. (2013) showed that the signal
supporting Metaves was almost exclusively associated with a single locus
(FGB/β-fibrinogen). Phylogenomics is based on the idea that analyses of many
loci can overcome the existence of misleading signal in any individual locus. The
preponderance of the phylogenomic data of Jarvis et al. (2014) clarified the phylog-
eny of metaves, identifying a cluster that includes flamingos and grebes along with
doves, sandgrouse, and mesites (collectively named Columbea by Jarvis et al. 2014)
and placing that clade sister to all other Neoaves (termed Passerea; Jarvis et al.
2014). In contrast, Prum et al. (2015) places Caprimulgiformes sister to all other
Neoaves (Fig. 2). Both Jarvis et al. (2014) and Prum et al. (2015) dispersed the other
metavian taxa across several other basal neoavian lineages.
Reddy et al. (2017) analyzed the topological conflicts between Jarvis et al. (2014)
and Prum et al. (2015) and concluded that the observed conflicts reflect “data-type
effects,” which they defined as the observation that there are “different signals
associated with analyses of subsets of the genome that can be defined a priori
using non-phylogenetic criteria.” The data used to generate the Prum et al. (2015)
tree were 82.5% exonic, and this very probably led to incorrect taxonomic groupings
on their tree, whereas the Jarvis et al. (2014) TENT reflects an analysis of 41.8 Mbp
that included a mixture of introns, coding exons (first and second codon positions
only), and noncoding UCEs. Reddy et al. (2017) argued that exons are more likely to
A Jarvis et al. TENT

7
B Prum et al. anchored hybrid enrichment tree
Flamingos Phoenicopteriformes* Nightjars & allies Caprimulgiformes*
Columbea
Grebes Podicipediformes* 5 Cuckoos Cuculiformes
Doves Columbiformes* 4 Bustards Otidiformes
Sandgrouse Pterocliformes* # Turacos Musophagiformes
6
Mesites Mesitornithiformes* # Doves Columbiformes*
5 Nightjars & allies Caprimulgiformes* Sandgrouse Pterocliformes*
6
Cuckoos Cuculiformes Mesites Mesitornithiformes*
Bustards Otidiformes Cranes, Rails Gruiformes
4
# Turacos Musophagiformes 7 Flamingos Phoenicopteriformes*
Hoatzin Opisthocomiformes* Grebes Podicipediformes*
Shorebirds Charadriiformes # Shorebirds Charadriiformes
Cranes, Rails Gruiformes 3 Tropicbirds Phaethontiformes*
3 # # Sunbittern Eurypygiformes*
Tropicbirds Phaethontiformes*
Sunbittern Eurypygiformes* Loons Gaviiformes
Loons Gaviiformes Expanded Pelicans & allies Pelecaniformes
#
Pelicans & allies Pelecaniformes Waterbird 2 Tubenoses Procellariiformes
2
Passerea
Tubenoses Procellariiformes cade Penguins Sphenisciformes
Penguins Sphenisciformes Hoatzin Opisthocomiformes*
New World vultures Accipitriformes New World vultures Accipitriformes
Eagles, Hawks Accipitriformes Eagles, Hawks Accipitriformes
Owls Strigiformes # Owls Strigiformes
Mousebirds Coliiformes Mousebirds Coliiformes
# Cuckoo-roller Leptosomiformes
Cuckoo-roller Leptosomiformes 1
Trogons Trogoniformes Trogons Trogoniformes
Hornbills & allies Bucerotiformes Hornbills & allies Bucerotiformes
1 #
Rollers & allies Coraciiformes Rollers & allies Coraciiformes
Woodpeckers & allies Piciformes Woodpeckers & allies Piciformes
Seriemas Cariamiformes Seriemas Cariamiformes
Falcons Falconiformes Falcons Falconiformes
Parrots Psittaciformes Parrots Psittaciformes
Passerines Passeriformes Passerines Passeriformes
Fig. 2 The Jarvis et al. (2014) TENT and the Prum et al. (2015) anchored hybrid enrichment
(sequence capture) tree exhibit many differences at the base of Neoaves. Both of these trees are
presented as rooted trees for Neoaves and taxa placed in “Metaves” (see text) are indicated with
asterisks. (a) Jarvis et al. (2014) TENT with low-support branches (branches with <100% bootstrap
support) indicated using thin lines. Very low-support branches (branches with <70% bootstrap
support) are indicated with a hash (#) below the relevant branch. The strongly supported basal
division of Neoaves into Columbea and Passerea is indicated. (b) Prum et al. (2015) tree with
low-support (branches with <70% bootstrap support) and very low-support (branches with <50%
bootstrap support) are indicated in the same manner. Taxa placed in Columbea and Passerea are also
indicated to side of the Prum et al. (2015) tree to emphasize their non-monophyly in that analysis; an
“expanded waterbird clade” (named Aequorlitornithes by Prum et al. 2015) is indicated using a gray
box. We used different bootstrap support cutoffs for the two trees because they are based on data
matrices of different sizes (more than 13,000 loci for the Jarvis et al. 2014 TENT but only 259 loci
for the Prum et al. 2015 tree)
be misleading than noncoding regions for two reasons: (1) coding exons exhibited
greater GC-content variation than noncoding regions and (2) the structure of the
genetic code combined with selection to maintain the amino acid sequence
represents a violation of most models used for phylogenetic analyses. However,
the fundamental observations underlying Reddy et al. (2017) were empirical:
(1) analyses of a largely noncoding 54-locus data matrix for 235 species supported
the same basal split in Neoaves as the Jarvis et al. (2014) TENT; and (2) trees based
on large-scale coding datasets exhibited more topological similarities to each other
than to trees based on noncoding data. Reddy et al. (2017) also found that trees based
on rare genomic changes, like TE insertions, were more congruent with the trees
based on noncoding data than with the trees based on coding data (see Fig. 6 in
Reddy et al. 2017). Taken as a whole, those results indicate that the observed
differences between Jarvis et al. (2014) and Prum et al. (2015) are more likely to
reflect data type than taxon sampling. We address below some of these remaining
conflicts revealed by these three studies as they represent some of the most important
puzzles in avian phylogenetics.
Phoenicopteromorphae (Clade 7; Also Called Mirandornithes)

and Columbimorphae (Clade 6) There is little question that a close relationship
between flamingos and grebes (clade 7 in Fig. 2) was astonishing to most
ornithologists when it was first proposed (Van Tuinen et al. 2001). However, that
node was relatively easy to resolve (even with mitochondrial DNA; Van Tuinen
et al. 2001; also see Cracraft et al. 2004, p. 476) and was present in the results of
analyses conducted by many different investigators in multiple labs (e.g., Chubb
2004; Fain and Houde 2004; Mayr 2004a; Ericson et al. 2006; Hackett et al. 2008).
From a result that few believed at the time, it is now solidly accepted. The closest
relative of the flamingo-grebe clade is another matter. Hackett et al. (2008) placed
them sister to a subset of metavian taxa, in a clade comprising doves, mesites, and
sandgrouse (clade 6 in Fig. 2) and tropicbirds. Jarvis et al. (2014) placed flamingos
and grebes sister to doves, mesites and sandgrouse, separating the tropicbirds from
both lineages. Importantly, the clade comprising doves, mesites, sandgrouse,
flamingos, and grebes (Columbea) was strongly (100% bootstrap) supported and
sister to all other Neoaves (Passerea). Reddy et al. (2017) also recovered the deep
division between Columbea and Passerea, albeit with lower bootstrap support
(95% for Columbea but only 50–90% for Passerea). In sharp contrast, Prum
et al. (2015) did not recover Columbea or Passerea. Instead, Prum et al. (2015)
placed flamingos and grebes sister to shorebirds in a “generalized waterbird clade”
(emphasized using a gray box in Fig. 2). Provocatively, many analyses of coding
exons in Jarvis et al. (2014) also supported a generalized waterbird clade, albeit with
rearrangements relative to Prum et al. (2015). Those results are consistent with the
data-type effects hypothesis advanced by Reddy et al. (2017). Prum et al. (2015) also
supported monophyly of doves, mesites, and sandgrouse but placed that clade sister
to cuckoos, bustards, and turacos; they named this large clade, which comprises
clades 4 and 6 from Fig. 2, Columbaves. We consider the latter relationship to be less
likely given the nature of character support discussed by Reddy et al. (2017), but it
seems clear that the Columbea and Columbaves hypotheses both deserve additional
study.
Caprimulgiformes (Clade 5; Also Called Strisores) Many current taxonomies

treat Caprimulgiformes (the clade comprising nightjars, nighthawks, oilbirds,
potoos, frogmouths, owlet-nightjars, swifts, and hummingbirds) as a single order
(Table 3), but those diverse taxa were split into at least two orders in older
classifications (e.g., hummingbirds and swifts in Apodiformes; Table 3). For this
reason, Reddy et al. (2017) viewed Caprimulgiformes as one of their “magnificent
seven” superordinal clades. As described above, many analyses of coding exons
(including Prum et al. 2015) place Caprimulgiformes sister to all other Neoaves.
There is also uncertainty regarding the relationships among the families within
Caprimulgiformes. This could reflect a data-type effect, since Reddy et al. (2017)
place the potoo-oilbird clade sister to all other caprimulgiforms (Fig. 3a), whereas
Reddy et al. (2017) Prum et al. (2015)

A (non-coding data) B (coding data)
Oilbird Nightjars
# Potoos Oilbird
Nightjars Potoos
Frogmouths Frogmouths
#
Owlet-nightjars Owlet-nightjars
Hummingbirds Hummingbirds
Swifts Swifts
Treeswifts Treeswifts
C
Hummingbirds
Trochilidae
Owlet-nightjars Swifts
Aegothelidae Apodidae
Treeswifts
Reddy root Hemiprocnidae
Frogmouths
Podargidae Potoos
Nyctibiidae
Prum root
Nightjars & Nighthawks

Oilbird
Caprimulgidae
Steatornithidae
Fig. 3 The position of the root of Caprimulgiformes (clade 5) is uncertain. (a) Most analyses
reported by Reddy et al. (2017) place the root between the oilbird + potoos clade and all other
Caprimulgiformes, although some analyses (see supporting material for Reddy et al. 2017) place the
root between potoos and all other Caprimulgiformes. (b) Prum et al. (2015) place the root between
the nightjar family and all other Caprimulgiformes. (c) The unrooted ingroup topology for
Caprimulgiformes is identical in the Prum et al. (2015) and Reddy et al. (2017) analyses, but the
position of the root differs. Support is indicated as above, with thin branches or thin branches and a
hash (#). We do not consider Jarvis et al. (2014) in this figure because that study only included three
Caprimulgiformes (a nightjar, a swift, and a hummingbird); relationships among those taxa are
strongly supported in many analyses (including analyses of individual genes; cf. fig. 1b in Hackett
et al. 2008)
Prum et al. (2015) place nightjars and nighthawks (Caprimulgidae) sister to all
other caprimulgiforms (Fig. 3b). Both studies support a clade comprising the
five remaining families (frogmouths, owlet-nightjars, swifts, tree-swifts, and
hummingbirds). The uncertainty actually reflects alternative placements of the root
since both studies supported the same unrooted ingroup topology (Fig. 3c). Obvi-
ously, this group is an excellent target for additional phylogenomic analyses. In fact,
phylogenomic analyses of caprimulgiforms could be especially informative given

their extensive Paleogene fossil record (Mayr 2009, 2004b). Those fossils have long
suggested that nightjars and their relatives arose early in the neoavian radiation so
they can provide an excellent source of calibrations for molecular clock studies.
Otidimorphae (Clade 4) A clade comprising cuckoos, bustards, and turacos has

emerged in only the most recent phylogenomic studies (Jarvis et al. 2014; Prum
et al. 2015), although we do not view this group as decisively established (Fig. 1).
Clade 4 has strong (100%) bootstrap support in the Jarvis et al. (2014) TENT, which
placed cuckoos sister to a turaco-bustard clade. However, the position of turacos
sister to bustards is the most poorly supported clade in the Jarvis et al. (2014) TENT
(only 55% bootstrap support). Prum et al. (2015) does not place turacos sister to
bustards; instead, the Prum et al. (2015) tree places turacos sister to a cuckoo-bustard
clade. Clade 4 does have strong support in the Bayesian analysis reported by Prum
et al. (2015), but it has very low support (only 41%) in their ML tree. The cuckoo-
bustard clade was strongly supported (98% bootstrap) in the Prum et al. (2015) ML
analysis. Reddy et al. (2017) did not recover clade 4. Instead, Reddy et al. (2017)
recovered a cuckoo-bustard clade (albeit with limited support in many analyses)
and placed turacos elsewhere, sister to cranes, rails, and allies (Gruiformes).
Provocatively, Reddy et al. (2017) also found the turaco-gruiform clade in their
reanalyses of the Prum et al. (2015) dataset after excluding data that was also present
in the Jarvis et al. (2014) TENT dataset (this was done to produce a coding exon
dataset that was truly independent of Jarvis et al. 2014). Clearly, relationships among
these taxa, their relationships to other clades, or whether they even form a clade is
still an open question that is ripe for additional phylogenomic exploration.
Shorebirds, Cranes, Rails, and the Hoatzin (the “Orphan Orders” in Reddy
et al. 2017) The three orders containing shorebirds (Charadriiformes); cranes, rails,
and allies (Gruiformes); and the Hoatzin (Opisthocomus hoazin, the only extant
species in the order Opisthocomiformes) form a clade on the Jarvis et al. (2014)
TENT, although this clade did not receive 100% bootstrap support in the TENT. In
contrast to the TENT, the larger Jarvis et al. (2014) “whole-genome tree” placed
shorebirds sister to core landbirds (core landbirds are clade 1 in Fig. 2) and placed
caprimulgiforms as sister to gruiforms, with the Hoatzin as their sister. The whole-
genome tree reflected an analysis of 322 Mbp, more than seven times larger than the
TENT dataset. However, Jarvis et al. (2014) expressed concern that the sequence
alignment and the assessment of orthology for the whole-genome tree dataset were
inferior to the TENT dataset. Prum et al. (2015) placed the Hoatzin sister to the core
landbirds (clade 1), naming that more inclusive clade Inopinaves. Prum et al. (2015)
also separated shorebirds and gruiforms, placing the former in a clade with flamingos
and grebes and the latter sister to a larger neoavian clade (Fig. 2). Reddy et al. (2017)
also separated all three of these orders, albeit with low support. Additional
phylogenomic analyses are clearly necessary to resolve these relationships.
Other than relationships among palaeognathous birds (see above), perhaps the
most long-standing problem in avian higher-level relationships has been that of the
Hoatzin. The Hoatzin exhibits peculiar specializations, including a folivorous diet

and foregut fermentation as well as the hypertrophy and use of forelimb claws by
nestlings. These features fueled fanciful speculations that the Hoatzin might be
primitive among extant birds (Feduccia 1996; Olson 1985). Historically, many
authorities placed Hoatzin close to fowl (Galliformes) or within a questionably
monophyletic circumscription of Cuculiformes (in that case defined as comprising
both cuckoos and turacos). Studies that supported the latter position placed the
Hoatzin sister to either cuckoos or turacos (see Sibley and Ahlquist 1990 for review;
also see Hughes and Baker 1999; Sorenson et al. 2003). This uncertainty continues
today; as stated above, no phylogenomic study upholds any of the previous
hypotheses. Moreover, none of the comprehensive phylogenomic studies (Jarvis
et al. 2014; Prum et al. 2015; Reddy et al. 2017) agree with one another regarding the
position of the Hoatzin. The McCormack et al. (2013) UCE study, which did not
include all major avian clades, placed Hoatzin sister to shorebirds, similar to the
Jarvis et al. (2014) TENT (Fig. 2a). However, it placed gruiforms (represented in
that study by the trumpeter, family Psophiidae) in another position. The difficulty
of resolving the position of the Hoatzin likely reflects the rapid radiation of
Neoaves itself and the fact that the Hoatzin lineage diverged very close in time to
all lineages that are putative close relatives. Coupled with that is the monotypy of
Opisthocomiformes, which eliminates the possibility of breaking up its long-branch
stem. The fossil record unfortunately sheds little light on the issue except to
document that early hoatzins of modern appearance (as far as it is known from
limb bones) were distributed in South America, Europe, and Africa during the Oligo-
Miocene (Mayr 2014; Mayr et al. 2011; Mayr and De Pietri 2014).
Phaethontimorphae (Clade 3), Aequornithia (Core Waterbirds; Clade 2),

and Telluraves (Core Landbirds; Clade 1) These clades comprise many pheno-
typically distinct lineages. Clade 3 comprises tropicbirds and Eurypygiformes (the
Sunbittern and Kagu), lineages placed in Metaves by early phylogenomic studies
(Fig. 2). Later studies resolved them as a distinct lineage that is either related to core
waterbirds (clade 2; Fig. 4a) or core landbirds (clade 1; Fig. 4b). This could also be a
data-type effect; analyses that include exonic data (including the Jarvis et al. 2014
TENT) place the tropicbird-eurypygiform clade as sister to waterbirds, whereas
analyses of exclusively or almost exclusively noncoding data matrices [the analyses
of introns and noncoding UCEs in Jarvis et al. (2014) and Reddy et al. (2017)] place
the tropicbird-eurypygiform clade sister to landbirds. However, if this is a data-type
effect, it is unusual. Reddy et al. (2017) proposed the terminology data-type effects
in order to discuss the conflicts between the Jarvis et al. (2014) TENT and the Prum
et al. (2015) tree with respect to the deepest divergence in Neoaves. In this case, the
Jarvis et al. (2014) TENT (which reflects a data matrix that is 68% noncoding)
supports monophyly of Columbea (clades 6 and 7) and places of Columbea sister to
Passerea (all other Neoaves). The Jarvis et al. (2014) TENT and intron trees as well
as the Reddy et al. (2017) tree support reciprocal monophyly of Columbea and
Passera; the Jarvis et al. (2014) UCE tree supports monophyly of Passerea. Thus, the
TENT resembles trees based on noncoding data. In contrast, the position of the
A “Core” waterbirds Aequornithes C α Pelicans Pelecanidae
Tropicbirds Phaethontiformes Hamerkop Scopidae
β
Sunbittern Eurypygiformes Shoebill Balaenicipitidae
Pelecaniformes
Kagu Eurypygiformes Herons Ardeidae
“Core” landbirds Telluraves Ibises, Spoonbills Threskiornithidae
Jarvis et al. (2014) - TENT, WGT Darters Anhingidae
Prum et al. (2015) - coding Cormorants Phalacrocoracidae
B Gannets, Boobies Sulidae
“Core” waterbirds
Frigatebirds Fregatidae
“Core” landbirds Storks Ciconiidae
Tropicbirds Tubenoses Procellariiformes
Sunbittern Penguins Sphenisciformes
Kagu (Only in Reddy et al. 2017) Loons Gaviiformes
Jarvis et al. (2014) - introns, UCEs

Reddy et al. (2017) waterbird topology
Reddy et al. (2017) - non-coding
Fig. 4 Relationships among core landbirds (clade 1), core waterbirds (clade 2), and the
tropicbird + eurypygiform clade (clade 3). (a) Phylogeny based on TENT, WGT, and coding
data. (b) Phylogeny based on introns, UCEs, and non-coding data. The position of the
tropicbird + eurypygiform clade sister to either core waterbirds or core landbirds depends on the
data type analyzed (analyses of coding data and mixtures of coding and noncoding data support a
waterbird sister hypothesis, whereas analyses of noncoding data alone support a landbird sister
hypothesis). (c) The inset shows the topology within core waterbirds. Most large-scale relationships
within core waterbirds are strongly supported and robust both to data type and to analytical
approach, but there are two exceptions (indicated using Greek letters, see text for details)
tropicbird-eurypygiform clade in the TENT corresponds to its position in analyses of

coding data. Another potential explanation for the difficulty placing the tropicbird-
eurypygiform clade is that they are long branches. This does not reflect length due to
an accelerated rate of molecular evolution; instead it reflects the fact that neither
lineage has close relatives. There are only three tropicbird species, all of which are
very closely related and are placed in a single genus (Phaethon). There is another
extant eurypygiform (the New Caledonian Kagu; Fig. 4), which is placed in a
different family. Reddy et al. (2017) did break up the long branch to the Sunbittern
as much as possible by including Kagu, and it remains possible that adding genome-
scale Kagu data will be helpful; however, adding Kagu represents the limit for the
addition of taxa to clade 3 in a meaningful way. Thus, the practice of adding taxa,
which many systematists believe to be one of the best ways to improves estimates
of phylogeny (Heath et al. 2008), is unlikely to be a way to improve placement of
clade 3. Overall, because tropicbirds and eurypygiforms are both long branches that
can only be subdivided near the tips, improved phylogenomic analyses may repre-
sent the only hope for a convincing resolution of the position of the tropicbird-
eurypygiform clade.
The relationships among core landbirds, core waterbirds, and the tropicbird-
eurypygiform clade are somewhat more complex than we indicate in Fig. 4. The
TENT and intron tree in Jarvis et al. (2014) both support a clade comprising core
landbirds, core waterbirds, and tropicbirds + eurypygiforms, but most other analyses
place other lineages within that clade. Analyses using exonic data typically nest the
core waterbird + (tropicbird + eurypygiforms) clade within a larger clade comprising
many aquatic and semiaquatic lineages (e.g., shorebirds, flamingos, and grebes);
analyses using noncoding data separate those lineages. We believe this “greater
waterbird clade” (named Aequorlitornithes by Prum et al. 2015) is unlikely to be a
true clade because analyses of rare genomic changes, which are likely to have
different strengths and weaknesses relative to analyses of either or coding and
noncoding sequences (see below, in Sect. 3), yield a tree topology closer to the
noncoding trees (see Reddy et al. 2017).
The core waterbirds [clade 2, called Aequornithes by Mayr (2011) and
Aequornithia by Cracraft (2013)] are perhaps one of the most exceptional groups
in Neoaves: almost all major groups within this clade have the same topology in all
recent phylogenomic analyses. There are, however, two nodes of interest (branches
α and β in Fig. 4). Branch α, which unites pelicans with the Hamerkop, is especially
surprising; the RAxML (Stamatakis 2014) analyses in both Prum et al. (2015) and
Reddy et al. (2017) support the resolution shown in Fig. 4 whereas the Bayesian
analyses conducted in both of those studies support a different resolution (Hamerkop
sister to pelicans + shoebill). The Bayesian analyses in Prum et al. (2015) reflect the
use of ExaBayes (Aberer et al. 2014) whereas Reddy et al. (2017) used both
ExaBayes and MrBayes (Ronquist et al. 2012); thus, these results are not associated
with specific software. It is unclear whether this topological difference reflects the
details of the specific programs used for analyses or more fundamental differences
between ML analyses (where the parameters used for analyses are assigned the
values that result in the optimal likelihood score) and Bayesian analyses (where the
method integrates over the uncertainty in those parameters, assuming some prior
distribution for the parameter values). Regardless, the observed differences between
ML and Bayesian analyses as implemented in commonly used phylogenetic
programs deserve further scrutiny. The other branch (β in Fig. 4) is somewhat
more variable across analyses. Both of these remaining questions regarding core
waterbird phylogeny deserve attention in coming analyses of phylogenomic data.
Monophyly of core landbirds (clade 1, also called Telluraves; Yuri et al. 2013)
is strongly supported in almost all multigene analyses with sufficient taxon sampl-
ing that have been published since Hackett et al. (2008). Like the core waterbirds,
core landbirds comprise some of the most widespread and familiar avian
lineages, including raptors (hawks, eagles, falcons, and owls), songbirds and allies
(Passeriformes, typically called passerines), parrots, and the woodpeckers and their
allies (such as rollers, kingfishers, bee-eaters, hornbills, hoopoes and woodhoopoes,
trogons, and other lineages). The Jarvis et al. (2014) TENT, like a number of prior
analyses (e.g., Ericson et al. 2006; Hackett et al. 2008; Kimball et al. 2013), splits
core landbirds into two clades that Ericson (2012) named Australaves and Afroaves
(Table 3). That split renders the raptorial lineages para- or polyphyletic. Specifically,
the falcons are placed in Australaves sister to parrots and passerines whereas the
hawks, eagles, New World vultures, and owls are placed in Afroaves as the succes-
sive sister groups of a clade comprising mousebirds, and a diverse assemblage that
includes woodpeckers and their allies (Fig. 2a). However, the Jarvis et al. (2014)
TENT conflicts with the Prum et al. (2015) topology with respect to the position of
the clade comprising hawks, eagles, and New World vultures (Accipitriformes); the
TENT (and Reddy et al. 2017) places accipitriforms sister to all other Afroaves
whereas Prum et al. (2015) places them sister to all other core landbirds (Fig. 2b).
The Jarvis et al. (2014) analysis of first and second codon positions supported a third
topology, with a clade comprising accipitriforms and owls sister to all other core
landbirds. Although there is some conflict between the Jarvis et al. (2014) exon
analysis and the Prum et al. (2015) tree, this suggests the position of accipitriforms
could reflect another data-type effect.
Although differences between the results of analyses using coding vs. noncoding
data have emerged as a major source of conflict in the avian tree of life, differences
due to data-type effects are not sufficient to explain all of the conflicts at the base of
core landbirds; analytical methods also play an important role (Fig. 5). Analyses of
noncoding data using multispecies coalescent (MSC) methods (“species tree”
methods; see below in Sect. 3) yield trees that are more congruent with standard
concatenated analyses of exon data, either placing an accipitriform-owl clade or
accipitriforms alone sister to all other landbirds (Fig. 5). Thus, the position of
accipitriforms and owls represents an interesting case of uncertainty related to two
different factors: (1) data type and (2) whether the analytical method assumes a
single underlying tree or a mixture of trees (for additional details regarding analytical
methods, see below in the next section).
Mousebirds represent an equally striking source of conflict (Fig. 5). Many
analyses, regardless of data type or analytical approach, place mousebirds sister to
the diverse clade comprising woodpeckers and their allies (named Cavitaves by Yuri
et al. 2013; see Fig. 5). The analysis of UCEs in Jarvis et al. (2014) and the analysis
of TE insertions Suh et al. (2015) are the major exception; analyses of both of those
data types support monophyly of Afroaves, but they place mousebirds sister to the
other taxa in that clade. However, earlier multigene analyses recognized mousebirds
as a rogue taxon, shifting to various positions within core landbirds depending on the
analytical approach, taxon sample, and data (Suh et al. 2011; Wang et al. 2012;
McCormack et al. 2013). Those analyses include positions sister to or even within
Australaves or sister all other landbirds. Like tropicbirds and eurypygiforms,
mousebirds represent a long branch that can only be subdivided close to the tip.
This led Suh (2016) to favor the Fig. 5d topology by arguing that the UCE and TE
analyses are more resistant to the long-branch attraction artifact (see below in Sect.
3). However, Gilbert et al. (2018) found that the position of mousebirds was unstable
when they applied data filtering approaches designed to reduce noise to the Jarvis
et al. (2014) UCE data. The observation that analyses of UCE data are sensitive to
filtering does not refute the Fig. 5d topology. However, it does question one of the
examples of congruence advanced by Suh (2016) for favoring that topology (i.e., the
congruence of the UCE and TE analyses). These conflicts regarding the position of
mousebirds are especially frustrating given the excellent fossil record of this lineage
(Ksepka and Clarke 2009; Ksepka et al. 2017). However, when the available
A New World vultures B New World vultures

Eagles, Hawks Eagles, Hawks
Owls Owls
Mousebirds Mousebirds
Cavitaves Cavitaves
Australaves Australaves
Jarvis et al. (2014) - TENT, introns Jarvis et al. (2014) - binned intron MSC
Reddy et al. (2017) - non-coding Prum et al. (2015) - coding
Kimball et al. (2013) - non-coding MSC*
Edwards et al. (2017) - non-coding MSC*
Owls Mousebirds
C D
New World vultures New World vultures
Eagles, Hawks Eagles, Hawks
Mousebirds Owls
Cavitaves Cavitaves
Australaves Australaves
Jarvis et al. (2014) - TENT MSC, intron MSC Jarvis et al. (2014) - UCEs
Reddy et al. (2017) - coding (Prum noJAR) Suh et al. (2015) - TE insertions
Fig. 5 Relationships within core landbirds depend on data type and analytical approach. We
present four candidate topologies that have been recovered when different data types and analytical
approaches are used. Multispecies coalescent (“species tree”) analyses are indicated using “MSC”;
all other analyses used concatenated data. (a) Division into two major clades (Australaves and
Afroaves; see Table 3), present in the Jarvis et al. (2014) TENT and the Reddy et al. (2017) analyses
of concatenated noncoding data. (b) Accipitriformes sister to all other core landbirds, found in
the binned MP-EST (Mirarab et al. 2014a) analysis of intronic data reported by Jarvis et al. (2014)
and the Prum et al. (2015) concatenated tree. The Kimball et al. (2013) NJst analysis had as
similar topology (the asterisk indicates a minor rearrangement placing mousebirds sister to owls).
This topology was also supported by unbinned MP-EST analyses of three different noncoding
datasets in Edwards et al. (2017), although the taxon sampling in that study was limited. (c) An
acciptriform + owl clade sister to all other core landbirds, found in binned MP-EST analysis of the
TENT data and unbinned MP-EST analysis of introns by Jarvis et al. (2014). Reddy et al. (2017)
also found this topology in their concatenated analyses of a 104-locus coding data matrix (their
“Prum noJar” tree). (d) Division into two major clades but mousebirds sister to all other Afroaves,
found in the Jarvis et al. (2014) concatenated UCE tree and the Suh et al. (2015) analysis of TE
insertions. Cavitaves is defined as Piciformes, Coraciiformes, Bucerotiformes, Trogoniformes, and
Leptosomiformes (Yuri et al. 2013)
analyses are taken as a whole, it seems the positions of acciptriforms, owls, and
mousebirds within core landbirds should all be approached with caution. Regardless
of the details, core landbirds appear to represent yet another major avian clade where
additional phylogenomic analyses will provide surprises and insights.
3 Why Are the Deep Branches in Neoaves So Difficult?
The motivation for developing phylogenomic methods was the hypothesis that
analyses of large data matrices would result in a fully resolved tree of life (Gee
2003). However, as described above, simply collecting more data does not appear to
be sufficient to resolve the tree of life with confidence. Arguably, the failure of
phylogenomics to provide a convincing and simple resolution of the tree of life
should have been expected. Long before it was possible to collect phylogenomic-
scale data, mathematical studies (e.g., Felsenstein 1978; Hendy and Penny 1989) and
simulations (e.g., Hillis et al. 1994) had revealed that some analyses of large-scale
data matrices fail to converge on the correct tree. Much of the early theoretical work
focused on identifying conditions where specific phylogenetic methods converge
on an incorrect topology with confidence when additional data are added. This
suggests that one might expect analyses of genome-scale data to exhibit a high
degree of support, at least when support is assessed using standard methods (i.e., the
nonparametric bootstrap; Felsenstein 1985). In sharp contrast to this simplistic
expectation, many analyses of large data matrices actually result in low support
and conflicting results.
In this section we provide a brief review of those foundational results in theoreti-
cal phylogenetics and connect those early results to observed conflicts in the bird tree
(see above). However, we emphasize that we do not view the base of Neoaves (or the
bird tree in general) as unique; indeed, these types of conflicts have been observed in
phylogenomic studies focused on other groups (e.g., Philippe et al. 2011; King and
Rokas 2017; Pease et al. 2018), and we expect many additional challenging nodes to
be identified across the tree of life during the phylogenomic era. Hinchliff et al.
(2015) synthesized published phylogenetic information for all organisms, including
taxa that range from microbes to mammals and birds, and they found 4610 nodes that
conflict with their taxonomy. Some of those conflicting nodes are likely to reflect
data-limited studies or cases in which the taxonomy was incorrect, but they also
highlighted a few cases that reflect conflicts among phylogenomic studies. The
Hinchliff et al. (2015) study underscores the work that still needs to be done to
resolve the tree of life, even in the phylogenomic era. However, the base of Neoaves
is among the best studied phylogenetic problems that remain unresolved. Therefore,
it seems likely that understanding the reasons for these continuing difficulties in
resolving the bird tree will have general implications for the resolution of other
challenging nodes on the tree of life.
Although many systematists have suggested that complex analytical methods
may be necessary to arrive at a satisfying resolution of the difficult nodes in the tree
of life (e.g., Philippe et al. 2011; Reddy et al. 2017; Steel 2005), the biological basis
of those difficult nodes is actually quite straightforward. The nodes most difficult to
infer are those associated with short internal branches (int in Fig. 6a). This reflects
the fact that, ultimately, all phylogenetic methods (including both parametric and
nonparametric approaches) rely on the existence of characters that unite taxa
(synapomorphies; cf. Hennig 1966). Given even the simplest models of evolution,
the probability that a synapomorphic substitution uniting a specific group exists is
outgroup
A
A B C D
term
Time before present

a
b int
c
True
species tree
outgroup
B C
outgroup
65% 40% 60% 45% 45%

C A B C D
D
B
40%
40%
45%
Phylogram with Species tree
molecular branch lengths showing GC-content
45%
A A
D D E
outgroup
outgroup
B
C
C
B D
Mixture of
gene trees with Mixture of
different gene trees with
relative rates deep coalescence
(heterotachy) (DC) trees
Fig. 6 Potential sources of nonhistorical signals relevant to phylogenomic analyses. (a) Almost all
of the most challenging nodes in the tree of life reflect short internal branches (int) that provide little
time for synapomorphic changes to accumulate. Long terminal branches (term) exacerbate the
directly linked to the length of the internal branch uniting that group (Braun and
Kimball 2001). However, the terminal branch lengths (term in Fig. 6a) also play an
important role. All other things being equal, longer terminal branches result in a
higher probability that (1) subsequent substitutions will obscure synapomorphic
substitutions and (2) convergent substitutions will create the appearance of
synapomorphies that unite other groups. Thus, correctly inferring the topology for
short internal branches deep in the tree will be more challenging than inferring short
branches closer to the tips. In fact, reconstructing the phylogeny for deep branches is
impossible if the rate of accumulation for substitutions exceeds a specific value
(Mossel 2003), a particularly pessimistic finding for phylogenomicists.
The extreme case in which phylogenetic reconstruction is impossible is unlikely
to be the case for the data types typically used for avian phylogenomics (i.e., those
used by Jarvis et al. 2014). Chojnowski et al. (2008) used simulations to show that
analyses of sequences evolving at a rate similar to avian introns could resolve a tree
with branch lengths similar to those at the base of Neoaves; even as little as 32 kb of
simulated intron data could yield trees with an average of only one rearrangement.
Introns are the most rapidly evolving data type analyzed by Jarvis et al. (2014).
However, examining the results of Jarvis et al. (2014), Prum et al. (2015), and Reddy
et al. (2017) in light of the earlier Chojnowski et al. (2008) study raises another
important question: why is there so much incongruence among those studies given
the large size of the data matrices in each study? The conflict within the Jarvis et al.
(2014) study is especially troubling. Chojnowski et al. (2008) found that analyses of
simulated exon data did not perform as well as analyses using simulated intron data.
However, that study simulated a maximum of 8000 base pairs (bp) of exonic data;
the Jarvis et al. (2014) exon datasets were three orders of magnitude larger than that.
Thus, the observed conflicts at the base of Neoaves must reflect much larger issues
than simply the overall rate of sequence evolution, the time between cladogenic
events at the base of Neoaves, or even the amount of available data.
Long-Branch Attraction, Changes in Patterns of Sequence Evolution, and Data

Types Two major phenomena with the potential to explain the observed conflicts
were identified long before the phylogenomic era using mathematical approaches
and simulations: long-branch attraction and shifts in the model of sequence evolu-
tion. Highly unequal rates of evolution (e.g., Fig. 6b) are thought to be a major
source of long-branch attraction (Felsenstein 1978). This potential source of
misleading signal is actually one of the reasons that many systematists advocate
Fig. 6 (continued) problems associated with short internal branches. (b) Example of long-branch
attraction. Branch lengths reflect numbers of substitutions per site (c) Changes in model parameters,
illustrated for this tree by focusing on shifts in the equilibrium GC-content. (d) Example of a
heterotachous tree mixture. The number of substitutions per site for the locus associated with the
black gene tree differs from the number of substitutions per site for the gray tree. (e) Example of a
tree mixture that has multiple topologies (potentially generated by the MSC). The black and gray
gene trees have the same topology but different branch lengths and the dashed gene tree has a
distinct topology
breaking up long branches by adding taxa (Bergsten 2005), although there are many
reasons that adding taxa is likely to be beneficial. However, highly unequal rates are
not absolutely necessary for long-branch attraction; Hendy and Penny (1989) found
cases where the MP criterion is inconsistent (i.e., it converges on an incorrect tree in
expectation) even for when the data conform to the molecular clock. Long-branch
attraction has often been viewed as a problem associated with the MP criterion that
can be solved by parametric methods (e.g., Huelsenbeck 1997; Swofford et al. 2001),
such as ML and Bayesian inference. However, a number of studies have shown that
long-branch attraction can mislead those parametric methods when the model used
for analyses is incorrect (e.g., Gaut and Lewis 1995; Lockhart et al. 1996), a fact
that should be troubling given that “true underlying models” of evolution are both
unknown and ultimately unknowable (Sanderson and Kim 2000). Regardless, the
fundamental finding of this theoretical work is that large amounts of sequence
data generated by a single model have the potential to converge on an incorrect tree
with high support. The body of older theoretical work should also give pause to
systematists who focus only on high support values in as much as those support
values could be inflated. However, the results of recent phylogenomic studies do not
conform to the expectation of relatively simple artifacts like long-branch attraction
because many challenging nodes on the tree of life receive low support even when
very large datasets are analyzed. It is now clear that the real-world evolution of
genomic data cannot be characterized by a single model, making it reasonable to
speculate the limited support observed in recent phylogenomic studies reflects the
existence of a complex mixture of evolutionary processes rather than a simple artifact.
Patterns of sequence evolution with the potential to mislead phylogenetic
methods are not limited to long-branch attraction; there are myriad model violations
that might mislead available analytical methods. The most obvious and easy model
violation to detect is a case in which the base composition changes across the tree
(Fig. 6c). Indeed, the results of some phylogenetic analyses appear to be driven
largely by convergence in base composition (e.g., Katsu et al. 2009; Phillips et al.
2004), and some authors have suggested that genes with variable base composition
(often called “nonstationary” base composition) should be excluded from phyloge-
netic analyses for this reason (e.g., Collins et al. 2005; Jeffroy et al. 2006). The
general time reversible (GTR) model is the most commonly used model in
phylogenomics; it assumes that base frequencies remain constant (i.e., stationary)
over time. However, many other shifts in the patterns of substitution are possible,
and some do have the potential to affect phylogenetic estimation. For example,
there is evidence that the rate matrix (the relative rates of various substitutions
types, including the transition-transversion ratio and the relative rates of different
transitions and transversions) can change across trees (e.g., Ota and Penny 2003).
Those changes in model parameters can degrade the performance of ML analyses
using standard models like GTR (Casanellas and Fernandez-Sanchez 2007). It is
unclear whether changes in base composition or in the rate matrix across the tree will
necessarily lead to nodes with limited support. It is clear, however, that the bird tree
exhibits both highly unequal branch lengths (Fig. 7) and variation in model
parameters (Fig. 7 shows variation in GC-content). This variation may, at least in
part, explain the limited support for specific avian clades in phylogenomic analyses.
43.6% - 34.9% (rhea)

50.2% - 53.1% (tinamou)
Palaeognathae
Galloanseres
45.2% - 38.4% (screamer)
45.4% - 40.9% (nighthawk) Neoaves

(Passerea)
45.3% - 38.4% (cuckoo)

Neoaves
(Columbea)
fftails
hemipode
“birds of prey”
(owls, hawks, eagles, & New World vultures)
49.4% - 47.1% (woodhoopoe)

49.8% - 54.2% (bee-eater)
0.05
substitutions per site woodpeckers,
honeyguides, & barbets
“birds of prey” (falcons & seriemas)
45.6% - 39.0% (parrot)
50% - 51.8% (lark)

Colors used to emphasize taxa: 50% - 52.4% (sparrow)
black -- evolutionary rate
red -- high GC taxa
blue -- low GC taxa
GC-content is reported for:

inf sites - fast sites
Fig. 7 Phylogram of the Prum et al. (2015) ML tree emphasizing shifts in GC-content and
evolutionary rate. Taxa with the most extreme values for the GC-content of the parsimony
informative sites and the “fast sites” (sites in the 95th percentile for number of MP steps given
this tree) are indicated using colored arrows (red for high values and blue for low values). The
median GC-content for informative sites was 46.8% (range 43.6–50.2%), and the median
GC-content of the fast sites was 44.7% (range 34.9–54.2%). The files used for these analyses can
be found in Braun (2018). Some taxa with very high GC-contents were also long branches (long
branches indicate lineages with elevated substitution rates). We also emphasize lineages with
branch lengths that differ substantially from their sister groups: (1) rails and allies (which are sister
to cranes and allies), (2) hemipodes (which are sister to the gulls, skuas, and allies), (3) a kingfisher
(nested within the order Coraciiformes), and (4) the woodpeckers and allies (sister to jacamars and
puffbirds). We also emphasize the “birds of prey,” as defined in Jarvis et al. (2014), because they
have shortest branches (i.e., lowest evolutionary rates) within Telluraves (core landbirds). Many
high-rate taxa are characterized by long branches in analyses of other data (e.g., compare this
phylogram to Fig. 4 in Reddy et al. 2017). We have indicated taxa in Columbea and Passerea, the
two major clades within Neoaves in the Jarvis et al. (2014) TENT, on the tree to emphasize that they
are non-monophyletic in the Prum et al. (2015) tree
Much of our discussion regarding conflicts within Neoaves (see above) focused
on data-type effects. Data-type effects are not a phylogenetic artifact like long-
branch attraction or base compositional shifts; they are simply a way to discuss
different topological signals that emerge in phylogenetic analyses using distinct
subsets of the genome that can be defined using non-phylogenetic criteria. The
fundamental idea is that distinct subsets of the genome might exhibit different
degrees of model violation. If the model used for analysis is not violated by a subset
of the genome (or, more likely, the model is only violated to a modest degree), then
analyses of that subset are likely to yield an accurate estimate of phylogeny; if the
model violation is strong, then analyses could yield an inaccurate estimate of
phylogeny. Reddy et al. (2017) defined their data types crudely (i.e., coding
vs. noncoding regions), but one could subdivide the genome more finely. For
example, it would seem logical to subdivide noncoding data into transcribed and
non-transcribed regions, whereas coding regions might be subdivided using protein
structure (in fact, Pandey and Braun 2018 recently reported a data-type effect linked
to protein structure for the base of Metazoa). Regardless, the fundamental reason that
Reddy et al. (2017) proposed data-type effects was to provide a framework for
exploring and discussing variation in the phylogenetic signal evident in different
parts of the genome; the actual reason(s) why analyses of any particular data type
might yield an incorrect estimate of phylogeny will relate to specific model
violations associated with each data type.
It might seem that one could overcome data-type effects by conducting analyses
that apply different models to each data partition. Partitioned analyses are common
practice in phylogenomics (Lanfear et al. 2014), and partitioned analyses could, at
least in principle, solve data-type effects if one could identify adequate models for
each partition. However, the idea that Reddy et al. (2017) articulated is that there
may not be any models that yield accurate estimates of phylogeny for a specific data
type (or, at the very least, none of the models that are “good enough” have been
implemented in a software package that is practical to use for phylogenomic
analyses). Most programs used in phylogenomics, like RAxML and MrBayes,
only implement the GTR model and its submodels (typically in combination with
methods to describe among-sites rate heterogeneity like Γ-distributed rates and/or
invariant sites). This has led to the use of the GTR model (or a submodel) in almost
all empirical studies (Sumner et al. 2012). If the GTR model is fundamentally
problematic for analyses of one or more of the data type(s), then partitioned analyses
that apply the GTR model to each partition will also be problematic. The only
solution would be excluding the problematic data or using models that differ from
the GTR model in a more fundamental way. Efforts to do the latter are ongoing; for
example, IQ-TREE (Nguyen et al. 2015) is fast enough for phylogenomic studies
(Zhou et al. 2018), and it implements a broader suite of models than many other
programs. If these newer models have a better fit to the data that are poorly described
by the GTR model and yield accurate estimates of phylogeny for those data, then
partitioned analyses (using the appropriate models) should ameliorate data-type
effects.
Mixtures of Gene Trees That Reflect Heterotachy, Incomplete Lineage Sorting,

or Reticulation Another challenge for phylogenomic studies is the fact that geno-
mic data may reflect a mixture of trees rather than a single tree (Maddison 1997). The
simplest case is a mixture of trees with the same topology but different branch
lengths (Fig. 6d), a phenomenon also called heterotachy (Lopez et al. 2002).
Heterotachy in protein-coding regions is often thought to reflect shifts in selective
constraints, causing sites to transition between a state in which substitutions can
accumulate, and a second state in which substitutions are removed by purifying
selection (Penny et al. 2001). However, regional variation in mutation rates is well
characterized in birds (e.g., Axelsson et al. 2005), and shifts in the mutation rate for a
specific neutral region will also result in heterotachy. Regardless of the biological
basis for heterotachy, analyses of heterotachous data using standard (i.e.,
non-heterotachous) ML methods can be misleading (Matsen and Steel 2007), some-
times resulting in a “mixed branch repulsion” analogous to long-branch attraction. A
more extreme tree mixture involves gene trees with different topologies (Fig. 6e).
Those tree mixtures are biologically realistic; a number of processes, such as
incomplete lineage sorting (ILS) and hybridization, can result in mixtures of trees
with different topologies (Maddison 1997). Edwards (2009) pointed out that ILS
actually results in both heterotachy and discordant trees; the heterotachy reflects
variation in coalescence times for gene trees with the same topology. In fact, some
gene trees with the same topology as the species tree still reflect a deep coalescence
(DC) in which the split in the gene tree occurs prior to multiple speciation events
(e.g., the gray gene tree in Fig. 6e); branch lengths of those trees will certainly differ
from the non-DC trees. Mixtures with multiple topologies arise when DC gene trees
are discordant with the species tree (e.g., the dashed gene tree in Fig. 6e). There is
strong direct evidence for heterotachy (e.g., note the very different terminal branch
lengths for BDNF and PPP2CB in Fig. 8), although the impact of heterotachy on
estimates of avian phylogeny using standard ML methods remains unclear. The
evidence for discordance among gene trees due to ILS is indirect, but there is a
strong theoretical basis for expecting ILS whenever there are short branches in a
species tree. The impact of ILS on estimates of the bird tree obtained using standard
ML methods also remains uncertain.
The theoretical and computational phylogenetics community has put a tremen-
dous amount of effort into the development of MSC (“species tree”) methods over
the past decade (reviewed by Edwards 2009; Edwards et al. 2016; Liu et al. 2009;
Warnow 2018). MSC methods are designed to infer the correct species tree given
mixtures of gene trees due to ILS. Standard ML analyses using concatenated gene
sequences implicitly assume a single underlying tree with a specific set of branch
lengths. Thus, standard ML analyses violate the MSC model, and those analyses will
converge on an incorrect estimate of the true species tree in certain parts of parameter
space (Kubatko and Degnan 2007; Mendes and Hahn 2017; Roch and Steel 2015).
Although the fact that MSC methods are consistent (i.e., they converge on the correct
tree in expectation) is viewed as a desirable property, some authors have raised
concerns about the criterion of statistical consistency. Warnow (2015) pointed out
that available proofs of consistency for MSC methods actually focus on a “weak
A Ostrich
Tinamou PALAEOGNATHAE
*
Duck
99 Chicken 55.4%
Turkey GALLOANSERES
* 43.6%
Dove 51%
75
Mesite
96 Sandgrouse
98 Flamingo
91 Grebe 50% NEOAVES (Columbea)
Cormorant
*
Tropicbird 41.5%
Cuckoo 42.1%
Bee-eater 42.1%
94
Budgerigar 42.2%
* Kea 42.6%
Woodpecker
Turkey vulture
Trogon
Sunbittern
Turaco
Owl
Loon
White-tailed eagle
85 * Bald eagle
Falcon
81 Hummingbird
* Swift
Egret
Fulmar
Rifleman
Manakin
* Crow
Darwin’s finch
* Zebra finch
*
Pelican
Emperor penguin
* Adelie penguin
Crane
Mousebird
PPP2CB Hornbill 58.4%
intron Nightjar
length = 1,872 nt 70
Seriema
Cuckoo roller
Killdeer
86
Ibis
0.07 Bustard 52.2%
substitutions per site NEOAVES (Passerea)
Hoatzin 49.9%
Fig. 8 Two examples of gene trees from Reddy et al. (2017), emphasizing differences in relative
rates and base composition. Support values from an ultrafast bootstrap (Minh et al. 2013) analysis in
IQ-TREE are shown next to branches when they are 70%. (a) Tree based on an intron in the
PPP2CB locus. This is one of the few individual gene trees that divides Neoaves into Columbea and
Passerea. As in Fig. 7, the GC-content for informative sites is indicated with colored arrows (red for
the six most GC-rich taxa and blue for the six least GC-rich taxa). Although the median GC-content
for informative sites (45.4%) does differ from the GC-content for constant sites (49.7%), this locus
exhibits limited GC-variation overall (range for informative sites ¼ 41.5–58.4%). (b) Tree based on
part of the BDNF coding exon. This locus exhibits substantial rate variation (note the very long
branches for the bee-eater, Darwin’s finch, and tinamou). Like PPP2CB, the median GC-content for
informative sites (47.5%) differs from the GC-content of constant sites (52.5%). However, this
region also exhibits substantial GC-content variation (range for informative sites ¼ 33.9–84.8%).
Most nodes in this gene tree have limited support, similar to the PPP2CB gene tree (and most trees
based on short gene regions), but the BDNF tree does includes some strongly supported clades.
However, some of those strongly supported clades contradict monophyly of Palaeognathae and
Neoaves, which are united by very long branches in all other estimates of the avian species tree. The
extreme GC-content and rate variation suggest that these conflicts may reflect a biased estimate of
phylogeny. All data supporting this figure is available in Braun (2018)
B 95 Duck
Ostrich 40.7% PALAEOGNATHAE
Chicken 34.8%
* Turkey GALLOANSERES
Loon
Bustard NEOAVES (Passerea)
77
Grebe NEOAVES (Columbea)
97 Hornbill
Killdeer
Fulmar
Ibis
82
Seriema
Woodpecker
Egret 42.4%
76
Cormorant
Trogon 41.5%
Pelican
Adelie penguin
74 Emperor penguin
71 Falcon
Cuckoo 42.4%
Sunbittern
84
Hoatzin
White-tailed eagle
99 Bald eagle
Turaco
Cuckoo roller
Turkey vulture
Mousebird
Manakin
Crow 33.9%
Zebra finch 53.4%
Mesite
Sandgrouse
Budgerigar
Owl
Kea
Crane
Dove
Flamingo
BDNF Tropicbird
coding exon Nightjar
length = 688 nt Hummingbird
Swift 56.8%
Rifleman 55.9%
Darwin’s finch 71.2%
0.03 Bee-eater 75.4%
substitutions per site
Tinamou 84.8%
Fig. 8 (continued)
version” of statistical consistency, showing only that “tree estimated by the species
tree method will converge in probability to the true species tree as the number of sites
per locus and the number of loci both increase.” Even this weak version of statistical
consistency assumes data were generated by gene trees that reflect the MSC model
along with a model of sequence evolution for which available methods are consis-
tent. Springer and Gatesy (2016) pointed out that the MSC model ignores important
aspects of genome evolution such as selection and linkage. Even as ubiquitous a
phenomenon as population subdivision will yield a different spectrum of gene trees
than expected given the simple MSC model (Slatkin and Pollack 2008). It is
certainly possible that the real-world processes underlying genome evolution are
close enough to the assumptions that underlie MSC methods that those methods will
yield an accurate estimate of the species tree; indeed, this is generally assumed to be
the case when those methods are applied. However, a recent study examined the fit
of 25 datasets to the MSC model, finding that 20 of those datasets violate the model
(Reid et al. 2013). Ultimately, it should be clear that the criterion of consistency only
yields guarantees in the abstract world of mathematics, not in the real world of
biology.
Given their growing use in phylogenomics, it is important to understand the types

of MSC methods available at this time, despite the concerns we expressed regarding
their justification by appealing to their statistical consistency. Modern MSC methods
often yield trees that are quite congruent with trees based on standard (concatenated)
ML analyses (Tonini et al. 2015), and some are actually less computational burden-
some than those ML approaches. Xu and Yang (2016) reviewed MSC methods,
highlighting two basic approaches: (1) full-likelihood methods and (2) gene tree
summary methods. Xu and Yang (2016) also described (but did not name) a third
approach that we call site pattern methods. Full-likelihood methods integrate over
the uncertainty in gene trees (this is the approach Maddison 1997 originally
suggested). At this time the full-likelihood approach has been implemented in
BEST (Liu 2008), *BEAST (Heled and Drummond 2010), RevBayes (Höhna
et al. 2016), and BPP (Rannala and Yang 2017); all of those programs use a
Bayesian Markov chain Monte Carlo approach, and none are able to scale to
phylogenomic analyses similar in size to Jarvis et al. (2014). Gene tree summary
methods involve two steps: (1) a standard method (e.g., ML) is used to generate gene
trees and (2) the estimated gene trees are combined to generate the species tree.
Examples of gene tree summary methods include MP-EST (Liu et al. 2010),
ASTRAL (Mirarab et al. 2014b; Mirarab and Warnow 2015; Zhang et al. 2018),
and NJst/ASTRID (Liu and Yu 2011; Vachaspati and Warnow 2015). There are
tools to visualize discordance among estimated gene trees, either as networks or
using other approaches (e.g., Ottenburghs et al. 2016b; Sayyari et al. 2018). Many
phylogenomic studies, including a number focused on birds (e.g., Kimball et al.
2013; McCormack et al. 2013; Jarvis et al. 2014; Edwards et al. 2017), have used
gene tree summary methods. The practice of estimating gene trees and then combin-
ing those trees is actually imposing less computational burden than standard
ML analyses of concatenated loci. Finally, site pattern species tree methods use a
concatenated data matrix as input. However, they differ from standard analyses of
concatenated data by decomposing the data matrix into quartets (SVDquartets;
Chifman and Kubatko 2014) or rooted triples (SMRT-ML; DeGiorgio and Degnan
2010) and identifying the optimal tree for those subsets of taxa. Obviously, the
method used to infer the quartet (or rooted triplet) subtrees must be consistent given
the MSC for the approach to be viewed as a species tree method. However, methods
that are consistent for those subtrees (under at least some circumstances) do exist
(DeGiorgio and Degnan 2010; Long and Kubatko 2017). Use of singular value
decomposition to choose the subtrees (the criterion used by SVDquartets) is also
very fast computationally. After generating the subtrees, they are combined using a
supertree method such as MRP (Baum 1992; Ragan 1992) or Quartet MaxCut (Snir
and Rao 2012) to generate the species tree. Although the use of site pattern methods
remains less common than the gene tree summary methods, they have begun to
attract attention in avian phylogenomics (e.g., Hosner et al. 2015b; Meiklejohn et al.
2016; Moyle et al. 2016; Sun et al. 2014).
Hybridization represents another major source of discordance among gene trees.
Hybrids have been documented in most bird orders (Ottenburghs et al. 2015, 2017b),
and hybridization can impact phylogenetic estimation for recent radiations (e.g.,
Lavretsky et al. 2014). Phylogenomics is likely to revolutionize the study of these
complex radiations (Lamichhaney et al. 2015; Grant and Grant 2016; Stryjewski and
Sorenson 2017). However, the amount of gene tree discordance due to introgression
in many lineages may be limited by the fact that hybrids often have lower fitness than
parental types (e.g., Bronson et al. 2003). Two phenomena which might be espe-
cially important for generating mixtures of gene trees that reflect hybridization are
(1) hybrid speciation and (2) despeciation. Hybrid speciation reflects cases where
hybrid populations become reproductively isolated from both parental species. The
Golden-crowned manakin appears to be an example of such a hybrid species; ~2/3 of
its genome is more closely related to the Opal-crowned manakin, whereas ~1/3 is
more closely related to the Snow-capped manakin (Barrera-Guzmán et al. 2018).
Despeciation refers to the fusion of two related species, resulting in a single species
and erasing the initial speciation event. Kearns et al. (2018) presented phylogenomic
evidence that Common ravens arose by fusion of two raven lineages that diverged
ca. 1.5 Mya and would likely be viewed as species if they were extant (“Holarctic”
and “California”). Chihuahuan ravens diverged from the California lineage, while it
was isolated from the Holarctic birds. Both of these phenomena lead to a spectrum of
gene trees that differ from the expectation given ILS alone (see Fig. 3 in Hahn and
Nakhleh 2016).
If we focus deeper in evolutionary history, the impact of hybridization is expected
to be more difficult to examine: gene tree estimation error might make it virtually
impossible to distinguish the descendants of lineages that underwent limited
amounts of ancient hybridization from those with purely treelike history (combined
with ILS). However, the expectation that sex-linked genes will exhibit less intro-
gression than autosomal genes (Rheindt and Edwards 2011) might be useful for
distinguishing conflict due to ILS from conflict due to introgression. Indeed, Fuchs
et al. (2013) used this to explain a difference between autosomal and Z-linked genes
they observed in woodpeckers. That study used seven autosomal loci, three Z-linked
loci, and mitochondrial sequences, so it would be interesting to reexamine it in a
phylogenomic framework. Another test uses the expectation that the two minority
topologies for rooted triplets in gene trees will be recovered in equal numbers if ILS
alone is responsible for discordance; given the failure of a collection of true gene
trees to observe this equality would lead one to reject treelike evolution under ILS
alone. However, errors in estimated gene trees can either produce or obscure
inequalities in the numbers of gene trees with each minority resolution, limiting
the utility of the inequality test. To address this, Zwickl et al. (2014) proposed a
“cumulative support distribution” test that incorporates information about support in
the gene trees. Developing practical tests that are able to establish whether the null
hypothesis of ILS alone can explain discordance among gene trees represents a
major challenge in the phylogenomic era.
Rare Genomic Changes A fundamental problem for studies focused on discor-

dance among gene trees is the indirect nature of the evidence. Unlike evolutionary
rate heterogeneity and heterotachy, in which it is relatively straightforward to find
direct evidence for the phenomena, the evidence for ILS and introgression is indirect
because it depends on the comparison of gene trees. However, phylogenetic trees
estimated using short sequences, like individual genes, are subject to substantial
estimation error (e.g., Chojnowski et al. 2008; Gatesy and Springer 2014; Mirarab
et al. 2014a; Patel et al. 2013) and could be subject to systematic error (e.g., if there is
strong evolutionary rate variation and/or nonstationary base composition). Thus,
observing incongruence among estimated gene trees does not provide direct evi-
dence of ILS or introgression because that incongruence could reflect error. This is
true even for loci with limited evolutionary rate and GC-content variation (e.g.,
Fig. 8a); it is certainly true for gene trees for loci with substantial rate and
GC-content variation (e.g., Fig. 8b).
Rare genomic changes, which correspond to a heterogeneous set of slowly
accumulating changes in the genome, provide an alternative means to examine
ILS and introgression that has the potential to be better than the use of estimated
gene trees. Ideally, rare genomic changes represent uniquely derived genomic
characters (i.e., homoplasy-free changes that are subject neither to reversal nor to
convergence). Genuinely homoplasy-free genomic characters would define a single
branch in their associated gene tree perfectly. Analyses of avian ILS have used three
types of rare genomic changes: (1) TE insertions, (2) numt insertions, and (3) indels
(insertions and deletions) as a whole. TE insertions are the most commonly used
(e.g., Haddrath and Baker 2012; Suh et al. 2011). Many conflicting TE insertions
have been identified in birds (Han et al. 2011; Jarvis et al. 2014; Matzke et al. 2012;
Suh et al. 2015, 2011, 2017); this observed conflict has typically been interpreted
as direct evidence of ILS. Unfortunately, precise quantification of ILS using TE
insertions is difficult because (1) the rate of TE insertion is quite variable over time
(Kapusta and Suh 2017); (2) informative TE insertions are relatively rare even
when they are scored at a whole-genome scale (Suh 2015); and (3) avian TE
insertions do not appear to be completely free of true homoplasy (Han et al. 2011).
Unlike TE insertions, the sole numt study (Liang et al. 2018) did not reveal any
homoplasy. However, Liang et al. (2018) only identified a small number of informa-
tive numt insertions and that study included a limited taxon sample. If we expand our
focus to indels as a whole, which are much more numerous than TE or numt
insertions, Jarvis et al. (2014) predicted that discordance due to ILS would yield a
positive relationship between internal branch lengths and the proportion of apparent
synapomorphies mapping to each of those branches that appear non-homoplastic.
That exact relationship was observed, although that approach cannot provide a
quantitative estimate of ILS. Regardless, the observed levels of conflict among
rare genomic changes indicate there was a relatively large amount of ILS near the
base of Neoaves.
Analyses of rare genomic changes could be revolutionized by truly whole-
genome phylogenetics. Many analyses reported by Jarvis et al. (2014) actually did
not require whole-genome sequencing. For example, sequence capture could
(at least in principle) have been used to generate the data for the TENT. In contrast,
generating rare genomic change data would have been much more difficult (poten-
tially even impossible) without genome sequencing. Identifying TE insertions prior
to the phylogenomic era involved labor-intensive methods necessary for their
identification (described by Haddrath and Baker 2012). Bioinformatic screens of
whole-genome assemblies can reveal TE insertions without the need for complex
laboratory methods. Repetitive sequences, like TEs, can be challenging to assemble,
but this can be solved by using platinum-quality genome sequences (Kapusta and
Suh 2017; Weissensteiner and Suh 2018). It is also challenging to target numt
insertions, but they are straightforward to identify by searching whole-genome
data. However, because numt insertions are nonfunctional mitochondrial sequences
in the nuclear genome, they have a relatively high likelihood of being misassembled
in genomes based exclusively on short-read data (because some numt reads are likely
to cluster with mitochondrial reads). Thus, platinum-quality genome sequences
should improve numt scoring relative to most currently available assemblies. Finally,
the availability of more avian genome assemblies could allow the use of other types
of rare genomic changes. Microinversions are one of these types of rare genomic
change that are impossible to identify without sequence data (Braun et al. 2011).
Microinversions technically include all inversions that cannot be identified cytolog-
ically (Chaisson et al. 2006). However, the sole avian microinversion study (Braun
et al. 2011) focused on short (<50 bp) inversions, finding that they accumulate at a
rate comparable to TE insertions and suggesting that they are likely to be as useful
for phylogenetics as TE insertions. Large-scale identification of microinversions will
revolutionize their use and provide another way to examine discordance among gene
trees. We anticipate that the phylogenomic era will lead to a flood of rare genomic
change datasets.
Rare genomic changes are interesting from a computational standpoint because
the optimal tree for ideal rare genomic changes is the MP tree (Steel and Penny
2004, 2005). MP is orders of magnitude more computationally efficient than ML
(or Bayesian) methods (Sanderson and Kim 2000). It is unclear whether the MP tree
for a collection of ideal rare genomic changes generated on the tree mixture
generated by the MSC will be the species tree. However, Mendes and Hahn
(2017) have shown that MP analyses of concatenated data are consistent given the
MSC (assuming certain assumptions are made), providing reasons for optimism if
one views the criterion of consistency as critical (for detailed arguments against the
position that consistency is a necessary feature of phylogenetic methods, see Brower
2018; Sanderson and Kim 2000). Of course, there are two reasons that empirical rare
genomic change datasets will not be “perfect” (i.e., absolutely homoplasy-free):
(1) the relevant type of rare genomic change could exhibit some true homoplasy
and (2) errors in the genome assembly and/or orthology detection pipeline. It may be
necessary to develop analytical methods that consider those sources of error. Never-
theless, it seems reasonable to postulate that rare genomic changes will make it
possible to estimate the species tree and amount of ILS accurately.
Assessing Support and Confronting Theory with Phylogenomic Data Despite

the large body of theoretical work, we do not have a complete explanation for the
observed results of avian phylogenomic studies. The strongest type of theoretical
studies, proofs of consistency or inconsistency (e.g., Felsenstein 1978; Hendy and
Penny 1989; Kim 2000; Matsen and Steel 2007; Roch and Steel 2015; Mendes and
Hahn 2017), provides information about the behavior of specific analytical methods
given an infinite amount of data that were generated under a specific model. Kim
(2000) discussed an elegant geometric interpretation of phylogenetic methods that
have the interesting corollary that nonparametric bootstrap support for clades should
increase to 100% as the sample size increases. If the analytical method is consistent
given the true underlying model of evolution, the correct clade will exhibit 100%
support given sufficient data; if the data reflect a part of parameter space where the
method is not consistent, an incorrect clade is expected to exhibit 100% support.
However, neither Jarvis et al. (2014) nor Prum et al. (2015) observed 100% bootstrap
support for all clades. This raises the question of how many sites are necessary to
provide a “sufficient amount” of data to observe the expected asymptotic behavior.
Many empirical systematists would have predicted that an alignment of 41.8 Mbp
(the size used to generate the Jarvis et al. 2014 TENT) would be sufficient for all
nodes to converge on 100% support (recognizing that some nodes might be resolved
incorrectly due to inconsistency). But there are nine nodes in the TENT that have
<100% support (and one has only 55% support). Increasing the data matrix size
more than sevenfold does not eliminate those low-support branches; six nodes with
<100% support remain present in the Jarvis et al. (2014) whole-genome tree (four of
those nodes have 62% support). Thus, the limited support for clades at the base of
Neoaves given available analytical methods appears to reflect an intrinsic property of
the data rather than a trivial limitation in the amount of data.
The fact that analyses of a very large (even genome-scale) dataset can yield 100%
bootstrap support for an incorrect clade may seem disturbing. However, it is a natural
outcome when the analytical method is not consistent. By definition, a consistent
estimator converges on the true value (in phylogenetics this would be the true tree)
as the amount of data available for analysis increases (as more of the genome is
sampled). If an estimator is based on a fundamental misunderstanding of the
processes that generated the data, then the method is unlikely to be consistent, at
least in some parts of parameter space. It should not be surprising that analyses using
such a method could lead to an incorrect conclusion with 100% support; after all, we
defined the analytical method as one based on fundamentally incorrect assumptions
regarding the processes that generated the data. This raises a question: are there
phylogenomic methods that are immune to this issue? There have certainly been
efforts to create metrics of support for the phylogenomic era. For example, Seo
(2008) extended the standard bootstrap to a multilocus framework (ASTRAL
includes an easy to use implementation of this multilocus bootstrap). The general
idea of subsampling genes has also attracted attention (Edwards 2016);
bootstrapping is often used to assess support in locus subsampling studies. There
are other methods like concordance factors (Ané et al. 2007; Baum 2007) and the
information theoretic measures of Salichos et al. (2014), which instead focus on
examining the agreement among gene trees. The latter is especially interesting
because it has pointed out that it can also be applied to rare genomic changes. It is
important to recognize that measures of agreement among gene trees do not provide
information regarding the support for clades in the species tree. If data fit the MSC,
one may still have a high degree of confidence regarding the presence of a specific
clade in the species tree even when a relatively small proportion of gene trees agree
with that clade (Pamilo and Nei 1988). If enough gene trees are sampled, multilocus
bootstrapping (Seo 2008) and local posterior probabilities (Sayyari and Mirarab
2016) can both yield strong support for clades that only agree with a plurality of
gene trees. However, all methods exhibit the same behavior as the standard bootstrap
when the analytical method is inconsistent (i.e., incorrect clades will receive 100%
support if sufficient data are analyzed and those data reflect a part of parameter space
where the method is inconsistent). But all is not lost; many trees are both robust to
the analytical method (e.g., Rindal and Brower 2011) and very likely to be correct. In
fact, the story of avian phylogenomics is arguably one of steady progress, with parts
of the tree that would have been viewed as hopeless a decade ago now emerging as
“solved” in a satisfying manner (e.g., Fig. 2). It is the parts of the tree that remain in
flux despite the availability of very large amounts of data that represent a problem:
we must either conclude that the 41.8 Mbp analyzed by Jarvis et al. (2014) is not
sufficient to observe the expected asymptotic behavior (i.e., 100% support at all
nodes) or that there is something about the data and available methods of phyloge-
netic analysis that we do not understand.
There are four basic hypotheses that can explain the limited support for major
groups at the base of Neoaves in multiple phylogenomic analyses (Fig. 2). First,
errors in the data matrices (e.g., assembly, orthology assignment, and alignment)
could introduce noise. If this hypothesis is correct, the limited support should
disappear as the quality of the aligned dataset is improved, either by removing
problematic regions (e.g., using alignment-filtering methods like DivA; Mendoza
et al. 2014) or by extracting data from improved genome assemblies that lead to less
downstream error. Second, the poor support could reflect limitations of the available
computational implementations of methods. If the bird data lies very close to
“boundaries” with even minor differences in numerical optimization during the
calculation of the likelihood, the methods might choose different trees in different
bootstrap replicates. However, this would require all of the very large Jarvis et al.
(2014) datasets to be in parts of parameter space where those computational issues
manifest themselves. Third, it could the case that the heterogeneous nature of the
data tends to obscure phylogenetic signal. If this hypothesis is correct, it might be
possible to improve estimates of the avian tree by focusing on less noisy parts of the
genome. Reddy et al. (2017) presented several arguments that noncoding data, such
as introns and UCEs, might perform better in phylogenetic analyses than coding
exons. The strongest empirical argument favoring noncoding data was the fact that
trees based on rare genomic changes (one for TE insertions and one for all indels) are
more similar to the intron and UCE trees. Nonetheless, it would clearly be more
convincing to identify additional data types that yield trees congruent with the
noncoding trees. Finally, as described above, the base of Neoaves could represent
a hard polytomy (Suh 2016). If Suh (2016) is correct, then individual gene trees will
be random for the lineages involved in the hard polytomy. The hard polytomy
hypothesis predicts that any relatively small set of gene trees are unlikely to be
very congruent. Suh (2016) proposed a nine-taxon hard polytomy, and there are
>135,000 possible rooted nine-taxon trees. However, Reddy et al. (2017) found
that analyses of a largely intronic 54-locus dataset yielded a tree similar to the Jarvis
et al. (2014) intron tree (and the TENT, which was 68% noncoding data). Reddy
et al. (2017) also observed that analyses of a largely coding 104-locus dataset that
did not overlap with any loci in Jarvis et al. (2014) results in a tree similar to the
Jarvis et al. (2014) exon tree. Those results seem unlikely if the low support at the
base of Neoaves reflects a hard polytomy; if the hard polytomy hypothesis is correct,
it would require that analyses of data reflecting relatively small sets of gene trees
coincidentally converge on two specific parts of tree space that were identified by
Jarvis et al. (2014) in this manner is correlated with data type. We suggest that
understanding the heterogeneity present in genomic datasets will ultimately be
necessary to obtain a well-supported estimate of the avian tree of life.
The Impact of Genome Assembly Quality in the Phylogenomic Era All of the
issues discussed above focus on the behavior of analytical methods, raising an
important question: what is the role for platinum-quality genome assemblies in
avian phylogenomics? After all, draft genome assemblies typically capture at least
90% of most bird genome sequences, even when they reflect very low-coverage
sequencing (e.g., Tiley et al. 2018). The increased amount of data available in
platinum-quality genome assemblies is unlikely to have much direct impact on
phylogenomic analyses. However, high-quality genome assemblies are likely to
improve dataset quality. Springer and Gatesy (2018) reported that many alignments
analyzed by Jarvis et al. (2014) had homology errors (e.g., the inadvertent alignment
of exons to introns due to incorrect gene annotation). The existence of problematic
alignments in a phylogenomic dataset is not unexpected; any computational pipeline
used to generate a phylogenomic data matrix will yield both false positives (i.e., it
will align some nonhomologous sequences) and false negatives (i.e., it will fail to
align some truly homologous sequences). The greater contiguity and smaller number
of misassemblies in platinum-quality genome assemblies could make it easier to
extract orthologous sequences. Improved genome annotations will also make it
easier to extract orthologous regions. Functional data, such as RNA-seq, can be
very important for genome annotation (Roberts et al. 2011), and it is accumulating at
a rapid pace (e.g., Seki et al. 2017). Iso-seq data have the potential to be even more
helpful because it reflects long-read sequencing technologies to generate full-length
mRNA sequences (Gonzalez-Garay 2016), unlike RNA-seq data that reflect short
reads that do not define complete transcripts in a direct manner. Both Iso-seq data
and platinum-quality genome assemblies are now being generated for birds (e.g.,
Korlach et al. 2017; Workman et al. 2017). Better annotation will also highlight
changes in gene structure (e.g., precise intron deletions; Coulombe-Huntington and
Majewski 2007); this will provide another type of rare genomic change (Bleidorn
2017). Finally, better genome assemblies will also permit better analyses of genome
structure and content (e.g., inversions, rearrangements, gene losses, and gene
duplications). Methods to use gene order information for phylogenetic estimation
already exist (Hu et al. 2014), and all that is needed are genome assemblies of
sufficient quality. Ultimately, resolving the most difficult questions in the avian tree
of life is likely to require improved genome assembly and annotation, the extraction
of multiple data types, and improved analytical methods.
4 Progress Toward Species-Level Avian Megaphylogenies
Resolving the bird tree of life actually involves two related but somewhat distinct
research programs: (1) the resolution of difficult nodes, like the base of Neoaves, and
(2) the generation of large-scale trees that include all bird species. The latter is
complicated by evidence that many avian taxa should be considered species but are
not currently recognized as such (Gill 2014; Barrowclough et al. 2016). The decision
to assign the rank of species to taxa depends on the species concept one chooses to
employ (see Ottenburghs 2019 for a recent review of species concepts). Nonetheless,
it is likely that the number of evolutionary entities (regardless of whether or not those
sometimes-cryptic taxa are assigned the rank of species) will ultimately increase
from the ~10,000 bird species that are recognized in most current taxonomies
(Table 3) by at least twofold (and possibly even increase threefold). The Brown
et al. (2017) megaphylogeny is the only existing bird tree with more than 10,000
taxa, although it is unclear how many of those taxa will ultimately be assigned a
status equivalent to species upon detailed study. Nevertheless, generating large-scale
avian trees (megaphylogenies or simply “big trees”) that include most or all of the
currently recognized avian species would represent an excellent starting point for the
next phase of avian phylogenomics.
The available avian megaphylogenies (Table 4) have two major limitations:
(1) most fail to include all named bird species and (2) none of them reflect analyses
of phylogenomic-scale data. The reason that many megaphylogenies exclude
species is simple: molecular data are absent for a number of species. The complete
megaphylogenies (Brown et al. 2017; Jetz et al. 2012) incorporate data-deficient
species using taxonomic information. Two megaphylogenies (Hedges et al. 2015;
Jetz et al. 2012) also used strong backbone constraints (i.e., a number of
relationships were fixed). Other big trees (Brown et al. 2017; Davis and Page
2014) lack meaningful branch lengths because they reflect the synthesis of published
trees rather than a direct analysis of any molecular (or morphological) data. Burleigh
et al. (2015) is the only avian megaphylogeny generated using a purely empirical
approach; it reflects an unconstrained ML analysis of orthologous avian sequences
published before June 2011. The newest megaphylogeny (Brown et al. 2017) reflects
a synthesis of published trees (i.e., a supertree) so it does incorporate phylogenomic
data in the form of source trees generated using phylogenomic data; in fact, it has a
backbone identical to the Prum et al. (2015) tree (Fig. 2b).
The value of phylogenomic data, especially whole-genome data, for the genera-
tion of a species-rich bird tree is actually an open question, especially if we choose to
include more than 10,000 terminal evolutionary lineages. The additional information
available in whole-genome sequences may not justify data collection costs or the
computational burden of assembling, annotating, and analyzing whole-genome data
when we are near the tips of the tree. Sequence capture methods (e.g., Table 2)
Table 4 Avian megaphylogenies generated by synthesizing data from multiple sources

Number of Branch
Study neornithine taxa Analytical approacha Methodb lengthsc
Jetz et al. (2012) 6670/9993d Supermatrix (15 regions) BI time
Davis and Page (2014) 5379 Supertree analysis MRP n/a
Burleigh et al. (2015) 6714 Supermatrix (29 regions) ML mol
Hedges et al. (2015) 7163 Synthesis of divergence times TTOL time
Brown et al. (2017) 13,579e Supertree analysis RH n/a
a
Analytical approaches are supermatrix (analysis of concatenated sequence data), supertree (com-
bination of published trees), and synthesis of divergence times (refining a taxonomy using
published timetree data)
b
BI Bayesian inference (with constraints in the case of Jetz et al. 2012), ML maximum likelihood,
MRP matrix representation with parsimony (Baum 1992; Ragan 1992), RH Redelings and Holder
(2017) supertree pipeline, TTOL “time tree of life” approach (Hedges et al. 2015)
c
Branch lengths available as estimates of absolute time or molecular change (substitutions per site).
“n/a” indicates that branch lengths are not available
d
Two Jetz et al. (2012) trees are available. One comprises 6670 taxa for which at least some
sequence data were available. The other comprises 9993 taxa, and it includes taxa for which no data
are available; taxa without any associated sequence data were placed using taxonomic information
e
The Brown et al. (2017) tree includes taxa that are not assigned the rank of species in current
checklists
sidestep problems associated with homology assignment to a fairly large degree

because they focus on assembling relatively short contiguous regions; there is no
need to annotate the genome and extract the relevant data types. Moreover, it is
usually possible to recover complete or virtually complete mitochondrial genome
sequences even if mitochondrial sequences are not targeted by probes (Meikejohn
et al. 2014; Raposo do Amaral et al. 2015; Wang et al. 2017); alternatively,
low-coverage genome sequencing can yield mitochondrial genome sequences
(Barker et al. 2015). Mitochondrial sequences are likely to be especially valuable
near the tips of trees (Barrowclough and Zink 2009). At least in the near term, it
seems likely that sequence capture will contribute substantial amounts of data to
avian megaphylogenies.
The data included in current megaphylogenies is heterogeneous in quality, and
this has no doubt led to topological (and, when they are available, branch-length)
errors in those trees. The impact of those errors on downstream analyses is unclear,
and it probably depends on the specific analyses that are conducted. Indeed, in a
study focused on a single family (woodpeckers; about 200 species), Dufort (2016)
found at least 28 sequences for 10 different species in Jetz et al. (2012) that have
been (or could possibly be) assigned to other species. Burleigh et al. (2015) fared a
little better, including only 14 problematic sequences for 5 species. These errors
largely appear to reflect difficulties associated with reconciling the NCBI taxonomy
with current species limits, although Dufort (2016) also noted that both big trees
included a cytochrome b sequence that Fuchs et al. (2008) deemed a pseudogene.
Although these findings should raise concerns, the more important question is
whether the phylogenetic errors caused by these issues influence downstream

analyses. Wang et al. (2016) provided empirical evidence that errors can influence
downstream analyses (in this case biogeographic inference) by showing that a single
misplaced species in the Jetz et al. (2012) megaphylogeny had a large impact on their
conclusions. The practice of placing data-deficient taxa using taxonomic informa-
tion, which has been criticized on theoretical grounds (Rabosky 2015; Title and
Rabosky 2017), is potentially a bigger problem. There are clades in Jetz et al. (2012)
with posterior probabilities of 1.0 that depend on the position of taxa with no data
that conflict with clades that received 100% bootstrap support in an ML analysis of
eight nuclear loci and three mitochondrial regions (Hosner et al. 2015a). These
problems are not unique to Jetz et al. (2012); even those megaphylogenies that
eschew the use of data-deficient taxa suffer from problems inherent to the synthesis
of diverse data sources. The only solution is the collection of much larger datasets
from all bird species; there are ongoing efforts, like the B10K project (Zhang et al.
2015) and the OpenWings project (Pennisi 2018), that aim to produce the requisite
sequence data.
The exercise of generating an accurate, taxon-rich phylogeny of birds is not an
end in itself. Rather, it is a necessary component of comparative studies that
addresses evolutionary pattern and process. Trees allow us to ask about the evolu-
tionary opportunities, constraints, and processes that led to the biodiversity we now
observe. Comparative methods reveal whether observed patterns in biodiversity data
or correlations among traits require a deeper explanation or have the potential to
be explained by simple null hypotheses. Studies using these methods have revealed
patterns in biogeography (e.g., Claramunt and Cracraft 2015; Field and Hsiang
2018; Moyle et al. 2016; Wang et al. 2016), rates of diversification (e.g., Ricklefs
2007; Jetz et al. 2012), and many other types of phenotypic changes (e.g., Cooney
et al. 2017; Hosner et al. 2017; Field et al. 2018; Stoddard et al. 2017). Phylogenetic
trees can inform conservation priorities (e.g., Diniz-Filho et al. 2013; Jetz et al.
2014). Trees are also a necessary component of analyses that range from those
focused on examining sequence conservation and the genomic landscape (e.g.,
Botero-Castro et al. 2017; Zhang et al. 2015) to the relationship between whole-
organism traits and patterns of molecular change, such as evolutionary rates, amino
acid substitutions, and base composition (e.g., Zhang et al. 2014; Berv and Field
2018; Weber et al. 2014a, b). Phylogenomic studies have proven to be the most
revealing, surprising, and reliable of all sources of phylogenetic information. This
assertion may seem surprising given the conflicts among phylogenomic studies that
we have highlighted (Fig. 2), but phylogenomic trees exhibit substantially more
topological similarities than trees generated before the phylogenomic era. Indeed, it
has been only by virtue of conflicts that we have better come to understand the nature
and complexity of the evolutionary and historical processes that appear to have
misled earlier studies, both molecular (e.g., heterotachy, nonstationarity, ILS, and
hybridization) and morphological (e.g., convergent evolution; cf. Mayr 2008;
Sackton et al. 2018).
5 Where Do We Go From Here? The Future of Avian

Phylogenomics
We anticipate many challenges as the field of phylogenetics moves from analyses of

“phylogenomic-scale” datasets, like those generated by sequence capture methods
(Table 2), to analyses of truly whole-genome scale. The Jarvis et al. (2014) analyses
of 48 bird genomes required more than 400 years of CPU time; 42 of those CPU
years were devoted to the 322-Mbp whole-genome tree. Obviously, using the same
methods of analysis for the ~10,000 bird species recognized at this time will be
impractical. Filtering genome alignments to focus on the type(s) of data that are most
likely to provide an accurate estimate of evolutionary history may prove to be
necessary, thus further exploration of data-type effects will be no doubt be helpful.
Of course, the assertion that a specific data type yields a topology close to the true
tree is a hypothesis; finding ways to corroborate specific hypotheses regarding data-
type effects represents a major challenge for the field of phylogenomics. In principle,
improved models of sequence evolution could address data-type effects, but efforts
to develop complex (and presumably very computationally-demanding) models may
actually take us in the wrong direction. We argued that rare genomic changes might
be an extremely valuable source of information; rare genomic change data could
provide another solution to the computational problem because it might be possible
to use MP for their analyses without sacrificing accuracy. Moreover, the use of
rare genomic change data intrinsically reduces whole-genome alignments to much
smaller and, therefore, easier-to-analyze data matrices. This shifts the computational
challenges away from the tree search and toward the identification of rare genomic
changes; those computational challenges are likely to be further ameliorated by the
availability of platinum-quality genome assemblies with high-quality gene annotations.
Obviously, there may be other solutions to the computational challenges associated
with phylogenetic analysis of complete avian genomes, but the fact that it is possible
to envision some practical ways forward makes us sanguine that the challenges can
be solved.
In summary, it is clear that the avian tree of life will grow substantially over the
next few years, and we can expect that nearly all named taxa will eventually be
included. As we alluded to in our discussion of megaphylogenies, there is now a
major impetus among systematists globally to identify more and more “cryptic taxa”
(whatever their rank). This might easily swell avian taxonomic diversity from the
~10,000 species that are currently recognized to more than 30,000 evolutionary
entities. Investigators of avian diversity and biology will want all of these taxa to find
a home on the avian tree. Throughout this review, we have generally been agnostic
regarding the direction that analytic methods will take in the future, simply describ-
ing the results of various analyses and, in some cases, the strengths and weaknesses
of those methods. However, we believe any comprehensive avian tree should
ultimately reflect the results of an empirical approach that links the data and the
tree in a direct manner, such as the global supermatrix approach akin to the Burleigh
et al. (2015) study. Ideally, that tree would incorporate data from rare genomic
changes as well as sequence information. This will present huge computational
challenges, and it is unclear, for reasons we articulated above, how the community
will meet these challenges. Increasing incorporation of more taxa and more
characters has always called for shifts in analytical thinking, and these shifts will
no doubt continue to happen. Important questions in avian biology will be asked and
answered using many different kinds and amounts of data. Some of those questions
will require genome-scale data; some will even require reference quality genome
assemblies. However, it will remain possible to address many innovative questions
without genome-scale data. With that in mind, it is important that ornithologists
focus on developing and framing the innovative questions in avian biology; there is
little doubt that genome-scale data for birds will become broadly available and be
combined with dense taxon sampling. We expect that genome-scale data will make it
possible to answer new questions and push the frontiers of avian biology forward
over the coming years.
Acknowledgments We are grateful to Robert Kraus for inviting this chapter and for his encour-
agement (and patience) while we were writing it. We would also like to express our gratitude to
Tom Gilbert and two anonymous reviewers for insightful comments that improved the manuscript.
E.L.B. acknowledges support from the US National Science Foundation grants DEB-1118823 and
DEB-1655683 (the “OpenWings” project) and a seed grant from the University of Florida Biodi-
versity Institute; J.C. acknowledges the US National Science Foundation awards DEB-1241066 and
DEB-1146423.
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Avian Species Concepts in the Light
of Genomics
Jente Ottenburghs
Abstract
What is a species? This seemingly simple question has occupied the minds of
numerous biologists and philosophers, resulting in the formulation of many
species concepts. From a theoretical point of view, the species problem has
been resolved by equating species with independently evolving lineages
(i.e. the evolutionary species concept or the general lineage concept). However,
the practical issues with describing and delineating species remain. The origin of
species is a gradual process that typically requires thousands to millions of years,
creating a grey zone of species delimitation in which taxonomy is often contro-
versial. To account for this, an integrative taxonomy has been proposed in which
different taxonomic concepts and methods are integrated in the delimitation of
species. In this chapter, I argue that genomics provides another line of evidence in
this pluralistic approach to species classification. Indeed, genomic data can be
combined with classical species criteria, such as diagnosability, phylogeny and
reproductive isolation. First, genomic data can provide an extra diagnostic feature
in species delimitation. Compared to ‘old-school’ genetic markers, the use of
genome-wide markers leads to a significant rise in statistical power. Second,
phylogenomic analyses can resolve the evolutionary relationships within rapidly
diverging or hybridizing groups of species while taking into account gene tree
discordance. Third, genomic data can be used to pinpoint the genetic basis of
reproductive isolation and provide a detailed description of the speciation pro-
cess. All in all, the genomic era will supply avian taxonomists with a new tool box
that can be applied to old concepts, leading to better informed decisions in
cataloguing biodiversity.
J. Ottenburghs (*)
Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden

https://doi.org/10.1007/978-3-030-16477-5_7
212 J. Ottenburghs
Keywords
Distinguishability · General lineage concept · Hybridization · Integrative
taxonomy · Introgression · Phylogenomics · Reproductive isolation · Speciation ·
Species delimitation · Tobias criteria
1 Introduction
The debate on the definition of a species, commonly known as the species problem
(Richards 2010), has produced an insurmountable quantity of literature. Yet,
Darwin’s (1859, p. 44) description still holds today: ‘No one definition has yet
satisfied all naturalists; yet every naturalist knows vaguely what he means when he
speaks of a species’. The debate continues because of two main difficulties: (1) the
confusion between species concepts and species criteria and (2) the continuous
nature of species.
The first difficulty highlights the fact that the species problem consists of two
major issues: the theoretical question of what species are (i.e. species concepts) and
the ways in which species can be delimited in practice (i.e. species criteria). It is
important to realize that species concepts are not species criteria (Hey 2006). Many
taxonomists have accepted the view that species are lineages (Mayden 1997; De
Queiroz 1998, 1999), thereby resolving the first theoretical issue. In contrast to this
theoretical triumph, the practical aspect of the species question is still an active field
of research. There are many ways in which species can be described, including
morphological, ecological and genetic approaches. How should these different lines
of evidence for the status of particular taxa be interpreted? And what is the role of
genomics in species discovery and delimitation?
The second difficulty, the continuous nature of species, is a direct consequence of
the gradual process of speciation. The origin of new species typically requires
thousands to millions of years, creating a grey zone of species delimitation in
which taxonomy is often controversial (Roux et al. 2016). In this grey zone, different
species criteria might result in diverse decisions on the assignment of species ranks.
The developing field of speciation genomics might provide solutions for taxonomists
dealing with the continuous nature of species (Seehausen et al. 2014; Campbell et al.
2018).
In this chapter, I will explore the role of genomics in the debate of species
concepts and species criteria. First, I will show how others have made substantial
progress on these issues by adopting theoretical monism—species as lineages—and
practical pluralism, paving the way for an integrative taxonomy (Padial et al. 2010).
Then, I will introduce the most commonly used species criteria in avian taxonomy
(distinguishability, reproductive isolation and monophyly) and how they can be
integrated in species delimitation (Sangster 2014). Finally, I will show how genomic
data can be incorporated into taxonomic decisions. Some authors have proposed a
‘genomic species concept’ (e.g. Jarvis 2016). I will argue that there is no need for
Avian Species Concepts in the Light of Genomics 213
another species concept; rather, genomic data can be incorporated in the existing
species criteria.
2 Species Concepts Are Not Species Criteria
During the twentieth century, there has been a proliferation of species concepts.
Mayden (1997) lists no less than 22 distinct species concepts, including some
familiar ones such as the biological species concept (Mayr and Ashlock 1991), the
phylogenetic species concept (Cracraft 1983) and the recognition species concept
(Paterson 1993). Despite this plethora of species concepts, the so-called silver bullet
species concept, one that is universally applicable, was not achieved. A universal
species concept could not be formulated because of two main issues: (1) the plural-
istic nature of species and (2) the tension between conceptualization and delimitation
(Hey 2006). First, the proliferation of species concepts is a direct consequence of the
diversity of life: different taxonomic groups require different species concepts
depending on their particular characteristics (Mayden 1997). For instance, the
biological species concept, which stresses reproductive isolation between members
of different species, cannot be applied to asexually reproducing organisms. Second,
the issue of species conceptualization is often confused with the issue of species
delimitation (Mayden 1999): concepts are theories or ideas that are general and may
or may not be based on empirical observations, while species delimitation requires a
prescribed set of repeatable operations that lead to the outcome of whether a certain
group of individuals represent a species or not. Hey (2006) summarized this issue
nicely: ‘As scientists we should not confuse our criteria for detecting species with
our theoretical understanding of the way species exist. Detection protocols are not
concepts’.
2.1 Theoretical Monism
To resolve these issues, Mayden (1997) proposed a hierarchy of species concepts,

with a primary theoretical species concept and several secondary operational species
concepts. He argued that only the evolutionary species concept is suitable as primary
concept, because it is theoretically robust and generally applicable. This concept
states that a species is ‘an entity composed of organisms which maintains its identity
from other such entities through time and over space, and which has its own
independent evolutionary fate and historical tendencies’ (Wiley and Mayden
2000). The remaining concepts are secondary, functioning as guidelines that are
essential for the study of species in practice (Wiens and Servedio 2000; Sites and
Marshall 2004). Together, the primary and secondary species concepts form a
hierarchical system.
Similarly, De Queiroz (1998, 1999) reviewed several existing species concepts
and argued that all existing species concepts are variants of a single general concept,
which he dubbed ‘the general lineage concept’. Species are considered separately
214 J. Ottenburghs
Fig. 1 This simplified

diagram represents a single
lineage splitting into two
independently evolving 2 Species
lineages (or species). The
horizontal lines represent the
times at which the lineages
acquire different properties SC9
(e.g. they become phenetically
SC8
distinguishable,
reproductively isolated, SC7
reciprocally monophyletic, Gray Zone SC6
etc.). This set of properties (1 vs.2 species)
SC5
(SC species concept)
coincides with a grey zone in SC4
which alternative species SC3
concepts come into conflict. SC2
On either side of the grey
zone, there is agreement on SC1
the number of species.
Adapted from De Queiroz
(2007)
1 Species
evolving metapopulation lineages. A lineage indicates ‘an ancestor-descendant

series’, and metapopulation refers to an ‘inclusive population made up of connected
subpopulations’ (De Queiroz 2007). The difference between the existing species
concepts lies in the specific criterion that they emphasize, such as reproductive
isolation (Mayr and Ashlock 1991), systems of mate recognition (Paterson 1993)
or diagnosability (Cracraft 1983). These criteria should not be regarded as necessary
for species delimitation, but rather as contingent upon the evolutionary history of the
taxa. Moreover, during the speciation process, there will be a grey zone in which
different species criteria come into conflict (Fig. 1). Here, a ‘life history approach’ is
warranted, in which different species properties correspond to different stages in the
life history (i.e. speciation process) of a species (Harrison 1998). It is important to
keep in mind that the order in which species properties arise is contingent upon the
speciation process. In some cases, morphological differentiation evolves first,
followed by reproductive isolation, while, in other cases, it might be the other way
around. By acknowledging the continuous nature of speciation and realizing that
consequently species criteria might lead to conflicting outcomes in this grey zone,
taxonomists can be better informed in decisions on the number of species.
So, both De Queiroz (1998, 1999) and Mayden (1997) reached a similar con-
clusion, albeit using a different approach (Hey 2006; Naomi 2011). The species
problem can thus be partly resolved by theoretical monism (the evolutionary species
concept or general lineage concept) in combination with practical pluralism, in
which different species criteria correspond to different stages during the speciation
process.
2.2 Practical Pluralism in Avian Taxonomy
The view that species are (segments of) lineages emphasizes the unity of various
rival approaches to species delimitation and recognizes that taxonomy is necessarily
pluralistic because species have different characteristics. Sangster (2014) examined
taxonomic bird studies published between 1950 and 2009. He compared the appli-
cation of six species criteria in the description of new species, proposals to change
the taxonomic rank of species and subspecies and the taxonomic recommendations
of the American Ornithologists’ Union Committee on Classification and Nomen-
clature. The six criteria can be divided in three classes. They are based on several
species concepts and are not viewed as necessary for the delimitation of species. I
will briefly introduce these criteria and the species concepts they relate to
(De Queiroz 1998, 1999; Sangster 2009).
1. Distinguishability
1a. Diagnosability
If a taxon has a unique, fixed character or a unique combination of character
states, it can be considered a separate species. This criterion is based on the
diagnostic approach (Baum and Donoghue 1995) of the phylogenetic species
concept (Cracraft 1983). Nixon and Wheeler (1990) describe diagnosability as
follows: ‘the smallest aggregation of populations (sexual) or lineages (asexual)
diagnosable by a unique combination of character states in comparable
individuals’.
1b. Degree of difference
Some authors argue that taxa should be recognized as distinct species if they
differ ‘too much’ from other related taxa. The differences may refer to morpho-
logical, biometric, behavioural or genetic characters. Hence, the degree of differ-
ence criterion can be traced back to several species concepts, such as the
recognition species concept (Paterson 1993), the phenetic species concept
(Sokal and Sneath 1963) or the genetic cluster concept (Mallet 1995).
2. Phylogeny
2a. Monophyly
The criterion that taxa should be separated to prevent the recognition of a
paraphyletic species taxon is based on the monophyletic version (de Queiroz and
Donoghue 1988) of the phylogenetic species concept (Cracraft 1983).
2b. Exclusive coalescence
Baum and Shaw (1995) provided a genealogical perspective to the species
problem and argued that a species is ‘a group of organisms [that] is exclusive if
their genes coalesce more recently within the group than between any member of
the group’. This species criterion is often considered a special version of the
phylogenetic species concept (Baum and Donoghue 1995; Luckow 1995).
216 J. Ottenburghs
3. Cohesion
3a. Adaptive zone
The adaptive zone criterion is based on ecological differences or the occupa-
tion of different niches, emphasizing the importance of ecologically based natural
selection in the maintenance of species. This criterion is related to the ecological
species concept which states that ‘a species is a lineage (or a closely related set of
lineages) which occupies an adaptive zone minimally different from that of any
other lineage in its range and which evolves separately from all lineages outside
its range’ (Van Valen 1976).
3b. Reproductive isolation
This criterion can take many forms and depends on the nature of reproductive
isolation. A distinction is made between pre- and postzygotic isolation
mechanisms (Coyne and Orr 2004): prezygotic isolation mechanisms act before
fertilization, whereas postzygotic isolation mechanisms act after fertilization and
can be either intrinsic or extrinsic. Intrinsic postzygotic isolation mechanisms
lead to sterility or inviability of the offspring, while extrinsic postzygotic isolation
mechanisms encompass lower fitness of the offspring for ecological or
behavioural reasons, not developmental defects. Depending on the specific repro-
ductive isolation mechanism, this criterion can be traced back to several concepts,
such as the recognition species concept (Paterson 1993), the biological species
concept (Mayr and Ashlock 1991) or the cohesion species concept (Templeton
1989).
The analysis of Sangster (2014) showed that the most frequently used species
criterion in avian taxonomy is diagnosability, followed by reproductive isolation and
degree of difference. Furthermore, reproductive isolation is less frequently applied in
studies of allopatric taxa compared to sympatric or parapatric taxa, suggesting that
avian taxonomists are reluctant to speculate on the degree of reproductive isolation
between allopatric populations. One approach of species delimitation of allopatric
populations concerns the so-called Tobias criteria, in which the divergence in a
sample of sympatric species is used to calibrate thresholds for species status in
parapatric or allopatric taxa (Tobias et al. 2010). This procedure was formally
introduced by Mayr (1969) and later recast in a more quantitative framework by
Isler et al. (1998), who provided guidelines for species delimitation in antbirds
(family Thamnophilidae) based on song divergence in several sympatric species
pairs. Tobias et al. (2010) expanded this procedure to more bird families and argued
that it ‘can be applied to the global avifauna to deliver taxonomic decisions with a
high level of objectivity, consistency and transparency’. Nevertheless, this system
has been criticized, and there is certainly room for improvement (e.g. Donegan
2018).
More importantly, however, the analysis of Sangster (2014) revealed that nearly
half of the studies (46.5%) applied multiple criteria in species delimitation,
supporting the notion that avian taxonomy is pluralistic. This pluralism is in line
with propositions for an integrative taxonomy (Dayrat 2005; Padial et al. 2010) in
which different taxonomic concepts and methods are integrated in the delimitation
and description of species. Within this context, two general frameworks are being
advocated: ‘integration by congruence’ and ‘integration by cumulation’ (Padial et al.
2010).
The congruence approach to species delimitation entails that different datasets,
such as molecular and morphological characters, support the decision to recognize
certain taxa as valid and distinct species (Dayrat 2005; DeSalle et al. 2005). For
example, Alström et al. (2008) used congruence between plumage, biometrics, egg
coloration, song, mitochondrial DNA and distribution to draw species limits in the
Bradypterus thoracicus complex. The main advantage of this approach is that most
taxonomists will agree on the validity of a species supported by several independent
datasets, leading to taxonomic stability. The limitation of integration by congruence
is the risk of underestimating species numbers, because speciation does not always
involve character change at all levels (Adams et al. 2009), specifically in adaptive
radiations where ecological divergence and reproductive isolation develop at differ-
ent rates (Seehausen 2004; Shaffer and Thomson 2007).
The alternative framework, integration by cumulation, is based on the assumption
that divergence in any of the datasets can be used as evidence for the delimitation of
species. Congruence is desired but not necessary (De Queiroz 2007). In practice,
evidence from different datasets is cumulated, concordances and discordances are
explained within the specific evolutionary context of the taxa under study, and based
on the available evidence a decision is made. An advantage of this approach is that
species delimitation is not restricted by a particular biological property. The main
disadvantage is that the uncritical use of a single line of evidence, such as mtDNA,
can lead to an overestimation of species numbers (Agapow et al. 2004; Isaac et al.
2004; Meiri and Mace 2007).
The occurrence of concordances and discordances between species criteria is
predicted by the general lineage concept (De Queiroz 1998, 1999), because the
continuous nature of speciation results in the occurrence of certain criteria at
particular phases during the speciation process. Discordance between species criteria
is to be expected in the early stages of speciation. Furthermore, which characters
diverge first is contingent on the evolutionary history of the taxa under investigation.
For example, in some cases, genetic divergence might precede morphological
differentiation, while in other cases, it might be the other way around. As speciation
proceeds, more characters may diverge, resulting in concordance among different
species criteria.
3 Enter Genomics
Genomics has become a standard practice in ornithology (Kraus and Wink 2015;
Oyler-McCance et al. 2016; Toews et al. 2016; Wink 2019). With genomics I refer to
the next-generation sequencing techniques discussed in Toews et al. (2016), namely,
whole genome sequencing and resequencing, reduced representation techniques
[genotype-by-sequencing (GBS) and restriction-site-associated DNA sequencing
(RADseq)], sequence capture and RNA sequencing. The impact of these novel
218 J. Ottenburghs
sequencing techniques on avian species delimitation has only started to be addressed

in the literature.
Several microbiologists have proposed a system of species delimitation solely
based on genomic data (Konstantinidis et al. 2006; Meier-Kolthoff et al. 2013).
Jarvis (2016) extended this line of thinking to vertebrates and argued that ‘with the
availability of extant genomes of all “species” of a vertebrate class, one could define
or redefine species based on genomic distances’. This approach is not feasible for
several reasons. First, what threshold should be applied? The decision for a certain
threshold of minimum divergence seems arbitrary. Furthermore, comparable to the
>2% divergence rule for mitochondrial DNA barcodes (Johns and Avise 1998;
Hebert et al. 2004), it might not be applicable to all bird taxa (Lovette 2004; Will
et al. 2005). Second, how will the genomic distance be calculated? An average over
the whole genome ignores heterogeneity in divergence across the genome (Ellegren
et al. 2012; Poelstra et al. 2014; Dhami et al. 2016; Wolf and Ellegren 2017). Indeed,
divergence is often concentrated in certain ‘genomic islands’ (Michel et al. 2010;
Cruickshank and Hahn 2014).
In the remainder of this chapter, I will argue that there is no need for a genomic
species concept in birds; rather, genomic data can be incorporated in the three classes
of species criteria described by Sangster (2014), namely distinguishability, phylo-
geny and cohesion (and particularly the reproductive isolation criterion).
3.1 Genomics and Distinguishability
Distinguishability encompasses two species criteria: diagnosability and degree of

difference. The former criterion emphasizes that a unique, fixed character or a unique
combination of character states can be used to delineate species, while the latter
criterion emphasizes that this character or combination of character states should be
sufficiently different from other related taxa. In both criteria, the main point is the
observation of certain diagnostic features, which can be morphological, biometric,
behavioural or genetic characters.
Genetic markers, such as mtDNA, microsatellites or AFLPs (amplification frag-
ment length polymorphisms), have often been used to discriminate between species
(Hebert et al. 2004; Sites and Marshall 2004). Genome-wide datasets add more
power and sensitivity to detect subtle genetic differentiation. For instance, using
RADseq data, Ruegg et al. (2014a, b) were able to more reliably distinguish between
eastern and western populations of the Wilson’s warbler (Cardellina pusilla) com-
pared to previous studies based on mtDNA (Kimura et al. 2002; Paxton et al. 2013)
and AFLPs (Irwin et al. 2011).
As discussed above, discordance between species properties is to be expected in
the early stages of speciation. Also genomic data might not be in line with other
datasets. For example, based on thousands of SNPs, Oyler-McCance et al. (2015)
found genetic differentiation between the main North American population of
greater sage-grouse (Centrocercus usophasianus) and a parapatric population in
California and Nevada (referred to as the ‘bistate’ population). However,
morphological and behavioural studies revealed little or no differences between

these populations (Taylor and Young 2006; Schroeder 2008). An opposite pattern
was uncovered in redpoll finches (genus Acanthis) where several putative species
differed phenotypically despite largely undifferentiated genomes (Mason and Taylor
2015).
However, as speciation proceeds and more characters diverge, concordance
among different datasets might arise. For example, a genomic analysis of two
subspecies of the willet (Tringa semipalmata) indicated that both subspecies are
genetically distinct, providing an extra line of evidence, next to ecological,
behavioural and morphological differences, to treat these subspecies as separate
species (Oswald et al. 2016). Similarly, Gohli et al. (2015) argue that several
subspecies in the Afrocanarian blue tit (Cyanistes teneriffae) complex should be
treated as distinct species because of concordance among different characters, such
as genomics, morphology, song and sperm morphology.
These examples show that genomic data is not a defining species criterion but can
provide an extra diagnostic feature in the description and delimitation of species.
Compared to ‘old-school’ genetic markers (e.g. mtDNA or microsatellites), genomic
data result in a significant rise in statistical power to make inferences about species
ranks.
3.2 Genomics and Reproductive Isolation
One of the most commonly applied species criteria in avian taxonomy is reproduc-
tive isolation. This criterion is actually closely related to the most frequently used
criterion of diagnosability because reproductive isolation is often necessary for the
fixation and maintenance of diagnostic differences in sympatric and parapatric taxa
(Sangster 2014).
Reproductive isolation is mostly caused by the combination of several pre- and
postzygotic isolation mechanisms. Because these mechanisms often interact, it may
be difficult to determine the relative importance of each mechanism. Furthermore,
the present importance of a mechanism might be different from its historical
importance (Coyne and Orr 2004; Lackey and Boughman 2017). The interplay of
different reproductive isolation mechanisms can be depicted as a continuum from a
panmictic population to two irreversibly isolated species (Seehausen et al. 2014).
Speciation can be driven by divergent sexual or ecological selection, in which case
extrinsic postzygotic and prezygotic mechanisms act first and intrinsic postzygotic
mechanisms come into play later in the speciation process (Fig. 2a). Alternatively,
speciation can be driven by intrinsic postzygotic mechanisms, such as genetic
incompatibilities. Extrinsic postzygotic and prezygotic mechanisms accumulate
and reinforce reproductive isolation at a later stage (Fig. 2b). Hendry et al. (2009)
recognize four stages across the speciation continuum: (1) continuous variation
without reproductive isolation; (2) discontinuous variation with minor reproductive
isolation; (3) strong, but reversible, reproductive isolation; and (4) strong and
irreversible reproductive isolation. It is important to keep in mind that movement
220 J. Ottenburghs
a Speciation driven by divergent selection b Speciation driven by intrinsic barriers

Barrier effects
Barrier effects
Extrinsic postzygotic
Intrinsic
and prezygotic barriers
postzygotic
barriers
Intrinsic Extrinsic
postzygotic postzygotic and
barriers prezygotic
barriers
Panmictic Speciation continuum Two irreversibly Panmictic Speciation continuum Two irreversibly
populations isolated species populations isolated species
Fig. 2 The speciation continuum of speciation processes driven by (a) divergent selection, where
prezygotic and extrinsic postzygotic barriers evolve before intrinsic postzygotic barriers, and (b) by
intrinsic barriers, where intrinsic postzygotic barriers evolve before prezygotic and extrinsic
postzygotic barriers. The shapes of the curves are hypothetical. Adapted from Seehausen et al.
(2014)
along the speciation continuum is not constant; speciation can go back and forth at
different speeds or come to a halt at certain stages (e.g. formation of a stable hybrid
zone).
In line with the general lineage concept, the speciation continuum emphasizes the
continuous and contingent nature of speciation. By studying how populations move
across the species continuum (i.e. by reconstructing their evolutionary history), one
gains more insights into the speciation process, and one can make more informed
decisions in the delimitation of particular species. In other words, one needs to study
the process of speciation.
The term speciation was first used by Cook (1906) to describe ‘the origination or
multiplication of species by subdivision, usually, if not always, as a result of
environmental incidents’. Traditionally, speciation models have been classified in
a spatial context, namely, the well-known Mayrian triumvirate consisting of allo-
patry, parapatry and sympatry (Bush 1975; Butlin et al. 2008). In allopatric speci-
ation, the geographic range of a species is split in two or more isolated populations
that diverge by natural selection and/or genetic drift. Parapatric speciation concerns
the evolution of reproductive isolation between geographically overlapping
populations that still exchange genes to a limited extent. Sympatric speciation refers
to the situation in which new species originate from a single ancestral population
while inhabiting the same geographical region. From a population genetic per-
spective, allopatric and sympatric speciation are the ends of a continuum of gene
flow (m), with parapatric speciation in between (Fig. 3, Gavrilets 2004). From this gene
flow perspective, the latter two geographic speciation models (parapatry and sympatry)
are combined under the heading divergence-with-gene-flow (Fitzpatrick et al. 2008;
Pinho and Hey 2010).
The geographical classification of speciation models has been useful and is still
widely applied today (Harrison 2012). In addition, some more refined
Fig. 3 Speciation models on the basis of geography and gene flow. Each circle represents the
geographical range of a species (in red or yellow). The orange colour indicates that two species
overlap in their ranges. Gene flow is expressed as the migration (m) of alleles or gene flow from one
population to the other and varies between 0 and 0.5. The parapatric and sympatric speciation
models are usually combined under the heading divergence-with-gene-flow
geographically inspired speciation models emerged, such as peripatric (Mayr 1982),

stasipatric (White 1969), centrifugal (Brown 1957), microallopatric (Smith 1965;
Paulay 1985) or allosympatric speciation (Coyne and Orr 2004; Mallet 2005).
However, this geographical classification does have its limitations (Butlin et al.
2008), and other ways to classify speciation models have been proposed (Templeton
1981; Kirkpatrick and Ravigne 2002; Gavrilets 2004).
New ways of classifying speciation may result in a proliferation of speciation
models, similar to the situation on species concepts. Indeed, Kirkpatrick and
Ravigne (2002) noted that ‘theoreticians have balkanized the subject of speciation’,
because each mathematical model focuses on a highly specific scenario. A promising
attempt at an overarching ‘process-based’ classification has been made by
Dieckmann et al. (2004). They envision speciation as a route through a three-
dimensional cube (which I will call the ‘speciation cube’), of which the axes
represent spatial, ecological and mating differentiation (Fig. 4). At the onset of
speciation, there is no differentiation between the populations, which corresponds
to the starting point at the origin (i.e. lower left corner). In the classic allopatric
model, an external cause (represented by a dotted line) leads to spatial differentia-
tion, and the populations consequently diverge under genetic drift (dashed line) or
selection (solid line), resulting in mating and/or ecological differentiation.
Sympatric speciation scenarios can also be depicted in this ‘speciation cube’.
Because no external causes lead to spatial differentiation, the lines are restricted to
the front plane of the cube. Divergence can be driven by sexual selection (leading to
assortative mating) or ecological differentiation (Bolnick and Fitzpatrick 2007). A
textbook example of differentiation by sexual selection has been documented in
Lake Victoria (East Africa), where several sympatric populations of cichlid fish
show divergence in male colouration and female preferences (Seehausen et al.
2008). Differentiation by ecology-based divergent selection is commonly referred
to as ‘ecological speciation’ (Rundle and Nosil 2005; Nosil 2012). Several classical
examples of ecological speciation involve sympatric phytophagous insect species
using different host plants (Berlocher and Feder 2002), such as the apple maggot
222 J. Ottenburghs
al n
ALLOPATRIC ati tio
Sp ntia
SPECIATION r e Driven externally
f e
dif Driven by genetic drift
Driven by selection
n
ntiatio
g
Before speciation
Matin
After speciation
differe
Eco
diffe logica
ren l
tiatio
n
al n
ati tio
SYMPATRIC Sp ntia
r e Driven by selection
f e
SPECIATION dif
n
Before speciation
ntiatio
After speciation
g
Matin
differe
Eco
diffe logica
ren l
tiatio
n
al n
ati tio
Sp ntia
TWO-PHASE fe r e
Driven externally
dif
SPECIATION Driven by selection
n
ntiatio
Before speciation
g
Matin
After speciaton
differe
Eco
diffe logica
ren l
tiatio
n
Fig. 4 A process-based representation of speciation as ‘speciation cubes’. In the cube the axes
represent ecological (x), mating ( y) and spatial (z) differentiation. Divergence between two
populations can be driven by external processes (dotted line), genetic drift (dashed line) or selection
(solid line). These speciation cubes can be used to visualize different speciation models (see text for
further explanation and examples). Adapted from Dieckmann et al. (2004)
(Rhagoletis pomonella), which specializes on hawthorns and apples (Feder et al.

1988, 2003).
Sympatric speciation in birds is rare and controversial (Phillimore et al. 2008;
Price 2008). One putative case concerns African indigobirds, which are host-specific
brood parasites targeting several species, such as red-billed firefinch (Lagonosticta
senegala) and African firefinch (L. rubricata). Males mimic the song of the host,
while females chose their mates based on song and the nests they parasitize. This
situation can lead to immediate reproductive isolation when a host-shift occurs,
culminating in a sympatric speciation event (Sorenson et al. 2003). Given that
female indigobirds choose their partner based on host song, this particular case
can be considered sympatric speciation by sexual selection. To my knowledge,
African indigobirds represent the only well-documented avian example of such as
scenario. Several macroevolutionary studies found no support for sexual selection
directly driving (sympatric) speciation (Morrow et al. 2003; Huang and Rabosky
2014; Cooney et al. 2017). In general, sympatric speciation in birds seems to rely on
additional factors, such as ecological differentiation.
Differentiation by ecology-based divergent selection—ecological speciation—

has been proposed for several bird taxa (e.g. Caro et al. 2013; Ryan et al. 2007;
Smith et al. 2011; Zhen et al. 2017). For example, populations of the little greenbul
(Andropadus virens) are distributed across an ecological gradient between rainforest
and savannah in sub-Saharan Africa. Along this gradient, or ecotone, selection
pressures are expected to vary due to differences in environmental factors, such as
rainfall and tree cover. The resulting divergent natural selection might lead to
phenotypic differentiation and ultimately speciation. Several studies have shown
that this is indeed the case for the little greenbul (Smith et al. 1997, 2005; Kirschel
et al. 2011; Zhen et al. 2017). Ecotones might provide the ideal conditions for
ecological speciation, as exemplified by other bird studies along similar gradients
(Ribeiro et al. 2011; Smith et al. 2011; Caro et al. 2013).
Ecological speciation can be facilitated by so-called magic traits, which refer to
the situation where traits under divergent natural selection and traits involved in mate
choice are the same or share a similar genetic background (Servedio et al. 2011). In
birds, beak morphology is an obvious ‘magic trait’ candidate: divergent selection
can lead to distinct beak morphologies. Different beaks produce different sounds,
providing the opportunity for assortative mating. This scenario has been described
for North American red crossbills (Loxia curvirostra). This species complex can be
divided into nine call types based on differences in vocalizations, bill size and palate
structure (Benkman 1993, 1999). These differences are the result of divergent
selection for foraging on different conifer species. Hence, each call type inhabits a
distinct ecological niche (Benkman 2003). Behavioural and genetic studies indicated
that this ecological specialization contributes to reproductive isolation between
several call types (Parchman et al. 2006; Smith and Benkman 2007; Smith et al.
2012). Other examples include subspecies of the swamp sparrow (Melospiza
georgiana, Ballentine et al. 2013) and populations of medium ground finch
(Geospiza fortis) on Santa Cruz Island in the Galápagos archipelago (Podos et al.
2013).
A special case of speciation in sympatry is allochronic speciation, in which
populations diverge because they breed in different times of the year (Taylor and
Friesen 2017). This scenario has been described for several members of the sub-
family Hydrobatinae (storm petrels), such as Madeiran storm petrels (Hydrobates
castro) on the Azores (Monteiro and Furness 1998; Friesen et al. 2007) or Ainley’s
storm petrel (H. cheimomnestes) and Townsend’s storm petrel (H. socorroensis) on
Guadalupe (Taylor et al. 2018). Other species pairs in this subfamily have probably
diverged in allochrony after an initial allopatric stage (Wallace et al. 2017).
Apart from allopatric and sympatric speciation scenarios, ‘speciation cubes’ can
also be used to depict more complex, often multiphase, speciation processes. For
example, Fig. 4 shows a scenario in which two populations are first geographically
isolated and develop partial ecological and mating differentiation in allopatry. Later
they re-establish contact and further ecological and mating differentiation occurs.
Such complex speciation scenarios seem to be common in birds: numerous cases of
secondary contact after a period of geographical isolation have been documented
(Price 2008; Ottenburghs et al. 2017). A classical example of such a secondary
224 J. Ottenburghs
contact is the hybrid zone between the carrion crow (C. corone corone) and
hooded crow (C. c. cornix) across central Europe (Meise 1928).
The process-based approach by Dieckmann et al. (2004) complements the life
history approach to the species problem, discussed above (Harrison 1998). The
‘speciation cube’ allows for the depiction of many different speciation scenarios,
each representing the specific life history of a particular species pair. In all cases,
however, there is the build-up of reproductive isolation between two or more
populations. Genomic data can be used to provide a genetic basis for these repro-
ductive isolation mechanisms (Wu and Ting 2004; Presgraves 2010). In birds,
reproductive isolation is generally driven by prezygotic isolation mechanisms.
Intrinsic postzygotic isolation mechanisms, such as hybrid sterility of unviability,
evolve at a slower rate (Fitzpatrick 2004). In mammals, male hybrid sterility has
been linked to PRDM9, a rapidly evolving zinc finger protein that probably plays a
role in modifying recombination hotspots (Payseur 2016). Interestingly, birds lack
PRDM9, which might explain the slow evolution of intrinsic postzygotic isolation
(Singhal et al. 2015; Vignal and Eöry 2019).
One of the newest genomic tools in speciation research are genome scans (Haasl
and Payseur 2016), which calculate divergence statistics, such as FST or nucleotide
diversity (π) in windows across the genome. Analyses of recently diverged species
have uncovered highly heterogeneous patterns of genetic divergence across the
genome (Harr 2006; Michel et al. 2010; Ellegren et al. 2012; Nadeau et al. 2012;
Ravinet et al. 2017; Wolf and Ellegren 2017): some genomic regions show little
genetic differentiation, while other regions show high levels of differentiation and
may even harbour fixed differences that could be used to distinguish between
species.
The observation of such heterogeneous genomic divergence is often linked to the
speciation model of divergence-with-gene-flow, during which genetic differentiation
accumulates in some genomic regions, while the rest of the genome (which is
assumed to be largely neutral) is homogenized by gene flow (Barton and Bengtsson
1986; Nosil et al. 2009). Assuming that genes in these differentiated regions may be
involved in reproductive isolation, these regions have been referred to as ‘genomic
islands of speciation’ (Turner et al. 2005).
However, heterogeneous patterns of genetic differentiation can also be explained
by alternative models in which there is no gene flow between recently diverged
species (Noor and Bennett 2009; Cruickshank and Hahn 2014; Ravinet et al. 2017;
Wolf and Ellegren 2017). In these models, reproductive isolation is instantaneous
and complete (e.g. allopatric speciation). Species pairs show low levels of differ-
entiation because they have recently split. Shared alleles are due to ancestrally
inherited variation, not introgression. Heterogeneity in the level of differentiation
across the genome can be explained by stochastic variation in coalescent times and
natural selection. Loci experiencing strong selection will share less ancestral vari-
ation and will appear to be more differentiated. Hence, genomic regions under
selection may be directly involved in reproductive isolation or they may be unrelated
to any trait associated with species divergence and are simply influenced by ‘back-
ground’ selection. In contrast to the previous model, there is no differential gene flow
across loci, and the regions of highest diversity do not necessarily house genes
involved in reproductive isolation (Turner and Hahn 2010).
When studying genomic differentiation between species, one must consider that
species divergence does not only involve changes in DNA sequences but also
rearrangements of genome architecture. Chromosomal rearrangements, such as
translocations and inversions, often comprise fixed differences between closely
related species (Coyne and Orr 2004). Once fixed between populations, inversions
could enhance divergence between populations and eventually lead to speciation
(Noor et al. 2001; Rieseberg 2001). The role of chromosomal inversions in avian
speciation has been given less attention compared to sequence divergence (Price
2008). Chromosomal inversions do have a long history in cytological studies
(Shields 1982; Hooper and Price 2015), which have recently been complemented
with studies that used genomic data to map the breakpoints of these inversions
(Volker et al. 2010; Kawakami et al. 2014). For example, Kawakami et al. (2014)
compared the genomes of collared flycatcher (Ficedula albicollis) and zebra finch
(Taeniopygia guttata) and found evidence for 140 inversions, resulting in an
estimated mean fixation rate of about two inversions per million years. This suggests
that inversions can evolve fast enough to play an important role in speciation, but the
relation between inversions and reproductive isolation remains unclear.
The genomic perspective on speciation has led to many important insights into
the origin and maintenance of species, but these insights can also be applied in
species delimitation. Indeed, genomic islands or inversions may be used as diagnos-
tic characters. For example, Poelstra et al. (2014) explored patterns of introgression
across the European hybrid zone between the carrion crow and hooded crow. A
small number genomic regions displayed resistance against introgression, including
one prominent inversion containing genes involved in pigmentation and visual
perception, reflecting ‘colour-mediated prezygotic isolation’. It is, however, chal-
lenging to confidently show that certain genomic islands or inversions house genes
that are related to reproductive isolation and are not just the result of background
selection (Ruegg et al. 2014a, b; Delmore et al. 2015; Bay and Ruegg 2017).
3.3 Genomics and Phylogeny
The final class of species criteria listed by Sangster (2014) concerns phylogeny. The
two species criteria in this class (monophyly and exclusive coalescence of gene
trees) are not called upon often in avian species delimitation. The application of
genomic data might even make these criteria obsolete. The advent of multilocus data
showed that the occurrence of phylogenetic incongruence (i.e. analyses of different
genes resulting in discordant gene trees) is a common and widespread phenomenon
(Rokas et al. 2003; Ottenburghs et al. 2016b; Braun et al. 2019). Such incongruence
can be caused by analytical shortcomings (Rokas et al. 2003; Davalos et al. 2012) or
can be the result of biological processes, such as horizontal gene transfer,
226 J. Ottenburghs
hybridization, incomplete lineage sorting, and gene duplication (Pamilo and Nei
1988; Maddison 1997; Degnan and Rosenberg 2009).
Incomplete lineage sorting (ILS) occurs when lineages fail to coalesce in the
ancestral population of two species (Pamilo and Nei 1988; Maddison 1997; Degnan
and Salter 2005). The probability of ILS depends on effective population size of the
ancestral population and the time between two successive speciation events (Degnan
and Salter 2005; Wakeley 2009). In chimpanzees, gorillas and humans—clearly
separate species—about 30% of the loci in the genome support a gene tree that
deviates from the species tree (Scally et al. 2012). Under a strict interpretation of the
genealogical species concept, which stated that ‘a group of organisms [that] is
exclusive if their genes coalesce more recently within the group than between any
member of the group’, chimpanzees, gorillas and humans would constitute one
species.
Apart from ILS, introgressive hybridization can cause gene tree discordance.
Hybridization is a widespread phenomenon in birds, about 16% of the bird species
hybridized with at least one other species (Ottenburghs et al. 2015). When
hybridization occurs, the genetic material from one species might introgress into
the genome of the other species (Anderson 1949; Arnold 2006). This genetic
exchange has the potential to hamper phylogenetic analyses in a number of ways
(Rheindt and Edwards 2011; Ottenburghs et al. 2017). First, a hybridization event
can shorten branch lengths, thereby disrupting or resetting the molecular clock.
Second, introgression can alter tree topologies. When non-sister species hybridized
in the past, an introgressed gene might yield a topology that depicts a faulty sister
relation between these species. This has been observed in several bird genera, such
as Manacus (Brumfield et al. 2008) and Icterus (Jacobsen and Omland 2012). In
addition, if the introgression event happened far back in time, it can lead to
misleading branching arrangements at deep phylogenetic nodes. For instance,
Fuchs et al. (2013) attribute a conflicting pattern between several loci to ancient
hybridization between members of the woodpecker genus Campephilus and the
melanerpine lineage (Melanerpes and Sphyrapicus).
Genomic analyses of several bird groups show that gene tree discordance—
caused by incomplete lineage sorting and/or introgression—is common in birds
(Kulikova et al. 2005; Rheindt et al. 2009; Kraus et al. 2012; Jarvis et al. 2014;
Nater et al. 2015; Suh et al. 2015; Ottenburghs et al. 2016a, b; Sonsthagen et al.
2016). Therefore, it seems that the criterion of ‘exclusive coalescence of gene trees’
becomes problematic in the genomic era of avian taxonomy. In a genome-scale
collection of gene trees, there will always be a subset of trees that show exclusive
coalescence. Are these trees taxonomically more informative than discordant gene
trees? Putting more weight on a subset of monophyletic gene trees can result in
cherry-picking to support a particular taxonomic arrangement. Hence, it is advisable
to report all gene trees and consider genomic evidence in combination with other
data, such as morphometrics, song or plumage.
4 Conclusions
The species problem has been partly resolved by combining theoretical monism (the
evolutionary species concept or the general lineage concept) and practical pluralism
(Mayden 1997; De Queiroz 1998). The general lineage concept shows that there is a
grey zone during the speciation process where different species criteria lead to
different decisions on the number of species. By acknowledging the continuous
nature of speciation and realizing that these species criteria are not necessary
properties of species, taxonomists can make better informed decisions in species
delimitation.
The practical pluralism is also apparent in avian taxonomy. An analysis by
Sangster (2014) showed that almost half of the taxonomic bird studies applied
multiple criteria and the proportion of studies applying multiple criteria is increasing.
The most frequently used species criterion is diagnosability, followed by reproduc-
tive isolation and degree of difference. Other species criteria concern the use of
different adaptive zones, monophyly and exclusive coalescence of gene trees.
Combining these—often conflicting—species criteria calls for integrative taxonomy,
which can either proceed by congruence or by cumulation of species criteria
(Padial et al. 2010).
The advent of genomic data has prompted some authors to propose a ‘genomic
species concept’ (e.g. Jarvis 2016). The analysis of the species problem presented
here shows that such a genomic species concept will not resolve conflicts in species
delimitation. Instead, genomic data should be incorporated into the existing species
criteria. With regard to distinguishability (i.e. the use of a unique, fixed character or a
unique combination of character states to delineate species), for example, genomic
data can provide an extra line of evidence.
Reproductive isolation is mostly caused by the combination of several pre- and
postzygotic isolation mechanisms. The interplay of different reproductive isolation
mechanisms can be depicted as a continuum from a panmictic population to two
irreversibly isolated species (Seehausen et al. 2014). Genomic data can be used to
provide a genetic basis for these reproductive isolation mechanisms (Wu and Ting
2004; Presgraves 2010). In addition, genomic islands or inversions, which may play
an important role in certain speciation processes, can be used as diagnostic
characters.
Finally, the application of genomic data might complicate the criterion ‘exclusive
coalescence of gene trees’. The advent of multilocus data showed that the occurrence
of phylogenetic incongruence (i.e. analyses of different genes resulting in discordant
gene trees) is a common and widespread phenomenon (Rokas et al. 2003;
Ottenburghs et al. 2016b), which can be the result of several biological processes,
such as horizontal gene transfer, hybridization, incomplete lineage sorting and gene
duplication (Pamilo and Nei 1988; Maddison 1997; Degnan and Rosenberg 2009).
Hence, it is advisable to report all gene trees and consider genomic evidence in
combination with other data sources.
228 J. Ottenburghs
In conclusion, the genomic era has provided avian taxonomists with a new tool
box that can be applied to old concepts, leading to better informed decisions on the
delimitation and description of species.
Acknowledgements I would like to thank Robert Kraus for giving me the opportunity to write this
chapter and share my ideas on the role of genomics in avian taxonomy. These ideas are based on a
chapter from my PhD thesis on hybridization in geese. I am grateful to my supervisors—Herbert
Prins, Ron Ydenberg, Pim van Hooft, Sip van Wieren, Hendrik-Jan Megens and Martien
Groenen—for letting me explore and develop these concepts during my PhD at Wageningen
University (the Netherlands). Finally, I would like to thank two anonymous reviewers whose
comments and suggestions have greatly improved this chapter.
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Population Genomics and Phylogeography
Jente Ottenburghs, Philip Lavretsky, Jeffrey L. Peters,

Takeshi Kawakami, and Robert H. S. Kraus
Abstract
Population genetics is the study of genetic variation within populations and how
allele frequencies change over space and time. This field largely focuses on the
five fundamental evolutionary processes that influence genetic variation: muta-
tion, genetic drift, gene flow, natural selection, and recombination. In this chapter,
we review how genomic data from avian species have advanced our understand-
ing of each of these five processes, including an emphasis on their interactions in
shaping contemporary genetic diversity on the scale of whole populations. In
general, genomic data has increased the potential for fine-scale resolution of
population structure and determination of population boundaries and population
membership. However, delineating populations is not always straightforward,
and populations tend to fall on a continuum from isolation to panmixia. Mutation
is the ultimate source of all genetic variation within populations. The ability to
sequence whole genomes resulted in better estimates of mutation and substitution
rates in particular genomic regions (e.g., coding vs. noncoding DNA) and along
J. Ottenburghs (*) · T. Kawakami

Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden
e-mail: takeshi.kawakami@ebc.uu.se
P. Lavretsky
Department of Biological Sciences, University of Texas at El Paso, El Paso, TX, USA
e-mail: plavretsky@utep.edu
J. L. Peters
Department of Biological Sciences, Wright State University, Dayton, OH, USA
e-mail: jeffrey.peters@wright.edu
R. H. S. Kraus
Department of Migration and Immuno-Ecology, Max Planck Institute for Ornithology, Radolfzell,
Germany
Department of Biology, University of Konstanz, Konstanz, Germany
e-mail: rkraus@orn.mpg.de

https://doi.org/10.1007/978-3-030-16477-5_8
238 J. Ottenburghs et al.
different avian lineages. The uncovered variation in these rates will further
advance our knowledge of bird evolution. A genomic perspective on other
evolutionary forces, such as genetic drift (tightly linked with the concept of
effective population size [Ne]), migration, and selection, allows for more detailed
reconstructions of demographic and phylogeographic history. In addition, the
estimates of genome-wide recombination rates and their relationship with linked
selection and GC-biased gene conversion will improve the match between popu-
lation genetic models and biological reality.
Keywords
Assortative mating · Demography · Effective population size · GC-biased gene
conversion · Gene flow · Linked selection · Natural selection · RADseq ·
Recombination · Substitution rates
1 Introduction
The field of population genomics, defined as the “process of simultaneous sampling

of numerous variable loci within a genome and the inference of locus-specific effects
from the sample distributions,” was first conceptualized by Black IV et al. (2001).
This initial conceptualization emphasized distinguishing between factors that influ-
ence unlinked loci independently (locus-specific effects), such as mutation, recom-
bination, nonrandom mating, and selection, from those factors that have a similar
influence on loci throughout the genome (genome-wide effects), such as genetic
drift, gene flow, and inbreeding. Rather than emphasizing locus-specific effects,
Luikart et al. (2003) defined population genomics more broadly as “the simultaneous
study of numerous loci or genome regions to better understand the roles of evolu-
tionary processes [. . .] that influence variation across genomes and populations”
(p. 981). In contrast to Black IV et al. (2001), Luikart et al. (2003) concluded that the
most important contribution of genomic sampling is to provide better inferences of
population demography and evolutionary history. Hartl and Clark (2007) similarly
adhered to a broader definition, “the application of population genetics on a genomic
scale” (p. 469). In this review, we use this more general definition of population
genomics and examine the fundamental evolutionary processes that influence
genetic variation: mutation, genetic drift, migration (i.e., gene flow), natural selec-
tion, and recombination (Sects. 3–7).
Genetic diversity within populations is the result of these five fundamental
evolutionary forces. For the most basic model, equilibrium values of genetic diver-
sity are a function of mutation and genetic drift, both of which are a function of
population size (N ). Because more mutations occur and genetic drift is less efficient
at removing variation in larger populations, genetic diversity should be directly
proportional to N (Wright 1931), a relationship that has been supported by empirical
studies (Soulé 1976; Frankham 1996, 2012). However, this simple model makes a
Population Genomics and Phylogeography 239
number of assumptions, including no immigration, no selection, constant N (i.e.,

drift-mutation equilibrium), nonoverlapping generations, and random mating
(Wright 1931, 1938; Frankham 1995). In these mathematical models, N is not
what ecologists would count when they go out in the field and ask “how many
individuals are there?” The latter question refers to the census population size,
usually specified as Nc. In population genetics, we typically calculate the effective
population size Ne, which is a rather abstract quantity that reflects the genetic
diversity of a population under study but includes the effects of inbreeding and
subdivision, among others (Hartl and Clark 2007). Typically, Ne is much smaller
than Nc (for details see Sect. 2 in this chapter). The effectiveness of selection is also
dependent on Ne (Ohta 1972, 1992; Gillespie 2001; Ellegren 2009). Specifically, if
the product 2Nes (where s is the selection coefficient) 1.0, selection will override
drift in determining the fate of mutations, whereas if 2Nes 1.0, drift will dominate.
The emerging field of population genomics has revealed compelling evidence that
directional selection, balancing selection, purifying selection, and hitchhiking are
pervasive throughout the genome, causing widespread departures from neutral
models (Hahn 2008; McVicker et al. 2009; Charlesworth 2012; Burri 2017a).
However, some genomic features can also be explained by nonadaptive processes,
such as genetic drift (Lynch 2007).
2 What Is a Population?
The term population has been defined in a variety of ways throughout the scientific
literature (Waples and Gaggiotti 2006). At one extreme, population is essentially
synonymous with sampling location, referring to a group of individuals sampled
from a single location. Hartl and Clark (2007) defined a population in a more
biologically relevant way: “a group of organisms of the same species living within
a sufficiently restricted geographical area so that any member can potentially mate
with any other member of the opposite sex” (p. 45). In an ideal population of
sexually reproducing individuals, mating is random, and any individual has an
equal probability of mating with any other individual from the same population
(i.e., the population is panmictic). However, it is questionable whether any popula-
tion is truly panmictic. Mating is rarely, if ever, completely random, but rather
individuals are more likely to mate with individuals in close proximity. In other
words, the probability of mating decreases with increasing distance between
individuals, and this nonrandom mating results in a spatial organization of genetic
variation (i.e., isolation by distance), even in the absence of any other factors such as
mate choice or mobility. When making inferences about demographic histories or
selection, the distribution of alleles across space becomes critically important.
Most definitions of a population are not operational in the sense that they fail to
provide quantitative criteria for determining which individuals belong to the same or
a different population. Waples and Gaggiotti (2006) suggested using the number of
effective migrants per generation (Nem, where Ne is the effective population size and
m is the migration rate) as an operational criterion for determining whether groups of
individuals could be considered a population. The threshold for this criterion is

somewhat arbitrary, and estimating Nem can be cumbersome, especially with geno-
mic datasets. Moreover, calculating Nem requires some a priori knowledge about
which individuals are grouped. Therefore, the first hurdle in delineating populations
is determining which individuals are sufficiently similar that they can be considered
part of the same population.
In the past, statistical power from only a small number of genetic markers from
distant regions of the genome has often been insufficient to unveil weak population
structure, and increasing the number of markers has clearly shown that more markers
give better signals (Kraus et al. 2015). Population genomics uses technology to
increase the number of genetic markers by orders of magnitude (Kraus and Wink
2015; Wink 2019), thereby offering the potential for fine-scale resolution of popula-
tion structure and determination of population boundaries and population member-
ship. Peters et al. (2016) conceptualized a quantitative framework for using large-
scale genetic datasets to delineate “conservation units.” This framework, largely
inspired by approaches in Harvey and Brumfield (2015), can be applied to
delineating populations. Using the mottled duck (Anas fulvigula) and genotypes
obtained from a reduced representation genomic approach, double-digest restriction-
associated DNA sequencing (ddRAD-seq), Peters et al. (2016) used a variety of
analytical methods to distinguish between apparent panmixia, discrete population
units, and isolation by distance. Specifically, they demonstrated that the Florida and
Western Gulf Coast populations of mottled ducks were discrete units—genotypes
were sufficiently similar within regions and different between regions that (1) all
individuals grouped together in population-specific clusters on the basis of ddRAD-
seq genotypes (Fig. 1a), (2) all individuals were assigned unambiguously to their
populations of origin, (3) the geographic area separating these populations was a
better predictor of allele frequency differences than geographic distance alone, and
(4) there was limited evidence of admixture and gene flow between these
populations. In contrast, there was no evidence of population structuring within
Florida or the Western Gulf Coast. Therefore, in the case of mottled ducks,
delineating population boundaries was unambiguous. Other studies of avian taxa
have used similar approaches with ddRAD-seq data to demonstrate discrete
differences in multilocus genotypes between geographic groups (Parchman et al.
2013; Harvey and Brumfield 2015; Kopuchian et al. 2016), and such discrete
population structure has been used as evidence for species delimitation (Oswald
et al. 2016).
In contrast to the discrete population units found in mottled ducks, studies of
some avian taxa found ambiguous evidence of population boundaries (Kraus et al.
2013; Lavretsky et al. 2015). For example, Lavretsky et al. (2015) used principal
component analyses (PCA) to cluster individuals based on ddRAD-seq genotypes in
mallards (Anas platyrhynchos) and Mexican ducks (A. diazi). Although there was
some evidence of discrete or nearly discrete populations (e.g., eastern and western
populations of mallards and mallards vs. Mexican ducks), individuals could not be
unambiguously assigned to populations and there appeared to be substantial admix-
ture. Overall, a pattern of isolation by distance seemed to describe the geographic
Fig. 1 Examples of the gradient of possible outcomes when applying genomic data to inferences of
population boundaries, including (a) discrete population units in mottled ducks (Peters et al. 2016),
(b) continuous variation with possible isolation by distance in mallards and Mexican ducks
(Lavretsky et al. 2015), and (c) apparent panmixia in turtle doves (Calderón et al. 2016)
distribution of alleles (Fig. 1b); for example, western mallards, which are geograph-
ically closer to Mexican ducks, were genetically intermediate between eastern
mallards and Mexican ducks, and Mexican ducks sampled from the United States
were genetically intermediate between mallards and Mexican ducks sampled from
Mexico. Within Mexico, there was a stepping-stone pattern of genetic differentia-
tion: individuals from the most geographically distant sampling locations were the
most genetically differentiated (e.g., Sonora vs. Puebla), whereas there was substan-
tial overlap in principal component (PC) scores among individuals from neighboring
sites (e.g., Puebla vs. the state of Mexico). A similar pattern of isolation by distance
was also found among subspecies of dark-eyed junco (Junco hyemalis) in North
America: the principal components showed a striking resemblance to geographic
distribution (Friis et al. 2016). Although the slate-colored junco (J. h. hyemalis) does
appear to be a discrete population, it is important to emphasize that all the individuals
examined were sampled from the same location; more comprehensive sampling
across their range will be necessary to determine whether this subspecies represents
a discrete population or if it fits within a broader pattern of isolation by distance.
Otherwise, for both juncos and Mexican ducks, the challenge is that delineating
population boundaries is not possible given the gradation in multilocus genotypes
over space, despite clear evidence of population structure. Thus, the use of popula-
tion genomics to infer aspects of population demography, history, and selection
necessitates models that incorporate isolation by distance.
Similar to Mexican ducks and dark-eyed juncos, population genomics suggests
that red crossbills (Loxia curvirostra) comprise a mix of discrete, nearly discrete, and
non-discrete ecotypes that loosely correspond to geographic populations (Parchman
et al. 2016). However, in this case, there was no overall pattern of isolation by
distance, at least partly as a result of their nomadic behavior. For example, PCA
clusters individuals from the western and eastern parts of the red crossbill’s range to
the exclusion of individuals from the interior. Parchman et al. (2016) concluded that
adaptation to conifer species, rather than geography, was a better explanation of the
observed genetic differentiation. In addition, the population from South Hills, Idaho,
USA, appeared to be a discrete population that was genetically distinct from other
crossbills, and these results coupled with differences in morphology and calls have
resulted in the recognition of a distinct species, the Cassia crossbill (L. sinesciuris)
(Chesser et al. 2017).
In some cases, population genomics might fail to reveal population structure,
even for species with broad geographic distributions. For example, Calderón et al.
(2016) sampled European turtle doves (Streptopelia turtur) from locations through-
out eastern and western Europe and obtained genomic data using ddRAD-seq. Using
PCA on single-nucleotide polymorphisms (SNPs; pronounced snips), they found
that PC scores overlapped substantially among individuals from different sampling
locations (Fig. 1c). Thus, despite its widespread distribution, genetic variation within
European turtle doves is consistent with a single, panmictic population. On ecologi-
cal timescales, populations from the different regions may or may not be demo-
graphically independent; however, on evolutionary timescales, there is sufficient
genetic connectivity (i.e., gene flow, range expansion) that detectable population
structure does not emerge. Population genomic data likewise failed to reveal popu-
lation structure in mountain chickadees (Poecile gambeli), despite geographically
structured phenotypic variation and evidence of local adaptation in life history
(Branch et al. 2017). Thus, for the purpose of population genomics, samples from
different regions could be pooled and analyzed as a single population for inferences
of evolutionary history.
The above case studies illustrate possible outcomes of inferring population
structure using genomic data and multivariate statistics. Waples and Gaggiotti
(2006) provided a visual representation of the continuum of population differentia-
tion, from isolation to panmixia, and PCA and other similar orthogonal
transformations (e.g., discriminant function analysis) offer the ability to visualize
where species of interest fall within this continuum. For instance, the examples
discussed above illustrate this continuum; mottled ducks (Fig. 1a) clearly fit the
scenario of isolation or “complete independence,” Mexican ducks (Fig. 1b) fit both
“modest connectivity” (Sonora, USA, and inland sampling locations) and “substan-
tial connectivity” (inland locations: Zacatecas, Guanajuato, Mexico, and Puebla),
whereas European turtle doves (Fig. 1c) best fit “panmixia.” Further advances could
be made by developing methods for quantifying this structure to facilitate
comparisons across taxa from different studies. Also, such approaches are applicable
to the opposite end of distribution of genetic variation when this leads into speciation
(Ottenburghs 2019).
3 Mutation
The ultimate source of all genetic variation within populations is mutation, which
changes the nucleotide sequences within a region of DNA through a point mutation
(a single base pair change), insertion or deletion of one or more nucleotides,
inversions, etc. Mutation is independent in different populations. In the absence of
homoplasy (i.e., recurrent mutations, back mutations to the previous state) and gene
flow, new mutations that arise after populations split will be unique to a single
population and cause populations to genetically diverge over time.
Mutation rates have been estimated across the tree of life, from simple RNA
viruses and bacteria to higher eukaryotes, and vary widely from 7.2 107 to
7.2 1011 per base pair per generation (Drake et al. 1998). In humans, this estimate
translates to a germline mutation rate of about 0.5 109 per base pair per year
(Scally 2016). The number of new mutations that enter a population each generation
is a function of Ne. However, many mutations are lethal or strongly deleterious and
are not passed to future generations. Therefore, in population genetics, we consider
the substitution rate, which is the rate at which new mutations accumulate over time.
The substitution rate depends on both the rate at which mutation adds new variants
and the rate at which natural selection removes deleterious or lethal mutations (see
Box 1 in Barrick and Lenski 2013). In the case of strictly neutral evolution, when
new variants do not affect biological fitness, the substitution rate is equal to the
mutation rate. However, with genomic data, the substitution rate is lower than the
mutation rate, and in the absence of mutation accumulation experiments (Barrick and
Lenski 2013) in birds, we can only measure the long-term substitution rates.
Genomic substitution rates vary considerably among lineages of birds. Substitu-
tion rates have been estimated for fourfold degenerate sites in coding regions.
Fourfold degenerate refers to the observation that each of the 4 nucleotides at a
site results in the same amino acid. A substitution at a fourfold degenerate site is also
referred to as a synonymous substitution. The substitution rate at these sites was
estimated to be approximately 3.3 109 substitutions per site per year (s/s/y) for
Passeriformes (perching birds) and <1.0 109 s/s/y for Struthioniformes
(ostriches) (Zhang et al. 2014). The global rate across all avian lineages was
approximately 1.9 109 s/s/y (Zhang et al. 2014). Similarly, Nam et al. (2010)
found a nearly twofold difference in substitution rates at fourfold degenerate sites
(1.23–2.21 109 s/s/y), with the lowest rates in ancestral bird lineages and the
highest rates in a representative of Passeriformes. The substitution rate estimated
from ddRAD-seq, which generates a pseudorandom sampling of the genome and
includes sequences from both coding and noncoding regions, was similar to that
found at fourfold degenerate sites—approximately 1.75 109 s/s/y for a lineage of
Anseriformes (waterfowl) (Peters et al. 2016). However, the substitution rate for
ultraconserved elements (UCEs) and their flanking regions was found to be about an
order of magnitude lower, 2.59 1010 s/s/y, in a lineage of Charadriiformes
(shorebirds) (Oswald et al. 2016).
Substitution rates also vary across the genome. As a general rule, substitutions
accumulate more rapidly in noncoding regions of the genome, such as introns and
intergenic regions, than in protein-coding exons. However, avian genomes contain
an estimated 3.2 million highly conserved elements (HCEs) interspersed throughout
both noncoding and coding DNA (Zhang et al. 2014), and these HCEs contribute to
high variation in substitution rates even within classes of DNA. Similarly, overall
substitution rates also vary among chromosomes. Based on analyses of
transcriptomes for ten species of birds, dS (divergence at synonymous sites) was
negatively correlated with chromosome size, suggesting that the synonymous sub-
stitution rate is lower for larger chromosomes than for smaller chromosomes
(Künstner et al. 2010). They also found that dS was higher for the Z chromosome
than for autosomes, a pattern that was also observed by Zhang et al. (2014) in a
comparative analysis of full genomes from 45 avian species.
In addition to providing information about the rate of evolution, estimates of
substitution rates are necessary to calculate demographic parameters from sequence
data. For example, percent sequence divergence (d ) can be calculated directly from
genomic data with the formula d ¼ 2μt, where μ is the substitution rate and t is the
time since divergence. Thus, having an estimate of μ (in substitutions per site per
year) allows us to estimate the number of years since two species or populations
began diverging. Similarly, genetic data can provide an estimate of the composite
parameter θ (theta), where θ ¼ 4Neμ, and an estimate of μ (in substitutions per site
per generation) can therefore be used to estimate effective population sizes. These
estimates of demographic parameters are important for making inferences about
evolutionary history, conservation priorities, and phylogeography.
4 Genetic Drift and Effective Population Sizes
Whereas mutation adds genetic variation to a population, genetic drift removes

it. Genetic drift is the stochastic fluctuation in allele frequencies over time that
results from the random survival of individuals and the random sampling of gametes
during reproduction. In an idealized population of size Nc, the probability that two
copies of a gene randomly sampled from a population are identical by descent (i.e.,
they were derived from the same ancestor in the previous generation) is 1/2Nc.
Lineages that fail to leave descendants go extinct, and any unique mutations within
those lineages are lost. Because the rate at which genetic variation is lost is inversely
correlated with population size, smaller populations lose variation more rapidly than
larger populations. However, this relationship assumes a constant population size
(i.e., population sizes remain the same between generations), generations that do not
overlap, 1:1 sex ratios, equal variance in reproductive success between the sexes, and
random mating. In reality, populations deviate from these assumptions, which
usually results in a faster rate of genetic drift than expected given Nc. The Ne is the
size of an ideal population that loses genetic variation at a rate equal to that of the
actual population (Wright 1931). In other words, Ne quantifies the rate at which
genetic drift decreases genetic diversity within a population. Across a wide range of
studies, Frankham (1995) estimated that Ne averaged about 0.1Nc.
Applications of genomics to inferences of Ne and the role of genetic drift have
primarily focused on fluctuations in population sizes over evolutionary time, with a
particular emphasis on the role of past climate changes. Calderón et al. (2016) used
approximate Bayesian computation (ABC) to fit ddRAD-seq data from European
turtle doves to five models of demographic history, including constant population
sizes and various scenarios of fluctuating population sizes. They found that their data
best fit a model that included a population expansion during the late Pleistocene
(~78,000 years before present; ybp) followed by a population decline during the
Holocene (~7600 ybp). Reductions in Ne have also been inferred from ddRAD-seq
data for various species of dry forest birds from South America (Oswald et al. 2017).
Interestingly, they found similar changes in Ne between ancestral and daughter
populations among the six species studied, despite considerable variation in popula-
tion divergence times. They attributed these long-term reductions in Ne to historical
reductions in the geographic extent of dry forests in this region. One of the main
strengths of these inferences lies within the hypothesis-driven framework that is
often used in population genomics and phylogeography (Carstens et al. 2017; see
Sect. 8). In particular, fitting the data to various models of population size changes
and using a Bayesian or likelihood approach to choose the best-fit model make it
possible to reject simpler models in favor of more complex models.
Whole-genome data from a single diploid individual can also provide information
about past population size changes. In a comparative study of 38 bird species,
Nadachowska-Brzyska et al. (2015) used the pairwise sequentially Markovian
coalescent (PSMC, Li and Durbin 2009) model to show that demographic histories
varied considerably among species and that the Ne of some species fluctuated by
orders of magnitude. One prominent pattern was a major reduction in population
sizes associated with the last glacial period (LGP; ~110–12 kya). Surprisingly,
however, Nadachowska-Brzyska et al. (2015) did not find a relationship between
the extent of the decline and whether current ranges overlapped with regions
severely influenced by glaciation (e.g., were formerly covered in ice or extreme
deserts). Similar patterns of demographic fluctuations and major reductions in Ne
associated with the last glacial period have been inferred from whole-genome
sequences and the PSMC for grouse (Lagopus spp.) (Kozma et al. 2018), black-
and-white flycatchers (Ficedula spp.) (Nadachowska-Brzyska et al. 2016), and geese
(genera Anser and Branta) (Ottenburghs et al. 2017b).
5 Gene Flow
When a population gets subdivided, random genetic drift and selection can lead to
genetic divergence among the subpopulations. Migration—the movement of
organisms among these subpopulations—can act as a kind of genetic glue that
binds the subpopulations genetically and sets a limit to the amount of genetic
divergence that can accumulate (Hartl and Clark 2007). In the literature, migration
and gene flow are often used interchangeably. However, there is an important
difference between both terms: migration refers to the movement of individuals
between subpopulations, while gene flow encompasses the movement of alleles and
their establishment into a different gene pool (Tigano and Friesen 2016). Hence,
migration does not necessarily result in gene flow (Verhulst and Van Eck 1996).
Direct estimates of migration often involve mark-recapture methods, which can
be impractical and labor-intensive for large populations with low migration rates.
Therefore, indirect measures based on genetic data are mostly preferred. Early
studies estimating gene flow—expressed as Nem—from genetic data relied on FST
or other measures of differentiation (Slatkin and Barton 1989). However, the
population genetic models for these estimations assume unrealistic conditions,
such as constant population size, symmetrical migration, and mutation-drift equilib-
rium (Whitlock and McCauley 1999; Wilson and Rannala 2003; Marko and Hart
2011). The development of non-equilibrium approaches provided the opportunity to
assess more realistic scenarios of gene flow. Specifically, isolation-with-migration
models enabled the joint estimation of gene flow, genetic diversity, and divergence
times within a maximum likelihood framework (Hey and Nielsen 2004; Hey 2006;
Hey et al. 2018). For example, isolation-with-migration analyses based on a
multilocus dataset indicated asymmetrical gene flow from indigo bunting (Passerina
cyanea) to lazuli bunting (P. amoena) (Carling et al. 2010). Alternative software
packages, such as migrate-n (Beerli and Palczewski 2010), have also been used to
quantify the degree of gene flow in migrating waterfowl populations of mallard
(Anas platyrhynchos) (Kraus et al. 2013) and barnacle goose (Branta leucopsis)
(Jonker et al. 2013).
Similar to the inference of genetic drift and effective population sizes, the
development of ABC models allowed population geneticists to probe more complex
models and evaluate the extent of gene flow by comparing simulated DNA sequence
evolution with empirical data (Beaumont 2010). For instance, a recent study com-
pared 15 models (with different patterns and levels of gene flow) to assess the
demographic history of pied flycatcher (Ficedula hypoleuca) and collared flycatcher
(F. albicollis). ABC modelling based on whole-genome re-sequencing data from
20 individuals supported a recent divergence with unidirectional gene flow from
pied to collared flycatcher after the Last Glacial Maximum (Nadachowska-Brzyska
et al. 2013). Similar analyses have been performed to assess the demographic history
of other bird species, such as Melospiza sparrows (Smyth et al. 2015), Myrmeciza
antbirds (Raposo do Amaral et al. 2013), and Platalea spoonbills (Yeung et al.
2011). These studies indicate that model-based approaches are a fruitful avenue for
the reliable estimation of gene flow (Ottenburghs et al. 2017a). Recently, machine
learning techniques are being applied to population genomic questions (Schrider and
Kern 2018), but this approach has not reached the ornithological community yet.
The development of more sophisticated tools in combination with the availability
of genomic data led to important insights into the role of gene flow in population
dynamics (Ottenburghs et al. 2017a). Similar to mutation, gene flow can introduce
novel alleles into a population. Even between species this can be shown when
modelling the probability of allele sharing between, e.g., related duck species with
or without assuming hybridization (Kraus et al. 2012). The main difference with
mutation is the speed at which this happens: the rate of migration is vastly greater
than the rate of mutation (Hedrick 2013). The fate of these novel alleles depends on
the specific genetic and environmental context in which they end up (Payseur 2010).
In general, alleles can be divided into three categories: (1) neutrally evolving alleles
that flow freely between populations, (2) alleles that confer an adaptive advantage
and flow quickly, and (3) alleles that are not adapted to local conditions and are
consequently selected against.
These allele-specific patterns of gene flow result in a heterogeneous genomic
landscape in which some genomic regions are more prone to be exchanged between
populations than others (Nosil et al. 2009; Ravinet et al. 2017; Wolf and Ellegren
2017). For example, a study comparing the genomes of hooded crow (Corvus corone
cornix) and carrion crow (C. c. corone), two subspecies that interbreed along a
narrow hybrid zone across Europe, uncovered a peculiar genomic landscape in
which gene flow was relatively unrestricted across the genome except for one
genomic region. This region harbored several genes involved in pigmentation and
visual perception, suggesting a role in reproductive isolation (Poelstra et al. 2014).
In recently diverged populations, reproductive isolation can be caused by assor-
tative mating, in which individuals with similar phenotypes mate with one another
more frequently than would be expected under a random mating pattern (Ritchie
2007; Uy et al. 2018). For instance, the alba and personata subspecies of the white
wagtail (Motacilla alba) mate assortatively based on head plumage patterns. This
nonrandom mating results in a reduction in gene flow—estimated using almost
20,000 SNPs—between these subspecies (Semenov et al. 2017). The traits underly-
ing assortative mating are various (e.g., song, plumage, behavior) and can originate
in different ways (Uy et al. 2018). Sexual selection can drive changes in mating
preferences and associated display traits (Ritchie 2007; Kopp et al. 2018).
Alternatively, natural selection can cause divergence in traits not related to mate
choice, which may later be co-opted as mating signals, so-called magic traits
(Servedio et al. 2011). In the end, natural and sexual selection can act in concert,
culminating in a barrier to gene flow (Servedio and Boughman 2017). This synergy
between natural and sexual selection is nicely illustrated by bird species in which
different subpopulations are adapted to different food sources. Divergent natural
selection can then result in distinct beak morphologies, which consequently produce
different acoustic signals, such as songs or call types. Assortative mating based on
song or call type can lead to a reduction in gene flow between subpopulations. This
scenario has been described for Loxia crossbills (Parchman et al. 2006), Melospiza
sparrows (Ballentine et al. 2013), and Aphelocoma scrub jays (Langin et al. 2015).
So far, genetic data has allowed population geneticists to document these patterns,
and genomics will lead to a more fine-grained picture of gene flow dynamics and
provide the opportunity to pinpoint the genetic basis of the traits underlying assorta-
tive mating.
In addition to assortative mating, barriers to gene flow can also be physical.
Numerous studies have documented how mountain ranges (Manthey et al. 2016;
Moyle et al. 2017; Machado et al. 2018; Padró et al. 2018), rivers (Maldonado-
Coelho et al. 2013; Fernandes et al. 2014), ecological transitions (Caro et al. 2013;
Zhen et al. 2017; Garg et al. 2018), and sea currents (Munro and Burg 2017) can
limit dispersal and act as barriers to gene flow. However, when assessing how
geographical and topological barriers influence patterns of gene flow, it is important
to keep the ecology and dispersal capacity of the species under investigation in mind.
A study on Pleistocene land bridges in Sulawesi emphasizes this point: using
ddRAD-seq data, the authors estimated the amount of ancient gene flow between
the island populations of two bird species, the henna-tailed jungle flycatcher
(Cyornis colonus) and the golden whistler (Pachycephala pectoralis). During the
Pleistocene, the islands Peleng and Taliabu were connected by land bridges allowing
animals to disperse from one island to the other. The analyses revealed little evidence
of genetic exchange between the jungle flycatcher populations on Peleng and
Taliabu, whereas there had been gene flow between island populations of golden
whistler. The differences in gene flow dynamics probably depended on the ecology
of the species: the jungle flycatcher is a specialized bird with poor dispersal
capacities and does not venture outside forests often. The golden whistler, however,
is a generalist that tends to explore new territories (Garg et al. 2018). Similarly,
research on the role of Amazonian rivers as barriers to gene flow has culminated in
contrasting results: some studies report clearly separated populations on each side of
the river (Maldonado-Coelho et al. 2013; Fernandes et al. 2014), while other studies
documented gene flow at headwaters (Weir et al. 2015; Sandoval-H et al. 2017). In
summary, what might be a barrier for one bird species is not necessarily a barrier for
another one.
6 Selection
Species are continuously adapting to ever-changing environments (Dobzhansky

1940; Bush 1975; Orr and Smith 1998). Genetic differences that arise through
mutation or enter a population by gene flow result in populations of individuals
with subtle morphological, ecological, or other differences (Coyne and Orr 2004). It
is this diversity that selection works with, and thus these differences among
individuals often dictate the “adaptability” of a species or population (Barton and
Hewitt 1989; Orr 2001). Specifically, selection favors morphological, ecological, or
other traits that increase survival and fecundity of an individual in a particular niche
space (Fischer 1930; Price 1998; Rundle and Nosil 2005; Via 2009; Sobel et al.
2010; Wolf et al. 2010). In fact, it was in 1859 that Charles Darwin determined that
the composition of a population or species changes (or evolves) due to the differen-
tial survival of individuals in varying environments and coined the responsible force
as “natural selection” (Darwin 1859). Thus, evolution proceeds through the selection
of traits that provide a competitive advantage, consequently increasing mating
success. Finally, since Charles Darwin established natural selection as a dominant
force in the evolutionary process, there has been a refinement regarding the types of
selection. For example, the elaborate feathers and mating displays of birds are
classical examples of sexual selection (Lande 1980; Andersson 1994; Johnsgard
1994; Promislow et al. 1994; Grant and Grant 1997; Price 1998; Clutton-Brock
2007; Krakauer 2008). In such a case, sexual selection confers higher mating success
for the displaying sex despite any negative impact the trait may have via natural
selection (i.e., predation).
Given that the number and survival of new mutations is largely dictated by
population size (i.e., more mutations enter and are maintained in larger populations),
selection is most effective in large populations (Ohta 1972, 1992; Gillespie 2001;
Ellegren 2009), whereas genetic drift will dominate in smaller populations (Sect. 4).
Thus, the probability of beneficial mutations to be lost due to genetic drift increases
as population size becomes increasingly small. Due to a lag effect on the influence
from selection on traits and associated genetic variation, the majority of new
mutations are often lost due to genetic drift as a result of their naturally low
frequency within any population if the selection favoring the new mutations is not
very strong (i.e., relatively small selection coefficient s). In short, higher individual
diversity increases the probability that a species survives challenges, such as changes
affecting their current environment or when invading novel niche space (Turelli et al.
2001; Wu 2001; Coyne and Orr 2004). For example, a species that is comprised of
largely clonal individuals (i.e., low genetic diversity) has little chance to survive new
ecological or other challenges because none of the individuals have variants that
would confer an adaptive response. Such scenarios are often an important issue for
endangered or highly specialized taxa with small population sizes or ranges (e.g.,
islands) (Dickerson 1973; Templeton 1986; Lacy 1987; Hughes et al. 1997; Oyler-
McCance et al. 1999; Mock et al. 2004). Captive breeding programs often need to
contend with this issue (Elsbeth McPhee 2004; Fraser 2008; Cassin-Sackett et al.
2019). Conversely, a species comprised of a diversity of individuals is more likely to
have a proportion of individuals that may have the (genetic) variation necessary to
survive the same challenges.
The emerging field of population genomics has revealed compelling evidence
that directional selection, balancing selection, purifying selection, and hitchhiking
are pervasive throughout the genome, causing widespread departures from neutral
models (Hahn 2008; McVicker et al. 2009; Charlesworth 2012; Burri 2017a). Thus,
in addition to variance in mutation (Sect. 3) and recombination (Sect. 7) rates, as well
as differential gene flow (Sect. 5), the variance in selection also contributes to the
heterogeneous nature of genomes. Importantly, just as with the other evolutionary
forces, selective processes leave traceable signatures across the genomes of
populations that researchers are now able to discern between (Wu and Ting 2004;
Sabeti et al. 2006; Wolf et al. 2010; Schoville et al. 2012; Keller et al. 2013; Wray
2013; Seehausen et al. 2014; Andrews et al. 2016; Van Belleghem et al. 2018).
Often, these genes or genetic regions are “needles in a haystack,” and thus, increas-
ing genomic coverage is essential for their discovery. Once a limitation by standard
Sanger sequencing methods, the genomic era now enables researchers to attain
sufficient genomic coverage required when searching for regions under selection
in a genome (Kraus and Wink 2015; Jax et al. 2018b). Thus, by accessing larger
portions of the genome, researchers are able to (1) determine how selection has
operated in the evolution of their taxonomic system, (2) find important genes
associated with adaptive traits in their systems, and (3) distinguish between differing
selective signatures.
The identification of putative genes or genetic regions under selection is often
accomplished through “genomic scans” in which markers are compared between
taxa of interest with various summary statistics, such as relative (e.g., FST, ΦST) and
absolute (e.g., dXY) genetic divergence, as well as other measures of genetic diver-
sity (e.g., pairwise nucleotide diversity π, Tajima’s D). For example, conducting
these genomic scans across ~3500 ddRAD loci, Lavretsky et al. (2015) were able to
demarcate putative outliers (demarcated as regions of elevated genetic divergence)
on the Z-sex chromosome and several autosomal chromosomes that may be linked to
genes important in the divergence process between mallards and Mexican ducks
(Fig. 2). Additionally, advances in Bayesian and maximum likelihood methods
capable of analyzing large genomic datasets now allow researchers to assign statis-
tical significance to each outlier (Pérez-Figueroa et al. 2010; Feng et al. 2015). In
short, these programs often test each marker by comparing it to the overall genomic
background to determine their statistical significance. For example, genomic scans
and statistical tests revealed that the evolution of high-elevation adaptation for
several Andean birds was due to the simple effect of positive selection on amino
acid changes in hemoglobin for higher oxygen affinity (McCracken et al. 2009;
Natarajan et al. 2015).
Sex-linked markers have been particularly interesting, as these have often been
found to have significantly higher divergence patterns as compared to autosomal
and/or mitochondrial markers. These patterns are especially detectable when specia-
tion is at the earliest stage (Haldane 1948; Frank 1991; Reeve and Pfennig 2003;
Phadnis and Orr 2009) and have been documented in birds (Minvielle et al. 2000;
Fig. 2 Distribution of ΦST values for chromosomes containing significant outliers

(chromosomes Z, 1, 2, 3, 4, and 14) for pairwise comparisons between mallards (MALL), American
black ducks (ABDU), Mexican ducks (MEDU), Western Gulf Coast mottled ducks (MODUWGC),
and Florida mottled ducks (MODUFL). Black dots denote markers identified to be putatively under
diversifying selection in each pairwise comparison when analyzed in BayeScan v. 2.1 (Foll and
Gaggiotti 2008). Such comparative analyses provide the opportunity to identify in which species
divergent selection may be occurring in. For example, the outlier region within an ~11 Mbp region
(1.0 x 108–1.2 x 108 bp) on chromosome 1 was found when comparing mallards to each of the
monochromatic taxa, suggesting divergent selection occurring in mallards. Similarly, an outlier
locus on chromosome 14 (position ~1.6 x 107; also see Lavretsky et al. 2015) was detected in all
four comparisons involving Mexican ducks, suggesting directional selection at this or a linked locus
in Mexican ducks only. The figure was adapted from Lavretsky et al. (2019)
Saether et al. 2007; Pryke 2010), insects (Phadnis and Orr 2009; Martin et al. 2013),
and mammals (Tucker et al. 1992; Sutter et al. 2013). For example, important
reproductive isolation mechanisms, such as male sterility, sexually selected male
plumage traits, and assortative mating, have all been linked to sex chromosomes
(Minvielle et al. 2000; Saether et al. 2007; Turelli and Moyle 2007; Carling and
Brumfield 2009; Phadnis and Orr 2009; Pryke 2010; Abbott et al. 2013; Pease and
Hahn 2013; Stölting et al. 2013). In general, recent work suggests that due to the
effect of recombination on the possible breakup of coadapted genes and admixture of
alleles between diverging populations via gene flow, selection is more likely to lead
to the adaptive divergence of traits linked to markers found in regions of low
recombination because these regions are shielded from maladaptive gene flow
from other populations (Delmore et al. 2015; Samuk et al. 2017). Thus, the proba-
bility of recovering markers linked to evolutionarily important regions on sex
chromosomes is likely the product of their smaller absolute and effective size, as
well as higher linkage disequilibrium as compared to autosomes (Bergero and
Charlesworth 2009). For example, conducting genomic scans using ddRAD-seq
data between mallards and Mexican ducks, Lavretsky et al. (2015) found 2–3% of
Z-linked loci, compared to <0.1% of autosomal loci as outlier loci under divergent
selection. Indeed, elevated Z-differentiation deviated from neutral expectations
when simulating data that incorporated demographic history and differences in

effective population sizes between marker types. In contrast, Z-linked and autosomal
differentiation (ΦST ¼ 0.017 and 0.013, respectively) were similar among the seven
Mexican duck sampling locations, following a scenario of genetic drift and isolation
by distance. Similar to Mexican ducks and mallards, Chaves et al. (2016) found that
key adaptive traits (e.g., beak size) in Darwin’s finches were also associated with a
few genes (11 of 32,569 SNPs) but found these putatively evolutionary important
genes across multiple chromosomes. Similarly, other studies also report genetic
regions involved in adaptive divergence and reproductive isolation to be scattered
throughout the genome (Parchman et al. 2013).
7 Recombination
Similar to other genomic parameters, such as gene density and mutation rate,
recombination rate is highly variable along a genome. Regardless of chromosome
size, at least one crossover per chromosome (or chromosome arm) is required for
proper segregation of homologous chromosomes during meiosis (Fledel-Alon et al.
2009; Wang et al. 2012). This obligatory crossover results in a negative correlation
between recombination rate and chromosomes size because the rate is calculated as a
total genetic distance (in centimorgans, cM) divided by physical size of a chromo-
some (in Mb). Because of the large differences in chromosome size in bird genomes
(Damas et al. 2019), recombination rate is an order of magnitude different between
the largest and smallest chromosomes in birds (Groenen et al. 2009; Backström et al.
2010; Kawakami et al. 2014; van Oers et al. 2014). In addition, recombination rate is
also variable within a chromosome, where the rate is lower near centromeres and
increases away from them (Choo 1998; Talbert and Henikoff 2010). At a finer scale,
birds and several other species have small genomic regions, referred to as recombi-
nation hotspots, where the rate is often hundreds or even thousands times higher than
the surrounding regions (reviewed in Stapley et al. 2017). Genomic locations of
recombination hotspots appear to be conserved over tens of millions of years during
bird evolution (Singhal et al. 2015; Kawakami et al. 2017). Furthermore, the pseudo-
autosomal region (PAR), the only recombining region on sex chromosomes in the
heterogametic sex (i.e., female birds with Z and W sex chromosomes), shows an
extremely high recombination rate (>700 cM/Mb) (Smeds et al. 2014). Therefore, a
highly heterogeneous recombination landscape is a hallmark of avian genomes, and
characterizing detailed variation of recombination rate is a necessary step toward the
understanding of how genetic variation changes over time in a genome.
There are at least two ways for recombination to affect genetic variation in a given
genomic region, namely, “linked selection” and GC-biased gene conversion
(gBGC). As discussed in Sect. 6, positive selection removes genetic variation at a
locus under selection by fixation of an advantageous allele, while negative selection
(purifying or background) reduces genetic variation because new mutations in
functionally important regions, such as protein-coding genes and regulatory
elements, cannot increase in frequency if they have a negative effect on fitness
(i.e., deleterious mutations). Removal of variants is not restricted to target loci under
selection (positive and negative); variants at neighboring loci can also be removed
from a population if those neighboring loci are physically linked to the target loci
(hence referred to as “linked selection”) (Cruickshank and Hahn 2014; Burri 2017b).
Since the extent of linkage between loci under selection and neighboring loci
depends on local recombination rate, there is a significant negative correlation
between genetic diversity and recombination rate (Burri et al. 2015; Vijay et al.
2017). Because recombination rate variation is likely conserved between species
(Singhal et al. 2015; Kawakami et al. 2017), patterns of genetic diversity along a
genome are also likely similar between species (Burri et al. 2015; Dutoit et al. 2017;
Vijay et al. 2017). Evaluation of baseline genetic diversity is particularly important
in genomic scan analyses because measurement of relative genetic divergence
between species is a function of genetic diversity within species and, consequently,
low recombination regions tend to stand out as highly differentiated outlier regions
even without direct involvement in the process of speciation.
Second, gBGC is a neutral, recombination-associated process that can leave a
similar genetic footprint as positive selection by distorting the allele frequency
distribution. Recombination is initiated by the formation of DNA double-strand
breaks (DSBs), which are subsequently repaired as crossovers or noncrossovers.
When crossovers occur, there is reciprocal exchange of DNA between homologous
chromosomes (Fig. 3). During these repair processes, G or C nucleotides are
preferentially transmitted over A or T nucleotides in regions close to DSBs with
G:C and A:T base mismatches between paternal and maternal chromosomes (Duret
and Galtier 2009; Mugal et al. 2015). Since gBGC takes place more frequently in
regions experiencing frequent DSBs and recombination, highly recombining regions
are more strongly affected by gBGC with stronger transmission bias toward G:C
Fig. 3 DNA double-strand G

C DSB formation
breaks (DSBs) are repaired as A
either crossovers (COs) or T
noncrossovers (NCOs).
G
GC-biased gene conversion C
(gBGC) results from biased DSB resection
T
incorporation of GC over AT
nucleotides in regions close to
G
DSBs with base mismatches
DSB repair
between paternal and maternal
T
chromosomes (red and blue)
G G
C C
C C
T T
GC-biased gene conversion

G G
C C
C C
G G
Crossover (CO) Non-crossover (NCO)

nucleotides. While positive selection increases allele frequency of “better-fit” alleles

by virtue of their selective advantages, gBGC spreads G:C alleles independent of
their effect on fitness. This causes a serious challenge in detecting a signature of
selection because the strong effect of gBGC in high recombination regions can drive
the fixation of potentially deleterious G or C alleles and, hence, counteract natural
selection. In addition, the skewed allele frequency distribution by gBGC relative to
neutral expectation can also affect inferences of demographic history and natural
selection based on various population genetic statistics (Bolívar et al. 2018; Pouyet
et al. 2018). Altogether, we must estimate the baseline genetic diversity by taking
into account the effect of linked selection and gBGC in order to infer demographic
history and detect signatures of selection (Mugal et al. 2015). Forward simulation
approaches that take into account the variation of recombination rate, gene density,
background selection, and demographic events can provide analytical framework to
simulate genome-wide patterns of genetic diversity and divergence, with which an
empirical data can be compared in order to detect outlier regions (Comeron 2017). In
addition, machine learning approaches can jointly estimate effective population sizes
and the impact of linked selection (both background selection and selective sweep)
on the pattern of genetic diversity (Schrider and Kern 2016, 2018; Schrider et al.
2016; Sheehan and Song 2016).
8 Phylogeography: The Interface Between Population

Genetics and Phylogenetics
The early study of mitochondrial DNA lineages when PCR and DNA sequencing
became available (Wink 2019) revealed that branches of intraspecific gene trees
often followed striking geographic patterns (Avise et al. 1987). The study of the
relationship between gene genealogies and geography became known as
phylogeography (Avise 2000). Some early examples of phylogeographic studies
on avian mtDNA include snow goose (Anser caerulescens) (Avise et al. 1992; Quinn
1992), northern flicker (Colaptes auratus) (Moore et al. 1991), and common grackle
(Quiscalus quiscula) (Zink et al. 1991). Phylogeography provides a bridge between
phylogenetics (i.e., the reconstruction of evolutionary relationships) and population
genetics, describing how genetic variation—introduced by mutation (see Sect. 3)—
is geographically structured within and between populations by population genetic
processes, such as genetic drift (see Sect. 4), gene flow (see Sect. 5), selection (see
Sect. 6), and recombination (Sect. 7). For populations that have been separated
historically and have experienced little or no gene flow, genetic differences can
accumulate by these evolutionary processes, potentially resulting in speciation
(Ottenburghs 2019).
Phylogeography relied heavily on non-recombining and rapidly evolving mtDNA
to match gene genealogies with geography (Avise 2000). The advent of genomic
data in combination with the development of coalescent theory (Kingman 1982a, b)
has revolutionized the field (Edwards et al. 2015). In general, the application of next-
generation sequencing technologies uncovers more detailed population structure that
is often missed by traditional markers, such as mtDNA and microsatellites. For

example, using RADseq data, Ruegg et al. (2014) were able to more reliably
distinguish between eastern and western populations of the Wilson’s warbler
(Cardellina pusilla) compared to previous studies based on mtDNA (Kimura et al.
2002; Paxton et al. 2013) and AFLPs (Irwin et al. 2011). In addition, the application
of multilocus datasets revealed that different genes often result in different gene trees
(Degnan and Rosenberg 2009). This phylogenetic incongruence can provide a more
detailed picture of population history because different gene trees capture particular
historical events and population genetic processes that have shaped the present
patterns of genetic diversity. However, recent work has also uncovered high levels
of reticulation due to recombination (see Sect. 7) and gene flow (Edwards et al.
2016). New statistical methods are being developed to deal with such reticulated
scenarios (Dai et al. 2010; Ottenburghs et al. 2017a; Zhu et al. 2018).
9 Conclusions and Outlook
In this chapter we mostly dealt with questions relating to which inferences we can
make from genetic variation data on a population scale, with respect to what we
know about geological events as well as current and past geography. Traditionally,
the study of phylogeography has a strong focus on demographic processes and
distribution of genetic variation in time and space. The introduction of genomic
techniques dramatically increases the statistical power with which we can answer
questions and describe systems. In sections about the source, maintenance, and loss
of genetic variation, we introduced the concepts of natural and sexual selection. This,
in contrast to neutral variation that is shaped by demography, is the second and
perhaps more innovative major addition that population genomics brings us com-
pared to population genetics.
Measuring genetic variation everywhere in the genome, including both neutrally
and adaptively evolving regions, allows us to understand demography and adapta-
tion concurrently. Studies into the functional variation have so far only been possible
on the interspecies level. Many studies in the past have analyzed the evolutionary
history of genes known to be involved in key adaptations of a certain lineage. For
instance, innate immunity in birds is well studied on the avian lineage scale. Cheng
et al. (2015) deciphered evolutionary signals in effector molecules of the immune
defense such as defensins and cathelicidins, and Velová et al. (2018) studied
membrane proteins that control the identification and recognition of pathogens, the
toll-like receptors. However, from our point of view, the really interesting studies are
those taking into account the population approach and using population genetic
theory to measure selection pressure. The first toll-like receptor population
re-sequencing paper on wader species revealed purifying selection and domain-
specific evolution (Raven et al. 2017). This was not a genome-wide study and lacked
comparative information from a number of related genes, so the final conclusions
remain tentative, but such studies are important next steps in understanding the
relationship between functional and adaptive variation and offer a glimpse of what
may become possible in the future. A similar study on bird species with rather
different phylogeographic histories curiously rejected the impact of natural selection
(here, supposedly pathogen pressure) on the molecular evolution on this receptor
family. Instead, the authors found that drift in small populations overrides the effects
of natural selection (Gonzalez-Quevedo et al. 2015) as is expected on theoretical
grounds under such conditions (Lynch 2007). Chapman et al. (2016) studied several
toll-like receptor genes both on the lineage and population scale to place their
evidence for diversifying selection into the broader evolutionary framework. Yet,
the individual gene was studied in isolation from the rest of the genome. Whole-
genome information on a population scale will make possible the study of selection
patterns and their interaction with phylogeography, as the costs of sequencing
continue to decline (Kraus and Wink 2015). New approaches to study functional
variation not only within gene families (gene-centric) but also within and across
biological pathways (pathway-centric) are now becoming possible. Jax et al. (2018a)
showcase interactions between the functional variation and molecular evolution of
more than 100 genes across several immune pathways in mallards and closely
related ducks around the world and their geographic origin. The future development
of population genomics might thus culminate in a more functional approach to avian
evolution.
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American Ornithological Society
Avian Population Studies
in the Genomic Era
Arild Husby, S. Eryn McFarlane, and Anna Qvarnström
Abstract
Long-term studies on birds have played a pivotal role in addressing important
questions in evolutionary biology, and avian biologists were quick at adopting
new genetic tools. The integration of genetic work on birds has revolutionised the
way we think about avian mating systems, for example. During the last decade,
we have seen a tremendous decline in the cost of sequencing, making it possible
to genotype or sequence hundreds or even thousands of individuals. These tools
are offering an exciting new array of questions to be asked from long-term
longitudinal studies on birds. We review here some of the genetic resources
currently available to researchers studying avian population samples, some sta-
tistical approaches to analyse population-level genomic data and future questions
that long-term studies on birds can provide insights into. It is clear that genomic
approaches on long-term studies on birds have played, and will continue to play,
an important role for addressing fundamental evolutionary questions.
A. Husby (*)
Department of Ecology and Genetics, Uppsala University, Uppsala, Sweden
Organismal and Evolutionary Biology Research Programme, University of Helsinki, Helsinki,
Finland
Centre for Biodiversity Dynamics, Norwegian University of Science and Technology, Trondheim,
Norway
e-mail: arild.husby@ebc.uu.se
S. E. McFarlane
Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK
e-mail: eryn.mcfarlane@ed.ac.uk
A. Qvarnström
e-mail: anna.qvarnstrom@ebc.uu.se

https://doi.org/10.1007/978-3-030-16477-5_9
268 A. Husby et al.
Keywords
Quantitative genetics · Microevolution · GWAS · Epigenetics · Heritability ·
Longitudinal studies · Polygenic · Chromosome partitioning · DNA methylation ·
SNP
1 Introduction
The technical ability to trace inheritance of phenotypic traits at the resolution of

single nucleotides opens up exciting novel possibilities for studies on ecology and
evolution. In this chapter, we outline some statistical methods associated with these
technical developments and discuss major advantages and obstacles faced by
researchers that apply these methods to wild populations of birds. We start with a
short description of the historical perspective where we argue that long-term popu-
lation studies on birds have played a central role for the development of the whole
field of studying inheritance of phenotypic traits in nature.
Long-term studies on birds have traditionally played an important role in gaining
insights into central theories of ecology and evolution. A major underlying reason
for the focus on birds is the relative ease by which large numbers of individuals can
be marked, measured, blood sampled as well as having their reproductive perfor-
mance followed in great detail under natural conditions. The first long-term individ-
ual-based field studies were typically run by highly skilled and enthusiastic
naturalists, who focused on particular species of birds that were easy to monitor.
These early scientists include Huibert Kluijver (1902–1977) and David Lack
(1910–1973) studying great tits (Parus major) in The Netherlands and England,
respectively, Margaret Nice (1883–1974) studying song sparrows (Melospiza
melodia) in the USA and Lars von Haartman (1919–1998) studying pied flycatchers
(Ficedula hypoleuca) in Finland. By collecting information on phenotypic traits
(e.g. morphology and behaviour) measured at the level of individual birds together
with information about their survival and reproductive performance (i.e. fitness), it
became possible to actually measure the strength and direction of the main drivers of
evolution, natural and sexual selection. Another major advantage with many long-
term studies of birds is that extensive pedigree information can be gathered, which is
a crucial asset for quantitative genetic approaches.
The opportunity to combine measures of natural and sexual selection with
estimates of genetic parameters means that evolutionary responses to environmental
change can be studied in real time. One of the best examples of how important this
type of individual-based field studies can be for increasing our general understanding
of evolutionary events taking place on a contemporary time scale comes from Peter
and Rosemary Grant’s long-term study on Darwin’s finches on the Galápagos
Islands. A population of medium ground finches (Geospiza fortis) living on the
Avian Population Studies in the Genomic Era 269
island of Daphne Major experienced two serious periods of drought (in 1976–1977
and 1984–1986), which inferred changes in food supply resulting in corresponding
changes in natural selection acting on beak morphology. Due to detailed collection
of data, the Grants were able to predict the phenotypic responses in beak morphology
observed in the population based on estimates of natural selection and heritability
(Grant and Grant 1995). This study hence nicely illustrates evolution in action. The
quantitative genetic approach used by the Grants and their colleagues (e.g. Boag and
Grant 1978) was based on the classical framework developed by Fisher, who made
the assumption that the genetic variance present in a population was based on a large
number of Mendelian factors, each having a small additive effect on the phenotype
(Fisher 1930). Together with Haldane and Wright, Fisher founded the discipline of
population genetics that formed an important part of the whole modern evolutionary
synthesis where several fields of biology, including ecology, genetics, systematics
and palaeontology, were brought together through a joint acceptance of evolution as
the central unifying theory of biology.
The quantitative genetic framework developed by Fisher, Haldane and Wright not
only played an important role for the development of modern evolutionary theory; it
also formed the basis of the discipline of conservation biology that focuses on
endangered organisms’ ability to adapt to human activities (e.g. Stockwell et al.
2003) and laid the foundation for applied animal- and plant-breeding programmes.
The latter applied fields, in turn, contributed to further development of statistical
tools like the animal model, which consider covariance between multiple sets of
relatives at the same time and thereby allows more detailed dissections of genetic
and environmental effects (Henderson 1950). In the animal model, phenotypic and
pedigree data is combined to estimate genetic parameters such as additive genetic
variance (VA) of traits or additive genetic covariances between traits. Not surpris-
ingly some of the first studies using the animal model in the context of natural
populations were based on long-term studies on birds, including collared flycatchers,
great tits, long-tailed tits and house sparrows (Jensen et al. 2003; MacColl and
Hatchwell 2003; Sheldon et al. 2003; Garant et al. 2004). One example of how the
animal model can be combined with long-term breeding data to test central evolu-
tionary theories comes from a study using 24 years of data on collared flycatchers
breeding on the Swedish island Gotland in the Baltic Sea. Qvarnström et al. (2006)
used this data set to test all the key genetic requirements of both Fisherian (Fisher
1930) and ‘good-gene” models (Zahavi 1975) on sexual selection. They found
significant heritability for all key components: the ornament (male forehead patch
size), female choice based on the ornament, male fitness and female fitness. How-
ever, when the required genetic correlations between these components were taken
into account, the estimated strength of indirect selection on female choice was
negligible (Qvarnström et al. 2006). This means that female mate choice based on
forehead patch size most likely evolves in response to direct selection,
e.g. depending on resources and parental care provided by the males. Selection of
female choice based on male forehead patch size moreover varies across years such
that females paired with large-patched males experienced high relative fitness only
270 A. Husby et al.
during dry summer conditions (Robinson et al. 2012). Additionally, a decline in the
mean size of this trait across the study period appears connected to the warmer
climate (Evans and Gustafsson 2017), although the physiological explanation to the
connection between weather and relative performance of large-patched males
remains unknown. Thus, the effects of environmental heterogeneity can be
quantified and taken into account in the animal model, something that can increase
accuracy in predicting evolutionary responses and in testing core evolutionary
theories.
Most quantitative genetic models (like the animal model) provide insights into the
genetic variance and covariance of traits but not the underlying loci. To identify the
QTL (quantitative trait loci) that contribute to genetic and phenotypic variation, we
need genotype information for each individual and phenotypic data.
1.1 What Do We Gain By Identifying Genes in Natural Bird

Populations?
Knowledge about the structure and function of genes at the molecular level was until
recently restricted to molecular genetic studies on model organisms in the lab.
However, the technical ability to trace inheritance of phenotypic traits at the
single-nucleotide resolution that is now emerging opens up the possibility to answer
novel sets of evolutionary questions (Ellegren and Sheldon 2008; Barrett and
Hoekstra 2011; Losos et al. 2013). For example, what is the number and effect
size of the loci involved in the determination of the trait in focus? Are genetic
correlations between traits a result of pleiotropic effects or due to linkage? Where are
these loci located in the genome? What are the local rates of recombination and
mutations at these sites? How is genetic variation maintained at the level of the loci?
What selective forces operate at the level of the loci? By answering this type of
questions, a more detailed understanding of the principles and mechanisms of
evolution and speciation is within reach.
2 Genomic Resources in Avian Population Research
Avian biologists were quick at adopting new genetic resources and used multilocus
DNA fingerprinting probes to examine mating system variation already in the 1980s
(Burke and Bruford 1987). Historically, birds were considered to be among the
animal groups with the highest levels of social and genetic monogamy (>90%; Lack
1968). The use of DNA fingerprinting, and subsequently microsatellites (SSR), have
however clearly shown that birds are far from monogamous; indeed less than 25% of
species are in fact genetically monogamous (Griffith et al. 2002). This finding has
revolutionised the field of avian behavioural ecology and our understanding of
sexual selection in birds.
While microsatellites continue to be the genetic marker of choice for many
applications, the decreasing costs of high-throughput sequencing mean large amount
Fig. 1 A selection of some long-term pedigree based studies on birds [(a) Collared flycatcher, (b)
great tit, (c) house sparrow, (d) barn swallow and (e) blue tit] for which genomic resources are
available. Photo credits: (a) Eryn McFarlane, (b and c) Martin Lind, (d and e) Arild Husby
of SNP markers are easy to generate, and they also have lower error rates than
microsatellites (Kraus et al. 2015). This means it is now possible to construct custom
SNP arrays for genotyping hundreds or even thousands of birds (e.g. Kraus et al.
2011, 2013; van Bers et al. 2012; Hagen et al. 2013; Kawakami et al. 2014;
Lundregan et al. 2018), much like that seen in the field of human genetics.
Nevertheless, independent of which markers are used, a key consideration in all
population studies is the need to genotype or sequence a large number of individuals
to have reasonable statistical power to study selection or for trait mapping (Kardos
et al. 2016). Hence, even a relatively low cost for genotyping or sequencing an
individual will quickly become expensive when hundreds or even thousands of
individuals are needed. For this reason there are still relatively few model species
where population scale genomic data are available (Fig. 1, Table 1). Below we
review recent developments of genetic tools within the field of avian genetics,
focusing on large-scale individual-based population studies.
2.1 Whole Genome Sequencing Efforts
The availability of sequenced bird genomes has increased exponentially over the last
few years, and several avian ecological model species have now been sequenced
(Ellegren 2013; Zhang 2014; Vignal and Eroy 2019). The collared and the pied
flycatcher were the first ecological model bird species to have their genome
272 A. Husby et al.
Table 1 Some examples of wild bird systems in which individual phenotypic and genomic data
have been collected
Species Available genomic resources Reference
Blue tits (Cyanistes Genome, transcriptome, 12 K Mueller et al. (2016) and
caeruleus) SNP array Szulkin et al. (2016)
Collared and pied flycatchers Genome, transcriptome, 50 K Ellegren et al. (2012), Uebbing
(Ficedula albicollis and Illumina SNP array et al. (2013) and Kawakami
F. hypoleuca) et al. (2014)
Red junglefowl and Genome, transcriptome, Gering et al. (2015)
domestic chickens (Gallus commercially available SNP
gallus) arrays
Darwin’s finches (Geospiza Genome Lamichhaney et al. (2015)
sp., including all species in
radiation)
Red grouse (Lagopus 384 SNP array Wenzel et al. (2015)
lagopus scotica)
Superb starling Transcriptome, 102 SNPs Weinman et al. (2015)
(Lamprotornis superbus)
Great tits (Parus major) Genome, transcriptome, van Bers et al. (2012) and
methylome, 10 K SNP array, Laine et al. (2016)
650 K SNP array
House sparrows (Passer Genome, transcriptome, 10 K Hagen et al. (2013) and Elgvin
domesticus) and 200 K SNP arrays et al. (2017)
Ruff (Philomachus pugnax) Genome, transcriptome Küpper et al. (2016) and
Lamichhaney et al. (2016a)
Attwater’s prairie chicken ~20 K SNP array Bateson et al. (2016)
(Tympanuchus cupido
attwateri)
Zebra finch Genome, transcriptome, Knief et al. (2017)
6000 K SNP array
sequenced (Ellegren et al. 2012), following sequencing of the chicken (Hillier et al.
2004), zebra finch (Warren et al. 2010), turkey (Dalloul et al. 2010) and domestic
duck (Huang et al. 2013) genomes. The sequencing of the collared and pied
flycatcher marked a significant milestone in avian genomics because it was done
by a small group of scientists rather than a large consortium. The genomes of other
important bird species in evolutionary ecological research have followed swiftly as
sequencing methodology has advanced and prices declined; this includes among
others great tit (Laine et al. 2016), Darwin’s finches (Lamichhaney et al. 2015), blue
tit (Mueller et al. 2016), barn swallow (Safran et al. 2016) and house sparrow (Elgvin
et al. 2017).
In addition to the species with long-term data listed above, bird genomes are
sequenced on a daily basis and the goal of the Avian Phylogenomics Consortium
Bird 10 K project (Zhang 2015) is to have a draft genome from all extant bird species
done before 2020 (https://b10k.genomics.cn), as of July 2017 (last time website has
been updated) about 300 species have had their genome sequenced.
Genome sequencing studies have been instrumental for the technological devel-
opment of avian genomics (e.g. Kraus and Wink 2015) and have generated many
important insights into avian genome evolution and provided insights into the
genetic basis of adaptation (Ellegren 2013, 2014). One illustrative example is the
use of whole genome sequencing of the ground tit (Parus humilis), an endemic
species in the Paridae family living in the Tibetan plateau, to gain insight into the
genetic basis of high-altitude adaptation. This species is found exclusively above the
tree line on rocky steppes and grasslands and has evolved a number of characteristic
features such as longer and distinctly downward curved bill, larger body size and
longer legs (used for foraging and digging burrows). Qu et al. (2013) used Illumina
HiSeq 2000 platform to sequence and assemble a female ground tit as well as a great
tit, a yellow-cheeked tit and a Mongolian ground jay. Using phylogenetic
reconstructions they demonstrated that the ground tit is indeed closer related to
other Parus species than jay, to which it is more phenotypically similar. Interest-
ingly, Qu et al. (2013) also found the ground tit genome showed gene expansion in
energy metabolism and contractions in immune and olfactory perception genes
compared to the other species, and there were signs of positive selection in genes
related to hypoxia response and skeletal development. This study demonstrates the
power of whole genome sequencing to detect the genetic basis of traits involved in
adaptation to, for example, high altitude in wild bird populations.
Whole genome sequencing has provided significant insights into avian genome
evolution, phylogenetics and demography (Ellegren et al. 2012; Nadachowska-
Brzyska et al. 2013; Burri et al. 2015). However, to study evolution in action
(i.e. microevolution across ecological time scales), large-scale population samples
are needed. For this purpose whole genome sequencing is still too expensive [but see
Kardos et al. (2016) for one example], and here resequencing data can be used to call
SNPs and design custom SNPs arrays that can be used for large-scale population
genotyping.
2.2 Development of SNP Arrays
SNP arrays can be very useful tools for large-scale population genetic studies and
trait mapping. SNP discovery can be done using transcriptomic data, sequence data
or RAD sequence data from a number of individuals such that SNPs can be identified
as polymorphic in the population and selected for inclusion on the SNP array (Hagen
et al. 2013; Kawakami et al. 2014; Malenfant et al. 2014). In birds high-density SNP
arrays were first developed for great tits (van Bers et al. 2012), followed by the house
sparrow (Hagen et al. 2013) and the collared flycatcher (Kawakami et al. 2014).
Small-scale custom arrays have also been used in red grouse (Wenzel et al. 2015)
and zebra finch (Knief et al. 2017).
Depending on the type of SNP to be included, either one or two probes per locus
are needed, where A/T and C/G SNPs require two probes (one probe for each allele,
so-called Infinium type I probe design in Illumina terminology). The cost of a
custom array depends on the number of probes (bead types) included rather than
274 A. Husby et al.
the number of loci, and thus the Infinium type II probe design is more common.
Following successful SNP identification, a list of SNPs with sequence information
50–100 bp up- and downstream of the SNP is sent to the company (Illumina/
Affymetrix) for quality control using their proprietary assay design tools to make a
final SNP selection to include on the array. Most custom SNP arrays on wild bird
populations have seen success rates above 90% (i.e. 90% of included SNPs on the
array passed the assay design and were polymorphic), with some exceptions (about
75% in house sparrows Hagen et al. 2013).
2.3 Reduced Representation Techniques or SNP Arrays?
For genotyping a large number of individual custom-designed SNP arrays have been
considered the medium of choice. However, reduced representation sequencing
techniques, where restriction enzymes are used to cut out parts of the genome
which are then sequenced, can also be a very cost-efficient way of obtaining
genotype information (Andrews et al. 2016). This cost is somewhat traded off with
the increased need to have bioinformatics skills and resources to do proper post-
processing of sequence data, which are more substantial compared to the genotype
data one obtains from SNP arrays. In particular, quality of genotype calling is lower
than in SNP arrays and depends critically on the coverage. However, the flexibility
of RAD seq to target varying amounts of loci and coverage means that the cost per
genotype might be lower than with SNP arrays and the flexibility higher.
Which technique is better suited is also a question of the available genomic
resources for the species in focus; RAD seq can be used even if no previous genomic
resources are available, whereas SNP array (preferably) needs information about
genomic location as well as sequence information around the SNPs.
3 Statistical Genetic Methods Used to Analyse Avian

Population Samples
3.1 The Decline of Linkage Analysis and Rise of GWAS (Genome-

Wide Association Studies)
A number of studies have used linkage analyses in natural populations of birds to

detect QTL of complex traits. For example, Tarka et al. (2010) genotyped 333 indi-
vidual great reed warblers for 45 autosomal and 6 sex-linked microsatellites/AFLP
markers and located a QTL for wing length on chromosome 2 that explained more
than a third of the phenotypic variance (Fig. 2). A series of studies on a captive
population of zebra finches have also used linkage mapping to detect QTL for beak
morphology (Knief et al. 2012), beak colouration (Schielzeth et al. 2011b) and wing
length (Schielzeth et al. 2011a).
Linkage mapping requires both a genetic map and a pedigreed population to be
able to determine co-segregation of markers with the phenotype (Slate 2005;
c)
a)
b)
-log(p-value)
Chromosome
Fig. 2 The long-term study of great reed warblers from lake Kvismaren, Sweden, is one of the few
studies that have attempted to replicate a QTL from a linkage analysis with a GWAS in a natural
population. (a) Linkage plot for wing length QTL on chromosome 2 in great reed warblers from
Tarka et al. (2010). (b) Manhattan plot for the same trait from the GWAS using RAD sequencing
data. Adapted from Hansson et al. (2018). (c) A great reed warbler from the long-term study
population at Kvismaren. Photo credit: William Velmala
Slate et al. 2010). Thus, in principle a linkage map to establish position of markers
would be needed for the species in which one aims to do linkage mapping. For
example, a three-generation, captive pedigree was used to establish a linkage map in
zebra finches (Stapley et al. 2008), and a nestbox population with a two-generation,
half sib design was used to design a linkage map in collared flycatchers (Backstrom
et al. 2006). However, work in the collared flycatchers has also demonstrated that
synteny (preservation of gene order among different species) with the chicken and
zebra finch genome is high (Backstrom et al. 2006, 2008a, b), something that
facilitates construction of genetic maps in species without a linkage map.
While linkage analysis has been successfully used in some bird species, it is
limited by the need for a pedigreed population to trace the co-segregation of marker
loci and phenotypes (Slate et al. 2010) and in general has low power to detect
anything but loci of major effect (Santure et al. 2015). Partly for this reason,
association mapping, which relies on historical recombination events rather than
recombination events within a pedigree, has become popular. The use of historical
recombination events also allows QTL region to be localised to a narrower region
than when utilising recombination events within a pedigree. The other main reason
for the rise in association mapping studies is no doubt that high-throughput sequenc-
ing costs have declined to the extent that it is feasible to resequence a number of
individuals to generate SNP arrays for large-scale genotyping (Ellegren 2014).
For example, SNP arrays used for association mapping purposes are now avail-
able in a number of species (Table 1), and GWAS have been applied to great tits
(Santure et al. 2015), collared flycatchers (Husby et al. 2015; Kardos et al. 2016), red
276 A. Husby et al.
grouse (Wenzel et al. 2015) and house sparrows (Silva et al. 2017). Generally, these
studies have found evidence for polygenic architectures of quantitative traits (Kardos
et al. 2016; Santure et al. 2015). The application of GWAS to wild bird populations
has allowed finer-scaled studies of genetic architectures than was possible with
linkage analyses because the identified regions are much smaller in association
mapping.
While many of the early avian studies cited above that have used dense marker
panels and GWAS have been in pedigreed populations, this need not be the case
(Speed and Balding 2015). Dense marker panels could be used to infer genomic
relatedness between individuals within the same generation (i.e. over only one
breeding season of study) and could lead to more precise, rather than inferred,
estimates of relatedness (Bérénos et al. 2014).
3.2 Chromosome Partitioning
In order to assess if the genetic architecture of traits are polygenic, Yang et al. (2011)
proposed partitioning genetic variance onto chromosomes and testing for a positive
correlation between the size of a chromosome and the amount of genetic variance
that it explained. If particular chromosomes explained more genetic variance than
would be expected based on chromosome size, then this could indicate either a
marker of large effect, or as cluster of markers each contributing a small effect, and
could point to chromosomes, and subsequently regions, that would be interesting for
further inspection (Schielzeth and Husby 2014). In birds, chromosome partitioning
has now been done in great tits (Robinson et al. 2013; Santure et al. 2015), red
grouse (Wenzel et al. 2015), collared flycatchers (Silva et al. 2017) and house
sparrows (Silva et al. 2017). The chromosome partitioning results from these studies
tend to conform to a polygenic basis to most traits, but there are large variations in
regression slopes and outlier chromosomes in all these analyses.
At present it is not clear if this variation is biologically interesting or a result of
variation in sample size and in marker density on the different chromosomes.
Chromosome partitioning can be a useful tool for traits for which there are no
clear markers of large effect from a GWAS, because a positive correlation between
chromosome size and proportion of variance explained would indicate a polygenic
basis to traits (Kemppainen and Husby 2018a). However, the power of this approach
depends on factors such as heritability of the trait, the QTL effect size distribution,
clustering of loci in the genome and sample size (Kemppainen and Husby 2018a),
and thus its suitability to infer a polygenic basis of traits should be carefully
evaluated on a per case basis. Also, variation in chromosome size and numbers
used in the analysis influences power with lower power when there is small variation
in chromosome sizes (Kemppainen and Husby 2018a). These factors, combined with
earlier studies using a biased test for inferring polygenic architecture (Kemppainen
and Husby 2018b), mean that careful consideration and interpretation of chromo-
some partitioning results is needed and that future studies should use the correction
introduced by Kemppainen and Husby (2018b).
3.3 Admixture Mapping
Admixture mapping is another genetic technique that can be useful in wild bird
populations if there is frequent hybridisation and backcrossing (Ottenburghs et al.
2015). As such it can be a useful tool if there is large-scale population data and
phenotype data in avian hybrid zones (Ottenburghs et al. 2017). For example,
Delmore et al. (2016) used hybridising Swanson’s thrushes to dissect the genetic
basis of migratory orientation and plumage colour. By using multiple-SNP Bayesian
models, these authors were able to consider all SNPs together and control for factors
that influence phenotypes and correlated with genotypes such as population struc-
ture. Several regions associated with migration and colour were reported including
TYRP1 (i.e. previously known to associate with plumage colour in quail; Nadeau
et al. 2007) and several genes involved in brain development and circadian locomo-
tor behaviours (Delmore et al. 2016). Another example of admixture mapping in
birds is a recent study on golden-winged (Vermivora chrysoptera) and blue-winged
(V. cyanoptera) warblers, which hybridise across a broad zone of eastern North
America. Toews et al. (2016) assayed SNPs in divergence peaks (i.e. genomic
regions of high divergence between these two species) in a few hundred individuals
with known phenotypes that were sampled from both allopatric and sympatric
geographic regions. One of the most intriguing findings from this study was a perfect
correlation between the agouti signalling protein ASIP region and throat colouration
(Toews et al. 2016). This black throat colouration of golden-winged warblers was
identified as a Mendelian recessive trait already in 1908 (Nichols 1908) and is absent
in blue-winged warblers and F1 hybrids. Interestingly, associations between ASIP
and recessive melanistic phenotypes have been found in other vertebrates (Hoekstra
2006).
4 What Have We Learned About the Genetic Architecture

and Evolution of Complex Traits from Studies on Birds?
4.1 Genomic Vs. Pedigree Heritability
The narrow sense heritability (h2), the proportion of the phenotypic variance
explained by additive genetic variance, is a central parameter in evolutionary biology
because it determines the expected response to selection. Traditionally, this has been
estimated using different breeding designs (parent offspring regression, full sib and
half sib analyses), but during the last 20 years, pedigree data has become the
preferred way to calculate h2 (Lynch and Walsh 1998). Collecting pedigree data is
of course a laborious process because all individuals in the population must be
marked and followed for generations so that relatedness can be inferred. Indeed
several active long-term studies on birds extend back for 30 or more years (e.g. great
tits in Hoge Veluwe in the Netherlands and in Wytham Woods in Oxford, UK,
collared flycatchers, Gotland, Sweden).
278 A. Husby et al.
It is now easy to obtain tens of thousands of markers on many individuals and use
this molecular information to recreate pedigrees or even use estimates of realised
relatedness (as opposed to expected relatedness) directly to estimate heritability
(Husby et al. 2015; Santure et al. 2015; Silva et al. 2017; Gienapp et al. 2017). Do
the possibilities to genotype individuals and assign relatedness based on molecular
markers mean that long-term pedigreed studies are becoming redundant? If the main
goal is to estimate evolution in action and/or to study mechanisms of selection in
detail, the answer to this question is clearly no. Long-term pedigreed populations are
also useful for estimating heritability because reliable estimates need large sample
sizes, preferably also of individuals that are distantly related and hence less likely to
share environment. Moreover, longitudinal pedigreed studies with repeated sam-
pling of the same individual in different environmental conditions also allow for
studies of genetics of plasticity (e.g. Husby et al. 2011), something that would not be
possible if sampling just a single time point.
On the other hand, genotyping populations and using the molecular markers to
estimate relatedness could improve pedigree estimates if these have been inferred
from observations (as is the case with many long-term studies on passerines). Using
marker information to estimate relatedness can also improve heritability estimates
because of variation in relatedness around expected IBD sharing due to recombina-
tion and Mendelian sampling (Visscher et al. 2008).
For the above reasons, it is not always clear what approach is best for estimating
heritability, and the choice in many ways probably comes down to practical factors.
Also, in cases where the two approaches have been compared (i.e. using pedigree or
using realised relatedness from marker data), they have been largely concordant
(Bérénos et al. 2014; Santure et al. 2015). It is important to keep in mind that such
comparison is valid only when there is strong family structure in the data. On
unrelated individuals, which is typically used in most human studies, the heritability
estimates using realised relatedness is much smaller because it no longer measures
pedigree relatedness, but the proportion of variance captured by the SNP array
(de Los Campos et al. 2015). In family data the SNPs capture the long-range LD
found between family members and hence reflect pedigree relatedness rather than
proportion variance explained by the SNPs. Thus, what the genomic heritability
measures depends on the pedigree structure in the sample.
4.2 Many Small or a Few Large Genes?
A fundamental assumption in quantitative genetics is the infinitesimal model (Fisher

1930), which postulates that traits are governed by many (infinite) genes of individ-
ually small effect. While this assumption is of course incorrect (after all there is a
finite number of genes in an individual), the infinitesimal model has played, and
continues to play, an important role in evolutionary theory (Nadeau and Jiggins
2010; Rockman 2011).
Linkage mapping studies on birds such as those by Tarka et al. (2010) on great
reed warblers and the series of studies on a captive population of zebra finches to
detect QTL for beak morphology (Knief et al. 2012), beak colouration (Schielzeth
et al. 2011b) and wing length (Schielzeth et al. 2011a) found QTL that explained a
sizeable portion of the phenotypic, and not least genetic, variance. Taken together
these studies suggested that the genetic architecture consists of at least some genes of
large effect that can be found even using sparse marker maps and moderate sample
size. Although the studies acknowledged there might be some inflation of effect sizes
due to the low number of individuals used (‘Beavis effect’; Beavis et al. 1994), the
extent of the proportion of variance explained by the QTL in these studies was
nevertheless a surprise. Not least as work on Drosophila had demonstrated that most
traits are governed by many genes of individually small effect (Mackay et al. 2009),
as expected (and assumed) in quantitative genetic models (Falconer and Mackay
1996).
The picture emerging from the latest association studies is however a very
different one because they almost all identify loci of smaller effect, if any significant
loci at all (Santure et al. 2013, 2015; Husby et al. 2015; Kardos et al. 2016; Silva
et al. 2017; Lundregan et al. 2018; Hansson et al. 2018).
This is not to say large effect QTL do not exist in birds; they obviously do
(e.g. Farrell et al. 2013; Küpper et al. 2016; Mundy et al. 2016; Tuttle et al. 2016).
However, when considering the inflation of effect sizes common to mapping studies
of most current avian studies (Slate 2013), it seems prudent to emphasise that
polygenic architectures seem to be the norm. For example, GWAS and chromosome
partitioning have been used to determine a polygenic architecture of eight morpho-
logical traits in two replicated populations of great tits (Santure et al. 2015).
Additionally, a recent study on a sexually selected trait (forehead patch size) in
collared flycatchers that used a combination of whole genome sequence analyses of
select individuals with extreme phenotypes and GWAS of a less dense SNP array on
a larger sample of individuals found no evidence for genes of large effect (Kardos
et al. 2016), in spite of using whole genome resequencing data. Given the short
chromosomal distances that LD typically reaches in bird populations (e.g. Kardos
et al. 2016), the somewhat low densities of markers used in many studies of wild bird
populations and the possibility for inflated effect sizes (i.e. the Beavis effect;
e.g. Slate 2013), caution should be advised when proposing oligogenic architectures
of traits.
4.3 Insights into Factors That Can Maintain Genetic Variance
While identifying QTL can inform us about the genetic architecture of traits, an even
more interesting aspect of detecting QTL is the possibility to examine the evolution-
ary forces acting on them (Ellegren and Sheldon 2008; Schielzeth and Husby 2014).
For example, a major unresolved question is how genetic variance can be maintained
in natural populations given that many traits seem to be under persistent directional
selection (Kingsolver et al. 2001). Providing further insights into this question
requires that we know the QTL and have allele-specific fitness estimates because
we can then test evolutionary hypotheses that can maintain genetic variance, such as
280 A. Husby et al.
Fig. 3 An example of a GWAS in collared flycatchers identifying some loci related to variation in
clutch size (Husby et al. 2015). (a) A female collared flycatcher alarms outside a nestbox. Photo
credit: Arild Husby. (b) A collared flycatcher nest from the long-term study population on Öland,
Sweden, where the median clutch size in the population is six eggs. Photo credit: Arild Husby. (c)
Manhattan plot for clutch size with genome-wide significant QTL on chromosome 18 and two
suggestive ones on chromosome 9 and 26. From Husby et al. (2015). (d) Fine-scale association plot
and patterns of linkage disequilibrium underlying the QTL on chromosome 18
intra-locus sexual conflict, heterozygote advantage (or overdominance in general) or

fluctuating selection. Long-term studies on birds are in a unique situation to answer
these questions because of the detailed fitness data and genotype data that is
available on the individual level.
To date most studies have focussed on detection of QTL rather than testing
evolutionary hypothesis of what can maintain variation at the QTL. One exception
is work on collared flycatchers where Husby and colleagues used an association
mapping approach to detect QTL for clutch size (Husby et al. 2015, Fig. 3). They
identified a genome-wide significant QTL on chromosome 18 (with three SNPs in
high LD) and a suggestive QTL on chromosome 26. Because the individuals in this
study are part of a population of collared flycatcher that is intensively monitored
(Qvarnström et al. 2010), information about lifetime reproductive success is avail-
able making it possible to test hypotheses for how genetic variation in QTL for
clutch size can be maintained. Interestingly, for the suggestive QTL on chromosome
26, there was evidence of intra-locus sexual conflict where females that were
homozygous for the A allele at the locus had lower fitness compared to males
homozygous for the same allele. In females, the lower fitness was a result of the
same allele having a negative effect on clutch size and therefore number of
fledglings, but the reasons for increased fitness in males homozygous for the same
allele are unclear. Males homozygous for the G allele had slightly higher annual
reproductive success and longer lifespan compared to males homozygous for the A
allele or heterozygotes. While this study would support intra-locus conflict as a

mechanism for maintenance of genetic variance of this clutch size QTL, it is
important to keep in mind that this was only a suggestive QTL and sample size
was low (n ¼ 309). More work is needed both to replicate the QTL and to confirm
the fitness patterns seen in this study. Moreover, there was also no indication of any
fitness differences among genotypes at the genome-wide significant QTL on chro-
mosome 18, suggesting that evolutionary mechanisms that can maintain genetic
variation are likely different among QTL for the same trait.
As more research groups genotype thousands of individuals from their long-term
populations on high-density SNP arrays, we will surely start to see not just the
discovery of more QTL but also tests of evolutionary mechanisms that maintain
genetic variation. It is worth keeping in mind though that most traits are polygenic,
and hence both identification of QTL and detecting fitness differences among
genotypes will be challenging. For example, the large fitness differences found
between horn genotypes in Soay sheep (Johnston et al. 2013) and armour plating
in three-spined stickleback (Barrett et al. 2008) are both near Mendelian traits
governed by a single locus of major effect (RXFP2 and EDA, respectively).
4.4 Replication of QTL
The considerable effort needed to collect large individual-based samples needed for
linkage mapping or association studies means that most detected QTL have not been
replicated and hence cannot be considered causal. The preferable association
mapping design would be to have a discovery sample and a validation/replication
sample, but for practical reasons this is difficult in avian studies. First of all most
researchers only work on a single population of a given species, and second, sample
sizes are often too small to allow subdivision into a discovery and replication
population.
Nevertheless, several studies have now attempted to replicate association signals
in wild bird populations. Korsten et al. (2010) used a candidate gene approach to
examine association between personality and a dopamine receptor gene (DRD4) in
four different great tit populations, of which one was the population where an initial
association was found. While the initial association was replicated in the discovery
population, there was no signal in two of the other populations and only a weak
association in the third population. One potential reason for this could be because of
differences in linkage disequilibrium around the associated SNP in the different
populations, but followed-up work has ruled out this explanation (Mueller et al.
2013). At the moment genetic heterogeneity among populations therefore
(i.e. different genetic variants involved) seems most likely as an explanation for
the role of DRD4 in individual variation in personality.
A very impressive replication study, also in great tits, was done by Santure and
colleagues (Santure et al. 2015). Using individuals genotyped on a 10 K custom SNP
array from a population in the Netherlands and in the UK, they carried out quantita-
tive genetic analyses, chromosome partitioning, linkage analyses and association
analyses for both population samples and eight different morphological traits. The
282 A. Husby et al.
quantitative genetic analyses show that, with the exception of fledgling mass, there
are no significant population differences in additive genetic variance. Surprisingly
however, regions of the genome associated with traits in one population were not
replicated in the other. Given the very weak genetic differentiation between these
two populations (FST ¼ 0.01), this is unexpected, and the authors suggest that the
most likely reason for this lack of replication might be low power to detect causal
variants in either population (i.e. that the detected regions are false positives) or that
different loci contribute to trait variation.
Finally, Knief et al. (2017) attempted to replicate results from earlier linkage
analyses of a captive population of zebra finches. They genotyped additional
individuals in the same captive population, from other captive populations as well
as a wild population of zebra finch for around 700 SNPs in and around linkage
regions previously identified for different morphological traits (Schielzeth et al.
2011a, b; Knief et al. 2012). While associations within the QTL regions were highly
repeatable using an independent set of individuals from the discovery population,
and also partly replicable in other captive populations, they were weak or
non-existent in the wild population. The most likely explanation seems to be, like
above, that the causal variants have not been detected, and thus the association signal
is rather in LD with a causal variant.
As these studies demonstrate, replication on QTL is nontrivial even in relatively
large-scale studies. The low LD in most bird populations poses serious challenges
not just for initial gene discovery but also for follow-up studies that aim to replicate
associations. Unfortunately, functional work in birds is still difficult to do, but
verifying that the QTL have an effect on the trait using knock-down or knock-out
techniques will be important for the field to move forward elucidating the functional
role of QTL.
5 Future Prospects in Avian Genomics
5.1 Development of Novel Statistical Genetic Methods
The statistical genetic machinery we currently use in avian genomics studies has
been developed with human studies in mind and as a consequence might not be
readily applicable to avian genomic studies. One example of this is association
studies which use linear mixed effects models with the kinship matrix as a random
effect to control for population stratification (Aulchenko et al. 2007) and a single
observation per individual. Association studies in natural populations also use linear
mixed effects models, but they generally include many more random effects to
control for factors such as yearly variation, spatial variation or the fact that we can
have repeated measures from the same individuals (Postma and Charmantier 2007).
Surprisingly, this is not possible in the association mapping software available today.
An exception to this is RepeatABEL which was developed specifically to handle the
longitudinal data structure common to many avian studies (Rönnegård et al. 2016).
This R package (built on the popular GenABEL R package used in human
association studies; Aulchenko et al. 2007) allows for two random effects (kinship
matrix and repeated measures matrix), which better reflect the data structure of
repeatedly sampled population data and can also result in higher statistical power
to detect genetic variants associated with the trait. Hopefully, in the future, this or
similar software can be modified to also allow other sources of variances to be
modelled explicitly.
Another example of a method developed in human genetics and then applied to
data on natural populations, including many avian study systems, is chromosome
partitioning (Yang et al. 2011). This method is explained above, but the principal
idea is to regress individual chromosome level heritability on chromosome size to
test for a positive association, indicative of a polygenic trait. While this has been
applied many times, there has been little evaluation of this method as used on natural
populations. For example, bird karyotypes are unique in that they have very many
small micro-chromosomes (10 Mb), and this makes estimation of the
chromosome-specific relatedness matrices challenging because of few markers on
these chromosomes (Silva et al. 2017). In addition, sample size of data from natural
populations is often orders of magnitude lower than in human studies, and it has not
been verified to what extent this influence our ability to draw inferences about a
polygenic basis of traits using this approach.
We are, of course, still in the early stages of applying large-scale genomic data in
avian population studies, but it is clear that we need to take care not to rush applying
methods from the human genetics field onto our own data without a careful consid-
eration as to the requirements and assumptions of those models. Future
modifications or development of new software that can better model the complex
data that arise from natural populations are likely to play a key part in the develop-
ment and progress of avian genomics.
5.2 Epigenetics
There is great interest in epigenetic mechanisms (structural adaptations of chromo-

somal regions so as to register, signal or perpetuate altered activity states; Bird 2007)
in evolutionary biological studies in general as well as within avian studies specifi-
cally at the moment (Derks et al. 2016; Laine et al. 2016). Part of the excitement has
to do with this being a new layer of complexity to gene regulation that we so far
know very little about and that now can be relatively easily targeted using high-
throughput sequencing technology. In particular, both alterations to chromatin
structure and DNA methylation, the two main epigenetic mechanisms, can be
screened using ChIP sequencing and bisulfite sequencing (whole genome or reduced
representation approach), respectively. So far just a few studies have examined DNA
methylation levels in natural bird populations, but this is changing rapidly. One early
example is a study on house sparrows in Kenya that used methylation-sensitive
AFLPs to score DNA methylation and sequence variation. Liebl et al. (2013) found
that variation in DNA methylation levels was higher than genetic diversity and
suggested that variation in DNA methylation could be important adaptive variation
284 A. Husby et al.
as house sparrows in Kenya have been introduced and therefore have low levels of
genetic diversity but yet have managed to spread over much of the country (Liebl
et al. 2013).
We know very little about potential adaptive epigenetic effects in wild bird
populations, and the epigenetic resources are limited for most species. However,
the great tit has had its full methylome sequenced recently (Laine et al. 2016). Laine
and colleagues found that CpG methylation patterns are very similar to that seen in
mammals (i.e. reduced methylation within CpG sites and around transcription start
sites) and there was also increased CpG methylation in regions inferred to be under
selection from sequence data (genes related to neuron development and learning).
This demonstrates a potential role for epigenetic regulation in adaptive evolution
(see also Verhulst et al. 2016).
Epigenetic studies on birds will no doubt increase in the near future. However,
like for association studies, a significant challenge will be to demonstrate causality.
This is particularly so for methylation analyses because it is almost certain to be
tissue specific as well as time dependent. To our knowledge no study has yet
examined temporal changes in DNA methylation within the same individuals, but
such changes would be needed if methylation should regulate seasonal variation in
expression patterns. Some insights into tissue specificity of DNA methylation
however have been done in the great tit for the DRD4 gene where methylation in
the brain and blood showed a very high correlation at CpG sites (rp ¼ 0.97, Verhulst
et al. 2016).
5.3 Detecting Signs of Polygenic Adaptation
It is becoming increasingly clear that most (avian) traits are polygenic (i.e. Robinson
et al. 2013; Santure et al. 2013, 2015; Kardos et al. 2016; Silva et al. 2017), and
hence adaptation is likely to take place through minor changes in allele frequencies
at many loci (Berg and Coop 2014), so-called polygenic adaptation, rather than rapid
allele frequency changes at a single locus or very few loci. Detecting polygenic
adaptation is challenging because statistical methods to detect selection in sequence
data have mainly focussed on identifying hard or soft sweeps where the signal of
allele frequency change is stronger than in polygenic adaption. In contrast, in test of
polygenic adaptation, loci might not go to fixation at all or will take a very long time
to do so. Hence, the signal of polygenic adaptation comes from examining the
covariance in allele frequency change among loci of similar effect (Berg and Coop
2014). Statistical methods to test for polygenic adaptation have been developed and
applied on humans (Berg and Coop 2014), but to our knowledge not yet on data from
natural bird populations. One reason for this is no doubt because this method relies
on having effect size estimates of individual loci identified in GWAS to compute a
genetic value that is then tested for local adaptation among replicate populations
(Berg and Coop 2014). However, most GWAS on natural populations are under-
powered to detect genome-wide significant effects and hence will only capture a
(very) small proportion of the genetic variance, something that has a negative effect
on the statistical power to detect polygenic adaptation (Berg and Coop 2014). Other
approaches to detect covarying loci, such as random forest algorithms (Laporte et al.
2016), may be more promising.
5.4 Microevolutionary Trends and Responses to Selection
As mentioned in the introduction, one of the general goals of evolutionary biology is

to demonstrate evolution in action, specifically in ecological time scales. This is
because only studies done at ecological time scales can reveal details about the
relative importance and interaction between sources of selection, random genetic
drift and processes such as genomic conflicts in driving evolutionary changes.
Genomic techniques not only allow better possibilities to investigate these questions
on a more fine scale but also facilitate dissections of possible links between evolution
in action (i.e. microevolutionary changes) and large-scale macroevolutionary
patterns. For example, it is possible to investigate whether genes underlying ecolog-
ical adaptations also are responsible for the build-up of genetic incompatibilities
between diverging populations and to follow the long-term fate of key complexes of
genes (e.g. with established adaptive functions) in large phylogenetic contexts.
A long-standing interest for avian biologists working on long-term individual-
based monitoring projects has been to demonstrate that particular traits are
(a) heritable, (b) under selection and (c) have undergone genetic changes, because
this would demonstrate microevolution. Demonstrating a response to selection in
wild populations is not straightforward, and this is especially true for studies aiming
to investigate responses to ongoing climate change where all three classical
requirements for detecting microevolution have rarely been met (Gienapp et al.
2008). Of course, genetic changes can also be due to drift, such that when examining
genetic trends over time, drift should be modelled explicitly. For example, a study by
Evans and Gustafsson (2017) found that collared flycatcher forehead patch size has
evolved to be smaller in response to increased temperatures on Gotland, most likely
because male flycatchers with small patches survive better over winter in warmer
years. This evolutionary change was larger than would be expected just due to drift
(Evans and Gustafsson 2017), but the underlying genomic changes remain
unknown.
Studying the genomic basis to variation in forehead patch size is complicated by
the highly polygenic nature of this trait (Kardos et al. 2016, Fig. 4), although this is
expected for a condition-dependent sexually selected trait (Rowe and Houle 1996).
By contrast, beak morphology in Darwin’s finches seems to be controlled by genes
with somewhat larger effect sizes (Fig. 4). Let us return to the example of medium
ground finches (Geospiza fortis) living on the island of Daphne Major that experi-
enced two serious periods of drought outlined in the introduction. A recent study
based on blood samples from individuals collected before and after a drought shows
that a specific gene associated with beak morphology, HMGA2, went through large
changes in allele frequency following the drought episode (Lamichhaney et al.
2016b). Individuals who survived drought tended to carry a different allele than
286 A. Husby et al.
a)
Fig. 4 Whole genome scans to identify loci controlling forehead patch size variation in (a) collared
flycatchers (from Kardos et al. 2016) and (b) beak size variation between groups of Darwin’s
finches with blunt (G. magnirostris and G. conirostris) versus pointed beaks (G. conirostris and
G. difficilis), from Lamichhaney et al. (2015)
those who succumbed. This study meets the first three conditions needed to demon-
strate evolution due to climatic conditions; beak size is a heritable trait (Keller et al.
2001), under selection, with the changes in the phenotype associated with underlying
genetic changes (Lamichhaney et al. 2016b). A model demonstrating that the
changes in HMGA2 allele frequency were not due to drift would further strengthen
the proof for adaptive evolutionary change at the gene level.
We are not aware of any individual-based studies, aside from the aforementioned
example, that have passed all of these conditions to demonstrate evolution in
response to ecological factors, but given the increasing resolution of genomic data
and larger-scale genotyping and sequencing efforts, it seems likely that we will see
more demonstrations of adaptive microevolutionary change in the near future.
6 Summary
The early pioneering work of Kluijver, Lack, Nice and von Haartman laid the
foundations for long-term studies on birds and has provided an immensely valuable
resource for future generations of avian biologists. The longitudinal data on parents
and their offspring over many generations have made quantitative genetic studies,
and now more recently genomic studies to map QTL and examine selection at the
QTL, feasible. During the last 20 years, we have seen a tremendous transformation
in the genetic resources that are available to the avian biologist, something that has
led to revision of many established ideas about avian mating systems, phylogenetic
relationships and demographic histories being overturned. Avian population studies
have been important contributors to this development and provided some of the most
well-known examples of natural selection and evolution in the wild, like the case
with Darwin’s finches (Grant and Grant 1995; Lamichhaney et al. 2015).
A long history of quantitative genetic studies on birds have clearly demonstrated
a genetic basis to nearly all avian traits and that natural and sexual selection is
ubiquitous (reviewed in Merilä and Sheldon 2001; Postma and Charmantier 2007).
More recently, genomic approaches have confirmed the genetic basis of avian traits
using marker-derived relatedness matrices in a mixed model framework
(e.g. Santure et al. 2013, 2015; Husby et al. 2015; Kardos et al. 2016; Silva et al.
2017). Moreover, some of the major insights provided by recent genomic work on
birds are that most traits are controlled by many genes of individually small effect
(Santure et al. 2015; Husby et al. 2015; Silva et al. 2017; Kardos et al. 2016). Despite
some early indications to the contrary from linkage studies (e.g. Tarka et al. 2010),
the polygenic nature of most traits will come as no surprise to evolutionary
geneticists familiar with gene mapping results in lab organisms, such as Drosophila
(Mackay 2001). This finding has important implications for future gene mapping
studies in birds because small effect sizes, combined with rapid decay in LD
(Poelstra et al. 2013; Kardos et al. 2016), mean that thousands of individuals and
hundred of thousands of markers are needed to identify QTL. While such studies
seemed fanciful just a few years back, they are already in the progress, and this opens
exciting new possibilities to address how populations adapt to changing environ-
mental conditions in a more realistic manner, i.e. through polygenic adaptation.
Acknowledgments We are grateful to Robert Kraus for the invitation to contribute to this book
and for his encouragement during the process. We would also like to thank Robert Kraus and three
anonymous reviewers for helpful comments and suggestions that improved the manuscript.
A.H. acknowledges support from the Norwegian Research Council grants 239974 and 223257;
A.Q. acknowledges the Swedish Research Council and the Knut and Alice Wallenberg Foundation;
and SEM acknowledges support from the Swedish Research Council (2017-00499).
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The Contribution of Genomics to Bird
Conservation
Loren Cassin-Sackett, Andreanna J. Welch, Madhvi X. Venkatraman,

Taylor E. Callicrate, and Robert C. Fleischer
Abstract
The world’s birds are in trouble, and scientific research, including genetic and
genomic methods, can play an important role in understanding and mitigating
these problems. In this review, we summarize several ways that the concepts and
methods of genomics can help with bird conservation and how the dramatically
increasing power and decreasing costs of these methods may allow an even
greater role in the future. We assess six primary, not exhaustive, and not mutually
exclusive research areas, including avian forensics, captive management, infec-
tious disease and vector interactions, metagenomic and microbiome applications,
systematics and the definition of conservation units, and the genomics of adapta-
tion. We conclude that the uses of genomics to identify, understand, and in some
cases reduce anthropogenic impacts on bird populations are well underway. And
the future holds great promise that developments in our understanding of avian
genomes and tools to modify them will play an increasingly important role in
future attempts to alleviate these impacts.
L. Cassin-Sackett
Department of Biology, University of Louisiana, Lafayette, LA, USA
A. J. Welch
Department of Biosciences, Durham University, Durham, UK
e-mail: a.j.welch@durham.ak.uk
M. X. Venkatraman · R. C. Fleischer (*)
Center for Conservation Genomics, Smithsonian Conservation Biology Institute,
National Zoological Park, Washington, DC, USA
e-mail: venkatramanm@si.edu; fleischerr@si.edu
T. E. Callicrate
Species Conservation Toolkit Initiative, Chicago Zoological Society, Brookfield, IL, USA
e-mail: taylor@scti.tools
This is a U.S. government work and not under copyright protection in the U.S.; 295
foreign copyright protection may apply 2019
https://doi.org/10.1007/978-3-030-16477-5_10
296 L. Cassin-Sackett et al.
Keywords
Conservation · Genomics · Birds · Wildlife forensics · Infectious disease ·
Population management · Evolutionarily significant units · Metabarcoding ·
Adaptation · Climate change
1 Introduction
Like the proverbial canary in the coal mine, birds serve as important model systems
and umbrella taxa for conservation of ecosystems. Since 1500 as many as 183 species
of birds have gone extinct, and currently about 23% of the nearly 11,000 species of
birds are considered endangered, vulnerable, or threatened, and their decline
continues at an alarming pace (Birdlife International 2018). While there are many
scientific fields involved in understanding and mitigating these threats to birds,
genomics and advanced genetic methods have much to contribute to the conser-
vation of birds. Here we summarize several of the recent advances in genomic
applications in conservation and provide examples of these approaches from the
literature. We also provide a prospective view of novel future applications and
approaches, given the rapidly increasing power arising from high-throughput
sequencing, synthetic biology technologies, and increased bioinformatics power.
Genomics in the Broad Sense Here we consider genomics in the broad sense, that
is, not just sequencing and characterizing full genomes but using genomic and
advanced (e.g., next-generation) sequencing methods that can provide useful data
for a wide range of applications in conservation biology. For example,
transcriptomics methods can be used to identify avian genes expressed in response
to environmental stressors such as disease, climate change, or pollution (Jax et al.
2018). In addition, use of metagenomic and microbiome methods can elucidate how
avian symbionts influence survival, life history, and population dynamics and
facilitate characterization of diets and dietary shifts (Trevelline et al. 2018). Genomic
methods can also be used in such classic applications of conservation genetics as
assays of genetic variation in small and captive populations (Harrisson et al. 2014),
determination of species and evolutionary significant units (Robertson et al. 2014;
Oyler-McCance et al. 2015; Peters et al. 2016; Ottenburghs 2019), inbreeding levels
(Li et al. 2014), movements (Kawakami et al. 2014), population sizes
(Nadachowska-Brzyska et al. 2015), and the presence of disease (Sun et al. 2015).
And layered upon these types of issues in conservation genetics, environmental and
noninvasive DNA approaches have proven useful for research on endangered and
difficult-to-study birds. Below we survey several of the areas of avian conservation
biology for which we believe genomic methods can and will play an important role.
The Contribution of Genomics to Bird Conservation 297
Genomics Methods These can involve assessment of whole genomes or just

sizable numbers of nuclear and plastid loci sampled from across the genome
(Lerner and Fleischer 2010; Toews et al. 2015). Early applications of genomics
have primarily involved reduced representation sequencing, in which short
sequences (e.g., SNPs) are sampled from throughout the genome to represent
genomic or evolutionary processes occurring across the genome (Huang et al.
2013; Kraus et al. 2011, 2012). Genomic methods can include whole-genome
“shotgun” sequencing, transcriptome characterization (usually via RNA-seq
methods; Wang et al. 2009), and reduced representation methods such as restriction
site-associated DNA (RADs, Baird et al. 2008), ultra-conserved elements (UCEs,
McCormack et al. 2012), exomes, and other sorts of capture approaches. They also
often include metagenomic (Riesenfeld et al. 2004) and metabarcoding (amplicon)
methods (Taberlet et al. 2012) such as those used in some eDNA (environmental
DNA or DNA sampled from an environmental substrate rather than a single organ-
ism) and microbiome assessments.
2 Applications of Genomics to Avian Conservation
2.1 Forensics
One of the seemingly simplest uses of genetic and genomic data is in avian forensic
analysis. Forensics involves using tools to make identifications that are relevant to
the solution of a public issue (such as identification of a bird involved in an airplane
bird strike) or a crime (such as poaching or illegal trade). The classical approaches
involve species identifications using comparisons of feather morphology or anat-
omy, but more recently simple DNA barcoding has become standard (Dove et al.
2008, 2009; Johnson 2011). In addition, microsatellite or simple tandem repeat
(STR) markers, which usually require higher-quality DNA samples, have been the
standard for identifying individuals within a species (e.g., Dawnay et al. 2009;
Bielikova et al. 2010; Jan and Fumagalli 2016; Coetzer et al. 2016) and population
of origin (Weissensteiner and Suh 2019). Usually these methods are used in identifi-
cation cases where individual birds have been the focus of poaching or theft. More
sophisticated DNA analyses have now increased resolution of population- and
individual-level identifications (Iyenegar 2014; Arenas et al. 2017), and next-
generation sequencing and genomic methods hold great promise for making more
accurate and inexpensive identifications.
In addition, the use of genomic methods in forensics includes identification of
species and of individuals from remnant and often degraded samples, which are not
usually considered “genome quality” and thus are amenable to only some next-
generation sequencing methods (Yang et al. 2014). In the past, simple PCR of
mtDNA and microsatellites have been the primary approaches in avian forensics
(e.g., Dove et al. 2008; Coetzer et al. 2017), but next-generation methods may have a
greater ability to contribute to forensic analyses. This is because very small
fragments of DNA are all that are needed via shotgun or hybridization capture
methods to characterize a SNP and many dozens to thousands of SNPs can be

assayed in a single analysis, assuming adequate coverage to call each SNP.
Recently, next-generation sequencing methods have been used to identify larger
numbers of avian microsatellites than can be obtained from standard methods, for
example, for use in parentage analyses of Bornean birds (Kaiser et al. 2015) and
forensics of potentially criminally obtained endangered New World parrots (Jan and
Fumagalli 2016). In this process, many thousands or even millions of random
sequences are obtained from shotgun sequencing of one or more individuals on a
high-throughput sequencer. Sequence platforms must be capable of producing
sequences of sufficient length to include sequence repeats capable of generating
variation (>10 dinucleotide to pentanucleotide repeats), plus adequate priming sites
(usually >150 bp in total length). From this approach, many hundreds or thousands
of loci can be identified that can then be developed and optimized for further use.
Another interesting application has been to use PCR with mammalian markers on
avian specimens to assess the identity of predators of birds such as sage grouse from
mammalian saliva or other remnants left behind (Hopken et al. 2016). And micro-
biomes may also be useful for identifying population of origin or perhaps may even
have signatures useful to identify individuals (Arenas et al. 2017).
Perhaps the biggest problem with forensics as applied to birds is more in terms of
policy, in that for criminal investigations specific protocols and constrained chains of
custody are required, and development, troubleshooting, and application of
novel methods tend to evolve very slowly in these communities because of
legal constraints.
2.2 Captive Population Management
Genomic techniques are ideally suited to advance the goals of captive population
management. Captive population managers seek to preserve the range of genetic
diversity of a species in order to preserve adaptive variations and adaptive potential.
Managers also try to avoid adaptation to captivity or other forms of selection which
might influence the allele frequencies of the population. Genomic data can be
applied to increase the accuracy and precision of current methods for maintaining
overall genetic diversity and has the potential to enable new methods/practices to
avoid adaptation to captivity and preserve adaptive variants.
2.2.1 Maintaining Genetic Diversity

One of the main goals of captive population management in zoos and other
institutions is to enable conservation in situ, by providing animals for reintroduction
or for release into declining wild populations (Ralls and Ballou 2013). Managers
may also maintain viable captive populations for display/public education or
research (Ralls and Ballou 2013). Typically, captive management includes manag-
ing both the demographic and genetic profile of the population as a whole to preserve
diversity and ensure that the population will grow or retain its size. The goal of
maintaining a stable or growing captive population is generally met through the use
of pedigree information and planned pairings (Ballou and Lacy 1995). Each wild-
caught founding member of the captive population is assumed to be unrelated. The

relationships (or kinship) between each animal and all the others in the population
are determined, and a breeding plan is devised to minimize the average mean kinship
within the population. This has been shown to be the optimal strategy to preserve the
original genetic diversity of the founders (Ballou and Lacy 1995). Care is also taken
to manage the demographic profile of the population to ensure that there will be
enough breeders of both sexes in the future to maintain the population.
However, most captive populations do not meet the assumption of unrelated
founders. For example, multiple individuals from the same clutch may inadvertently
be collected, and multiple members of the same natural population are often selected
as founders without knowing their relatedness (Bergner et al. 2014). Genetic data
can be used to identify related individuals in some cases, but this is more difficult in
small populations that have been subject to multiple generations of inbreeding.
Historically, genetic markers could be limited in their ability to provide sufficient
resolution to resolve relationships (e.g., allozymes), or large numbers of markers
were required to be developed de novo for each species due to low genetic variability
(microsatellites). Genome-wide data allows calculation of kinships between both
larger numbers of and more related individuals and can be used to resolve or revise
pedigrees (Bergner et al. 2014). Genome-wide sequencing in California condors, for
example, has resulted in revised breeding strategies by revealing unknown related-
ness structures between founders (Romanov et al. 2009; Ryder et al. 2016). Simi-
larly, the ability to easily generate large number of microsatellite markers using next-
generation sequencing methods (as described above) will also allow greater resolu-
tion of pedigrees and relatedness within captive populations.
For captive populations where collection of new founders is still possible, the
ability to compare genomic diversity in captive and wild populations is invaluable
(e.g., Rascha et al. 2016). These data would help conservation managers preserve as
much wild genetic diversity as possible (Fig. 1, top right) by allowing them to target
underrepresented genetic profiles for collection or translocation (Mounce et al.
2015). Even if no captive genomic data exist, studies of wild populations that
identify genomic diversity or population structure can greatly benefit collection
efforts (Mounce et al. 2015). Additionally, captive managers benefit from studies
of wild population genomics as they learn about potential subspecies or other groups
that should possibly be managed separately.
Birds that live and breed in groups, like flamingoes, also present a problem for
traditional methods, as individual parents cannot be tracked to create accurate pedi-
grees. Parentage assignments of individuals can be done with traditional genetics
techniques, but determining the relatedness between individuals in a given group or
between groups has been more challenging (Smith 2010). Large numbers of
genome-wide markers could be used to improve group management by allowing
comparison of individuals or subgroups without needing to reconstruct a compli-
cated pedigree using traditional genetic methods.
Fig. 1 Conceptual figure depicting a few ways in which genomics contributes to avian conserva-
tion. Clockwise from top right: (a) Individuals (rows) from a given species can be evaluated for
similarity to a target sample at loci across the genome (more blue ¼ higher similarity, more
orange ¼ low similarity, gray ¼ no data) in forensic analysis [Section 1]. (b) Kinship matrix for
individuals within a species, where green shows low relatedness (i.e., potential breeding pairs) and
purple represents high relatedness (i.e., relatives or other individuals to avoid breeding in captive
propagation) [Section 2]. (c) Differential expression of immune function genes can be analyzed in
resistant and susceptible individuals to determine which genes control infection (greater than
1 ¼ over-expression, less than 1 ¼ under-expression) [Section 3]. (d) The distribution of bacterial
OTUs in decaying chicken over time; each polygon represents an OTU, where early time points
(0–2 days) indicate shortly after death and later time points are indicative of carrion [Section 4]. (e)
Three species of variably threatened Hawai’ian honeycreepers, the Hawai’i ‘amakihi
(Chlorodrepanis virens, green), ‘i’iwi (Drepanis coccinea, curved bill), and ‘apapane (Himatione
sanguinea, red with black bill). (f) Clustering of individuals (rows) across genomic loci (columns)
can inform scientists of the loci involved in species divergence and reveal cryptic species [-
Section 5]. (g) Outlier analyses can reveal loci showing significantly higher divergence (blue
circles) than the rest of the genome, indicating potential adaptation to local climatic regimes or
other conditions [Section 6]
2.2.2 Adaptive Variation

As more is understood about adaptive variation, genomic data could potentially be
used to help preserve adaptive diversity beyond the mean-kinship method. Selection
based on traits in conservation breeding has potential downfalls, such as (1) we do
not know which traits will be adaptive when the captive-bred animals are released,
(2) interactions between multiple loci are not well-understood for traits of interest,
and (3) selection for some traits could reduce other genomic variation. Genomic
studies will allow deeper investigation of some of these concerns.
With the advent of the use of genomics in evaluating adaptive variation, captive
population managers will have to develop methods for integrating that information
into management decisions. A first step in this direction, which is already being
undertaken, is understanding adaptation to captivity. This occurs when selection
pressures are unintentionally applied in the captive environment and result in genetic
shifts in the population which may then be maladaptive upon release of individuals
to the wild. Currently, adaptation to captivity has been identified in several species
(notably fish in aquaculture; Mäkinen et al. 2015), but this work remains to be done
in birds. Learning which genomic features are susceptible to selection in the
captive environment will help managers reduce those effects while maintaining
overall genetic diversity.
2.2.3 Future Directions

Using genomic tools for captive management is still an emerging practice, and many
challenges have yet to be addressed. A few avian species, such as California condors
and whooping cranes, are already being managed using data from genomic studies
(Ryder et al. 2016). These methods are becoming more accessible to the conser-
vation community, and several groups are developing consistent workflows for
marker development and genotyping. However, the following questions still need
to be addressed: (1) how many markers are needed to resolve pedigrees and answer
kinship questions? (2) If some of the pedigree is known, how can an optimal set of
individuals be chosen for genotyping? (3) In which cases is it more efficient to use
genomic data to reconstruct a pedigree vs. calculate genomic kinships? As genomic
tools are applied to the captive management of more species, answers to these
questions should emerge.
2.3 Avian Infectious Disease
Genomic tools have enhanced our ability to understand avian disease dynamics,
coevolution between avian hosts and pathogens, and the evolution of pathogen
virulence and host resistance and tolerance. These technologies have enabled
scientists to push the boundaries of knowledge in infectious disease research.
Although many of the major questions in avian disease ecology and evolution
remain the same, our ability to answer them in more nuanced ways has improved.
Biologists seek to explain patterns of host susceptibility, pathogen virulence, parasite
specificity, coevolution, and parasite transmission dynamics across complex
landscapes and host communities. Prior to the genomics era, studies of avian disease
were focused on single “candidate” host genes thought to play a role in resisting
infection (e.g., Jarvi et al. 2004), single parasite genes posited to confer virulence,
reliance on parasite morphology or single genes to describe taxonomy
(Križanauskienė et al. 2006), and phenotypic disease pathology in the host.
These approaches have led to significant advances in our understanding of host
and pathogen ecology and evolution; however, each poses barriers to a complete
understanding of host-pathogen dynamics. Prior to the advent of genomic
technologies, avian disease biology experienced several primary limitations: (1) vari-
ation in virulence among parasite strains was cryptic; (2) disease pathology results
from the interaction between parasite virulence and host susceptibility and thus
cannot be used to infer exclusively host or pathogen processes; (3) alleles conferring
both pathogen virulence and host resistance/tolerance were usually unknown; (4) a
lack of variation in commonly sequenced genes hindered comparative study; and
(5) individual species contributions to disease transmission were difficult to quan-
tify. With genomic tools, avian biologists are on the cusp of making large
breakthroughs in these and other areas. Here, we outline a few outstanding questions
in avian disease biology and highlight some key studies that have capitalized on
genomic approaches.
2.3.1 Host Susceptibility and Pathogen Virulence
Host Susceptibility
Single-gene studies, primarily focused on major histocompatibility complex (MHC,
Weissensteiner and Suh 2019) genes and other genes known a priori to be part of
normal immune system processes, have resulted in slow discovery of novel disease-
related genes. In turn, this has biased our understanding of disease response such that
non-MHC genes have largely been ignored. Negative results have likely gone
unpublished, leading to an artificial inflation of the generality of importance of
very few genes. Until recently, scientists had little ability to discover novel genes
invoked in the infection process, as this work was economically inefficient. The
single-gene approach is still of great use in systems where particular genes are
known to be involved in the infection process (Chapman et al. 2016). However,
there is growing evidence that a large number of host genes may be under selection
from pathogens (Cheng et al. 2015; Wang et al. 2016). Of these, only some are
conserved across populations (Connell et al. 2012; Videvall et al. 2015; Cassin-
Sackett et al. 2018); furthermore, genetic variation at these loci can be shaped by
both selection (Raven et al. 2017) and drift (Gonzalez-Quevedo et al. 2015). Thus, a
priori hypotheses of relevant genes will resolve only a limited picture of the
evolutionary response to infection. Moreover, with the global increase in wildlife
diseases, many of which are introduced, pathogen invasion of novel hosts and host
response to novel pathogens may invoke genes that co-opt cellular machinery for
other purposes. A classical example of such a solution to disease is the sickling of red
blood cells that prevents replication of Plasmodium falciparum in humans. The gene
causing this abnormal hemoglobin likely would have been overlooked with a target
gene approach. Recent studies have assessed the potential for using reciprocal
translocations as a means to reintroduce genetic diversity at disease resistance
genes into wild populations (Grueber et al. 2017); this type of effort could be
aided by a more complete understanding of which disease-related genes play a
role in each system (Fig. 1, bottom right).
Pathogen Virulence
Traditionally, pathogen virulence in bacteria has been assumed to arise as a result of
virulence genes contained on plasmids. This is often the case, but genomic tools
have allowed us to understand that virulence is often conferred by multiple genes
acting in tandem, not only on plasmids but also localized on “pathogenicity islands”
(Pilo et al. 2005) or elsewhere in the genome. Often, virulence genes control some
other cellular function such as metabolism and lead to virulence as a by-product (Pilo
et al. 2005; Szczepanek et al. 2010; Tulman et al. 2012). Comparative genomics has
been used in studies of commensal and pathogenic bacterial strains to identify
unique genes as putative causes of virulence. For example, genomic tools have
enabled scientists to subtract the genomes of nonpathogenic strains of Escherichia
coli from the genomes of avian pathogenic strains, leading to the confirmation of
existing virulence plasmids as well as identifying novel virulence factors
(Schouler et al. 2004). The subtractive genomic approach can also be used to
subtract pathogen genomes from host genomes in a single host tissue.
The identification of virulence factors in non-bacterial pathogens is even more
complex. Mechanisms of host red blood cell invasion, for instance, are not
conserved across Plasmodium species: diversification of particular host invasion
mechanisms may have occurred only in mammal-infecting Plasmodium lineages and
thus lend limited insight into invasion of avian blood cells by P. gallinaceum
(Lauron et al. 2015) or P. relictum. Candidate gene studies of Plasmodium infection
have typically focused on a small number of putative virulence genes such as the
merozoite surface protein 1 (msp1), as one of the first steps in host cell invasion
(Hellgren et al. 2013). However, transcriptome sequencing of P. gallinaceum
illuminated additional parasite genes necessary for avian infection and revealed
evidence of diversifying selection as a result of the host immune response
(Lauron et al. 2014). As in host resistance/tolerance, parasite virulence may likewise
be conferred by multiple genes, especially in systems where a coevolutionary arms
race is occurring or where multiple host species exist that differ in susceptibility. For
instance, attenuation of a Mexican West Nile virus lineage was conferred by both
pre-membrane and envelope proteins, while either gene alone did not confer attenu-
ation (Langevin et al. 2011).
In addition to characterization of genes involved in virulence, genomics techno-
logies facilitate pathogen discovery when the cause of wildlife disease outbreaks is
unknown. Some diseases have unknown etiologic agents: a panviral microarray
led to discovery of an avian bornavirus as the previously uncharacterized causative
agent of proventricular dilatation disease and mortality events in both the
United States and Israel (Kistler et al. 2008). Genomics tools allow similar promise
for pathogen discovery (Borner and Burmester 2017).
2.3.2 Host Specificity and Coevolution

Accurate description of host specificity relies on correct delineation of host and
parasite species. Historically, parasite taxonomy was studied using parasite morpho-
logy, parasite life cycle, host species, or host pathology. More recently, mtDNA
haplotypes have been used to describe taxonomic relationships. Both of these
approaches have yielded great insights into pathogen specificity and coevolutionary
dynamics, yet they have often led to spurious conclusions resulting from incomplete
information. For instance, malaria parasites (including Plasmodium, Leucocytozoon,
and Haemoproteus species) were traditionally classified based on their morpho-
logical characteristics and host species, leading to inferences of high host specificity.
However, Beadell et al. (2006) and Martinsen et al. (2006) demonstrated that
morphospecies are not always linked to particular genotypes, suggesting potentially
widespread cryptic diversity or morphological convergence among haemosporidian
species. The use of mitochondrial genes modified our understanding of parasite
taxonomy and demonstrated wide variation in host specificity (Beadell et al. 2006,
2009) in this group, as dozens of cryptic lineages were discovered (e.g., Palinauskas
et al. 2015; Nilsson et al. 2016). As more host species have been sampled and similar
lineages detected across taxa, we have come to understand that many blood parasites
display high host generality (Iezhova et al., 2011; Nilsson et al. 2016). Nonetheless,
haemosporidians display greater levels of host specificity than other pathogens
such as West Nile virus, in which lineages correspond to geography rather than
host species (May et al. 2011).
In addition to their range of host specificity, pathogens also exhibit varying
degrees of vector specificity that was largely undetectable prior to the genetics era.
Martinsen et al. (2008) were among the first to demonstrate parasite-vector specific-
ity: they found that major clades of malaria parasites were associated with shifts into
new vectors, which then permitted weaker associations with major vertebrate host
clades. While some parasites are capable of infecting multiple vector families, others
may be specific to particular genotypes. For instance, the existing population of
introduced Culex quinquefasciatus in Hawaii (a mix of North American and mostly
Austral-Pacific lineages; Fonseca et al. 2006) successfully transmits the local strain
of Plasmodium relictum, GRW4, to local avian species, whereas the North American
lineage of Cx. quinquefasciatus was experimentally shown to be refractory to GRW4
(Fonseca, Fleischer, unpublished). The identification of these two strains was
facilitated by genetic studies, and genome-wide scans of the distinct vector lineages
would lend insight into the functional differences between genotypes. Wilkinson
et al. (2014) used a combined 16S and targeted gene approach to reveal that
pathogenic bacteria display vector specificity in seabird ticks [although this may
be the case only for certain tick symbionts, as Duron et al. (2016) found minimal
evidence of host specificity and a high degree of horizontal transfer]; this combined
approach adds power to avian disease systems for which genomic resources have yet
to be developed. Indeed, next-generation sequencing technologies can be used to
develop Sanger sequencing assays (Hellgren et al. 2013).
In complement to pathogen vector specificity, host preference of vectors has been
illuminated by bloodmeal analysis (Kilpatrick et al. 2006; Savage et al. 2007),
reinforcing the idea that hosts are not chosen from a community randomly (Paull
et al. 2012). Genomics offers the potential for addressing specificity at various levels
using high-throughput analysis of bloodmeals in both hosts and vectors.
Cryptic variation in avian host species (Stervander et al. 2016) could lead to
erroneous conclusions such as overestimating host specificity (e.g., one parasite
lineage in two cryptic host species) or the diversity of parasites infecting host species
(e.g., two parasite lineage in two cryptic host species). In addition, host-switching
events would not be detected when parasite lineages infected a new, cryptic host
taxon. In the case of multiple parasite lineages infecting cryptic host species, an
incomplete understanding of host taxonomy could beget the false inference of stable
parasite species coexistence when in fact two parasite lineages competitively
exclude each other in different host species (specialization on cryptic host taxa).
Using genomic data to inform avian and parasite taxonomy solves many of these
problems; in fact, whole-genome information resolves many taxonomic uncer-
tainties (Jarvis 2014; Hug et al. 2016; Ottenburghs 2019) and should enable a better
understanding of host specificity, host and vector preference, and parasite diversity.
With the ever-increasing amount of genomic data available on parasite lineages,
more variation among strains will be detected, posing the problem of how to classify
parasite species. Historically, species were delineated based on the existence of one
or a few nucleotide differences in mitochondrial genes, but it is now trivial to
discover dozens of novel variants within a single parasite species, lending the
problem of delineation based on molecular information. Thus, biologists studying
parasites will have to find common ground on which to define and delineate taxa
(Outlaw and Ricklefs 2014) to facilitate comparative studies of host specificity and
coevolution. It is now possible to identify specific parasite genes that are differ-
entially expressed in different host species (Videvall et al. 2017), allowing for a
functional understanding of host specificity that may contribute to our understanding
of parasite taxonomy.
2.3.3 Phylogeography, Population Genetics, and Within-Host

Evolution
After some limitations of mtDNA for resolving phylogeographic patterns became
evident (e.g., the introgression of entire mitochondrial genomes (Bellemain et al.
2008) can lead to discordance between mtDNA and species trees), scientists started
leveraging virulence genes or host-specific parasite attack genes to describe host-
parasite relationships, host-switching events, parasite phylogeny, and parasite phylo-
geography (Taubenberger et al. 2005; Hellgren et al. 2013, 2014; Harkins and Stone
2015). These single-gene studies have begun to clarify some phylogenetic relation-
ships but have obscured others (Harkins and Stone 2015).
In contrast to drawbacks with morphological approaches, single-gene studies
confront the problem of poor detection of mixed infections (Valkiūnas et al.
2006). The cost efficiency of multi-gene Sanger sequencing is declining, particularly
in systems where suitable genes for taxonomy, phylogeography, or functional
ecology and evolution have not yet been identified. However, single-gene studies
can overlook functional diversity at loci not typically used for taxonomy. These
drawbacks have the potential to be resolved with parasite genomics (Nilsson et al.
2016). Genomic tools can reveal cryptic diversity that can be leveraged for fine-scale
studies of parasite phylogeography and within-host evolution.
Introduced pathogens can be traced using genomics to identify the origin of
introduction. For instance, whole-genome sequencing of West Nile virus strains in
the western Mediterranean revealed that the present diversity stems from a single
introduction followed by local maintenance in the region (Sotelo et al. 2011). This
study also revealed a meaningful insight about pathogen evolution: a single mutation
linked to virulence (Brault et al. 2007) occurred multiple times in distinct geographic
regions during the evolutionary history of West Nile virus (Sotelo et al. 2011). The
ability to trace pathogen introduction history both to a particular host species and
geographical location enables quarantine or vector control measures to be put in
place to conserve vulnerable avian species. For example, Usutu virus strains from
various locations in Africa were subjected to whole-genome sequencing, and the
country of origin of European introduced Usutu virus was inferred due to high
similarity between a native strain from Senegal and introduced strains in Europe
(Nikolay et al. 2013).
2.3.4 Insights from Genomics

In summary, many broad evolutionary patterns have been revealed by the avail-
ability of genomic data, including several major insights in avian disease. Table 1
presents a selection of several major themes in host and parasite biology for which
our understanding has changed as a result of genomic advances.
2.3.5 Limitations
One area in avian disease research that still lags behind, as in other fields in avian
biology, is verification of gene function in non-model organisms. Experimental
infection studies are often impractical or unethical in non-model species, particularly
those of conservation concern. As a result, most of the data on genes underlying
pathogen and host phenotypes are correlational. Comparative genomics with well-
annotated genomes, in conjunction with experimental challenges of widespread
related species, will assuage this shortcoming. In addition, improvements in gene
prediction, genome annotation, and characterization of noncoding DNA will facili-
tate prediction of how genetic changes will interface with different genomic
backgrounds. These improvements can be made not only via experimental gene
knockout studies and germ cell editing to assess gene function (Park et al. 2014) but
with a better understanding of genome evolution. Synteny is higher in avian
genomes than in other vertebrate taxa (Zhang et al. 2014), enabling these types of
advances more readily in birds.
A limitation that is biological and not technical emerges from recent findings in
genomics studies that resistance is conferred by multiple, often unpredictable loci
that vary across populations (Connell et al. 2012; Videvall et al. 2015; Cassin-
Sackett et al. 2018). This suggests a continual need for species-specific studies of
host and parasite evolution. However, this shortcoming is ameliorated by genomics,
Table 1 Summary of applications of genomics to major themes in the study of avian disease
Previous
Topic understanding Current understanding Relevant contributions
Parasite Morphology- A large degree of cryptic Beadell et al. (2006),
taxonomy or diversity exists, some of Martinsen et al. (2006),
cytochrome which may have functional Palinauskas et al. (2015) and
b-based consequences Nilsson et al. (2016)
Host Most There is wide variation in the Fonseca et al. (2006),
specificity parasites are degree of host specificity; Martinsen et al. (2008),
host-specific some parasites exhibit vector Iezhova et al. (2011), May
rather than host specificity et al. (2011), Wilkinson et al.
(2014), Nilsson et al. (2016),
Duron et al. (2016) and
Videvall et al. (2017)
Mechanisms Primarily Many additional immune Cheng et al. (2015), Wang
of host MHC-based genes are involved (and some et al. (2016) and Cassin-
resistance/ genes not in the immune Sackett et al. (2018)
tolerance pathway)
Evolution of Diversifying Diversifying selection, Grueber et al. (2013), Raven
MHC selection balancing selection, neutral et al. (2017) and Gonzalez-
evolution Quevedo et al. (2015)
Pathogen Decreases Increases, decreases, or Szczepanek et al. (2010),
virulence over remains stable over Tulman et al. (2012), Murray
evolutionary evolutionary time et al. (2017a, b) and Fan et al.
time (2017)
which enables whole-genome and transcriptome data to be gathered from increas-

ingly smaller biological samples. As whole-genome data are acquired with increas-
ing efficiency and decreasing cost, this limitation is expected to decline.
Although genomic technology is changing rapidly, it remains difficult to isolate
parasite DNA from host tissue due to the lower cellular representation of parasite
DNA. Nonetheless, some promising approaches have proven successful in recent
parasite studies: Plasmodium relictum was isolated from blood smears using lasers
and subsequently subjected to whole-genome sequencing (Lutz et al. 2016). This
technology offers the potential to acquire genomic information from archived blood
smears and other valuable sources.

Future genomics work on parasites and vectors of avian disease are needed to
characterize the ecological and evolutionary linkages in vector-borne diseases.
These studies will illuminate the mechanisms underlying vector adaptation to
parasite lineages as well as parasite influence on vector behavior (e.g., how Plasmo-
dium relictum invokes a feeding preference of infected birds on uninfected
mosquitoes; Cornet et al. 2013) and therefore disease dynamics.
The unprecedented ability to obtain whole-genome sequence data rapidly and
with increasing cost efficiency paves the way for effective conservation actions to
take place in near real time. In the coming years, whole-genome sequencing or
reduced representation screening at disease resistance loci can be carried out prior to
management actions such as captive breeding or relocation, effectively increasing
the disease resistance or tolerance of vulnerable host populations.
Exciting opportunities for conservation have emerged with the discovery of
CRISPR (clustered regularly interspaced short palindromic repeats) defense systems
in bacteria and archaea and the associated advent of genome-editing technologies
(Jinek et al. 2012). With CRISPR-associated (Cas) systems, genetic information can
be permanently edited, which can be leveraged in disease systems by modifying
pathogen virulence, vector competence, or host tolerance. Indeed, disease systems
have been some of the first real-world applications of this tool (Kistler et al. 2015;
Hammond et al. 2016). Moreover, the technology allows for multiple loci to be
edited simultaneously (Jao et al. 2013), which would simplify genome editing in the
case of multilocus tolerance or virulence. Recent developments facilitate CRISPR-
Cas gene editing in non-model avian species (Cooper et al. 2017, 2018). As
pathogens are increasingly moved around the globe and naïve host populations are
exposed to novel pathogens, CRISPR could allow for emergency management of
critically endangered populations when there is not sufficient time for captive
breeding or other conservation strategies.
2.4 Avian Conservation Genomics and Heterogeneous Samples:

Metagenomics and Metabarcoding
In the traditional sense, genomics has involved the discrete analysis of one or a few
samples, usually collected from individuals with known taxonomy or other
characteristics of particular interest. Recently, however, there has been increasing
interest in the simultaneous analysis of mixed samples that represent the “commu-
nity” (Xu 2006). These heterogeneous community samples can include, for example,
feces, microbiota, or mixtures of the remains of several individuals/species. High-
throughput sequencing of these samples provides the power and sequencing depth
required to obtain genomic data from those individuals, without the necessity of
time-consuming approaches such as molecular cloning to first separate them by
species or genotype. These data can be used for a wide variety of purposes in avian
conservation, which can include (but are not limited to) biodiversity detection,
investigating bird diets (which may limit aspects of the annual cycle such as breeding
or survival during migration), detecting endangered birds as dietary items of
predators, and understanding how avian microbiota contribute toward adaptation
and health (see below).
In the strictest definition, metagenomics involves shotgun (i.e., theoretically
random) sequencing of DNA—potentially to the scale of whole genomes—directly
from a heterogeneous sample (Taberlet et al. 2012). This approach can be used to
characterize the (often microbial) community in terms of taxa present and investigate
the content and function of the genes sequenced. Similar approaches analyzing RNA
instead of DNA can investigate further questions such as how gene expression
changes in interacting communities under different conditions or states (Fierer
et al. 2012; Poretsky et al. 2009). In a related technique, often termed metabarcoding,
DNA from an environmental sample is first PCR amplified for a particular region
(e.g., 16S ribosomal RNA or COI, the cytochrome oxidase I gene), and then these
amplicons are sequenced on a high-throughput platform, usually with the aim of
deeply characterizing the community composition (Taberlet et al. 2012).
Below we describe some recent and potential applications of these two general
approaches with birds, some strengths and limitations of each, and discuss possible
future directions for the field.
2.4.1 Metabarcoding
Perhaps one of the greatest potential areas of impact for metabarcoding in avian
conservation is through biodiversity monitoring and discovery. Theoretically, any
sample mixture could be screened to identify the presence of avian species. This type
of work is routinely taking place for mammals and aquatic animals (e.g., Bohmann
et al. 2014), but so far has been less often applied to birds. One current application is
“bulk-bone” metabarcoding of partial, undiagnosable bones from archeological and
paleontological sites (Murray et al. 2013; Honka et al. 2018). This work allows
identification of the presence of previously unreported bird species through time and
may be particularly beneficial in documenting changes in diversity of small-bodied
birds whose bones are easily fragmented and therefore often unrecognized. Thus,
metabarcoding may help in determining a baseline for conservation actions, esta-
blish the former presence of currently extinct or extirpated species, and identify the
former range of species for possible reintroduction. Another potential application
could arise from metabarcoding remains after bird collisions. In cases where colli-
sions with aircraft result in samples lacking sufficient feather material to allow
morphological analyses, or in cases where mixed species flocks were involved in
the strike, metabarcoding may provide important information on the avian taxa
involved in collisions, thus allowing implementation of more effective management
strategies (Dove et al. 2008). Metabarcoding could also be implemented to study
collisions with turbines and wind energy technologies, where multiple birds may be
struck through time and where the individuals struck did not die within the imme-
diate vicinity and therefore cannot be identified through visual monitoring.
The approach could also be applicable to medicines and foods derived from
illegal harvests of birds, where DNA is present from multiple species or too
degraded for traditional barcoding (Staats et al. 2016).
Another area for which metabarcoding demonstrates great potential to contribute
to avian ecology and conservation is through analysis of diets. A better understand-
ing of diets could provide key insights, for example, about interspecies competitive
interactions and predator-prey dynamics, which would in turn inform our under-
standing of factors limiting reproductive success or survival. Ornithologists have
long struggled to characterize diets by making foraging observations and analyzing
gut contents or regurgitation. Metabarcoding can potentially yield vast quantities of
data on these questions in a relatively short amount of time. Such analyses, however,
have only recently been successfully applied to birds (Vo and Jedlicka 2014), likely
due to the high acidity of bird feces, which can make obtaining DNA a challenge.
Jedlicka et al. (2017) used a metabarcoding approach to characterize the diets of

western bluebirds (Sialia mexicana) in vineyards and found that they consumed
primarily herbivorous insect taxa, rather than the predator or parasitoid taxa that may
also be contributing to pest control. Also, McClenaghan et al. (2019) investigated the
diet of a declining avian insectivore, the barn swallow (Hirundo rustica). They found
that this species is a broad-scale generalist and is able to feed nestlings a varied diet
and take advantage of opportunistic food resources. Thus, this species should
theoretically be resilient to changes in food availability. Other studies have
investigated the use of aquatic insect prey sensitive to pollution by Louisiana
waterthrush (Parkesia motacilla) (Trevelline et al. 2016), as well as prey usage by
semipalmated sandpipers (Calidris pusilla) during migratory stopover (Gerwing
et al. 2016). Alternatively, diet metabarcoding could help identify when birds are
the prey items, for example, in the diets of felines, mustelids, rodents, and other
invasive predators (Zarzoso-Lacoste et al. 2016).
Metabarcoding has also been used to understand the bacterial communities of
birds and impacts on their physiology and health (Waite and Taylor 2015). For
example, hoatzins (Opisthocomus hoazin) consume leaves, and fermentation is
thought to take place in their enlarged crops, reminiscent of cattle and horses rather
than other birds. A comparison of hoatzin and cow foregut and hindgut bacterial taxa
suggested that indeed, the foregut taxa of hoatzin and cow were more similar to each
other than to their own hindgut community (Godoz-Vitorino et al. 2012). This
suggests that the bacterial community of the hoatzin crop may represent a conver-
gent, evolutionary adaptation for dealing with a herbivorous diet. In contrast to this,
the critically endangered kakapo (Strigops habroptilus), which also consumes plant
material, but not entire leaves, has a very different foregut bacterial community and
therefore is unlikely to perform fermentation and has adapted to its diet in different
ways (Waite et al. 2013, 2014). Similarly, black (Coragyps atratus) and turkey
vultures (Cathartes aura), which scavenge decaying carcasses rife with toxic bacte-
rial compounds, host a unique gut microbiome, which likely originates from their
diet (Roggenbuck et al. 2014). Additional analyses of the avian microbiome could
also play a role in understanding, for example, the bacterial communities which may
be critical for coloration and degradation of feathers, mate attraction, nestling
development, reproductive investment, as well as migratory ability (Jacob et al.
2014, 2015). All of these aspects influence survival and reproduction and therefore
would be particularly important to understand for endangered avian taxa.
2.4.2 Metagenomics
While implementation of metabarcoding approaches is becoming more common,
few metagenomics studies have involved birds, particularly wild birds. One such
recent paper examined the cecal metagenome of the greater sage grouse
(Centrocercus urophasianus, Kohl et al. 2016). The greater sage grouse regularly
(and at some points in the annual cycle exclusively) consumes sagebrush (Artemisia
sp.), a plant that contains toxic secondary compounds. Kohl et al. (2016) sequenced
total DNA from the cecal microbiota and assembled gene sequences (rather than full
genomes). They found that the sage grouse cecal metagenome was enriched for
genes related to breakdown of these compounds, as compared to the microbiota of

chicken and mammalian herbivores. This suggests that greater sage grouse and their
microbiota may be specially adapted for a diet of sagebrush and other plants with
similar toxic secondary compounds.
Metagenomics could be particularly useful for understanding pathogens and
disease in wild birds. Disease is an important factor limiting population growth
rates and carrying capacity; therefore, gaining a better understanding of the
infections that birds carry and how they spread could have important management
and conservation implications. Beyond this, wild birds may serve as the reservoir for
diseases that impact humans (e.g., avian influenza and West Nile virus), so a better
understanding of bird diseases would benefit people as well (Kapgate et al. 2015). In
these cases, having a more complete, less-biased picture of the bacteria, viruses, and
fungi present (and potentially accurate representations of their abundance) would be
especially important. A metagenomics approach was recently taken to investigate
the virus community in domestic turkeys (Day et al. 2010), and a similar approach
could be taken for monitoring and surveillance of wild and endangered birds.
2.4.3 Strengths and Challenges

While the above examples provide insights into the current and potential future
applications of metagenomics and metabarcoding studies, it is useful to have an
understanding of the challenges these studies face.
In any metabarcoding study, one of the early steps involves PCR amplification
with theoretically universal primers. Unfortunately no primer is truly universal, and
this step may introduce taxonomic biases during amplification (Deagle et al. 2014),
which can be difficult to predict ahead of time. These biases include not only
amplification success of some species and failure for others but also biases in the
strength of amplification with some species amplifying strongly and others
amplifying weakly. This makes interpretation of metabarcoding sequence abun-
dance challenging (Elbrecht and Leese 2015). Beyond this, sequences obtained
from metabarcoding are often compared to available DNA barcode sequence
databases, such as the Barcode of Life (Ratnasingham and Hebert 2007). Taxonomic
biases in the database can lead to biased sequence identification or identification to
higher taxonomic levels only (such as family or order; Kvist 2013). A related
problem is that DNA barcoding loci may in some cases lack sufficient resolution
(particularly for degraded samples which make use of shorter barcode loci), and thus
two or more taxa may share the same barcode sequence and thus could not be
identified uniquely to the species level. This is a common issue for bird diet items
such as plants and may exist for arthropods and some higher vertebrates as well
(Starr et al. 2009; Janzen et al. 2005). In general, data on presence of taxa may be
more reliable than data on absence or abundance of taxa.
The benefits of the metabarcoding approach, however, offset the challenges. If
characterizing biodiversity is the goal, then this approach targets sequencing to
limited areas of the genome that are comparatively well covered in online databases
(e.g., the COI gene, Ratnasingham and Hebert 2007). Also, by targeting the
sequencing effort, many samples can be combined within a single sequencing run,
and smaller instruments with a cheaper total run cost may be sufficient (e.g., the
Illumina MiSeq), making this work feasible for projects with limited budgets or very
large sample sizes. Beyond this, because the protocols generally start with a PCR
amplification step, it becomes possible to append overhanging sequences to the
primers that match sequencing adapters and thus more quickly and efficiently
conduct library preparation for sequencing, though these overhangs may also
cause taxonomic biases during amplification (Berry et al. 2011).
One of the benefits of the metagenomics approach is that it removes the initial
PCR step that generates much amplification bias. Because sequencing may yield
multiple and/or longer regions, taxonomic resolution may be increased with this
approach, and therefore result in more comprehensive characterization of the com-
munity of interest (Srivathsan et al. 2015). However, it should be noted that few
extensive tests of metagenomics sequencing for community characterization have
been conducted and these approaches may still suffer from technical problems, such
as the presence of inhibitors or biased ligation of sequencing adapters. Another
strength of the metagenomics approach, as mentioned above, is that when sequenc-
ing is no longer targeted to one or a few genes, a wider range of questions may be
addressed, leading to deeper insights about microbial community interactions, gene
expression, and gene function (Fierer et al. 2012; Poretsky et al. 2009).
Metagenomics studies also face several challenges. Because sequencing is not
targeted, more sequencing is necessary, and therefore run costs are likely to be
higher. In addition to this, datasets are likely to be less complete, as compared to a
traditional genomic study, because rather than sequencing a single individual,
hundreds or thousands of individuals/species may be sequenced at the same time.
This also leads to bioinformatic and computational challenges of assembling reads
from many individuals into useful datasets (Wooley et al. 2010).

There are several areas where advances in metagenomics and metabarcoding could
make a significant impact on our knowledge and understanding of avian conser-
vation. Gaining a better understanding of the factors that influence the accuracy of
the relationship between abundance in the sample and the proportion of sequencing
reads would yield many new insights. For example, the inclusion of accurate
abundance information into analyses of food web dynamics would indicate not
only that a prey taxon was consumed but its relative importance in the diet.
Similarly, for biodiversity monitoring, abundance of a species’ sequences could
indicate the relative numbers of individuals living in different areas or the relative
abundance of pathogenic microorganisms compared to commensal or beneficial
taxa. For metabarcoding, many factors may influence abundance, including but not
limited to storage and DNA extraction methods, primer bias, polymerase bias, and
even bioinformatics approaches for quality control and taxonomic assignment
(Vo and Jedlicka 2014; Deagle et al. 2013, 2014; Kopylova et al. 2016;
Krehenwinkel et al. 2017; Nichols et al. 2018). Metagenomic studies may suffer
from less-biased sequence abundance, but little is known. Potential confounding
factors could include copy number issues, biased digestion, and biased databases for
taxonomic comparison and functional annotation. Despite these potential con-

founding factors, several studies are beginning to suggest that a relationship may
exist between abundance and sequence proportions (e.g., Srivathsan et al. 2015;
Evans et al. 2016, and others). Further, for metagenomic studies, improvements in
sequencing (number, quality, and length of reads) and reduction in cost would be
valuable, but perhaps the most beneficial would be continued improvements in our
computational abilities, both with regard to the raw computing power and software
to handle complex datasets.
2.5 Systematics and Species Limits
To conserve species, scientists and managers need to know what are distinct
species—that is, how do data inform us of species status and limits based on
accepted species definitions. In addition, an understanding of hierarchical levels of
genetic structure below the species level is necessary to also optimally conserve
genetic variation. Thus we also need to have clear-cut concepts and definitions of
such entities as subspecies, evolutionarily significant units (ESU), and the
Endangered Species Act (ESA) legally defined “distinct population segments”
(DPS). While there are problems with each of these categories because of differences
in the definitions that have been proposed (Frankham et al. 2012; Garnet and
Christidis 2017; Haig et al. 2006; Funk et al. 2012) and difficulties obtaining relevant
data (such as hybrid fitness and local adaptation), the roles of genetic, and now
genomic, data have proven useful to elucidate these categories for policy and
management decisions. Note, the chapter “Avian Species Concepts in the Light of
Genomics” in this volume is devoted to species concepts and how genomic data can
be incorporated into criteria to distinguish species, so our discussion here will largely
be concerned with their applications to conservation.
2.5.1 Species Definitions and Limits

These can be tested with genomic data to determine species boundaries and
relationships and amounts and directions of introgression at contact zones
(Ottenburghs et al. 2017). The expansion of data to the genomic level has greatly
increased our ability to determine these variables (Toews et al. 2016) to better test
species limits. The ability to discover chromosomally local “islands of divergence”
between taxa that are otherwise very closely related genetically can reveal the
presence of barriers to unrestricted gene flow, the directionality and timing of
introgression, and the roles of sexual selection and local adaptation in speciation.
In addition, these data are also very useful for assessment of different subspecies,
ESU and DPS definitions, and could also provide information on the role of adaptive
divergence that might limit a taxon or populations to a particular range or habitat.
Although biologists have defined a plethora of different species concepts
(Frankham et al. 2012; Garnett and Christidis 2017), one in particular, the Biological
Species Concept (BSC, Mayr 1942), has largely dominated the systematics of birds.
More recently, a set of determined ornithologists have advocated for the application
of the Phylogenetic Species Concept (PSC, Eldridge and Cracraft 1980), which
would potentially double the number of described avian species to over 20,000
(Barrowclough et al. 2016). Conservation biologists argue that species definitions
and designations need standardization and better control (Mace 2004; Frankham
et al. 2012), even proposing an official body of scientists be established to carry out
this role (e.g., Garnett and Christidis 2017).
For both of ornithology’s primary species concepts and others that could be
applied (Hill 2017; Kraus et al. 2012), genomic approaches would provide greater
resolution of species limits. Assessment of fine-grained genome-wide sequence
variation across avian contact zones would provide information on divergence
(Fig. 1, middle left) but also on the level and pattern of introgression across the
zone and into the parental taxa (e.g., Poelstra et al. 2014; Baldassare et al. 2014;
Toews et al. 2016; Kearns et al. 2017). For example, only six small genomic regions
were differentiated between the morphologically and mitochondrially (Gill 1997;
Shapiro et al. 2004) distinct blue-winged (Vermivora cyanoptera) and golden-
winged (Vermivora chrysoptera) warblers (Toews et al. 2016), and in proximity to
four of those regions were genes potentially involved in the plumage differences
between the two taxa. Based on the genomic sequence analyses, these two “species”
have had a long and complicated history of interaction and, despite this interaction
and their high genomic similarity, are likely best viewed as distinct species. There
are a number of other cases emerging for birds in which evidence of apparently
reproductively isolated taxa show minor, but detectable, overall genomic-level
differentiation (e.g., differentiated subspecies in the barn swallow Hirundo rustica,
Safran et al. 2016) or only a few islands of differentiation (as the above warbler
case), and these will likely necessitate modification in the criteria used for species
and subspecies designations.
2.5.2 ESU and DPS Definitions

Other definitions of importance in conservation management are what have been
defined as evolutionarily significant units (ESU) and distinct population segments
(DPS). The latter is actually incorporated as a defined unit of conservation for only
vertebrates under the US Endangered Species Act (see Pennock and Dimmick 1997
and follow-ups for a discussion of DPS and how employing ESU alternatives could
muddy the waters).
For a DPS to be defined and listed, the population under consideration must be
discrete and significant and have an appropriate level of threat or endangerment.
Although little guidance on these three criteria were provided in the act itself,
additional legislation and discussion have indicated that the discrete population
segment should “differ markedly from other populations of the species in its
genetic characteristics” and genetics has often played a role in these designations.
There have been many definitions of ESU proposed since it was first discussed in
a meeting review by Ryder (1986), including ones that involved mitochondrial DNA
reciprocal monophyly and significant allele frequency differences (Moritz 1994) and
identification of loci (and/or morphological or ecological traits) that reflect adapta-
tion to local environments (Waples 1995; Crandall et al. 2000).
While genetic markers have been used in ESU and DPS approaches to a great
extent (see examples in Fleischer 1998; Phillimore and Owens 2006), only recently
have genomic methods been applied to confirm subspecies or ESU designations. A
nice example of genomic differentiation and even potential local adaptation among
subspecies and populations is that of barn swallows in Eurasia and North America
(Safran et al. 2016; Scordato et al. 2017). These studies show clear genomic-level
divergences based on PCA and structure analyses, with varying levels of
hybridization or gene flow among the taxa based on hybrid analyses. Isolation by
distance versus isolation by adaptation analyses suggest a strong component of the
divergence is due to the latter, but only on a macrogeographic level. Another recent
study showed only minor genomic-level differentiation using RAD-seq markers
(and morphology) between Japanese and Hawaiian populations of the black-footed
albatross (Dierickx et al. 2015), although the authors could not exclude the possi-
bility that the slight differentiation detected might be meaningful for adaptation and
thus conservation management. In most cases, genomic markers enable a much
greater degree of resolution of taxon- or population-level differentiation than ana-
lyses based on mtDNA or nuclear sequences or microsatellites, but we must caution
that the greater resolution of genomic differences attained by large numbers of
markers may not necessarily imply biological significance.

Fitting structured and legal definitions like species, ESU, and DPS to biological data
can be difficult, and genomic data may not really add to our ability to elucidate these
concepts. Perhaps the key point of utility for avian conservation is really to under-
stand how populations or species are related to each other, how they may be locally
adapted, and what are the current and historical levels of gene flow among them.
Population genomics is enhancing our ability to quantify these aspects and would
allow us to determine how these entities would need to be managed. It is now
possible to genotype hundreds of individuals at thousands of markers without
needing a reference genome (although many more reference genomes are becoming
available; Callicrate et al. 2014; Jarvis et al. 2014; Zhang 2015). Such a plethora of
information enables us to explore how genome structure and evolution contributes to
differentiation and speciation. We are now able to assess functional and adaptive
differences between populations using genomic and transcriptomic data (e.g., Safran
et al. 2016; Taylor and Mason 2015). Genome-wide markers such as single nucleo-
tide polymorphisms (SNPs) can be used to genotype the same loci in ancient and
modern samples, and although working with ancient DNA presents many chal-
lenges, genomics helps us overcome some of the problems with fragmented DNA.
Thus, ultimately we will be able to document the historical contexts (past population
size, structure, migration levels) using approaches such as Pairwise Sequentially
Markovian Coalescent (PMSC; Li and Durbin 2011) and similar analyses for
present-day situations (e.g., Nadachowska-Brzyska et al. 2015, Murray et al.
2017a, b). These can provide information useful for the management and recovery
of endangered avian species.
2.6 Adaptation to Climate Change and Other Stressors
Adaptation occurs in response to various biotic and abiotic factors. As anthropogenic

stressors increase, studying species response has become essential. Understanding
how populations react to climate, habitat loss, and other compounding stressors can
help us conserve species as a whole. In the past, studying adaptation required
previous knowledge of genes involved in specific pathways. Early work focused
on identifying candidate genes responsible for the change in phenotype through
methods such as quantitative trait locus (QTL) analyses. With the advent of next-
generation sequencing, our capacity to study adaptation in response to anthropo-
genic stressors has drastically improved. The field has expanded to include
multiple pathways of adaptation, such as transcriptional regulation, selection in
exonic regions, and posttranscriptional regulation. Next-generation sequencing
allows us, without any a priori knowledge, to identify loci under selection that
may be important in adaptation (Fig. 1, top left; Stillman and Armstrong 2015).
Additionally, it enables us to study responses within a single generation (e.g.,
phenotypic plasticity) using methods such as RNA-seq (Wang et al. 2009;
avian transcriptomics review: Jax et al. 2018).
2.6.1 Species Response to Climate Change

When faced with a stressor, species respond in one of four ways: (1) evade, (2) die
(local extinction), (3) acclimate, or (4) adapt. While it is more straightforward to test
for evasion or local extinction, the latter two have been historically more difficult to
tease apart (Gienapp et al. 2008). Climate change-related range shifts entail an
overall shift toward higher latitudes and higher elevations (Chen et al. 2011).
Range shifts are considered to have two components—the cool-edge expansion
(evasion) and the warm-edge contraction (local extinction; Wiens 2016). Range
expansion and local extinction have been a primary focus in the response to climate
change, whereas the importance of evolution occurring at the warm edge is often
overlooked (Hoffmann and Sgrò 2011; Vedder et al. 2013).
Population response in the warm edges of a species’ range can occur through
adaptation and acclimation or genotypic specialization and phenotypic plasticity
(Box 1). A well-documented example of population response to climate change is
the occurrence of earlier mean breeding dates in many bird taxa (Charmantier and
Gienapp 2013). Breeding timing is often based on prey abundance, which is in turn
dependent on climatic factors. In great tits (Parus major), breeding season depends
on a brief annual peak in caterpillar abundance, their offspring’s food source, which
is determined by spring temperatures (Visser et al. 2004). Vedder et al. (2013) found
evidence of plastic response in breeding time in Wytham Woods great tits. Based on
these data, the authors modelled extinction risk and found that the absence of this
phenotypic plasticity would increase the likelihood of population extinction by
500-fold. They, however, note that little is known about the underlying causes and
limits of this plasticity. The usage of next-generation sequencing methods could help
identify the genomic basis of this response.
Fig. 2 Representation of
reaction norms of three
genotypes for a single trait.
Genotypes A and B show
discontinuous and continuous
phenotypic plasticity,
respectively. Genotype C
shows no plasticity for this
trait since its phenotype
remains consistent across
environments
Box 1: Genotypic Specialization Vs. Phenotypic Plasticity

Genotypic specialization is the process by which canalized, locally adapted
phenotypes are present in an environment, and phenotypic specialization
(or plasticity) is differentiation by regulated gene expression in response to
the given environment (Wahl 2002). Genotypic specialization involves the
genetic makeup of a population changing over generations due to the differ-
ential survival or reproductive success of certain genotypes in an environment
or natural selection (DeBiasse and Kelly 2016). Conversely, phenotypic
plasticity involves a change in phenotype in response to the environment
within an organism and within a single generation (DeBiasse and Kelly
2016). Historically, the influence of the environment on the genome was
assigned primarily to natural selection or gene-by-environment (GxE)
interactions. A GxE interaction is when two genotypes respond to the same
environment differently. Recently, however, the response of a single genotype
in different environments has been attributed a more dynamic role (Fig. 2). For
example, given the same genotype, an individual’s phenotype can be a result
of the environment it is exposed to from development through adulthood
(Fusco and Minelli 2010); this is phenotypic plasticity.
Phenotypic plasticity comes with a cost, as species have to maintain the
genetic and cellular mechanisms required to produce plastic responses, e.g.,
regulatory genes and/or enzymes (Scheiner 1993). Additionally, there could
be costs associated with information gathering about environmental conditions
and genetic costs involving linked genes, pleiotropy, and epistatic interactions
(DeWitt et al. 1998). Therefore, plasticity is not likely to evolve in a popula-
tion that does not experience a variety of environments or does not possess the
genetic variance required for a plastic response in that specific trait. When
environmental heterogeneity is fine-grained, individuals will experience vari-
ous environmental conditions during their lifespan, requiring an increased
(continued)
acclimation capacity, and therefore phenotypic plasticity is more likely to

evolve (Storz et al. 2010). However, when environmental heterogeneity is
coarse-grained, individuals experience a limited range of environmental
conditions, and therefore phenotypic plasticity is less likely to evolve
(Storz et al. 2010).
When an individual’s phenotype is altered in the direction of the
local optima, plasticity is said to be adaptive. An adaptive plastic response
can later become genetically encoded via natural selection through a process
known as genetic assimilation (Ghalambor et al. 2015). This process allows
an organism exposed to a constant stressor to eventually develop a bio-
logically robust response in the direction of its previously plastic response
(DeBiasse and Kelly 2016).
2.6.2 Approaches for Assessing Response to Climate Change

The genomic basis of response to climate change can be studied via multiple
approaches (Table 2). It can also be studied on multiple levels: genomic, tran-
scriptomic, and epigenomic. Reduced representation methods, such as RAD-seq
(Baird et al. 2008), and sequence capture provide genomic information, whereas
RNA-seq and reduced representation bisulfite sequencing (RRBS; Meissner et al.
2005) are used for transcriptomics and epigenomics, respectively. Multilevel
approaches are important in parsing the relative effects of adaptation and acclimation
or genotypic specialization and phenotypic plasticity.
In populations where candidate genes are responsible for most of the variation in
potentially evolving traits, sequence capture can be employed to isolate and enrich
those genes for analysis. This approach can be used to identify genotypic special-
ization if there is a priori knowledge of the species’ genome. A classic example of
this method is the usage of exome capture to identify the genetic basis of high-
elevation adaptation in Tibetans (Yi et al. 2010). Long-term study of a single popu-
lation allows researchers to differentiate between genetic and environmental contri-
butions to trait change. Using whole-genome resequencing or reduced representation
genomics, researchers can look for signatures of selection that correspond to climate-
related phenotypic changes. This method, while effective, requires studying a popu-
lation over multiple years, which is not always possible.
Common garden experiments and transplant experiments offer a method by
which one can tease apart the roles of local adaptation and plasticity in adaptive
success. In a single generation, RNA-seq and RRBS can be used to identify plastic
responses to environmental change. In a multi-generation experiment, researchers
can identify signatures of selection and heritability of epigenetic modifications. For
example, to test for plasticity in high and low elevation populations of the rufous-
collared sparrow (Zonotrichia capensis), Cheviron et al. (2008) transplanted indi-
viduals to a low elevation common garden. None of the transcripts that were
Table 2 Next-generation approaches for assessing the genetic basis of response to climate change
in wild avian populations (Adapted from Hoffman and Sgro 2011)
Next-gen
sequencing
Approach Advantages Limitations method
Assessing variation in Useful when candidate Requires a priori Sequence
candidate genes for genes are responsible knowledge of both the capture
relevant traits for most of the variation loci and the mechanism
in relevant trait of adaptation
Useful only if few
genes are responsible
for most of the variation
in the relevant trait
Long-term study of a Useful in long-term Time-consuming Sequence
single population study populations because it requires capture
Can parse out genetic repeated sampling over RAD-seq
and environmental multiple years/seasons Genome
contributions resequencing
Common garden and Can differentiate Not all species can be RNA-seq
transplant experiments between genotypic transplanted and/or live RRBS
across a climatic gradient specialization and in captivity
(single generation) phenotypic plasticity
Common garden and Can identify signatures Not all species can be Genome
transplant experiments of selection transplanted and/or live resequencing
across a climatic gradient in captivity RAD-seq
(multi-generation) RNA-seq
RRBS
Experimental evolution Can differentiate Not all species can be Genome
in artificial environments between genotypic transplanted and/or live resequencing
designed to simulate specialization and in captivity
climate change phenotypic plasticity
Can identify signatures Difficult to extrapolate RAD-seq
of selection to wild populations RNA-seq
experiencing RRBS
compounding stressors
differentially expressed at native elevations remained different in the common

garden, demonstrating a considerable level of phenotypic plasticity in cold and
hypoxia response (Cheviron et al. 2008). While common garden and transplant
experiments yield valuable insights into population response to stressors, they are
limited to species that can be transplanted and survive in a captive environment.
Experimental evolution in artificial environments designed to simulate climate
change provides a unique opportunity to study adaptation and acclimation to a
controlled stressor. For example, wild guinea pigs that were exposed to heat
treatments showed epigenetic modifications that were passed on to their offspring,
providing them with improved resilience to environmental temperature increase
(Weyrich et al. 2016). Simulated environment experiments allow researchers to
study response along a single axis; however, whether these data can be extrapolated
to wild populations experiencing compounding stressors remains to be seen.

Studying the genomic basis of population response to climate change is a relatively
new field in ornithology. Future studies that use transplant and/or simulated
experiments and next-generation sequencing are needed to understand the relative
roles of adaptation and acclimation in response to climate change. Additionally,
research on rapid evolution in invasive species could provide some insight into how
species will respond to climate change in the future (Moran and Alexander 2014;
Chown et al. 2015). Nonetheless, the function of many avian genes is still unknown,
particularly for genes with multiple functions and small effect sizes. Studies that
work to characterize gene function across avian taxa are greatly needed. Still, as
sequencing becomes more cost-efficient, multilevel studies that compare genomics,
transcriptomics, and epigenomics of a population will become possible, thereby
creating a more complete picture of the avian evolutionary response to climate
change.
3 General Conclusions
The advent of genomics and next-generation sequencing methods has enabled

significant advances in our understanding of avian conservation. It has the potential
to facilitate the preservation of adaptive diversity—particularly as technology
progresses and our understanding of gene function improves. In combination with
new computational approaches, we have gained incredible power to reconstruct
population and evolutionary histories of endangered species and to define units for
conservation. In addition, new high-throughput methods increase our ability to learn
about the biology of endangered species often without handling or disturbing them
(such as noninvasive analyses of population size, inbreeding, movements, diet, and
disease). Some challenges remain, most notably that we lack detailed information
about gene function across taxa. Nonetheless, the increasing availability and
decreasing cost of obtaining large-scale genomic data are resulting in a burgeoning
number of studies that address this shortcoming, and our understanding of gene
function stands to improve dramatically in the next few years. Our ability to harness
the power of genomics will be amplified by using multiple genomic approaches to
creatively answer pressing questions in avian conservation.
Acknowledgments We thank three anonymous reviewers for their insightful comments on the
manuscript and Carly Muletz-Wolz for providing Fig. 1d.
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Jurassic Park: What Did the Genomes
of Dinosaurs Look Like?
Darren K. Griffin, Denis M. Larkin, and Rebecca E. O’Connor
Abstract
Recent palaeontological evidence is clear that birds are extant dinosaurs. Evolving
along the lineage Diapsida—Archelosauria—Archosauria—Avemetatarsalia—
Dinosauria—Ornithoscelida—Theropoda—Maniraptora—Avialae, birds are the
latest example of dinosaurs emerging from catastrophic extinction events as
speciose and diverse. Indeed, rather than being wiped out by the Cretaceous-
Paleogene meteor strike, they are the survivors of at least three extinction events.
Dinosaurs capture the public imagination through art, literature, television and film,
most recently through the Jurassic Park/World franchise. Claims in the scientific
literature of isolating dinosaur DNA (from amber-preserved insects or elsewhere)
have largely been debunked. Nonetheless, the overall structure of dinosaur
genomes along the above lineage can be determined by inference from
chromosome-level genome assemblies. Our work focused first on determining
the likely karyotype of the avian ancestor (probably a small, bipedal, feathered,
terrestrial Jurassic dinosaur) finding great similarity to the chicken. We then
progressed to determining the likely karyotype of the diapsid ancestor and the
changes that have occurred to form extant animals. A combination of bioinformat-
ics and molecular cytogenetics indicates considerable interchromosomal rearrange-
ment from a “lizard-like” karyotype of 2n ¼ 3646 to one similar to that of certain
turtles from 275 to 255 million years ago (mya). Remarkable karyotypic similarities
between some turtles and chicken suggest identity by descent, in other words that,
Author names listed in alphabetical order to indicate equal contribution.
D. K. Griffin (*) · R. E. O’Connor

School of Biosciences, University of Kent, Canterbury, UK
e-mail: d.k.griffin@kent.ac.uk; r.o'connor@kent.ac.uk
D. M. Larkin
Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London,
London, UK

https://doi.org/10.1007/978-3-030-16477-5_11
332 D. K. Griffin et al.
aside from ~7 fissions, there were few interchromosomal changes from the
archelosaur (bird-turtle) ancestor to the Avemetatarsalia (dinosaurs and pterosaurs),
through the theropods to modern birds. Indeed, a similar rate of change beyond
255 mya would have meant that the avian-like karyotype was in place about
240 mya when the first dinosaurs and pterosaurs emerged. We mapped
49 intrachromosomal changes in the intervening period, finding significant gene
ontology enrichment in homologous synteny block and evolutionary breakpoint
regions. The avian-like karyotype with its many chromosomes provides the sub-
strate for variation (the driver of natural selection) through increased random
segregation and recombination. It thus may impact on the ability of dinosaurs to
survive and thrive, despite multiple extinction events.
Keywords
Dinosaur · Theropod · Chromosome · Karyotype · Molecular cytogenetics ·
Comparative genomics · Genome evolution
This is a book about dinosaur genomes. Everything you’ve read so far represents an
exposition and analysis of dinosaur genomics. Birds did not evolve from dinosaurs;
birds are not related to dinosaurs. On the contrary, the latest palaeontological
evidence is very clear that birds are dinosaurs. Dinosaurs have been ever present
in popular culture and the creative arts since the earliest fossil discoveries, promoted
through film, television, press, art and literature. And, rather than being a group of
animals that were wiped out by a very well-known extinction event caused by a
meteor, they are in fact the survivors of several extinction events. Each time they
emerge more diverse and are probably more likely to survive whatever each extinc-
tion event might throw at them. In a recent study, we suggest this may be due, in part,
to their unique genomic structure, i.e. their karyotype.
1 What Are Dinosaurs and Where Do They Fit in Vertebrate

Evolution?
Amniota is the vertebrate clade that includes mammals, birds and non-avian reptiles.
It is a remarkably diverse group of terrestrial tetrapods believed to have shared a
common ancestor 325 mya (million years ago) in the Permian period (Shedlock and
Edwards 2009). Amniotes diverged into the lineages that ultimately became
mammals (Synapsida) and reptiles (Diapsida) during this time. Diapsida has
~17,500 extant members (~10,500 of which are birds) and is subdivided into two
groups (Fig. 1). Lizards, snakes and tuataras form a monophyletic group—
Lepidosauromorpha—that is the sister group to the Archelosauria (crocodilians,
dinosaurs, pterosaurs, turtles, birds), all of which shared a common ancestor
275 mya (Hedges et al. 2015; Shedlock and Edwards 2009). The stem groups of
Lepidosauria and Archelosauria also include several extinct lineages that existed in
Jurassic Park: What Did the Genomes of Dinosaurs Look Like? 333
Permo-Triassic Carnian–Norian End-Triassic mass Cretaceous-Paleogene (K-

extinction event extinction event extinction event Pg) mass extinction event
Timeline is not
scaled to allow
275 mya >255 mya <255 mya 251 mya 245-240 mya 240-235 mya 228 mya 201 mya 170-165 mya 66 mya Present day more clarity
Squamates
(Snakes & Lizards)
Lepidosauromorpha
Sphenodontia
(Tuatara)
Diapsida
Testudines
(Turtles) Pseudosuchia
Archelosauria (Crocodilians)
Archosauromorpha/ Pterosauria
Archosauria
Saurischia
Avemetatarsalia Ornithischia
Dinosauria
Ornithoscelida
Theropoda Maniraptora Avialae Neornithines (Aves)
Fig. 1 The lineage from the diapsid ancestor to modern birds including the major extinction events
the Triassic period including the rhynchosaurs (Ezcurra et al. 2014). Of the
lepidosaurs, the tuatara diverged 272 mya making them an extraordinarily ancient
species and the only extant example of its order, the Rhynchocephalia (Rauhut et al.
2012). Assuming that the majority of recent molecular phylogenies of amniote
interrelationships are correct, turtles (Testudines) diverged from Archelosauria first
(around 255 mya), forming the crown-group Archosauromorpha (crocodilians,
pterosaurs and dinosaurs, including birds). Prolonged debate previously surrounded
the phylogenetic relationship of turtles (Testudines). The lack of temporal fenestrae
in the skull led for many years to their assumed placement as a primitive anapsid;
however, molecular evidence now places the Testudines as a sister group to the
Archosauromorpha (Chiari et al. 2012; Crawford et al. 2012; Shaffer et al. 2013).
The earliest recorded Testudine fossil (Odontochelys semitestacea) is Late Triassic
in age (237–223 mya) (Benton et al. 2015; Nicholson et al. 2015) although stem
turtle species (Eunotosaurus africanus) have been dated even further back to
260 mya (Lyson et al. 2010). It is reasonably well established therefore that the
Testudine divergence occurred around 255 mya (Fig. 1).
Archosauromorpha exhibits a basal split into the crocodile-line (Pseudosuchia or
Crurotarsi) and dinosaur (bird)-line (Ornithodira or Avemetatarsalia) clades. The
group of animals most commonly known as dinosaurs is formally defined as the
clade including Triceratops, Passer (songbirds) and all of the descendants of their
common ancestor, with birds nested within the theropod clade Maniraptora (Fig. 1).
Previous research dated the oldest unequivocal dinosaur fossils to 230 mya
(Martinez et al. 2011); however, recent fossil finds now indicate that, although
reported divergence times of major clades vary between different studies, the earliest
dinosaur divergence from non-dinosaur dinosauromorphs was approximately
240–245 mya (Nesbitt et al. 2013). Pterosauria, the sister group to Dinosauromorpha
diverged around 245 mya, although an earlier origin is possible. The ornithodiran-
crurotarsan (bird-crocodilian) divergence occurred in the Lower Triassic period, or
potentially earlier around the time of the Permo-Triassic boundary 252 mya, despite
older studies dating the crocodilian split from birds to 219 mya (Shedlock and
Edwards 2009). Species numbers appear to have remained relatively low in both
the lepidosaurs and the archosauromorphs until the Permo-Triassic mass extinction
event (PTME) devastated the synapsids around 251 mya (Benton and Twitchett
2003). Massive volcanic eruptions in the Siberian Traps are thought to have initiated
the conditions that created the PTME. These eruptions led to a prolonged period of
global warming, creating anoxic conditions that devastated 80–90% of life on land
and in the oceans (Benton and Twitchett 2003). The subsequent period of climate
change led to increasingly arid conditions that marked out the beginning of the
Triassic as a period of extraordinary ecological change. Estimates indicate that it
took 10–15 million years before ocean reefs, forests and vertebrates were
re-established after the PTME (Benton et al. 2013).
2 Dinosaurs Were (and Are) Speciose and Abundant
In terms of species diversity and abundance, dinosaurs were still relatively low in
number over the first 30 million years of their evolution but, by the mid Jurassic,
began to increase vastly in abundance, geographical spread and body size (Benton
et al. 2014). The following 135 million years is remarkable for being a period not
only for when dinosaurs were the dominant vertebrates but also for being a time
when they displayed an extraordinary range of species diversity. Throughout this
period the dinosaurs survived further extinction events including the Carnian-Norian
(CNEE) 228 mya that saw the end of the rhynchosaurs and dicynodonts (Brusatte
et al. 2008) and the End-Triassic mass extinction event (ETME) 201 mya that also
devastated the Pseudosuchia (or Crurotarsi—the crocodilian ancestors), leaving only
23 extant species. The period after the ETME corresponded with a steady increase in
dinosaur diversity and abundance arguing against the widely held belief that the
release of an ecological niche by the extinction of their competitors led to a surge in
dinosaur disparity (Brusatte et al. 2008). There are now over 1000 known species of
dinosaur that appear in the fossil record with around 30 more being identified each
year particularly in regions rich in newly discovered fossils such as China. They
were significantly decimated by the Cretaceous-Paleogene (K-Pg) extinction event
66 mya but emerged again as modern birds, with over 10,000 phenotypically diverse
representatives.
The extraordinary species diversity and abundance seen in the dinosaurs is often
attributed to the eradication of competitor species that allowed the dinosaurs to
flourish. However, it has also been suggested that these high levels of diversity
and abundance reflect adaptations unique to dinosaurs that enabled them to survive
through such harsh conditions, while other species perished. For example, the
extraordinary growth rates evidenced by bone growth patterns along with highly
adapted respiration systems such as pneumatised bones (Farmer and Sanders 2010)
and unidirectional respiration are both considered to be key features that enabled the
dinosaurs to flourish (O’Connor and Claessens 2005). Interestingly, these very
adaptations that may have led to the success of the dinosaurs are also key features
that contribute to the success of birds.
There is now little doubt that modern birds are the latest in a long line of dinosaurs
with fossil evidence showing that both groups shared features such as feathers,
oviparity, brooding behaviours and skeletal similarities (Varricchio et al. 2008). The
question thus arises whether non-avian dinosaurs also exhibited distinctively avian
features at a genetic level. Originating around 150 mya in the late Jurassic, birds
evolved from a theropod lineage (Chiappe and Dyke 2002) at a time when the
supercontinent Pangaea was separated into two landforms—Laurasia and
Gondwana. The fossil Archaeopteryx lithographica dating back to 150 mya and
found in the nineteenth century in late Jurassic limestone in Germany (Meyer 1861)
provides evidence of a transitional species between the non-avian and avian
dinosaurs. Although previously considered to be the fossil representative of an
early modern bird, features such as a bony tail and teeth rule A. lithographica out
from being considered a true avian ancestor (Mayr et al. 2007). As the oldest
unambiguous fossil representative of modern birds Vegavis is an aquatic bird
classified within Anseriformes and most closely related to Anatidae—ducks, geese
and swans (Clarke et al. 2005). Dating back to ~67 mya, the discovery of this fossil
supports the notion that representatives of modern birds were co-extant with
non-avian dinosaurs prior to the Cretaceous-Paleogene (K-Pg) boundary 66 mya
(Clarke et al. 2005). The inherent difficulties in fossil dating due to geographic and
depositional sampling bias, however, have led to much controversy in the field of
palaeontology (Chiappe and Dyke 2002), meaning that analyses at a genomic level
are a useful complement to a fossil record that could represent actual avian ancestors
imperfectly. Interestingly, the dinosaur ancestor of birds is generally considered to
be a bipedal, terrestrial, relatively small (small size being a pre-adaptation to flight)
Jurassic dinosaur with limited flying ability, not dissimilar to the Galliformes
(Witmer 2002).
Until the publication in 2014 of a revised avian phylogeny, the timing of avian
diversification has been a subject of much debate (Jarvis et al. 2014). The first avian
divergence is now considered to have taken place around 100 mya when the
Palaeognathae (ratites and Tinamous) diverged from the Neognathae (Galloanseres
and Neoaves). Within the Palaeognathae, the ratites and Tinamous then diverged
84 mya, while the Neognathae diverged into its stem lineages, the Galloanseres and
Neoaves, 88 mya. The Galloanserae divergence into the Galliformes (landfowl) and
Anseriformes (waterfowl) occurred around the time of the K-Pg. The major
divergences of the Neoaves into Columbea and Passerea are now dated to before
the K-Pg boundary (67–69 mya). The rest of the divergences within Neoaves were
largely complete at the ordinal level by 50 mya with the Passeriformes basal split
estimated to be approximately 39 mya (Jarvis et al. 2014). The K-Pg event was of
course a period of abrupt, mass global extinction and extreme climate change
coinciding with the Chicxulub asteroid impact in Mexico (Schulte et al. 2010). It
was a significant event for archaic birds (Ornithurae), of which modern birds are
descendants. Recent fossil evidence points to a major radiation of advanced
ornithurines occurring prior to the end of the Cretaceous period. The same group
then suffered an abrupt extinction around the K-Pg event with their disappearance
from the fossil evidence from the Paleogene period onwards (Longrich et al. 2011).
Data from the Jarvis et al.’ (2014) analysis also suggests that the K-Pg transition
period was one of the rapid neornithine speciation with 36 lineages radiating over a
period of 10–15 million years. Genomic studies have therefore, in recent years,
revolutionised our understanding of avian genomics and its relationship to pheno-
type and diversity. Genomic structure and organisation can be studied from a
number of perspectives, e.g. genome size, karyotype and nuclear organisation, and
what the actual overall genomic structure of dinosaurs looked like therefore warrants
further investigation.
3 Genome Size Calculated from Direct Fossil Evidence

and Why There Will Be No Jurassic Park
When Jurassic Park hit the screens in 1993 (following, in one of the authors’
opinion, the even better Michael Crichton novel in 1990), it was a cultural phenom-
enon and an outstanding story. It was of course just fiction. Finding reasonably
credible (albeit subsequently disproved) claims of isolating dinosaur DNA is diffi-
cult (there are a lot of clearly bogus claims that do not warrant mention here), but
they include Scott Woodward and colleagues who isolated DNA molecules from
two ancient bone fragments and produced nine readable sequences from a strand of
DNA for a particular gene (Woodward et al. 1994). This was, we believe, the first
report an authoritative journal has published about an apparent success in isolating
what is presumably dinosaur DNA. In the event, however, the fragments were too
small to be identified unequivocally as dinosaurian and were probably mammalian
(and, if so, represented human contamination) (Gibbons 1994; Hedges et al. 1995;
Zischler et al. 1995; Allard et al. 1995). In a reanalysis of DNA sequence data from a
dinosaur egg fossil from Xixia, Henan Province, China, Wang and colleagues
claimed to identify the real source of two putative dinosaur 18S rDNA sequences
(Wang 1996) but subsequently found them to be of fungal and plant origin (Wang
et al. 1997). Despite further claims (DeSalle et al. 1992; Cano et al. 1992), it seems
that the preservation of DNA from dinosaurs is not likely to be possible, and
therefore attempts to study the dinosaur genome directly are unlikely (Penney
et al. 2013). In Jurassic Park, dinosaur DNA is extracted from the blood isolated
from amber-preserved remains of ancient blood-sucking insects. In reality although
it may be theoretically possible to extra the DNA of the insect itself (there are
uncorroborated claims of this), any dinosaur DNA is likely to have quickly
degraded.
Nevertheless, using osteocyte size as an indicator of genome size from fossilised
bones, Organ et al. (2007) were able to identify a clear distinction between the small,
characteristically avian genomes identified in the saurischian-theropod dinosaur
lineage (that gave rise to modern birds) and the larger inferred genome seen in the
ornithischian dinosaur lineage (that led to the lineage that includes stegosaur and
Triceratops) that is comparable in size to those of modern crocodiles. They provided
evidence that saurischian dinosaur genomes averaged around 1.78 pg compared to
ornithischian dinosaur genomes of about 2.49 pg. Their findings suggest that much
of the genomic adaptation widely considered to be an adaptation for the metabolic
demands of flight in birds, such as small genome size and low repeat content, are
features that evolved between 250 and 230 mya and, in this lineage, have changed
little since (Organ et al. 2007). These results were initially interpreted as supporting
the hypothesis that small genome size and low repeat content were a genomic
exaptation that preceded and facilitated the endothermic metabolic demands of
birds, e.g. for flight (Hughes and Hughes 1995). It was further hypothesised that
the avian karyotype evolved in response to a reduction in genome size in birds
(Hughes and Hughes 1995). This theory was subsequently challenged by a study that
suggested that a decline in overall genome size occurred in non-volant dinosaurs
(Organ et al. 2007). As we will see in subsequent sections, however, we have
evidence that an avian-like karyotype emerged well before the origin of flight and
was independent of genome size reduction.
4 Karyotype Formation and Evolution in Dinosaurs
In the absence of cellular material or even relatively intact DNA molecules (from
amber or any other biological source), data from genome sequence assemblies of
extant species provide us with the ability to reconstruct karyotypic structures of
extinct lineages by inference. We can do this on the proviso that genomes are
assembled at, or close to, chromosome level, i.e. one contiguous length of sequence
per chromosome (Zhang et al. 2014). In a study that coincided with the publication
of the multiple avian genomics and phylogeny papers (Jarvis et al. 2014; Zhang et al.
2014), we analysed (near) chromosome-level assemblies from six living birds. Using
an Anolis carolinensis lizard outgroup, we inferred the most likely ancestral karyo-
type of all birds for the macrochromosomes and, because outgroup chromosome-
level assemblies were relatively sparse for the smaller chromosomes, for the
Neognathae ancestor for the microchromosomes. We then went on to reconstruct
the most likely sequence of events that led to contemporary karyotypes in birds. We
provided evidence that the chicken (Gallus gallus) was the closest karyotypically to
the reconstructed ancestral pattern, with budgerigar (Melopsittacus undulatus) and
zebra finch (Taeniopygia guttata) experiencing the greatest number of inter- and
intrachromosomal rearrangements, respectively (Romanov et al. 2014).
Last year we (O’Connor et al. 2018) applied a similar approach to that of 5 years
ago (Romanov et al. 2014) to recreate the most likely ancestral karyotype of
Diapsida. We used both the multiple genome rearrangement and analysis
(MGRA2) tool and molecular cytogenetics. That is, focusing our attention on the
best-quality chromosome-level assemblies of avian (chicken (G. gallus) (Hillier
et al. 2004; Warren et al. 2017), mallard (Anas platyrhynchos) (Rao et al. 2012),
zebra finch (T. guttata) (Warren et al. 2010)), reptilian [Carolina anole lizard
(A. carolinensis) (Alföldi et al. 2011)] and mammalian [grey short-tailed opossum
(Monodelphis domestica) (Mikkelsen et al. 2007)] genomes, we supplemented
bioinformatic data with novel molecular cytogenetic approaches including a univer-
sally hybridising BAC probe set (Damas et al. 2017). Indeed, one of the principal
technical advances that made this study possible was the identification of a probe set
that would hybridise directly across species that diverged hundreds of millions of
years ago (O’Connor et al. 2018). We chose these species as they have robust
chromosome-level assembled genomes and because M. domestica has a karyotypic
structure thought to resemble the mammalian ancestor most closely (Mikkelsen et al.
2007). Among other genome assemblies that might have proven useful in our
analyses, those generated from alligators and turtles (St John et al. 2012; Shaffer
et al. 2013) were discounted as they are too fragmented, i.e. not close to chromosome
level. Also, near chromosome-level assemblies for turkey, budgerigar and ostrich
were ultimately excluded because our studies in these species (not shown) revealed
that the level of fragmentation in these genomes had the potential to introduce false
evolutionary breakpoint regions. Finally, we discounted crocodilians, partly because
of a relative lack of FISH success after multiple attempts and partly because, in any
event, all crocodilian species studied do not have a typical archelosaur karyotype,
i.e. no microchromosomes (Srikulnath et al. 2015). The BACs that we isolated gave
strong hybridisation signals on two turtles Trachemys scripta (red-eared slider) and
Apalone spinifera (spiny soft-shelled turtle) and some signals on A. carolinensis
metaphases. Although these two turtles do not have chromosome-level assemblies,
molecular cytogenetic analysis allowed us to anchor the series of events from the
perspective of an archelosaur ancestor such as Eunotosaurus. A combination of
molecular cytogenetics and bioinformatics therefore allowed us to recreate the inter-
and intrachromosomal changes that occurred from the ancestral diapsid ancestor, to
the archelosaur (bird-turtle) ancestor (Benton et al. 2015), and thereafter through the
theropod dinosaur lineage to birds.
Our data, and interpretations from it, provide strong evidence that most features
that we now associate with a “typical avian karyotype” (see our previous chapter)
were established before the Testudine divergence 255 mya. That is, zoo-FISH
[taking FISH probes isolated from one species and hybridising to the metaphases
of another—in this case chicken probes (Damas et al. 2017)] was largely successful
on the chromosomes of A. carolinensis and especially both turtles (Fig. 2). The data
clearly indicates that most chicken (and by inference, ancestral avian) chromosomes
1–28 + Z are syntenic to those of the spiny soft-shelled turtle A. spinifera (2n ¼ 66).
Exceptions included fusions of chromosome 4 in chicken and chromosome 22 in
turtles. Of the microchromosomal probes, 29 of the original 36 (81%) worked
successfully on chromosomes 10–15 and 17–28. Chromosomes 16 and 28–38
were not included because of the lack of sequence coverage. Analysing probes
that worked on all species revealed that the orthologues of chicken chromosomes
12 and 13 were fused and chromosome 26 was attached to chromosome 4 in
T. scripta and A. carolinensis but represented as separate microchromosomes in
A. spinifera. The chromosome 22 orthologue appeared as a separate chromosome in
A. carolinensis but as fused to the centre of a macrochromosome in T. scripta. Eight
bird microchromosome orthologues (10, 11, 15, 17, 19, 21, 23 and 24) were also
single microchromosomes in all three reptiles studied. To this list we added the
orthologues of chromosomes 14, 18, 25 and 28 when considering turtles only and
added chromosomes 12, 13 and 26 as single microchromosomes in A. spinifera
alone. Hybridisation of some probes to the chromosomes of T. scripta (2n ¼ 50) and
A. carolinensis metaphases (2n ¼ 36) revealed some chromosomes with
Fig. 2 (a) FISH experiment for chicken chromosome probes derived from chromosome
19 hybridising to Trachemys scripta (red-eared slider) turtle chromosomes. Avian and some turtle
karyotypes are very similar. (b) FISH experiment for chicken chromosome probe derived from
chromosome 26 on Anolis carolinensis (Carolina Anole lizard) indicating a “proto-
microchromosome”
microchromosomal homologues attached. This means that they have either fused to
macrochromosomes or, probably more likely, retained the ancestral state present in
the diapsid ancestor. These we termed “proto-microchromosomes” as seen in
Fig. 2b. Indeed, the A. carolinensis karyotype had some broad similarities to the
diapsid ancestor established by the bioinformatic (MGRA) approach. Looking at the
chromosomes of T. scripta (2n ¼ 50) in isolation, there are broad similarities to birds
but with more microchromosomal homologues attached to larger chromosomes than
in A. spinifera. This would either indicate that T. scripta has a karyotype that
represents an earlier stage of differentiation to the “bird-like” turtle pattern or that
it subsequently underwent a series of fusions (such as in the crocodilians). The first
of these two options is the most parsimonious as it requires fewer events. Moreover,
the range of diploid numbers in turtles is 2n ¼ 26–68; the majority are more “avian-
like” than their other reptilian counterparts, inspiring us to further study turtles in
order to shed increasing light on the evolution of the dinosaur pattern. Among
macrochromosomes, chromosome painting data indicates that chromosomes 1–9
+Z are also all precise counterparts, with the avian W chromosome evolving after the
Palaeognathae-Neognathae divergence since dimorphic sex chromosomes are absent
from turtle and ratite karyotypes. Given that we are talking about an overall pattern
involving multiple chromosomes, rather than individual events, convergence (homo-
plasy) seems to be unlikely, this leaving us with the only option of identity by
descent leading us to the inescapable conclusion that the “avian-like pattern” (at least
as far as 2n ¼ 66 with chromosomes syntenic to modern birds) was in place ~255
mya.
A narrative emerges (Fig. 3) therefore of a diapsid ancestral karyotype (~275
mya) with a chromosome number of 2n ¼ 36–46—roughly half would have been
macro- and half microchromosomes (Beçak et al. 1964; Alföldi et al. 2011). This is
the same as that which has been previously reported (Organ et al. 2008) and with
Diapsid Archelosaur Gallus Diapsid Archelosaur Gallus

Ancestor Ancestor gallus Ancestor Ancestor gallus
275mya 255mya 275 mya 255 mya
11
7
12
1
13
14
7 15
16
17
18
2
19
20
21
5
22
23
3
24
5 25
26
27
4 28
26
20 5
20
6
13
7
8
27
9
4a
10
Fig. 3 Overall evolution of chromosomes from the diapsid ancestor, to the archelosaur ancestor to
modern birds
most other reptiles apart from birds and turtles. Rapid rearrangement appears to have
then occurred over a period of 20 million years leading to a pattern similar to that
seen in A. spinifera. We know this because Alföldi et al. (2011) found direct synteny
at the sequence level between the microchromosomes of A. carolinensis and
G. gallus, with all but one lizard microchromosome corresponding to a single
chicken microchromosome. Given that the A. carolinensis genome has
12 microchromosomal pairs compared to the 28–30 seen in most birds, the most
likely explanation is that these 12 were present in the diapsid ancestor, the remainder
evolving thereafter by fission (Alföldi et al. 2011) to at least 2n ¼ 66 by 255 mya.
These conclusions are also consistent with previous studies using chicken
macrochromosome paints on Chinese soft-shelled turtle (Pelodiscus sinensis)
(2n ¼ 66) (Matsuda et al. 2005), T. scripta (Kasai et al. 2012) and the painted turtle
(Chrysemys picta) chromosomes (both 2n ¼ 50) (Badenhorst et al. 2015) which
provide direct evidence that turtle and bird macrochromosomes are precise
counterparts of one another. Since 255 mya, only ~7 fissions are sufficient to form
the pattern that we see in ratites, Galliformes, Anseriformes, Columbea and Passerea
(among other birds). Determining how soon these changes occurred and the modern
bird karyotype (2n~ ¼ 80) was complete is difficult, particularly as the various
crocodilian karyotypes with their many fused chromosomes do not help us. If a
similar rate of fission that occurred from 275 to 255 mya carried on for another
15 million years, a complete bird-like karyotype would have emerged before the
appearance of the earliest dinosaurs and pterosaurs 240 mya (Baron et al. 2017). At
the other extreme, a complete cessation of fission events 255 mya would indicate that
the earliest dinosaur and pterosaur karyotypes were more similar to that of
A. spinifera or P. sinensis. Either way, these two scenarios are very similar. David
Burt (2002) suggested that most avian microchromosomes were present in the avian
common ancestor >80 mya (Cracraft et al. 2015), arguing that it probably had the
small genome size characteristic of birds and a karyotype of around 2n ¼ 60. In the
light of our recent data, we would disagree with aspects of this as we suggest that the
karyotype came before the reduction in genome size (Burt 2002). Indeed Uno and
colleagues (Uno et al. 2012) suggested that the archelosaur ancestor probably had
microchromosomes like turtles.
It cannot be ignored, however, that there is an association between genomes with
fewer chromosomes (and no microchromosomes) and larger genome sizes around
2.5–3Gb, as in mammals (Kapusta et al. 2017) and crocodilians (St John et al. 2012).
More repetitive elements could provide substrates for interchromosomal rearrange-
ment, which is commonplace in mammals but rare in birds, and it has been suggested
that an avian karyotype provides fewer opportunities for interchromosomal rear-
rangement due to the existence of fewer recombination hotspots (despite a higher
overall recombination rate) (Kawakami et al. 2014; Smeds et al. 2016), repeat
structures (Gao et al. 2017; Warren et al. 2017; Mason et al. 2016), endogenous
retroviruses (Cui et al. 2014; Farré et al. 2016; Romanov et al. 2014) and more
conserved noncoding elements (Damas et al. 2017). Fission and intrachromosomal
rearrangement are not precluded in this model, however. Returning to the discussion
about the relationship between genome size, flight and karyotype therefore, the
evidence suggests that the avian-like karyotype came first, followed by a reduction
in genome size, followed by flight. Moreover, although flight evolution might be
correlated with smaller genome size [consider pterosaurs vs. other avemetatarsalians
(Organ and Shedlock 2009); bats vs. other mammals (Hughes and Hughes 1995);
strong vs. weak flying/flightless birds (Gregory 2005)], other mechanisms are almost
certainly involved.
5 Intrachromosomal Rearrangement and the Role of Gene

Ontology Analysis
Our results suggest that, aside from ~7 fissions, the primary mechanism for chromo-
somal rearrangement in the avian stem lineage after 255 mya was intrachromosomal
(most likely chromosome inversions). Using MGRA2, we generated 19 contiguous
ancestral regions (CARs). To all intents and purposes, these represented the
chromosomes of the diapsid ancestor. That is, while we could not be entirely certain
that each CAR represented a whole chromosome as MGRA2 will; inherently
“break” the chromosome if it cannot find synteny; a total of 19 diapsid ancestral
CARs probably represented a similar number, if slightly fewer, chromosomes.
Reconstructed CARs, when compared to extant genomes, resulted in the identifica-
tion of rearrangements between the diapsid ancestor and chicken (G. gallus)
genomes. Doing this, we inferred 49 intrachromosomal (chromosome inversion)
events. This, however, must be an underestimate due to the lack of sequence
coverage in some areas, particularly the smallest bird microchromosomes. Deter-
mining rates of change is little more than educated guesswork, but there is some
evidence of intrachromosomal change speeding up in modern birds, even in the
chicken, which is thought to be very similar chromosomally to the avian common
ancestor (Romanov et al. 2014). Increased intrachromosomal change has been
reported in specific groups, with several studies suggesting that the greatest rates
would be found in songbirds (Skinner and Griffin 2011; Farré et al. 2016; Zhang
et al. 2014), the bird group with the most species. Similarly, bursts of speciation may
have also been accompanied by increased rates of chromosome inversion in other
dinosaur groups.
In our recent study of dinosaur karyotypes, we found nearly 400 HSBs (homolo-
gous synteny blocks) and EBRs (evolutionary breakpoint regions) in our analysis
(O’Connor et al. 2018). The number of HSBs per CAR (“chromosome”) was
between 2 and 59 depending on the chromosome. In total, 17 chicken chromosomes
were aligned to these CARs, namely, chromosomes 1–8, 11–13, 15, 18, 24, 26,
27 and Z; some microchromosomes could not be represented. Reconstructed CARs
were then mapped to the extant genomes. From the diapsid ancestor to the chicken
genome, 49 inversions were identified plus 10 interchromosomal changes including
a translocation between the orthologues of chicken chromosomes 5 and 20, consis-
tent with the FISH results. By combining this with the molecular cytogenetic (FISH)
data adds to our existing narrative, suggesting that interchromosomal
rearrangements formed the turtle-like (2n ¼ 66) pattern 275–255 mya with most
intrachromosomal rearrangements (inversions) occurring after. For simplicity of
presentation in Fig. 3, all intrachromosomal changes are shown after formation of
the basic archelosaur common ancestor pattern 255 mya, although we cannot
preclude the possibility that some happened before. Between the diapsid and
archelosaur common ancestors, a fusion most likely occurred to form chromosome
1, and translocations/fissions occurred between avian ancestral CARs that became
chromosome 7 (Fig. 3).
Studies of the best assembled genomes also indicate that EBRs are located within
gene-dense loci and are enriched with genes related to lineage-specific biology,
transposable elements and other repetitive sequences (Farré et al. 2016; Damas et al.
2017; Alföldi et al. 2011; Hillier et al. 2004). Conversely, sequences that stay
together during evolution (HSBs) are enriched for developmental genes and regu-
latory elements (Hillier et al. 2004). While random breakage during karyotype
evolution (Nadeau and Taylor 1984) cannot be excluded completely, especially
when they have a neutral effect on phenotype, there is mounting evidence that the
larger HSBs and selected EBRs (at least in animal genomes) are maintained
nonrandomly (Pevzner and Tesler 2003; Larkin et al. 2003; Farré et al. 2016;
Damas et al. 2017). There are certainly regions more prone to breakage (e.g. in
recombination hotspots or open chromatin areas), and those chromosome breaks not
disturbing essential genes or providing a selective advantage are more likely to be
fixed in populations, thereby becoming EBRs (Farré et al. 2016). In other words,
chromosome rearrangement may serve a functional purpose.
When we examined GO (gene ontology) terms in HSBs, significant enrichments
were observed for those relevant to amino acid transmembrane transport and signal-
ling as well as synapse/neurotransmitter transport, nucleoside metabolism and use,
cell morphogenesis and cytoskeleton and sensory organ development. HSBs are
often enriched for GO terms related to phenotypic features that remain constant
(Larkin et al. 2009), and the results presented here are consistent with this hypothe-
sis. Sankoff (2009) however proposed that EBRs are where the “action” in genome
evolution lies, and, previously, we found that GO terms in avian EBRs are associated
with specific adaptive features, e.g. enrichment for forebrain development in the
budgerigar EBRs (consistent with vocal learning) (Farré et al. 2016). Among the
EBRs, we identified significant enrichments in genes and single GO terms relevant
to chromatin modification and chromosome organisation as well as proteasome/
signalosome structure. Specifically, our first GO annotation cluster consisted of six
genes related to proteasome/signalosome structure within EBRs located on six
chicken chromosomes. The second annotation cluster included 15 genes relevant
to chromatin modification within 12 EBRs on seven chicken chromosomes. These
results illustrated some parallels to recent findings in rodents where chromosomal
changes were associated with open chromatin (Capilla et al. 2016) and the majority
of the 15 genes found in this GO term were related to control of gene expression.
Transcription factors modify chromatin by making it accessible during transcription,
and this is relevant because EBRs rearrange transcription factor genes. This might
affect expression of other genes of the same pathway. It is interesting to note that two
of these genes showed a different expression pattern when comparing birds and
mammals: HDAC8 functioning in early embryo development (Murko 2010) and
PRMT8 expressed in the brain (Wang et al. 2017). Our results thus suggest a
correlative link between chromosomal and morphological changes among species,
in some way mediated by rearranging genes controlling the expression of develop-
mental pathways.
6 Karyotypic Stability Leading to Phenotypic Diversity?
The fact that this avian-like karyotype has remained quite unchanged for such a long
evolutionary period suggests a manner of genome organisation that might have
provided the raw materials for evolutionary success. Reasons for such success
might, we have suggested, be due to its ability to generate variation, the basis of
natural selection. That is, when a karyotype has many chromosomes, including
microchromosomes with high recombination rates, this inherently generates greater
variation through increased genetic recombination and increased random chromo-

some segregation. David Burt (2002) also suggested that a higher recombination rate
has contributed to the unique genomic features seen in microchromosomes such as
high GC content, low repeat content and high gene density, which subsequently led
to its maintenance. Phenotypic variation, in turn, promotes rapid adaptation and may
therefore have contributed to the >10,500 species of birds and who knows how
many species of non-avian dinosaurs. Of course, a karyotype with many tiny
chromosomes is not the only means by which variation can be generated (genic,
epigenetic and interchromosomal variation all come to mind), and amphibians have
enormous phenotypic variation but few chromosomes. We nonetheless think that
this may explain the apparent paradox of a group with very little interchromosomal
change but incredible phenotypic diversity. Thus, while we have previously thought
that the characteristic avian karyotype might help explain the differences between a
hummingbird and an ostrich (and most things in between), we perhaps should
broaden our minds and think that it might help explain the difference between a
hummingbird and a tyrannosaur.
So, although there will be no Jurassic Park, or Jurassic World for that matter, we
can reconstruct the overall likely karyotype of common ancestors inferring the
sequence of events that led to living animals. Armed with that information, it
becomes easy to suggest that if we had the opportunity to make metaphase chromo-
some preparations from the tissue of some of our favourite theropod dinosaurs
(Tyrannosaurus and Velociraptor are both genera of the group), then karyotype
and zoo-FISH results would reveal little difference from a modern chicken, pigeon
duck or ostrich. Of course, it is probably the case that there are some groups which
underwent significant interchromosomal change: kingfishers (Christidis 1990)
(many fissions), parrots (Nanda et al. 2007) and falcons (Damas et al. 2017)
(many fusions) are modern examples of this, but it would be hard to guess what
they were.
In discovering that the avian karyotype had deeper origins than previously
thought, this echoes other recent discoveries about dinosaur morphology,
demonstrating that features hitherto believed to be characteristic of crown-group
birds only (e.g. feathers and pneumatised skeletons) arose first among more ancient
dinosaur or archosaurian ancestors (Zhou 2004; Baron et al. 2017). “Everyone”
loves dinosaurs. They were the dominant group of animals for nearly 200 million
years; their radiations following two mass extinction events and, despite being
almost wiped out by a third (the K-Pg meteor impact), their resilience as a highly
diverse and speciose clade (extant birds) (Barrowclough et al. 2016) have fascinated
scientists since they were first discovered. Of course, many of the evolutionary
changes were in response to a rapidly changing environment, and many would
have involved individual genes. A possible “next step” in our research therefore
might be to infer individual genic sequences and their possible functions in individ-
ual animals.
In conclusion, dinosaur karyotypes are not just descriptions (and inferred ones at
that). The gross genome organisation and evolution of dinosaur chromosomes
(including the success of modern birds) might have been a major factor contributing
to their phenotypic diversity, unique physiology and ability to adapt (Berv and Field
2018) and, ultimately, to survive. Jurassic Park surely would have not been quite so
interesting.
Acknowledgements We are grateful to a number of our colleagues and co-authors who made this
work possible: Marta Farre-Belmonte, Joana Damas and Michael Romanov for providing bioinfor-
matic analysis for our various studies, Nicole Valenzuela for excellent turtle metaphases, Malcolm
Ferguson-Smith for samples and sagely insight, Paul Barrett for much needed palaeontological
input as well as Rebecca Jennings and Lucas Kiazim for FISH analysis.
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ISBN 978-3-030-16476-8 ISBN 978-3-030-16477-5 (eBook)

# Springer Nature Switzerland AG 2019

An Introduction to “Avian Genomics in Ecology and Evolution: From

# Springer Nature Switzerland AG 2019 1

The recent rise of next-generation DNA sequencing technologies has transformed

genomics, whether technical or conceptual, require some time for familiarization.

1 Ornithology: From Aristoteles to Linné

# Springer Nature Switzerland AG 2019 7

1. Accipitres: raptors, owls, parrots, shrikes and waxwings

2 Towards a Natural System of Birds in the Twentieth

Ornithologists after Darwin tried to establish a “natural system” of birds, which

Taxonomy and systematics were mainly based on morphological characters, such

3 Systematics and Phylogeny in the Age of DNA Analysis

5 Comparison of the Sibley Phylogeny with NGS Data (See

6 The Era of DNA Sequence Analysis

STRUTHIONIFORMES RHEIFORMES CASUARIIFORMES APTERYGIFORMES

GALLIIFORMES Megapodiidae Cracidae Numididae

EURYPYGIFORMES Eurypygidae Rhynochetidae

Psophiidae Aramidae Gruidae

APODIFORMES Aegothelidae Hemiprocnidae Trochilidae Apodidae

PELECANIFORMES Balaenicipitidae Threskiornithidae

Charadrii Lari Scolopaci

ACCIPITRIFORMES Cathartidae Sagittariidae Pandionidae Accipitridae

STRIGIFORMES Tytonidae Strigidae

BUCEROTIFORMES Upupidae Phoeniculidae Bucerotidae Bucorvidae

PSITTACIFORMES Psittacidae Cacatuidae Strigopidae

PASSERIFORMES Suboscines Oscines

Alain Vignal and Lel Eory

# Springer Nature Switzerland AG 2019 21

1 Introduction: Chicken as a Model Organism for Birds

Fig. 1 A sample of selected phenotypic variation in chicken

groups collaborating with one or several high-capacity sequencing centres (Mullikin

2 The Dawn of Chicken Genomics: From Maps to Genome

2.1 Genomics for Improving Breeding Practices

Interestingly, the ﬁrst coordinated efforts towards genome-wide studies in chicken

B: maps, sequence and structural annotation

Radiation hybrid map

BAC contig map

Alignement to genome: structural annotation

Tissues Protein identification

common duck genome assembly is composed of 227,597 contigs assembled into

2.2 The Definition of the Karyotype and Cytogenetic Maps

The deﬁnition of the karyotype, a description of the number and appearance of

assignment of DNA fragments to chromosomal regions. Without this, genome

2.3 Genetic Maps and the Initial Mapping of Phenotypic Traits

2.4 Physical Maps: A Step Towards Positional Cloning

2.5 Expression Maps, Comparative Maps and Low Chromosomal

2.6 The First Chicken Genome Assembly

2.7 The Missing Microchromosomes and Towards a Complete

Table 1 Gallus gallus genome assembly statistics

and as a consequence the inclusion of chromosome 31, in Galgal5 (Warren et al.

2.8 Statistics on the Chicken Genome Assemblies

2.9 Understanding the Genome: The Annotation

towards detecting functional elements in the genome is to detect constrained

3 The Breeder’s Perspective on the Genome

3.1 Marker-Assisted Selection, Genomic Selection and QTL

3.2 Identification of Genomic Events Causing Variation

autosomal albinism (Tobita-Teramoto et al. 2000). The lavender phenotype is due to

3.3 The Complex Case of Quantitative Trait Loci (QTLs)

Identifying the molecular mechanisms underlying QTLs is much more challenging

4 Exploiting Breeding Populations and Model Organisms

4.1 Domestication Syndrome