Download A pragmatic approach for selecting evo

CHAPTER

A pragmatic approach for selecting evo-devo
model species in amniotes
Athanasia Tzika and Michel C. Milinkovitch
One major classical justification of using a model metazoan species for
experimentation has been that discoveries of biological phenomena in
that species could be extrapolated to other multicellular species. Because
the chances that this extrapolation is valid in humans depend on the phylogenetic distance between humans and the model species, many researchers
have somewhat sacrificed the major benefits of small size, short generation
time and ease of manipulation that characterise some invertebrates in order
to use species that humans can more readily relate to, such as the laboratory
mouse (Mus musculus). However, the community of biologists has continued to use additional model species because each of the selected
taxa have specific features that make experimental manipulation easier
(e.g. easy-to-score morphological, variation and giant polytene chromosomes in Drosophila melanogaster, or accurate description of the largely
invariant complete cell lineage and full neural connectivity of the roundworm Caenorhabditis elegans).
Ever since the molecular genetic revolution, a constant concern has been
the possibility of manipulating the genome of model species. For example,
generations of Drosophila scientists have developed and applied ingenious
approaches that allow, in principle, screening for any phenotype at any
stage of development (reviewed in St Johnston ). Even for the
mouse model, multiple techniques, such as homologous recombination,
tissue-specific activation/inactivation techniques, cloning and RNA interference (RNAi), have been developed for performing genotype- or
phenotype-driven experiments. Furthermore, recent access to full genome
sequences makes genome engineering of some model species easies.
However, one of the most important limiting factors to the utility of
genome engineering approaches is the difficulty with which phenotypes
can be identified. Indeed, many genes exert functions that cannot be
Evolving Pathways: Key Themes in Evolutionary Developmental Biology, ed. Alessandro
Minelli and Guiseppe Fusco. Published by Cambridge University Press. # Cambridge
University Press .


ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
investigated by simple examination of a few general morphological
and/or physiological parameters. Fortunately, imaging technologies and
physiological measuring techniques have recently been miniaturised and
adapted for use with small species. Another source of difficulty for phenotyping is due to the epistatic effects among genes. For example, multiple
studies have demonstrated that a full knock-out mouse for a gene supposedly essential for a given major process can yield no visible phenotype
because another gene has become involved to fulfil the function of the
invalidated gene.
MEDICINE-DRIVEN MODELS
(SEARCHING
FOR EVOLUTIONARY
CONSERVATION) VS. EVO-DEVO-DRIVEN MODELS
(UNDERSTANDING
EVOLUTIONARY CHANGE)
As indicated above, most of the research performed so far with model
species has been justified by the potential power of these species for understanding human biology. However, in the context of evo-devo, it is the
massive realm of living species that should, ideally, be opened to
genome manipulation and phenotypic investigation. Indeed, the interests
of evolutionary developmental biologists go well beyond conserved physiologies and developmental processes or patterns as they seek to understand
the generative mechanisms underlying biological diversity (Minelli ).
Uncovering these mechanisms will require the merging of several disciplines (Milinkovitch and Tzika, in press), including molecular developmental biology, evolutionary molecular genetics, paleontology (Wagner
and Larsson ) and ecology (Dusheck , Gilbert and Bolker
). Hence, one major challenge in evo-devo will be to adapt the tremendous knowledge and sophisticated technologies accumulated on ‘classical’ model species to model organisms from a wider assortment of
lineages on the tree of life in a wider set of environmental conditions.
However, as demonstrated in the past, promoting the use of the same
set of model species increases the efficiency with which techniques and
analytical approaches are developed simply through collaboration, emulation and establishment of public databases. For example, given the necessity to optimise the interplay between in-silico analyses and in-vivo
experiments, it is crucial to establish the list of preferred species for
which the genome will be sequenced in priority, and this should be
done with more input from the evo-devo community (Milinkovitch and
Tzika, in press).
Model species in amniotes

CRITERIA FOR CHOOSING NEW MODEL SPECIES
Beside the practical criteria of small size, short generation, abundant
progeny, ease of manipulation and of housing/breeding, accessibility of phenotyping and genome manipulation techniques, etc., there are other parameters that should be considered when listing preferred model species.
An intuitive and simple criterion to guide the choice of model species is
the evolutionary divergence among these species (Figure .). However, this
phylogenetic distance criterion is limited by, among others, two parameters:
() the rate of phenotypic transformation is highly variable among lineages
and () variation worth investigation exists at multiple phylogenetic levels
(one should not focus only on major transformations). Intermingled with
the phylogenetic distance criterion, the ancestrality of a model species is a
decisive factor. For example, the zebrafish is often considered a canonical
vertebrate (Fishman ) because the common ancestor of all vertebrates
was fish-like. Although intuitively appealing, this statement is of limited
value because, on average, no extant species is intrinsically more ancestral
than any other. So, the real, non-trivial, question is: what is the considered
species a model of (i.e. at what hierarchical level(s) of the phylogeny)? The
answer depends heavily on what characters one is interested in. For example,
as far as anatomy is concerned, the zebrafish might be a better model for
teleost fishes than for vertebrates because the species exhibits multiple characters that seem ancestral for the former group, whereas it is very derived (as
all teleost fishes are) in respect to the vertebrate ancestor (Metscher and
Ahlberg ). Furthermore, although we agree that structural and
genomic simplicity can help in guiding the choice of a model species (as
in the case of the cephalochordate amphioxus; Holland et al. ), it
should be carefully investigated whether the observed simplicity is ancestral
for the group and not a secondary (derived) simplification, as is likely to be
the case for flatworms and myzostomes, for example (Eeckhaut et al. ).
The availability of robust and extensive (molecular) phylogenetic
hypotheses constitutes an important criterion for choosing evo-devo
model species: evolutionary trees constitute the basic framework on
which character changes are mapped. For example, mapping of phenotypic
characters on robust molecular phylogenies have demonstrated extensive
and multiple convergences of ecologically specialised species (ecomorphs),
e.g. of cichlid fishes (Kocher et al. , Ruber et al. ), ranid frogs
(Bossuyt and Milinkovitch ), Anolis lizards (Losos et al. ) and
mammals (e.g. between some Afrotherian and Eulipotyphla insectivores,
see below). Similarly, the snake-like body form has evolved multiple times

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
300
250
200
150
100
50
0 mya
Chiroptera
Cetartiodactyla
Perissodactyla
Pholidota
Carnivora
Eulipotyphla
(1) LAURASIATHERIA
(2) EUARCHONTOGLIRES
(3) AFROTHERIA
(4) REPTILIA
Rodentia
Lagomorpha
Primates
Scandentia
Dermoptera
(1)
(2)
Xenarthra
Eutheria
Sirenia
Hyracoidea
Proboscidea
Tubulidentata
Macroscelidea
Afrosoricida
Mammalia
(3)
Amniotes
Marsupialia
Monotremata
Lizards
Snakes
Testudines
Aves
Palaeozoic
Mesozoic
Cenozoic
Crocodylomorpha
(4)
Figure . Right: cladogram showing the best estimate of the phylogeny of amniotes
based on molecular data. The Eutheria cladogram is based on Bayesian and Maximum
Likelihood analyses of  bp of DNA from  nuclear and  mitochondral genes
from  placental and  marsupial extant species (Murphy et al. b). All nodes are
supported by posterior probabilities >% except the nodes indicated with an asterisk.
Note that current analyses of morphological data still disagree with a significant portion
of this cladogram. Left: phylogram showing the time of divergence among major
amniote lineages. Divergence times (and % credibility intervals, grey bars) within
Eutheria were obtained (Springer et al. ) by analysing the  bp dataset cited
above under a Bayesian approach allowing rates variation among lineages and
incorporating fossil information in the form of constraints on divergence times (Kishino
et al. ). Some paleontologists favour a model of radiation of placental mammals
after the Mesozoic/Cenozoic boundary. Grey bars on the Marsupials versus Eutherians,
and Monotremes versus Theria splits are the lowest and highest estimates of divergence
time (i.e. considering standard errors) obtained with two molecular data sets of  bp
for  taxa and  bp from  taxa, respectively (van Rheede et al. ). Divergence
time for other nodes on the tree (arrows) are minimum age estimates obtained from
palaeontological data (Benton and Donoghue ). Dotted lines indicate that no
reliable estimate for the divergence of snakes from lizards is available. Two lineages of
lizards are indicated to underline the likely paraphyly of that group (with respect to
snakes). Geological eras are indicated (Paleozoic, – mya; Mesozoic, – mya;
Cenozoic, – mya).
Model species in amniotes

independently in squamate reptiles (Wiens et al. ). Investigating the
development of such convergent traits in different lineages could form
the basis for understanding possible general mechanisms involved in convergence (see Jenner, Chapter  of this volume, for a discussion on nomothetic
versus idiographic approaches to evo-devo). Such analyses will require a
strategy of choosing model organisms based on their traits rather than
phylogenetic position per se (Figure .).
A MULTIDISCIPLINARY PRAGMATIC OPTIMISATION
APPROACH TO THE SELECTION OF MODEL SPECIES
As further developed elsewhere (Milinkovitch and Tzika, in preparation),
the criteria that are relevant to the choice of a set of model species are multiple and can even be contradictory. This is intrinsic to the highly multidisciplinary nature of evo-devo. For example, a lineage can be characterised by a
unique and dramatic set of derived character states that makes it particularly
appealing for evo-devo studies, but the group might lack representatives
that could reasonably constitute a widespread model species in the laboratory
(e.g. take the extreme case of cetaceans). We think that the only possibility is
the use of a pragmatic (and partly subjective) optimisation approach, incorporating criteria such as phylogenetic position (Figure .) as well as number
and nature of the ancestral/derived character states of the model species, level
of diversity within a relevant higher taxon to which the chosen species
belongs, ease with which the representative species can be handled, housed
and bred, and their protection status. Compromises will have to be made,
as it is simply impossible to find species that combine all possible advantageous features. We apply such an approach below for a set of species
that could serve as the workhorses for evo-devo research within amniotes.
We shortly discuss the advantages and disadvantages of some candidate
species and hope this will be used as a starting point for an in-depth analysis
with input from morphologists, paleontologists, animal breeders, physiologists, developmental biologists and molecular phylogeneticists.
REPTILIA
Several lineages within Reptilia (including birds) are more genetically
diverse than mammals such that using a single reptilian species from a
single lineage seems insufficient, especially given that the Mammalia is
already represented by several model species (see below).

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
Anapsida
Testudines
This clearly monophyletic group (turtles, tortoises and terrapins) includes
only about  extant species in  families and is characterised by several
synapomorphies of which the most obvious is the shell (enclosing front
and hind limb girdles) comprising a dorsal carapace (made from dermal
bones associated with endochondral modified vertebrae and ribs) and a
ventral plastron (made of clavicles, interclavicles and abdominal ribs).
They have relatively low metabolic rates and are dependent on external
sources of body heat, although the leatherback turtle (Dermochelys
coriacea) seems to show some degree of endothermy. High longevity might
also be a character of interest. Long generation time is an obstacle to using
testudines as models: in most species, males and females reach sexual maturity
not before  and  years, respectively. Analysis of development is feasible as
eggs can be incubated artificially (Gilbert et al. ).
Two species have been suggested as candidates for full genome sequencing (www.reptilegenome.com): Trachemys scripta (red-eared slider) and
Chrysemys picta (painted turtle). Individual size, and housing, handling
and breeding techniques, are very similar for the two species but the
latter is much more difficult to breed and much less prolific than the
former. Shell morphogenesis has been investigated in Trachemys
(Gilbert et al. ), whereas a BAC library is available for Chrysemys
(www.nsf.gov/bio/pubs/awards/bachome.htm), aiding the sequencing of
its genome. For sure, only one of these two species should be sequenced
as they are redundant phylogenetically (they belong to the same subfamily
Deirochelyinae, in the family Emydidae). These species are closely related to
the tortoises (family Testudinidae) (Krenz et al. ), of which several
species in the genus Testudo are successfully bred around the world and
detailed husbandry information is available (Lapid et al. ).
We think that an additional species, Pelodiscus sinensis (Chinese softshell turtle, see Figure .A), deserves attention from the evo-devo community. This species is the smallest soft-shell turtle, and belongs to a superfamily (Trionychoidea) phylogenetically distant from the species discussed
above. Housing and breeding of Pelodiscus sinensis are very well documented
because numerous farms exist, mainly in Asia, with a production exceeding
six million individuals per year. Furthermore, Hox genes have been characterised and their expression patterns investigated (Ohya et al. ), details
of the shell development are known (Kuraku et al. ) and a turtle–
chicken chimera has been generated (Nagashima et al. ). In addition,
Model species in amniotes

Figure . Some of the proposed new model species for amniotes: A, Pelodiscus sinensis,
the Chinese soft-shell turtle; B, Pogona vitticeps, the bearded dragon; C, Elaphe guttata, the
corn snake; D, Crocodylus niloticus, the Nile crocodile; E, Petaurus breviceps, the sugar glider
(the left and right insets show the patagium and the embryo in the mother pouch,
respectively); F, Echinops telfairi (the lesser hedgehog tenrec); G, Atelerix albiventris, the
African pygmy hedgehog. A colour version of this figure is available at www.ulb.ac.be/
sciences/ueg/modelsp.

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
several cDNA libraries have been developed (Kuraku et al. ) and ideal
incubation conditions have been identified (Du and Ji ).
All species mentioned above belong to the Cryptodira. Less information
is available for members of the Pleurodira. Possibly the best candidate as a
Pleurodira model species is Emydura subglobosa, the ‘pink-bellied sideneck’,
a mid-sized (about  cm) aquatic turtle native to Australia and Papua New
Guinea. The species is robust and prolific, and is becoming an increasingly
popular pet around the world.
Lepidosauria
Lizards
Sequencing of the full genome of Anolis carolinensis, the green anole lizard,
has recently been initiated (www.genome.gov/). Individuals are
small (average cm for females and cm for males), and their life span
is – years. Their maintenance is, however, not trivial. As most lizards,
they are oviparous although their reproduction presents a peculiar feature:
females produce a single egg every  to  weeks with successive alternation
of the offspring’s sex, a phenomenon that seems hormonally controlled by
the female (Lovern and Passek ). However, only an average of seven
eggs are laid per year (the maximum being ), limiting the material available
for developmental biology studies. Extensive literature on the species
(especially behavioural and ecological studies) has accumulated since the
s, mainly owing to the animal’s small size and abundance of the
species in North America (see Lovern et al.  for a review).
We consider, however, that an alternative species, Pogona vitticeps
(bearded dragon; see Figure .B), deserves attention from the evo-devo
community. Bearded dragons are larger than anoles as they have an
average adult size of about  cm, and they have recently become a
common pet all around the world. Indeed, these animals are tame, easy
to handle and feed, and much information is accessible on their maintenance and reproduction. Female bearded dragons are significantly more prolific than the green anole as the former can lay about  eggs up to six times
a year.
Snakes
The Reptilian Genomics Working Group has suggested the sequencing of
the full genome from Thamnophis sirtalis (the common garter snake).
This is a relatively small-sized species (– cm) easy to maintain in
Model species in amniotes

captivity. Individuals reach sexual maturity at about  cm (about  years
old) and live in captivity up to  years. After a three-month gestation,
females produce clutches of  to  offspring, depending on the size of
the mother. Given its accessibility in nature in multiple areas of the
United States, this species has been widely used for physiology and
ecology research programs, but not in developmental biology. A key reproductive feature could prove problematic: Thamnophis sirtalis is ovoviviparous. Although ovovivaparity (and associated placental membranes) is by
itself a subject worth investigating (Krohmer ), this feature seriously
limits the accessibility of embryos at late developmental stages unless the
mother is sacrificed.
This is why we suggest that Elaphe guttata (the corn snake, see
Figure .C) constitutes a valid alternative to the common garter snake. It
has the great advantage of being oviparous, such that embryos can be incubated artificially for investigation at any stage of the – days of development after egg laying (i.e. about  days after fertilisation) (Kohler ).
Archosauria
Crocodylomorpha
The large size and long generation time (sexual maturity depends on size
but in no species do individuals reach sexual maturity before the age
of ) is a discouraging factor for their use in evo-devo studies, but we
think the task is not impossible because eggs are available in crocodile
farms all around the world, mostly for Alligator mississippiensis and Crocodylus niloticus. Little information is available on the development of crocodiles (Guillette and Gunderson , Tissir et al. , Milnes et al. ),
and their sister-group relationship with birds makes this group especially
interesting for evo-devo studies. The need to collaborate with crocodile
farms is probably the most important constraint on the choice of species.
Not surprisingly, the US-based Reptilian Genomics Working Group recommends the sequencing of the Alligator mississippiensis (American alligator) genome, but this species is unfortunately common only in the
United States, whereas Crocodylus niloticus (Nile crocodile, see Figure .D)
is much easier to find in European and African farms and zoos and is phylogenetically closely related to Crocodylus porosus, the species most studied
by Australian laboratories. Furthermore, A. mississippiensis could be advantageously replaced by another representative of the Alligatorinae subfamily:
the Cuvier’s dwarf caiman (Paleosuchus palpebrosus), which has the great

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
advantage of reaching a small adult size of about  cm. However, its generation time is not significantly shorter than those of larger species and it
remains a dangerous animal to handle (Grenard ).
Aves
Birds, the sister group to Crocodylomorpha, constitute a highly diverse group
comprising about   extant species in about  orders. It is very likely
that Gallus gallus, the domestic fowl (Galliformes), will remain the
obvious focal representative model species of Aves because it exhibits a
short generation time (sexual maturity is reached at the age of about 
months), its reproductive output is staggering (a single hen can lay about
 eggs a year and a single rooster can produce   offspring a year),
much information is available on the embryonic development, captive
holding and breeding techniques incorporating automated feeding and egg
collection are very well documented and used all around the world, numerous
inbred strains (exhibiting different phenotypes) are available, and regulations
associated with captive holding/breeding are well known and much simpler
than for other farm animals. The domestic fowl has served as a model system
since Aristotle, and new technologies, such as in vivo electroporation and
transgenesis, have been continuously incorporated. The development of
chicken embryonic stem (ES) cells is an active topic and the genome of
the species has been sequenced (Burt , Stern ).
Despite the overwhelming advantages discussed above for the domestic
fowl, other species have been used as models. For example, multiple species
and strains of quails (genus Coturnix, Galliformes) have been used in behavioural studies (Mills et al. ) and reproductive ageing studies
(Ottinger et al. ). The Passeriformes, accounting for about  % of
the extant bird species (Poole ), also includes several model species,
especially popular with students of sexual selection and animal communication, such as Sturnus vulgaris (starling) (Gentner et al. ), Passer domesticus (house sparrow) (Bonneaud et al. ) and Taeniopygia guttata (zebra
finch) (Johnson and Whitney , Birkhead ). Captive handling information is available for representatives of a few additional groups, which
should aid comparative developmental studies undertaken by evo-devo scientists: Tyto alba (barn owl) and Athene noctua (little owl) of the Strigiformes
are successfully bred in captivity, the widespread Columbia livia (pigeon)
would be an obvious model species for Columbiformes, whereas Branta bernicla (brent goose) and numerous pet parrot species could be used as models
of Anseriformes and Psittaciformes, respectively (Poole ). Finally,
Model species in amniotes

Struthio camelus (the ostrich, Struthioniformes) is now bred in farms worldwide, making it possible to investigate development in this species (e.g. in
relation to the loss of flight abilities).
MAMMALIA
Monotremata
Egg-laying mammals include only two genera comprising one species of
Ornithorhynchidae and two species of Tachyglossidae. Ornithorhynchus
anatinus (the platypus) is very difficult to maintain and breed in captivity
despite efforts to do so starting as early as the s (Temple-Smith and
Grant ). On the other hand, Tachyglossus aculeatus (short-beaked
echidna) is a reasonably common animal in zoos throughout the world
and can be bred reasonably easily in captivity. Echidnas could serve as
model species not only for their peculiar reproductive features but also to
investigate other spectacular traits, such as the presence of spines and the
characters associated with their ability to feed on ants (Jackson ).
Obviously, the accessibility to a Monotremata species for evo-devo studies
would be of tremendous interest given their phylogenetic position and
the retention of ancestral traits in their anatomy in general and their reproductive system in particular. Unfortunately, many zoos are disinclined
to provide material given the restricted availability of animals outside
Australia. The low-coverage assembly of the platypus genome has recently
been made available.
Marsupialia
Monodelphis domestica (the grey short-tailed opossum) is the marsupial
species most often used in laboratories because of its small size, large reproductive output and short generation time (males and females reach sexual
maturity before  months). The species has been used to study the development of some marsupial traits, such as the formation of the placenta (Freyer
et al. ), of the median vagina and the pouch (Regli and Kress , Kress
et al. ), of the neural plate, neural crest and facial region (Smith ),
and of male morphology (Wilson et al. ). It is also used as a model for
studying cutaneous melanoma induced by ultraviolet radiation (Wang et al.
). One of the great advantages of using marsupials as model species is the
accessibility, from an early developmental stage, of the embryo in the mother
pouch. Finally, Monodelphis domestica has the great advantage of having
had its genome completely sequenced (www.broad.mit.edu/mammals/

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
opossum). Disadvantages of the species are its aggressive behaviour beyond
the age of  months, its preference for solitariness (Poole ) and the
difficulty of obtaining animals outside the United States.
We suggest that Petaurus breviceps (the sugar glider, see Figure .E)
should also be considered as an additional valid model species. Its size and
reproductive output are similar to those of the grey short-tailed opossum
but, unlike the latter, it is a social animal so that large groups can be maintained in captivity. Sugar gliders can breed all year around through artificial
regulation of the photoperiodicity associated with their reproductive cycle
(Jackson ). As they are becoming popular pets all around the world,
they are much easier to find than short-tailed opossums. Finally, sugar
gliders have a skin membrane, called patagium, extending from the forelimb
to the hind foot and used to glide between trees. This anatomical feature is of
interest in the evo-devo context because it evolved independently in three
marsupial families, two rodent families and the flying lemurs.
Eutheria
Afrotheria
The representatives of this major clade (Figure .) of mammals exhibit a
spectacular diversity of sizes and morphologies, from the tiny Microgale
species (about  grams) to the Asian and African elephants (about
 tons). Despite the fact that there is, to our knowledge, no known single
morphological character that would clearly support that clade, analyses of
multiple molecular sequence datasets unambiguously support its existence
(Stanhope et al. , Murphy et al. a, van Dijk et al. ) as a
lineage grouping elephants, sea cows, hyraxes, aardvarks, elephant shrews,
golden moles and tenrecs. Literature on afrotherian mammals is scarce,
but reliable data (originating mainly from zoos all around the world) are
available on their maintenance and reproduction, such that evo-devo
studies would almost certainly require collaborations with zoological institutions. Given their size and long generation time, the Tubulidentata (aardvark), Proboscidea (elephants) and Sirenia (dugongs, sea cows and
manatees) will obviously be the most difficult to investigate. On the
other hand, representatives of the Afrosoricida (tenrecs), Macroscelidea
(elephant shrews) and Hyracoidea (hyraxes) are much more amenable to
housing and breeding in the laboratory. Tenrecs are particularly interesting
as they retain some primitive mammalian features, such as the lack of
scrotum in the male or the presence of a cloaca in females (as in marsupials
Model species in amniotes

and monotremes). In addition, several species of tenrecs have spines, a character that has convergently evolved in representatives of other mammalian
lineages. Finally, tenrecs could constitute useful models for understanding
the mechanisms associated with endothermy as their thermoregulation is
less efficient than in other eutherians. We suggest Echinops telfairi (lesser
hedgehog tenrec, see Figure .F) as the model species of choice because
() it is one of the smallest species for which breeding colonies already
exist in research facilities, () it has spines macroscopically very similar to
those of hedgehogs, and () extensive studies have been undertaken on
the development of the placenta (Carter et al. ), as well as the
brain structure and physiology (Kunzle ). Furthermore, the genome
of E. telfairi has been recently sequenced.
For elephant shrews ( living species), only some anatomical and physiological studies are available (e.g. organisation of the somatosensory cortex
in Elephantulus edwardii [Dengler-Crish et al. ] and thermoregulation
in E. myurus [Mzilikazi and Lovegrove , ]). Very little information
on the reproduction of these species is available, although they have been
bred in the past (Rathbun et al. ). An alternative could be Macroscelides
proboscideus; less information is available on its biology but established
colonies can be found in numerous zoos around the world.
For the Hyracoidea, Procavia capensis seems the obvious choice given that
it is successfully bred in zoos (although gestation takes about  months), it is
relatively small, it is a social animal, and its full genome might soon be
sequenced (www.genome.gov/).
Xenarthra
Representatives of this mammalian group (anteaters, sloth and armadillos) are
confined to South America and are highly specialised animals. Anteaters and
sloths are very difficult to study owing to their large size and special requirements for maintenance, whereas armadillos could be bred in laboratory facilities. The armour shell of armadillos, a structure consisting of bony scutes
covered with keratinous plates, is of interest for evo-devo research, as is an idiosyncrasy of the reproductive strategy of the genus Dasypus: litters typically
consist of four clones as the offspring develop from a single fertilised egg. Breeding in captivity has proven difficult, although not impossible (Carvalho et al.
). The nine-banded armadillo (Dasypus novemcinctus) genome sequence
has recently been made available, although many genes are interrupted or
missing because of the low-coverage (×) assembly: each base in the final

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
sequence is present, on average, in two reads only, as opposed to more than six
reads for other sequenced species such as human or mouse.
Euarchontoglires
This large grouping (Figure .) includes the best-studied mammals as it
comprises the Rodentia (mice, rats, squirrels, gophers, porcupines, beavers,
etc.), Lagomorpha (rabbits, hares and pikas), Primates (lemurs, tarsiers,
new and old-world monkeys), Scadentia (tree shrews) and Dermoptera
(flying lemurs). Only the latter group has not been extensively studied
given the difficulty of keeping these animals in captivity.
Rodentia. Obviously, the laboratory mouse and rat dominate the literature in numerous fields of experimental biology but quite a series of
additional species are used as model species for medical and academic
research such as, among others: Microtus agrestis (short-tailed field vole),
Meriones unguiculatus (laboratory gerbil), and Mesocricetus auratus and
Cricetus grisues (Syrian and Chinese hamster, respectively) (Poole ,
Cavanagh et al. , Mand et al. , Shimozuru et al. ). Finally,
members of the genus Cavia (guinea pigs) have been used in sociophysiology
studies (Sachser ) and are very valuable models for ascorbic acid metabolism because they are the only non-primate laboratory animals that require
a dietary source of vitamin C (Burk et al. ). Guinea pigs are of particular
interest in evo-devo studies because they are representatives of the hystricomorph rodents with the largest amount of available information regarding
their biology as well as captive holding and breeding. The full genome
sequences of the laboratory mouse and rat are available, whereas the genome
of one Cavia species will soon be undertaken (www.genome.gov/).
Lagomorpha. This group is represented by a widely used model species,
Oryctolagus cuniculus (the laboratory rabbit) for which the full genome
sequence is available. Rabbits are widely used for antibody production
and have served as a model organism in many physiological and immunological studies. An emerging additional lagomorph model species is
Ochotona curzoniae (the black-lipped pika) which could be useful for
comparative studies given that it belongs to a different family from the laboratory rabbit. Females can breed and produce litters every three months in
the summer, with the size of the litter depending on environmental conditions (Dobson et al. ). Little empirical information is available
regarding its captive breeding, but specimens are available in the pet
trade. Genome sequencing has been proposed for this species (www.
genome.gov/).
Model species in amniotes

Primates. The most frequent and obvious justification for the use of
primate species as models is their close phylogenetic relationship with
humans. However, beside multiple and significant ethical problems associated with their use as model species (owing to this close relationship with
humans), the high demands in maintenance of primates, as well as their vulnerable status in nature, make them difficult and expensive to acquire, breed
and rear. Full genome sequences are available for human the chimpanzee
(Pan troglodytes), the rhesus macaque (Macaca mulatta) and the bushbaby
(Otolemur garnettii).
Scandentia. Tree shrews form a small group ( species in five genera) of
small-sized mammals that are native to tropical Southeast Asia. The vast
majority of experimental work has been done on their visual system
because tree shrews are considered good models for understanding the
neural organisation of the early primate visual system (Drenhaus et al.
). The focal tree shrew model species has always been Tupaia belangeri,
colonies of which have been successfully established in laboratories (Poole
). Its value as a medical research model animal and its phylogenetic position as an outgroup of primates are the two main reasons for Tupaia belangeri being a genome sequencing target.
Laurasiatheria
This large grouping (Figure .) comprises Carnivora, Pholidota, Perissodactyla, Cetartiodactyla, Chiroptera and Eulipotyphla.
Pholidota. Pangolins are very difficult (if not impossible) to breed in captivity, so that observations can only be based on opportunistic sampling.
The situation is particularly unfortunate from an evo-devo perspective
because the representatives of this extraordinary group (with only seven
living species found in the tropical regions of Africa and Asia) show spectacular features such as, among others, a highly modified skull, an extraordinarily long and muscular tongue, and a set of overlapping scales covering
their head, back and tail.
Perissodactyla (horses, tapirs and rhinoceroses) and Cerartiodactyla
(camels, ruminants, pigs, hippopotamuses, whales, dolphins and porpoises).
The large size of most of these mammals complicates their use as evo-devo
model species. The situation is, however, easier for families represented by
domesticated species (horses, cows, sheep, goats) for which multiple
inbred lines, much developmental information, genome engineering techniques and full genome sequences (for pig and cow) are available. Furthermore, a recent investigation (Thewissen et al. ) of gene expression

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
during early development of hind-limb buds in embryos of the pantropical
spotted dolphin, Stenella attenuata (providing data on the molecular basis
for hind-limb loss during cetacean evolution), demonstrates that a good
museum collection of embryo specimens can partially but efficiently compensate for the lack of laboratory animal models.
Carnivora. The diversity of phenotypes among the multiple inbred lines
of dogs (Canis familiaris) has already been successfully exploited in evo-devo
studies (e.g. Fondon and Garner ), and representatives of other families
(such as mustelids) are becoming increasingly available as possible laboratory
model species. Dog and cat full genome sequences are available in public
databases.
Eulipotyphla. True insectivores are small, highly mobile animals with
long, narrow and often elaborate snouts, adapted to eating insects. Lowcoverage assemblies of the European shrew (Sorex araneus) and the
western European hedgehog (Erinaceus europaeus) genomes have recently
been made available. The African pygmy hedgehog (Atelerix albiventris,
Figure .G) would have probably been a better choice of target species
for genome sequencing because representatives of this species are small
(average size of  cm) and tame animals that can be bred all year around
in laboratory conditions. They are partially covered with spines, opening
the possibility of investigating the development of this trait convergent
with the situation observed in some tenrec species (see above). In fact,
multiple convergences have evolved between representatives of
Eulipotyphla and Afrotheria: Erinaceidae (hedgehogs) versus Echinops
(Madagascar ‘hedgehogs’), Soricidae (shrews) vs. Microgale (shrew-like
tenrecs), and Talpidae (moles) vs. Chrysochloridae (golden moles).
Finally, it would be particularly appealing to identify a model species for
the subterranean insectivores. These species exhibit derived traits such as
reduced eyes (sometimes covered by skin) and lack of external ears, as
well as modified limb morphology. Unfortunately, no information is available on the maintenance of breeding colonies for any of these species,
although it is possible to keep them for long periods in captivity
(Borroni et al. ).
Chiroptera. Bats have acquired a number of spectacular evolutionary
innovations associated with their adaptation to active flight. Microbats
have a number of interesting features including heavily modified limbs
(Sears et al. ), the ability to echolocate (Smotherman and
Metzner ), precise control over both thermoregulation and gestation (Badwaik and Rasweiler ), and an unexpectedly long life
span (Dobson ). Thus far, the little brown bat (Myotis lucifugus)
Model species in amniotes

has been proposed as a model species because of its small size and its
availability. BAC libraries already exist for the little brown bat, and
its full genome sequence has recently been assembled with low coverage.
An alternative valid model species for bats would be Carollia perspicillata
(short-tailed fruit bat) whose maintenance can be readily adjusted to
laboratory facilities and whose reproduction takes place at any time of
the year (Kiefer ). Its embryonic developmental stages have been
recently described and illustrated, a fundamental tool for evo-devo
studies (Cretekos et al. ). This basic information allowed further
analyses of the development of bat wing digits, providing a plausible
scenario as to how bats aquired powered flight soon after their
divergence from other mammals about  million years ago (Sears
et al. ).
ACKNOWLEDGEMENTS
We thank Alessandro Minelli and Giuseppe Fusco for organising the EvoDevo workshop in Venice in May  and giving us the opportunity to
contribute to the present book. Our manuscript has benefited from discussions with Denis Headon, Olivier Lambert, Christophe Rémy and Günter
Wagner. Galen B. Rathbun provided information on Macroscelidea. We
thank Alessandro Minelli, Giuseppe Fusco and Ronald Jenner for reviewing
the first version of the manuscript. This work was supported by grants
from the ‘Communauté Français de Belgique’ (ARC /),
and the National Fund for Scientific Research Belgium (FNRS). A.Tzika
is a Ph.D. candidate at the Fonds pour la formation à la Recherche dans
l’Industrie et dans l’Agriculture (FRIA), Belgium.
REFERENCES
Badwaik, N. K. & Rasweiler, J. J. IV . Altered trophoblastic differentiation and
increased trophoblastic invasiveness during delayed development in the shorttailed fruit bat, Carollia perspicillata. Placenta , –.
Benton, M. J. & Donoghue, P. C. J. . Paleontological evidence to date the tree
of life. Molecular Biology and Evolution , –.
Birkhead, T. R. . Sperm competition: evolutionary causes and consequences.
Reproduction, Fertility and Development , –.
Bonneaud, C., Chastel, O., Federici, P., Westerdahl, H. & Sorci, G. . Complex
Mhc-based mate choice in a wild passerine. Proceedings of the Royal Society.
Biological Sciences , –.
Borroni, A., Loy, A. & Capanna, E. . A flexible arrangement for the study of
moles in captivity. Acta Theriologica , –.

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
Bossuyt, F. & Milinkovitch, M. C. . Convergent adaptive radiations in
Madagascan and Asian ranid frogs reveal covariation between larval and adult
traits. Proceedings of the National Academy of Sciences of the USA , –.
Burk, R. F., Christensen, J. M., Maguire, M. J. et al. . A combined deficiency of
vitamins E and C causes severe central nervous system damage in guinea pigs.
Journal of Nutrition , –.
Burt, D. W. . Chicken genome: current status and future opportunities.
Genome Research , –.
Carter, A. M., Blankenship, T. N., Kunzle, H. & Enders, A. C. . Structure
of the definitive placenta of the tenrec, Echinops telfairi. Placenta ,
–.
Carvalho, R. A., Lins-Lainson, Z. C. & Lainson, R. . Breeding nine-banded
armadillos (Dasypus novemcinctus) in captivity. Contemporary Topics in
Laboratory Animal Science , –.
Cavanagh, R. D., Lambin, X., Ergon, T. et al. . Disease dynamics in cyclic
populations of field voles (Microtus agrestis): cowpox virus and vole tuberculosis (Mycobacterium microti). Proceedings of the Royal Society. Biological
Sciences , –.
Cretekos, C. J., Weatherbee, S. D., Chen, C. H. et al. . Embryonic staging
system for the short-tailed fruit bat, Carollia perspicillata, a model organism
for the mammalian order Chiroptera, based upon timed pregnancies in
captive-bred animals. Developmental Dynamics , –.
Dengler-Crish, C. M., Crish, S. D., O’Riain, M. J. & Catania, K. C. . Organization of the somatosensory cortex in elephant shrews (E. edwardii). Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
, –.
Dobson, G. . On being the right size: heart design, mitochondrial efficiency
and lifespan potential. Clinical and Experimental Pharmacology and Physiology
, –.
Dobson, S. E., Smith, A. T. & Gao, W. X. . Social and ecological influences on
dispersal and philopatry in the plateau pika (Ochotona curzoniae) Behavioral
Ecology , –.
Drenhaus, U., Rager, G., Eggli, P. & Kretz, R. . On the postnatal development
of the striate cortex (V) in the tree shrew (Tupaia belangeri). European
Journal of Neuroscience , –.
Du, W.-G. & Ji, X. . The effects of incubation thermal environments on size,
locomotor performance and early growth of hatchling soft-shelled turtles,
Pelodiscus sinensis. Journal of Thermal Biology , –.
Dusheck, J. . It’s the ecology, stupid! Nature , –.
Eeckhaut, I., McHugh, D., Mardulyn, P. et al. . Myzostomida: a link between
trochozoans and flatworms? Proceedings of the Royal Society. Biological Sciences
, –.
Fishman, M. C. . Genomics. Zebrafish: the canonical vertebrate. Science ,
–.
Model species in amniotes

Fondon, J. W. III & Garner, H. R. . Molecular origins of rapid and continuous
morphological evolution. Proceedings of the National Academy of Sciences of
the USA , –.
Freyer, C., Zeller, U. & Renfree, M. B. . Ultrastructure of the placenta of thetammar wallaby, Macropus eugenii: comparison with the grey short-tailed
opossum, Monodelphis domestica. Journal of Anatomy , –.
Gentner, T. Q., Fenn, K. M., Margoliash, D. & Nusbaum, H. C. . Recursive
syntactic pattern learning by songbirds. Nature , –.
Gilbert, S. F. & Bolker, J. A. . Ecological developmental biology: preface to the
symposium. Evolution & Development , –.
Gilbert, S. F., Loredo, G. A., Brukman, A. & Burke, A. C. . Morphogenesis of
the turtle shell: the development of a novel structure in tetrapod evolution.
Evolution & Development , –.
Grenard, S. . Handbook of Alligators and Crocodiles. Malaba: Krieger Publishing Company.
Guillette, L. J. Jr. & Gunderson, M. P. . Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrinedisrupting contaminants. Reproduction , –.
Holland, L. Z., Laudet, V. & Schubert, M. . The chordate amphioxus: an emerging model organism for developmental biology. Cellular and Molecular Life
Sciences , –.
Jackson, S. . Australian Mammals: Biology and Captive Management.
Collingwood, Australia: CSIRO Publishing.
Johnson, F. & Whitney, O. . Singing-driven gene expression in the developing
songbird brain. Physiology and Behavior , –.
Kiefer, J. C. . Emerging developmental model systems. Developmental
Dynamics , –.
Kishino, H., Thorne, J. L. & Bruno, W. J. . Performance of a divergence time
estimation method under a probabilistic model of rate evolution. Molecular
Biology and Evolution , –.
Kocher, T. D., Conroy, J. A., McKaye, K. R. & Stauffer, J. R. . Similar morphologies of cichlid fish in Lakes Tanganyika and Malawi are due to convergence. Molecular Phylogenetics and Evolution , –.
Kohler, G. . Incubation of Reptile Eggs: Basics, Guidelines, Experiences. Malaba:
Krieger Publishing Company.
Krenz, J. G., Naylor, G. J., Shaffer, H. B. & Janzen, F. J. . Molecular phylogenetics and evolution of turtles. Molecular Phylogenetics and Evolution , –
.
Kress, A., Regli, C., Spornitz, U. M. & Morson, G. . Changes in the epithelium
of the vaginal complex during the estrous cycle of the marsupial Monodelphis
domestica. . Scanning electron microscopy study. Cells Tissues Organs ,
–.
Krohmer, R. W. . The male red-sided garter snake (Thamnophis sirtalis parietalis): reproductive pattern and behavior. Ilar Journal , –.

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
Kunzle, H. . Thalamo-striatal projections in the hedgehog tenrec. Brain
Research , –.
Kuraku, S., Usuda, R. & Kuratani, S. . Comprehensive survey of carapacial
ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution. Evolution & Development , –.
Lapid, R., Nir, I., Snapir, N. & Robinzon, B. . Reproductive traits in the spurthighed tortoise (Testudo graeca terrestris): new tools for the enhancement of
reproductive success and survivorship. Theriogenology , –.
Losos, J. B., Jackman, T. R., Larson, A., Queiroz, K. & Rodriguez-Schettino, L.
. Contingency and determinism in replicated adaptive radiations of
island lizards. Science , –.
Lovern, M. B., Holmes, M. M. & Wade, J. . The green anole (Anolis carolinensis): a reptilian model for laboratory studies of reproductive morphology and
behavior. Ilar Journal , –.
Lovern, M. B. & Passek, K. M. . Sequential alternation of offspring sex from
successive eggs by female green anoles, Anolis carolinensis. Canadian Journal of
Zoology , –.
Mand, S., Specht, S., Zahner, H. & Hoerauf, A. . Ultrasonography in
filaria-infected rodents: detection of adult Litomosoides sigmodontis and
Brugia malayi filariae. Tropical Medicine and International Health ,
–.
Metscher, B. D. & Ahlberg, P. E. . Zebrafish in context: uses of a laboratory
model in comparative studies. Developmental Biology , –.
Milinkovitch, M. C. & Tzika, A. in press. Escaping the mouse trap: the selection of
Evo-Devo model species. Journal of Experimental Zoology Part B: Molecular
and Developmental Evolution, Published online  May , DOI:
./jez.b..
Mills, A. D., Crawford, L. L., Domjan, M. & Faure, J. M. . The behavior of the
Japanese or domestic quail Coturnix japonica. Neuroscience and Biobehavioral
Reviews , –.
Milnes, M. R., Allen, D., Bryan, T. A., Sedacca, C. D. & Guillette, L. J. Jr .
Developmental effects of embryonic exposure to toxaphene in the American
alligator (Alligator mississippiensis). Comparative Biochemistry and
Physiolology. C. Toxicology & Pharmacology , –.
Minelli, A. . The Development of Animal Form: Ontogeny, Morphology, and
Evolution. Cambridge: Cambridge University Press.
Murphy, W. J., Eizirik, E., Johnson, W. E. et al. a. Molecular phylogenetics
and the origins of placental mammals. Nature , –.
Murphy, W. J., Eizirik, E., O’Brien, S. J. et al. b. Resolution of the early
placental mammal radiation using Bayesian phylogenetics. Science ,
–.
Mzilikazi, N. & Lovegrove, B. G. . Daily torpor in free-ranging rock elephant
shrews, Elephantulus myurus: a year-long study. Physiological and Biochemical
Zoology , –.
Model species in amniotes

Mzilikazi, N. & Lovegrove, B. G. . Noradrenalin induces thermogenesis in
a phylogenetically ancient eutherian mammal, the rock elephant shrew,
Elephantulus myurus. Journal of Comparative Physiology B , –.
Nagashima, H., Uchida, K. et al. . Turtle–chicken chimera: an experimental
approach to understanding evolutionary innovation in the turtle. Developmental Dynamics , –.
Ohya, Y. K., Kuraku, S. & Kuratani, S. . Hox code in embryos of Chinese softshelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in
the turtle. Journal of Experimental Zoology Part B (Molecular and Developmental Evolution) , –.
Ottinger, M. A., Abdelnabi, M., Li, Q. et al. . The Japanese quail: a model for
studying reproductive aging of hypothalamic systems. Experimental Gerontology , –.
Poole, T. . The UFAW Handbook on the Care and Management of Laboratory
Animals. Oxford: Blackwell Science.
Rathbun, G. B., Beaman, P. & Maliniak, E. . Capture, husbandry and breeding
of rufous elephant-shrews (Elephantulus rufescens). International Zoo Yearbook
, –.
Regli, C. & Kress, A. . Changes in the epithelium of the vaginal complex
during the estrous cycle of the marsupial Monodelphis domestica. . Transmission electron microscopy study. Cells Tissues Organs , –.
Ruber, L., Verheyen, E. & Meyer, A. . Replicated evolution of trophic specializations in an endemic cichlid fish lineage from Lake Tanganyika. Proceedings
of the National Academy of Sciences of the USA , –.
Sachser, N. . Of domestic and wild guinea pigs: studies in sociophysiology,
domestication, and social evolution. Naturwissenschaften , –.
Sears, K. E., Behringer, R. R., Rasweiler, J. J. IV & Niswander, L. A. .
Development of bat flight: morphologic and molecular evolution of bat
wing digits. Proceedings of the National Academy of Science of the USA ,
–.
Shimozuru, M., Kikusui, T., Takeuchi, Y. & Mori, Y. . Scent-marking and
sexual activity may reflect social hierarchy among group-living male Mongolian gerbils (Meriones unguiculatus). Physiology and Behavior , –.
Smith, K. K. . Early development of the neural plate, neural crest and facial
region of marsupials. Journal of Anatomy , –.
Smotherman, M. & Metzner, W. . Auditory-feedback control of temporal call
patterns in echolocating horseshoe bats. Journal of Neurophysiology , –
.
Springer, M. S., Murphy, W. J., Eizirik, E. & O’Brien, S. J. . Placental mammal
diversification and the Cretaceous–Tertiary boundary. Proceedings of the
National Academy of Sciences of the USA , –.
St Johnston, D. . The art and design of genetic screens: Drosophila melanogaster. Nature Reviews Genetics , –.
Stanhope, M. J., Waddell, V. G., Madsen, O. et al. . Molecular evidence for
multiple origins of Insectivora and for a new order of endemic African

ATHANASIA TZIKA AND MICHEL C. MILINKOVITCH
insectivore mammals. Proceedings of the National Academy of Sciences of the
USA , –.
Stern, C. D. . The chick; a great model system becomes even greater. Developmental Cell , –.
Temple-Smith, P. & Grant, T. . Uncertain breeding: a short history of
reproduction in monotremes. Reproduction, Fertility and Development ,
–.
Thewissen, J. G., Cohn, M. J., Stevens, L. S. et al. . Developmental basis for
hind-limb loss in dolphins and origin of the cetacean bodyplan. Proceedings
of the National Academy of Sciences of the USA , –.
Tissir, F., Lambert De Rouvroit C., Sire, J. Y., Meyer, G. & Goffinet, A. M. .
Reelin expression during embryonic brain development in Crocodylus niloticus. Journal of Comparative Neurology , –.
van Dijk, M. A., Madsen, O., Catzeflis, F. et al. . Protein sequence signatures
support the African clade of mammals. Proceedings of the National Academy of
Sciences of the USA , –.
van Rheede, T., Bastiaans, T., Boone, D. N. et al. . The platypus is in its place:
nuclear genes and indels confirm the sister group relation of monotremes and
therians. Molecular Biology and Evolution , –.
Wagner, G. P. & Larsson, H. C. E. . What is the promise of developmental
evolution? III. The crucible of developmental evolution. Journal of Experimental Zoology B (Molecular and Developmental Evolution) , –.
Wang, Z., Dooley, T. P., Curto, E. V., Davis, R. L. & VandeBerg, J. L. .
Cross-species application of cDNA microarrays to profile gene expression
using UV-induced melanoma in Monodelphis domestica as the model
system. Genomics , –.
Wiens, J. J., Brandley, M. C. & Reeder, T. W. . Why does a trait evolve
multiple times within a clade? Repeated evolution of snake-like body form
in squamate reptiles. Evolution , –.
Wilson, J. D., Shaw, G., Leihy, M. L. & Renfree, M. B. . The marsupial model
for male phenotypic development. Trends in Endocrinology and Metabolism
, –.