RESEARCH ARTICLE

Transient role of the middle ear as a

lower jaw support across mammals
Neal Anthwal1, Jane C Fenelon2, Stephen D Johnston3, Marilyn B Renfree2,
Abigail S Tucker1*
1
Centre for Craniofacial and Regenerative Biology, King’s College London, London,
United Kingdom; 2School of BioSciences, University of Melbourne, Victoria,
Australia; 3School of Agriculture and Food Sciences, University of Queensland,
Gatton, Australia

Abstract Mammals articulate their jaws using a novel joint between the dentary and squamosal
bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation
from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before
the bones of the novel mammalian jaw joint form. These mammals need to rely on other
mechanisms to allow them to feed. Here, we show that this vital function is carried out by the
earlier developing, cartilaginous incus of the middle ear, abutting the cranial base to form a cranio-
mandibular articulation. The nature of this articulation varies between monotremes and marsupials,
with juvenile monotremes retaining a double articulation, similar to that of the fossil mammaliaform
Morganucodon, while marsupials use a versican-rich matrix to stabilise the jaw against the cranial
base. These findings provide novel insight into the evolution of mammals and the changing
relationship between the jaw and ear.

Introduction
In non-mammalian vertebrates, the jaw joint is formed between the quadrate (or palatoquadrate) of
*For correspondence: the upper jaw and the articular part of Meckel’s cartilage, a rod of cartilage that runs through the
[email protected] lower jaw (Figure 1A). This is known as the primary jaw joint. In mammals, this function is carried out
by a new joint between the dentary and squamosal bones, known as the temporomandibular joint or
Competing interests: The
TMJ in humans, and is referred to as the secondary jaw joint. In mammals, the bones of the original
authors declare that no
primary jaw joint have been incorporated into the ear and play a role in hearing (Anthwal et al.,
competing interests exist.
2013). In addition to forming a joint with the articular as part of the primary jaw joint, the amniote
Funding: See page 19 quadrate also articulates with the cranial base. During the evolutionary transition that gave rise to
Received: 14 April 2020 mammals, the connection between the quadrate and the cranial base simplified (Luo and Cromp-
Accepted: 17 June 2020 ton, 1994). The robust quadrate of reptiles moved from being attached to up to five separate skele-
Published: 30 June 2020 tal elements, able to bear the mechanical force of feeding, to become the diminutive mammalian
incus, suspended by a ligament from a single cranial base bone, the petrosal, in an air-filled cavity
Reviewing editor: Min Zhu,
Chinese Academy of Sciences,
allowing sound transmission (Kemp, 2005; Kielan-Jaworowska et al., 2004). At the same time,
China Meckel’s cartilage lost its permanent nature, separating the incus and neighbouring malleus from
the rest of the jaw in adults (Figure 1B; Anthwal et al., 2017; Urban et al., 2017).
Copyright Anthwal et al. This
Early mammal-like reptiles had a permanent Meckel’s cartilage and joints between the quadrate
article is distributed under the
and articular (Q-A), and posteriorly between the quadrate and cranial base – similar to extant rep-
terms of the Creative Commons
Attribution License, which tiles (Figure 1A). In mammaliaforms, such as Morganucodon, both a primary Q-A and a secondary
permits unrestricted use and dentary squamosal joint were present, in addition to a joint between the quadrate (incus) and the
redistribution provided that the paraoccipital process of the petrosal (Figure 1C). This petrosal and incus joint precedes detachment
original author and source are of the middle ear from Meckel’s cartilage in mammal evolution (Luo and Crompton, 1994). A con-
credited. nection between the future middle ear bones and the cranial base is therefore a feature of fossil

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eLife digest The defining feature of all mammals is how the jaw works. Fish, reptiles and other
animals with backbones have a lower jaw made of many bones fused together, one of which
connects to the upper jaw. The lower jaw in mammals, however, is made of a single bone that
connects with the upper jaw using a completely unique jaw joint. This new joint emerged as the
ancestors of all mammals split from the reptiles around 200 million years ago. The bones that
formed the original jaw joint ended up in the middle ear in mammals and switched to a role in
hearing.
Nowadays, there are three types of mammals: the placentals, marsupials and monotremes (the
egg laying mammals). In mice, humans and other placental mammals, the skeleton of the adult jaw
joint forms in the embryo before birth. However, marsupials (such as kangaroos and opossums) and
monotremes (platypuses and echidnas) are born at a much earlier embryonic stage, before the adult
jaw joint has formed. It is therefore unclear how newborn marsupials and monotremes are able to
move their jaws to feed on milk from their mother.
Anthwal et al. compared how the jaw develops in mice, opossums, platypuses and echidnas
before and after the adult jaw joint becomes functional. The experiments showed that young
echidnas, platypuses and opossums use their middle ear bones to articulate the lower jaw with the
head before the adult jaw joint forms. In young opossums, the ear bones form a cushion to support
the jaw. In juvenile platypuses a double joint is evident, with the ear bones forming a joint at the
same time as the newly formed adult jaw joint, similar to the situation observed in fossils of mammal
ancestors. The experiments also indicated that mice and other placental mammals may potentially
use their ear bones to support the jaw before birth.
These findings shed light on why the ear and jaw have such a close connection in mammals. In
humans, the ear and jaw bones are still connected by ligaments, explaining why trauma to the jaw
joint can cause dislocation of the ear bones. Similarly, defects in the development of the jaw can
impact the ear, such as in Treacher Collins Syndrome, where in some cases the jaw joint fails to form
and the ear bones appear to try and take this role. Understanding how the ear and jaw evolved will
help us understand why they look like they do and why a defect in one can have knock-on
consequences for the other.

mammaliaforms. In extant mammals, the proposed homologue of the paraoccipital process is the
crista parotica, which forms as a cartilaginous spur off the petrosal and is derived from neural crest
cells, distinct to the rest of the petrosal and otic capsule, which are mesodermally derived (O’Gor-
man, 2005; Thompson et al., 2012). Modern mammals have separated the middle ear from the jaw
in adults, and the ossicles (malleus, incus and stapes) are now suspended by ligaments from the cra-
nial base to allow free vibration during sound transmission from the ear drum to the inner ear
(Figure 1B). Paleontological evidence indicates that the evolution of the definitive mammalian mid-
dle ear (DMME) occurred at least twice, once in the lineage that gave rise to monotremes and once
in the therian (marsupial and eutherian) mammals (Meng et al., 2018; Rich et al., 2005), while new
developmental data suggests that the two groups of therian mammals may have each independently
acquired the DMME (Urban et al., 2017). Here, we refer to eutherian mammals rather than placental
mammals, as marsupials have a yolk-sac placenta (Renfree, 2010).
Marsupials (Allin, 1975; Filan, 1991) and monotremes (Griffiths, 1978), exhibit extreme altricial-
ity, greater than is seen in any eutherian (Werneburg et al., 2016). This has profound consequences
for early feeding as the bones that form the mammalian jaw joint, the dentary and squamosal, have
not fully ossified by the time of birth/hatching. The dentary-squamosal joint forms prior to birth in
eutherian mammals, and begins to function in the embryo (Habib et al., 2007; Jahan et al., 2014).
In the mouse, gestation is approximately 20 days, with breakdown of Meckel’s cartilage, to separate
the lower jaw from the ear bones, following during early postnatal stages (Anthwal et al., 2013). In
contrast, the opossum Monodelphis has a short gestation of just 13 days (Keyte and Smith, 2008),
and is born before development of the dentary-squamosal articulation, which forms between
14 and 20 days after birth (Filan, 1991; Maier, 1987). Monotremes hatch out of the egg after 10
days post-oviposition (Griffiths, 1978). The formation of the dentary-squamosal joint in monotremes

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A Gecko

B Opossum

C Morganucodon

Meckel’s Cartilage
Cranio-mandibular Joint Malleus / Articular
Incus / Quadrate
Stapes
Gonial / Prearticular
Tympanic / Angular
Middle Ear Surangular
Otic Capsule / Petrosal
Squamosal
Dentary

Figure 1. Schematic of cranial-mandibular jaw articulation showing the roles of the quadrate/incus and articular/malleus in the hearing and jaw joint
modules in (A) reptile gecko, (B) mammal opossum, (C) mammal-like reptile Morganucodon.

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has recently been followed and shown to form from 10 days after hatching in the platypus
(Anthwal and Tucker, 2020). Breakdown of Meckel’s cartilage in both marsupials and monotremes
occurs relatively late postnatally (Urban et al., 2017), with a robust Meckel’s still evident in nest
young platypuses (Zeller, 1993). There is, therefore, a significant gap between birth and the advent
of a functional mammalian jaw joint in both marsupials and monotremes.
The feeding strategies of new-born mammals vary in extant members of each group of mammals.
Compared to eutherian mammals, marsupials rely on placental support for a relatively short period
of time (Renfree, 2010) and consequently receive the nutrition required for their development via a
lengthy and sophisticated lactation (Tyndale-Biscoe and Janssens, 1988; Tyndale-Biscoe and Ren-
free, 1987). During their early postnatal life marsupials attach to the mother’s teat and use the com-
paratively early developed tongue musculature to suck (Smith, 1994). In the grey short-tailed
opossum, Monodelphis domestica, pups are born after 13 days of embryonic development, which is
followed by around 14 days permanently attached to the mother’s teat, after which they detach
intermittently from the mother but continue to suckle. Weaning occurs around postnatal day 60
(Keyte and Smith, 2008). In contrast to therian mammals, young monotremes do not obtain milk in
quite the same way as therian mammals due to the absence of teats in the mother (Griffiths, 1978).
Instead young monotremes suck up milk vigorously from the flattened but protuberant nipple-like
areola on the mother’s abdomen (Griffiths, 1978). In the case of echidnas, these areolae are within
the pouch.
Given the lack of a jaw joint at birth, it has been proposed that marsupials and monotremes use
the connection between the middle ear bones and cranial base to permit feeding prior to the forma-
tion of the articulation between the dentary and squamosal and cavitation of the middle ear
(Crompton and Parker, 1978; Maier, 1987; Sánchez-Villagra et al., 2002; Zeller, 1993). To investi-
gate this idea further, we have analysed the articulations that link the lower jaw to the cranial base
(cranio-mandibular joints) in monotremes (platypus Ornithorhyncus anatinus and short-beaked
echidna Tachyglossus aculeatus) as they develop from hatching, and compare them to a marsupial
(grey short-tailed opossum, Monodelphis domestica), and a eutherian (mouse, Mus musculus), with
additional comparison to the gecko, guinea pig and bat. We show that in early post-hatching life the
monotreme incus and cranial base fuse, and later form an articulation, creating a double cranio-man-
dibular articulation, similar to the jaw anatomy of fossil mammal-like reptiles. This close association
of the incus and cranial base is also observed at embryonic stages in eutherians and is reflected in
mouse cell lineage studies. In contrast, opossums at birth utilise a cushion of extra-cellular matrix-
rich mesenchyme in between the incus and petrosal to provide an articulation point. Marsupials and
monotremes, therefore, have different strategies for coping with an early birth. Our research sug-
gests that the incus retains a transient lower jaw support role across extant mammals but at different
stages of pre and postnatal development.

Results
The primary jaw joint (malleus-incus) does not provide a site of
articulation in marsupials and monotremes at birth
It has been suggested that the joint between the malleus and incus might act as the jaw joint early
on in marsupial postnatal development, thereby recapitulating the reptilian function of these bones
in mammals (Müller, 1968; Crompton and Parker, 1978). Alternatively, it has been suggested that
the actual articulation point in marsupials is between the incus and the cranial base (Maier, 1987;
Sánchez-Villagra et al., 2002). Less information is available regarding monotreme development,
however, the incus has been described as being in cartilaginous connection with the cranial base
during early post hatching development (Watson, 1916; Zeller, 1993). The development of the mal-
leus and incus, and incus and cranial base, was therefore investigated across the three groups of
mammals, with the gecko as an outgroup.
In the ocelot gecko (Paroedura picta), the quadrate and articular (the homologous elements to
the incus and malleus respectively in non-mammal amniotes) form a clear synovial joint in the embryo
at mid-gestation (Figure 2A). In mice (Mus musculus), the malleus and incus are initially formed from
a single cartilaginous condensation that separates, by the formation of a joint, at Embryonic (E) day
15.5 (Amin and Tucker, 2006). At birth, therefore, the incus and malleus are evident as distinct

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Figure 2. Timing of the development of the quadrate-articular/malleus incus, and cranio-incudo joints. Histological sections stained with alcian blue
and picrosirius red. (A) The primarily jaw articulation is formed by 35 days of post-oviposition (35dpo) during in ovo development in geckos. (B) The
malleus-incus joint, the homologue of the quadrate-articular joint, is formed during in utero development in mice, and is fully formed at birth (Postnatal
day (P) 0). (C-D) The malleus incus joint is still partially fused in 4 day postnatal (P4) opossum pups (C) and 1 day post-hatching echidna young (P1) (D).
(E) During development the gecko quadrate forms a joint with the opisthotic (structurally equivalent to the mammalian petrosal). (F) At birth there is no
articulation between the crus breve of the incus and the surrounding crista parotica of the petrosal in mice (P0). (G) The crus breve of the incus sits in
close proximity to the petrosal in opossums at P4 (G). (H-I) The incus is fused with the petrosal in both P1 echidna (H) and the P2 platypus (I). Green
arrows highlight Q-A/M-I interaction. Red arrows highlight Incus/Q-petrosal/opisthotic interaction. a. articular; c.b.i crus breve of the incus; (i) incus; m.
malleus; opo, opisthotic; ptr. petrosal; q. quadrate. Scale in A = 200 microns, same scale in E. Scale bar in I = 100 microns, same scale in B-D, F-H.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1. 3D Reconstruction of cartilaginous middle ear ossicles and cranial base from histological sections shows differences in anatomy
in different groups of mammals during development.

cartilages (Figure 2B). In Monodelphis domestica, the malleus and incus are still connected at birth
at the dorsal end by a ridge of cartilage (Filan, 1991; Figure 2C). We observed a similar connection
between the malleus and incus in the echidna (Tachyglossus aculeatus) just after birth. Like the opos-
sum, the middle ear ossicles were fused dorsally, indicating that they function as a unit (Figure 2D).
These findings demonstrate that, like opossums, monotremes do not use the primary jaw joint as
the craniomandibular articulation before the development of the dentary-squamosal joint.
We therefore investigated the relationship between the incus and the petrosal in the cranial base
in mice, opossums, platypus and echidna, comparing the interaction to the developing joint between
the quadrate and opisthotic in embryonic geckos. In many reptiles, as shown in the gecko, the quad-
rate (incus homologue) forms a synovial joint with the opisthotic (also known as the otoccipital) in
the cranial base during embryonic development (Figure 2E). The opisthotic/otoccipital is architectur-
ally equivalent to the petrosal of mammals. In mice, the crus breve (short process) of the incus nes-
tled in a fossa created by the crista parotica of the petrosal, but was separated by a region of
mesenchymal cells, highlighting the lack of a clear articulation point between the two elements
(Figure 2F). The incus at birth, therefore only articulated with the adjacent middle ear bones, the
malleus and stapes. Similar to the mouse, the crus breve in neonatal opossums, fitted into a fossa
created by the crista parotica, but abutted the petrosal on the inferior aspect of the crista parotica
(Figure 2G). The incus and petrosal were therefore positioned much closer than in the mouse.
The relationship between the incus and crista parotica in the two monotreme species was signifi-
cantly different from the therian mammals. In both platypus (Ornithorhynchus anatinus) and echidna
(Tachyglossus aculeatus), the incus appeared to be fused with the crista parotica at birth (Figure 2H,
I), agreeing with Watson, 1916. The lower jaw, via Meckel’s cartilage, would therefore be physically

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connected to the upper jaw, via the incus at this timepoint. 3D reconstructions of the incus, malleus
and petrosal, showing the relationship of these different elements in the different species is shown
in Figure 2—figure supplement 1. The relatively small size of the incus in both monotremes is strik-
ing, as is the extended and tapered crus breve of the incus in the opossum.

Development of an incus-petrosal joint in monotremes during early

feeding
To investigate the monotreme relationship between the incus and crista parotica further we followed
development of these two cartilages from birth to functional use of the dentary-squamosal joint, but
before complete cavitation of the middle ear space. Due to the scarcity of available specimens very
little is known about monotreme ear and jaw development. In adult platypuses, the incus appears in
contact with the crista parotica, forming a fibrous articulation (Zeller, 1993; Luo and Crompton,
1994). Similarly, in the adult echidna, the incus has been described as tightly attached to the petro-
sal (Aitkin and Johnstone, 1972).
At 2 days and 6.5 days the platypus incus was fused to the crista parotica by immature chondro-
cytes (Figure 3A,B). Between 10 days and 30 days the connection was difficult to make out, with the
two cartilages almost completely integrated together (Figure 3C,D). Strikingly, by 80 days, when
the dentary-squamosal joint would have started to become functional, the incus and crista parotica
were no longer fused, with the two distinct cartilages abutting each other (Figure 3E). At this stage,
in contrast to the other stages investigated, the ear ossicles and petrosal had begun to ossify. How-
ever, the regions forming the malleus-incus joint, and the incus-petrosal articulation remained carti-
laginous. A cartilaginous articular surface between the incus and petrosal was maintained at 120
days, a period when the young would have started to leave the burrow (Figure 3F; Holland and
Jackson, 2002). A similar move from early fusion, to articulation was observed in the echidna
(Figure 3F–J). No evidence of a synovial capsule, however, was identified at any stage.
The fusion of the incus and crista parotica coincides with the period when the young would have
been feeding from milk, while the move to an articulation was associated with periods when the den-
tary-squamosal was fully formed and functional. After separation of the incus and petrosal, there was

Figure 3. Development of the incus-petrosal joint in monotremes. (A-B) The platypus incus is fused to the petrosal by immature chondrocytes at 2 days
(A) and 6.5 days (B) post-hatching. (C) At 10 days post-hatching, the fusion persists, with mature chondrocytes forming the connection. (D) A similar
morphology is seen at 30 post-hatching. (E) At 80 days post-hatching the incus and petrosal are no longer fused, but instead the two cartilages abut
each other. (F) At 120 days post-hatching the incus and petrosal have begun to ossify, but the region of articulation in between the two elements
remains cartilaginous. (G-H) In echidna the incus is fused to the petrosal by immature chondrocytes at 3 days (G) and 10 days (H) post-hatching. (I-J) By
18 days post-hatching the two elements are separated but remain abutted (I), This connection remains though to 55–65 days post-hatching (J). i: incus;
ptr. petrosal. Scale bar = 100 microns.
The online version of this article includes the following figure supplement(s) for figure 3:
Figure supplement 1. The middle ear and jaw joints of a 50 day old platypus.

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a period where two cranial-mandible articulations were evident in the platypus - between Meckel’s
cartilage and the petrosal, via the malleus and incus, and between the dentary and squamosal (Fig-
ure 3—figure supplement 1C). The chain of elements linking Meckel’s cartilage to the petrosal in
the platypus is shown in Figure 3—figure supplement 1A, B, D at 50 days post-hatching.
Middle ear cavitation occurred very late in the monotreme specimens analysed, with only the 120
day platypus showing partial cavitation around the hypotympanum, but this did not extend upwards
to where the ossicles are housed. Hearing, thus, must be a very late developing sense in the
platypus.

Upregulation of Wnt signalling initiates joint formation between the

ossicles and cranial base in echidna
Limited expression analysis has been performed in monotremes, with no previous expression data
performed in the ear or jaw during development. In order to further understand the change in the
relationship between the incus and petrosal, immunohistochemistry staining was carried out in
echidna samples 0 and 3 days post hatching.
In the fused incus-petrosal region of 0-day-old echidna (Figure 4A), the expression of both a mas-
ter regulator of cartilage development, Sox9, and a principal component of cartilage extra cellular
matrix, Collagen Type 2, were continuous between the incus and the crista parotica of the petrosal,
as well as between the incus and the malleus (Figure 4B). Since the connection between these ele-
ments is lost later in post-hatching development, IF for beta-catenin was carried-out. Nuclear local-
ised beta-catenin is a readout of canonical Wnt signalling, and is known to negatively regulate
chondrocytes differentiation and promote joint formation (Hartmann and Tabin, 2001). Few beta-

Figure 4. Fusion of the Incus with the petrosal in Echidna pouch young. (A) Alcian blue/picrosirius red staining on the fusion between the incus and
petrosal observed in the newly hatched echidna. (B) Immunofluorescence staining against the regulator of chondrogenesis Sox9 (red) (B,B’) and the
marker of mature cartilage Collagen type 2 (green) (B,B”) demonstrates that the cartilaginous incus and petrosal bones are fully fused at post-hatching
day 0/1 (P0/1). (C) Immunohfluorescence against b Catenin (green) shows no activity within the cartilages at this timepoint. Expression is observed in the
neuroepithelium of the inner ear. (D) Alcian blue/picrosirius red staining on the fusion between the incus and petrosal observed in 3 day post-hatching
echidna (P3) shows that the elements are now fused by fibrocartilage. (E) Immunofluorescence staining against the regulator of chondrogenesis Sox9
and the marker of mature cartilage collagen type 2 (E,E”). Sox9 is still continuously expressed between the elements (E,E’), but collagen type 2 is down
regulated in the incus-petrosal and incus-malleus articulation region (E,E”). (F) Immunofluorescence against b Catenin shows nuclear localisation within
the incus-petrosal and incus-malleus articulation regions, indicating active canonical Wnt signalling, an important step in suppression of chondrogenesis
during joint formation. i. incus; m. malleus; ptr. petrosal.

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catenin positive cells were observed within the cartilage of the middle ear and petrosal at 0 days,
though beta-catenin was strongly expressed in the neuro-epithelium of the inner ear (Figure 4C). At
post-hatching day 3, the incus and crista parotica were still fused, although the cells joining the two
elements resembled fibrocartilage or immature chondrocytes (Figure 4D). Expression of Sox9 was
still strong and continuous throughout all elements (Figure 4E,E’), however Collagen Type 2 expres-
sion was weaker in the fusion region (Figure 4E,E”), possibly indicating a change in cartilage type
from hyaline cartilage to fibrocartilage. Interestingly nuclear beta-catenin, suggestive of active Wnt
signalling, was observed in two stripes, in the chondrocytes between the incus and petrosal, and
within the malleus-incus joint, indicating suppression of cartilage fate in these regions (Figure 4F).
Upregulation of Wnt signalling between the incus and petrosal therefore, may play a role in forma-
tion of a joint between these two, initially fused, structures.

Interactions between the petrosal and incus are also observed

prenatally in eutherian mammals
While the fusion between the incus and petrosal in echidna and platypus could be explained by the
evolutionary distance between monotremes and therian mammals, it has also been suggested that
the incus is transiently attached to the cranial base in 7-week-old human fetuses (Rodrı́guez-
Vázquez et al., 2018). This suggests that the potential for fusion may be a default state in mammals.
In order to examine this, we next undertook fate mapping experiments in the mouse, and investi-
gated the relationship between the incus and petrosal in other eutherian mammals during embryonic
development.
Sox9 expressing cells were fate mapped by tamoxifen induction at E14.5 in Sox9CreERT2; tdTo-
mato mice, which were then collected at P0 (Figure 5A). At this stage Sox9 (green) was expressed
in the petrosal and incus and suspensory ligaments, overlapping with the red fluorescent Protein
(RFP) marking the Sox9 lineage cells. In addition, the red Sox9 lineage cells were found in the Sox9
negative mesenchymal cells, in the gap between the petrosal and incus (Figure 5A). A pre-cartilagi-
nous bridge is therefore evident in the mouse between the incus and the crista parotica. Next,
expression of Sox9 was investigated at E14.5. The incus, and the crista parotica are both neural crest
derived (O’Gorman, 2005; Thompson et al., 2012), while the rest of the petrosal is mesodermal.
We therefore looked at the expression of Sox9 (red) in Mesp1Cre;mTmG mice, where mesoderm-
derived tissue can be detected by anti-GFP IF (Figure 5B). Since tissue processing and wax embed-
ding removes endogenous fluorescence, the membrane RFP that is expressed in the non-meso-
dermal tissue of Mesp1Cre;mTmG mice was not detectable in these slides. Consequently, all red
signal was Sox9 immunofluorescence staining. Sox9 protein was expressed continuously between
the incus and the petrosal. The incus Sox9 expression domain was continuous with the expression
domain of the neural crest -derived crista parotica, which in turn was fused to the mesodermal por-
tion of the petrosal. Since the incus does not fuse with the petrosal in the mouse, despite the
expression of Sox9 between the elements, we next looked at the mRNA expression of joint markers
Gdf5 and Bapx1 between the incus and petrosal of mice by in situ hybridisation (Figure 5C–
E; Storm and Kingsley, 1999; Francis-West et al., 1999; Tucker et al., 2004). Gdf5 was expressed
in the mesenchyme between the incus and petrosal, as well as in the malleus-incus joint (Figure 5D).
Bapx1, which specifies both the malleus-incus joint and the quadrate-articular joint (Tucker et al.,
2004), was not expressed in between the incus and the petrosal (Figure 5E). In the mouse, therefore
there is a potential for the incus and crista parotica to fuse but they are prevented from doing so by
the upregulation of the joint marker Gdf5.
Very close associations between the incus and crista parotica during development were also
observed in other eutherian mammals via PTA stained microCT (see bat in Figure 5—figure supple-
ment 1), suggesting that interactions between these two elements are observed as a feature prena-
tally in eutherian mammals, similar to post-hatching monotremes. The function of this prenatal
connection between the upper and lower jaw is unclear but may act as a brace to buffer movement
during this period.

Petrosal-incus relationships in marsupials

Next we investigated the articulation between the incus and petrosal observed in the developing
opossum. It was originally suggested that the marsupial incus forms a joint with the crista parotica

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Figure 5. Mouse fate mapping studies demonstrate developmental fusion between incus and petrosal. (A) Genetic tracing of chondrogenic Sox9
expression cells by inducible reporter mice at postnatal day 0 (P0). Sox9 lineage cells (red) (RFP) are observed in the mesenchyme and developing
ligaments between the crus breve of the incus and the petrosal. Sox9 protein (green) is not expressed in the mesenchyme surrounding the incus at P0
(arrowhead). (B) Genetic tracing of mesoderm lineage cells (green) (GFP) and immunohistochemistry against Sox9 protein (red) at embryonic day 14.5
Figure 5 continued on next page

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Figure 5 continued
(E14.5). Sox9 expression at E14.5 confirms that the incus and petrosal are formed from a continuous chondrogenic mesenchyme, and that the incus
joins with the petrosal at the crista parotica, which is not of mesodermal origin. (C-E) Expression by in situ hybridisation of joint markers in sagittal
section of E14.5 mouse middle ears. Gdf5 mRNA is expressed with the malleus-incus joint, and between the incus and the petrosal (D), potentially
acting to inhibit the Sox9 expressing mesenchyme between the ear and the cranial base from differentiating into cartilage. The middle ear joint marker
Bapx1 is not expressed between the incus and the petrosal (E). * indicates space between of incus and petrosal in C-E. i. incus; m. malleus; ptr.
petrosal. Scale bar in A,B = 100 microns.
The online version of this article includes the following figure supplement(s) for figure 5:
Figure supplement 1. Contrast enhanced mCT of embryonic bat (Pterobnotus quadridens) middle ear at Carnegie Stage21, showing abutment of the
crus breve of the incus against the petrosal.

(Maier, 1987), although this was disputed in Monodelphis (Filan, 1991). Although this latter paper
found no evidence of a joint they did show the mesenchyme between the crista parotica and incus
as being condensed (Filan, 1991). We therefore investigated the extra cellular matrix (ECM) compo-
nents of the mesenchyme surrounding the opossum incus in more detail (Figure 6). It was noted
that mesenchyme surrounding the crus breve and superior portion of the body of the incus had a
more intense staining with alcian blue compared to those regions around the inferior border of the
incus and the other ossicles (Figure 2C,G). This pattern was observed throughout ossicle develop-
ment (Figure 6A–C). In order to further characterise the differences in the ECM in the different
regions of the middle ear mesenchyme, immunohistochemistry for versican was carried out. Versican
is a large proteoglycan with side chains of glycosaminoglycans (GAGs), such as hyaluronic acid (HA).
Proteoglycan complexes act to attract water, and are held in place by collagen fibres to stiffen the
matrix in hyaline cartilage, and act to lubricate articular cartilage (Wu et al., 2005). Versican is
required during the initial condensation of mesenchyme but is absent from mature cartilage, where
aggrecan is expressed (Kamiya et al., 2006). Versican expression is maintained in the joint region
during limb cartilage development, acting to inhibit maturation of the mesenchyme to form cartilage
(Choocheep et al., 2010; Snow et al., 2005).
Versican was strongly expressed in the mesenchyme surrounding the short arm of the incus at 5
days, 10 day and 27 days, correlating with the region of strong alcian blue expression (Figure 6D–
F). The high level of versican around the crus breve therefore suggests a role for the ECM in provid-
ing a buffering function in this region. Cell density of the mesenchyme was measured in regions with
strong alcian blue/versican staining and compared against the cell density of regions with low alcian
blue/versican staining. Unpaired two-tailed t-test demonstrated that the regions with high alcian
blue had a significantly higher (p=0.0152) cell density than those regions with lower alcian staining
(Figure 6G).
Versican is processed by ADAMTS family members for clearing and remodelling
(Nandadasa et al., 2014). While the full-length form of versican is thought to have a structural role,
the cleaved form has an active role in signalling, influencing morphogenesis and tissue remodelling
(Nandadasa et al., 2014). Interestingly when we analysed the cleaved form of versican, using anti-
bodies against DPEAAE, the expression was largely reciprocal to that of uncleaved versican, with
lower levels specifically around the crus breve (Figure 6—figure supplement 1A). This suggests that
versican around the incus is protected from cleavage allowing it to maintain its structural role. The
lack of cleaved versican around the crus breve, suggests the lack of a signalling role in this region, in
agreement with the low level of expression of CD44, a cell surface receptor and binding partner of
versican-hyaluronan complexes. CD44 was not associated with the mesenchyme around the crus
breve, but was instead restricted to the perichondrium of the cartilaginous elements and periosteum
of the skeletal elements of the ear (Figure 6—figure supplement 1B).

Discussion
Ear ossicles as transient jaw support in mammals
The incus of adult mammals plays a key role in hearing. Our data here suggest that the incus also
plays a transient role supporting the lower jaw against the cranial base during both marsupial and
monotreme postnatal development. The role of the incus and the points of jaw articulation are

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Figure 6. Specialist mesenchyme supports incus-petrosal connection in juvenile opossums. (A-F) Mesenchyme surrounding the crus breve of the incus
is rich in the proteoglycan Versican (Vcan) at postnatal day (P)5 (A,D) and P10 (B,E). During cavitation of the middle ear at P28 versican rich mesenchyme
is concentrated between the crus breve of the incus and the petrosal (C,F). (G) At P5 the proteoglycan-rich regions surrounding the crus breve have a
Figure 6 continued on next page

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Figure 6 continued
significantly greater cell density than the regions with less proteoglycan. *p=0.0152 unpaired two-tailed t-test. Error bars = 1 standard deviation. c.b.i
crus breve of the incus; i. incus; ptr. petrosal. Scale bar in A = 100 microns, same scale in B-F.
The online version of this article includes the following source data and figure supplement(s) for figure 6:
Source data 1. Statistical analysis of cell number in specialised mesenchyme surrounding the opossum incus.
Figure supplement 1. Versican signaling is not evident in the mesenchyme around the opossum incus.

summarised in Figure 7A. The incus and petrosal were found to be fused at hatching in both monot-
remes. During this early fusion period, the puggle would be feeding exclusively on milk and Meckel’s
cartilage could therefore act as a flexible elastic strut to facilitate jaw movement (Zeller, 1993).
Interestingly, a potential role of the ear ossicles in jaw support was also observed in eutherians
during prenatal development. Fate mapping and gene expression studies in mice indicated that the
crus breve of the incus and the crista parotica were formed from a continuous region of Sox9
expressing chondrogenic cells (Figure 5A, B), separated by expression of the joint marker Gdf5
(Storm and Kingsley, 1999; Figure 5C). Furthermore, the incus and cranial base temporarily fuse
during the development of the human middle ear region (Rodrı́guez-Vázquez et al., 2018), and
abut during bat development (Figure 5—figure supplement 1). Together these data indicate that
the relationship of the incus to the cranial base is not a derived feature of monotremes, and that the
common mammal-like reptile ancestors of both monotremes and therian mammals may have formed
an articulation between the quadrate/incus and petrosal through fusion of the elements followed by
joint formation though Wnt and Gdf5 signalling.
The current study indicates that the first pharyngeal arch-derived incus forms a continuous field of
chondrocytes with the second arch-derived crista parotica, which in turn is fused with the meso-
derm-derived body of the petrosal. The borders between these developmentally distinct popula-
tions are, therefore, not always reflected by the mature anatomy.
For young monotremes and marsupials, the middle ear must function as part of the mandible
postnatally until the dentary-squamosal bones have formed. This is similar, but not identical to the
situation in cynodont ancestors of mammals. In these animals, the quadrate/incus articulated with a
number of cranial elements, including the quadratojugal, to stabilise the jaw articulation. These con-
nections and many elements like the quadratojugal have been lost in extant mammals in order to
free the incus and increase its mobility during sound transmission (Luo and Crompton, 1994). The
mechanical requirements for feeding placed upon the middle ears in monotremes and marsupials
during early life have resulted in the fusion of the incus and petrosal in monotremes, and the elon-
gated contact supported by a proteoglycan matrix in marsupials. These adaptations allow for stabili-
sation of the middle ear before the development of the dentary-squamosal joint and separation of
the middle ear from the mandible, but do not compromise the effectiveness of the middle ear in
later life. The changing connections between the middle ear ossicles and the cranial base in the dif-
ferent groups are highlighted in Figure 7B.

The opossum has a specialised anatomy to brace the middle ear against
the cranium during sucking
The crus breve of the incus is elongated in the developing opossum compared with other species
analysed (Figure 2—figure supplement 1). In order to feed by suckling in the absence of a dentary-
squamosal joint we propose that this anatomy allows for an increased surface contact with the cra-
nial base during postnatal development, which, in combination with the proteoglycan-rich surround-
ing mesenchyme, acts to stabilise the mandible against the rest of the head. It is noted that many
adult marsupials have a relatively elongated crus breve of the incus compared to eutherian species,
for example the bare-tailed woolly opossum Caluromys philander, and the grey short-tailed opos-
sum, Monodelphis domestica (Sánchez-Villagra et al., 2002). Even when eutherian mammals have a
longer crus breve, such as in Talpid moles, the process is thinner and more finger-like compared to
that of marsupials (Segall, 1973; Segall, 1970). This may be a consequence of the developmental
requirement for an elongated short process to facilitate feeding before the development of the
mature mammalian jaw articulation.

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A Prebirth Birth / Hatching Juvenile Adult

Eutherian
Mouse

Marsupial
Opossum

Monotreme
Platypus

Morganucodon

Articulation point
Meckel’s Cartilage
Malleus / Articular
Incus / Quadrate
Stapes
Gonial / Prearticular
Tympanic / Angular
Surangular
Otic Capsule / Petrosal
Squamosal
Dentary
Middle Ear Mesenchyme

B Prebirth
Joint
Birth / Hatching Juvenile Adult
Suspensory Ligament

Eutherian
Mouse

Specialized Mesenchyme
Suspensory Ligament

Marsupial
Opossum

Fusion Joint Fibrous Joint

Monotreme
Platypus

Morganucodon

Figure 7. Summary of involvement of middle-ear ossicles in jaw articulation during development. (A) The location of the jaw articulation in developing
living mammals and in the extinct mammal-like reptile Morganucodon. Arrows indicate jaw articulation points. Eutherian mammals are born with a
functional dentary-squamosal joint (TMJ), while young marsupials and monotremes use the middle ear bones due to a lack of this joint, which develops
later. During postnatal development monotremes show evidence of a double jaw articulation, similar to fossil mammal-like reptiles such as
Figure 7 continued on next page

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Figure 7 continued
Morganucodon. (B) The connections between the middle ear ossicles and the cranial base in developing mammals. The connections between the incus
and cranial base differ in young marsupials and monotremes. The fusion followed by a joint seen in monotremes is also observed in pre-natal
eutherians. Neonatal marsupials support the incus with a specialised middle-ear mesenchyme.

In the majority of adult marsupials, including Monodelphis, the incus is suspended from the cra-
nial base by suspensory ligaments, and the crus breve extends into a fossa. One interesting excep-
tion is the marsupial mole, in which the crus breve has a connective tissue attachment to a lamella
on the petrosal (Archer, 1976). This results in the middle ear ossicles being affixed to the cranial
base, an adaptation to a fossorial niche found in other mammals such as in true moles. In light of the
current study, the absence of an incudal fossa in the marsupial mole may be interpreted as a reten-
tion of the juvenile petrosal morphology (paedomorphy).

Consequence of ECM in opossum middle ear

In adult non-mammalian amniotes the homologue of the incus - the quadrate - and cranial base are
strongly attached by fibrous syndesmoses or cartilaginous synchondroses (Payne et al., 2011), and
we show that a synovial joint appears to form in geckos during development (Figure 2). In the neo-
natal opossum neither type of connection is observed. In neonatal marsupials Sánchez-Villagra and
colleagues describe the connection between the incus and petrosal as being an ‘immature syndes-
mosis’, which acts as a ‘supportive strut’ during sucking (Sánchez-Villagra et al., 2002). In the cur-
rent study, we demonstrate a specialised condensed mesenchyme surrounds the incus of opossum
postnatal juveniles. We show that this condensed mesenchyme is rich in the proteoglycan versican
(Figure 6). In contrast expression studies in human foetuses demonstrate that versican is restricted
to the perichondrium of Meckel’s cartilage (Shibata et al., 2014; Shibata et al., 2013), with high
hyaluronic acid levels within the joints but not surrounding the incus (Takanashi et al., 2013). This
concentration of versican around the crus breve therefore may be a feature of Monodelphis, and
perhaps marsupials in general.
The versican-rich mesenchyme may act to either stabilise the incus by increasing the tension of
the surrounding mesenchyme during feeding, ‘lubricate’ the articulation between the incus and cra-
nial base by increasing the hydration of the ECM, or both. In keeping with this role, versican is
dynamically expressed at the pubic symphysis during pregnancy in mice (Rosa et al., 2012), during
which time the mouse pubic symphysis forms a fibrous joint or syndesmosis (Ortega et al., 2003).
Significantly, there is little cleaved versican (DPEAAE) around the crus breve of the incus, suggesting
a mechanical, rather than a signalling role (Figure 6—figure supplement 1. A). Overall it is likely
that this mesenchyme is supporting the incus, rather than enabling mobilisation, with the high level
of uncleaved versican acting to increase fibroviscocity while also elevating hydration of the ECM. In
this way, the mesenchyme around the incus acts as a cushion during the mechanical stress of
suckling.

A double jaw articulation during monotreme development

Meckel’s cartilage persists to at least 50 days post-hatching in the platypus. At this timepoint, juve-
nile monotremes have two connections between the lower and upper jaw. The first connection is
through the middle ear, which in juveniles remains attached to the mandible and articulates with the
cranial base via the incus. The second is the later developing novel mammalian jaw joint. Only much
later in the life of the young does it appear that the connection between the middle ear and mandi-
ble is lost, and the malleus and incus act as a DMME. The connection of the incus to the cranial base
appears to be maintained in the adult echidna and platypus (Luo and Crompton, 1994; Aitkin and
Johnstone, 1972). This would be expected to impact on the movement of the incus, and therefore
the efficiency of hearing, reflected in the poor hearing reported for monotremes (Aitkin and John-
stone, 1972; Gates et al., 1974).
This novel finding of a double cranial articulation in the juvenile has significant implications for the
evolution of the middle ear and jaw joint in mammals. Fossil evidence indicates that mammalian
ancestors had a persistent connection between the middle ear ossicles and the jaw, as evidenced by
the presence of an ossified Meckel’s element, or a dentary groove and post dentary trough,

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supporting a persistent Meckel’s cartilage (Luo, 2011; Rich et al., 2005; Urban et al., 2017). For
these animals, the connection of the middle ear with the jaw took one of two forms, in each case the
mammalian secondary jaw joint was present. The first was a more basal mandibular middle ear
where the incus and malleus were firmly attached to the cranial base and dentary respectively. More
derived fossils had a partial, or transitional mammalian middle ear (PMME or TMME), where the mid-
dle ear was medially inflected away from the dentary, presumably allowing for improved vibration,
but the malleus was still connected to the jaw, via Meckel’s cartilage (Luo, 2011). In these fossils
with a PMME, little is understood of the rear of the ossicular chain, where the incus meets the petro-
sal, due to the poor and rare preservation of middle ear ossicles in the fossil record, a consequence
of their small size. For example, only recently has a multituberculate with a complete incus been
described (Wang et al., 2019). Our data suggest that even in these transitional mammals with a
PMME, the incus would have still articulated with the cranial base via the crista parotica, at least at
some point during the animal’s life history.
The DMME appears to have evolved independently in monotremes and therian mammals
(Rich et al., 2005). Due to the absence of evidence we do not know if the incus articulation in ani-
mals with a PMME varied in a lineage specific manner, with the therian lineage resembling juvenile
marsupials, and monotremaformes resembling juvenile platypuses and echidna, or if both lineages
had similar articulations. The data from transgenic reporter mice (Figure 5), along with data from
humans (Rodrı́guez-Vázquez et al., 2018) and non-model therians (Figure 5—figure supplement
1) suggests that the monotreme-type fusion and articulation of the incus with the cranial base may
have been common in mammal like-reptiles. As such, the developing monotreme, with a double jaw
articulation and a fused or articulated incus and petrosal, provides an exciting model for the study of
the developmental basis of mammalian evolution.

Materials and methods

Key resources table
Reagent type
(species) or Source or Additional
resource Designation reference Identifiers information
Strain, strain short-tailed Anthwal et al., 2017.
background opossum DOI: 10.1038/s41559-017-0093;
(Monodelphis Urban et al., 2017.
domestica) DOI: 10.1098/rspb.2016.2416
Strain, strain Guinea pig Anthwal et al., 2015.
background DOI: 10.1186/s13227-015-0030-6
(Cavia
porcellus)
Strain, strain ocelot gecko Zahradnicek et al., 2012.
background (Paroedura picta) DOI: 10.1111/j.1469-7580.2012.01531.x
Strain, strain CD1 King’s College London
background
(Mus musculus)
Strain, strain short-beaked University of
background echidna Melbourne
(Tachyglossus
aculeatus)
Genetic Sox9CreERT2: Other RRID:MGI:4947114 Prof Robin Lovell-
reagent tdTomato Badge, Francis
(Mus musculus) Crick Institute
Genetic Mesp1Cre; RIKEN Mesp1tm2(cre)Ysa::
reagent mTmG Gt(ROSA)26S
(Mus musculus) ortm4(ACTB-tdTomato,-EGFP)Luo;
RRID:MGI:3702469
Biological sooty Other Karen Sears,
sample mustached UCLA
(Pterobnotus bat mCT scan
quadridens)
Continued on next page

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 Research article Developmental Biology Evolutionary Biology

Continued

Reagent type
(species) or Source or Additional
resource Designation reference Identifiers information
Biological platypus Hill Collection, Museum Specimen W; M45; Museum Samples
sample histological für Naturkunde, Leibniz Specimen Delta;
(Ornithorhynchus slides Institute for Research on M038; HP; Specimen
anatinus) Evolution and Beta; HX
Biodiversity, Berlin;
Cambridge University
Museum of Zoology;
Green, 1937; Presley and
Steel, 1978; Watson, 1916
Biological short-beaked Cambridge University HX; Echidna H.SP Museum
sample echidna Museum of Zoology; EC5; Echidna Samples
(Tachyglossus histological Green, 1937; Presley H.SP EC4
aculeatus) slides and Steel, 1978;
Watson, 1916
Antibody rabbit Millipore ab5535; IF 1/200
polyclonal RRID:AB_2239761
anti Sox9
Antibody chicken Abcam ab13970; IF 1/500
polyclonal RRID:AB_300798
anti GFP
Antibody Rat Chromotek 5f8-100; IF 1/200
monoclonal RRID:AB_2336064
anti RFP
Antibody Rabbit polyclonal Santa Cruz sc-7199; IF 1/200
anti Beta-catenin RRID:AB_634603
Antibody mouse monoclonal DSHB M1B4; IF 1/40
anti Tenascin C RRID:AB_528488
Antibody mouse monoclonal DSHB II-II6B3; IF 1/50
anti type 2 collagen RRID:AB_528165
Antibody mouse monoclonal DSHB HERMES-1; IHC 1/50
anti CD44 RRID:AB_528148
Antibody mouse monoclonal DSHB 12C5; IHC 1/50
anti Versican RRID:AB_528503
Antibody rabbit polyclonal Abcam ab19345; IF 1/400
anti Versican RRID:AB_444865
V1 (DPEAAE)
Antibody Donkey Polyclonal Invitrogen A10042; IF 1/300
Alexa568 RRID:AB_2534017
conjugated
anti-Rabbit
Antibody Donkey Polyclonal Invitrogen A-21206; IF 1/300
Alexa 488 RRID:AB_2535792
conjugated
anti-Rabbit
Antibody Donkey Polyclonal Invitrogen A10037; IF 1/300
Alexa568 RRID:AB_2534013
conjugated
anti-Mouse
Antibody Goat Polyclonal Invitrogen A-11039; IF 1/300
Alexa488 RRID:AB_2534096
conjugated
anti-Chicken
Antibody Goat Polyclonal Invitrogen A-11077; IF 1/300
Alexa568 RRID:AB_2534121
conjugated
anti-Rat
Antibody Goat Polyclonal Dako E0433; IHC 1/400
Biotin Anti-Mouse RRID:AB_2687905
Continued on next page

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Continued

Reagent type
(species) or Source or Additional
resource Designation reference Identifiers information
Recombinant Mouse Gdf5 in situ Tucker et al., 2004.
DNA reagent hybridisation probe DOI: 10.1242/dev.01017
Recombinant Mouse Bapx1 in situ Tucker et al., 2004.
DNA reagent hybridisation probe DOI: 10.1242/dev.01017
Commercial ABC-HRP Vector Labs PK-6100;
assay or kit streptavidin kit RRID:AB_2336819
Commercial ImmPACT DAB Vector Labs SK-4105;
assay or kit Peroxidase RRID:AB_2336520
Substrate
Software, FIJI Schindelin et al., 2015; RRID:SCR_002285
algorithm Schindelin et al., 2012
Software, Prism 8 Graphpad RRID:SCR_002798
algorithm
Other Aqueous mounting Abcam ab104139
medium with DAPI
Other Picro-sirius red, CCRB Histology
haematoxylin and Core at King’s
alcian blue College London
trichrome staining

Animal tissues
Opossum (Monodelphis domestica) tissue was collected as previously described (Anthwal et al.,
2017; Urban et al., 2017).
Archival platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus)
slides were imaged from the collections at the Cambridge University Museum of Zoology, and the
Hill Collection, Museum für Naturkunde, Leibniz Institute for Research on Evolution and Biodiversity,
Berlin. Details of samples imaged are in Table 1. All museum samples have been studied in previ-
ously published works (Green, 1937; Presley and Steel, 1978; Watson, 1916). Stages for platypus
are estimated based on Ashwell, 2012. Staging of echidna H.SP EC5 and H.SP EC4 are estimated
by cross-referencing (Griffiths, 1978; Rismiller and McKelvey, 2003). Post-hatching day 0 to 3
echidna samples were collected by Marilyn Renfree and Stephen Johnston.
Wildtype and Mesp1Cre; mTmG were kept at the King’s College London Biological Services Unit.
Sox9CreERT2:tdTomato embryos were a gift of Prof Robin Lovell-Badge and Dr Karine Rizzoti at the
Francis Crick Institute, London.
Phosphotungstic acid (PTA) contrasted embryonic Pterobnotus quadridens bat mCT scans were
provided by Prof Karen Sears and Dr Alexa Sadier at the University of California Los Angeles.

Table 1. Museum held monotreme specimens used in the current study.

CRL – Crown rump length. *Estimate based (Ashwell, 2012). †Estimate based on Griffiths, 1978 and Rismiller and McKelvey, 2003.
Species Collection ID Estimated age CRL/Max length
*
Ornithorhynchus anatinus Cambridge Specimen W 2 days 16.5 mm
*
Ornithorhynchus anatinus Berlin M45 6.5 days 33 mm
*
Ornithorhynchus anatinus Cambridge Specimen Delta 10 days 80 mm
Ornithorhynchus anatinus Berlin M038 30 days Unknown
Ornithorhynchus anatinus Cambridge HP 50 days * 200 mm
Ornithorhynchus anatinus Cambridge Specimen Beta 80 days* 250 mm
Ornithorhynchus anatinus Cambridge HX 120 days 295 mm
Tachyglossus aculeatus Cambridge Echidna H.SP EC5 18 days† 83 mm
†
Tachyglossus aculeatus Cambridge Echidna H.SP EC4 55–65 days 174 mm

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Guinea pig (Cavia porcellus) displays samples were collected as previously described
(Anthwal et al., 2015).
Gecko and mouse samples were investigated during embryonic development (35 days post ovi-
position (dpo) and E16.5 respectively). The gestation for geckos is around 60 days, and mice have a
gestation of 20–21 days. Much of opossum and echidna development occurs during early post-ges-
tation/hatching life, including formation of the dentary-squamosal joint, and so 4-day-old opossums,
and 0- to 3-day-old echidnas were investigated before the onset of this joint.
All culling of mouse, opossum, guinea pig and reptile tissue followed Schedule One methods as
approved by the UK Home Office and was performed by trained individuals. Use of genetically mod-
ified mice was approved by the local GMO committee at King’s, under personal and project licences
in accordance with the Animal (Scientific Procedures) Act of 1986, UK.

Tissue processing and histological staining

All tissues for histological sectioning were fixed overnight at 4˚C in 4% paraformaldehyde (PFA),
before being dehydrated through a series of graded ethanol, cleared with Histoclear II, before wax
infiltration with paraffin wax at 60˚C. Wax-embedded samples were microtome sectioned at 8 mm
thickness, then mounted in parallel series on charged slides.
For histological examination of bone and cartilage, the slides were then stained with picrosirius
red and alcian blue trichrome stain using standard techniques.

Immunofluorescence
For immunofluorescence staining slides were rehydrated through a graded series of ethanol to PBS.
Heat induced antigen retrieval was carried out by microwaving the samples for 10 min in 0.1M
Sodium citrate pH6 buffer. Slides were then blocked in 1% Bovine serum albumin, 0.1% cold water
fish skin gelatine, 0.1% triton-X for 1 hr. Sections were then treated over night at 4˚C with primary
antibodies. The following primary antibodies were used, rabbit anti Sox9 (Chemicon) at a dilution of
1/200, chicken anti GFP (Abcam) at a dilution of 1/500, rat anti RFP (Chromotek) at a dilution of 1/
200, Rabbit anti Beta-catenin (Santa Cruz) 1/200, mouse anti type 2 collagen (DSHB) at 1/50, mouse
anti CD44 (DSHB) at 1/50, mouse anti Tenascin C (DSHB) at 1/40, mouse anti versican (DSHB) at 1/
50, rabbit anti versican V1 (Abcam) at 1/400. Following repeated PBS washes, secondary antibodies
were added. For fluorescent labelling the following antibodies were used at 1/300: Alexa568 conju-
gated Donkey anti-Rabbit, Alexa 488 conjugated Donkey anti-Rabbit, Alexa568 conjugated Donkey
anti-Mouse, Alexa568 conjugated Donkey anti-Rat, Alexa488 conjugated Donkey anti-Chicken (all
Invitrogen). Secondary antibodies were added in the blocking buffer for 1 hr at room temperature in
the dark. The secondary antibody was then washed off with PBS, and the slides mounted with Flur-
oshield mounting medium containing DAPI (Abcam). Sections were visualised by Leica SP5 confocal
microscopy. For Versican and CD44 slides, secondary biotinylated goat anti-mouse antibody (Dako)
was added to the slides 1/400 in blocking buffer. Slides were then washed in PBS before being
treated with ABC-HRP streptavidin kit (Vector Labs), and then revealed with DAB (Vector Labs).
Monotreme immunofluorescence staining was carried out in technical replicates due to the rare
nature of the samples. Mouse and opossum analysis was carried out in biological triplicates.

In situ hybridisation
Radioactively labelled antisense RNA probes were made against mouse Gdf5 and Bapx1 mRNA,
and radioactive in situ hybridisations were carried out to detect the expression of these genes in
sagittal plain cut sections of wildtype mice, as previously described (Tucker et al., 2004). All in situ
staining was carried out in biological replicates.

3D reconstruction
Three-dimensional reconstructions of middle ear and surrounding cranial base cartilages were gener-
ated from serial histology images in FIJI (ImageJ 1.47 v), using the Trackem2 Plugin
(Schindelin et al., 2015; Schindelin et al., 2012).

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 Research article Developmental Biology Evolutionary Biology

Cell density counts

Cell density was counted in a DAPI stained sections of 5-day-old opossums (n = 3). In FIJI, 20 sepa-
rate 80 mm2 fields were randomly placed across the mesenchyme surrounding the incus across 5 sec-
tions. The total number of nuclei were counted if they were located wholly within the field, or where
more than 50% of the nuclei intersected the upper or right hand margin of the field. Next by looking
at parallel alcian blue stained sections, the fields were scored as being in proteoglycan-rich or weak
regions. Two fields were ambiguous, and so were removed from the analysis. The user did not know
the proteoglycan status of the field at the time of counting. Next the mean cell number in each field
was calculated in the remaining 8 proteoglycan-rich (alcian blue stained) regions and 10 proteogly-
can weak (weak alcian blue stain) regions, and compared by unpaired two-tailed students t-test in
Prism statistical analysis software (Graphpad), with p<0.05.

Acknowledgements
Thanks to Karen Sears (UCLA) for the bat microCT scan. Thanks to Robert Asher for access to mono-
treme samples held at the Zoological Museum in Cambridge University, and to Andrew Gillis for
imaging. Thanks to Peter Giere for access to the Hill Collection at the Berlin Museum fur Natur-
kunde. Thanks to Prof Robin Lovell-Badge and Dr Karine Rizzoti at the Francis Crick Institute, London
for provision of the Sox9cre/Tom mice. Mesp1cre mice were provided through an MTA agreement
with RIKEN, Japan. Thanks to Currumbin Wildlife Sanctuary for the echidna pouch young samples.

Additional information
Funding
Funder Grant reference number Author
Wellcome 102889/Z/13/Z Abigail S Tucker
Australian Research Council Linkage Grant Stephen D Johnston
Marilyn B Renfree

The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.

Author contributions
Neal Anthwal, Conceptualization, Formal analysis, Investigation, Methodology, Writing - original
draft, Writing - review and editing; Jane C Fenelon, Stephen D Johnston, Marilyn B Renfree, Resour-
ces, Writing - review and editing; Abigail S Tucker, Conceptualization, Supervision, Funding acquisi-
tion, Investigation, Writing - review and editing

Author ORCIDs
Neal Anthwal https://orcid.org/0000-0002-4104-7839
Jane C Fenelon https://orcid.org/0000-0001-8771-5196
Marilyn B Renfree https://orcid.org/0000-0002-4589-0436
Abigail S Tucker https://orcid.org/0000-0001-8871-6094

Ethics
Animal experimentation: This study was performed under UK Home Office licence and regulations in
line with the regulations set out under the United Kingdom Animals (Scientific Procedures) Act 1986
and the European Union Directive 2010/63/EU. The University of Queensland Animal Experimenta-
tion Ethics Committees approved all sampling for echidnas, in accordance with the National Health
and Medical Research Council of Australia guidelines 2013.

Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.57860.sa1
Author response https://doi.org/10.7554/eLife.57860.sa2

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 Research article Developmental Biology Evolutionary Biology

Additional files
Supplementary files
. Transparent reporting form

Data availability
All data generated or analysed during this study are included in the manuscript.

References
Aitkin LM, Johnstone BM. 1972. Middle-ear function in a monotreme: the Echidna (Tachyglossus aculeatus).
Journal of Experimental Zoology 180:245–250. DOI: https://doi.org/10.1002/jez.1401800210, PMID: 5025443
Allin EF. 1975. Evolution of the mammalian middle ear. Journal of Morphology 147:403–437. DOI: https://doi.
org/10.1002/jmor.1051470404, PMID: 1202224
Amin S, Tucker AS. 2006. Joint formation in the middle ear: lessons from the mouse and guinea pig.
Developmental Dynamics 235:1326–1333. DOI: https://doi.org/10.1002/dvdy.20666, PMID: 16425222
Anthwal N, Joshi L, Tucker AS. 2013. Evolution of the mammalian middle ear and jaw: adaptations and novel
structures. Journal of Anatomy 222:147–160. DOI: https://doi.org/10.1111/j.1469-7580.2012.01526.x,
PMID: 22686855
Anthwal N, Peters H, Tucker AS. 2015. Species-specific modifications of mandible shape reveal independent
mechanisms for growth and initiation of the coronoid. EvoDevo 6:35. DOI: https://doi.org/10.1186/s13227-015-
0030-6, PMID: 26568815
Anthwal N, Urban DJ, Luo Z-X, Sears KE, Tucker AS. 2017. Meckel’s cartilage breakdown offers clues to
mammalian middle ear evolution. Nature Ecology & Evolution 1:0093. DOI: https://doi.org/10.1038/s41559-
017-0093
Anthwal N, Tucker AS. 2020. The TMJ disc is a common ancestral feature in all mammals, as evidenced by the
presence of a rudimentary disc during monotreme development. Frontiers in Cell and Developmental Biology
8:356. DOI: https://doi.org/10.3389/fcell.2020.00356, PMID: 32509783
Archer M. 1976. The basicranial region of marsupicarnivores (Marsupialia), interrelationships of carnivorous
marsupials, and affinities of the insectivorous marsupial peramelids. Zoological Journal of the Linnean Society
59:217–322. DOI: https://doi.org/10.1111/j.1096-3642.1976.tb01016.x
Ashwell KW. 2012. Development of the hypothalamus and pituitary in Platypus (Ornithorhynchus anatinus) and
short-beaked echidna (Tachyglossus aculeatus). Journal of Anatomy 221:9–20. DOI: https://doi.org/10.1111/j.
1469-7580.2012.01508.x, PMID: 22512474
Choocheep K, Hatano S, Takagi H, Watanabe H, Kimata K, Kongtawelert P, Watanabe H. 2010. Versican
facilitates chondrocyte differentiation and regulates joint morphogenesis. Journal of Biological Chemistry 285:
21114–21125. DOI: https://doi.org/10.1074/jbc.M109.096479, PMID: 20404343
Crompton AW, Parker P. 1978. Evolution of the mammalian masticatory apparatus. American Scientist 66:192–
201. PMID: 646211
Filan SL. 1991. Development of the middle ear region in Monodelphis domestica (Marsupialia, Didelphidae):
marsupial solutions to an early birth . Journal of Zoology 225:577–588. DOI: https://doi.org/10.1111/j.1469-
7998.1991.tb04326.x
Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, Allen S, MacPherson S, Luyten FP, Archer
CW. 1999. Mechanisms of GDF-5 action during skeletal development. Development 126:1305–1315.
PMID: 10021348
Gates GR, Saunders JC, Bock GR, Aitkin LM, Elliott MA. 1974. Peripheral auditory function in the Platypus,
Ornithorhynchus anatinus. The Journal of the Acoustical Society of America 56:152–156. DOI: https://doi.org/
10.1121/1.1903246, PMID: 4853586
Green H. 1937. VIII—The development and morphology of the teeth of Ornithorhynchus *. Philosophical
Transactions of the Royal Society of London. Series B, Biological Sciences 228:367–420. DOI: https://doi.org/
10.1098/rstb.1937.0015
Griffiths M. 1978. The Biology of the Monotremes. London: Academic Press.
Habib H, Hatta T, Rahman OI, Yoshimura Y, Otani H. 2007. Fetal jaw movement affects development of articular
disk in the temporomandibular joint. Congenital Anomalies 47:53–57. DOI: https://doi.org/10.1111/j.1741-
4520.2007.00143.x, PMID: 17504387
Hartmann C, Tabin CJ. 2001. Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing
appendicular skeleton. Cell 104:341–351. DOI: https://doi.org/10.1016/S0092-8674(01)00222-7, PMID: 112393
92
Holland N, Jackson SM. 2002. Reproductive behaviour and food consumption associated with the captive
breeding of Platypus (Ornithorhynchus anatinus). Journal of Zoology 256:279–288. DOI: https://doi.org/10.
1017/S0952836902000328
Jahan E, Matsumoto A, Rafiq AM, Hashimoto R, Inoue T, Udagawa J, Sekine J, Otani H. 2014. Fetal jaw
movement affects ihh signaling in mandibular condylar cartilage development: the possible role of ihh as

Anthwal et al. eLife 2020;9:e57860. DOI: https://doi.org/10.7554/eLife.57860 20 of 22

 Research article Developmental Biology Evolutionary Biology

mechanotransduction mediator. Archives of Oral Biology 59:1108–1118. DOI: https://doi.org/10.1016/j.

archoralbio.2014.06.009, PMID: 25033382
Kamiya N, Watanabe H, Habuchi H, Takagi H, Shinomura T, Shimizu K, Kimata K. 2006. Versican/PG-M regulates
chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation. Journal of Biological
Chemistry 281:2390–2400. DOI: https://doi.org/10.1074/jbc.M509341200, PMID: 16257955
Kemp TS. 2005. The origin and evolution of mammals. Oxford, UK: Oxford University Press.
Keyte AL, Smith KK. 2008. Opossum (Monodelphis domestica): A marsupial development model. Cold Spring
Harbor Protocols 2008:emo104. DOI: https://doi.org/10.1101/pdb.emo104
Kielan-Jaworowska Z, Cifelli R, Luo Z-X. 2004. Mammals from the age of dinosaurs : origins, evolution, and
structure. Columbia University Press.
Luo Z-X. 2011. Developmental patterns in mesozoic evolution of mammal ears. Annual Review of Ecology,
Evolution, and Systematics 42:355–380. DOI: https://doi.org/10.1146/annurev-ecolsys-032511-142302
Luo Z, Crompton AW. 1994. Transformation of the quadrate (incus) through the transition from non-mammalian
cynodonts to mammals. Journal of Vertebrate Paleontology 14:341–374. DOI: https://doi.org/10.1080/
02724634.1994.10011564
Maier W. 1987. The angular process of Monodelphis domestica (Didelphidae, Marsupialia) and its relation to the
middle ear: an ontogenetic and evolutionary morphologic study]. Gegenbaurs Morphologisches Jahrbuch 133:
123–161. PMID: 3569814
Meng J, Bi S, Zheng X, Wang X. 2018. Ear ossicle morphology of the jurassic euharamiyidan Arboroharamiya and
evolution of mammalian middle ear. Journal of Morphology 279:441–457. DOI: https://doi.org/10.1002/jmor.
20565, PMID: 27228358
Müller F. 1968. Zur phylogenese des sekundären kiefergelenks. Rev Suisse Zool 75:373–413.
Nandadasa S, Foulcer S, Apte SS. 2014. The multiple, complex roles of versican and its proteolytic turnover by
ADAMTS proteases during embryogenesis. Matrix Biology 35:34–41. DOI: https://doi.org/10.1016/j.matbio.
2014.01.005, PMID: 24444773
O’Gorman S. 2005. Second branchial arch lineages of the middle ear of wild-type and Hoxa2 mutant mice.
Developmental Dynamics 234:124–131. DOI: https://doi.org/10.1002/dvdy.20402, PMID: 15861402
Ortega HH, Muñoz-de-Toro MM, Luque EH, Montes GS. 2003. Morphological characteristics of the interpubic
joint (Symphysis pubica) of rats, guinea pigs and mice in different physiological situations a comparative study.
Cells Tissues Organs 173:105–114. DOI: https://doi.org/10.1159/000068947, PMID: 12649588
Payne SL, Holliday CM, Vickaryous MK. 2011. An osteological and histological investigation of cranial joints in
geckos. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology 294:399–405.
DOI: https://doi.org/10.1002/ar.21329
Presley R, Steel FL. 1978. The pterygoid and ectopterygoid in mammals. Anatomy and Embryology 154:95–110.
DOI: https://doi.org/10.1007/BF00317957, PMID: 677485
Renfree MB. 2010. Review: marsupials: placental mammals with a difference. Placenta 31 Suppl:S21–S26.
DOI: https://doi.org/10.1016/j.placenta.2009.12.023, PMID: 20079531
Rich TH, Hopson JA, Musser AM, Flannery TF, Vickers-Rich P. 2005. Independent origins of middle ear bones in
monotremes and therians. Science 307:910–914. DOI: https://doi.org/10.1126/science.1105717, PMID: 15705
848
Rismiller PD, McKelvey MW. 2003. Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus
aculeatus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 136:851–865.
DOI: https://doi.org/10.1016/S1095-6433(03)00225-3
Rodrı́guez-Vázquez JF, Yamamoto M, Abe S, Katori Y, Murakami G. 2018. Development of the human incus with
special reference to the detachment from the chondrocranium to be transferred into the middle ear. The
Anatomical Record 301:1405–1415. DOI: https://doi.org/10.1002/ar.23832
Rosa RG, Akgul Y, Joazeiro PP, Mahendroo M. 2012. Changes of large molecular weight hyaluronan and versican
in the mouse pubic symphysis through pregnancy. Biology of Reproduction 86:44. DOI: https://doi.org/10.
1095/biolreprod.111.093229, PMID: 22011392
Sánchez-Villagra MR, Gemballa S, Nummela S, Smith KK, Maier W. 2002. Ontogenetic and phylogenetic
transformations of the ear ossicles in marsupial mammals. Journal of Morphology 251:219–238. DOI: https://
doi.org/10.1002/jmor.1085, PMID: 11835361
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S,
Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source
platform for biological-image analysis. Nature Methods 9:676–682. DOI: https://doi.org/10.1038/nmeth.2019,
PMID: 22743772
Schindelin J, Rueden CT, Hiner MC, Eliceiri KW. 2015. The ImageJ ecosystem: an open platform for biomedical
image analysis. Molecular Reproduction and Development 82:518–529. DOI: https://doi.org/10.1002/mrd.
22489, PMID: 26153368
Segall W. 1970. Morphological parallelisms of the Bulla and auditory ossicles in some insectivores and
marsupials. Fieldiana Zool 51:1–52. DOI: https://doi.org/10.5962/bhl.title.2899
Segall W. 1973. Characteristics of the ear, especially the middle ear in fossorial mammals, compared with those
in the Manidae. Cells Tissues Organs 86:96–110. DOI: https://doi.org/10.1159/000144112
Shibata S, Sakamoto Y, Baba O, Qin C, Murakami G, Cho BH. 2013. An immunohistochemical study of matrix
proteins in the craniofacial cartilage in midterm human fetuses. European Journal of Histochemistry 57:39.
DOI: https://doi.org/10.4081/ejh.2013.e39

Anthwal et al. eLife 2020;9:e57860. DOI: https://doi.org/10.7554/eLife.57860 21 of 22

 Research article Developmental Biology Evolutionary Biology

Shibata S, Sakamoto Y, Yokohama-Tamaki T, Murakami G, Cho BH. 2014. Distribution of matrix proteins in
perichondrium and periosteum during the incorporation of Meckel’s Cartilage into Ossifying Mandible in
Midterm Human Fetuses: An Immunohistochemical Study. The Anatomical Record 297:1208–1217.
DOI: https://doi.org/10.1002/ar.22911
Smith KK. 1994. Development of craniofacial musculature in Monodelphis domestica (Marsupialia, Didelphidae).
Journal of Morphology 173:149–173. DOI: https://doi.org/10.1002/jmor.1052220204
Snow HE, Riccio LM, Mjaatvedt CH, Hoffman S, Capehart AA. 2005. Versican expression during skeletal/joint
morphogenesis and patterning of muscle and nerve in the embryonic mouse limb. The Anatomical Record Part
A: Discoveries in Molecular, Cellular, and Evolutionary Biology 282A:95–105. DOI: https://doi.org/10.1002/ar.a.
20151
Storm EE, Kingsley DM. 1999. GDF5 coordinates bone and joint formation during digit development.
Developmental Biology 209:11–27. DOI: https://doi.org/10.1006/dbio.1999.9241, PMID: 10208739
Takanashi Y, Shibata S, Katori Y, Murakami G, Abe S, Rodrı́guez-Vázquez JF, Kawase T. 2013. Fetal development
of the elastic-fiber-mediated enthesis in the human middle ear. Annals of Anatomy - Anatomischer Anzeiger
195:441–448. DOI: https://doi.org/10.1016/j.aanat.2013.03.010, PMID: 23706648
Thompson H, Ohazama A, Sharpe PT, Tucker AS. 2012. The origin of the stapes and relationship to the otic
capsule and oval window. Developmental Dynamics 241:1396–1404. DOI: https://doi.org/10.1002/dvdy.23831,
PMID: 22778034
Tucker AS, Watson RP, Lettice LA, Yamada G, Hill RE. 2004. Bapx1 regulates patterning in the middle ear:
altered regulatory role in the transition from the proximal jaw during vertebrate evolution. Development 131:
1235–1245. DOI: https://doi.org/10.1242/dev.01017, PMID: 14973294
Tyndale-Biscoe CH, Janssens PA. 1988. The Developing Marsupial: Models for Biomedical Research. Berlin,
Heidelberg: Springer. DOI: https://doi.org/10.1007/978-3-642-88402-3
Tyndale-Biscoe H, Renfree M. 1987. Reproductive Physiology of Marsupials, Reproductive Physiology of
Marsupials. Cambridge University Press. DOI: https://doi.org/10.1017/CBO9780511623493
Urban DJ, Anthwal N, Luo Z-X, Maier JA, Sadier A, Tucker AS, Sears KE. 2017. A new developmental mechanism
for the separation of the mammalian middle ear ossicles from the jaw. Proceedings of the Royal Society B:
Biological Sciences 284:20162416. DOI: https://doi.org/10.1098/rspb.2016.2416
Wang H, Meng J, Wang Y. 2019. Cretaceous fossil reveals a new pattern in mammalian middle ear evolution.
Nature 576:102–105. DOI: https://doi.org/10.1038/s41586-019-1792-0, PMID: 31776514
Watson DMS. 1916. The monotreme skull: a contribution to mammalian morphogenesis. Phil Trans R Soc Lond B
207:311–374. DOI: https://doi.org/10.1098/rstb.1916.0007
Werneburg I, Laurin M, Koyabu D, Sánchez-Villagra MR. 2016. Evolution of organogenesis and the origin of
altriciality in mammals. Evolution & Development 18:229–244. DOI: https://doi.org/10.1111/ede.12194,
PMID: 27402569
Wu YJ, La Pierre DP, Wu J, Yee AJ, Yang BB. 2005. The interaction of versican with its binding partners. Cell
Research 15:483–494. DOI: https://doi.org/10.1038/sj.cr.7290318, PMID: 16045811
Zahradnicek O, Horacek I, Tucker AS. 2012. Tooth development in a model reptile: functional and null
generation teeth in the gecko Paroedura picta. Journal of Anatomy 221:195–208. DOI: https://doi.org/10.
1111/j.1469-7580.2012.01531.x
Zeller U. 1993. Ontogenetic Evidence for Cranial Homologies in Monotremes and Therians, with Special
Reference to Ornithorhynchus. In: Szalay F. S, Novacek M. J, McKenna M. C (Eds). Mammal Phylogeny. New
York: Springer. p. 95–107.

Anthwal et al. eLife 2020;9:e57860. DOI: https://doi.org/10.7554/eLife.57860 22 of 22