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Brain shapes of large-bodied, flightless ratites (Aves: Palaeognathae) emerge through distinct developmental allometries

The Royal Society
Royal Society Open Science
Authors:
  • Richard Gilder Graduate School
  • New York Institue of Technology College of Osteopathic Medicine

Abstract and Figures

Comparative neuroanatomical studies have long debated the role of development in the evolution of novel and disparate brain morphologies. Historically, these studies have emphasized whether evolutionary shifts along conserved or distinct developmental allometric trends cause changes in brain morphologies. However, the degree to which interspecific differences between variably sized taxa originate through modifying developmental allometry remains largely untested. Taxa with disparate brain shapes and sizes thus allow for investigation into how developmental trends contribute to neuroanatomical diversification. Here, we examine a developmental series of large-bodied ratite birds (approx. 60–140 kg). We use three-dimensional geometric morphometrics on cephalic endocasts of common ostriches, emus and southern cassowaries and compare their developmental trajectories with those of the more modestly sized domestic chicken, previously shown to be in the same allometric grade as ratites. The results suggest that ratites and chickens exhibit disparate endocranial shapes not simply accounted for by their size differences. When shape and age are examined, chickens partly exhibit more accelerated and mature brain shapes than ratites of similar size and age. Taken together, our study indicates that disparate brain shapes between these differently sized taxa have emerged from the evolution of distinct developmental allometries, rather than simply following conserved scaling trends.
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Brain shapes of large-
bodied, flightless ratites
(Aves: Palaeognathae)
emerge through distinct
developmentalallometries
Meghan R. Forcellati1,2, Todd L. Green4,5 and Akinobu
Watanabe5,3,6
1Department of Ecology, Evolution, and Environmental Biology, Columbia University, New
York, NY 10027, USA
2Richard Gilder Graduate School, and 3Division of Paleontology, American Museum of Natural
History, New York, NY 10024, USA
4Biomedical and Anatomical Sciences, New York Institute of Technology, College of Osteopathic
Medicine at Arkansas State University, Jonesboro, AR 72401, USA
5Department of Anatomy, New York Institute of Technology, College of Osteopathic Medicine,
Old Westbury, NY 11568, USA
6Life Sciences Department, Natural History Museum, London SW7 5BD, UK
MRF,0000-0001-5886-4686; TLG,0000-0003-1407-6738;
AW,0000-0001-5057-4772
Comparative neuroanatomical studies have long debated
the role of development in the evolution of novel and
disparate brain morphologies. Historically, these studies have
emphasized whether evolutionary shifts along conserved
or distinct developmental allometric trends cause changes
in brain morphologies. However, the degree to which
interspecific differences between variably sized taxa originate
through modifying developmental allometry remains largely
untested. Taxa with disparate brain shapes and sizes thus
allow for investigation into how developmental trends
contribute to neuroanatomical diversification. Here, we
examine a developmental series of large-bodied ratite
birds (approx. 60–140 kg). We use three-dimensional
geometric morphometrics on cephalic endocasts of common
ostriches, emus and southern cassowaries and compare their
developmental trajectories with those of the more modestly
sized domestic chicken, previously shown to be in the same
allometric grade as ratites. The results suggest that ratites
and chickens exhibit disparate endocranial shapes not simply
accounted for by their size differences. When shape and age
are examined, chickens partly exhibit more accelerated and
© 2024 The Author(s). Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
Research
Cite this article: Forcellati MR, Green TL,
Watanabe A. 2024 Brain shapes of large-bodied,
flightless ratites (Aves: Palaeognathae) emerge
through distinct developmental allometries. R.
Soc. Open Sci. 11: 240765.
https://doi.org/10.1098/rsos.240765
Received: 10 May 2024
Accepted: 31 July 2024
Subject Category:
Organismal and evolutionary biology
Subject Areas:
evolution
Keywords:
allometry, micro-CT imaging, endocasts,
geometric morphometrics, Palaeognathae,
ontogeny
Author for correspondence:
Akinobu Watanabe
e-mail: awatanabe@nyit.edu
Electronic supplementary material is available
online at https://doi.org/10.6084/
m9.figshare.c.7431955.
mature brain shapes than ratites of similar size and age. Taken together, our study indicates that
disparate brain shapes between these differently sized taxa have emerged from the evolution of
distinct developmental allometries, rather than simply following conserved scaling trends.
1. Introduction
Identifying the underlying mechanisms which generate neuroanatomical diversity is an enduring goal
in comparative neuroanatomy. Seminal works have analysed total brain volume, brain regional volume
and body size measurements of adult vertebrate samples in order to investigate whether evolution
follows (i) an integrated or concerted pattern, involving changes in clade-wide scaling relationships
(allometry) of brains [1–4] or (ii) a modular or mosaic pattern, where individual brain regions evolve
quasi-independently from one another potentially due to loss of genetic, developmental, functional
and/or spatial constraints on the overall brain morphology [5,6]. Despite being seemingly dichoto-
mous, evidence has supported a combination of both concerted and mosaic trends occurring simulta-
neously within the same lineages [7–10].
Greater degree of concerted evolution is characterized by similar wholesale allometric changes
to the brain across species [1], which could happen through shared developmentally constrained
processes or allometric effects acting across the brain. Studying development and evolution together
would elucidate whether conserved or distinct ontogenetic and allometric factors contribute to brain
morphology divergence between species [11–17]; however, comparative neuroanatomical studies have
typically focused on evolutionary allometric patterns across taxa without examining developmental
trends. For instance, in bats, carnivorans and primates, body size accounts for vast majority of brain
volume variation, with some notable deviations within each clade [13]. Similar analysis on avian
brain volume and body size data demonstrated that these two traits are highly correlated across the
avian phylogeny, and decoupling of their scaling relationship has contributed to identifying notable
brain size differences among major clades [16]. Yet, without developmental sampling, the cause of
interspecific allometric trends remains largely unknown.
Most comparative neuroanatomical studies have taken a volume-based approach to studying trait
variation of brains. This has produced rich bodies of work which have yielded powerful insights
into how brain scaling relationships may evolve, but implementation of geometric morphometric
techniques allows for richer characterization of neuroanatomy. Shape can indicate changes that may
be overlooked by measuring relative volumes because structures with the same volume can still have
different morphologies, which point to the occurrence of important differences in pattern and process.
It is worthwhile to investigate whether patterns in brain volumetric changes across lineages are similar
to patterns in brain shape changes. For example, if volumetric and shape data both indicate that larger
taxa align with smaller taxa in their allometric trends, then we can infer that the brain morphology
of larger taxa represents an extension of conserved developmental trajectories. Alternatively, if shape
trajectories between larger and smaller taxa differ fundamentally, then their neuroanatomical variation
probably arose through evolution of unique developmental allometric trajectories.
In this study, we further elucidate how developmental and evolutionary allometric trajectories
underlie brain evolution by sampling an ontogenetic series of taxa with differences in overall size
and brain morphology. Among birds, the large, flightless palaeognaths, referred to as ratites (moas,
ostriches, rheas, elephant birds, emus, cassowaries and kiwis), are of particular interest as candidates
for examining the influence of size on neuroanatomical evolution [16–19]. Ratites are thought to have
evolved from volant ancestors resembling the small-bodied Eocene palaeognath Lithornis, and then
secondarily lost their flight repeatedly across their evolutionary history [20–22]. Even with the advent
of modern micro-computed tomography (µCT) methods, most neuroanatomical descriptions of adult
palaeognaths were performed during the early twentieth century without using modern CT scan
methods (see [23]). In addition, the literature describing palaeognath cranial ontogeny is even more
scarce (but see [17,18,24–27]). Studies have found that ratites differed substantially from members of
other early diverging birds such as Galliformes in their relative brain volume proportions and in their
brain composition [12,28]. In contrast, a recent comparative volumetric analysis more broadly sampling
the avian phylogeny identified large, flightless palaeognaths as sharing a statistically similar allomet-
ric relationship between brain volume and body size as some non-avian dinosaurs and other early
diverging birds, including galliforms such as the domestic chicken Gallus gallus [16]. From the former
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observation, ontogeny of ratite brain morphology would probably be distinct from that of galliforms,
in line with regional volume differences [12,28]. From the latter observation, ratite brain morphology
might represent a larger end of a continuum along the same developmental and allometric trajectory
shared with other early diverging birds with more modest, typical avian body sizes such as chickens
[16].
To investigate these possible scenarios, we utilize a comprehensive ontogenetic series of three ratite
species: common ostrich (Struthio camelus), emu (Dromaius novaehollandiae) and southern cassowary
(Casuarius casuarius), with the avian model system domestic chicken (G. gallus) as comparison. We
reconstruct endocasts (internal moulds of the brain cavity) from µCT imaging data and provide a new
description of the neuroanatomical changes occurring throughout ontogeny in these birds. We then use
a high-density geometric morphometric approach to test whether the patterns identified from brain
volume and body size data extend to patterns in brain shape, and how developmental trajectories may
relate to differences in endocranial shape between adult birds. We refer to endocranial regions by the
name of the corresponding neuroanatomical features that are reflected on the surface (e.g. ‘cerebrum’
for impressions of the cerebrum on the endocranial surface).
2. Material and methods
2.1. Specimens
Sampling included captive-bred late-stage embryonic to adult specimens of 14 S. camelus, 14 D.
novaehollandiae and 18 C. casuarius from the American Museum of Natural History (AMNH; New
York, NY, USA), Museum of Osteology (MOO; Oklahoma City, OK, USA) and T. L. Green Research
Collection (TLG; Denver, CO, USA). We combined this new sampling with an ontogenetic series of 14
G. gallus collected from Charles River Laboratory (North Franklin, CT, USA) and originally published
in Watanabe et al. [29]. A full list of specimens with specific institution, preservation type, age, sex
and µCT scanning parameters can be found in the electronic supplementary material, table S1. All
specimens had intact skulls with adequate cranial ossification to reconstruct cephalic endocasts. The
age range for each taxon is as follows: S. camelus, approximately HH39 (embryo) to at least 20 years
old; D. novaehollandiae, approximately HH40 (embryo) to 16 years old; C. casuarius, approximately
HH41 (embryo) to 35 years old; G. gallus, 1 day old neonate to greater than 8 weeks old. The stages
of embryonic ratite specimens were determined using the criteria previously described in Green and
Gignac [24]. Because ratite specimens were collected opportunistically, only an age range was given
for some specimens and the mid-point of the range was used as the age for these specimens. Age data
were recorded and converted to number of days, and this dataset was used to create additional age
data that added the incubation periods of each taxon to the age (21 days for G. gallus, 48 days for C.
casuarius, 50 days for D. novaehollandiae and 42 days for S. camelus), as well as age data standardized
for the respective age of sexual maturity (7 months for G. gallus, 5 years for C. casuarius, 2 years for D.
novaehollandiae and S. camelus) [30,31]. We refer to the sum of the age and incubation period as ‘total
age’, and to the total age standardized with respect to age of sexual maturity as ‘standardized age’. An
institutional animal care and use protocol was not required for ratites as all specimens were collected
and sampled as cadaveric specimens after death. No ratite individuals were harmed or sacrificed for
the purpose of this study. G. gallus specimens from previously published research were euthanized
through cervical dislocation by Charles River Laboratory, following approved ethical standards. This
protocol is described in [29] and was approved by the AMNH Institutional Animal Care and Use
Committee in 2014 at the time of specimen collection.
2.2. Imaging and endocranial reconstructions
The heads of ratite specimens were µCT-scanned on the following institutional systems: (i) a 2010
GE phoenix v|tome|x s240 high-resolution microfocus CT system (General Electric, Fairfield, CT,
USA) located in the Microscopy and Imaging Facility of the AMNH, (ii) a Mediso nanoScan PET/CT
(Arlington, VA, USA) located in the Genome Research Center at the Cold Spring Harbor Laboratory
(CSHL; Woodbury, NY, USA), (iii) a 2012 Nikon XT H 225 ST µCT system (Nikon Metrology, Brighton,
MI, USA) located at the Dentsply Research and Development Office (Dentsply; Tulsa, OK, USA),
and (iv) a 2018 Nikon XT H 225 ST µCT system located at the Micro-CT Imaging Consortium for
Research and Outreach (MICRO; Fayetteville, AR, USA). Electronic supplementary material, table S1
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lists scanner used for each specimen. Larger specimens with multiple scans had separate image stacks
which were imported into ImageJ (FIJI) v1.53c [32] and fused using the ‘3D Stitching’ function. Full
image stacks of ratite data were imported for the virtual segmentation into Dragonfly v. 2021.1.0.977
(Object Research Systems, Inc., Montreal, Canada) and Amira v. 2019.1 (Thermo Fisher Scientific,
Waltham, MA, USA). Endocranial segmentation was conducted following recommendations proposed
by Balanoff et al. [33]. Reconstructed endocasts were then exported as three-dimensional polygon mesh
(PLY) files to be further processed. Endocranial reconstructions of the G. gallus are from a previous
study [29,34].
We considered these to be reasonable proxies of internal moulds of the brain after [29]. Although
Watanabe et al. [29] did not sample palaeognaths, (i) they found that even in crocodiles, much more
distant outgroups to chickens than the ratites we sampled, there was still decent fidelity between the
endocranium and the forebrain and midbrain early in ontogeny, (ii) our immature ratites sampled,
starting after about 5.5 months in C. casuarius, about 11 months in S. camelus and about 3 months in
D. novaehollandiae, preserved impressions of blood vessels on their cerebra throughout the rest of their
ontogeny, which other researchers before Watanabe et al. [29] used as an indicator of high soft tissue-
endocranial correspondence [35], (iii) research in brain volume and skull volume across many species
of birds has suggested close correspondence [36], and (iv) we observed that the brain of embryonic
C. casuarius closely corresponded to the brain cavity (electronic supplementary material, figure S1), in
line with the interpretations of Watanabe et al. [29]. Thus, we considered brain shape and centroid size
(CS) data to closely be reflected by endocranial shape data and we interpret changes in the endocasts
as broadly reflecting changes in the brain in the discussion [29,37]. After endocasts were generated, we
processed the data in GeoMagic Wrap v. 2020 (3D Systems, Rock Hill, SC, USA). First, we removed
small, isolated components from the mesh file using the ‘Mesh Doctor’ tool in the program. Then,
we used its ‘QuickSmooth’ tool to globally smooth the endocranial meshes and thus remove noise
artificially introduced during segmentation. Research has suggested surface simplification techniques
affect a very small fraction of the variation in geometric morphometric shape data for Type I landmarks
[38], and it appears this holds true for semi-landmarks as well [39]. After this procedure, to facilitate
importation into the software we used for landmarking, we decimated the specimens in Meshlab v.
2022.02 [40] to 1.5 million faces for models with more than this number of polygons (figure 1).
2.3. Geometric morphometric data
We performed high-density three-dimensional landmark-based geometric morphometrics to character-
ize endocast shapes, sizes and regional morphologies (figure 2). Our landmarking scheme excludes the
olfactory bulb due to the difficulty in consistently identifying and delineating this structure through
ontogeny. Instead, we briefly commented on possible differences between taxa in our morphological
description of the endocasts. Likewise, we excluded a few important structures from the hindbrain,
such as the flocculus and the pituitary gland, because these could not be reliably landmarked. While
similar to a previous landmarking scheme [19,29,34], we used a different approach for placing surface
semi-landmarks to achieve a more even distribution of surface semi-landmarks than previously. We
used Landmark Editor v. 3.6 [41] to place discrete landmarks on the right side of the endocasts
and curve semi-landmarks that define the major brain regions. Then, the curve semi-landmarks
were resampled using previously customized code [39,42]. For placing surface semi-landmarks, the
‘placePatch’ function from the ‘Morpho’ R package [43] was used to project surface semi-landmarks
from a template using a somatically mature domestic G. gallus.
After landmark data were collected, we performed generalized Procrustes alignment while
minimizing total bending energy and allowing semi-landmarks to slide on the mesh surface using
a combination of ‘slider3d’ function in the ‘Morpho’ package [43] and the ‘gpagen’ function in the
‘geomorph’ R package [44–49]. To avoid artefacts from aligning one side of a bilaterally symmetric
structure [50,51], we mirrored the right landmarks onto the left sides of the endocasts across the
midsagittal landmarks used for our analysis with the ‘mirrorfill’ function from the ‘paleomorph’
R package [52]. After alignment, the mirrored landmarks on the left side of the endocast were
removed. The final shape data comprise 13 fixed landmarks, 66 curved sliding semi-landmarks
and 140 sliding surface semi-landmarks (electronic electronic supplementary material, table S2). We
also acquired CS, the square root of the sum of the squared distances of landmarks from their
centroids [53].
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2.4. Analysis
All analyses were performed using R v. 4.2.1 [54]. We used the ‘gm.prcomp’ function in the ‘geomorph’
R package v. 4.0.6 [48] and base ‘prcomp’ function to perform principal components analysis (PCA)
on the endocranial shape data to create morphospaces that visualize overall shape differences across
taxa and ontogenetic stages. This was conducted for two datasets: one including all ratite and G. gallus
specimens, and one with exclusively ratites to target neuroanatomical differences among ratite species.
First, we plotted CS against combined age (postnatal age plus incubation age) to confirm whether
ratites exhibit larger brains that go beyond G. gallus brain sizes during development. Next, to
Embryonic + Neonate Adult
(e)(d)(c)(b)(a)
(f) (g) (h) (i) (j)
(k) (l) (m) (n) (o)
(t)(s)(r)(q)(p)
Gallus
Struthio
Dromaius
Casuarius
Figure 1. Exemplar endocasts of (a–e) G. gallus, (fj) S. camelus, (ko) D. novaehollandiae and (pt) C. casuarius sampled for
this study in right lateral (above) and dorsal (below) views. Ontogenetic sequences are ordered from most immature (left) to most
mature (right) for the following specimens: (a) TLG GG015 (immature, 1 day); (b) AWRC Gg014 (immature, 1 week); (c) AWRC Gg017
(immature, 6 weeks); (d) TLG GG017 (immature, 8 weeks); (e) AWRC Gg020 (adult); (f) TLG SC094 (embryonic, approx. HH39); (g)
TLG SC032 (immature, 1 day); (h) TLG SC030 (immature, 11 months); (i) TLG SC083 (adult, approx. 4.0–5.0 years); (j) TLG SC080
(adult, approx. 19.5–21.5 years); (k) TLG E139 (embryonic, approx. HH40); (l) TLG E093 (immature, 5 days); (m) TLG E115 (immature,
12 months); (n) TLG E114 (adult, greater than or equal to 3.0 years); (o) TLG E167 (adult, approx.y 14.8–16.8 years); (p) TLG C030
(embryonic, approx. HH41); (q) TLG C024 (immature, 9 days); (r) TLGC031 (immature, 14 months); (s) TLG C069 (adult, 6.2 years); (t)
MOO 8031 (adult, 35.7 years). Scale bar = 2 cm.
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graphically compare developmental trajectories between taxa, we used regression scores against
log-transformed CS. In addition, we used a procedure proposed by Mitteroecker et al. [55], that
involves a pooled regression of the shape variables on log-transformed CS and age, using the ‘cac’
function from ‘Morpho’ package [43]. For visualizing the differences, we plotted this vector against
the first PC of the residual values (RSC1) for each species from that line. This first PC of the residuals
captures the deviations from the overall allometric trend shape against log-transformed CS. As in our
PCA for studying endocast shape, we repeated this analysis for two subsets of the data: all specimens,
and ratites exclusively. We also repeated this analysis using size as the allometric variable and log-
transformed total age (postnatal age plus incubation age). Finally, to explore potential mechanisms
responsible for variation in developmental patterns, we plotted shape against age standardized by
approximate age of sexual maturity for each species, with the acknowledgement that the age of sexual
maturity is intraspecifically variable in the taxa we studied.
We next performed non-parametric multivariate analysis of variance (MANOVA) to elucidate
group-level differences between nested pairs of taxa [49,56,57]. We did not perform statistical analyses
with phylogenetic correction due to relatively poor understanding of the timing of evolutionary
divergences among ratite species. Instead, we compared ontogenetic trajectories between nested
phylogenetic pairs of taxa (i.e. G. gallus and ratites, S. camelus and D. novaehollandiae + C. casuarius;
D. novaehollandiae and C. casuarius). When using MANOVA, we statistically tested for differences in the
(a)
(b)
(e)(d)(c)
cer.bl. cer. o.b.
op.l
br.st.
Figure 2. Landmark scheme used in this study shown on the endocranial reconstruction of D. novaehollandiae (TLG E054). Red, blue
and green points represent fixed, curve and surface landmarks, respectively, in (a) right oblique (c) lateral, (d) dorsal and (e) ventral
views. (b) is a right lateral two-dimensional schematic diagram illustrating the main neuroanatomical region divisions being defined
for this study. cer. cerebrum; cer.bl., cerebellum, o.b., olfactory bulb, br.st., brainstem, op.l., optic lobe. Figure is not to scale.
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trajectories across the entire shape space, not just the RSC1 or regression scores we used to visualize
shape differences between species.
For our morphological description, we visualized specimens in Blender v. 2.93.3 (Stichting Blender
Foundation, Amsterdam, The Netherlands). We examined the major brain regions in three dimensions
and noted any differences in the brain regions, the suture lines and the blood vessel impressions we
observed between the species studied.
3. Results
3.1. Neuroanatomical description
3.1.1. Endocranial size
From 1 day old to over 8 weeks old, G. gallus endocasts nearly doubled in rostrocaudal length, from
approximately 16.7 mm in the youngest specimen (figure 1a) to 28.6 mm in the oldest (figure 1e). As
they progress from embryo to adult, S. camelus endocasts also nearly double in rostrocaudal length,
from approximately 25.4 mm in the HH39 embryonic specimen (figure 1f) to 53.4 mm in the adult
over 20 years of age (figure 1j). From embryo to adult, D. novaehollandiae endocasts nearly double in
rostrocaudal length, from approximately 25.3 mm in an HH40 embryo (figure 1k) to 55.2 mm in an
adult between 14.8 and 16.9 years old (figure 1o). From embryo to adulthood, C. casuarius endocasts
nearly triple in rostrocaudal length, from approximately 23.9 mm in an HH41 embryo specimen (figure
1p) to 61.8 mm in the 35.7-year-old adult (figure 1t). While the ontogenetic range is not equivalent
across taxa sampled in this study, the growth data indicate that cassowaries undergo greater growth
during ontogeny.
3.1.2. Forebrain
Across all sampled specimens, the cerebrum expands in each primary anatomical plane during develop-
ment, and the Wulst becomes more prominent, which previous research has noted undergoes the greatest
change in surface area during ontogeny for S. camelus [58]. In all taxa, the sulcus between the cerebrum and
the cerebellum increases because of increasing distances between the development of the two structures,
which starts approximately as the Wulst begins expanding. Adult S. camelus differs from D. novaehollandiae,
C. casuarius and G. gallus in (i) having proportionately smaller olfactory bulb endocranial regions, a
structure that was not characterized in the shape data owing to the difficulty of segmenting it in a systematic
and comparable way; (ii) in having slightly more globular cerebra; and (iii) in having greater dorsoventral
flexion (which is observed in our shape analysis reported below). Casuarius casuarius are unique amongst
the sampled taxa in their rostrocaudally elongate and dorsoventrally wide olfactory tract bases (figure
1t). This appears to begin at some point between 1.5 and 5.5 months in postnatal development; before this
point, C. casuarius olfactory bulbs are remarkably similar to those of D. novaehollandiae (figure 1k,l,p,q). Gallus
gallus differ from ratites in having a cerebellum that is more horizontally level with the cerebrum; these
two regions are separated by a deep gap which emerges between 6 and 8 weeks of postnatal development,
and the difference in positioning of these structures between G. gallus and ratites is obvious even from
late-embryonic stage (figure 1a,f,k,p).
The species further differ in the development of the Wulst. In adult ratites, the Wulst is more
prominent than in adult G. gallus. The Wulst also spans a greater relative mediolateral width in D.
novaehollandiae and C. casuarius than in S. camelus. Furthermore, it is known that the angle of the left
and right Wulsts are distinct between S. camelus and the greater rhea (Rhea americana) compared with
D. novaehollandiae, with S. camelus and R. americana possessing a parallel orientation of the Wulst and D.
novaehollandiae possessing a caudally divergent configuration [18]. We find that this caudally divergent
morphology is also shared between D. novaehollandiae and C. casuarius (figure 1m–o,rt). Second, we
find that these differences are noticeable starting from when the Wulst first becomes externally visible
on the surface of the endocast: in embryonic stages for S. camelus and D. novaehollandiae, and within the
first day post-hatching for C. casuarius (figure 1f,k,p).
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3.1.3. Midbrain
For embryonic and perinatal specimens we examined, it is difficult to distinguish the optic lobe from
the cerebrum in lateral view (i.e. figure 1a,f,k,p). The optic lobe generally elongates along the rostrocau-
dal axis in ratites and along the oblique axis in G. gallus, where it is ventrally slanted rostrally. The
optic lobe is rostrally shifted in ratites compared with that of G. gallus. In perinates through to adults,
the optic lobe in G. gallus is slightly caudal to or at the maximum caudal extent of the cerebrum (figure
1a–e). Indeed, its dorsalmost extent wraps around the caudal cerebrum, nearly reaching the sulcus
between the cerebrum and cerebellum (figure 1a–e). However, in ratites, the optic lobe terminates
either at the level of the maximum caudal extent of the cerebrum or rostrally to it (figure 1f–t). Another
key difference in the optic lobes of G. gallus and ratites is the major axis of the ellipsoid contour
in lateral view. In the optic lobes of G. gallus, it is much more strongly downturned rostroventrally
than those of ratites, which are instead angled more horizontally level with respect to the rest of the
endocast and the sagittal plane (e.g. figure 1d,i,n,s).
3.1.4. Hindbrain
The hindbrain lengthens rostrocaudally during ontogeny for ratites and G. gallus. Its relative medio-
lateral width declines, but this is largely because of the increased lateral prominence of the Wulst
and cerebrum and the rostrocaudal lengthening of the brainstem. This lengthening is accompanied
by a general loss of convexity of the brainstem, instead becoming more flattened in all specimens.
Meanwhile, the flocculus projects farther caudolaterally and becomes more arcuate, as can be seen in
dorsal view (e.g. figure 1a,e).
For all of the endocasts we examined, the cerebellum in the endocast lacks preservation of the
cerebellar foliation, despite it being an important characteristic of cerebellar neuroanatomy in birds
[59]. This structure failing to preserve in endocasts has been documented in other palaeognaths, and
is probably because of a large venous sinus overlying the area which restricts this region from contact
with the remainder of the endocast [18,58].
As previously noted, the hindbrain in ratites is ventrally deflected compared with that of G. gallus,
such that the forebrain is situated relatively more dorsally (figure 1e,j,o,t). In G. gallus, the dorsal
surface of the cerebellum is almost level with the cerebrum (e.g. figure 1d). This difference appears
to emerge early in development and is already visible between the 1-day-old specimens of G. gallus
and three perinatal ratites sampled. It becomes more exaggerated in immature chickens as the sulcus
between the cerebrum and cerebellum deepens, but slightly less exaggerated in adult G. gallus as the
Wulst continues expanding dorsally (figure 1e). Concomitantly, ratites possess a ventral rotation of the
foramen magnum relative to the rest of the endocast: in G. gallus this structure angles approximately
horizontally with respect to the rest of the endocast, while in ratites, it tends to face more ventrally
(figure 1d,i,n,s). The shape of the caudal cerebellum varies between taxa in lateral profile. For instance,
G. gallus specimens possess a shallow but smooth caudal curve of the cerebellum (figure 1d,e, while it
is more linearly slanted in D. novaehollandiae (figure 1m–o) and vertical and perpendicular in both S.
camelus and C. casuarius (figure 1h,i,rt).
3.1.5. Sutures
We observed a general pattern of the suture lines for the overlying frontal and parietal bones being
poorly visible or not visible at all in embryonic and perinatal endocasts (figure 1a,f,k,p). This is
probably because the overlying bones are partially ossified with fontanelles, and because the brain
probably occupies proportionately less of the brain cavity in immature individuals [29]. These suture
lines then become strongly developed in more mature specimens, with lines clearly delimitating
the frontoparietal suture along the boundary between the cerebrum and cerebellum and the caudal
parietal suture on the cerebellum visibly prominent in dorsal view of endocasts (figure 1b,g,l,q) [60].
Finally, in adults, these suture lines become much less prominent on the surface of the endocasts
probably due to further closure of the suture (figure 1e,j,o,t). Typically, this change tends to occur in
the frontoparietal suture first and then the parietal suture. It coincides with both the ossification of
these bones and an overall dorsoventral elaboration of brain in the endocast, resulting in the increased
development of the Wulst and enlargement of the cerebrum and cerebellum.
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3.2. Centroid size data
Based on CS extracted from our coordinate data, we find that endocasts of ratites surpass the size
range observed in the G. gallus sampled in this study (figure 3). The youngest specimens of C. casuarius
and D. novaehollandiae overlap slightly with adult G. gallus in endocast size. When CS of endocasts are
plotted against log-transformed age, the plot indicates that ratites and G. gallus have similar slopes
but distinct intercepts (figure 3). These results are consistent with the notion that ratites may obtain
their larger endocast, and thus, brain sizes, through acceleration or extension of shared developmental
scaling trends. Other researchers have similarly found that chickens and ratites are on a common
allometric grade in terms of their relative brain-to-body size [12,16,28]. If this is true, we may hypothe-
size that the youngest ratites should also have similar brain shapes to G. gallus, because they may also
share a common shape-to-size trajectory. The difference in intercepts could then be explained by longer
or accelerated prenatal development, since we only sampled late-embryonic specimens. We evaluate
this hypothesis below with shape data.
3.3. Shape data
We performed PCA on the endocast shape data and created PC morphospaces to visualize patterns of
endocast shape variation across the entire ontogenetic series of all taxa (figure 4). For the full dataset,
PC1 accounted for 37.6% of the total shape variation, and PC2 accounted for 20.3% of the total variation
(figure 4a). For ratites exclusively, PC1 and PC2 encompass 48.2% and 10.7% of the total variation,
respectively (figure 4b). Because the ontogenetic series are oriented along PC1 axes in both morphospa-
ces and a positive correlation between PC1 and log CS (R2 = 0.493; p < 0.0001) exists, we interpreted PC1
as strongly associated with growth, with more mature, larger specimens possessing higher PC1 values
and younger specimens possessing lower values along the PC1 axis. This close association between
PC1 and size is expected and what has been reported in previous studies examining endocranial
shape in birds and non-avian archosaurs [15,17,29,34,61]. The shape changes associated with PC1
include overall rostrocaudal lengthening of the endocast; increased midbrain flexion in the endocast
between the cerebrum and cerebellum; ventral shift in the optic lobe and increased separation between
it and the cerebrum; rostrocaudal lengthening of the cerebellum and brainstem; and an increased
convex curvature of the cerebellum. The PC2 axis separates G. gallus and ratites in the full dataset
(figure 4a), and a more positive PC2 is associated with more rostrocaudally oriented cerebrum and
cerebellum; sharper dorsal curvature along the cerebrum and cerebellum; caudal shift in and increased
relative size of the optic lobe along with decreased separation between it and the cerebrum; and a
more horizontally oriented caudodorsal surface of the cerebellum. The morphospace shows extensive
overlap between S. camelus and D. novaehollandiae + C. casuarius during early ontogenetic stages, but
with more divergent morphology along PC2 as they grow (figure 4). PC2 is also associated with
log-transformed CS (R2 = 0.388; p < 0.0001), probably driven by the size divergence between G. gallus
and ratites.
In the ratites-only dataset, changes in PC1 mirror distribution of shape variation identified in the
full dataset and similarly are associated with developmental changes occurring in endocast shape
(figure 4b). Struthio camelus occupies a smaller proportion of the PC1 morphospace than the other two
taxa. Conversely, PC2 reflects changes associated with differences between the two lineages, with D.
novaehollandiae and C. casuarius sharing similar, although not entirely overlapping, PC2 values, to the
exclusion of S. camelus throughout all but the earliest stages of their entire ontogeny, during which S.
camelus overlaps with D. novaehollandiae (figure 4b). A more positive PC2 is associated with increased
prominence of the Wulst, especially rostrally; differences in orientation and position of the optic lobe
(both more horizontally oriented with the rest of the forebrain and more ventrally positioned); and a
more vertically sloping caudal portion of the hindbrain (figure 4b).
Statistically, results from the non-parametric MANOVA on the entire Procrustes shape data (i.e.
not PC scores) support the graphical results portrayed by the morphospaces. We statistically tested
whether the aggregate shape data across ontogeny for each of these three groups are significantly
different from each other. First, G. gallus and ratite endocasts differ in shape when their entire
ontogenetic sampling is compared (R2 = 0.189; p < 0.001). Similarly, comparing shape variation between
S. camelus and the combined D. novaehollandiae + C. casuarius data also resulted in significant differences
in the shapes between these two different evolutionary radiations of ratites (R2 = 0.120; p < 0.001).
Conversely, our analysis failed to identify significant differences between D. novaehollandiae and C.
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casuarius (R2 = 0.043; p = 0.217). As expected by our volumetric data, this suggests that ratites are
different from chickens, but also that even within ratites of similar sizes or with convergently evolved
similarities in traits (e.g. flightlessness and large body size), there are still neuroanatomical differences,
except for emus and southern cassowaries, which are sister taxa.
3.3.1. Developmental trajectories
We graphically compared the ontogenetic trajectories of endocranial shape changes associated with
size by plotting the PC1 of residuals (residual shape component, RSC1) from the common allometric
component (CAC), or the pooled allometric trends across all the specimens sampled (figure 5). Similar
to the morphospace results, we find distinct allometric trajectories between G. gallus and ratites (figure
5a), as well as between S. camelus and the other two ratites (figure 5c), but there is some overlap in
the ontogenetic shape changes occurring in C. casuarius and D. novaehollandiae (figure 5c). When we
visualize the regression score against CS for all the lineages, we find that there is minimal overlap
between G. gallus and ratites (figure 6a,b). With this said, we still see considerable differences within
ratites, which generally overlap in size with respect to age (figure 6c,d) but have different shape
and size regressions (figures 4–6). Specifically, they all overlap early in development, but S. camelus
quickly diverges from D. novaehollandiae + C. casuarius. This can be interpreted as a fundamental
difference in the resultant adult brain morphology between the S. camelus and D. novaehollandiae + C.
casuarius trajectories, possibly explained by their greater phylogenetic difference. The major differences
in trajectories can be visualized from the inset image of figure 6c. Here, we can see that S. camelus
possesses a more globular adult brain morphology, particularly in its cerebrum, and a rostrocaudally
compressed cerebellum and optic lobe. When we correct for standardized age (i.e. how old individuals
500
400
300
200
500
400
300
200
500
400
300
200
500
400
300
200
0 5000 10 000 0 5000 10 000
4 6 80 2.5 5.0 7.5
Log Age (Days Post-Hatching)
Age (Days Post-Hatching)
Log Total Age
Total Age
Centroid SizeCentroid Size
Key
Gallus gallus
Casuarius casuarius
Dromaius novaehollandiae
Struthio camelus
(a) (b)
(c) (d)
Figure 3. Plots of endocast centroid size against (a) days post-hatching, (c) log-transformed days post-hatching, (b) total
(post-hatching age plus incubation age) and (d) log-transformed total age between ratites and G. gallus throughout ontogeny. Note
that ratites (C. casuarius, D. novaehollandiae and S. camelus) encompass endocranial volumes larger than that of G. gallus sampled for
this study. Common ostriches (S. camelus) have the largest adult brains but otherwise generally overlap in size with the other ratites
during ontogeny.
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are relative to age of sexual maturity), we see similar results, except that G. gallus seems to have a
greater slope, indicating accelerated development, probably associated with shorter incubation and
life history (electronic supplementary material, figure S2). This result counters the hypothesis that
ratites exhibit more ‘mature’ brain morphology compared with G. gallus from extending along a shared
developmental trajectory.
For our analytical procedure comparing developmental trajectories between G. gallus and ratites
and within ratites, we find statistically significant differences between taxa at all phylogenetic levels
0.075
0.050
0.025
–0.025
–0.050
0
0.04
0.02
–0.02
–0.04
0
–0.05 0 0.05 0.10
–0.05 0 0.05
8 weeks
1 day
~15 years
~15 years
~HH41
~HH41
~35 years
~35 years
~HH40
~HH40
~20 years
~20 years
~HH39
~HH39
PC1 (48.2%)
PC1 (37.6%)
PC2 (10.7%) PC2 (20.3%)
(a)
(b)
Key
Gallus gallus
Casuarius casuarius
Dromaius
novaehollandiae
Struthio camelus
Figure 4. Morphospace of endocast shapes in (a) G. gallus and ratites; (b) within ratites (C. casuarius, D. novaehollandiae and S.
camelus). Non-parametric MANOVA shows G. gallus and ratites significantly differ in endocast shapes (n = 60; R2 = 0.189; p <
0.001). Non-parametric MANOVA shows within ratites, S. camelus significantly differs in endocast shape from D. novaehollandiae and C.
casuarius (n =46; R2 = 0.120; p < 0.001). In contrast, D. novaehollandiae and C. casuarius were found with non-parametric MANOVA to
lack significant differences in shape (n = 32; R2 = 0.043; p = 0.217). Inset images depict extreme shapes along PC1 and PC2 in lateral
view. The oldest and youngest specimens of each group, with approximate ages labelled, are indicated with arrows.
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examined (figures 5 and 6). Specifically, we find that ratites are statistically significantly different
from G. gallus in their developmental trajectories after allometry is accounted for (non-parametric
MANOVA: R2 = 0.175; p < 0.001). We similarly find that S. camelus is significantly different in its
developmental trajectory from D. novaehollandiae + C. casuarius (non-parametric MANOVA: R2 = 0.182;
p < 0.001). Finally, we find that D. novaehollandiae and C. casuarius are significantly different from
each other in their developmental trajectories (non-parametric MANOVA: R2 = 0.050; p < 0.001).
When regressing shape over total age (i.e. age including incubation period), non-parametric MAN-
OVA indicates significant correlation between endocast shape and log-transformed age for combined
ratites and G. gallus (R2 = 0.273; p < 0.001), as well as within ratites (R2 = 0.343; p < 0.001). After the
pooled allometric trend is accounted for, G. gallus and ratites are shown to exhibit statistically distinct
trajectories (R2 = 0.273; p < 0.001), as are S. camelus and D. novaehollandiae + C. casuarius (R2 = 0.487; p =
0.0067).
4. Discussion
Our results show that (i) there is a continuous developmental size distribution between brain size in G.
gallus and ratites, (ii) the brain shapes of ratites are significantly different from each other and G. gallus
throughout development from late-stage embryos to fully mature individuals, and (iii) the develop-
mental allometry of ratite brain shape is distinct from G. gallus, and is not simply an extension of a
common shape-to-size scaling relationship. Examining the developmental endocranial shape changes
with age reveals that this is partly due to accelerated shape change in G. gallus compared with other
ratites. As such, based on our endocast data, there is significant dissociation between brain size and
age between G. gallus and ratites, and even within ratites when comparing S. camelus with the clade
RSC1
0.1
0
–0.1
Key
Gallus gallus
Casuarius casuarius
Dromaius novaehollandiae
Struthio camelus
0.05 00.05
RSC1
0.025
0
0.025
0.050
0.075
0.05 0 0.05
Common Allometric Component (Centroid Size)
0.10
0.05
0
0.05
0.05
0 0.05
0.00
0.03
0.03
0.06
0.09
0.05 0 0.05
Common Allometric Component (Total Age)
(a) (b)
(c)(d)
Figure 5. Plots of PC1 of residuals (RSC1) from the common allometric component (CAC) of endocast shape against log-transformed
log-centroid size for (a) G. gallus and ratites and (c) only ratites (C. casuarius, D. novaehollandiae, S. camelus), and against
log-transformed total age for (b) G. gallus and ratites and (d) only ratites. Grey bands represent 95% confidence intervals for each
taxon.
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comprising C. casuarius + D. novaehollandiae (figure 3). We also find a significant dissociation between
brain shape and age between all the taxa we compared (figures 5 and 6). The pattern of developmental
shape changes agrees with the findings of Corfield et al. [28,62], which identified differences in brain
and body size allometry and total brain to regional brain allometry among adult ratites, suggesting
that the shape data are capturing these more regional patterns.
4.1. Unique brain shapes in ratites evolved through the acquisition of distinct allometric
trajectories
A goal for our study was to identify whether brain shape changes between differently sized taxa—
ratites and G. gallus—follow an extension of conserved developmental scaling trends, as may be
expected by total brain volume and body size relationships [16], or different allometric trajectories, as
implied by relative brain volume measurements [12,28,63]. By analysing a densely sampled
ontogenetic series of ratites, we find that different endocranial morphologies observed in the adults of
these early diverging birds seem to have evolved through the acquisition of distinct developmental
allometry. For instance, S. camelus differed in its immature and adult endocranial morphology from the
other sampled ratites despite largely overlapping in early, postnatal endocast shape and body size
(figures 1 and 4). This result implies that the brain morphology of ratites did not evolve simply
through increase in size along a shared brain shape-to-size relationship as predicted by concerted brain
Regression Score Regression Score
Key
Gallus gallus
Casuarius casuarius
Dromaius novaehollandiae
Struthio camelus
0.05
0
–0.05
–0.10
0.05
0
–0.05
–0.10
4.8 5.2 5.6 6.0
Log Centroid Size
5.25 5.50 5.75 6.00
0.10
0.05
0
–0.05
0.15
0.10
0.05
0
–0.05
Log Total Age
64 8
6
4 8
(a) (b)
(c) (d)
Figure 6. Plots of regression score against log-transformed log-centroid size for (a) domestic chickens (G. gallus) and ratites (C.
casuarius, D. novaehollandiae and S. camelus), with the predicted minimum and maximum shapes along the regression embedded in
the figure, (c) only ratites, with the predicted minimum and maximum shapes along the regression and the shape changes between
D. novaehollandiae + C. casuarius and S. camelus embedded in the figure, log centroid size and against log total age for (b) G. gallus
and ratites, and (d) only ratites. The inset figures in (c) overlying the regression line represent the predicted minimum and maximum
shapes along the pooled regression line of D. novaehollandiae and C. casuarius, while those below reflect the same but for the ostrich
regression line. The boxed inset image shows the shape difference between the predicted shape of the largest C. casuarius + D.
novaehollandiae along their pooled regression line (grey spheres) and the predicted shape of largest ostrich along its own regression
line (end of black lines).
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evolution [1]. In other words, our shape analysis contrasts with the shared allometric trends noted
among G. gallus and extant ratites based on volumetric data [16]. However, these outcomes do not
contradict each other, but are rather complementary because they separately examine evolutionary
versus developmental trends and volumetric versus shape trends in allometry. Collectively, the
analyses imply that evolutionary allometric trends may be shared across somatically mature G. gallus
and extant ratites, but the developmental trends leading to evolutionary patterns in shape may be
divergent. It is important to note that we specifically obtain this result in the context of shape against
endocranial size, rather than incorporating body size into our regression. Studying how these changes
map to body size would be valuable for future research.
The relationship between RSC1 and age suggests that ratites and G. gallus significantly differ from
one another in the way brain shape develops (figure 5). This outcome agrees with the qualitative
and quantitative evidence that the brain shape of G. gallus and large-bodied ratites are fundamentally
different throughout development—the shape of the brain of adult chickens does not converge to that
of ratites, even in immature individuals (figure 1). This contradicts the hypothesis that shared brain
and body size allometry begets shared brain shape and brain size allometry between these different
taxa. In plots of regression scores against log age, the species overlap most closely in the younger
part of their shape-size trajectories, such that adult G. gallus seem to grade into the ratite regression
lines (figure 6). This visual overlap is probably due to an artefact of regression score projection of
data. Because there is minimal size overlap and age range overlap between ratites and G. gallus, a
pooled regression including these two lineages will arrange the shape changes occurring between
the smaller, shorter lived G. gallus and larger, longer lived ratites along what would appear to be a
singular regression line (figure 6). In contrast, it is easy to compare within ratites for this type of a
regression given their considerable size and age overlap, and these findings strongly imply divergent
morphologies arising through early shifts in developmental shape trajectories.
What may explain the differences observed in brain morphologies between taxa across develop-
ment? Future research must rule out body size as a confounding factor, but our findings suggest
that even species along a common allometric grade which possess similarly large body sizes possess
differences in developmental trajectories. For instance, ostriches are thought to have independently
evolved their large body size from other ratites, and ratites themselves have large body size variation,
even among closely related groups [20,22,64–66]. Naively, by studying allometric grades, one may
expect similarly sized, closely related taxa with similarly sized brains to have convergent brain shapes,
but our research suggests this is not always the case, as has been suggested by other studies looking at
regional brain volume [6,12,13]. Instead, ostriches have distinct brain morphologies and developmental
shape trajectories from C. casuarius + D. novaehollandiae. This implies that body size alone does not lead
to convergent changes in brain shape evolution, even when brain sizes may be similar. Besides body
size, the deviation of S. camelus from the other two sampled ratites may also be attributable to greater
abundance of proliferating neurons found in the telencephalon of adult S. camelus brain compared with
D. novaehollandiae [67].
Another explanation of shape differences observed between taxa may be that G. gallus, through
domestication, has been selected for accelerated growth and development compared with the ancestral
condition, unlike the ratites in our study. Domestication of G. gallus is associated with both
size-independent and size-dependent brain shape changes relative to red junglefowl, which are
considered to resemble the wild-type condition [68]. This means G. gallus exhibits derived traits
introduced through domestication, although red junglefowl are still fairly similar to G. gallus in their
brain shape [68]. Furthermore, as a result of their domestication, G. gallus have selectively larger
cerebra and cerebella than the red jungle fowl when the genetic independence between body size and
brain size is taken into account, and these differences apparently emerge through distinct loci rather
than through shared genes [69,70]. Sampling red junglefowls in a future study would help corroborate
the relationship we have already identified with our dataset and help researchers better understand
the relationship between domestication and brain shape and scaling changes.
Interestingly, we find that C. casuarius and D. novaehollandiae are not significantly different in their
brain morphology when comparing across their entire distribution of developmental stages, from
late-stage embryo to adult (figure 4), but they are different in their developmental trends (figures 5
and 6). This is a striking finding given that emus and cassowaries are estimated to have diverged
approximately 30 million years ago, and possess ecological and behavioural differences [22,30,71–73].
The fossil record of emus and cassowaries is relatively poor, making it difficult to ascertain how
their similar endocast shapes relate to body size evolution [22]. For example, if emus and cassowaries
evolved large body size independently, then we could investigate the extent to which phylogenetic
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inertia or their similar allometric trends contributed to their brain evolution, depending on whether
the ancestral brain morphology was similar or different in shape. There is greater body size and
ecological diversity in extinct casuariids. The oldest Dromaius fossil known, D. arleyekweke, is estimated
to have been less than 20 kg in body size and was probably even more strongly adapted for cursorial
lifestyle than modern emus [22,66,74]. Other smaller bodied, (e.g. less than 25 kg), recently extinct
emu subspecies (e.g. D. novaehollandiae minor and D. novaehollandiae baudinianus), extinct cassowaries
(C. lydekkeri), and extant dwarf cassowaries (C. bennetti) also suggest a substantial body size variation
within this taxonomic family [22,64–66,74–76]. Because there are few cranial remains from extinct
taxa, it may be worthwhile to compare the endocast ontogeny patterns of larger bodied species from
the present study with those of smaller bodied C. bennetti to study the relationship between brain
morphology and body size within this clade [22,64,76,77].
Still, phylogenetic inertia is probably at least a partial reason for brain shape similarities, because
the magnitude of differences we observe in endocast shape and developmental trajectories between
taxa are concordant with phylogenetic distance, where Dromaius and Casuarius are sister groups among
extant ratites and thus exhibit the closest allometric trajectories and brain morphologies. In contrast
to their similar endocast shape, D. novaehollandiae and C. casuarius possess statistically significantly
different developmental trajectories. While statistically significant at the 0.05 level, the results (e.g.
relatively low R2 value) suggest that very similar shape-to-size scaling relationships govern the
developmental brain shape changes in D. novaehollandiae and C. casuarius. Why, besides the aforemen-
tioned body size evolution or ecological specialization, are these taxa statistically indistinguishable in
gross morphology, but significantly different in their development? The subtle differences between
their trajectories, which includes variation in curvature of the dorsal aspect of the cerebrum, could
be due to differences in other areas of the skull during ontogeny between these two species, such as
the development of an enlarged casque with a neomorphic bony element (median casque element) in
C. casuarius [24] (figure 6c). Species of Casuarius are currently the only avian taxa known to exhibit
disunited casque development, in which unpaired midline elements participate in casque formation
[25]. Notably, certain elements which participate in the C. casuarius casque formation, including the
paired frontals, form the roof of the forebrain [24,25]. This may explain some of the differences in the
thickness of the region immediately caudal to the olfactory bulb (figure 1).
4.2. General trends in avian evolution identified by our results and their possible implications for
studying non-avian dinosaur evolution
Due to their relatively large and flightless condition, ratites have often been considered analogues to
non-avian theropod dinosaurs (e.g. [78]). We observed neuroanatomical traits in developing ratites and
G. gallus which may yield fruitful hypotheses about how evolutionary changes to the developmental
process proceeded along the transition from non-avian theropods to birds. We find that the optic
lobes are never caudodorsally positioned with respect to the cerebrum across late-embryonic to adult
stages as in non-avialan maniraptorans [79]. What is considered to be the crown bird arrangement
of the optic lobes and cerebrum is present even in the youngest embryonic specimens we sampled
[79]. This observation corroborates the previous finding that crown birds exhibit distinct allometric
trajectories that define their brain evolution and development, compared with non-avian dinosaurs
[34]. Conversely, in our late-embryonic and hatchling samples of S. camelus and C. casuarius (figure
1a,f,k,p), the Wulst is barely distinguishable, even as the optic lobe is clearly rostroventrally deflected
with respect to the ancestral non-avian condition. This is in line with observations made by prior
studies that the Wulst undergoes the greatest proportional amount of size change in development
compared with any other part of the endocast in S. camelus [58]. This result implies that the Wulst may
not be visible in immature bird fossils even if it does in fact emerge in adulthood, possibly obscuring
the evolutionary origin of the Wulst when sampling stem birds that are ontogenetically immature, such
as certain Archaeopteryx lithographica specimens used in other studies (e.g. [80,81]). In other words, we
might not expect to be able to identify this structure in stem birds unless they are near or entirely
mature specimens.
4.3. Future directions
Palaeognaths, while understudied, represent the outgroup to other extant birds (i.e. neognaths), and
thus serve to contextualize how avian brain evolution proceeds. In this study, we did not sample all
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extant palaeognath species, including large-bodied rheas (Rhea), the smaller bodied kiwis (Apteryx),
and the smaller bodied and volant tinamous (Tinamus, Nothocercus, Crypturellus, Rhynchotus, Notho‐
procta, Nothura, Taoniscus, Eudromia and Tinamotis). One avenue for further testing whether body size
has led to unique brain shapes in the large-bodied ratites would be to include developmental series
of kiwis and tinamous in a comparative analysis with our dataset. Because these two palaeognathous
groups are both smaller bodied and differ in their volancy, they may provide excellent context for
avian neuromorphology and neurodevelopment. However, these taxa are challenging to sample,
especially along an ontogenetic series. Also related to our taxonomic sampling, it is worth noting
that G. gallus is a highly derived lineage that exhibits neurocranial changes introduced as a result of
domestication [68]. Therefore, although G. gallus is considered an indispensable model organism in the
biological sciences and is of modest body size more typical of extant birds, sampling other neognath
species along the same allometric grade as the ratites we sampled [16] would clarify whether the
pattern observed here extends across the avian phylogeny.
Finally, the birds in our study radically differ in body size and developmental stages, so it is
difficult to adjust for this completely in any single regression of their shape. Following up to study
how body size directly relates to differences in brain morphology between these taxa would help
further elucidate how brain shape developmental allometry may relate to brain volume allometry. For
example, developmental series of these taxa are difficult to access, but future studies could sample
smaller species of extant cassowaries such as C. bennetti, since they exhibit some of the greatest amount
of size variation within a genus of palaeognaths [64]. Additionally, although generally endocranial
shape and volume tightly correlate with brain shape and volume changes throughout ontogeny in
birds, prior research has suggested that for shape this tends to hold out better in the forebrain than in
the hindbrain and in later ontogenetic stages in G. gallus [29,36]. Likewise, we did not fully quantify all
regions of the brain, such as the flocculus, which may be functionally relevant in explaining differences
between lifestyles of our taxa (although see [82]). Further sampling these regions would enable us to
investigate whether there are other structures in the brain, not quantified by our study, which may be
affected by the differences observed between emus and southern cassowaries.
5. Conclusions
Our study utilized the large adult size difference between our sampled ratites and G. gallus as
a case study for investigating whether disparate brain morphologies originate from extension of
shared scaling relationships or through different allometric trends. A previous large-scale compara-
tive analysis of avian brain and body sizes suggests that ratites and G. gallus fall under the same
evolutionary allometric regime [16], but other studies of relative brain volumes have suggested ratites
differ substantially from these early diverging birds [12,28,63]. With three-dimensional shape analysis
and developmental sampling, we find that the brain morphology, as represented by endocasts, of
large-bodied ratites does not conform to the same developmental scaling continuum as G. gallus. In
other words, these ratites did not acquire their different brain shapes from simply extending the
allometric trajectory of G. gallus as volumetric data may indicate. Rather, our sampled ratites show
a distinct shape–age relationship where shape changes occur more gradually during equivalent age
ranges compared with G. gallus. This results in distinct allometric trends where G. gallus exhibit more
mature endocranial shapes for a given size, and through extension of their own distinct shape-to-size
trajectory, large-bodied ratites attain their distinct neuroanatomy. Within ratites, S. camelus exhibit
their own allometric and developmental trajectories that differ significantly from C. casuarius and
D. novaehollandiae. Taken together, our study demonstrates that taxa along the same interspecific
allometric continuum do not necessarily overlap in developmental shape allometries. These results
oppose our expectation of conserved evolutionary allometry that larger species may follow and extend
the developmental trajectory of smaller taxa to obtain different brain morphologies.
Ethics. A care and use protocol was not required for ratites as all specimens were collected and sampled as cadaveric
specimens after death. No ratite individuals were harmed or sacrificed for the purpose of this study. Gallus gallus
specimens were euthanized through cervical dislocation by Charles River Laboratory, following ethical standards
approved by the AMNH IACUC in 2014.
Data accessibility. Three-dimensional coordinate data of avian endocasts sampled in this study and code are available
via the Dryad Digital Repository [83].
Supplementary material is available online [84].
Declaration of AI use. We have not used AI-assisted technologies in creating this article.
16
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 11: 240765
Authors’ contributions. M.R.F.: conceptualization, data curation, formal analysis, funding acquisition, investigation,
methodology, validation, visualization, writing—original draft; T.L.G.: conceptualization, data curation, funding
acquisition, investigation, methodology, project administration, resources, supervision, visualization, writing—
review and editing; A.W.: conceptualization, data curation, formal analysis, funding acquisition, investigation,
methodology, project administration, supervision, visualization, writing—review and editing.
All authors gave final approval for publication and agreed to be held accountable for the work performed
therein.
Conflict of interest declaration. We declare we have no competing interests.
Funding. This study was funded by the Laidlaw Foundation (to M.R.F.), the Richard Gilder Graduate School
Fellowship (to M.R.F.), the Columbia University Travel Grant and the Charlotte Mangum Student Support Program
to present this work at the Society for Integrative and Comparative Biology Meeting in 2022 held in Phoenix,
Arizona (to M.R.F.), Mary R. Dawson Predoctoral Fellowship Grant through the Society of Vertebrate Paleontology
(to A.W.), the Doctoral Dissertation Improvement Grant (DEB-1406849 to A.W.), New York Institute of Technology
College of Osteopathic Medicine (to A.W.), and Western Interior Paleontological Society (2016 and 2019 Karl Hirsch
Memorial Scholarships to T.L.G.). We also thank the Jackson School of Geosciences Travel Grant (to M.R.F.) for
providing funds to attend and present part of this study at the Society of Vertebrate Paleontology Meeting in 2022
held in Toronto, Canada.
Acknowledgements. The authors thank Bentley Bird, Joel Cracraft, Paul Sweet, Thomas Trombone (American Museum
of Natural History, New York, NY, USA); Ellen Dreyer, M. David Quavillon, Michelle Ferguson, Michelle Smurl
(Brevard Zoo, Melbourne, FL, USA); R. Glenn Hood, Scott Snedeker (Cassowary Conservation Project, Fort Pierce,
FL, USA); Jay Young (Colorado Gators, Mosca, CO, USA); Doug Warner (Charles River Laboratory, North Franklin,
CT, USA); Mark Corbridge, Tish Corbridge (Dream Acres Emu Ranch, Cheyenne, WY, USA); Andrew Doll, Garth
Spellman, Jeff Stephenson (Denver Museum of Nature and Science, Denver, CO, USA); Yulia Brockdorf and Rich
McClure (Hillsboro, OR, USA); Stan Barenberg, Samantha Potts (Longneck Ranch, Rose Hill, KS, USA); Michelle
Hayer, Jay Villemarette (Museum of Osteology, Oklahoma City, OK, USA); Ryan Reins (North Florida Wildlife
Center, Lamont, FL, USA); Ashley Bowen, Katie Glatfelter, Kathy Wolyn (Pueblo Zoo, Pueblo, CO, USA); Linn
Turner, Terry Turner (Rabbit Creek Emu Ranch, Livermore, CO, USA); Heather Arens, Phillip Horvey, Scott
Newland (Sedgwick County Zoo, Wichita, KS, USA); Joylene Reavis (Sugar Maple Emus, Monroe, WI, USA);
and Betty Lou Cauffman (Valley View Emus, Fennimore, WI, USA) for access to and/or donation of specimens
for this project; and Comet Technologies Canada, Inc. for providing free, non-commercial license for the software
Dragonfly. We appreciate assistance in specimen µCT imaging from Morgan Hill, Andrew Smith, Henry Towbin
(American Museum of Natural History, NY, USA); Scott Lyons, Joseph Merrill (Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY, USA); Steven Rigsby (Dentsply Research and Development Office, Tulsa, OK, USA);
Manon Wilson (Micro-CT Imaging Consortium for Research and Outreach, Fayetteville, AR, USA); and Kelsi
Hurdle (New York Institute of Technology College of Osteopathic Medicine, Old Westbury, NY, USA). For
additional assistance and/or discussions regarding the study, we thank Paul Gignac, David Kay, Larry Witmer,
Ariella Lang, Catherine Early, Sara Oppenheim, Christopher Raxworthy, James Napoli, and Kaiya Provost. Finally,
we thank three anonymous reviewers, Vera Weisbecker, and editors for their helpful feedback on previous versions
of this manuscript.
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