Osteohistological description of ostrich and emu long bones, with comments on markers of growth

Abstract

Ostriches and emus are among the largest extant birds and are frequently used as modern analogs for the growth dynamics of non-avian theropod dinosaurs. These ratites quickly reach adult size in under 1 year, and as such do not typically exhibit annually deposited growth marks. Growth marks, commonly classified as annuli or lines of arrested growth (LAGs), represent reduced or halted osteogenesis, respectively, and their presence demonstrates varying degrees of developmental plasticity. Growth marks have not yet been reported from ostriches and emus, prompting authors to suggest that they have lost the plasticity required to deposit them. Here we observe the hind limb bone histology of three captive juvenile emus and one captive adult ostrich. Two of the three juvenile emus exhibit typical bone histology but the third emu, a 4.5-month-old juvenile, exhibits a regional arc of avascular tissue, which we interpret as a growth mark. As this mark is not present in the other two emus from the same cohort and it co-occurs with a contralateral broken fibula, we suggest variable biomechanical load as a potential cause. The ostrich exhibits a complete ring of avascular, hypermineralized bone with sparse, flattened osteocyte lacunae. We identify this as an annulus and interpret it as slowing of growth. In the absence of other growth marks and lacking the animal's life history, the timing and cause of this ostrich's reduced growth are unclear. Even so, these findings demonstrate that both taxa retain the ancestral developmental plasticity required to temporarily slow growth. We also discuss the potential challenges of identifying growth marks using incomplete population data sets and partial cortical sampling.

Full text

Journal of Anatomy. 2022;00:1–9. wileyonlinelibrary.com/journal/joa
|
1© 2022 Anatomical Society.
1 | INTRODUCTIO N
Osteohistological studies of extant aves, and in particular the pa-
leognaths, illuminate aspects of extinct dinosaur biology previously
thought unattainable, like metabolic regime (Castanet et al., 2000;
Cubo et al., 2016), biomechanics (de Ricqlès et al., 2000), and even
fossil preservation rate (Marshall et al., 2021). These insights are
predicated on studies demonstrating a one- to- one correspon-
dence bet ween arrested growth lines and chronological age (e.g., de
Buffrenil & Castanet, 200 0; Kohler et al., 2012; Montes et al., 2007).
Even so, there is a growing chorus of studies reporting difficulties in
differentiating between annually deposited, accurate skeletochro-
nological indicators, and growth marks associated with exogenic
factors (Heck & Woodward, 2021; Schucht et al., 2021; Starck &
Chinsamy, 2002).
Paleognath birds are a group of primarily large and flightless
taxa, which include ratites such as ostriches and emus. The exact
phylogenetic position of these taxa within Palaeognathae is the
Received: 26 October 20 21
|
Revised: 16 Marc h 2022
|
Accepted: 28 March 2022
DOI : 10.1111/ joa.1 3665
ORIGINAL ARTICLE
Osteohistological description of ostrich and emu long bones,
with comments on markers of growth
Nathan Ong1 | Brenna Hart- Farrar1 | Katie Tremaine2,3 | Holly N. Woodward1
1Depar tment of Anatomy and Cell Biology,
Oklahoma State University Center for
Health S ciences, Tulsa , Oklahoma, USA
2Depar tment of Ear th Science, Montana
State University, Bozeman, Montan a, USA
3Museum of t he Rockies, Montana State
University, Bozeman, M ontana, USA
Correspondence
Nathan Ong, Depar tment of Anatomy and
Cell Biology, Oklaho ma State Unive rsity
Center for Health Sciences, Tulsa,
Oklahoma, USA.
Email: nong@okstate.edu
Abstract
Ostriches and emus are among the largest extant birds and are frequently used as
modern analogs for the growth dynamics of non- avian theropod dinosaurs. These
ratites quickly reach adult size in under 1 year, and as such do not typically exhibit
annually deposited growth marks. Growth marks, commonly classified as annuli or
lines of arrested growth (LAGs), represent reduced or halted osteogenesis, respec-
tively, and their presence demonstrates varying degrees of developmental plasticity.
Growth marks have not yet been reported from ostriches and emus, prompting au-
thors to suggest that they have lost the plasticity required to deposit them. Here we
observe the hind limb bone histology of three captive juvenile emus and one captive
adult ostrich. Two of the three juvenile emus exhibit typical bone histology but the
third emu, a 4.5- month- old juvenile, exhibits a regional arc of avascular tissue, which
we interpret as a growth mark. As this mark is not present in the other two emus
from the same cohort and it co- occurs with a contralateral broken fibula, we sug-
gest variable biomechanical load as a potential cause. The ostrich exhibits a complete
ring of avascular, hypermineralized bone with sparse, flattened osteocyte lacunae.
We identify this as an annulus and interpret it as slowing of growth. In the absence
of other growth marks and lacking the animal's life history, the timing and cause of
this ostrich's reduced growth are unclear. Even so, these findings demonstrate that
both taxa retain the ancestral developmental plasticity required to temporarily slow
growth. We also discuss the potential challenges of identifying growth marks using
incomplete population data sets and partial cortical sampling.
KEY WORDS
annuli, emu, growth marks, lines of arrested growth, osteohistology, ostrich

2
|
Ong e t al.
subject of ongoing debate, but consistently ostriches are recovered
as one of the earliest- diverging extant palaeognaths, whereas emus
are recovered as one of the latest- diverging extant palaeognaths
(Mitchell et al., 2014). Ratites are of interest to paleohistologists be-
cause, like their non- avian dinosaur ancestors, these rapidly growing
birds exhibit vascular architectures that span the full range between
slowly deposited longitudinal vascularization and rapidly deposited
radial vascularization. In extant ratites, these architectures can be
correlated with long bone osteogenesis rates ranging from 20 to
80 μm/d (Castanet et al., 2000). Because non- avian dinosaurs like
Maiasaura and Tyrannosaurus exhibit comparable vascular archi-
tectures, it follows that these taxa and their relatives were able to
sustain osteogenesis rates equivalent to avians (Cooper et al., 2008;
Horner et al., 1999; Padian et al., 2001; Woodward et al., 2015).
Unlike their extant avian descendants, most non- avian dinosaurs
required multiple years to attain adult size, indicated by the pres-
ence of annually deposited arrested growth rings within the bone
cortex (de Buffrénil et al., 2021). In some dinosaur taxa such as
Plateosaurus (Klein & Sander, 2007) and Tyrannosaurus (Woodw ard
et al., 2020) variable spacing between annual cortical growth rings
reflects plasticity in osteogenesis, possibly in response to nutrient
availability (Cullen et al., 2014; Woodward et al., 2020). Despite their
rapid osteogenesis, extant paleognaths remain smaller than many of
their non- avian dinosaur ancestors because their growth duration
is significantly shorter; adult size is reached within 180– 190 days
(Cooper, 2005). As a byproduct of this truncated growth window,
ostrich, and emu bones do not typically exhibit annually depos-
ited arrested growth rings. Because of this, it is currently unclear
if ostriches and emus have retained the ability to arrest growth and
deposit growth marks, or if this ancestral plasticity is lost (Starck &
Chinsamy, 2002). Here, we describe for the first time grow th marks
discovered in one ostrich and one emu and discuss the implications
of their presence.
2 | MATERIALS AND METHODS
2.1 | Specimens used
For this study, we borrowed a set of transverse diaphyseal thin sec-
tions from a 3- year- old adult male ostrich, from the collections of the
Museum of the Rockies (MOR 1707). The two samples each spanned
two slides due to the large size of the femur in transverse section. In
total, this produced four petrographic thin sections, two from each
sample. One set was stained using Toluidine blue, the other was not.
The ostrich was farm- raised in Montana, but no additional informa-
tion about its provenance or life history accompanied the specimen.
All three emu specimens hatched within the same year at the
Montana Emu Ranch (Kalispell, MT). The individuals died on the
ranch prematurely of unknown causes at 3.5, 4.5, and 5.5 months of
age. The emu cadavers had been frozen and stored, and eventually
hind limbs and sacra were shipped to OSU- CHS overnight to avoid
thawing. Specimens are accessioned into the Sam Noble Oklahoma
Museum of Natural Histor y osteolog y collections under specimen
numbers OMNH RE 864, OMNH RE 865, and OMNH RE 866 re-
spectively. Transverse mid- diaphyseal blocks of right femora, tibiae,
and fibulae were prepared as ground sections. The right fibula of
the 4.5- month- old emu (OMNH RE 865) exhibits a fracture callus,
but all other emu bones appear healthy. Because of this, slides were
also prepared from the left side of the 4.5- month- old emu (OMNH
RE 865) to control for potential variance induced by the pathol-
ogy found in the specimen's right fibula. All specimens died prior
to their donation for this study, so approval from the Institutional
Animal Care and Use Committee was unnecessary (Oklahoma State
University Policy 1– 0505).
2.2 | Methods
Because the specimen was already prepared by an unknown inves-
tigator for an unknown reason, the methods used to produce the
ostrich petrographic thin sections (MOR 1707) are also unknown.
Emu bones were prepared using methods established in Schweitzer
et al. (2007) and modified in Woodward et al. (2014). In summation:
specimens were skeletonized, preserved in a solution of 10% buff-
ered formalin, dehydrated in a 70%, 85%, and 100% ethanol series
over the course of 1 week, embedded in polyester resin, wafered
and mounted, and ground into petrographic thin sections ranging
between 20 and 100 microns in thickness. We also report that the
ostrich slides range in thickness between 90 and 230 μm. All slides
were imaged using a Nikon DS- Ri 2 camera mounted to a Nikon
Eclipse petrographic microscope with 2x and 5x objectives and an
ASI automated stage. Slides were imaged under cross- polarized,
full- wave plate, or plane- polarized light. Photomosaic images were
assembled using Nikon Elements: Documentation version 5.20.02,
and figures were compiled in Adobe CC Photoshop and Illustrator.
Once imaged, MOR 1707 ostrich sections spanning two slides were
reassembled using Photoshop. Roughly 1 mm of material was lost
when blocks were subdivided via tile saw to be mounted across mul-
tiple slides, so a 1 mm gap was added bet ween photomosaic images
when they were combined in Photoshop. Once fully reconstructed,
the section was digitally traced in Photoshop, and the silhouette
was impor ted into ImageJ, a product of Fiji (Schindelin et al., 2012).
Using the BoneJ plugin (Doube et al., 2010), histomorphometric data
like the centroid, circumference, and cross- sectional area of corti-
cal tissues were taken. Centroid coordinates were transferred to
Photoshop, where measurements were taken.
2.3 | Terminology
To facilitate the accurate identification of a growth mark, we use the
following three qualitative diagnostic criteria: (1) parallel or lamellar
fiber orientation that displays high anisotropy under cross- polarized
light, (2) reduced density and cross- sectional area of osteocyte la-
cunae, and (3) radially flattened osteocyte lacunae and canaliculi.

|
3
Ong et al.
These criteria are qualitative and comparative, meaning that they
are not explicitly measured, but rather described in comparison to
the tissues deposited before and after it.
Once identified in a general sense, the “clarity” of growth
marks can delineate them into two broad categories: Lines of
Arrested Growth (L AGs) and annuli. A LAG presents as a distinct
line, while an annulus presents as a diffuse ring of avascular tissue
(Francillon- Vieillot VdB et al., 1990). Note that “annulus” here re-
fers to the ring- like shape of the structure, as opposed to potential
annual deposition of the structure. Although LAGs and annuli are
presented herein as two distinct categories for descriptive pur-
poses , these terms ex ist as th e en d point s of a s pec tr um ( Atterhol t
et al., 2021), and the clarity of growth marks can transition from
one end of the spectrum to the other, depending on localized
variance in growth rate. The standard physiological interpreta-
tion of these marks is that LAGs represent the complete cessa-
tion of growth, while annuli represent a period of slowed growth
(Francillon- Vieillot VdB et al., 1990). Ultimately, growth marks are
delineated into these categories for descriptive precision, but the
physiological significance of their identification (i.e., the demon-
stration of slowed growth and developmental plasticity) is the
same for both categories.
To discuss the distribution of growth marks along the radius of
bone shafts, the terms “cyclical grow th marks” (CGMs) and “non-
cyclical growth marks” (NCGMs) are typically used, for which Padian
and Lamm (2013) offer this definition: “CGMs can be distinguished
from nonc yclical marks b ecause the appe arance of the latte r tends to
be haphazard rather than regular (i.e., they do not reflect a particular
spacing or rhythm) and the latter tend not to encircle the entire shaft
but tend to be locally confined to an arc.” (p. 196). Interpretation of
unevenly spaced growth marks as non- c yclical may be incorrect as
alligator osteohistological studies have demonstrated that uneven
spacing may result from variable duration of growth hiatuses as op-
posed to irregular (i.e., non- cyclical) timing (Woodward et al., 2014).
Regardless, both growth marks described herein occur in isolation,
so it is not possible to determine if they are evenly spaced relative
to other marks. Regardless of their radial distribution, growth marks
typically present as fully enclosed circles, but due to cortical remod-
eling and anisometric growth, they can also present as incomplete
arcs.
Transverse, mid- diaphyseal sections of adult animals often ex-
hibit other markers of variable growth, like the outer circumferential
layer (OCL), inner circumferential layer (ICL), and bright lines, all of
which must be distinguished from growth marks to facilitate their
accurate identification. During deposition of fibrolamellar cortical
tissue, hypermineralized bright lines are formed, which can some-
times be erroneously misidentified as the hypermineralization asso-
ciated with growth marks. Unlike growth marks, these bright lines
are highly conformable with surrounding laminar vasculature, and
their deposition via static osteogenesis yields a disorganized woven
fiber organization that is distinct from the lamellar fiber organiza-
tion of growth marks deposited via dynamic osteogenesis (Prondvai
et al., 2014; Stein & Prondvai, 2014).
The OCL and ICL are both deposited as growth slows in senes-
cence, but the diagnostic criteria used to identify and distinguish
them differ slightly. Differentiating the ICL from cor tical tissue (and
by extension potential grow th marks) is straightforward because the
ICL deposits onto a resorptive surface that lines the medullary cav-
ity, thus producing a non- conformable “tide line” (Francillon- Vieillot
VdB et al., 1990, p. 505). In contrast, the OCL is always found be-
neath the periosteal surface, but differentiation of the OCL from
appositional cortical tissue is more challenging because underlying
resorptive surfaces are not always present. In the case of rapidly
growing ver tebrates such as large ratites and non- avian dinosaurs,
the OCL can generally be distinguished from appositional primary
tissue by a pronounced reduction in relative vascular density in the
outermost cortex. This region consists of slowly deposited parallel-
fibered or lamellar tissue with an increasingly tight spacing of growth
marks (Cullen et al., 2021; Woodward et al., 2020) but in the absence
of a resorptive surface, drawing a sharp “line” between cortical tis-
sue and the OCL requires the alignment of multiple lines of evidence,
as opposed to the identification of a single line of direct evidence.
For more general descriptions of these structures and others
not explicitly described above, we follow standard terminology put
forth by Francillion- Viellot VdB et al. (1990, pgs 509– 512), Chinsamy
et al. (2013), and O'Connor et al. (2018).
3 | RESULTS
3.1 | 4.5- month- old emu
The juvenile emu tibia of OMNH RE 865 shows cortical thickness
that varies from 2.6 mm along the anterior aspect of the cortex to
5.3 mm along the posterolateral aspect (Figure 1a). The endosteal
surface is mostly smooth and uninterrupted, except for large nutri-
ent foramina along its posterolateral border (Figure 1b). Tissue fib-
ers appear isotropic under cross- polarized light, suggestive of woven
tissue (Figure 1c). In the anterior innermost cortex, a distinct sliver
of compact coarse cancellous bone (CCCB) (Enlow & Yaeger, 1963)
measuring approximately 1.9 mm thick can be seen (Figure 1d).
These regions show increased reticular vascularization and enlarged
osteocyte lacunae (70 μm vs 100 μm). Fiber bundles in this region are
isotropic, reflecting their disorganization (Figure 1e). Vascularization
remains reticular along the full thickness of the lateral portion of the
cortex. Vascular orientation across the rest of the cor tex is highly
variable but ranges from reticular at the inner cortex to sublaminar
toward the periosteal surface (Figure 1f). Wherever sublaminar pri-
mary vascularization is present across the section, locally hypermin-
eralized ‘bright lines’ are present within tissue laminae (Figure 1g).
Periosteal surface topography is continuous along most of its cir-
cumference, with sparse irregular vascular canals open to the sur-
face (Figure 1h).
A growth mark is clearest along the anterior side of the corti-
cal bone, between 260 μm and 350 μm from the periosteal surface
(Figure 2a). It cannot be readily traced completely around the bone

4
|
Ong e t al.
circumference. The terminal edges of the mark are ill- defined, and
show no scalloping indicative of active secondary resorption. The
mark is clearest under plane light, where a modest darkening of the
bone is present (Figure 2b). Under a full- wave plate (Figure 2c) and
cross- polarized light (Figure 2d), changes in brightness and fiber ori-
entation are inconsistently present along its length. The most strik-
ing feature of the mark is a change in osteocy te lacunar density and
morphology. Osteocyte lacunae are oblong here, with their long axes
aligned parallel to the periosteal surface. Communicating canaliculi
between osteocyte lacunae are sparser in the regions surrounding
the structure, and the channels that are present are more likely to
connect adjacent osteocytes (Figure 2e). On aggregate, this localized
osteocyte variance gives the mark its definition on a macroscopic
scale. Radial offshoots of reticular vascularization terminate in con-
tact with this horizon (Figure 2f). Sparse longitudinal and latitudinal
vascular c anals run parallel to the mark (Figure 2f).
3.2 | Three- year- old ostrich
The adult femur of MOR 1707 shows a cortical thickness between
3.9 mm and 5.8 mm, with its thickest region corresponding to tro-
chanter minor (Vijayan et al., 2019) along the posteromedial as-
pect of the bone (Figure 3a). Another, more modest thickening is
FIGURE 1 Mid- diaphyseal sec tion of 4.5- month- old emu tibia, imaged under multiple light conditions, showing various structures
discussed in “Results” section. (a) Photomosaic scan imaged under 20x magnification with anatomic directions. (b) Photomosaic of endosteal
surface imaged under 20x magnification and cross- polarized light, (c) CCCB/cortex interface imaged under 50x magnification and full- wave
plate light, (d) CCCB/cortex interface imaged under 50x magnification and full- wave plate light, (e) CCCB imaged under 10x magnification
and full- wave plate light, (f) photomosaic of reticular to sublaminar vascularization imaged under 20x magnification and cross- polarized
light, (g) bright lines imaged under 100x magnification and cross- polarized light, and (h) photomosaic of periosteal surface imaged under 20x
magnification and polarized light

|
5
Ong et al.
present along the anterolateral surface, and likely corresponds with
the anterior intermuscular line (Figure 3a) (Vijayan et al., 2019). The
endosteal surface is continuous, except for two prominent tra-
beculae located anteriorly and extending into the medullary cavity
(Figure 3a). A complete, non- conformable layer of avascular tissue
separates the innermost cortex from the medullary cavity (the inner
circumferential layer, ICL), and averages about 200 μm in thickness
(Figure 3b). Sparse vascular canals radially intrude through the ICL,
providing communication between the medullary cavity and the
cortex, the region is otherwise avascular (Figure 3b). Under cross-
polarized light, the ICL exhibits anisotropic birefringent fiber orien-
tation (Figure 3b).
Throughout most of the cortex, vascular canal orientation is lam-
inar, though areas of sublaminar tissue are sparsely present along
the inner regions of the cor tex (Figure 3c). Secondary osteons are
absent throughout the cortex. Under cross- polarized light, regions
with laminar and sublaminar vascularization also exhibit localized
lines of hypermineralized matrix that lie at the midpoint between
primary laminar vascularization (Figure 3d). Unlike hypermineraliza-
tion typically seen in growth marks, these lines undulate alongside
local primary vascularization (Figure 3d). These lines also exhibit dis-
ordered f iber direction, suggesting that they are composed of woven
tissue (Figure 3e).
Wide, reticular vascularization comprises trochanter minor
throughout the full thickness of the cortex (Figure 3f). These re-
gions are also non- conformable with surrounding vascularization,
suggesting that the area experienced secondar y remodeling during
ontogeny (Figure 3f). Likewise, the cortex consisting of the ante-
rior intermuscular line shows vascularization that is predominantly
sublaminar with isolated gradations to subreticular vascularization.
Volkmann's canals in this region become wider in diameter and ra-
dially elongated adjacent to the feature's surface (Figure 3f). A thick
layer of avascular, lamellar tissue approximately 150 μm thick com-
pletely encloses the outer cortex (outer circumferential layer, OCL)
(Figure 3g).
A growth mark can be traced around the entire circumference
of the bone (Figure 4a). It travels largely uninterrupted within
heavily remodeled regions like the reticular vascularization form-
ing trochanter minor (Figure 3f) and is only occasionally inter-
rupted by sparse Volkmann's canals (Figure 4a). The morphology
FIGURE 2 Mid- diaphyseal sec tion of 4.5- month- old emu tibia, imaged under multiple light conditions. (a) Photomosaic with anatomic
directions. Black region is a preparation artifact caused by partial delamination of specimen from the slide. Red box indicates region enlarged
in b– d. (b– d) photomosaic of the growth mark imaged under 20x magnification and imaged under (b) plane- polarized, (c) full- wave plate, and
(d) cross- polarized light. (e) Osteocyte lacunae at the growth mark, imaged under 100x magnification and polarized light and (f) osteocyte
lacunae at the growth mark, imaged under 100x magnification and cross- polarized light.

6
|
Ong e t al.
of the mark is ovate, ranging from a 1.9 cm radius along its minor
axis to a 2.6 cm radius along its major axis. Cortex deposition from
the mark to the periosteal surface is consistently 1.4 mm in thick-
ness (Figure 4a). Under plane- polarized light, the mark is slightly
lighter in appearance (Figure 4b). Stained with Toluidine blue, ad-
jacent tissue is slightly lighter in plane light. Under cross- polarized
(Figure 4c) and waveplate- retarded light (Figure 4d), the mark is
modestly brighter and fiber orientation is consistently distinct
from surrounding tissue. Osteocy te lacunae within the structure
are sparse, radially flattened, and less interconnected by commu-
nicating canalicular channels (Figure 4b).
4 | DISCUSSION
4.1 | Emu interpretations
With the exception of the mark found in the 4.5- month- old emu
tibia, all other bone histology is typical of rapidly growing juvenile
emus (Cooper, 2005). In this tibia section, the mark shows modest
hypermineralization, parallel- fibered bone, sparse vascularization,
and flattened osteocy te lacunae. As expected, there is no ICL or
OCL in this juvenile, so this avascular tissue cannot be associated
with these layers. Likewise, this structure does not undulate with
FIGURE 3 Mid- diaphyseal sec tion of femur from 3- year- old ostrich, imaged under multiple light conditions, showing various structures
discussed in “Results” section. (a) Photomosaic scan imaged under 20x magnification and plane- polarized light, with anatomic directions. (b)
ICL/cortex interface, imaged under 20x magnification and cross- polarized light. (c) Photomosaic scan of toluidine- blue stained slide, imaged
under 20x magnification and plane- polarized light, note sublaminar to laminar vascularization throughout the cortex. (d) Photomosaic was
taken at 20x magnification under cross- polarized light to illuminate hypermineralized vascular laminae. (e) Sublaminar vascularization with
sparse woven matrix, imaged under 50x magnification and waveplate retardation. (f) Photomosaic scan of toluidine- blue stained slide,
imaged under 20x magnification and polarized light. Note reticular vascularization deep to trochanter minor. (g) Outer circumferential layer
(OCL) imaged under 50x magnification and cross- polarized light

|
7
Ong et al.
nearby primary laminar vascularization, and so cannot be described
as a bright line. Because this mark meets our diagnostic criteria and
cannot be identified as any alternatives, we identify it as a growth
mark. This mark presents as a distinct line as opposed to a diffuse
ring and therefore fits the anatomical diagnostic criteria of a LAG.
LAGs are typically interpreted to represent full cessation of growth,
and while we agree with this interpretation, we note that this ces-
sation was highly localized to a regional arc. The edges of the mark
are diffuse until no longer distinguishable but exhibit no obvious
interruptions due to resorption or primary vascularization. There
are also no other marks present within the cortex, so we cannot
comment on its potential as a cyclically deposited skeletochrono-
logical indicator. Because the animal is only 4.5 months of age, this
mark was unlikely to be deposited as part of an annual fluctuation
in osteogenesis rates. Fur thermore, this mark is not present in the
femur or contralateral tibia, which does not support physiologically
induced systemic growth cessation seen in, for example, alligators
(Woodward et al., 2014).
Vertebrates undergoing anisometric cortical growth often pro-
duce cortical drift lines similar to this (Enlow & Yaeger, 1963), but
the absence of this mark in the other emus suggests that it formed
not as a byproduct of standard ratite growth dynamics, but rather
as a produc t of individual pathological history. Unlike the two other
individuals raised alongside it, the 4.5- month- old emu presented
with a broken left fibula and a growth mark in its right tibia, but not
in corresponding contralateral elements. Biomechanically adaptive
bone modeling has been hypothesized as a response to broken right
fibulae in two juvenile Maiasaura (Cubo et al., 2016) but these pre-
sented as distinct crescents of radial fibrolamellar tissue in the adja-
cent right tibia. Alternatively, biomechanical stress associated with
fibular injury may have caused a temporary arrest of growth in the
emu. The co- occurrence of this growth mark with pathology pro-
vides only circumstantial evidence, and a controlled experiment is
required for testing this hypothesis.
Regardless of our growth mark interpretations, its inconsistent
presence underscores the importance of using complete transverse
sections and controlling for individual outliers by examining multiple
specimens. If this thin section was prepared from only a core drilled
from the cortex, as is sometimes done in paleohistological studies,
then this mark could be interpreted as a complete ring, as opposed to
an incomplete arc. Likewise, if presumably uninjured emu individuals
were not included in this dataset, then the pathological present ation
of a growth mark could be erroneously interpreted as a population-
wide phenomenon.
4.2 | Ostrich interpretations
With the e xception of the grow th mark, the ost eohistology d escribed
herein is as expected for an adult ostrich, showing predominately
FIGURE 4 Mid- diaphyseal sec tion of MOR 1707, a femur from 3- year- old ostrich. Photomosaic shows the growth mark imaged under
multiple light conditions and magnifications. (a) Photomosaic scan of entire specimen, with region in red box expanded to see the potential
mark. Imaged under 20x magnification and plane- polarized light. Red box in the expanded region indicates regions imaged by b- d. red
arrows indicate mark. (b- d) growth mark imaged at 100x magnification and various light conditions. Red arrows indicate the mark. (b) Mark
imaged under polarized light. Note flattened osteocyte lacunae and canaliculi. (c) Mark imaged under cross- polarized light. Note inconsistent
hypermineralization of the region. (d) Mark imaged under cross- polarized light and wave- plate retardation. Note distinct fiber orientation

8
|
Ong e t al.
laminar to sublaminar vascularization, woven fiber matrix, an OCL,
and an ICL. Because the mark shows (1) hypermineralization, (2) par-
allel fibered bone, (3) sparse vascularization, and (4) flattened os-
teocyte lacunae, we confidently identify it as a growth mark. A clear
resorptive surface distinguishes the ICL from primary cor tical tissue
(Figure 3b), so we do not identify the mark as being part of the ICL.
A non- conformable resorptive surface that separates the OCL from
the cortex is not consistently present, but a thick layer of fibrola-
mellar tissue separates the mark from incontrovertible OCL tissue
(Figure 3c ,d), and so we feel confident that the mark is not part of an
early onset of the OCL. The mark is also non- conformable with sur-
rounding vasculature and does not show a woven fiber organization,
which distinguishes it from hyperminerized bright lines. The growth
mark is occasionally interrupted by primary vascularization, but it is
still identifiable as a complete ring. Some regions of the mark closely
resemble a clearly defined LAG, but most regions present as a dif-
fuse annulus. From this, we interpret that growth slowed in most
regions along the cortex, and entirely stopped in others. We hypoth-
esize that due to its load- bearing role, the presence of a growth mark
in the femur is reflective of system- wide slowed growth, but cannot
test this hypothesis, as additional skeletal elements for the individ-
ual are unavailable. Furthermore, because we observe only a single
mark, we cannot comment on the potential periodicity of growth
arrest. Additionally, Schutch et al. (2021) have demonstrated that
the final number of visible growth marks may be at least partially
determined by methods with which the specimen was prepared,
and so a slide prepared via microtome sectioning may yield more
marks that are currently invisible in our data. Unfortunately, because
we lack the original, undehydrated specimen, this study cannot be
undertaken.
The unknown individual life histories of these specimens pre-
clude the definitive identification of underlying causes, but the
presence of a growth mark within the cortex of an ostrich and emu,
reported here for the first time, remains significant. Our findings add
ostriches and emus to the list of Aves that retain an ancestral non-
avian dinosaur capacity to temporarily slow or stop osteogenesis,
which also includes Diatryma, Amazon amazonica, the New Zealand
moa, and the New Zealand kiwi bird (Bourdon et al., 2009; Heck &
Woodward, 2021). Due to the infrequency with which growth marks
are obser ved and reported in Neornithine taxa, fur ther investigation
is required to pinpoint their cause(s).
5 | CONCLUSIONS
Growth plasticity can be obser ved in all vertebrates (de Buffrénil
et al., 2021). More specifically, growth marks have been reported
in crocodilians (Woodard et al., 2014), non- avian dinosaurs, extant
neognaths (De Ricqlès et al., 2001), paleognathous kiwi birds (Heck
& Woodward, 2021), moa (Turvey et al., 2005), and the elephant bird
(De Ricqlès et al., 2016). Thus, capacity for growth plasticity, ubiq-
uitous in Vertebrata, is to be expected in ostriches and emus. Even
so, because they typically reach skeletal maturity within 1 year and
therefore lack annual markers of decreased osteogenesis, our study
is the first to report that these large extant ratites still maintain the
ability to reduce growth rates in response to external or physiologi-
cal stimuli.
ACKNOWLEDGMENTS
We thank the Museum of the Rockies for loaning the ostrich slides
for examination. Oklahoma State University Center for Health
Sciences provided funding as well as facilities for the histological
studies and the imaging instruments. We also thank Dana Rashid
(Montana State University) and the Montana Emu Ranch for sup-
plying the emu specimens and answering our questions concerning
them. We thank Haley O'Brien, Daniel Barta, and Christian Heck for
their insightful and thoughtful feedback while drafting this manu-
script. Finally, we thank Jessie Atterholt and Edward Fenton for peer
review of our manuscript.
AUTHOR CONTRIBUTIONS
H. N. W. conceived the experiment, prepared emu slides, and re-
viewed figures, tables, and drafts of the paper, and supplied the
reagents, materials, and analysis equipment. N. S. O. formatted the
manuscript, performed the experiment for the emu individuals, ana-
lyzed their data, wrote the paper, prepared figures and/or tables,
and reviewed drafts of the paper. B. H- F. performed the experiment
for the ostrich individual and analy zed the data, wrote the paper,
aided in figure preparation, and reviewed drafts of the paper. K. T.
edited a manuscript draft, provided initial identification of ostrich
LAG, and provided initial digital imaging for analyses.
DATA AVAIL ABILI TY STATEMENT
N/A
ORCID
Nathan Ong https://orcid.org/0000-0002-4968-3338
REFERENCES
Atterholt, J., Poust , A .W., Erickson, G.M. & O'Connor, J.K. (2021)
Intraskeletal Osteohistovariability reveals complex growth strate-
gies in a late cretaceous enantiornithine. Frontiers in Earth Science,
9, 118. https://doi.org/10.3389/feart.2021.6 40220
Bourdon, E., C astanet , J., de Ricqles, A., Scofield, P., Tennyson, A.,
Lamrous, H. et al. (20 09) Bone grow th marks reveal protracted
growth in New Zealand kiwi (Aves, Apterygidae). Biology Letters, 5,
639– 642.
Castanet, J., Curry Rogers, K., Cubo, J. & Jacques- Boisard, J. (2000)
Periosteal bone grow th rates in ex tant ratites (ostriche and emu).
Implications for assessing growth in dinosaurs. Comptes Rendus
de l'Académie des Sciences - Series III - Sciences de la Vie, 323,
54 3– 550 .
Chinsamy, A ., Chiappe, L.M ., Marugan- Lobon, J., Chunling, G. & Fengjiao,
Z. (2013) Gender identification of the Mesozoic bird Confuciusornis
sanctus. Nature Communications, 4, 1381.
Cooper, L.N., Lee, A.H., Taper, M.L. & Horner, J.R. (2008) Relative growth
rates of predator and prey dinosaurs reflect effects of predation.
Proceedings of the Biological Sciences, 275, 2609– 2615.
Cooper, R.G. (2005) Growth in the os trich (Struthio camelus var. domesti-
cus). Animal Science Journal, 76, 1– 4.

|
9
Ong et al.
Cubo, J., Le Roy, N., Martinez- Maza, C. & Montes, L. (2016)
Paleohistological estimation of bone growth rate in extinct archo-
saurs. Paleobiology, 38, 335– 349.
Cullen, T.M., Brown, C.M., Chiba, K., Brink, K.S., Makovicky, P.J. & Evans,
D.C. (2021) Growth variability, dimensional scaling, and the inter-
pretation of osteohistological growth data. Biology Letters, 17(11),
20210383. https://doi.org/10.1098/rsbl.2021.0383
Cullen, T.M., Evans, D.C., Ryan, M.J., Currie, P.J. & Kobayashi, Y. (2014)
Osteohistological variation in growth marks and osteocyte lacunar
density in a theropod dinosaur (Coelurosauria: Ornithomimidae).
BMC Evolutionary Biolog y, 14, 231.
de Buf frenil, V. & C astanet, J. (2000) Age estimation by skeletochronol-
ogy in the Nile monitor (Varanus niloticus), a highly exploited spe-
cies. Journal of Herpetology, 34, 414– 424.
de Buffrénil, V., Zylberberg, L., de R icqlès, A.J., Padian, K., Laurin, M.
& Quilhac, A. (2021) Vertebrate skeletal histology and paleohistology.
Boca Raton, FL: CRC Press.
De Ricqlès, A., Bourdon, E., Legendre, L .J. & Cubo, J. (2016) Preliminar y
assessment of bone histology in the extinct elephant bird Aepyornis
(Aves, Palaeognathae) from Madagascar. Comptes Rendus Palevol,
1 5 ( 1 – 2 ) , 1 9 7 – 2 0 8 .
De R icq lès , A. , Pad ian , K. , Hor ner, J.R . & Gau thier, J. (20 01) The bo ne hi s-
tology of basal birds in phylogenetic and ontogenetic perspectives.
In: New perspe ctives on the origin a nd early evolution o f birds: proceed-
ings of the international symposium in honor of John H. Ostrom. New
Haven, CT: Peabody Museum of Natural Histor y, Yale Univer sity,
pp . 411– 4 26 .
de Ricqlès, A.J., Padian, K., Horner, J.R . & Francillon- Vieillot, H. (2000)
Palaeohistology of the bones of pterosaurs (Reptilia: Archosauria):
anatomy, ontogeny, and biomechanical implications. Zoological
Journal of the Linnean Society, 129, 349– 385.
Doube, M., K losowski, M.M., Arganda- Carreras, I., Cordelieres, F.P.,
Dougherty, R.P., Jackson, J.S. et al. (2010) BoneJ: free and extensi-
ble bone image analysis in ImageJ. Bone, 47, 1076– 1079.
Enlow, D.H. & Yaeger, J.A. (1963) Principles of bone remodeling; an account
of post- natal growth and remodeling processes in long bones and the
mandible. Journal of the American Medical Association, 184(11), 906.
https://doi.org/10.1001/jama.1963.03700 24009 8035
Francillon- V ieillot VdB, H., Castanet, J., Géraudie, J., Meunier, F. J., Sire,
J.Y., Zylberberg, L . et al. (1990) Microstructure and Mineralization
of Vertebrate Skeletal T issues. In: Skeletal biomineralization: pat-
terns, processes and evolutionary trends, Vol. 5. Washing ton, DC:
American Geophysical Union.
Heck, C .T. & Woodward, H.N. (2021) Intraskeletal bone growth pat terns
in the Nor th Island Brown kiwi (A pteryx mantelli ): growth mark dis-
crepancy and implic ations for ex tinct taxa. Journal of Anatomy, 239,
1075– 1095.
Horner, J., De Ricqlès, A . & Padian, K . (1999) Variation in dinosaur skele-
tochronology indicators: implications for age assessment and phys-
iolog y. Paleobiology, 25, 295– 304.
Klein, N . & Sander, M. (20 07) Bone histology and growth of the prosau-
ropod dinos aur Plateosaurus engelhardti von Meyer, 1837 from the
Norian bonebeds of Trossingen (Germany) and Frick (Switzerland).
Special Papers in Palaeontology, 77, 169.
Kohler, M., Marin- Moratalla, N., Jordana, X. & Aanes, R. (2012) Seasonal
bone grow th and physiology in endotherms shed light on dinosaur
phy sio log y. Nature, 487, 358– 361.
Marshall, C.R., Latorre, D.V., Wilson, C.J., Frank, T.M., Magoulick, K.M.,
Zimmt, J.B. et al. (2021) Absolute abundance and preservation rate
of Tyrannosaurus rex. Science, 372, 284– 287.
Mitchell, K.J., Llamas, B., Soubrier, J., Rawlence, N.J., Wor thy, T.H.,
Wood, J. et al. (2014) Ancient DNA reveals elephant birds and
kiwi are sister taxa and clarifies ratite bird evolution. Science, 3 44,
898– 90 0.
Montes, L ., Le Roy, N., Perret, M., De Buf frenil, V., Cas tanet, J. & Cubo,
J. (2007) Relationships between bone growth rate, body mass and
resting metabolic rate in growing amniotes: a phylogenetic ap-
proach. Biological Journal of the Linnean Society, 92, 63– 76.
O'Connor, J., Erickson, G.M., Norell, M., Bailleul, A.M., Hu, H. & Zhou,
Z. (2018) Medullar y bone in an early cretaceous enantiornithine
bird and discussion regarding its identification in fossils. Nature
Communications, 9, 5169.
Padian, K., de Ricqles , A.J. & Horner, J.R. (2001) Dinosaurian growth
rates and bird origins. Nature, 412, 405– 408.
Padian , K. & Lamm, E.- T. (2013) Bone histol ogy of fossil Tetrapods. Berkeley
and Los Angeles, CA: Universit y of California Press.
Prondvai, E., Stein, K.H., De Ricqlès, A. & Cubo, J. (2014) Development-
based revision of bone tissue classification: the importance of se-
mantics for science. Biological Journal of the Linnean Society, 112(4),
7 9 9 – 8 1 6 .
Schindelin, J., Arganda- Carreras, I., Frise, E., Kaynig, V., Longair, M.,
Pietzsch, T. et al. (2012) Fiji: an open- source platform for biological-
image analysis. Nature Methods, 9, 676– 682.
Schucht , P.J., Klein, N. & Lambertz, M. (2021) What's my age again? On
the ambiguity of histology- based skeletochronology. Proceedings of
the Biological Sciences, 288, 20211166.
Schweitzer, M.H., Elsey, R.M., Dacke, C.G., Horner, J.R . & Lamm, E.T.
(2007) Do egg- laying crocodilian (Alligator mississippiensis) archo-
saurs form medullary bone? Bone, 40, 1152– 1158.
Starck, J. M. & Chinsamy, A. (2002) Bone microstructure and develop-
mental plasticity in birds and other dinosaurs. Journal of Morphology,
254, 232– 246.
Stein, K. & Prondvai, E . (2014) Rethinking the nature of fibrolamellar
bone: an integrative biological revision of sauropod plexiform bone
formation. Biological Reviews, 89(1), 24– 47.
Turvey, S.T., Green, O.R. & Holdaway, R.N. (2005) Cor tical grow th marks
reveal extended juvenile development in New Zealand moa. Nature,
435(7044), 940 – 943.
Vijayan, R.C., Sudheer, P.H., Maria, W.H. & Kalarippar ambath, S. (2019)
Comparative morphological studies of the pelvic limb bones in os-
trich (Struthio camelus), emu (Dromaius novaehollandiae), domestic
fowl (Gallus gallus domesticus) and duck (Anas platyrhynchos domes-
ticus). Indian Journal of Animal Research, 53, 1445– 1449.
Woodward, H.N., Freedman Fowler, E.A., Farlow, J.O. & Horner, J.R.
(2015) Maias aura, a model organism for extinct vertebrate popu-
lation biology: a large sample st atistical assessment of growth dy-
namics and survivorship. Paleobiology, 41, 503– 527.
Woodward, H.N., Horner, J.R. & Farlow, J.O. (2014) Quantification of
intraskeletal histovariabilit y in Alligator mississippiensis and implica-
tions for vertebrate osteohistology. Pee rJ, 2, e422.
Woodward, H.N., Tremaine, K., Williams, S.A ., Zanno, L.E., Horner, J.R. &
Myhrvold, N. (2020) Growing up Tyrannosaurus rex: Osteohistology
refutes the pygmy "Nanotyrannus" and suppor ts ontogenetic
niche partitioning in juvenile tyrannosaurus. Science Advances, 6,
eaax6250.
How to cite this article: Ong, N., Hart- Farrar, B., Tremaine, K .
& Woodward, H.N. (2022) Osteohistological description of
ostrich and emu long bones, with comments on markers of
growth. Journal of Anatomy, 00, 1– 9. Available from: h t t p s ://
doi .org/10.1111/joa.136 65