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ONTOGENY OF THE ENDOCASTS OF OSTRICHES (AVES: STRUTHIO CAMELUS) WITH IMPLICATIONS FOR INTERPRETING EXTINCT ENDOCASTS

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A Thesis Presented to

The faculty of

The College of Arts and Sciences

Ohio University

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In Partial Fulfillment

of the Requirements for Graduation

with Honors in Biological Sciences

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By

Cheyenne Ariel Romick

April 2013

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TABLE OF CONTENTS

Abstract…………………………………………………………………………...……4

Introduction…………………………………………………………………………….5

Why Study Ostriches?...... 6

Endocasts as a Tool for Study………………………………………………….7

Function and Anatomy…………………………………...... 9

Overview of Current Knowledge……………………………………………..11

Holes in the Research…………………………………………………………16

Potential Benefits……………………………………………………………..17

Materials and Methods………………………………………...... 19

Materials………………………………………………………………………19

Table 1………………………………………………………………………...19

CT Scanning…………………………………………………………………..19

Generation of Digital Endocasts………………………...... 19

Results………………………………………………………………………………...20

Adult Endocast Introduction………………………………………………….20

Figure 1a……………………………………………………………………...21

Figure 1b……………………………………………………………………...22

Visible Qualitative Results……………………………………………………22

Figure 2……………………………………………………………………….25

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Figure 3 and Figure 4…………………………………………………………26

Figure 5……………………………………………………………………….27

Quantified Relative Surface Area…………………………………………….27

Table 2………………………………………………………………………..29

Lugol’s Analysis………………………………………...... 29

Figure 6……………………………………………………………………….30

Discussion…………………………………………………………………………….30

Prioritization…………………………………………………………………..31

Ontogeny Recapitulates Phylogeny…………………………………………..32

Figure 7……………………………………………………………………….33

Implications for Ostrich Development………………………………………..33

Implications for Non-Avian Dinosaur Development…………………………35

Future Direction………………………………………...... 37

References…………………………………….….….……..…………………………39

Appendix A…………………………………………………………………………...44

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Abstract

The goal of this project is to document the ontogeny of the cranial (brain) endocast of Struthio camelus, the African ostrich. Comparison of size and shape via

3D reconstruction from CT scans provided the data needed to study the ostrich endocast in various stages of life. Endocasts of seven specimens were generated for analysis. The specimens consisted of two embryos, three juveniles of differing ages, and two adults. Comparison of the shape and size of the endocasts was done using relative and absolute scaling. Surface area comparisons between specimens were digitally performed to determine the changes in nine brain regions from embryo to adult. Results suggest a definite change in shape as the organism shifts from embryo to adult. The most notable change in surface area is seen in the Wulst region of the cerebrum, implying a prioritization of that region at little expense to the other regions.

The Wulst is located dorsally on the telencephalon and is composed of two distinct bumps on either side of the dorsal cerebrum. Additional analysis of one of the specimens with the Lugol’s iodine staining method revealed that the flocculus of the endocast is mainly composed of venous rather than neural tissue. The Lugol’s analysis also revealed that the cerebellar foliation present in the ostrich brain is not detectable in the endocast, because a large venous sinus overlies that area. The inner ear changes mainly in size during development, with a fairly constant shape throughout life. My results suggest that the Wulst is eventually prominent in the ostrich yet is not present at hatching, perhaps reflecting a parallel between ontogeny and phylogeny in that the 5

Wulst also appears later in avian brain evolution. In addition, the fact that the inner ear maintains its shape is indicative of its importance throughout the history of non-avian and its immediate importance after hatching in a precocial species.

Introduction

The goal of this project is to create an informative ontogenetic series for the endocast of the brain of the African ostrich, Struthio camelus, and to apply this information to the inferences of non-avian dinosaur endocasts as well as extant taxa.

In this text, non-avian dinosaur refers to those dinosaurs which are not members of

Aves, or more simply, not birds. The study of the brain is important for multiple reasons. The brain acts as a control center and behavior generator. Its role as an information processing and motor control center can give us clues to the physical capabilities of extinct species in addition to their cognitive-behavioral faculties. The lack of soft-tissue preservation in most relegates comparative studies to the endocast rather than to the actual brain. Little is known about the ontogeny of the endocast of non-avian dinosaurs (e.g., Evans, Ridgely, & Witmer, 2009), primarily because most species lack enough specimens to generate a growth series. This project can help advance the scientific understanding of the development of the endocasts of non-avian dinosaurs in addition to that of Struthio camelus. The ostrich was chosen as the study organism due to its relatively basal position on the phylogenetic tree of Aves

(Burish, Kueh, & Wang, 2004), placing it evolutionarily closer to non-avian dinosaurs than most other extant avian taxa. A growth series of heads and skulls was readily available to be analyzed or had already been analyzed and rested in the specimen 6

freezer or in the Ohio University Vertebrate Collection. Two embryonic heads were available, along with one each of a 14-day old, a 2-month old, and a 4-month old, and several adult heads were accessible. Please note that the term “head” refers to a specimen that had not been skeletonized.

Why Study Ostriches? Ostriches are members of the ratite clade, which is considered part of the earliest-branching clade of modern birds (Burish et al., 2004).

Tinamous are the closest volant relatives to the ratites, and together, these form

Palaeognathae. All other extant birds are called neognaths. Please note that in this paper, the term non-avian dinosaur is used preferentially to just dinosaur because birds are dinosaurs.

Palaeognaths may have a similar shape of the brain to that of neognaths, but they have smaller compared to their body size (Iwaniuk & Nelson, 2003).

Unfortunately, it is difficult to determine the significance of this difference, because most palaeognaths are so large, and the smallest, kiwis, are highly specialized

(Corfield, Wild, Cowan, Parsons, & Kubke, 2008), which can confound conclusions

(Iwaniuk & Nelson, 2003). All birds’ brains are fairly easy to study from skeletal remains, because birds, like mammals, fill most of their braincase with actual brain

(Emery & Clayton, 2005), and so their endocasts tend to faithfully reflect basic brain structure. This is not true for most non-avian dinosaurs. Non-avian dinosaur brains are often a challenge to study because they usually do not fill their endocranial space with neural tissue, such that, aside from the theropod lineage (Evans, 2005), the endocast can have a rather ambiguous, amorphous shape. Much of the empty space in the skull 7

not occupied by the brain is instead home to what is called cerebrospinal fluid (Evans,

2005). This makes it difficult to infer what the actual brain looked like, whereas in birds and mammals, it is relatively straightforward. Non-avian theropods in general tend to have filled the endocranial volume with neural structure, which is consistent with the theory that this group gave rise to modern birds (Witmer, Chatterjee,

Franzosa, & Rowe, 2003).

Non-avian dinosaurs fall between more basal reptiles and extant avians on the phylogenetic tree, a situation referred to as the “extant phylogenetic bracket” (Witmer,

1995, p. 19) of birds and crocodilians. The bird brain is thought to be similar yet uniquely derived from the basal reptilian brain (Cragie, 1940). More exploration into both bird and crocodilian brains and endocasts would optimally help us understand non-avian dinosaurs. When studying the brain, it is important to remember the principle of proper mass (Jerison, 1973). This rule states that size indeed matters for the processing load of a given brain structure. The more space devoted to a region of the brain, the more work that structure is doing, which indicates that the function accomplished by this region must be more important. (Jerison, 1973). For example, the importance of sight in most birds is reflected by their enlarged optic tecta.

Endocasts as a Tool for Study. Endocasts are the main scientific approach to understanding non-avian dinosaur brains. An endocast is the volume that can be discerned from the bony boundaries of the braincase in an organism. Scientists want to learn about the non-avian dinosaur brain, but we have only their endocasts to study.

This obstacle is the reason for creating endocasts for the ostriches in the first place. If 8

we merely wished to study extant organisms, scientists would preferentially look at the actual brain, but the endocasts of dinosaurs are the only available indications of their brain anatomy. Therefore, when comparing birds to extinct dinosaurs, the endocast is actually more appropriate for comparative analysis. Obtaining endocasts in the past was achieved by creating rubber molds of the braincases of the skulls of specimens, a practice still employed today. It is ever more common, however, to scan the fossilized skull, dried skull, or head of a given specimen with a computed tomographic (CT) scanner. A CT scanner outputs a specimen as a series of slices, in the xy, xz, and yz planes, which allows for 3D reconstruction. The number of slices is determined by the resolution of the scanner itself. The higher the number of slices, the more refined the data. The technology of CT scanning has been improving due to its medical uses, making it an excellent scientific resource when research is in need of such a tool.

An important limitation to computed tomography is that bone, the densest tissue, is the only definitively resolved tissue in a standard scan. Bone appears as white in a CT scan, whereas other tissues register as a very uniform gray, and empty space (e.g., air) is black. Methods do exist to resolve other types of tissue, but using them permanently alters the specimen. For example, in order to detect blood vessels preferentially, a specimen must be carefully injected with a barium-latex mixture. The density of this compound makes the vessels it fills appear as a bright white against the other gray tissues when scanned and viewed. Another method for differentially resolving the soft tissue in general is the use of Lugol’s iodine solution. The specimen is submerged in a solution of iodine potassium iodide for a varying period of time and 9

then scanned. The resulting scan shows a remarkable contrast between several types of tissues and allows the distinct separation of brain from braincase.

The data obtained from the CT scan of a specimen is analyzed with a dedicated

3D visualization program. Once interpreted by the software, the data can be used to generate a 3D model of the tissues imaged by the CT scan. For this study, the endocast was the target of reconstruction. This method allows direct comparison and manipulation of multiple specimen models through 3D rotation, creation of “line ups,” and various other types of comparative analysis that are made significantly easier through use of this technology. For example, a model created from a rex can be loaded and analyzed alongside that of a modern bird, lizard, mammal, etc.

Function and Anatomy. A 3D endocast model is not interpretable without knowledge of the structure and function of its parts. (See Figures 1a and 1b for examples of adult endocasts.) The brains of birds and non-avian dinosaurs are shaped differently from those of mammals. The brain of a dinosaur (including birds) does not display the numerous characteristic wrinkles in the cerebral region, the way a mammal brain does (Orosz, 1996). The cerebrum in both types of animal still serves similar functions, despite being different in appearance (Orosz, 1996). The cerebrum is responsible for several processes related to information processing. It is the large rostrodorsal region of the ostrich brain. The endocast region known as the Wulst can be described as a rostrodorsal bulge of the cerebrum. This region is formed from the hyperpallium and is considered important in both cognitive ability and the processing 10

of bodily sensation (Picasso, Tambussi, & Degrange, 2010), and it is visually discernible in the brain of Struthio camelus (Peng, Feng, Zhang, Liu, & Song, 2010).

The Wulst is also commonly referred to as the eminentia sagitallis (Picasso et al.

2010).

The avian cerebellum, like the mammalian cerebellum, exhibits foliation, which means transverse folds are present in its structure (Iwaniuk, Hurd, & Wylie,

2006). The extent of this foliation varies greatly within Aves, and across ecological types (Iwaniuk et al., 2006, Ksepka, Balanoff, Walsh, Revan, & Ho, 2012). The cerebellum and flocculus (cerebellar auricle) are tasked with processing information on the movement and position of the body, and the flocculus is known for allowing an animal to “lock on” to an object of interest while moving (Witmer et al., 2003, Peng et al., 2010, Picasso et al., 2010, Walsh & Milner, 2011b, Ksepka et al., 2012). The cerebellum is positioned behind the cerebrum and above the medulla, and in the ostrich, the flocculi are small bumps on either side (Peng et al., 2010).

Ratites are noted for their poor sense of smell (Ashwell & Scofield, 2008), and the ostrich is no exception with its diminutive olfactory bulbs, slightly protruding rostrally from the telencephalon (Peng et al., 2010). The optic tecta (lobes) are associated with processing visual information. They are quite defined in the ostrich, located to either side of the brain, just ventral to the cerebral lobes, and rostral to the cerebellum and medulla (Peng et al., 2010). The optic chiasm is a large nerve bundle converging between the two optic tecta in the ostrich brain. This grouping of nerves diverges and leads to either eye as the associated optic nerve (Peng et al., 2010). The 11

optic chiasm is the channel through which visual information is transported to the optic tecta to be interpreted. Located ventral to the optic chiasm is the pituitary, the master gland of all other glands in the body. Caudal to the pituitary and ventral to the cerebellum lies the medulla region, which controls homeostatic functions such as breathing and pulse. It also houses most of the cranial nerves (Witmer et al., 2003).

Finally, the endosseous labyrinth (inner ear) was analyzed along with the brain endocast. The endosseous labyrinth is used in the senses of hearing and balance. These organs sense how quickly an organism’s head turns and transmit this information to the brain (Walsh & Milner, 2011b). The floccular region of the endocast actually protrudes through the anterior canal and ends within the center of the arcs formed by the three canals (Witmer et al., 2003).

Overview of Current Knowledge. Rapid growth in birds is well known.

Comparing the 14-day-old, 2-month-old, and 4-month-old ostrich specimens, the head of the 4-month-old is nearly the size of an adult head. This rapid growth pattern is also observed in Picasso’s analysis of the ontogeny across three Rhea americana specimens (Picasso et al., 2010). Picasso also notes that the endocast appears to change the most between the chick and juvenile stages. The detailed study of the brain anatomy of Struthio camelus performed by Peng et al. (2010) revealed one of the shortcomings of endocasts. The endocast is not a perfect mimic of the brain in birds, a fact easily forgotten, considering how often it is described as good enough or

“interchangeable” (Picasso et al., 2010). 12

Witmer et al. (2003) described pterosaur endocasts and included inferences on the function of different structures of the brain used in flight. While flight does not concern the ostrich, agility certainly does. An animal capable of the speeds of an ostrich must be able to react quickly to obstacles and dangers. The pterosaurs appear to approach flight in a fairly similar manner to modern birds, but the pterosaur brains are significantly smaller than those of extant avians. Pterosaur evolution did not favor a large brain, however, and these creatures are suggested to have dealt with flight by immensely expanding their endosseous labyrinths (inner ears) and drastically enlarging their floccular region (Witmer et al., 2003). These changes would render both structures more sensitive to motion and position of the animal, aiding in flight in the absence of an overall larger brain (Witmer et al., 2003).

Picasso et al.’s (2009) study of the endocranial anatomy of an extinct eagle from the late Miocene provides an example of a fossil bird capable of flight. The endocast of this bird displays an obvious Wulst, and Picasso et al. (2009) argued that this supports the idea of its evolutionary emergence occurring in the Lower Eocene.

They also remarked how similar the endocast is to extant taxa, suggesting that current bird brain architecture has changed little overall for several million years. Indeed, what is shown of the eagle endocast is clearly similar to even the flightless ostrich’s brain (Peng et al., 2010).

Outside the realm of flight, yet still similar, is aquatic acrobatic capability.

Endocast analysis of an extinct penguin lineage by Ksepka et al. (2012) discussed the similarity between penguin brains and the brains of flying birds. Ksepka et al. (2012) 13

suggested that the large flocculi, inner ear canals, and well-developed Wulst allowed for underwater maneuverability analogous to aerial flight. Ksepka et al. (2012) allowed, however, the possibility that the Wulst served other functions, such as cognitive functioning or visual assistance rather than agility in hunting. While all these characteristics are attributed to aiding in penguin diving and pursuit skill, the endocasts’ appearances are quite similar to the ostrich brains described in Peng et al.

(2010). The largest apparent difference is the extension of the floccular regions in the penguin endocasts (Peng et al., 2010, Ksepka et al., 2012). It is remarkable how similar the endocasts of a flier, swimmer, and runner can be, given their vastly different locomotion and ecology.

Ashwell & Scofield (2007) published a large study focusing on the endocasts of a New Zealand moa and other paleognaths. This paper provides information for comparison between basal taxa, with a primary focus on encephalization (Ashwell &

Scofield, 2007). Ashwell & Scofield (2007) determined that the moa, fairly isolated in

New Zealand, possesses similar olfactory and Wulst proportions and shape to non- isolated ratites. On the other hand, the kiwi (Apteryx owenii) has an obvious specialization in olfaction, with an enlarged olfactory processing region to complement this ability. Again, the general shape of the moa endocast shown by

Ashwell & Scofield (2007) is similar to the ostrich brain in Peng et al. (2010).

Corfield et al. (2008) analyzed palaeognath brain size throughout their evolutionary history (Corfield, et al., 2008). The researchers pointed out that nine of the 10 species examined appeared similar in anatomy to typical neognath brains, with 14

the kiwi species (Apteryx mantelli) exhibiting divergent morphology and a relatively much larger brain for its body size (Corfield et al., 2008). However, they noted that size of the entire brain does not necessarily indicate intelligence, since the kiwi does not display increased cognitive faculties compared to other ratites. Morever, the kiwi appears to have increased its olfactory perception at the loss of its visual acuity, perhaps indicating a trade-off within the brain (Corfield et al., 2008).

Despite the ambiguity of brain size relative to body size in birds as an indicator of “intelligence,” it has still been a subject of investigation. Brain size and its relation to developmental strategy have been studied in birds (Iwaniuk & Nelson, 2003).

Studies focused on intelligence can provide relevant information concerning brain anatomy of birds and therefore their ancestors (Burish et al., 2004, Emery & Clayton,

2005). In general, the more precocial a bird is, the smaller its brain is for its body size

(Iwaniuk & Nelson, 2003). Thus, the small relative size of an ostrich brain, perhaps surprising at first, makes sense in the context of the Iwaniuk & Nelson (2003) study.

While the study advances anatomical developmental understanding, it does not assert information regarding avian intelligence because overall size may not relate directly to intelligence (Corfield et al., 2008), and apparently neither does external surface complexity in mammals, as proposed by Emery & Clayton (2005).

The organization of the avian brain was previously misunderstood and mis- categorized as arising from neural tissue associated with instinct (striatum) (Cragie,

1940) rather than learning (pallium). Now, we understand the physical differences 15

between mammalian and bird brains, which are well characterized by the following quote from Emery & Clayton (2005):

“…rather than producing a layered cortex as in mammals, in birds the result was a nucleated structure with pockets of grey matter. The analogy would be to compare a club sandwich (mammalian) to a pepperoni pizza (avian) (p. R947).”

This pizza-like arrangement of the processing centers of birds is important for veterinarians and biologists alike to understand (Orosz, 1996). Despite the difference in organization, the brains of mammals and birds have similar potential for cognition.

Indeed, all vertebrate brains seem to be composed of the same basic working parts, with some modifications and generally strict conservation of others, namely the medulla and cerebellum (Northcutt, 2002). Not all mammals or birds are extremely intelligent, but both types of animals have members that rival the cognitive abilities of their counterparts (Emery & Clayton, 2005). If modern birds possess this potential for intelligence, is it fair to discount the cognitive functioning of dinosaurs so readily?

Certainly it would be wiser to give at least the theropods more fair consideration. On the other hand, other studies have deemed the social intelligence of Archaeopteryx to be on the level of the domesticated chicken (Burish et al., 2004). Still, the size of

Archaeopteryx’s brain compared to its body size lined up more closely with birds than reptiles (Burish et al., 2004). Perhaps the 150-million-year-old bird (Burish et al.,

2004) will be discovered to have been underestimated, once a more complete data set of endocasts has been assembled.

Non-avian dinosaur endocasts must also be studied if this project is to aid in understanding them, and they have been investigated with increasing frequency 16

(Evans, 2005, Evans, 2009, Zelenitsky et al., 2011). Impressions of blood vessels left behind in fossils are thought to indicate that the brain was pushing them against the bone, which means the brain in the areas with these impressions was filling the cavity closely (Evans, 2005). The study by Evans (2005) on ornithischian dinosaur fossil braincases casts doubt on the assumption that only 50% of a typical dinosaur’s endocranial space is occupied by brain (Evans, 2005). This assumption has been used since approximately the 1970s (Evans, 2005). This study of ornithischian dinosaurs’ braincases helps with reaching a more accurate method of estimating how much of a dinosaur endocast is actually composed of brain. Clearly, the 50% rule is not equally and globally applicable to such an enormous variety of taxa which occupied this planet and evolved for over 165 million years.

Holes in Previous Research. Note the paucity of ontogenetic analyses among the studies recounted here. This huge gap in analysis is part of the motivation for this project. By understanding bird endocranial development, we can begin to understand it in non-avian dinosaur species. The need for more study of palaeognaths is clear

(Corfield et al., 2008, Picasso et al., 2010), and embryonic studies of modern birds should be in high demand (Iwaniuk & Nelson, 2003). Without these facets of knowledge at our disposal, extrapolating to non-avian dinosaur endocasts and even their brains will remain challenging. We need more comprehensive data on bird brains in order to aid in inferring extinct forms as well as understanding evolutionary patterns leading up to extant taxa. While some very old useful exhaustive studies of Struthio camelus do exist (Cragie, 1940, Webb, 1957), they do not focus on endocasts or non- 17

avian dinosaurs, and they were still operating under the assumption that a “bird brain” is not capable of the feats of a mammalian one (Cragie, 1940).

Potential Benefits. This study is in part meant to provide information that will improve the understanding of extinct taxa. Currently, interpretation is mainly limited to theropods, as they are most closely related to modern birds. This ontogenetic series can inform us of many aspects of dinosaur development, such as structure prioritization and when the Wulst emerged in phylogeny. In addition, it will inform scientists regarding confounding factors such as large venous imprints in the endocast that take up space but are not truly neural. Another fact is that the absence of a structure in an endocast is not absolutely indicative of its absence in the actual brain.

The science and art of figuring out what dinosaur brains actually looked like will take a vast amount of time and effort, and this study will aid that effort.

This study also holds virtue in understanding extant avian brain development in general. The information we gain from such studies can be applied in contemporarily practical ways, such as conservation and breeding programs or improving the overall health of a species. Throwing different taxa into contrast has often been a strong aid in more intimately understanding the taxa of focus. For example, a future researcher studying the ontogeny of passerine endocasts may find the contrasts between their specimens and the palaeognathine ostrich very useful in highlighting the unique aspects of the passerine endocasts. It could also lead to new realizations about Struthio while it is being compared to other avian and non-avian endocasts. 18

In addition to being a potential launch pad for understanding other taxa, both extinct and extant, this study can help open the gateway to using ontogeny as a means of informing phylogeny. The timing of detectability of certain structures, such as the

Wulst, holds potential indicators for the structure’s evolutionary emergence. The debate over when the Wulst first emerged is a prominent one among those people concerned with avian evolution. One estimation is that it first evolved during the end of the (Walsh & Milner, 2011a), but pinpointing its origin is a challenge.

We must be able to determine when absence of structural imprint in an endocast truly indicates absence of the brain region itself. We must also learn how the changing selective pressures driving the evolution of the Wulst affected the rate of its emergence. These are concerns for just one area of the avian brain. Consider the possibility of understanding multiple features and knowing when they each appeared.

The question of whether avian brains took on their characteristics all at once or bit by bit is an important question to answer as well (Witmer, 2004), and while it may still be a long time coming, this study can help advance that goal as well.

This study is also a model approach to the study of the ontogeny of avian endocasts, should other researchers wish to supplement this data set. Providing a template from which more research can be conducted is necessary to facilitating its widespread inception. This project also provides an understanding of basal avian endocast ontogeny beyond the lone Rhea analysis (Picasso et al., 2010). Hopefully, many more such ontogenetic studies will emerge into the scientific community soon.

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Materials and Methods

Materials. Seven heads and skulls of individuals of the species Struthio camelus, the African ostrich, were used in this project. All specimens were obtained legally as natural casualties from breeders in the United States or as specimens salvaged from processing centers. The specimen numbers, CT scan parameters, and other information are listed in Table 1.

Table 1 OUVC Age Description Scanning Types OUVC 10521 39 days incubation head & neck µCT OUVC 10518 40 days incubation head & neck µCT OUVC 10463 ~ 14 days dried skull with neck µCT OUVC 10519 ~ 2 months head & neck µCT and µCT Lugol's OUVC 10517 ~ 4 months head & neck µCT OUVC 10630 young adult braincase µCT OUVC 10661 young adult head & neck µCT Table 1. List of all specimens used in this study.

CT Scanning. Scanning was done at the Ohio University micro-CT scanner at the Ridges in Athens Ohio.

Generation of Digital Endocasts. The software Avizo, versions 6 and 7, was used to generate 3D models of the endocasts of each specimen using the imported data from the CT scans. Avizo can be used to generate a Label Field, which takes each CT slice and stacks them one after the other, making a 3-dimensional canvas on which a model can be generated through a process called segmentation. This model can include bone, endocast, inner ear, nerves, blood vessels, empty space, or any combination thereof. Segmentation can be described as a type of painting. Using the xy, xz, and yz planes of individual slices of a specimen, I use the program Avizo to 20

identify and color in areas and structures of interest. Each major piece of the endocast is colored in three dimensions and assigned its own material in Avizo. These materials can be made into a 3D visible model within Avizo which can be freely rotated and studied.

Once a 3D model was created in Avizo, the endocast portion was transferred to

Maya, a different 3D manipulation program. In Maya, the object was painted by endocast region as discernible by physical features of the endocast itself. The painted areas as well as the entire object were turned into separate objects and returned to

Avizo for analysis. In Avizo, the number of triangles comprising the surface area of each material was counted, totaled, and compared to the original total.

In addition to the creation of a standard endocast for specimen OUVC 10519, the 2-month-old, a Lugol brain model was also generated. OUVC 10519 was submerged in Lugol’s iodine solution for a period of nine months before additional scanning took place. With the soft tissues now more discrete from one another, the segmentation of the actual brain, commonly referred to as a “Lugol brain,” was completed in Avizo.

Results

Adult Endocast Introduction. Figures 1a and 1b display one of the adult endocasts with nerves, veins, and arteries included in yellow, dark blue, and red respectively. The endocasts of the adult specimens have well defined Wulst, cerebrum, flocculi, optic chiasm, optic tecta, and pituitary regions. The cerebellar region is discrete from the cerebrum, flocculi, and optic tecta, but not from the medulla region. 21

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Figures 1a and 1b. Lateral and rostral (1a), dorsal and ventral (1b) views of the endocast of OUVC 10661 adult Struthio camelus specimen.

Visible Qualitative Results. The cerebellum is difficult to differentiate from the medulla region, even in adult specimens. In the 14-day-old specimen, the cerebellum becomes somewhat discrete from the cerebral region, maintaining this 23

minor yet discrete boundary throughout the older specimens. In the embryos, the optic tecta and cerebral hemispheres have almost indiscernible boundaries, save for a few

guiding features already present in the end ocasts. In the 14-day-old specimen, the cerebral hemispheres are notably discreet from most surrounding features. The floccular regions are identifiable in all specimens, but they exhibit obvious change in shape throughout development. A striking shape emerges in the 14-day-old specimen: an odd shape with an exaggerated base, marked by a red star in Figure 2 and is visible in Figures 3 and 4. The lack of ossification in the olfactory region rarely permits accuracy of the model in that area. Thus, its change in definition is not a dependable source of information at this time. The region of the optic chiasm is not sufficiently ossified to be resolvable in specimens other than the two adult specimens, leaving the models of the younger endocasts with largely flat and unresolved regions from which the optic chiasm could otherwise be discerned. In the embryos, as stated previously, the optic and cerebral lobes have few features that allow their shape to be approximated. In the 14-day-old specimen, however, the optic and cerebral lobes are easily identifiable, maintaining this status through the adults. The optic tecta also become more discernible from other surrounding regions in the 14-day-old specimen, maintaining this through to the adults. While the bony boundary of the pituitary region is not shown in the scan of the 39-days-incubation embryo, this feature is readily apparent in the 40-days-incubation embryo. This difference is intriguing, considering the two were scanned together at the same resolution and at the same time. The Wulst is another feature which begins as barely discernible in the embryos and quickly 24

reaches the status of moderately discrete in the 14-day-old individual. The Wulst continues to grow much more than the cerebral hemispheres and optic lobes, for example. It grows from a slight rise in the cerebrum in the two embryos to a large, bulbous protrusion resting atop the cerebral region in the adults. From the 39-days- incubation embryo to the 40-days-incubation embryo to the 14-day-old specimen, the

Wulst is detectable progressively further caudally in the direction of the cerebellum.

While the definition of the endosseous labyrinth is lacking in certain sections in the embryonic specimens due to incomplete ossification, the missing pieces can be inferred quite easily. The endosseous labyrinths of the endocasts appear to change very little in shape throughout development. The primary change is in size, with a major growth spurt evident between 14 days and 2 months, which can be seen in

Figure 5. 25

Figure 2: Lateral, rostral, ventral, dorsal, and scaled lateral views of endocasts colored according to the nine brain regions studied: Cerebellum(yellow), Flocculi(red), Cerebrum(light blue), Wulst(pink), Olfactory bulbs(light purple), Optic tecta(green),

Optice chiasm(orange), Pituitary(dark blue), Medulla(dark purple), for five specimens. 26

Figures 3 and 4. Displaying lateral(3) and rostral(4) views depicting endocast with endosseous labyrinth, nerves, and blood vessels for each specimen. 27

Figure 5. Lateral, rostral, dorsal, and scaled lateral views of left endosseous labyrinth by age, left to right, of five specimens.

Quantified Relative Surface Area. Comparing the relative surface area of different brain regions over time is a method which attempts to quantify brain regions, which is a difficult task due to the continuous nature of tissues. While assessing volumes would be more neurologically appropriate, the issue of somewhat arbitrary boundaries is far more problematic with a volume-focused method than with relative surface area comparison. Furthermore, due to the nature of this analysis, in which regions of an endocast are painted virtually, variation that seems to form a trend is not 28

necessarily doing so. Therefore, only large and consistent change in percentage of overall surface area through time was given true consideration. There appears to be a general trend of the cerebellar region occupying less relative surface area as the specimens increase in age. No trend appears to exist for a significant change in relative surface area for the cerebral region. There appears to be an extremely slim trend for the flocculi to decrease in relative surface area in the endocasts as the specimens increase in age, but the change is probably not significant. No pattern appears to emerge in the relative surface area occupied by the medulla region as the specimens increase in age. Due to the poor resolution of the ostrich’s olfactory region in all specimens, any pattern that may appear to present itself is likely a result of a less resolved region in a number of specimens. No notable change seems to occur in the relative surface area of the optic chiasm with age, even considering its poor definition up until adulthood. No notable change seems to occur in the relative surface area of the optic tecta with age, considering the range of difference between the embryos and between the adults. The pituitary does not present a change in relative surface area through time. As noted above, however, its definition is lacking in the 39-days- incubation embryo, but, interestingly, the pituitary region is adequately resolved in the

40-days-incubation specimen. The Wulst appears to have a more marked increase from embryo to adult, rising from 7.47% and 7.54% for the two embryos to 15.04% and 13.80% for the two adults (See Table 2). This increase in relative surface area is the most noticeable among the various regions. 29

Table 2. List of relative surface area occupied by the indicated endocast region of the colored endocast for all specimens.

Lugol’s Analysis. Thanks to Lugol’s iodine analysis, shown in Figure 6, it is clear that the floccular region is largely composed of venous tissue, not neural as implied by the CT scans. Thus, information gained from an endocast floccular region can be misleading. The upper area of the cerebellum and a large portion of the lower medulla sections of the regular endocast also appear to have a thick layer of venous network contributing to their volumes, as seen in Figure 6. This discovery also shows the shortcomings of pure endocast analysis, even in birds, which are so often considered as needing only endocast-analyzing methods. These confounding factors mean that calculating the actual surface areas of these regions is much less accurate when using an endocast. Thus, the endocast data on these regions should be treated with caution. The olfactory bulbs are much more easily resolved in a Lugol brain, furthering the statement that standard CT scanning tends not to show accurate borders of the olfactory neural region. The boundary between cerebrum, olfactory bulb, and 30

olfactory nerve tract is imperceptible in a standard CT scanned sample, yet easily picked out in a Lugol-treated individual’s scans.

Figure 6. Comparison between Lugol brain with blood vessels and endocast.

Discussion

It is important to remember that the capacity to detect certain brain regions and structures is dependent on the presence of the surrounding ossified tissue, not the neural or other soft-tissue structures. While a structure may not show up in an endocast, this does not mean it is absent. Rather it is merely lacking surrounding bone to reveal its existence. The ossification of particular regions at certain times could relate to importance and priority level, given that converting tissue into bone obviously takes cellular effort. 31

Prioritization. From youngest to oldest in the first three ontogentic samples, the caudal boundary of the Wulst becomes detectable progressively further caudally, as seen in the lateral and dorsal views of Figure 2. This expands upon Picasso et al.’s

(2010) observation that the Wulst in Rhea, (their eminentia sagittalis), developed a more pronounced rostral boundary throughout development. The phenomenon described by Picasso et al. (2010) is more difficult to observe gradually in the ostrich endocasts, but a comparison between the embryo and adult specimens makes the increase in rostral boundary definition apparent. The rest of the Wulst can be seen to gradually emerge from the cerebrum over time, while the rostral boundary consistently has the least definition. This large and obvious change in size and shape of the endocast suggests that the development of the Wulst is a high priority during development. The near doubling of the relative surface area of this region from embryo to adult provides support for this hypothesis. It is the only region that displays both a pronounced shape and size change as well as a clear increase in relative surface area.

No other region showed a noticeable increase or decrease in relative surface area over time. Though a slightly consistent decrease seems to appear in the cerebellum, the change is far too small to be considered significant, given the qualitative nature of this project. In addition, any apparent change in the cerebellum region is confounded by the discovery that it is partially composed of venous tissue, as revealed by the Lugol’s iodine staining analysis of the two-month-old specimen,

OUVC 10519. (See Figure 6.) Also, in Peng et al. (2010), the figures, which are 32

photographs of an actual adult ostrich brain, reveal that the cerebellum is much more defined in the true brain than in the endocast, along with several other features, to be discussed later. While the Wulst increases in surface area, other regions do not appear to be decreasing proportionally. This suggests that the increase in relative surface area of the Wulst comes at little cost of area to other regions of the endocast. The capacity to develop the Wulst at little cost to surrounding brain regions would suggest that the ostrich’s ontogeny is designed to maintain stability of all regions during the Wulst’s increase in size.

Ontogeny Recapitulates Phylogeny. We see a general increase in definition of the endocast’s features as the specimens increase in age, with most regions becoming more discrete, particularly the optic tecta and cerebrum. The same could be said when describing the phylogeny of theropod endocasts seen in Witmer & Ridgely (2009) and

Figure 7, in which the representative endocasts seem to show more features of the underlying brain during progression from older to more recent taxa. The increase in definition goes along with an increase in the appearance of the endocast as “birdlike,” or taking on a shape more akin to a typically avian endocast. That is to say, the ancestors appear to lead up to the modern avian condition of having an endocast strikingly similar to the brain (Picasso et al., 2010). A phylogeny composed of some representative theropods is shown in Figure 7. The increased avian character and more brain-like definition of the ostrich endocast throughout development can be said to recapitulate phylogeny to a noticeable extent, mirroring the evolutionary development of the theropods. 33

Figure 7. Shows a phylogeny of some selected members of non-avian theropods, with Archaeopteryx as most derived.

In contrast to the notable structural alteration of the endocast, the inner ears remain very similar in shape throughout development, suggesting a high level of morphogenetic and/or functional constraint. Such an important organ, which is relevant from birth to death, is not likely to show major changes—beyond size— throughout development. While the bony boundary is not seen as complete in the embryonic specimens, ossification is obvious within 14 days, when using OUVC

10463 as an indicator. A feature of the inner ear that is important, however, is the shape and proportions of its canals. The shape of the inner ear during development appears quite constant. Its change in size follows the change in size of the endocast, showing the largest increase between the 14-day-old and the 2-month-old, minor growth between the 2-month-old and the 4-month-old, and another noticeable size increase from the 4-month old to the adults.

Implications for Ostrich Development. If the missing portions of the embryonic specimen ears are taken as valid, very little change is apparent in the 34

endosseous labyrinths of the inner ears of the specimens as they increase in age. This implies that hearing and especially the sense of equilibrium (balance) are vital from birth, which makes sense for a precocial bird such as the ostrich. The main change seen during development is the predictable one: size. See figure 5 for visual detail.

The flocculi across specimens loosely share a common shape and orientation, with the moderate anomaly of the 14-day-old presenting an interesting conundrum. The discrepancy is best seen in the lateral view of Figure 2, in which the 14-day old specimen possesses the common extension along with a raised and widened secondary base, a feature no other specimens possess in their floccular endocast regions. The wide variety of shape among the specimens can be attributed to the discovery that this region is composed primarily of venous tissue, rather than neural. The apparent anomaly in OUVC 10463 is less alarming considering this confounding factor. In addition, Peng et al.’s (2010) figures depict the flocculus, which they call the cerebellum auricle, as a small, simple bump emerging from the cerebellum

The importance of vision in the ostrich based on this analysis is also an interesting consideration. Even in the youngest embryo, it is possible to discern something of an optic structure from the cerebral and cerebellar regions. The boundaries are difficult to detect, but an optic tectum is clearly present. Given the marked emergence of the optic tecta from surrounding regions even at 14 days, sight would likely be considered a high priority in these birds, especially considering their precocial nature and enormous eye size. However, in contrast to the early appearance of the optic tecta in the endocast, the optic chiasm is not determinable in any 35

specimens but the two adults, suggesting that the ossification of the structure surrounding this area has relatively low priority. This disparity between optic tecta and chiasm resolution is strange, considering that ostriches have such massive eyes compared to their head.

The pituitary’s importance seems clear during development. Between the 39 and 40 days incubated embryos, the pituitary region becomes more defined, and it can be seen as fully discrete from the medulla in the 2-month-old specimen. Since the pituitary is an essential hormone-control center, it makes perfect sense that the section in which it rests in the endocast would be detectable early in development. Protection of this essential region through ossification of surrounding tissue makes sense from a resource expenditure standpoint given that it requires a considerable investment of energy to ossify tissues.

Implications for Non-Avian Dinosaur Development. This study advances potential understanding of dinosaur development, particularly in the theropods. The partial recapitulation of theropod phylogeny through ostrich development raises many new questions. The phenomenon of late ossification of the region adjacent to the optic chiasm has many possible implications. Perhaps the theropods and other dinosaurs lacked the excess energy (or the energy efficiency) to devote to this process. Perhaps protection of this area was sufficiently achieved with non-ossified hard tissue. The chiasm is not displayed in the Archaeopteryx specimen endocast from Domínguez

Alonso et al., 2004 (Domínguez Alonso, Milner, Ketcham, Cookson, & Rowe, 2004)

Nor is it depicted in the pterosaur endocast sketches of Witmer et al., 2003. Obviously, 36

Rhamphorhynchus is not a theropod, but it is an alternate example of an archosaur without ossification surrounding its optic chiasm. On the other hand, the second pterosaur endocast sketch presented in Witmer et al. (2003) included the optic chiasm.

Two pterosaurs that are relatively closely related either exhibit or lack ossification of the surroundings of the optic chiasm. This presents a number of interesting avenues for speculation, given the results of this ontogenetic ostrich endocast analysis. Does this feature vary for a reason beyond phylogeny? Was one specimen an adult and the other younger? This is just speculation regarding the two pterosaurs. The

Archaeopteryx specimen could lack ossification due to youth or perhaps the feature would never have developed at all. Much more intensive and extensive research is called for before such questions can be answered.

The Wulst being detectable even in the embryos of the ostrich has potential implications for its evolutionary emergence. However, this has been a topic of uncertainty for some time. In their paper on the brain anatomy of a late Miocene eagle,

Picasso et al. (2009) suggested that the earliest the Wulst appears is in the Eocene, not in the Cretaceous. Of course, given that the endocast is increasingly less informative of underlying brain structure as research moves to more ancient specimens, the absence of an impression in the bone of fossils of a Wulst does not necessarily preclude its presence. Accordingly, Walsh & Milner (2009) concluded that it was present but not apparent at the end of the Cretaceous. Discriminating lack of definition from absence of structure will take much more research into the endocast-brain relationship of fossils, including development of new techniques for gaining more 37

accurate brain anatomical structure from frustratingly vague fossil braincases. This endeavor has already begun to be undertaken by PhD candidate Ashley Morhardt. The technique is known as GABRA, and it seeks to create a means of extrapolating the true brain from the endocranial space that can apply across varying taxa (Morhardt,

2012).

Future Direction. Further testing the concurrence of modern brain development with phylogenetic patterns in dinosaurs would be beneficial for making inferences about extinct taxa. Whether this pattern holds in other basal taxa warrants investigation. In Picasso et al. (2010), the definition of the underlying brain structure does appear to increase in clarity during development, though an embryonic specimen would be an ideal inclusion in the data set. It would also help to create an ontogenetic endocast data set with a representative species of each extant order or superorder of birds to create a continuum of easily comparable features from which informative conclusions may be drawn. The other half of the extant phylogenetic bracket cannot be neglected either. Proceeding in accurately analyzing non-avian dinosaur ontogeny will require additional investigation of crocodilian endocast ontogeny. Learning that the

50% rule (which assumes that non-avian dinosaurs only filled 50% of their endocranial space with neural tissue) likely has limited applicability in certain cases

(Evans, 2005) is a major first step, to be sure, but developing a picture of the trend of how the brain of crocodilians fill their endocast with brain versus fluid over time can be useful in comparing extinct and modern birds with non-avian dinosaurs. 38

Coupling fossil analysis with extant research will naturally be necessary as well. Assembling what we can of extinct avian as well as non-avian theropod endocasts at different periods in development will be an important initial step in the paleontological arena, followed by the natural extension of scope beyond exclusively theropods. Non-avian members of this group can tell us much about modern and extinct avian taxa through potential contrast analyses. Creating a full picture of endocast evolution as well as ontogeny for each possible extinct species and a satisfactory amount of living ones allows for detailed inferences of the dinosaur brain on a level previously unavailable.

39

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44

Appendix: Procedure Details

In Avizo, the priority for each specimen is to generate a model of the endocast, inner ear, blood vessels, and nerves. In order to differentiate the parts of the desired soft tissue, “Materials” are created in addition to the defaults “Exterior” and “Interior.”

Exterior need not be modified. Interior’s name and color can safely be changed, for example, to “Endocast.” Additional materials can be generated, each with a unique name and color, which is ideal for keeping track of one’s work. For the basic procedure, Materials for Endocast, Inner Ear (also called Endosseous Labyrinth),

Nerves, Veins, and Arteries are created. Generally, the first area segmented is the endocast, which, according to lab protocol, is assigned a blue material.

Avizo is equipped with various Tools, each of which serves a different function related to generating a model. The most useful tools for the purposes of segmenting CT data are the Brush and the Magic Wand. The brush operates intuitively, displaying an adjustable area of effect, and shading that area when Left- clicked. “Ctrl + Left-click” will erase areas previously selected. The magic wand selects all voxels (3D pixels) within a certain range of brightness. The range can be narrowed or widened depending on the clarity of the scan and the level of detail desired. What cannot be picked up by the magic wand due to homogeneity of voxel brightness in a given specimen is manually segmented using the brush tool.

While Avizo does not possess an “Undo” function due to the amount of data it must constantly process, a “Clear” function exists to delete what has been brushed or selected with the magic wand, provided the selection has not yet been added to any 45

Materials, at which point it must be reselected and added to Exterior. In order to clear selected space, either the C key was pressed while in the viewer window or the eraser icon was clicked while in the Label Field tab of the Main Panel. It is best to not wait to segment out a large amount of tissue before adding your selection to a material. Much work will be lost if the system crashes as one tries to save, which sets the user back several minutes. It is extremely advisable to save often. With the new features of

Avizo 7 came the “Save as Pack and Go Network” option, which is very helpful when a different computer must be used.

Once an endocast was segmented, the caudal border was trimmed to the opening of the foramen magnum. Without saving the network, the materials other than the Endocast material were deleted, and a new surface file was generated which was comprised of only the Endocast. This Endocast-only surface file was saved separately from the network and reloaded on its own in a new instance of Avizo. The surface was then Remeshed, which forces the surface to be made of equilateral triangles, rather than variable isosceles triangles. This allows for smoother work in Maya, the

Autodesk program used to color the various brain regions. Files that are not remeshed in Avizo waste memory and slow Maya to unwieldy speeds.

After Remeshing, the .surf file was saved as an .obj file, a format Maya is able to open. Next, the remeshed file is loaded into Maya as a plain gray, hollow object.

The object is painted in a similar manner to assigning Materials in Avizo: assigning

Materials to painted sections on the object’s surface. The parts of the endocast to be shaded and their respective colors are: Cerebrum/blue, cerebellum/yellow, 46

medulla/dark purple, optic tectum/green, Wulst/pink, flocculus/red, olfactory region/light purple, pituitary region/blue, and optic chiasm/orange. Creation of all materials before starting to paint the respective brain regions is advisable.

In order to create materials in Maya, the Hypershade command was used, which opens the materials interface. Clicking on a type of material in the left of the window created a plain gray material in the work area on the right of the window.

Selecting the new material allowed color manipulation. The F key was tapped to center the camera on the object. If the object appeared as a dense, dark wireframe, the

“6” key was tapped to toggle to the simple gray surface. In order to paint the object, the arrow-shaped Select tool on the left of the work screen was highlighted, then the object was clicked once. This resulted in a green framework overlaying the object.

Next, the right mouse button was held down and moved slightly. Once the circular interface appeared, the mouse was dragged over “Face” and the right button was released. This overlaid the object with a blue framework. Next, the paintbrush symbol, located below the Select tool to the left of the work screen, was clicked. A purple framework appeared over the blue. As before, the mouse icon was placed over the object, the right mouse button was clicked, the mouse was dragged to Face, and the right button was released. The blue framework reappeared.

A region was chosen to begin shading. In order to adjust the size of the brush, the Ctrl+Right Mouse Buttons were held down and moved either left or right to enlarge or shrink the radius of the brush. When Face Mode (the blue framework) hindered the ability to discern boundaries, the right click and drag process used to 47

display the blue framework was repeated, but the button was released over “Object

Mode” instead. The object appeared with no framework, and any coloration so far applied was then clearly visible. Once the desired area was highlighted with the Brush tool, placing the mouse over the appropriate material holding down the middle mouse button, dragging the mouse over the painted area, and releasing the button assigned the shaded region to that material. The new color showed up under the brushed areas, which remained highlighted until erased or the entire object was selected with the

Select tool. This shading process was repeated for each region until the entire surface of the endocast was shaded and assigned appropriate regions. Once painted, the materials of the Maya object were divided into separate objects of their own. This was done by right-clicking a color in the Hypershade interface window and selecting

Select Objects with Material. Next, in the Mesh menu, Extract was selected, and

Object Mode was entered. Then, in the File menu, Export Selection was clicked, and the file type to export was chosen as .obj. All File Specific Options were disabled. The

.obj files were converted to .stl files using the program Deep Exploration. The .stl files were then opened and analyzed in Avizo.