an investigation of the odontocete inner ear

Is The Hearing of Whales and Dolphins Fully Developed at Birth?:

An investigation of the odontocete inner ear

A thesis submitted for the fulfilment of the degree of

Master of Science in Marine Science

At the University of Otago, Dunedin,

New Zealand

Tiffany Plencner August 2017

Acknowledgements

There has been a huge amount of support from many different people

during the course of my MSc thesis. Firstly, a big thank you to my supervisors

Ewan Fordyce and Steve Dawson for continued encouragement, support and

guidance throughout my time at the University of Otago. Thank you Museum of

New Zealand Te Papa for providing access to a massive cetacean collection and to

Andrew Stewart, who was wonderfully helpful and always made me feel welcome

during my visits. Thank you Emma Betty at Massey University in Auckland for

providing rare foetal material that proved to be really exciting to x-ray.

A key aspect of my thesis would not have been achievable without the help

of Te Upokorehe iwi of Ohiwa Harbour. Your hospitality was overwhelmingly

generous and it was a privilege to meet and work together during our 10 days

collecting data in the pop-up lab at Roimata marae. Special thanks to Wallace

Aramoana (Snr), Puti Aramoana, Trevor Ransfield, Lance Reha, Sandra Aramoana,

Irene Almond, Gaylene Kohunui, Glen Aramoana, Christine Martin, Kahukore Baker

and Te Upokorehe whānau for their leadership and kindness. To Ramari Stewart,

‘Tohunga Tohorā’ for her life-long expertise, initiative and guidance in processing

the Upokorehe customary collection, her commitment and dedication made this

collection possible. I am looking forward to visiting again with an update after

submitting.

This thesis came with some huge learning curves in terms of new imaging

software and analysis programs. So a huge thank you to everyone who helped me

wade through all of it: Andrew McNaughton and Karla Rovaris for teaching me

micro-CT, Luke Easterbrook and Hamish Bowman for helping with all the various

imaging software, and Tim Jowett for all the statistics guidance and help with the R

language.

My friends Anna Meissner, Sophie White, and Shaun Wilson, you were also a

massive part of the work at Ōhiwa Harbour. I want to thank you and all my

Dunedin friends for the support, advice and keeping me sane throughout the

course of my thesis.

Abstract

Sound is an essential component of toothed whale and dolphin

(odontocete) biology. The advanced hearing-dependent activities of precocial

calves lead us to believe that hearing is fully functional by birth. Nevertheless, few

studies have investigated the ontogeny of cetacean ears. This thesis investigates

the odontocete ear region (bones of the tympanoperiotic complex, TPC),

particularly the organ of hearing (cochlea) in the inner ear. I used radiography to

examine ossification in six odontocete foetuses, and micro-CT scans to reconstruct

3D cochlear models for four life stages (foetal, neonate, juvenile and adult) from

four ecologically divergent species from New Zealand: Hector’s dolphin

(Cephalorhynchus hectori), bottlenose dolphin (Tursiops truncatus), long-finned

pilot whale (Globicephala melas), and Gray’s beaked whale (Mesoplodon grayi).

Individuals ranging from newborns to adults showed no significant intraspecific

differences in the size and shape of their cochlear canals (p= 0.998) and TPCs, but

there were significant interspecific differences (p<0.001). Consistent with their

phylogeny, M. grayi cochleae significantly differed from the three delphinid study

species (p<0.05), while the cochlea of the three delphinids did not significantly

differ from each other (p>0.05). No ecological parallels were apparent within

cochlear structures. Radiographs of foetuses ranging from Stages 7-12 (of Štěrba et

al., 2000 classification) did not show evidence of ossification. The more advanced

Stage 11 G. melas foetuses, however, showed minor mineralisation of the TPC, and

a cochlear canal could be seen in the largest foetus (69cm). Cochlear and TPC size

and shape results suggest rapid prenatal development of hearing structures and

are consistent with the hypothesis that calves have fully functional hearing

abilities at birth.

ACKNOWLEDGEMENTS II

ABSTRACT III

CHAPTER 1: INTRODUCTION 1

1.1 Evolution of Cetacean Hearing and its Research 1

1.1.1 Evolution 1

1.1.2 Research History 2

1.1.3 Significance of Micro-CT 3

1.1.4 Significance of Morphometrics 4

1.2 Components of the Hearing Pathway 5

1.3 The Inner Ear in Detail 7

1.3.1 Cochlear Canal and Factors that Impact Hearing 9

1.3.2 Vestibular Complex 10

1.4 Significance of Age and Ecology 10

1.4.1 Age 11

1.4.2 Ecology 12

1.5 Study Aims: Is Hearing Fully Developed at Birth? 14

2 CHAPTER 2: METHODS 17

2.1 Sources of Material 17

2.2 Aging and Staging 20

2.3 Skull and TPC Measurements and Analysis 21

2.3.1 Measurements 21

2.3.2 Analysis 22

2.4 Building 3D Cochlear Models 24

2.4.1 Micro CT Scanning 24

2.4.2 3D Modeling Software 24

2.5 Inner Ear Measurements and Analyses 28

2.5.1 Measurements 28

2.5.2 Inner Ear Analyses 29

3 CHAPTER 3: RESULTS 33

3.1 Tympanoperiotic Complex and Skull Measurements 33

3.2 Radiography of Foetal Material 34

3.3 Inner Ear Measurements 35

3.3.1 Geometric Morphometrics 35

3.3.2 Linear Measurements and Ratios 37

4 CHAPTER 4: DISCUSSION 40

4.1 Significance to Age- Ontogeny 40

4.2 Significance to Species- Phylogeny and Ecology 42

4.3 Observational Notes 44

4.4 Future research 45

5 CONCLUSION 46

6 REFERENCES 47

Appendix A 67

Appendix B 75

Chapter 1: Introduction| 1

Chapter 1: Introduction

The following is an investigation into the ontogenetic development of the

hearing apparatus of toothed whales and dolphins (odontocetes). While we know

how dolphins receive sound (e.g. Brill et al., 1988), the current literature reveals

little of when these crucial hearing structures become fully developed. This study

focuses on development of the inner ear’s cochlear canal from four different age

classes of odontocetes. In this chapter, I first outline the current understanding of

how cetacean hearing has evolved. I describe the hearing pathway, with an

explanation of the essential parts of the inner ear, and finish with a brief

description of the study species and aims. Chapter 2 explains methods for

acquiring the material analysed, the collection of data and analyses including the

use of micro-CT, 3D models, and geometric morphometrics. Chapter 3 provides the

results, and Chapter 4 discusses their meaning with suggestions for future

research.

1.1 Evolution of Cetacean Hearing

1.1.1 Evolution

Hearing in cetaceans (whales and dolphins) has a fascinating evolutionary

history. Deriving from land mammals, cetaceans had to adapt the morphology,

physiology and behaviour to live successfully in an aquatic environment. Amongst

many changes, there was a streamlining of the body, loss of pinnae (external part

of the ear), telescoping of the skull and a shift in their hearing mechanism (Kellogg,

1928; Miller, 1923; Nummela et al., 2007; Thewissen et al., 2009). Terrestrial

mammals rely on acoustic vibrations from the air to hear. Cetaceans on the other

hand rely on acoustic vibrations that travel through water almost five times faster

than air (Reysenbach De Haan, 1960). Early cetaceans thus underwent some major

changes in their hearing structure and pathway to accommodate for the physical

properties of living an aquatic lifestyle.

This transition is displayed throughout the fossil record. Cetacean ancestors

likely shifted to marine living around 50 million years ago during the Eocene

Epoch (Reysenbach De Haan, 1957; Thewissen et al., 2009; Thewissen & Williams,

2002). Evidence suggests that ancestral cetaceans like basilosaurids,

mammalodontids and aetiocetids had low frequency hearing (less than 1000Hz;


Ekdale & Racicot, 2015; Park et al., 2017), as inferred from the long cochlea and

number of turns similar to those found in modern baleen whales (mysticetes),

which have had a remarkably consistent hearing morphology over the last 34-45

million years (Ekdale & Racicot, 2015; Kellogg, 1936; Lancaster, 1990). On the

other hand, toothed whales (odontocetes) experienced a drastic shift into

specialised high frequency hearing (more than 30,000 Hz; Churchill et al., 2016;

Hemilä et al., 2001; Nachtigall et al., 2007; Ridgway et al., 1981). This likely

occurred early in the odontocete-mysticete divergence, during the Eocene-

Oligocene boundary around 34 million years ago (Fordyce & Muizon, 2001; Park et

al., 2017). Fossil specimens have revealed that some of the earliest diverging

odontocete stem groups had ear morphology already adapted for high-frequency

specialisation (Churchill et al., 2016; Ekdale & Racicot, 2015; Park et al., 2016).

In addition to a shift in frequency specialisation, evolution within an aquatic

environment resulted in parts of the ear region becoming reduced, vestigial or lost

completely in cetaceans. For example, pinnae have disappeared (Cozzi et al., 2017;

Thewissen et al., 2009), the ear canals have become vestigial (McCormick et al.,

1970; Yamada, 1953) and there have been some intriguing changes to the inner

ear as well. The vestibular complex, known for its role in spatial perception and

balance (Angelaki & Cullen, 2008; Cawthorne et al., 1956), involves three semi-

circular canals whose sizes have also been greatly reduced in Cetacea (Hyrtl, 1845;

Spoor et al., 2002). The cause and significance of this reduction is still uncertain,

and it is not clear whether the function attributed to the vestibular complex has

also been reduced or lost altogether.

1.1.2 Research History

Hearing has been a topic of scientific interest since the 1700s. Petrus

Camper was first to publish research into sperm whale’s organ of hearing (Camper,

1765). John Hunter recorded many detailed necropsy observations, some of which

focused on cetacean hearing structures (Hunter & Banks, 1787). Due to

accessibility to cetacean material, early research on hearing often focussed on the

ears of terrestrial mammals, with opportunistic findings from cetaceans (Gray,

1908; Hyrtl, 1845; von Békésy, 1960). Research on various mammalian species has

provided a solid framework for advancing our knowledge of cetacean hearing


structures. Additionally, Munesato Yamada and Gerald Fleischer provided a

foundation of detailed observations, and established anatomical terminology and a

standardised procedure for investigating the ear region (Fleischer, 1976; Yamada

& Yoshizaki, 1959).

In the 1960s, a major paradigm shift completely changed our understanding

of the odontocete hearing pathway. Contrary to the belief at the time, that the ear

canal was believed to be the primary source of sound reception (Fraser & Purves,

1960), Ken Norris’s revolutionary ‘jaw-hearing’ hypothesis proposed that sound

reception starts with sound entering a thin part of the lower jaw, nicknamed the

‘acoustic window’ (Norris, 1964, 1968). We now know that specialised fats behind

the acoustic window also help to channel sound into the Tympanoperiotic complex

(TPC) and inner ear (Koopman et al., 2006; Norris, 1964, 1968; Zosuls et al., 2015).

Experiments by Brill (1988) supported the jaw-hearing hypothesis, finding that

the hearing abilities of captive bottlenose dolphins (Tursiops truncatus) were

significantly impaired when a neoprene hood masked the lower jaws. Additionally,

a study of evoked potentials found that hearing was less sensitive near the ear

canal than it was next to the jaw (Brill, 1991). Today, the jaw-hearing hypothesis is

widely accepted as the primary channel for sound reception in toothed whales and

dolphins (odontocetes; Ketten, 2000).

1.1.3 Significance of Micro-CT

Recent advancements in the understanding of the inner ear reflect the

positive impact of developments of micro Computed Tomography (micro-CT) and

associated analysis software (eg. Costeur et al., 2017; Grohé et al., 2016; Thean et

al., 2016). Initially, research on the inner ear was restricted to destructive

sampling, which usually involved cutting through the pars cochlearis with a bone

drill to access and observe the interior of the bony labyrinth (eg. Fleischer, 1976).

However, this was not ideal because vibrations from the drill could damage the

delicate structures inside. Fortunately, micro-CT was developed in the 1980s by

Jim Elliott (Elliott & Dover, 1982) and was commercially available by the 1990s.

Since then, ease, cost and accessibility to micro-CT machines have quickly

improved for a wider range of users. Conceptually, micro-CT is the same as the

more familiar Computerised Axial Tomography (CAT) scans used in medicine, but


with specialisation to image smaller objects at higher resolutions. CT scans use X-

rays to take a series of digital “slices” of an object (Abel et al., 2012). These slices

can then be stacked to form a 3D model that can be manipulated, examined and

analysed by the researcher. Technology including both the micro-CT machine and

various manipulation software packages are still improving. Although this study

used the SkyScan 1172 model, which can achieve 0.5 µm resolution, new models

have been developed to image at an even smaller scale. In addition to the

avoidance of destructive sampling, CT-scanning also offers the opportunity to

share specimens between scientists and museums digitally rather than physically.

1.1.4 Significance of Morphometrics

One way to utilise micro-CT models and investigate structure is through the

application of geometric morphometrics. Morphometrics is described as the study

of shape variation and its covariation with other variables (Bookstein, 1997; Rohlf

& Marcus, 1993). Initially, morphometric analysis was done through statistical

analysis of linear measurements from morphological points, ratios or angles

(Adams et al., 2004). Geometric morphometrics on the other hand, takes a more

robust and dynamic approach by running statistical analyses after the

incorporation of a standardised landmark coordinate system (Corti, 1993). This

method has been described as a revolution in the field of morphometrics (Rohlf &

Marcus, 1993).

First, the researcher places homologous landmarks in noteworthy places

that can be identified on all the included 2D images or 3D-models. The landmarks

may be anchors for series of semi-landmarks, indistinct surface points that are

computer generated at equally spaced positions. Then the combined landmark and

semi-landmark coordinate sets are run through a superimposition process called a

Generalised Procrustes Analysis (GPA), which calculates a ‘mean’ shape that allows

for multivariate statistic analyses. Although there are other approaches to

completing geometric morphometrics, this is one of the most common due to the

high statistical power of the ideal shape-space (Kendall’s shape-space) used to

create the ‘mean’ shape in GPA (Kent, 1994; Rohlf, 1999, 2000).

Ultimately, GPA is a statistical method for finding the optimum placement in

space for multiple superimposed objects (Rohlf, 1999; Slice, 2001) and allows for


complex shape analysis. It has become fundamental to modern shape analyses

(Adams et al., 2004) and a valuable tool to a range of disciplines (e.g. Adams et al.,

2013; Ekdale, 2016; Tsai & Fordyce, 2014).

1.2 Components of the Hearing Pathway

Since the jaw-hearing hypothesis of the 1960s we have established a much

better understanding of the odontocete hearing pathway and the inner

mechanisms of their hearing complexes. The hearing pathway can be thought of as

a series of steps that eventually reach the organ of hearing (cochlea) and the focus

of this thesis (Figure 1).

[1] Sound is first received through a thin posterior portion of the lower jaw,

called the pan bone or “acoustic window” (Norris, 1964, 1968).

[2] Specialised acoustic fats, located medial to the pan bone, channel these

sound vibrations towards the tympanoperiotic complex (earbone; Koopman et al.,

2006; Norris, 1964, 1968; Zosuls et al., 2015). Mandibular acoustic fats are similar

to those found in the melon and, biochemically, are unlike fats found anywhere

else in the body (Koopman et al., 2006; Litchfield et al., 1973). These specialised

fats are structured to act as a funnel, in which the outermost mandibular fats (long-

chain fatty acids) can receive fast moving sounds from a broad area and transfer

them to the slower inner mandibular fats (short-chain fatty acids), which are

directly adjacent to the tympanoperiotic complex (TPC; Hustad et al., 1971;

Koopman et al., 2006). At a size roughly the size of a golf ball in dolphins, the TPC

is largely made up of two parts, the ventral conch-like tympanic bulla, and the

dorsal periotic that houses the inner ear. Since the TPC is extremely dense and

almost completely isolated by the air-filled peribullary sinus, interference between

hearing and the vibrations of self-produced sound emissions are minimised

(Fraser & Purves, 1960; Norris, 1964; Nummela et al., 1999).

[3] Once they reach the TPC, sound vibrations are transferred from the

tympanic bulla into a series of three middle ear bones called the ossicular chain.

This is made up of the malleus (hammer), incus (anvil) and stapes (stirrup), which


concentrate sound into the small opening of the oval window, the fenestra ovalis

(Hemilä et al., 1999). Sound pressure increases when going from a large area to a

smaller one, and the ossicular chain’s function is to transmit energy with a particle

velocity that is suitable for the inner ear to receive (Nummela, 2009). Frequency

range is largely governed by the structure and elasticity of the middle ear and its

ability to transmit energy (Fleischer, 1978; Nummela, 2009; Plassmann & Brandle,

1992; Rosowski, 1992). The cetacean malleus is fused to the tympanic bulla,

producing a sound path from the sound-receiving outer lip of the bulla to the

middle and inner ear (Lancaster et al., 2015) and is significant in preventing the

attenuation of propagating sound frequencies before they reach the cochlea

(Zosuls et al., 2015).

[4] After being transferred via the stapes through the oval window,

vibrations reach the fluid-filled bony labyrinth. A general overview is given here,

and a closer look at properties of the bony labyrinth is provided in the following

Figure 1: Anatomical parts of the cetacean hearing pathway. Areas of interest are highlighted in red. Note the tpc is laterally displaced from the skull. Starting from the skull (top), sound travels through the pan bone (pb) and is channelled towards the middle ear bones and tympanoperiotic complex (tpc; bottom left). The cochlea (co; bottom right) is housed within the periotic (pr; bottom centre), and transforms sound vibrations into electrical signals for the brain. Abbreviations: anterior (ant), fenestra cochleae (fc), lateral (lat), laminar gap (lg), pars cochlearis (pc), posterior (pos), semicircular canals (sc), stapes (st), tympanic bulla (tb), vestibular complex (vc), ventral (ven)


section. Vibrations enter the portion of the bony labyrinth called Rosenthal’s canal

(or the cochlear spiral), where they propagate along the basilar membrane that

supports the organ of Corti. As vibrations follow the cochlear spiral ventrally from

base to apex, the paddle-like basilar membrane increases in width and flexibility,

while thickness decreases (Morell et al., 2015). The rigidity of the basilar

membrane is critical in identifying frequencies (Echteler et al., 1994); higher

frequencies typically stimulate hair cells towards the basal end and lower

frequencies stimulate hair cells closer towards the apex (Hudspeth, 1989).

[5] Finally, vibrations are transduced from mechanical energy into electrical

signals for the brain to process (Fleischer, 1976; Wartzok & Ketten, 1999). This

occurs when soundwaves bend and stimulate sensory hair cells on the organ of

Corti, which then send signals to the auditory nerve leading to the brainstem (von

Békésy, 1960). The spiral ganglion (modiolus) runs up the centre of the cochlear

spiral and generally gets wider as it travels from the ventral apex towards the

dorsal base (Ekdale, 2016). Auditory signals are processed via a feedback loop,

where afferent neurons carry signals to the brainstem, and efferent neurons send

signals back to the ear (Raphael & Altschuler, 2003).

1.3 The Inner Ear in Detail

The bony labyrinth is an intricate space. It is made up of two main parts, the

organ of hearing (cochlea) and the organ of balance (vestibular complex).

Additionally, there are more than 75,000 mechanical and electrical parts that fit

within a single cubic centimetre (Reynolds et al., 2000). Needless to say, the inner

ear is a complex and intricate organ.

In odontocetes, the larger portion of the bony labyrinth is the cochlear

canal. It is a fluid-filled spiral that is divided into three sections: the scala vestibula,

scala tympani, and scala media (Kirk & Gosselin-Ildari, 2009; Slepecky, 1996;

Figure 2). Progressing ventrally from base to apex, the walls separating the

cochlear turns get thinner (Ekdale & Racicot, 2015). In therian mammals, cochlear

coiling likely originated during the Cretaceous period (Manley, 2012), but the

reason behind it is still unclear. Some convincing arguments are that it may have

been a more efficient way to fit a longer cochlea into a smaller space (Meyer, 1907;

Manley, 2016), or that it could function in reducing length differences in acoustic


nerve fibres (West, 1985).

There are two types

of odontocete cochlear

spirals, Type I and Type II,

which have been defined

from properties of the

basilar membrane (Ketten,

2000). The basilar

membrane (Figure 2)

follows the curve of the

cochlea down from base to

apex, almost splitting the

canal in half. Its role is to

provide support for the

organ of Corti, and to adjust

rigidity, so that higher-

frequency sounds can be

received towards the

narrow and stiff base, and

lower frequency sounds

can be received towards

the wide and flexible apex

(Ketten & Wartzok, 1990).

The two different types of

odontocete cochleae that

have been observed are: (Type I) short, thick basilar membranes typical of inshore

or riverine species that detect ultra-high frequencies (over 100kHz), or (Type II)

long, thin basilar membranes typical of lower ultrasonic frequencies (under

100kHz; Ketten, 2000).

The hearing element of the cochlea is the organ of Corti (Figure 2). It rests

on the basilar membrane, which travels the length of the cochlear spiral, and is

made up of both supportive and sensory cells (Echteler et al., 1994). Sensory hair

cells, namely the inner hair cells (IHCs), contribute the large majority of afferent

Figure 2: Diagram showing major elements of the cochlear structure (C hectori- MM2410): (top) Cochlear canal sliced to reveal three fluid-filled compartments, the scala vestibuli (SV), scala media (SM) and scala tympani (ST); (bottom) Magnified region containing primary elements of the organ of hearing. The Organ of Corti is supported by the basilar membrane (BM), and contains sensory inner hair cells (IHC) and outer hair cells (OHC). The tectorial membrane (TM) covers their apical surfaces. Bottom schematic redrawn from Slepecky (1996).


signals to the cochlear nerve fibres (Ryugo, 1992). High densities of nerve cells in

the organ of Corti are throught to result in improved frequency discrimination

(Ekdale, 2015).

It is also important to note that although there is visible asymmetry in

odontocete skulls (Fahlke et al., 2011; Ness, 1967), there has been no significant

variation reported between left and right bony labyrinths. Ear symmetry has been

reported in cetacean research (Ary et al., 2016; Schnitzler et al., 2017), as well as in

studies on various other mammals (Mennecart & Costeur, 2016; Welker et al.,

2009).

1.3.1 Cochlear Canal and Factors that Impact Hearing

The proportions of the cochlea are thought to have a large influence on

hearing bandwidth and sensitivity (Ekdale, 2015; Greenwood, 1990).

Measurements such as volume, length, width, height to diameter ratio, number of

turns and graded curvature of the cochlear spiral are believed to correlate with

hearing ability and frequency discrimination (Fleischer, 1976; Kirk & Gosselin-

Ildari, 2009; Manoussaki et al., 2008; West, 1985; Wever et al., 1971). In general, a

shorter length and a low height to diameter ratio aids in detecting high

frequencies, and vice versa for low frequencies. Furthermore, the lower limits of

hearing are extended by an increase in the number of turns and a higher grade of

curvature, while higher limits are inversely correlated to volume.

As described earlier, the basilar membrane is an important structure, and

commonly used by researchers for deducing frequency sensitivities. West (1985)

established that there is a negative correlation between basilar membrane length

and the detection of frequencies, meaning that shorter membranes tend to be

more attuned to higher frequencies and longer membranes are more attuned to

lower frequencies. It is also believed that odontocetes with narrower basilar

membranes are higher frequency specialists (Reysenbach De Haan, 1957; Wever et

al., 1971). However, we need to be careful when interpreting results from basilar

membrane measurements because cetaceans do not necessarily follow a linear

cochlear frequency-place map, as most terrestrial mammals do. Terrestrial

mammals are predominantly considered hearing generalists, with frequency

sensitivity evenly distributed along the organ of Corti (Echteler et al., 1994;


Manley, 1972). Odontocetes on the other hand are often hearing specialists, with

tightly focused frequency sensitivity along certain regions of the organ of Corti

(Echteler et al., 1994; Parks et al., 2007).

1.3.2 Vestibular Complex

In terrestrial mammals, the vestibular complex functions in detecting

accelerations of the body and angular movements of the head relative to the force

of gravity (Angelaki & Cullen, 2008; Cawthorne et al., 1956). It is made up of the

utricle, saccule and three semi-circular canals (i.e. lateral canal, anterior canal and

posterior canal). It is an especially interesting part of the ear complex because, by

comparison, the size of the cetacean vestibular complex is about three times

smaller than those of mammals with the same body mass (Fleischer, 1976; Spoor

et al., 2002; Yamada & Yoshizaki, 1959). Generally, mammalian canals are more

sensitive to head movements when two semi-circular canals approach angles close

to 90 degrees (Berlin et al., 2013; Malinzak et al., 2012). In humans, there is little

change in the shape of the vestibular complex once it reaches an adult size, but

there are small changes in orientation relative to the cochlea, and the semi-circular

canals keep opening laterally until complete ossification of the compartment (

(Jeffery & Spoor, 2004; Spoor, 1993). Interestingly, the lateral canal seems to

develop at a slower rate than the other semi-circular canals (Rinkwitz et al., 2001).

Current knowledge of the vestibular complex in cetacean species remains limited.

1.4 Significance of Age and Ecology

Age- and ecology-dependent structural changes could affect a cetacean’s

ability to detect certain frequencies. Ketten (1997) suggested that body size,

behaviour and habitat could influence auditory anatomy, and thereby create

variation in hearing ability. Cetaceans are distinctive amongst mammals in that

their environment requires them to be precocious in water; they must swim

independently and surface immediately after birth (Dearolf et al., 2000;

Rauschmann et al., 2006). Additionally, hearing is a vital part of odontocete

ecology, playing a central role in navigating the environment, identifying and

communicating with conspecifics and mother-calf imprinting (Fripp & Tyack,


2008; King et al., 2016; Mooney et al., 2012), all of which are critical from a very

early age.

1.4.1 Age

Even without these unique pressures for aquatic precocial development,

many terrestrial mammals display a pattern of advanced ear development. Rabbits

have been found to have fully grown bony labyrinths at birth, with tympanic bullae

that increase in width for the following 22 days (Hoyte, 1961). Humans have a

bony labyrinth that reaches an adult size and shape well before birth and before

ossification approximately halfway through gestation (17-19 weeks; Bast, 1930;

Jeffery & Spoor, 2004; Spector & Ge, 1993). The development of the cochlea

progresses from base to apex, and it is suspected that cochlear function begins

with the opening of the organ of Corti around gestational week 20 (Pujol et al.,

1991). Humans have been documented to be functionally capable of hearing for at

least two and a half to three months before birth (Pujol et al., 1991).

Ruminants, which are close terrestrial relatives to cetaceans belonging to

the clade Cetartiodactyla, have similar patterns in their hearing complexes. The

bony labyrinth has been shown to ossify in the fourth gestational month and

reaches an adult size around that same mid-gestational timeframe (Costeur et al.,

2017; Mennecart & Costeur, 2016). Around mid-gestation, the total volume of bony

labyrinths is similar to that of adults, and there is even evidence to suggest that

they are slightly larger than the adult counterparts (Mennecart & Costeur, 2016).

However, there are still some uncertainties over observed variability in semi-

circular canals and slower development of open features like the cochlear

aqueduct of ruminants.

Hypothesized first by Perrin (1975), recent research on cetaceans has

indicated a very similar pattern of precocial hearing development to what we

observe in other mammals. The tympanoperiotic complex is undeniably the

densest part of a newborn odontocete skeleton, and changes little in size from

juvenile to adult (Cozzi et al., 2012; Kasuya, 1973; Lancaster et al., 2015). Middle

earbones are also known to rotate into their adult position during early stages of

foetal development (Reidenberg & Laitman, 2009). A recently published study has

given us a particularly interesting insight into the development of the cetacean


bony labyrinth. Thean et al. (2016) found that cetacean inner ears may ossify and

reach adult size as early as 32% through the gestational period, and as a whole had

earlier precocial inner ear development when compared against suid artiodactyls.

Contrary to other mammalian studies, ossification of the cetacean bony labyrinth

seems to begin before the labyrinth reaches an adult size. In addition, the study

found that the earliest embryos displayed a more flattened cochlear spiral,

compared to later stages and adult counterparts, although the implications are

unknown. There is the potential for small age-related differences in the bony

labyrinth, but ecological factors have been known to influence structure and

orientation of the bony labyrinth as well.

Almost half the world’s cetacean species can be sighted in New Zealand

waters. With eight species of mysticetes and thirty species of odontocetes, there is

more variety here than almost any other single region in the world (Baker, 1990;

Baker & van Helden, 1999). Odontocetes are heavily reliant on their hearing as a

means to navigate, hunt for prey, and communicate with others (Richardson et al.,

2013; Tyack, 2008). With the exception of Gray’s beaked whale, all the species

included in this study are dolphins from the Family Delphinidae, and all have

different ecologies. Due to the potential for ecological factors to influence hearing

ability (Ketten, 1997), below I will introduce our species of interest and summarise

their hearing limit and ecology.

1.4.2 Ecology

Hector’s dolphins

Hector’s dolphins (Cephalorhynchus hectori) also known as NZ dolphins, are

probably the most iconic cetacean species in New Zealand, and are well known for

their ‘Endangered’ classification (Reeves et al., 2013). The species comprises two

subspecies. Cephalorhynchus hectori maui is found only off the North Island west

coast, numbers around 51 individuals (Baker et al., 2016), and is considered

Critically Endangered. The South Island subspecies, Cephalorhynchus hectori

hectori numbers at least 7000 (Slooten et al., 2004). The subspecies are visually

identical. Hector’s dolphins tend to aggregate in pods of 2-8 individuals in shallow

turbid waters, often within eight kilometres of the coast (Dawson, 2009). Adult

individuals usually have a body length within the range of 117-145cm with a


weight of about 50 kilograms (Dawson & Slooten, 1988; Folkens & Randall, 2002).

They also prey on a variety of benthic and pelagic fish and cephalopods (Miller et

al., 2013). Hector’s dolphins can be quite vocal, with high frequency clicks around

125 kHz (Dawson & Thorpe, 1990).

Pilot whales

There are two species of pilot whales, both of which reach a length of about

six meters (Olson, 2009; Wilson & Mittermeier, 2014). The short-finned pilot

whales (Globicephala macrohynchus) favour warmer waters, while the long-finned

pilot whales (Globicephala melas) favour cooler temperate waters (Olson, 2009).

This study collected data from the latter. Pilot whales are highly social and

cohesive with their pod, usually consisting of about 20-100 individuals (Wilson &

Mittermeier, 2014). This cohesive nature also means that they are prone to mass

stranding (Kritzler, 1952; Perrin & Geraci, 2002). Generally, they live offshore and

deep-dive to feed on squid nocturnally (Baird et al., 2002). Sometimes these deep

dives can separate group members by hundreds of meters, requiring vocalisations

for the whales to relocate one another (Visser et al., 2016). Adults hear best at

frequencies ranging from 11-50kHz (Pacini et al., 2010). There is insufficient data

to assess the species’ conservation status, and it remains in the IUCN “Data

Deficient” category (Taylor et al., 2008).

Gray’s beaked whales

Gray’s beaked whales (Mesoplodon grayi) were recently reassessed and

classified as IUCN ‘Not Threatened’ due to the large number of records and high

genetic diversity (Thompson et al., 2013). Beaked whales in general tend to be

difficult to sight in the wild (Barlow, 1999) and most of our current knowledge has

been compiled from stranding records. They live offshore in waters deeper than

200m and in pods of five or less individuals; they are evasive of boat activity and

dive for extended periods of time (Zimmer et al., 2008). Despite this, Gray’s beaked

whales are still the most common beaked whales to strand in New Zealand (Baker

& van Helden, 1999), and have mostly been found in areas with deep, cold waters

south of 30°S (Folkens & Randall, 2002). Gray’s beaked whales feed on deep-water

fish and cephalopods, foraging in depths that reach 500 meters or more (MacLeod


et al., 2003; Pitman, 2009; Wilson & Mittermeier, 2014). Adults reach a body

length of over five meters (Baker & van Helden, 1999), and although audiograms

are yet reported, we know that close relatives like Blainville’s beaked whales

(Mesoplodon densirostris) make sounds around 30-50kHz (Johnson et al., 2004).

Bottlenose dolphins

The decision to collect data on bottlenose dolphins (Tursiops truncatus) was

largely because material is common in collections and there is an excellent

literature base. Although the number of species is currently controversial, the

genus is comprehensively studied and has a world-wide distribution of both

nearshore and offshore populations (Hoelzel et al., 1998). Presence of both

populations is unconfirmed in New Zealand but there have been occasional

sightings of non-local bottlenose dolphins in addition to the more common coastal

populations (Constantine, 2002). On average, groups contain 15 individuals and

adults reach a length of about 235-270cm (Sergeant, 1973; Wursig & Wursig,

1977). In general, coastal populations spend the majority of their time in waters

less than ten meters in depth (Wursig & Wursig, 1979) and have displayed both

cooperative and individual feeding behaviours in shallow waters (Leatherwood,

1975). Bottlenose dolphins feed on a variety of fish and squid (Wilson &

Mittermeier, 2014). They also develop their own vocal signatures in the first few

months after birth, which helps facilitate mother-offspring recognition and

reunion (Sayigh et al., 1990; Smolker et al., 1993; Tyack, 1997). Bottlenose

dolphins have a rich repertoire of sounds within the human hearing range (i.e.

<20kHz), and make broadband echolocation clicks extending to beyond 120kHz

(Au, 2009; Au et al., 1974). Their hearing is better studied than that of any cetacean

and shows good sensitivity to about 140kHz (e.g. Au et al., 2002; Johnson, 1967)

1.5 Study Aims: Is Hearing Fully Developed at Birth?

The aim of this study is to identify the degree to which odontocete hearing

structures are developed at birth. By tracing back the origins of principal

structures we can identify which parts develop first and are therefore fundamental

towards hearing ability. Even though there are many reasons why one might

assume that hearing is fully developed prenatally, few studies have investigated


the ontogeny of key hearing-dependent structures such as the cochlea. Even fewer

studies have been able to analyse multiple samples from the same species.

In this study I have analysed a range of different-aged individuals from four

different species local to New Zealand waters. Samples from multiple species give

the opportunity to identify whether patterns of ear development are taxonomic

(reflecting phylogeny), or ecological, or both. Through the use of micro-CT, I take a

closer look at the inner ear, and by utilising geometric morphometrics, I can gain

more robust and accurate results for reliable interpretation.

As discussed above, precocial ear development is reported in various

mammalian species, and some recent studies (Lancaster et al., 2015; Thean et al.,

2016) propose the same in odontocetes. Additionally, newborn cetaceans are

required to be precocious in many other ways in order to survive as an air-

breather in an oceanic environment. Evidence generally suggests that odontocetes

do indeed develop their hearing structures early in life, I seek to evaluate this

suggestion, elucidate the timing of development, and investigate if any components

of the cochlea take longer to develop than others.

As we improve our knowledge of the cochlea, we may be able to use its

structure with more confidence as a proxy for hearing ability. In turn, this would

make it easier to learn about rare or extinct species, for which there are no means

of using auditory evoked potentials. The hard tissues of the ossified bony labyrinth

have a direct structural relationship with the soft tissue. Although the hard tissue

of the bony labyrinth cannot represent the cochlear and vestibular complexes

exactly, it does show a maximum size and accurate shape of the soft tissue

structures it contained (Spoor et al., 1994; Walsh et al., 2009). The structure of the

basilar membrane, in particular, has been critical in deducing hearing ability

(Echteler et al., 1994; Ketten & Wartzok, 1990). Further, the membrane can be

measured by the impression it leaves in the bony labyrinth, as a laminar gap.

Palaeontologists increasingly use features of the middle and inner ear to

reconstruct the phylogenies of extinct species (e.g. Benoit et al., 2015; Tanaka &

Fordyce, 2016; Tsai & Fordyce, 2016). Earbones are extremely dense and can

therefore be more likely to survive burial and fossilisation than other parts of a

skeleton, which makes them an especially useful tool.


Our understanding of the structural aspects of odontocete hearing is

steadily improving due to the relatively recent utilisation of non-destructive

micro-CT and development of more robust analyses. The key steps now are to

identify factors that affect how sound is processed and transduced internally, and

to ascertain how these factors develop and affect hearing ability in ontogeny. My

hope is for this study to build a better understanding of the development of

hearing in modern odontocetes, perhaps ultimately helping to provide insight into

mass strandings or the effects of widespread oceanic noise pollution.

Chapter 2: Methods| 17

2 Chapter 2: Methods

2.1 Sources of Material

When studying cetacean morphology, there is a heavy reliance on the

availability and accessibility to stranding material for research. This makes it

difficult to obtain large sample sizes and even more uncommon to find and use

material from early age classes like neonates and foetuses. For this project, skull

and tympanoperiotic complex (earbone) measurements were collected from two

primary sources (Table 1 and 2). Most material came from the National Museum of

New Zealand Te Papa Tongarewa in Wellington. Additional contributions were

from a collection created and maintained by Te Upokorehe iwi, tāngata whenua

tūturu of the Ōhiwa Harbour in the Bay of Plenty region.

Together with Te Upokorehe and under a Memorandum of Understanding

(MOU; Appendix A), we established a novel research partnership to support

scientific engagement in the community and provide access to rare material for

this study. Material came from a stranding involving over 40 long-finned pilot

whales at the entrance of Ōhiwa Harbour in November of 2014. Under the

guidance of well-known tohunga tohorā (cetologist) Ramari Stewart, forty skulls

were collected and prepared by Te Upokorehe iwi to create a unique and well-

preserved community collection.

For my study, standard external measurements were taken from the skulls

and earbones (tympanoperiotic complexes; TPCs) of 60 individuals (15 individuals

per study species). Due to the cost of micro-CT and some incomplete specimens,

only earbones from the right side of each individual were included in this study.

Recent study of labyrinth symmetry has found however, that mammals (including

cetaceans) have no significant variations between the left and right bony

labyrinths (Ary et al., 2016; Mennecart & Costeur, 2016; Schnitzler et al., 2017;

Welker et al., 2009). Species of odontocetes used in this study are long-finned pilot

whale (Globicephala melas), bottlenose dolphin (Tursiops truncatus), Gray’s beaked

whale (Mesoplodon grayi), and Hector’s dolphin (Cephalorhynchus hectori).


Table 1: List of all skulls and TPC material used with the source and type of analysis conducted. Four species of odontocete total 61 individuals, and 14 specimens were additionally scanned with Micro CT. Abbreviations: Average mandible length (AML), condylobasal length (CBL), Museum of New Zealand Te Papa Tongarewa (MoNZ), total body length (TBL).

Location Species ID Number Analysis Age Class CBL (mm) AML (mm)

MoNZ Cephalorhynchus hectori MM001615 External Measurements, Adult 305 243 MoNZ Cephalorhynchus hectori MM002007 External Measurements, Micro CT Juvenile 259 203 MoNZ Cephalorhynchus hectori MM002014 External Measurements Neonate - 162 MoNZ Cephalorhynchus hectori MM002019 External Measurements Adult 284 230 MoNZ Cephalorhynchus hectori MM002067 External Measurements Neonate - 156 MoNZ Cephalorhynchus hectori MM002282 External Measurements Juvenile 264 210 MoNZ Cephalorhynchus hectori MM002286 External Measurements Adult 279 224 MoNZ Cephalorhynchus hectori MM002290 External Measurements Adult 277 220 MoNZ Cephalorhynchus hectori MM002410 External Measurements, Micro CT Adult 298 247 MoNZ Cephalorhynchus hectori MM002834 External Measurements Neonate 249 198 MoNZ Cephalorhynchus hectori MM002838 External Measurements Neonate - 190 MoNZ Cephalorhynchus hectori MM002839 External Measurements, Micro CT Neonate - 150 MoNZ Cephalorhynchus hectori MM002842 External Measurements Adult 294 216 MoNZ Cephalorhynchus hectori MM002846 External Measurements Juvenile 271 213 MoNZ Cephalorhynchus hectori MM002863 External Measurements Neonate 305 146 MoNZ Globicephala melas MM001840 External Measurements, Micro CT Neonate - 250 MoNZ Globicephala melas MM001848 External Measurements, Micro CT Juvenile 529 419 MoNZ Globicephala melas TMP25921 External Measurements, Micro CT Adult 619 495 Upokorehe Globicephala melas T1W2 External Measurements Adult 710 570 Upokorehe Globicephala melas T2W4 External Measurements Adult 657 535 Upokorehe Globicephala melas T3W1 External Measurements Adult 679 546 Upokorehe Globicephala melas T4W1 External Measurements Adult 695 550 Upokorehe Globicephala melas T4W2 External Measurements Adult 643 517 Upokorehe Globicephala melas T6W5 External Measurements Adult 605 485 Upokorehe Globicephala melas T7W1 External Measurements Adult 587 465 Upokorehe Globicephala melas T8W3 External Measurements Adult 568 451 Upokorehe Globicephala melas T8W4 External Measurements Adult 537 424 Upokorehe Globicephala melas T9W2 External Measurements Neonate 444 343 Upokorehe Globicephala melas T9W3 External Measurements Neonate 455 356 Upokorehe Globicephala melas T9W4 External Measurements Neonate 441 348 Upokorehe Globicephala melas T9W5 External Measurements, Micro CT Foetus - 85


Location Species ID Number Analysis Age Class CBL (mm) AML (mm) MoNZ Mesoplodon grayi MM002130 External Measurements Adult 722 636 MoNZ Mesoplodon grayi MM002234 External Measurements Adult 607 527 MoNZ Mesoplodon grayi MM002610 External Measurements Adult 613 515 MoNZ Mesoplodon grayi MM002624 External Measurements, Micro CT Adult 882 778 MoNZ Mesoplodon grayi MM002626 External Measurements Adult 711 631 MoNZ Mesoplodon grayi MM002890/1 External Measurements Neonate 444 379 MoNZ Mesoplodon grayi MM002891 External Measurements, Micro CT Neonate - 335 MoNZ Mesoplodon grayi MM002896 External Measurements Adult 818 723 MoNZ Mesoplodon grayi MM002935 External Measurements, Micro CT Juvenile 485 409 MoNZ Mesoplodon grayi MM002938/1 External Measurements Adult 724 623 MoNZ Mesoplodon grayi TMP013512 External Measurements Neonate 383 338 MoNZ Mesoplodon grayi TMP025923 External Measurements Juvenile 511 416 MoNZ Mesoplodon grayi TMP025924 External Measurements Adult 881 771 MoNZ Mesoplodon grayi TMP025925 External Measurements Neonate 484 401 MoNZ Mesoplodon grayi TMP025926 External Measurements Juvenile 487 430 MoNZ Tursiops truncatus MM000688 External Measurements Neonate 331 268 MoNZ Tursiops truncatus MM001872 External Measurements Juvenile 459 390 MoNZ Tursiops truncatus MM002867 External Measurements Adult 525 440 MoNZ Tursiops truncatus MM002868 External Measurements Adult 554 487 MoNZ Tursiops truncatus MM002974 External Measurements Adult 559 485 MoNZ Tursiops truncatus MM002975 External Measurements Adult 543 470 MoNZ Tursiops truncatus MM002979 External Measurements Adult 531 418 MoNZ Tursiops truncatus MM002980 External Measurements Adult 554 476 MoNZ Tursiops truncatus MM002981 External Measurements Adult 543 477 MoNZ Tursiops truncatus MM002982 External Measurements, Micro CT Juvenile 421 358 MoNZ Tursiops truncatus MM002983 External Measurements, Micro CT Neonate - 251 MoNZ Tursiops truncatus MM002984 External Measurements, Micro CT Adult 561 476 MoNZ Tursiops truncatus MM002985 External Measurements Adult 509 432 MoNZ Tursiops truncatus MM002986 External Measurements Adult 547 477 MoNZ Tursiops truncatus TMP012839 External Measurements Juvenile 426 340

Chapter 2: Methods 20

Table 2: List of all radiographed foetal material. Two species of odontocete total 6 individuals. Foetal stages follow criteria from Štěrba et al. (2000). Abbreviations: Institute for Applied Ecology New Zealand of Auckland University of Technology (AUT), Museum of New Zealand Te Papa Tongarewa (MoNZ), total body length (TBL).

Location Species Type ID Number Analysis Stage TBL (cm)

AUT Globicephala melas Whole Frozen GM230a X-ray 11 42.5 AUT Globicephala melas Whole Frozen GM247a X-ray 11 47 AUT Globicephala melas Whole Fixed GM254a X-ray 7 17 AUT Globicephala melas Whole Fixed GM274a X-ray 12 28.6 AUT Globicephala melas Whole Frozen GM406a X-ray 11 69 MoNZ Mesoplodon grayi Whole Fixed MM002875 X-ray 8 18.5

Further odontocete foetuses were obtained from Massey University in

Auckland and the Museum of New Zealand, Te Papa Tongarewa. Since specimens were

still whole, I chose to maintain the integrity of the material and avoid destructive

sampling by using radiography to identify if ossification was present before further

investigation. Three frozen and two fixed pilot whales were supplied from Massey

University, and one fixed Gray’s beaked whale was supplied from Museum of New

Zealand, Te Papa Tongarewa. Radiography was completed in the Dunedin Hospital’s

Radiology Department, and images were stored as DICOM files on CD-ROMs provided

by the hospital.

2.2 Aging and Staging

There are several methods for determining age in odontocetes, and one of the

most common, primarily in adults, is tooth sectioning. Cetacean teeth have growth

bands called Growth Layer Groups (GLGs), which can be counted to determine the age

of an individual in years (Hohn et al., 1989). Teeth were sent to the University of

Otago’s Histology Services Unit within the Department of Pathology, where they were

decalcified, sliced at 4m and stained with a standard Toluidine Blue. Unfortunately,

in this case, sections did not produce well-defined GLGs clear enough to determine

accurate ages.

If GLGs are too unclear, or specimens are too young to have GLGs, other

methods may be used to approximate age instead. The amount of closure and fusion in


the sutures of a skull (Jordan et al., 2015; Perrin, 1975; Figure 3), consideration of

skull size and closure of pulp cavities in the teeth can also indicate age class. As an

animal ages the sutures between its skull bones close and eventually fuse, creating a

strong seam. Therefore, open sutures can indicate neonates and young juveniles.

Additionally, because tooth pulp cavities build layers over the course of years, adults

tend to have full and dense pulp cavities, whereas young animals have thin and hollow

teeth (Besharse, 1971; Kasuya & Marsh, 1984). For this study, I relied on the above

methods to establish age classes: neonate, juvenile and adult. Individuals included in

the foetal age class were identified by total body length and stranding record

information. Staging of foetuses followed criteria from Štěrba et al. (2000).

2.3 Skull and TPC Measurements and Analysis

2.3.1 Measurements

The main focus of this study, to analyse differences in the inner ear, was

limited by the cost of micro-CT per specimen. Therefore, along with the standard skull

measurements, I also collected measurements from the tympanoperiotic complexes

(TPCs) to analyse earbone growth from external dimensions and make the overall

Figure 3: Four age classes represented by Globicephala melas specimens: Foetus (T9W5), Neonate (T9W3), Juvenile (MM1848), Adult (TMP25921). Photo credit for the foetus and neonate: Shaun Wilson.


study more robust. Measurements were taken from 15 individuals per species, plus an

additional foetal specimen for Globicephala melas. TPCs

for later micro-CT scanning were chosen from these same individuals.

Standard measurements followed those of Kasuya (1973) for the skulls and those of

Gutstein et al. (2014) for the TPCs (Figure 4, Table 3). Skulls were placed on a flat

surface with the rostrum propped upwards to lie parallel with the table surface.

Measurements were taken to the nearest millimetre using standard callipers, straight

edges and rulers. Skulls and TPCs have many rounded and irregular surfaces that

make accurate measurements difficult. To increase the accuracy of my data, I collected

the measurements of each feature on three separate occasions and took the average.

2.3.2 Analysis

Traditionally, the most widely used age-related dimensions have been condylobasal

length (CBL) and bizygomatic width (See Figure 4 and Table 3; Perrin, 1975; Pyenson

Figure 4: External measurements displayed with an adult Globicephala melas (OU22805): (A) Skull and mandible, (B) Top: Left tympanoperiotic complex resting on the ventral surface of the tympanic bulla; Bottom: Left isolated periotic resting on its dorsal surface. Measurements 2 and 4 are transverse. Refer to Table 1 for measurement descriptions.

A B


& Sponberg, 2011). However, young individuals have unfused skulls that are often in

disarticulated pieces, which makes traditional age-related dimensions difficult. To

analyse measurements across all age classes, I decided to use the readily available

average mandible length (AML) as a proxy for age in this study.

Nevertheless, it is important to express caution when interpreting results from

mandible growth, as feeding apparatus development is known to show allometric

growth that follows cranial length in cetaceans (Perrin, 1975).

Using the Akaike information criterion (AIC) I chose to run a Generalised Least

Squares Model (GLS) with residuals weighted by species and measurement name.

Weighted measurement names allow for correlation caused by using the same set of

bones for each measured feature. To compare between species, I also standardised the

x and y-axes by making each value a percentage of the species’ maximum value for

that measurement. Simply put, both axes represent a percentage of maximum size.

The percentage of maximum mandible size and proxy for age is on the x-axis, and the

percentage of maximum size for each TPC feature is expressed on the y-axis. This is

done because the size range of each measurement differs between each species and

measurement type.

Table 3: List of external measurements taken from 15 individuals in each of the four species. Measurements follow those of Kasuya (1973) and Gutstein et al. (2014).

Measurement # Description 1 Skull condylobasal length (CBL) 2 Skull bizygomatic width 3 Skull height 4 Rostrum width (left to right antorbital notch) 5 Rostrum length 6 Mandible length (left and right) 7 Mandible width (left and right) 8 Length of tympanic bulla 9 Maximum width of tympanic bulla 10 Width of internal acoustic meatus (IAM) 11 Length of internal acoustic meatus (IAM) 12 Length of periotic 13 Length of the pars cochlearis from anteriormost point to cochlear window 14 Width of the pars cochlearis (medio laterally) 15 Cochlear window diameter (medio laterally) 16 Width of the fossa for the stapedial muscle


2.4 Building 3D Cochlear Models

2.4.1 Micro CT Scanning

Thirteen TPCs were scanned with micro Computed Tomography (micro-CT):

one neonate, one juvenile and one adult sample from each of the four target species,

plus an additional foetal TPC from Globicephala melas. Scans were run using a SkyScan

1172 model (Control Program v. 1.5, build 21A from Bruker-MicroCT). The machine

uses X-rays from different angles to gather a set of 2D density image slices, which are

later stacked to form a 3D model. Samples were limited to a 60 x 60 mm cube and

mounted securely with radiotransparent material (If not mounted properly, slight

rotation wobbles can show up as artefacts in the scan and disrupt image clarity.).

Scanning time varied depending on the size of the specimen and the resolution

required. This study used medium resolution for all micro-CT scans. For more

information on the micro-CT process see Abel et al. (2012).

2.4.2 3D Modeling Software

Micro-CT images were processed to generate 3D models of the inner ear using

a combination of freeware techniques. To simplify and keep the methods clear I have

divided the of 3D model preparation into steps, with a list of the tools used in each

software program (Table 4). It was important reduce noise left by the micro-CT scan

and to reduce file sizes so that final models could be properly measured and analysed

without crashing the computer. Careful processing was essential to avoid

compromising the true structure of the inner ear.

Step 1: Reconstruction (NRecon)

Residual noise can be minimised by reconstructing the sample’s image series.

NRecon Reconstruction Software (v. 1.6.10.2) adjusts factors like beam hardening (the

reflection of photons from the object surface) and ring artefacts (rings that are created

from the rotation of the object during the scan) that may have impaired the final


image. After settings were adjusted appropriately, the programme was run to export a

new clearer (.tif) image series.

Step 2: Isolate Region of Interest (CTan)

CTanalyser (v.1.14.41; CTan) was used to isolate the cochlear canal located

within the scanned TPC. The top and bottom of the image series were selected and

extra slices that did not contain the cochlear were cut to reduce file size. I used the

following task list to isolate the cochlear canal and create a binary .tif image series

output file: Reload Thresholding Morphological Operations Despeckle

Bitwise Operations Reload Thresholding Bitwise Operations Save Bitmaps.

Step 3: Scale and Finalise (Fiji, Meshlab, Meshmixer)

The final steps in producing each 3D model make the model more presentable

and easier to measure during analysis. It is essential to scale the file by converting

pixels to millimetres for later measurements. When finished, save as an .obj file. Fiji

6.2 (Schindelin et al., 2012) was used to scale and reduce excess file size and noise.

Meshlab (v.1.3.3)(Cignoni et al., 2008) was used to invert the model surface and

export an .obj file. Meshmixer (v.3.1.377)(Schmidt & Singh, 2010) was used to remove

and smooth out any of the remaining noise.


Table 4: List of software and tools used create 3D cochlear models

Software Tool Description/Command String

Step 1:

Reconstruction NRecon

Smoothing Pixels in the image series are smoothed

Ring Artifacts

Reduction

Corrects for rings artifacts that develop when

the object is rotated during the scan.

Beam-

Hardening

Reduction

Corrects for beam-hardening that occurs when

x-rays are reflected off the surface of the

object.

Output Set destination of .tif image series output file

Step 2:

Region of

Interest

CTanalyser

(CTan)

Reload Loads fresh data series from the micro-CT scan

Thresholding Segmentation process that establishes a cut-off

value along the grayscale. Depending on this

value, the pixels in the image will be included

as bone or excluded as empty space, thereby

turning the dataset into a simple binary black

and white configuration.

Morphological

Operations

Establishes the Region of Interest (ROI) using a

series of shapes and/or drawing tools. Since

the cochlear canal is not an entirely enclosed

space (there are openings for the cochlear

window, and vessels), the ROI border for each

image was manually adjusted to close it off.

Despeckle Removes obvious artefacts or ‘speckles’ from

the images

Bitwise

Operations

Isolates the ROI from the rest of the image

through the operation “Image= ROI – Image”

Save Bitmaps “Apply to the image inside the ROI” and save as

a discrete data series file

Step 3:

Scale and

Finalise

Fiji

Set Scale Under the ‘Analyze’ tab, click ‘Set Scale’. The

parameters of these scans are equivalent to

0.01734mm for every one pixel and there is an

aspect ratio of one. These details are recorded

in the information log that comes with the

micro-CT image stack.


Step 3:

(Continued)

Fiji

(Continued)

Crop Draw a box around the area of interest and

under ‘Image’ click ‘Crop’. This reduces the file

size by removing surrounding pixels that don’t

contribute towards the final model.

Remove

Outliers

Under the ‘Process’ tab, click ‘Noise’ and then

‘Remove Outliers’. Repeat for both ‘Bright’ and

‘Dark’ at 10 pixels. This removes most noise

around the model and fills small holes.

Save Open ‘Plugins’, click ‘3D Viewer’. Display as a

surface by clicking on the ‘Edit’ tab. Then save

as a waveform/object file (.obj).

Meshlab

Invert Under ‘Filters’ and ‘Normals, Curves and

Orientation’, click ‘Invert faces orientation’.

This makes sure that the triangles making up

the model surface face outwards.

Meshmixer

Shell Under the Edit tab, click ‘Separate Shells’ and

delete everything in the list except for the first

item, which is the core object model. This

function deletes volumes that are not

connected to the main body of the model.

Make Solid Under the Edit tab, click ‘Make Solid’ and adjust

conditions to 128 and an ‘accurate’ type. This

minimizes file size while still keeping an

appropriate resolution.

Select Any major lumps or unwanted parts of the

bony labyrinth (e.g. nerve canal) that don’t

belong in the final model were removed by

selecting ‘Erase and Fill’ or ‘Remove’. The

‘Inspector’ tool under the ‘Select’ button was

also useful for filling any breaks in the surface

mesh.

Sculpt The robust smooth brush was used to target

and reduce any leftover noise.


2.5 Inner Ear Measurements and Analyses

2.5.1 Measurements

Linear measurements of the

cochlear spiral (Table 5) were taken

from 3D models (Figure 5) to the closest

tenth of a millimetre using tools from

Meshmixer (v.3.1.377) and Avizo (v.

9.3.0). There are no readily available

tools for measuring curves, so I hand-

placed a line of 40 points down the

midline of the cochlear lumen, following

the bony laminae for reference. Cochlear

length was measured by resampling this

40-point line into a 100 point equally

spaced line who’s length was calculated

with MATLAB (v. 9.2). Following the

methods of Geisler & Luo (1996), the

number of turns were counted using a

reference line that reached from the

Table 5: List of inner ear measurements and calculations taken from cochlear spiral 3D-models. Measurements 1-16, for the skull and tympanoperiotic complex, are in Table 3.

Measurement # Description

17 Number of turns 18 Cochlear width (wt) 19 Cochlear height (ht) 20 Cochlear length following the midline of the lumen (cl) 21 Base radius (br) 22 Apex radius (ar) 23 Axial pitch (cochlear height/ number of turns) 24 Radii ratio (base radius/apex radius) 25 Arch diameter of the posterior semicircular canal 26 Arch diameter of the anterior semicircular canal 27 Arch diameter of the lateral semicircular canal

Figure 5: Inner ear measurements involving the cochlear canal, using an adult Cephalorhynchus hectori (MM2410): apex radius (ar), base radius (br), canal length (cl), cochlear height (ht), cochlear width (wt). Dimensions follow Ekdale (2013).


laminar gap at the base of the first turn to the centre of apex coiling. Turns were

rounded to the closest eighth of a turn. All measurements were taken three times and

then averaged for a more accurate value.

2.5.2 Inner Ear Analyses

Two forms of analysis were used for the inner ear:

1) a geometric morphometric shape analysis using structural landmarks

to interpret general morphology (Adams et al., 2013; Zelditch et al.,

2012), and

2) plots for standard cochlear dimensions.

Line plots were used to observe potential patterns in cochlear measurements

because there were not enough samples available to run a statistical analysis

confidently. These preliminary plots can be used to identify areas of interest that are

worth investigating in future research.

1. Geometric Morphometrics

Geometric morphometric analysis relies on a common coordinate system to

find a statistically optimum placement in space for multiple superimposed 2D or 3D

objects (Corti, 1993; Rohlf, 1999; Slice, 2001). Each 3D model requires the placement

of fixed landmarks, which are placed at distinguishable homologous points, and semi-

landmarks that are placed equidistantly along the model. Landmark and semi-

landmark coordinate sets are then superimposed through a General Procrustes

Analysis, which allows for complex shape analysis with a choice of statistical tests.

Step 1: Landmarking

Three sets of coordinates were collected from each 3D model using Avizo:

1) semi-landmarks for the cochlear midline

2) semi-landmarks for the semicircular canal midlines, and

3) landmarks for distinctive locations on the cochlear model that can be

easily identified between specimens.


1

2

Figure 6: Diagram of landmarks (1-10) and semi-landmarks used for shape analysis. Landmarks are shown as yellow squares, and the two cochlear and vestibular splines are represented through an orange curve and spherical points. (See Table 6 for landmark position descriptions)


Unforeseen compatibility issues with my 3D models restricted me from the

Avizo tools: ‘Measure’, ‘Slice’ and ‘Spline Probe’, used in landmarking. Similar Avizo

tools (‘B-Spline’ and ‘Landmark’) however, allowed me to follow essentially the same

landmark-sampling methodologies from Ekdale (2016).

Modules ‘B-Spline’ and ‘Landmark’ are tools used to create lists of 3D-

coordinates for each inner ear model (Figure 6). Following the same method

described previously to measure cochlear length, points were hand-placed along the

midline of each lumen forming a spline (i.e. curve formed by joining designated

points). The cochlear splines and vestibular splines were then resampled in Matlab (v.

9.2) to create 100 equally spaced curve coordinates for the cochlea and 60 for the

vestibular complex. Additionally, landmarks were hand-placed at homologous

locations on the surface of the 3D model (Table 6). These mark key locations that

assist with superimposition in the next step.

Step 2: Generalized Procrustes Analysis (GPA)

Shape-analysis for all coordinate sets were run using the Geomorph package

(Adams & Otárola-Castillo, 2013; Adams et al., 2017) in R-Studio (v. 1.0.136). A

Generalized Procrustes Analysis (GPA), superimposed each 3D model’s landmarks and

semi-landmarks to a common coordinate system, minimizing residuals from rotation,

translation, and scaling to form the most ideal shape-space (Kendall's shape-space;

Gower, 1975; Rohlf & Slice, 1990). By taking this ‘mean’ shape and running it through

additional statistical analyses as routinely done in other work (e.g. Ekdale, 2016;

Table 6: List of bony labyrinth fixed landmarks. See Figure 6 for visual placement.

Landmark # Placement Description 1 Junction between the anterior and posterior semicircular canal 2 Junction between the posterior and lateral semicircular canal 3 Junction between the lateral and anterior semicircular canal 4 Base of the ampulla, under the junction between the anterior and posterior

semicircular canal 5 Crux between the fenestra cochlearis and the cochlear aquaduct 6 Base of fenestra cochlearis, at the bony lamina impression 7 One full turn from landmark 6, placed along the bony lamina impression 8 Apex 9 One half turn from landmark 6, placed along the bony lamina impression 10 Centroid


Grohé et al., 2016; Schnitzler et al., 2017), I could identify significant differences

between bony labyrinth shapes.

Step 3: Supplementary Statistics

Finally, I analysed changes in cochlear morphometrics with a Procrustes

analysis of variance, pairwise comparisons, and a descriptive principal component

analysis (PCA). All statistics were run through R-Studio (v. 1.0.136). The Procrustes

analysis of variance is similar to the more familiar permutation analysis of variance

(PERMANOVA), and was used to identify any differences in cochlear shape as a factor

of ontogeny or as a factor of phylogeny. Subsequently, any detected differences were

run through pairwise comparisons to statistically pinpoint where they differed within

age class or species. A principle component analysis was then used to visualise how all

the cochlear shapes related to one another and to see how different they were from

each other.

2. Linear data plots

Data were standardised as the percentage of maximum mandible size (here,

taken as a proxy for age) plotted on the x-axis, and the percentage of maximum size

for each cochlear dimension expressed on the y-axis. Plots display potential trends in

size change of different cochlear dimensions. Results can guide areas for future

research, but more samples would need to be collected to make statistical claims.

Chapter 3: Results| 33

3 Chapter 3: Results

3.1 Tympanoperiotic Complex and Skull Measurements

There were minimal changes in size and shape of the tympanoperiotic complex

(TPC) relative to age. Measurements were standardised to be percentages of

maximum size within the sample set. A Generalized Least Squares Model was used to

identify the rate of ontogenetic change in specific TPC measurements (via mandible

measurements). The statistical three-way interaction between measurement, species

and age was significant (p= 0.0001; Table 5). This indicates that at least one species

had a growth pattern significantly different from the other species: Cephalorhynchus

hectori ontogenetic size change of the pars cochlearis width significantly differed from

all three other species (p<0.05; Figure 7), and its tympanic bulla width size change

differed from both those of Globicephala melas and Tursiops truncatus (p<0.05; Figure

7).

Table 5: ANOVA results comparing tympanoperiotic complex measurements between species and/or mandible lengths (age proxy). Note the significant p-value for the 3-way interaction at the bottom of the table. Abbreviation: number of degrees of freedom (numDF).

numDF F-value p-value (Intercept) 1 191226.26 <.0001 Measurement 8 26.02 <.0001 Species 3 11.34 <.0001 Average Mandible Length 1 81.84 <.0001 Measurement: Species 24 5.40 <.0001 Measurement: Average Mandible Length 8 5.53 <.0001 Species: Average Mandible Length 3 2.04 0.1069 Measurement: Species: Average Mandible Length 24 2.57 0.0001

With the minor exceptions found within Cephalorhynchus hectori, features of

the TPC held very similar trend lines overall. There was a slightly positive correlation

between age and features of the TPC, but for the most part, size changed at a rate close

to zero (Figure 7). This indicates minimal changes in TPC features between younger

animals and adults of the same species.


3.2 Radiography of Foetal Material

The pilot whales and single Gray’s beaked whale foetuses spanned the

developmental Stages 7-12 of Štěrba et al. (2000), with Stage 11 being the most

advanced. These foetal stages in turn are based on the Carnegie staging system

(O’Rahilly, 1973) which follows generalised mammalian criteria. Štěrba et al.

rearranged the usual stage numbers to accommodate premature opening of eyelids

relative to the average mammal (see explanation by Štěrba et al. 2000, page 41). For

clarification, I have included chronological stage numbers that are consistent with the

progression of cetacean development (See Table 6).

Figure 7: Nine measured features of the tympanoperiotic complex, plotted as a function of age (via mandible length). Axes are standardized by calculating the percentage of the species’ maximum size. Abbreviations: internal acoustic meatus (IAM), muscle fossa (MF).


Table 6: List of observations from the six foetuses and corresponding radiography. Chronological stages are the cetacean- specific stages proposed by Štěrba et al. (2000) labelled in chronological order. For supporting images see Appendix B. Abbreviations: total body length (TBL), tympanoperiotic complex (TPC).

ID GM254a GM274a GM230a GM247a GM406a MM002875 Species G. melas G. melas G. melas G. melas G. melas M. grayi Stage (Chronological) 7 10 12 12 12 8 Stage (Štěrba) 7 12 11 11 11 8 TBL (cm) 17 28.6 42.5 47 69 18.5 % Birth length 11% 18% 27% 29% 43% 8% Umbilical stalk closed Y Y Y Y Y Y Eyelids open N Y Y Y Y N Elongated fluke N Y Y Y Y N Skin pigment N Y Y Y Y N Blue/black skin pigment N N Y Y Y N Ossified ribs N Y Y Y Y Y Ossified vertebrae N Y Y Y Y N Ossified braincase N Y Y Y Y N Ossified rostrum N Y Y Y Y N Ossified flippers N N Y Y Y N Outline of TPCs N N Y Y Y N Dense TPCs N N N N N N Cochlea spiral visible N N N N Y N Teeth visible N N N Y Y N

Radiography revealed questionable signs of ossification in the ear region, with

clear ossification seen only in the oldest (Stage 11, TBL= 69cm) G. melas foetus.

Although the TPCs were not distinctly more dense than the rest of the skull, they were

mineralised enough to detect the cochlear canal void (Appendix B). Additionally, faint

outlines of the tympanoperiotic complexes were detected in the smaller Stage 11

foetuses (Table 6; Appendix B). To keep the integrity of rare foetal specimens, I

decided not to dissect and isolate the ears for further micro-CT scanning. For images

of the foetuses and X-rays see Appendix B.

3.3 Inner Ear Measurements

3.3.1 Geometric Morphometrics

Landmarks from 3D cochlear models were run through a General Procrustes

Analysis (GPA), which creates a ‘mean shape’ formed by superimposing models into a

common coordinate system with the least amount of residuals (Kendall's shape-space;


Gower, 1975; Rohlf & Slice, 1990). Post-GPA analyses uses this ‘mean shape’ to detect

morphological differences as well as calculate the degree of variation. Based on the

results of a Procrustes ANOVA, I found that the shape of the cochlear spiral is not

significantly different between age classes (p= 0.998), but is significantly different

between species (p<0.001; Table 7). There were not enough samples to properly

compute a further two-way interaction with statistical confidence (that is, to establish

the combined effect of age and species on shape).

Table 7: Results of the Procrustes ANOVA main effect model at 10,000 permutations. Note there is a significant p-value for species, but not age class. Abbreviations: degrees of freedom (Df), sum of squares (SS), mean squares (MS), residuals squared (Rsq). Df SS MS Rsq F Z Pr(>F) Age 3 0.013 0.004 0.100 0.848 0.380 0.998 Species 3 0.086 0.029 0.666 5.670 2.797 1.00E-04*** Residuals 6 0.030 0.005 Total 12 0.130

Table 8: List of pairwise comparison results analysing cochlear morphology between species. Distance and Z-Score show the degree to which these cochlear comparisons differ. Note the p-values for M. grayi that show cochlear shapes to be significantly different to all the other species. P-values C. hectori G. melas M. grayi T. truncatus C. hectori 1.000 0.121 0.002 0.220 G. melas 0.121 1.000 0.000 0.336 M. grayi 0.002 0.000 1.000 0.005 T. truncatus 0.220 0.336 0.005 1.000 Distance (d) C. hectori G. melas M. grayi T. truncatus C. hectori 0.000 0.103 0.174 0.095 G. melas 0.103 0.000 0.174 0.082 M. grayi 0.174 0.174 0.000 0.164 T. truncatus 0.095 0.082 0.164 0.000 Z-Score C. hectori G. melas M. grayi T. truncatus C. hectori 0.000 1.292 2.050 1.111 G. melas 1.292 0.000 2.181 1.032 M. grayi 2.050 2.181 0.000 1.928 T. truncatus 1.111 1.032 1.928 0.000


Following the results of the Procrustes ANOVA, I ran a pairwise-comparison

(Table 8) to find which species significantly differed from one another. Results

showed that the shape of M. grayi cochlear spirals were significantly different from all

the other study species (p<0.05), while C. hectori, G. melas, and T. truncatus did not

differ significantly from one another. Based on pairwise distance results, G. melas and

T. truncatus were found to be the most similar in shape (d= 0.082), whereas G. melas

and M. grayi were the most different (d= 0.174). The degree of difference between all

individual cochleae can also be visualised through a Principal Component Analysis

(PCA) diagram (Figure 8). Note the clustering of each species and how M. grayi

cochleae lay far away from the rest of the samples.

3.3.2 Linear Measurements and Ratios

Statistics on linear measurements of the bony labyrinth were limited by sample

size. It can be difficult to acquire rare foetal material, and even if there were plenty of

samples to choose from, the cost of micro-CT can severely limit sample size. Despite

C. hectori

G. melas

M. grayi

T. truncatus

Figure 8: Principal Component Analysis (PCA) diagram of bony labyrinth shape variation. As PC1 increases, the cochlear canal forms a tighter spiral and increases in height. The semicircular canals also form longer and wider arches.


these caveats, it can still be useful to plot raw data as a means of identifying potential

patterns to further investigate in future research. I would just like to express caution

when drawing conclusions from the following plots (Figure 9).

Overall, linear measurements of the bony labyrinth seem to follow similar

growth patterns to the TPC features discussed earlier. There is little to no slope as age

increases, and even the youngest possible individuals have dimensions that are

already at 70% or more of their expected maximum size. However, some

measurements have a somewhat positive slope as well. Cochlear height, apex radius,

axial pitch and the diameter of the posterior semicircular canal arch all increase

slightly with age (Figure 9, Table 9). On the other hand, the radii ratio is the only

calculation that seems to decrease with age on average.

Figure 9: Plots of bony labyrinth dimensions and ratios as a function of age (via mandible length). Axes are standardised by calculating percentages from the species’ maximum size.

Chapter 3: Results 39

Table 9: List of all cochlear measurements to the closest one hundredth of a millimetre. Abbreviations: anterior semicircular canal width

(Ant-SC), average mandible length (AML), lateral semicircular canal width (Lat-SC), and posterior semicircular canal width (Post-SC).

AML Turns Cochlear

width

Cochlear

height

Cochlear

length

Base

radius

Apex

radius

Axial

Pitch

Radii

ratio

Post-

SC

Ant-

SC

Lat-

SC

C.

hec

tori

Adult 247 2.125 8.37 3.88 26.12 5.50 0.93 1.83 6.15 1.03 1.40 1.71

Juvenile 203 2 8.20 3.53 25.29 5.03 1.12 1.77 4.49 1.06 1.59 1.99

Neonate 150 2 8.39 3.70 26.33 5.14 1.09 1.85 4.76 0.93 1.40 2.01

G.

mel

as

Adult 495 2.125 11.08 5.17 36.31 7.76 1.15 2.43 6.86 1.21 1.88 2.44

Juvenile 419 2.125

11.40 5.05 38.67 7.79 1.33 2.38 5.94 1.25 1.90 2.51

Neonate 250 2.125

11.28 4.62 37.05 7.69 1.18 2.17 6.55 1.17 1.99 2.44

Foetal 85 2.125

11.27 4.26 37.05 7.68 1.00 2.00 7.77 1.04 2.01 2.71

M.

gra

yi Adult 778

1.875 11.21 5.68 32.10 7.18 1.14 3.03 6.31 2.33 3.03 3.29

Juvenile 409 1.75 11.97 5.70 28.50 7.41 1.17 3.26 6.50 1.71 2.49 2.52

Neonate 335 1.75 11.22 5.75 27.47 7.11 0.94 3.29 7.57 1.94 2.77 2.91

T.

tru

nca

tus Adult 476 2 10.76 4.85 33.78 6.92 1.60 2.42 4.37 1.53 1.76 2.71

Juvenile 358 2 9.92 4.46 32.72 6.23 1.52 2.23 4.21 1.30 1.82 2.58

Neonate 251 2.125 10.05 4.62 32.36 6.74 1.12 2.18 6.01 1.08 1.41 1.97

Chapter 4: Discussion 40

4 Chapter 4: Discussion

Newborn odontocetes display many forms of precocial development. From

swimming and surfacing right after birth, to navigating and communicating with

conspecifics, it should be no surprise that their primary sensory system, hearing, is

also well developed at an early stage. The goal of this study was to establish if the

cochlea hearing organ (and the tympanoperiotic complexes which house them) is fully

developed at birth, both in terms of size and morphology. Below I will discuss my

results for analysing size, shape and timing of ossification in the tympanoperiotic

complex (TPC) and its relevance to age-class and species. I will also review some

observational notes and discuss applications to conservation and the direction of

future research.

4.1 Significance to Age- Ontogeny

Overall, features of the TPC and morphology of the bony labyrinth (i.e. cochlear

canal and vestibular complex) did not significantly differ between age classes for any

one species. This was made clear by the statistical analysis of both TPC linear

measurements and bony labyrinth geometric morphometrics. Additionally, non-

statistical plots of the cochlear proportion data gave some preliminary insight into

growth patterns (Figure 9). Most cochlear dimensions showed minor changes in size,

which indicated minimal differences between age classes. The radii ratio however,

differed from the other plots, and with the exception of Cephalorhynchus hectori

tended to decrease with age. This could mean that younger animals are more sensitive

to lower frequency limits, a finding congruent with Greenhow (2013) who reported an

increased hearing sensitivity at the upper and lower frequency limits for a captive

short-finned pilot whale (Globicephala macrorhynchus) calf. High frequency hearing

loss with age has also been reported in captive bottlenose dolphins (Ridgway &

Carder, 1997).

The minimal size and shape differences between age classes are consistent

with other research, but it was unusual to not see a statistically significant difference

Chapter 4: Discussion| 41

in growth rates between features of the tympanic bulla and periotic as has been

reported in a range of odontocetes (Kasuya, 1973). Although the length and width of

the studied tympanic bullae visually appear to show growth, there was not significant

difference when comparing the rate of their size change to other TPC measurements.

This could be because I analysed differences based on the percentage of their

maximum size rather than raw unstandardized measurements. Nevertheless, I believe

this was a more useful comparison because it put all species and measurement

features on the same scale, and allowed me to identify common growth patterns

between variables. A larger sample size would help clarify my results.

Additionally, radiography revealed that the most advanced foetus (i.e.

Globicephala melas, Stage 11, 69cm) had visible cochlear canals, and the two other

Stage 11 foetuses had faint outlines of their TPCs (Appendix B) showing that there is

some degree of early ossification. All other foetuses studied were younger than Stage

11 and did not have ossified TPCs that could be definitively identified. These findings

indicate that there is some degree of ossification by or at the time of Stage 11 foetal

development, and supports the hypothesis (Ridgway & Carder, 2001) of precocial

hearing development in young odontocetes. Cochlear ossification is presumed to be

essential for the development of the basilar membrane and the Organ of Corti. Recent

studies have reported similar observations, where ossification seems to occur just

after Carnegie Stage 23 (Sterba Stages 10 and 11; Moran et al., 2011), or as early as

32% of the gestation period in various cetacean species (Thean et al., 2016). TPCs

continue to ossify and become the densest part of the skeleton by the time of birth

(Cozzi et al., 2015). Research has also found volumes of foetal and juvenile bony

labyrinths to be the same (if not slightly larger) than their adult counterparts

(Mennecart & Costeur, 2016).

This precocial development is significant because it implies that the presence

of fully functional hearing is important for postnatal survival. Whether it is

communicating with their conspecifics or navigating a poorly lit environment,

proficient hearing is a critical sense. Prenatal hearing has been suggested to influence


early imprinting in humans (DeCasper & Fifer, 1980; DeCasper & Spence, 1986), and it

is possible that a similar process could be happening in Cetacea. We know that human

hearing becomes fully functional around the start of the third trimester (Forbes &

Forbes, 1925; Pujol et al., 1991; Sontag & Wallace, 1935), and although it is unclear

when hearing becomes fully functional in odontocetes, ossification does begin earlier

than in humans (Cozzi et al., 2015; Thean et al., 2016). It would be interesting to know

if intrauterine imprinting occurs, as this might help us to better understand mother-

calf interactions postnatally.

4.2 Significance to Species- Phylogeny and Ecology

Even though there was no age-related change found in the features of TPCs or

bony labyrinths, there were statistically significant shape differences found between

species. The morphologies of Mesoplodon grayi cochleae were considered to be

significantly different from all three other odontocete species, while Cephalorhynchus

hectori, Globicephala melas and Tursiops truncatus all had cochleae that did not

significantly differ from each other. Congruent with their phylogeny, these

morphological patterns in cochleae could, hypothetically, be consistent with ecological

factors.

Ketten (2000) used cochlear shape changes to establish what is now a widely-

cited system of three cetacean ear types and associated ecotypes:

Type I: short, thick basilar membranes representative of inshore or

riverine species that detect ultra-high frequencies (over 100kHz),

Type II: long, thin basilar membranes representative of lower ultrasonic

frequencies (under 100kHz), and

Type M: large cochlea with flexible basilar membrane specific to

mysticetes.

All species of this study had cochleae considered to be Type II. Other research

has also shown a relationship between inner (cochlear) and outer morphologies of the

pars cochlearis and different (marine versus riverine) environments (Gutstein et al.,


2014). If ecology played a role in bony labyrinth shape, my study might have revealed

a close relationship between the cochleae of the two offshore, deep-diving species G.

melas and M. grayi, or similarities related to social behaviour like pod size or how

‘talkative’ they are. No ecological parallels were apparent between the study species

that would also fit the PCA descriptive plot, though.

A phylogenetic explanation would certainly make sense. In addition to having

the most morphologically distant cochleae, M. grayi is also phylogenetically the most

distantly related out of the four species studied. M. grayi belongs to the Family

Ziphiidae, whereas the other three species are Delphinidae. Furthermore, my results

(Figure 8, Table 8) found T. truncatus and G. melas bony labyrinth shapes to be the

most closely related of the four study species, which is phylogenetically congruent as

well (McGowen et al., 2009). The morphology of the tympanic bullae and periotics is

sufficiently distinct that the TPC has been used to identify specimens down to the

genus (Kasuya, 1973). I suggest that, based on my current results, it is more likely that

odontocete bony labyrinth morphology is only dependent on phylogenetic differences,

not ecological ones. Further samples with more species are needed to clarify these

results.

Specific cochlear measurements can give some insight into odontocete hearing

properties. For instance, axial pitch (= cochlear height/ number of turns) is negatively

proportional to frequency, and the radii ratio (= base radius/ apex radius) is strongly

correlated to the low frequency limits of hearing (Ketten & Wartzok, 1990). Cochlear

length however, does not correlate with frequency like it does in terrestrial mammals

(Ketten, 1992a; West, 1985). It is, instead, found to relate strongly with animal size in

cetaceans (Ketten, 1992a). A useful addition to this study would have been to add

bony laminae measurements, as they show stiffness of the basilar membrane (Ekdale

& Racicot, 2015) and is therefore a good indicator of hearing frequency.


4.3 Observational Notes

This was a study focused on establishing the structural changes of odontocetes

from different age classes, but it also proved to be an opportunity for observational

notes of material rarely seen from the perspective of a micro-CT scan. One of the more

interesting features to observe has been the vestibular complex. Here, both the lumen

thickness and arch diameter have been significantly reduced compared to that of land

mammals. Orientations of semicircular canals relative to the cochlea are also different.

I observed lateral canals that open out to a wider angle, and have shifted the start and

end points of the arch relative to the other semicircular canals.

The vestibular complex is comprised of the fluid-filled utricle, saccule and

semicircular canals, and is known for its role in balance perception (Cozzi et al., 2017).

Cetacean semicircular canals have been of particular interest because of their notable

reduction in size relative to the cochlea and body mass, compared to those of land

mammals (Hyrtl, 1845; Spoor et al., 2002). There have even been some reports of

incomplete semicircular canals in some adult odontocete species (Ketten, 1992b;

Ketten & Wartzok, 1990), which questions the structure’s functionality in cetaceans in

general. My observations from four ecologically diverse species of different age

classes found that semicircular canals were indeed reduced but not absent or

incomplete. Overall, of the four odontocete species, this study found the semicircular

canals to be the largest and most defined in Gray’s beaked whales (M. grayi).

Currently, the leading hypothesis to explain reduced semicircular canals is

attributed to limited head movements (shortened and/or fused cervical vertebrae)

and slower velocity of body movements due to life in a dense aqueous environment

(Gray, 1908; Ketten, 1992a). However, evidence suggests increased angular head

rotations due to inflexibility of the neck do not significantly justify the size of cetacean

semicircular canals (Kandel & Hullar, 2010). The vestibular complex plays a role in

compensating for uncontrolled head movements to stabilise gaze (Berthoz, 1997).

Perhaps the reduced reliance on eyesight (and therefore, need to stabilise it)


contributed to the smaller semicircular canals. The cause and significance of the

cetacean vestibular size reduction remains very much a mystery.

4.4 Future research

Micro-CT and geometric morphometrics have been valuable tools in analysing

inaccessible, complex shapes. There are many more tools available in the R package

‘Geomorph’ that were not covered in this thesis, but could help with a more detailed

shape analysis. The package includes analyses for shape allometry, (ontogenetic)

shape trajectories and phylogenetic signal (Adams & Otárola-Castillo, 2013). The key

limitation is the need for a large sample size and the cost of micro-CT scanning. To

understand more about the ontogeny of hearing in odontocetes, much more

investigation into foetal samples is needed. Nevertheless, micro-CT is getting

increasingly affordable with time and I believe that we will be able to clarify much of

the current research with these valuable, non-destructive tools.

Researching the basics of hearing structure development is an important first

step to learning more about odontocete hearing abilities in general. For example,

there is wide interest in the cause of mass strandings in New Zealand (e.g. Brabyn &

Mclean, 1992). Golden Bay is a particularly interesting location where there is usually

at least one pilot whale mass stranding event annually. This year, 2017, there was a

particularly large stranding event where more than 400 whales were re-floated and

more than 200 whales died. The cause of such mass strandings is debated, but one

leading hypothesis is that the shallow water of this area and flat ocean floor causes

higher noise reflection and disorientation in the animal. To fully understand this

phenomenon there is a need to learn how odontocete ears function in different

environments and, in this case, find what makes pilot whales different from other

odontocetes and more vulnerable to mass stranding.

Other whale strandings have been linked to military exercises and SONAR (e.g.

Rommel et al., 2005). Most of the time, it is unlikely that the sound itself is causes

damage to ears, but that the sudden sound may startle and cause erratic behaviour

Conclusion| 46

like surfacing too quickly, leading to the bends and/or haemorrhaging with air

bubbles in the blood stream (Cox et al., 2006; Weilgart, 2007). Additionally,

anthropogenic noise like boat traffic can cause displacement of cetacean populations

(Bejder et al., 2006; Nowacek et al., 2007). The results of this study show that young

whales have fully developed ears that are just as vulnerable to anthropogenic noise as

adults. I would caution these activities around near shore areas where calving and

nurseries often occur.

5 Conclusion

Overall, this micro-CT study showed little to no change in the size and shape of

odontocete ears from birth to adulthood. With minor exceptions, tympanoperiotic

complex (TPC) measurements had minimal size changes as a factor of age, and general

growth patterns were similar between the four study species. Morphometrically the

bony labyrinth only showed shape changes between species, not age classes.

Furthermore, although X-ray images of foetuses did not display dense ossification of

the ear region, there was enough to detect cochlear canals (Appendix B) in a 69cm G.

melas foetus at 43% of its newborn length. Further investigations should focus on

whether all of the parts and proportions of the TPC reach maturity at the same stage,

and if this is a universal pattern that can be observed across all odontocete species.

References| 47

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Adams, D. C., Collyer, M. L., Kaliontzopoulou, A., & Sherratt, E. (2017). Software

for geometric morphometric analyses. R package.

Adams, D. C., & Otárola-Castillo, E. (2013). Geomorph: An R package for the

collection and analysis of geometric morphometric shape data. Methods in

Ecology and Evolution, 4(4), 393–399.

Adams, D. C., Rohlf, F. J., & Slice, D. E. (2004). Geometric morphometrics: Ten

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Appendix A: Eight page Memorandum of Understanding (MOU) made with Te Upokorehe iwi regarding research on their pilot whale (Globicephala melas) material. This includes a two page Research Agreement Proposal. The Upokorehe collection is composed of 40 whales, including three neonates and a foetus, collected during the 2014 Ōhiwa Harbour stranding.

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Appendix B: Foetal material X-rayed to check for ossification of the ear region. Six specimens were observed, five Globicephala melas and one Mesoplodon grayi all ranging from stages 7-11 using Štěrba et al. (2000) criteria. Note that Stage 12 comes before Stage 11 for cetaceans in this system. Also note the cochlear spiral (arrows) seen in individual GM406a. (Left) foetus photos, (Middle) lateral X-ray images, and (Right) dorsoventral X-ray images of the head.

GM254a: Globicephala melas Stage 7 (Total body length= 17cm)

GM274a: Globicephala melas Stage 12 (Total body length= 28.6cm)

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GM230a: Globicephala melas Stage 11 (Total body length= 42.5cm)

GM247a- Globicephala melas Stage 11 (Total body length= 47cm)

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GM406a- Globicephala melas Stage 11 (Total body length= 69cm)

MM002875- Mesoplodon grayi Stage 8 (Total body length= 18.5cm)

an investigation of the odontocete inner ear

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