Ear Structures of The Naked Mole-Rat, (Rodentia: Bathyergidae)

RESEARCH ARTICLE
Ear Structures of the Naked Mole-Rat,

Heterocephalus glaber, and Its Relatives
(Rodentia: Bathyergidae)
Matthew J. Mason1*, Hannah L. Cornwall1, Ewan St. J. Smith2
1 University of Cambridge, Department of Physiology, Development & Neuroscience, Cambridge, United

Kingdom, 2 University of Cambridge, Department of Pharmacology, Cambridge, United Kingdom
* [email protected]
a11111
Abstract
Although increasingly popular as a laboratory species, very little is known about the periph-
eral auditory system of the naked mole-rat, Heterocephalus glaber. In this study, middle and
inner ears of naked mole-rats of a range of ages were examined using micro-computed
tomography and dissection. The ears of five other bathyergid species (Bathyergus suillus,
OPEN ACCESS
Cryptomys hottentotus, Fukomys micklemi, Georychus capensis and Heliophobius argen-
Citation: Mason MJ, Cornwall HL, Smith ES. J.
teocinereus) were examined for comparative purposes. The middle ears of bathyergids
(2016) Ear Structures of the Naked Mole-Rat,
Heterocephalus glaber, and Its Relatives (Rodentia: show features commonly found in other members of the Ctenohystrica rodent clade, includ-
Bathyergidae). PLoS ONE 11(12): e0167079. ing a fused malleus and incus, a synovial stapedio-vestibular articulation and the loss of the
doi:10.1371/journal.pone.0167079 stapedius muscle. Heterocephalus deviates morphologically from the other bathyergids
Editor: Heike Lutermann, University of Pretoria, examined in that it has a more complex mastoid cavity structure, poorly-ossified processes
SOUTH AFRICA of the malleus and incus, a ‘columelliform’ stapes and fewer cochlear turns. Bathyergids
Received: September 26, 2016 have semicircular canals with unusually wide diameters relative to their radii of curvature.
Accepted: November 8, 2016 How the lateral semicircular canal reaches the vestibule differs between species. Heteroce-
phalus has much more limited high-frequency hearing than would be predicted from its
Published: December 7, 2016
small ear structures. The spongy bone forming its ossicular processes, the weak incudo-sta-
Copyright: © 2016 Mason et al. This is an open
pedial articulation, the columelliform stapes and (compared to other bathyergids) reduced
access article distributed under the terms of the
Creative Commons Attribution License, which cochlear coiling are all potentially degenerate features which might reflect a lack of selective
permits unrestricted use, distribution, and pressure on its peripheral auditory system. Substantial intraspecific differences were found
reproduction in any medium, provided the original in certain middle and inner ear structures, which might also result from relaxed selective
author and source are credited.
pressures. However, such interpretations must be treated with caution in the absence of
Data Availability Statement: All relevant data are experimental evidence.
within the paper and its Supporting Information
files.
Funding: Support was provided by a John Ray

Trust Summer Research Grant (2015) to HLC
[http://johnraytrust.com/] and a Royal Society
Research Grant (RG130110) to ESS [https:// Introduction
royalsociety.org/grants-schemes-awards/grants/?
Bathyergid mole-rats, endemic to Africa, are members of the Ctenohystrica clade of rodents, a
gclid=CObSzoidrc8CFeQp0wodk4QENg]. The
funders had no role in study design, data collection
group which also contains the caviomorphs of South America [1]. The most famous bathyer-
and analysis, decision to publish, or preparation of gid must be the eusocial naked mole-rat, Heterocephalus glaber. Heterocephalus lives in under-
the manuscript. ground colonies of, on average, 75–80 individuals, among which reproduction is usually
PLOS ONE | DOI:10.1371/journal.pone.0167079 December 7, 2016 1 / 28

Ear of the Naked Mole-Rat
Competing Interests: The authors have declared restricted to just one female and one to two males (see [2] for a review). Naked mole-rats are
that no competing interests exist. weaned at around 3–6 weeks of age, can become reproductively active at 7–12 months, reach
full adult size at about 2 years and can live for more than 28 years (reviewed by [3]).
Visual cues are very limited in mole-rat burrows. The small eyes of naked mole-rats can
apparently detect the difference between light and dark [4], as can the eyes of other bathyergids
[5], but they are ill-equipped for image formation. There are challenges to audition under-
ground too: airborne sound, particularly at high frequencies, has been shown to be rapidly
attenuated with distance within the burrow systems of another mole-rat [6]. Low frequencies
of a few hundred Hertz propagate most effectively. It has been suggested that certain frequen-
cies may be selectively amplified in a “stethoscope effect” [7].
Despite the limits to sound propagation underground, vocalisations appear to be important
in the social communication of Heterocephalus. The naked mole-rat has an extensive repertoire
of at least 17 distinct sounds, including five juvenile-only vocalisations. Many are shorter than
200 ms duration but most are repeated. Atonal sounds contain frequencies from 0.2 to over 40
kHz, while tonal sounds fall between 1 and 9 kHz. Naked mole-rats can also scrape their upper
and lower incisors together to generate click-like sounds with most energy between 1 and 20
kHz, but it is not known if these sounds are used to communicate [8].
The hearing of Heterocephalus has been examined behaviourally, using a conditioned
avoidance method [9]. For airborne sound presented at 60 dB SPL, the hearing range of this
animal extends between 65 Hz and 12.8 kHz, which is an extremely restricted, low-frequency
range for a small mammal. Thresholds between 125 Hz and 8 kHz are rather flat and unusually
high, the lowest being 35 dB SPL. Together with its very poor sound localization ability, these
characteristics led Heffner & Heffner [9] to dub the hearing of the naked mole-rat “degener-
ate”. Heffner & Heffner suggested that the limitations on hearing in this animal are central
rather than peripheral, based on the fact that auditory thresholds are reduced with increased
sound duration. A recent examination of central auditory structures in the naked mole-rat has
shown that the binaural auditory brainstem nuclei lack the ion channel HCN1 [10]. This could
result in slowed integration of inputs in binaural auditory brainstem neurons, which could
impede the analysis of interaural time differences, potentially contributing to poor localization
abilities [10]. Insensitive hearing restricted to low frequencies is also a feature of a Zambian
bathyergid originally referred to as Cryptomys sp. but subsequently reidentified as Fukomys
anselli [11, 12], and has also been found in the more distantly-related spalacid mole-rat Spalax
[13, 14] and the pocket gopher Geomys [15]. It appears to be a general feature of subterranean
rodents. The somewhat wider hearing range of the coruro Spalacopus, a fossorial caviomorph,
is thought to relate to an increased amount of above-ground behaviour [16].
The fact that underground tunnels represent a ‘low-frequency’ environment has been used
to explain why vocalizations of subterranean rodents tend to emphasize such frequencies [6,
16–19]. This suggests that the peripheral auditory systems of these animals might show conver-
gently-derived, low-frequency adaptations, and many similarities have indeed been found
between the ear structures of distantly-related groups. Most subterranean mammals have
‘freely mobile’ ossicles which are relatively loosely articulated with the skull, reduced or miss-
ing middle ear muscles, relatively large stapes footplates and tympanic membranes lacking a
pars flaccida [20–24]. Freely-mobile ossicles may improve low-frequency sound transmission
through their reduced stiffness, but the physiological significance of the other features remains
unclear. The anatomical characteristic most closely linked to improved low-frequency hearing
in small mammals seems to be an enlarged middle ear cavity volume [25], but subterranean
species do not, in general, have markedly expanded middle ear cavities compared to terrestrial
species [21]. It has been argued that, despite the selective pressure towards low-frequency hear-
ing, hearing sensitivity need not be enhanced in these animals because of the so-called

“stethoscope effect” operating in their tunnels; the notion that the hearing of these animals is
degenerate has been strongly rejected [7, 20].
Among bathyergids, the middle and inner ears of species in the Cryptomys/Fukomys clade
have been examined in detail [26–35]. The ears of other bathyergids have received much less
attention, although brief descriptions exist of the middle ear apparatus in Bathyergus, Geory-
chus [20] and Heliophobius [36, 37]. The lack of information on Heterocephalus is surprising:
the only description of the middle ear of the naked mole-rat which we are aware of comes
from the unpublished PhD thesis of one of the present authors [38], and we have found no
information in the literature relating to its inner ear.
The purpose of the current paper is to produce the first detailed anatomical description of
the peripheral auditory system of Heterocephalus, considering not just adult morphology but
also postnatal ontogeny. Heterocephalus is increasingly used as a laboratory model species in
research into cancer, ageing and pain [39]. This description of its auditory apparatus comple-
ments the increasing body of knowledge being gathered about this strange and enigmatic
mammal, and allows us to investigate the extent to which its peripheral auditory system con-
tributes towards its restricted hearing. Other bathyergid species were examined for compara-
tive purposes: this is the first ear study to include representatives of all six currently-recognised
bathyergid genera.
Materials and Methods

15 corpses of Heterocephalus glaber, all non-breeders, were obtained from a laboratory colony
housed in the University of Cambridge (Table 1); most were preserved frozen until use. As
detailed in Table 1, these animals were either members of the original Cambridge colony or
offspring of these original members. The first Cambridge colony was formed of animals taken
from another captive colony, but data on the number of generations bred there were unfortu-
nately unavailable. The four youngest mole-rats used in this study had died naturally, perhaps
as a result of maternal neglect. The older animals had been sacrificed and in most cases their
brains removed as part of another research project, which sometimes resulted in damage to
the ear regions. Although most specimens were obtained unfixed, two of the adult Heteroce-
phalus had been anaesthetised with pentobarbitone and transcardially perfused with phos-
phate-buffered saline (pH 7.4), followed by 4% paraformaldehyde (at around pH 7.4). All
experiments on Heterocephalus had been conducted in accordance with the United Kingdom
Animal (Scientific Procedures) Act 1986 under a Project License (70/7705) granted to E.S.J.S.
by the Home Office; the University of Cambridge Animal Welfare Ethical Review Body also
approved procedures. A further three newborn Heterocephalus specimens, all from the same
litter, had been obtained in 1997 as perinatal casualties from a colony in Bristol Zoo and stored
frozen. One of these had been histologically sectioned. Having been left in Shandon Formal-
Fixx fixative (10% neutral buffered formalin) for six days, it was put into Shandon standard
decalifier for three days. Following dehydration and embedding in paraffin wax, sections were
taken at 10 μm thickness and stained with haematoxylin and eosin.
Dissections of the Heterocephalus specimens were performed using a Motic binocular light
microscope fitted with a GX-CAM 5 digital camera. GXCapture 8.0 software (GT Vision) was
used to take images and make scaled measurements from them. Light microscope images were
merged using Helicon Focus 4.10 (Helicon Soft Ltd., 2007), to increase apparent depth of
focus. Dissection and CT images originally of right ear structures were laterally inverted, to
facilitate comparison.
Three skulls of Cryptomys hottentotus (UMZ E3116, E3118 and E3119, originally collected
in Pietermaritzburg, South Africa) and one skull of Georychus capensis (UMZ E3110, originally

Table 1. Details of specimens examined in this project, including CT scan information.

Species Spec. No. Age, days Litter Body mass, g Sex Scan made Scanned Voxel side length, μm
Heterocephalus glaber CU1 1166 B1 44.5 M Cambridge Bulla 5.4
CU2 1316 A1 54.5 M - - -
CU3 1358 A1 63.5 M Cambridge Head 17.8
CU4 1 C3 1.8 F Cambridge Head 7.5
Bulla 6.9
CU7 1753 A1 58.0 M - - -
CU8 37 D3 2.3 M Cambridge Head 7.8
CU9 37 D3 1.1 M - - -
CU10 2 E2 1.0 ? - - -
CU15 204 D3 44.5 F - - -
CU16 212 D3 38.5 M - - -
CU17 194 D3 38.0 M Cambridge Bullae 6.0
CU18 176 D3 36.0 F Cambridge Bulla 4.8
Bulla 6.1
CU20 118 F3 23.0 M Cambridge Head 13.6
Bulla 4.9
BZ3 ?1 G 1.4 ? - - -
BZ4 ?1 G 1.1 M - - -
BZ5 ?1 G 1.5 M Cambridge Head 7.6
Bathyergus suillus 178 ? N/A ? ? Hull Skull 48.4
618 ? N/A 650 M Hull Skull 45.1
Cryptomys hottentotus E3116 ? N/A ? ? Cambridge Skull 12.6
E3118 ? N/A ? ? Cambridge Skull 13.3
E3119 ? N/A ? ? Cambridge Skull 15.3
Fukomys micklemi F1 ? N/A ? F Cambridge Head 21.5
Bulla 6.8
F2 ? N/A ? M Cambridge Head 22.5
Georychus capensis E3110 ? N/A ? ? Cambridge Skull 21.2
Heliophobius argenteocinereus Ha25 ? N/A 198 F Hull Skull 30.9
Ha38 ? N/A 148 F Hull Skull 33.2
Heterocephalus ‘CU’ specimens were from the colony in the University of Cambridge while ‘BZ’ specimens came originally from Bristol Zoo. The litter
lettering indicates which naked mole-rats were siblings. The associated number 1 refers to a member of the original Cambridge colony, 2 to an animal bred
from the colony queen, and 3 to animals bred from a male and female which had been separated from the original colony. CT scans from the University of
Hull were made by Dr. Phil Cox as part of a separate research project.
doi:10.1371/journal.pone.0167079.t001
collected at the Cape of Good Hope, South Africa), from the collections of the University
Museum of Zoology, Cambridge, were CT-scanned.
Two heads of Fukomys micklemi, formalin-preserved and stained with iodine for use in
another project, were donated by Dr. Phil Cox. The specimens had originally been obtained
from a colony at the University of Ghent, belonging to Prof. Dominique Adriaens. Dr. Cox
also kindly provided us with computed tomograms of prepared skulls of Bathyergus suillus and
Heliophobius argenteocinereus, which had been made at the University of Hull. The two Bath-
yergus specimens had been collected in Cape Town, South Africa, by Prof. Nigel Bennett,

while the two Heliophobius specimens had been collected in Blantyre, Malawi, by Prof. Radim
Šumbera.
Micro-computed tomography and reconstruction

CT scans were made of intact heads and dissected-out auditory regions of Heterocephalus and
Fukomys specimens, and of the intact, dried skulls of Cryptomys and Georychus specimens
(Table 1). Wet specimens were wrapped in cellophane to minimise drying during the scans,
which were all made using a Nikon XT H 225 scanner. The settings used were 125–130 kV and
110–130 μA. Images were constructed from 1080 projections, each with 1000 ms exposure and
two frames averaged per projection. The scan data were processed with CT AGENT XT. 3.1.9
and CT PRO 3D XT 3.1.9 (Nikon Metrology, 2004–2013). Cubic voxel side-lengths are given
in Table 1.
Tiff stacks from the micro-CT scans were converted to jpegs in Adobe Photoshop CS 8.0
(Adobe Systems Inc., 2003). MicroView 2.5.0 (Parallax Innovations Inc., 2016) was used to
produce 3D surface reconstructions and to reorient tomograms. Other reconstructions were
made using WinSurf 4.0 (Eric Neufeld, 2001), in a process requiring manual identification of
boundaries. The middle ear cavity reconstructions were based on its internal boundaries,
while inner ear reconstructions followed the inner walls of the bony labyrinth. Cartilage, soft
tissue and fluid were impossible to distinguish from each other in the CT scans, which made it
difficult to identify some of the structural boundaries in the neonatal Heterocephalus speci-
mens, prior to ossification. The formalin-preserved Fukomys specimens proved to have under-
gone some demineralisation and crystallisation within the auditory bullae, but it was possible
to distinguish and model the ear structures. Each recorded measurement was obtained from
one ear only per animal.
In order to make cochlear measurements, tomograms were reoriented in MicroView such
that the z-axis ran through the centre of the modiolus. The 3D coordinates of multiple points
along the centre of the cochlear canal, starting from the beginning of the primary spiral lamina
near the round window and extending to the apex, were then recorded. At least one point was
recorded every 10˚ along the spiral. The total length of the cochlear canal was estimated as the
sum of the linear distances between consecutive points. The number of spiral turns and the
radii of curvature at base (Rbase) and apex (Rapex) were established from the x-y coordinates of
these points. The radii of curvature were calculated following the method of Manoussaki et al.
[40], except that, for simplicity, the five points used were separated by 22.5 degree steps from
the estimated centre of the cochlear spiral, rather than being equally spaced. Measurements of
semicircular canal radii of curvature were derived from measurements of the ‘height’ and
‘width’ of the arcs, following Ekdale [41]. The internal diameter of each bony semicircular
canal was calculated as the mean of two orthogonal measurements, made at the point on the
canal arc where the ‘height’ measurement was taken.
Because they were originally made for the purposes of another project, the scans of the
Bathyergus and Heliophobius specimens were at a lower resolution than those of the other spe-
cies. Semicircular canal measurements were not made because the resolution was insufficient.
The descriptions of the inner ear and ossicles in these animals should be considered less reli-
able than those of the other species.
Results
The anatomical description in the first two sub-sections below is restricted to older Heteroce-
phalus specimens (>100 days) unless otherwise noted. The ears of the younger specimens
are described in the third sub-section, and the ears of other bathyergids in the fourth.

Measurement data are presented in the tables below; additional data are presented as support-
ing information (see S1 Table).
The external and middle ears of Heterocephalus

The naked mole-rat has no pinnae; the only sign of the external ear on the surface of the head
is a raised annulus (Fig 1). The entrance to the meatus in the centre of this annulus was typi-
cally semi-occluded with fine hairs and a small amount of cerumen. The meatus projects ven-
tromedially into the head to reach the auditory bulla. It is around 6 mm long with a very
narrow but patent lumen, diameter around 0.6 mm. Away from the surface of the head, the
meatus is largely free of hairs and cerumen.
The auditory bulla of Heterocephalus is relatively small (Fig 2). The walls of the middle ear
cavity were well-ossified in the adult specimens, in which tympanic and petrosal components
were indistinguishably fused.
The middle ear cavity can be divided into tympanic, epitympanic and mastoid components.
The tympanic cavity is by far the largest of these (Table 2). A small diverticulum extends lat-
erally from just caudal to the tympanic membrane but otherwise the tympanic cavity is roughly
ovoid. The tympanic membrane (Fig 3) forms part of its lateral wall: this averages 4.55 mm2 in
area (n = 4) and lacks any obvious pars flaccida. Within the tympanic cavity, the cochlear
promontory forms a rounded prominence medially. There are usually two, very small, partial
septa ventral to this. A thin lamina of bone extends from the promontory towards the rostro-
medial tip of the bulla, just above which is a channel which becomes the Eustachian tube. Dor-
sal to the promontory, this channel becomes covered over with a thin layer of bone, behind
which is located the tensor tympani muscle belly. The tensor tendon emerges posteriorly and
inserts on the manubrium of the malleus (Fig 3B). The oval window is just caudal to the muscle
belly. There is a strikingly wide gap between the stapes footplate and the oval window perime-
ter (Fig 4A). The fibrous annular ligament would normally occupy this space, but in the naked
mole-rat the stapedio-vestibular articulation was found to be synovial (see later). Immediately
below the oval window is the round window, within a shallow recess. No stapedius muscle was
found in any specimen of any age.
Dorsal to the tympanic cavity is the roughly pyramidal epitympanic recess, containing the
bodies of the fused malleus and incus. The manubrium of the malleus and the incudal long
process extend through the small, triangular opening between the two cavities. The short pro-
cess of the incus projects into a blind-ending, posterior diverticulum of the epitympanic recess,
where it is attached to the skull by means of ligamentous material inserting around its tip. The
facial nerve passes just under the tip of the short process, in a bony canal which is open to the
middle ear cavity over some of its length.
Three subcavities separately extend from the posterior part of the tympanic cavity into the
mastoid region of the skull (Figs 5 and 6). Two of them show a similar morphology to cavities
described in the gerbil Meriones unguiculatus [42], and so the same nomenclature has been
adopted. The dorsal mastoid cavity (DMC) emerges through the window framed by the lateral
semicircular canal and occupies most of the space between the three canals. The entrance to
the ventral mastoid cavity (VMC) is framed by the lateral and posterior semicircular canals,
the canal for the facial nerve and a partial septum dividing it from the tympanic cavity. The
VMC was internally divided by a septum in one 54-month animal. The posteromedial mastoid
cavity (PMC; not described in Meriones) is the smallest of the subcavities. It emerges through a
narrow gap left between the lateral and posterior semicircular canals (Fig 6). These three mas-
toid subcavities appear to ‘jostle for position’ around the semicircular canals and there were
individual differences in their relative shapes and sizes. However, there was no clear


Fig 1. The external ear of Heterocephalus. A: Lateral view of a live, adult animal. The position of the external ear,
which lacks a pinna, is indicated by an arrow. B: Photomicrographic close-up of the left external ear opening of a
different adult specimen, post-mortem. Note that the lumen is semi-occluded with hairs and cerumen. Scale bar for B
is 1 mm.
doi:10.1371/journal.pone.0167079.g001
relationship between age and the volumes of the middle ear subcavities in specimens older
than 100 days (Table 2).
The malleus and incus of the adult Heterocephalus are fused together (Figs 3A and 7A),
although a trace of the articulation zone can be discerned. The malleus is the larger ossicle. It
has a relatively short manubrium with prominent muscular and lateral processes. Internally,
Fig 2. MicroView reconstruction of the skull of an adult Heterocephalus (65 months). A: Ventral view of
skull. B: Lateral view, the left mandible having been digitally removed. The approximate extent of the left
middle ear cavity is indicated in both cases by red shading. Scale bar 10 mm.

Table 2. Measurements of middle ear subcavity and bony labyrinth volumes.

Species Spec. Age, ER volume, TC volume, VMC volume, DMC volume, PMC volume, Bony labyrinth
No. days mm3 mm3 mm3 mm3 mm3 volume, mm3
Heterocephalus glaber CU4 1 0.51 5.42 0 0 0 3.91
BZ5 ?1 0.65 7.20 0 0 0 4.48
CU8 37 0.60 5.79 0 0 0 4.02
CU20 118 1.47 16.76 3.08 4.14 0.35 4.22
CU17 194 1.74 (damaged) 6.23 6.93 0.83 (damaged)
CU1 1166 1.57 18.33 3.59 1.81 0.14 3.56
CU5 1656 1.99 21.06 4.88 (damaged) (damaged) (damaged)
CU19 1997 1.70 17.37 5.44 4.66 0.46 3.53
Bathyergus suillus 178 ? 111.66 318.63 7.41 0 17.79
618 ? 112.16 322.69 4.89 0 18.29
Cryptomys hottentotus E3116 ? 9.92 48.74 2.19 0 5.01
E3118 ? 14.39 48.27 2.85 0 6.87
E3119 ? 12.84 43.32 1.97 0 6.88
Fukomys micklemi F1 ? 19.28 74.16 0.83 0 7.60
F2 ? 19.50 75.31 1.27 0 N/A
Georychus capensis E3110 ? 25.37 101.53 1.77 0 9.35
Heliophobius Ha25 ? 24.16 79.45 2.30 0 9.20
argenteocinereus Ha38 ? 33.62 99.04 4.06 0 8.53
Note that the ventral mastoid cavity (VMC) could be distinguished from the tympanic cavity only in Heterocephalus, and only this species has a
posteromedial mastoid cavity (PMC). In some Heterocephalus specimens, ear structures were damaged and some measurements therefore could not be
made. In the very young specimens the outlines of the cavities were sometimes very unclear, so these values should be considered estimates.
the manubrium consists of spongy bone surrounding large, open spaces, presumably vascular
spaces (Fig 3B), which are not readily visible in a fresh specimen because of the periosteum
covering the ossicle. The distal tip of the long process of the incus is similarly developed, but
the bone is more compact elsewhere in the ossicular chain. The rounded malleus head, as its
name suggests, does resemble the head of a hammer. It expands much more rostrally than cau-
dally. Just below the head is a thin, bony lamina which represents a much abbreviated anterior
process. The free edge of this lamina joins the bony ridge which supports the tympanic mem-
brane and helps to divide the tympanic cavity from the epitympanic recess. The articulation
here between malleus and skull appears to be ligamentous and it is very flexible. The incus has
a long, narrow short process, which articulates with the skull at its tip, and a thicker long pro-
cess which abruptly tapers and turns medially to support a very tiny lenticular apophysis, for
articulation with the stapes. The connection between long process and lenticular apophysis is
narrow and flexible (Fig 4A). The zone of fusion between malleus and incus extends half-way
down the long process.
The stapes footplate is approximately oval in shape and averages 0.240 mm2 in area (n = 8).
It is very thin centrally with a thickened ridge around its perimeter (Figs 4A and 7A). Its ves-
tibular surface is quite flat. Instead of being stirrup-shaped with two crura, as would be typical
for a placental mammal, a triangular, bony plate extends out of the middle of the tympanic
side of the footplate. This very thin plate is strengthened by a low ridge on its dorsal aspect. Its
apex expands slightly to form the head of the stapes, for articulation with the incus. There is no
muscular process, consistent with the absence of a stapedius muscle.

Fig 3. Middle ear structures of Heterocephalus. A: Photomicrograph of a dissected left ear of an adult Heterocephalus (54 months), medial
view. The malleus, incus and tympanic membrane are visible; the stapes and tensor tympani muscle have been removed. Scale bar 1 mm. B:
Tomogram section through the left malleus of an adult mole-rat (65 months), posterior view. Note the difference between the compact bone which
forms the head and the spongy bone which forms the manubrium. The tensor tympani tendon is also visible. Scale bar 0.5 mm. APSC = ampulla for
posterior semicircular canal; ASC = anterior semicircular canal; C = cochlea; DMC = dorsal mastoid cavity; ED = bony tube for endolymphatic duct;
ER = epitympanic recess; FP = footplate of stapes; HM = head of malleus; LA = lenticular apophysis of incus; LPI = long process of incus;
LSC = lateral semicircular canal; MI = malleoincus; MM = manubrium of malleus; OW = oval window; PC = posterior crus of stapes; PD = bony tube
for perilymphatic duct (canaliculus cochleae); PMC = posteromedial mastoid cavity; PSC = posterior semicircular canal; RW = round window;
SPI = short process of incus; TC = tympanic cavity; TM = tympanic membrane; TT = tensor tympani tendon; V = vestibule of inner ear;
VMC = ventral mastoid cavity.
The bony labyrinth in Heterocephalus

The morphology of the inner ear was examined using CT reconstructions of the bony laby-
rinth (Fig 8). The semicircular canals are very wide relative to their length. The posterior end
of the lateral canal opens directly into the vestibule, just above the ampulla of the posterior
canal. The window framed by the arc of the posterior canal is divided by the lateral canal into a
larger upper fenestration and a smaller lower one. An extension of the DMC passes through
the upper fenestration in some specimens, while the PMC passes through the lower one (Fig
6). The narrow, orthogonal parts of the lateral and posterior canals approach each other
extremely closely. In one 37-day and one adult specimen they were separated by a very thin,
bony septum, whereas in other specimens the two canals were confluent at this point, although
their contents were probably separated by soft tissue in life.

Fig 4. The stapes of Heterocephalus, in situ. A: MicroView reconstruction of the left stapes region in an adult
mole-rat (65 months), ventrolateral view. The malleoincudal complex has been coloured blue, the stapes yellow.
Note the very wide gap between stapes footplate and the rim of the oval window. This gap and the opening of
the round window (both shaded grey) are covered over by membranes, but these do not show up on the CT
reconstruction. Scale bar 0.5 mm. B: Histological section of the stapes of a neonatal Heterocephalus, showing
the synovial structure of the stapedio-vestibular articulation. The red arrows indicate the synovial joint capsule,
which lies between the stapes footplate and the rim of the oval window. Scale bar 0.1 mm. APSC = ampulla for
posterior semicircular canal; ASC = anterior semicircular canal; C = cochlea; DMC = dorsal mastoid cavity;
ED = bony tube for endolymphatic duct; ER = epitympanic recess; FP = footplate of stapes; HM = head of
malleus; LA = lenticular apophysis of incus; LPI = long process of incus; LSC = lateral semicircular canal;
MI = malleoincus; MM = manubrium of malleus; OW = oval window; PC = posterior crus of stapes; PD = bony
tube for perilymphatic duct (canaliculus cochleae); PMC = posteromedial mastoid cavity; PSC = posterior
semicircular canal; RW = round window; SPI = short process of incus; TC = tympanic cavity; TM = tympanic
membrane; TT = tensor tympani tendon; V = vestibule of inner ear; VMC = ventral mastoid cavity.
There are on average 2.74 cochlear turns; the length of the cochlear canal averages 5.79 mm
(Table 3). The canaliculus cochleae, the very narrow channel for the perilymphatic duct, leaves
the bony labyrinth just posterior to the round window, while the channel which carries the
endolymphatic duct emerges from the vestibule at the base of the crus commune (the short
canal segment shared by both anterior and posterior semicircular canals) and travels adjacent
to the crus commune, widening just before it opens into the cranial cavity.
The ear region in very young Heterocephalus (<100 days old)

In the neonatal specimens (1–2 days old), the external ear canals were externally sealed and the
middle ear cavities were filled with fluid. By 37 days, the external ears of the larger specimen
were open, and the middle ear had begun to cavitate.
CT scans of the neonatal mole-rats showed that the tympanic bone remained unfused with
the petrosal. The middle ear cavity had a very simple structure consisting of a small, rounded
tympanic cavity with an epitympanic recess above. The mastoid cavities had not yet developed.
The walls of the epitympanic recess had not yet ossified and the position of the tympanic mem-
brane could not be discerned, so the recessus meatus could not be separated out from the mid-
dle ear component of the bulla. As a result, measurements of middle ear cavity volume
(Table 2) could only be crude estimates. By 37 days, the boundaries of the middle ear cavity
were clearer, although some small fenestrations between the bony elements remained. The air
within the recessus meatus and tympanic cavity helped to define the position of the tympanic
membrane in-between. Mastoid subcavities were still lacking, and middle ear morphology was
overall very similar to that of the neonatal specimens.

Fig 5. Middle ear cavities of bathyergids. WinSurf reconstructions of the left middle ear cavities and associated structures in four species of
bathyergids (Heterocephalus glaber, Fukomys micklemi, Cryptomys hottentotus and Georychus capensis), from lateral and slightly anterior views.
The walls of the middle ear cavities are shown semitranslucent. Positions of the tympanic membranes are indicated by brown shading. The
epitympanic recess is colour-coded red, the dorsal mastoid cavity blue and the ventral mastoid cavity green. The ossicles are yellow. The
posteromedial mastoid cavity, present only in Heterocephalus, is not visible in this view. The posterior parts of the middle ear cavities of the other
species extend into the mastoid region and are equivalent to the ventral mastoid cavity of Heterocephalus, but they lack constricted entrances and
so have not been separately coloured. Not to scale. APSC = ampulla for posterior semicircular canal; ASC = anterior semicircular canal;
C = cochlea; DMC = dorsal mastoid cavity; ED = bony tube for endolymphatic duct; ER = epitympanic recess; FP = footplate of stapes; HM = head
of malleus; LA = lenticular apophysis of incus; LPI = long process of incus; LSC = lateral semicircular canal; MI = malleoincus; MM = manubrium of
malleus; OW = oval window; PC = posterior crus of stapes; PD = bony tube for perilymphatic duct (canaliculus cochleae); PMC = posteromedial
mastoid cavity; PSC = posterior semicircular canal; RW = round window; SPI = short process of incus; TC = tympanic cavity; TM = tympanic
membrane; TT = tensor tympani tendon; V = vestibule of inner ear; VMC = ventral mastoid cavity.
The ossified parts of the malleus and incus were of the same shape and size in the neonates
as in the older specimens. CT scans revealed them to be composed of a shell of cortical bone
surrounding a large marrow space, although there were some bony trabeculae within the mar-
row space. Most of the manubrium, including the lateral process, remained cartilaginous, as
did the head and part of the neck of the stapes. There was no lenticular apophysis of the incus
and the position of the incudo-stapedial articulation was not clearly identifiable; instead, the

Fig 6. Bony labyrinths and mastoid cavities of Heterocephalus. WinSurf reconstructions of the left bony labyrinths and
mastoid cavities of three Heterocephalus specimens of different ages, seen from approximately posterior views. In the
youngest specimen on the left, coloured arrows indicate the future directions of expansion of the middle ear cavity into the
mastoid region. Blue = dorsal mastoid cavity; green = ventral mastoid cavity; orange = posteromedial mastoid cavity. The
38-month-old specimen had smaller mastoid cavities than many younger specimens, so this diagram should not be taken to
indicate a strict temporal sequence. APSC = ampulla for posterior semicircular canal; ASC = anterior semicircular canal;
C = cochlea; DMC = dorsal mastoid cavity; ED = bony tube for endolymphatic duct; ER = epitympanic recess; FP = footplate of
stapes; HM = head of malleus; LA = lenticular apophysis of incus; LPI = long process of incus; LSC = lateral semicircular
canal; MI = malleoincus; MM = manubrium of malleus; OW = oval window; PC = posterior crus of stapes; PD = bony tube for
perilymphatic duct (canaliculus cochleae); PMC = posteromedial mastoid cavity; PSC = posterior semicircular canal;
RW = round window; SPI = short process of incus; TC = tympanic cavity; TM = tympanic membrane; TT = tensor tympani
tendon; V = vestibule of inner ear; VMC = ventral mastoid cavity.
long process of the incus simply tapered into a cartilage bar which was continuous with the
bony body of the stapes. This bar proved to be flexible and tough. The malleus and incus were
tightly articulated but could be separated with a scalpel blade.
The ossified part of the stapes body in the neonates was relatively squat and broad, but the
bony part of the stapes footplate was smaller in area (0.166 mm2, n = 4) than in the adult speci-
mens. In some specimens there was an unossified region at the centre of this ossicle, represent-
ing an intercrural foramen (Fig 7A). This was covered over with periosteum such that it was
not patent. Histological sections of a neonatal Heterocephalus revealed the stapedio-vestibular
articulation to be synovial (Fig 4B). The joint capsule extended between the stapes footplate
and the rim of the oval window on both the tympanic and vestibular sides of the articulation,
with an open space for synovial fluid in-between.
In the larger of the 37 day-old specimens, the manubrium was ossified but there was still
cartilage between incus and stapes; the ossicles of the smaller specimen were less well-devel-
oped. The vascular cavities within the ossicles remained prominent, but malleus and incus
were now fused. The stapedes lacked intercrural foramina.
The inner ears of the neonatal specimens were almost completely surrounded by a shell of
bone. Although still poorly calcified, this allowed an accurate CT reconstruction of all parts
except for the medial side of the lateral semicircular canal, where no ossification was visible
and the boundaries of the canal had to be estimated. The inner ears were very similar in shape
and volume to those of the adult specimens examined, but the volumes (Table 2) are overesti-
mates because of the lack of ossification of internal structures such as the modiolus of the
cochlea, which could not be subtracted from the total volume. The bone around the inner ear
was more compact in the 37-day-old specimen and the boundaries of the lateral semicircular

Fig 7. Auditory ossicles of bathyergids. Left auditory ossicles of bathyergid mole-rats, drawn to scale. The mallei and incudes, which are fused
in these animals, are shown from an internal view in each case, the stapedes from a dorsal view. A: Heterocephalus glaber; B: Fukomys micklemi;
C: Heliophobius argenteocinereus; D: Bathyergus suillus; E: Georychus capensis; F: Cryptomys hottentotus. Two stapedes are shown for
Heterocephalus: the lower one comes from a neonate and has a small intercrural foramen and a cartilaginous head and neck (indicated by a
dashed outline). Two stapedes are also shown for Cryptomys, in this case illustrating individual variation in the adult ossicle. Scale bar 2 mm.
APSC = ampulla for posterior semicircular canal; ASC = anterior semicircular canal; C = cochlea; DMC = dorsal mastoid cavity; ED = bony tube for
endolymphatic duct; ER = epitympanic recess; FP = footplate of stapes; HM = head of malleus; LA = lenticular apophysis of incus; LPI = long
process of incus; LSC = lateral semicircular canal; MI = malleoincus; MM = manubrium of malleus; OW = oval window; PC = posterior crus of
stapes; PD = bony tube for perilymphatic duct (canaliculus cochleae); PMC = posteromedial mastoid cavity; PSC = posterior semicircular canal;
RW = round window; SPI = short process of incus; TC = tympanic cavity; TM = tympanic membrane; TT = tensor tympani tendon; V = vestibule of
inner ear; VMC = ventral mastoid cavity.
canal could be clearly distinguished (Fig 6). The internal structures of the cochlea were also
better ossified. The reconstructed inner ear was of adult form.
The ears of other bathyergid species

The middle ear cavities of the other bathyergids examined were more similar to each other
than any of them were to that of Heterocephalus (Fig 5). The epitympanic recess was in all
cases much larger, extending in a cranio-caudal direction. In Heliophobius, a septum just dor-
sal to the heads of the malleus and incus partially divided the epitympanic recess into rostral
and caudal compartments; a less prominent version was present in Georychus and Fukomys,
while in Bathyergus and Cryptomys the subcompartmentalization of the recess was irregular.
The VMC was in all cases well-developed but in wide communication with the tympanic cav-
ity, such that the two volumes could not be separated for individual measurement. The DMC,
extending through the lateral semicircular canal, was always present but relatively small
(Table 2). It was partially divided by a septum in all Cryptomys specimens and one Bathyergus.

Fig 8. Bony labyrinths of three bathyergid species. WinSurf reconstructions of the left bony labyrinths of Heterocephalus glaber, Cryptomys
hottentotus and Fukomys micklemi, seen from approximately lateral (left column), posterior (middle column) and medial (right column) views. Grey
shading has been added to indicate the positions of the openings into the labyrinth. The stapedes are shown in yellow. Note that Heterocephalus
has fewer cochlear turns than Cryptomys and Fukomys. The lateral semicircular canal of Heterocephalus reaches the vestibule directly, whereas in

Fukomys it merges with the ampulla of the posterior canal; the condition in Cryptomys is intermediate. Not to scale. APSC = ampulla for posterior
semicircular canal; ASC = anterior semicircular canal; C = cochlea; DMC = dorsal mastoid cavity; ED = bony tube for endolymphatic duct;
ER = epitympanic recess; FP = footplate of stapes; HM = head of malleus; LA = lenticular apophysis of incus; LPI = long process of incus;
LSC = lateral semicircular canal; MI = malleoincus; MM = manubrium of malleus; OW = oval window; PC = posterior crus of stapes; PD = bony
tube for perilymphatic duct (canaliculus cochleae); PMC = posteromedial mastoid cavity; PSC = posterior semicircular canal; RW = round
window; SPI = short process of incus; TC = tympanic cavity; TM = tympanic membrane; TT = tensor tympani tendon; V = vestibule of inner ear;
VMC = ventral mastoid cavity.
Posteromedial mastoid cavities were lacking. Three or four small septa were present ventral to
the inner ear as it projected into the tympanic cavity.
The mallei and incudes were similar in all bathyergids, although the shapes of the malleus
heads differed (Fig 7), as did the width of the manubrial blades, as seen from rostrally (widest
in Bathyergus). The zone of fusion between malleus and incus always extended down the incu-
dal long process but it was not continuous: the two ossicles separated slightly somewhere in
the middle of the articulating region, to a variable extent between individuals. The manubria
and incudal long processes were made of compact bone, as opposed to the spongy structure
found in Heterocephalus. The stapedes all possessed intercrural foramina, the size of which var-
ied among Cryptomys specimens (Fig 7F). The gap separating the footplate from the rim of the
oval window was particularly wide in this species. It was not possible to discern the nature of
the articulation in most of the CT scans, but in one Fukomys and one Cryptomys specimen a
synovial cavity could be seen within the zone of articulation. All species showed signs of a ten-
sor tympani muscle, but no stapedius was found.
The number of cochlear turns in one Heliophobius specimen was in the Heterocephalus
range, but in this animal both left and right cochleae were unusually loosely coiled: it looked
like the apical coils had failed to develop properly. In all other bathyergids, including the other
Heliophobius, the numbers of turns were greater (Table 3), reaching a maximum value of 4.00
in one specimen of Bathyergus. How the posterior limb of the lateral semicircular canal
reached the rest of the inner ear also varied between species. In Heliophobius it entered the ves-
tibule directly, as in Heterocephalus. In Fukomys (Fig 8) and Bathyergus it joined the ampulla
of the posterior canal. The morphology of Georychus was intermediate: although the lateral
canal converged with the posterior ampulla before reaching the vestibule, the two remained
Table 3. Inner ear measurements, made from CT scan data.

Species Cochlear Cochlear canal Cochlear Cochlear Anterior SCC Posterior SCC Lateral SCC Mean internal
turns length, mm Rbase, mm Rapex, mm radius, mm radius, mm radius, mm diameter (all
SCCs), mm
Heterocephalus 2.74 ± 0.10, 5.79 ± 0.28, 0.50 ± 0.07, 0.13 ± 0.02, 0.78 ± 0.04, 0.69 ± 0.02, 0.66 ± 0.01, 0.36 ± 0.02, n = 4
glaber n=6 n=6 n=6 n=6 n=4 n=4 n=4
Bathyergus suillus 3.90 ± 0.15, 16.80 ± 0.61, 0.90 ± 0.06, 0.31 ± 0.00, N/A N/A N/A N/A
n=2 n=2 n=2 n=2
Cryptomys 3.53 ± 0.08, 10.04 ± 0.47, 0.76 ± 0.04, 0.13 ± 0.03, 1.04 ± 0.07, 0.85 ± 0.04, 0.82 ± 0.10, 0.33 ± 0.02, n = 2
hottentotus n=3 n=3 n=3 n=3 n=3 n=3 n=3
Fukomys micklemi 3.84, n = 1 10.87, n = 1 0.57, n = 1 0.13, n = 1 0.94, n = 1 0.79, n = 1 0.83, n = 1 0.43, n = 1
Georychus 3.50, n = 1 11.92, n = 1 0.76, n = 1 0.28, n = 1 1.18, n = 1 0.84, n = 1 0.86, n = 1 0.42, n = 1
capensis
Heliophobius 3.02 ± 0.26, 10.23 ± 0.39, 0.84 ± 0.11, 0.17 ± 0.01, N/A N/A N/A N/A
argenteocinereus n=2 n=2 n=2 n=2
Values cited are means ± standard deviation. The measurements of radii of curvature of the cochlear turns at base and apex (Rbase, Rapex) follow the
method given in [40]; measurements of semicircular canal (SCC) radii of curvature follow [41]. Heterocephalus measurements were from specimens over
100 days old only. The semicircular canals were not measured in Bathyergus and Heliophobius due to poor scan resolution.

externally demarcated. The three Cryptomys specimens varied between the Heterocephalus and
Georychus conditions.
Discussion
Ctenohystrica ear features in bathyergids
The Ctenohystrica rodent group, to which the bathyergids belong, has some very characteristic
middle ear features [43, 44]. These include a malleus with a ‘bullet-shaped’ head and a very
reduced anterior process, malleo-incudal fusion and a relatively loose stapedio-vestibular artic-
ulation, which has been found to be synovial rather than fibrous in the species examined. Cer-
tain species such as chinchillas have very capacious middle ear cavities. Loose articulations and
large cavities increase middle ear compliance, which aids low-frequency sound transmission.
This is thought to be advantageous to arid-region species because low-frequency sound propa-
gates best in such environments [45, 46]. It is therefore possible that the ancestral Ctenohy-
strica rodents evolved in an arid habitat. However, ‘low-frequency’ ears are also considered to
be advantageous in subterranean environments (see Introduction). It has been noted that cer-
tain features of Ctenohystrica rodents, including their generally blunt body-shapes and small
pinnae and tails, are also characteristic of subterranean mammals, while a unique carpal bone
found in the hystricognath subgroup is considered an adaptation to digging [47]. This raises
the possibility that the ancestral Ctenohystrica rodents were fossorial. Whether or not this was
the case, it is no surprise that tuco-tucos (Ctenomys species: [24, 48]) and the coruro (Spalaco-
pus: [22]), which are subterranean, retain typical, low-frequency, Ctenohystrica-type ears. Our
findings, which confirm and extend previous descriptions of the ear in the Bathyergidae (see
Introduction), lead to the conclusion that the ears of this family of mole-rats also retain many
ancestral Ctenohystrica features. However, bathyergids are unique in that their malleo-incudal
fusion zone extends down the long process of the incus [36].
The tensor tympani muscle can be well-developed in Ctenohystrica rodents, but the stapedius
is typically reduced or absent [43, 44]. In the present study, a stapedius was not found in any
bathyergid, consistent with several previous descriptions [20, 34, 35, 49]. However, some other
reports suggest that a small stapedius may be retained in some individual mole-rats [26, 27, 36]
Mason [43] hypothesized that a very compliant stapedio-vestibular articulation could not
be stiffened to a functionally relevant extent by the stapedius muscle. This may have led that
muscle to degenerate, while a compensatory role of the tensor tympani could in principle be
augmented by malleo-incudal fusion. These Ctenohystrica characteristics may therefore be
functionally linked. Not only is the stapedio-vestibular articulation synovial in at least some
bathyergids (this could not be assessed in all species) but the gap between stapes footplate and
oval window is unusually wide (Fig 4A), which is expected to increase compliance still further.
A very wide gap has previously been documented in the spalacid mole-rat Spalax [23] and the
gerbil Gerbillurus [42].
Among the Ctenohystrica, the cochleae of Ctenodactylus and Petromus have 3.5 turns [34],
and a ‘high-spiralled’ cochlea with a similarly large number of turns is also characteristic of
caviomorph rodents [22, 41, 50, 51]. We found up to 4 cochlear turns in the bathyergids we
examined (Table 3), while Müller et al. [30] found 4 to 4.5 turns in Fukomys anselli. High-spi-
ralled cochleae therefore appear to be widespread within the Ctenohystrica, and may well be
primitive both for this group and for the Bathyergidae.
Unusual features of the Heterocephalus ear

Although bathyergid ears have many features in common, the naked mole-rat stands apart
from the others in terms of the smaller relative size of its epitympanic recess, the elaboration of

its mastoid subcavities (Figs 5 and 6), the spongy bone of its ossicular processes (Fig 3B) and
the structure of its stapes, which lacks an intercrural foramen in the adult (Fig 7A). Its inner
ear is unusual in its low number of cochlear turns (Fig 8) and, probably related to this, its short
cochlear canal length (Table 3).
A ‘columelliform’ stapedial morphology was found in one of two specimens of Georychus
examined by Burda et al. [20], but not in the specimen that we examined. We are unaware of
any other report of a columelliform stapes in a rodent but they are found in certain other
mammals including monotremes, some marsupials, pangolins, some edentates and many
aquatic species [36, 52, 53]. The existence of a tiny foramen in some neonatal Heterocephalus
specimens (Fig 7A) may reflect the presence of a stapedial artery in the embryo. This artery
regresses in many mammals, typically leaving an empty intercrural foramen [52] although a
bony spicule passes through it in caviomorph rodents [54]. The arch made by the stapedial
crura is thought to be mechanically advantageous in resisting bending [53], and the relative
development of the stapes crura may be influenced by the pull of the stapedius muscle [55].
Heterocephalus lacks this muscle, but so too do many other Ctenohystrica rodents which none-
theless retain normally-shaped stapedes, including other bathyergids.
Molecular phylogenetic studies suggest that Heterocephalus forms a basal outgroup to the
rest of the Bathyergidae [1, 56]. This genus is sometimes placed in a separate subfamily, the
Heterocephalinae, with the other genera forming the Bathyerginae [57]. It is therefore possible
that Heterocephalus retains the primitive ear characteristics for its family, while the bathyer-
gines are derived. However, the bathyergines are more similar to other Ctenohystrica species
in terms of their ossicular process ossification, stapedial morphology and cochlear coiling.
This suggests that, in these respects at least, it is Heterocephalus which shows the derived con-
dition. It is not clear what the ancestral middle ear cavity morphology would be for the Cteno-
hystrica because this varies so much among different rodent species, even within the same
family [23, 34, 42]. Heterocephalus is the smallest bathyergid: although cochlear coiling is unre-
lated to body size among mammals in general [41], it is possible that within the Bathyergidae
the number of turns is somehow restricted by size, and that size affects middle ear cavity
structure.
These unusual characteristics of the naked mole-rat ear are discussed again later, in the con-
text of possible degeneration of its peripheral auditory system.
Post-natal ear development in Heterocephalus

At birth, the naked mole-rat has a simple middle-ear cavity structure based on a tympanic cav-
ity and epitympanic recess only. The posterior part of the tympanic cavity goes on to invade
the area around the semi-circular canals, creating the three mastoid subcavities (Fig 6). Post-
natal expansion of the middle ear cavity into the mastoid region has been documented in the
kangaroo-rat Dipodomys [58], and a general increase in middle ear cavity volume has been
found in murid rodents [59–61]. Subcavities were not distinguished in the murid studies. The
extent of expansion of the mastoid subcavities varies between individual naked mole-rats in a
way that does not relate closely to age (Table 2), and septa were also found to be variably devel-
oped. Intraspecific variation in mastoid cavity structure has been noted in gerbils [62] and dor-
mice [63], but in these studies the ages of the specimens were not recorded so the differences
could, in principle, have been ontogenetic.
The malleus and incus of the naked mole-rat appear to be better-developed at birth than
those of other altricial rodents [58, 59, 61, 64], but the manubrium of the malleus and distal
long process of the incus remain cartilaginous. Once ossified, these processes are composed of
spongy bone with large vascular spaces, and they were not remodelled into compact bone even

in our oldest specimen (65 months). Stapes structure changes significantly post-natally: the
head and neck ossify, the bony footplate expands slightly and the body is slimmed down. Dur-
ing this process the intercrural foramen, if still present, is lost. Substantial postnatal remodel-
ling of the stapes has also been observed in the rat, kangaroo rat and rabbit, but in these
animals the intercrural foramen widens [58, 59, 65].
Unlike the middle ear cavity, the inner ear of Heterocephalus had achieved its adult dimen-
sions in the neonatal specimens. It seems to be a common trend among mammals that the
bony labyrinth changes little, post-ossification (see e.g. [65–67]). The membranous semicircu-
lar canals of Fukomys mole-rats have also been found not to expand postnatally [31].
The effect of the middle ear on hearing range

An electrical analogue model of the middle ear constructed by Hemilä et al. [68] proved to be
surprisingly accurate in predicting high-frequency hearing limits in a wide range of mammals.
The three anatomical parameters used in the model were tympanic membrane area, ossicular
mass and a measure of malleus length. Mason [21] put measurements from Heterocephalus
(taken from [38]) into this model and predicted a high-frequency hearing limit of 99.7 kHz at
60 dB SPL, a high value reflecting the small size of the ear. This prediction greatly exceeds the
mole-rat’s behavioural hearing limit of 12.8 kHz [9]. These results indicate that the gross struc-
tural dimensions of the Heterocephalus middle ear should be adequate for the transmission of
high, ultrasonic frequencies, suggesting that the limitation to high-frequency hearing in this
animal lies elsewhere.
In terms of low-frequency hearing, middle ear cavity volume is particularly important to
small mammals [25]. This is because cavity compliance, which is proportional to volume,
often dominates overall middle ear compliance in these animals [69], and compliance deter-
mines sound transmission at low frequencies. A laboratory rat, which is not a low-frequency
specialist, has a middle ear cavity volume around 60 mm3 [59]. The spalacid mole-rat Spalax
ehrenbergi has a middle ear cavity which is of moderate size (around 100 mm3: [23]). The hear-
ing of Spalax is more acute than that of rats at frequencies below around 1 kHz, but the low-
frequency hearing of arid-region rodents such as gerbils, kangaroo rats and chinchillas tends
to be better still [9, 13, 70]. These species have much more capacious middle ear cavities: ger-
bils have cavity volumes ranging from 145–1017 mm3 [42, 62], kangaroo rats of the genus
Dipodomys have cavity volumes from 430–1410 mm3 [71], while the cavity volume of the chin-
chilla can exceed 1500 mm3 [54, 72].
Despite the expansion of its middle ear cavity into the mastoid region, the total middle ear
cavity volume in Heterocephalus is only around 25–30 mm3 (Table 2), which is under the value
expected for a terrestrial mammal of its body size [21]. Bathyergus suillus, which is ten times
the weight of the naked mole-rat, has a cavity volume around 440 mm3, while the other bath-
yergids examined are of intermediate size and have intermediate cavity volumes (Table 2).
Assuming that cavity compliance is limiting, we would expect low-frequency hearing limits to
correlate inversely with body size in this group, but the data required to test this hypothesis are
lacking. Although postnatal expansion of the cavity into the mastoid region in bathyergids
might be the result of selective pressure to improve low-frequency sound transmission, the
small middle ear cavity of the naked mole-rat does not suggest that its low-frequency hearing
could be particularly acute. This conclusion is borne out by behavioural hearing studies, which
have found the thresholds of Heterocephalus at frequencies under 1 kHz to be somewhere
between those of laboratory rats and the arid-region rodents mentioned above [9]. The fact that
the rat has a larger total cavity volume than Heterocephalus but poorer low-frequency hearing
suggests that the rat’s much stiffer ossicular suspension dominates its middle ear compliance.

Subcompartmentalisation of the middle ear is not expected to have a significant impact at

low frequencies but might result in mid-frequency resonances. One significant resonance has
been observed and modelled in the cat, the middle ear of which is partially divided into two
large subcavities [73], and more resonances have been found in the middle ear response of the
chinchilla, which has many smaller subcavities [45]. However, no such resonances were
observed in the ear of the gerbil Meriones [69], which has a similar cavity structure to bathyer-
gids [42]. While it is possible that the partial divisions in gerbil and bathyergid middle ears
serve purely structural purposes, further experimental work is needed to confirm that they
have no significant functional effect.
Bone conduction
Foot-drumming and stomping are used for intraspecific communication in several species of
bathyergids, but these behaviours have not been observed in Heterocephalus or Heliophobius
(see [74] for a review). Ground vibrations generated in this way could be detected by the
somatosensory system, or alternatively by the ear through a form of bone conduction. Certain
species of golden moles [75] and talpid moles [76] have relatively enormous mallei which likely
underpin a form of inertial bone conduction. The mole-rat Spalax lacks notably enlarged ossi-
cles, but other features of its ear region may augment alternative modes of bone-conducted
hearing [23, 77].
The malleoincus unit of Heterocephalus weighs around 0.47 mg (n = 2), while its stapes
weighs around 0.026 mg (n = 1) [38]. These values are slightly smaller than expected for its
body mass [21], which is quite unlike the situation in golden moles. Heterocephalus shares cer-
tain features of its middle ear with that of Spalax, however, notably a loose articulation between
malleus and skull, a flexible incudo-stapedial articulation and a particularly wide annular liga-
ment of the stapes. It has been suggested that these features may reduce constraint to the point
where the ossicular chain could vibrate in response to skull vibrations [23]. However, any role
of this in promoting bone conduction will be limited by the small mass of the ossicles in these
animals.
Inner ear structure and function

Ekdale [41] found that bony labyrinth volume and cochlear canal length both correlate with
body mass among mammals in general. The volumes measured here in bathyergids fit his
mammalian trend-line well (Fig 9A). The cochlear canal length of Heterocephalus also falls
very close to Ekdale’s trend-line, but canal lengths in the other mole-rats examined (the bath-
yergines) fall above it (Fig 9B). This likely relates to increased cochlear coiling in the bathyer-
gines. However, cochlear canal length should approximate basilar membrane length, and for
the five species in common, Ekdale’s cochlear canal length measurements average 65% of the
basilar membrane length measurements collected from the literature by Manoussaki et al.
[40]. If Ekdale’s values are all revised upwards, the cochlear canal lengths of the bathyergines
would be very close to expected, while that of Heterocephalus would be shorter than expected
for its body size.
West [80] identified strong correlations between upper and lower hearing limits and inner
ear dimensions, for mammals in general:
log10 ðLFLÞ ¼ 1:76 1:66log10 ðBML CTÞ
log10 ðHFLÞ ¼ 2:42 0:994log10 ðBML=CTÞ
where LFL = low-frequency hearing limit at 60 dB SPL (kHz), HFL = high-frequency hearing

Fig 9. Relationships between inner ear measurements and body mass. Relationships between the natural logarithms of
inner ear measurements and body mass. Plot A considers the total volume of the bony labyrinth. Plot B considers the length of
the cochlear canal. Plots C, D and E consider the radii of curvature of the anterior semicircular canal (SCC), lateral semicircular
canal and posterior semicircular canal respectively. Plot F shows the mean cross-sectional diameter of all three semicircular
canals. In each plot, data for 28 extant mammalian species (black crosses) were taken from Ekdale (2013), and a least squares
regression line fitted as in that paper. Data-points representing mean values for bathyergids were superimposed as red symbols:
Heterocephalus individuals over 100 days old (square), Bathyergus (open triangle), Cryptomys (solid triangle), Fukomys (circle),
Georychus (diamond) and Heliophobius (plus). Body mass data, where available, were taken from Table 1 for the individual
animals concerned. Unrecorded body masses were estimated as follows: Fukomys 89 g, a mean value for animals from the same
laboratory colony [78]; Cryptomys 82 g, Georychus 211 g [79]. Inner ear data were taken from Tables 2 and 3.

limit at 60 dB SPL (kHz), BML = basilar membrane length (mm) and CT = number of cochlear
turns. Using these equations, and using cochlear canal length as an estimate of basilar mem-
brane length, one can predict a low-frequency hearing limit of 585 Hz and a high-frequency
limit of 125 kHz in Heterocephalus (Table 4). These values compare poorly with the naked
mole-rat’s behavioural hearing limits of 65 Hz and 12.8 kHz respectively [9]. Manoussaki et al.
[40] found a relationship between the radii of curvature at the cochlear base and apex and low-
frequency hearing limits in mammals:
LFL ¼ 1:507 exp½ 0:578ðRbase =Rapex 1Þ
where Rbase is the radius of curvature of the cochlear basal turn (mm) and Rapex is the radius of
curvature of the cochlear apical turn (mm). Using this equation, the low-frequency hearing
limit of Heterocephalus should be 291 Hz (Table 4), which is still much higher than the
observed value. These results suggest that normal mammalian structure-function relationships
do not hold for Heterocephalus, which has better low-frequency hearing but much poorer
high-frequency hearing than expected. Audiograms are not available for the other bathyergids
examined here, but predictions from the three equations are included in Table 4 for compara-
tive purposes.
Soft tissue structures of the cochlea could not be observed in our CT scans, but were
described in Fukomys anselli by Müller et al. [30]. Unusual features included a scala tympani
and helicotrema of very small cross-sectional areas, and a very low spiral ganglion cell density.
The basilar membrane is also of almost constant width and thickness over nearly half of its
length, and frequencies between 0.6 and 1 kHz were found to be tonotopically over-repre-
sented. However, there is no evidence for the particularly sharp tuning which would be
expected of a low-frequency “acoustic fovea” [81]. Müller et al. [30] noted that F. anselli has a
more limited range of hearing than predicted from anatomical correlations.
Turning to the vestibular system, the posterior limb of the lateral semicircular canal may
enter the vestibule directly (e.g. in Heterocephalus), merge with the posterior ampulla (e.g. in
Fukomys) or show an intermediate morphology (Fig 8). Direct entry into the vestibule was
found in the mouse (Mus) and guinea pig (Cavia), and was reconstructed as primitive both for
Rodentia and placental mammals as a whole [41]. This may well be so, but the diversity of phe-
notypes found here in just one family illustrates the need for caution in the interpretation of
the direction of inner ear evolution based on the sampling of a few, isolated species. The nature
of the entry of the lateral canal into the vestibule influences whether there is a space between
Table 4. Predictions of hearing limits in bathyergid mole-rats.

Species LFL from Manoussaki et al. (2008) equation, LFL from West (1985) equation, HFL from West (1985) equation,
kHz kHz kHz
Heterocephalus glaber 0.291 0.585 125
Bathyergus suillus 0.502 0.056 61.6
Cryptomys hottentotus 0.092 0.154 93.1
Fukomys micklemi 0.213 0.117 93.5
Georychus capensis 0.559 0.118 77.8
Heliophobius 0.154 0.194 78.2
argenteocinereus
LFL = 60 dB SPL low-frequency hearing limit; HFL = 60 dB SPL high-frequency hearing limit. Mean values taken from Table 3 were used in the calculations;
the length of the cochlear canal was used as an estimate of basilar membrane length. A comparison of the predictions for Heterocephalus with audiogram
data suggests that there is little reason to be confident about these values (see text).

lateral and posterior canals through which a posteromedial mastoid cavity can emerge, but the
functional implications of these differences in lateral canal morphology are otherwise
unknown.
One of the most striking features of the bony labyrinth of bathyergids is the ‘short and fat’
appearance of the semicircular canals (Fig 8). Our measurements (Table 3) may be compared
to values for a wide range of mammals tabulated by Ekdale [41]. The radii of curvature of each
of the semicircular canals of bathyergids are smaller than expected for their body masses (Fig
9C,9D and 9E), but the canal cross-sectional diameters are substantially greater than expected
(Fig 9F). The ratios of canal diameter to radius of curvature are as a result very high. From
Ekdale’s data, only the pangolin Manis and an extinct elephantimorph of uncertain identity
have canals of similar proportions. Among subterranean mammals, data tabulated by Crump-
ton et al. [82] show that certain golden moles and the marsupial mole Notoryctes can be added
to that list, while the membranous semicircular canals of the spalacid mole-rat Spalax have
similar dimensions to those of Fukomys [33]. ‘Short and fat’ canals have evidently evolved sev-
eral times in parallel among subterranean mammals, although they are not found in all
species.
According to the theoretical model developed by Oman et al. [83], mechanical sensitivity
of a given semicircular canal, in terms of displacement of the cupula per unit head angular
velocity, will be proportional to an ‘average radius of curvature’ of the entire fluid stream-
line and to its average squared cross-sectional area, weighted according to deviations from
perfect circularity. These measurements should include not just the narrow section of duct
but the entire circuit, including ampulla and utricular cavity, and should be based on the
membranous labyrinth rather than the bony labyrinth considered here. The Oman et al.
model has been used to predict that the sensitivities of the wide semicircular canals of bath-
yergid and spalacid mole-rats are enhanced in comparison to those of murid rats and guinea
pigs [32, 33].
Does the ear of Heterocephalus show signs of degeneration?

Anatomical measurements of peripheral auditory structures can be used to predict the upper
and lower hearing limits of mammals. In the cases of the Hemilä et al. [68] model and the
West [80] correlation equations, smaller ears generate higher-frequency predictions. Smaller
animals (with smaller ears) do indeed tend to have hearing ranges shifted to higher frequen-
cies, but the predictions are strikingly inaccurate when it comes to the naked mole-rat. Hetero-
cephalus has an ear which, from its small dimensions, would appear suitable for the
transmission of high, ultrasonic frequencies, but a hearing range restricted to very low fre-
quencies. It has been suggested that the central auditory system is responsible for the high
thresholds and rudimentary localisation abilities observed in this animal [9, 10], in which case
the structure of its peripheral auditory system might give no indication of its hearing limita-
tions. However, we have identified some features of the ear of the naked mole-rat which might
contribute to poor hearing and/or could be considered degenerate:
1. Heterocephalus lacks pinnae to channel sound into the ear and provide ‘pinna cues’ to aid
localisation [9]. Its external ear canals are very narrow and semi-occluded (Fig 1B), which
will reduce the sound energy reaching the tympanic membrane. Cerumen has been found
in the bony external meatus of several other bathyergid species [26].
2. The manubrium of the malleus and the distal tip of the incudal long process have the
spongy and poorly-ossified appearance of newly-developed bone, even in adult animals
(Fig 3B).

3. The columelliform stapes has a thin, flexible body which appears to lack the structural stiff-
ness typical of this ossicle. It articulates with a tiny lenticular apophysis mounted on a
weakly-developed long process of the incus (Fig 4A). Increased flexibility within the ossic-
ular chain can result in reduced sound energy transmission at high frequencies [84].
4. Reduced cochlear coiling and cochlear canal length in Heterocephalus might conceivably
represent a degenerate feature, if having a ‘high-spiralled’ cochlea was the ancestral condi-
tion for bathyergids.
Like that of Heterocephalus, the hearing range of Fukomys anselli is known to be very lim-
ited [11, 12], and it may be that hearing is poor in all members of the Bathyergidae. Points 2 to
4 above suggest that the hearing of Heterocephalus might be worse than that of the other bath-
yergids examined. However, these anatomical differences could be influenced by the small size
of the naked mole-rat, its phylogenetic position as the most basal offshoot of the family or the
fact that our specimens came from laboratory colonies rather than the wild. Our specimens
were also relatively young for naked mole-rats, albeit of comparable ages to the approximately
one-year-old animals which had their hearing tested by Heffner & Heffner [9].
Increased intraspecific variability is a possible indicator of a lack of selective pressure con-
straining ear structure. If the variation in the extent of pneumatisation of the mastoid region
among adult Heterocephalus proves to exceed that found in other small mammals, this feature
could be added to the list above. Our animals were from captive colonies, but the differences
in stapes structure found here in Cryptomys specimens and the differences in cochlear struc-
ture in Heliophobius specimens, all collected from the wild, could be interpreted along similar
lines. Significant intraspecific variability in stapes structure has previously been recorded in
Georychus [20] and in the Spalax ehrenbergi superspecies [85, 86]; Spalax is known to have
‘degenerate’ hearing similar to that of Heterocephalus [13]. We did not find the same level of
intraspecific variability in all middle and inner ear structures, however, which could indicate
that some features retain more functional importance than others.
Although the anatomical features noted above are suggestive of degeneration, in accordance
with what is known about the hearing of the naked mole-rat, none are conclusive. Given the
dangers of post-hoc interpretations of this nature, experimental studies are necessary to test
these ideas.
Supporting Information
S1 Table. Middle and inner ear measurements. Worksheet 1 contains the measurements
made on individual animals from which mean values were calculated. Worksheet 2 contains
the x,y,z coordinates of points along the cochlear ducts, which were used to calculate some of
the inner ear parameters represented in Table 3.
(XLSX)
Acknowledgments
The authors would like to record their immense gratitude to Phil Cox for sending us speci-
mens and CT scans, and to Dominique Adriaens, Paul van Daele, Nigel Bennett and Radim
Šumbera for originally providing the specimens concerned. Matt Lowe and Robert Asher at
the University Museum of Zoology, Cambridge, kindly allowed access to that collection. We
thank Mrs. Anita Shelley for making the histological slides, members of the Smith lab for help
with specimen preparation, the Cambridge Biotomography Centre for the use of their scanner
and the two reviewers for commenting on this paper. This work was supported by a John Ray

Trust Summer Research Grant to H.L.C. and a Royal Society Research Grant (RG130110) to E.
S.J.S.
Author Contributions
Conceptualization: MJM.
Formal analysis: MJM.
Funding acquisition: ESJS HLC.
Investigation: MJM HLC.
Methodology: MJM HLC.
Project administration: MJM.
Resources: ESJS.
Supervision: MJM.
Visualization: MJM.
Writing – original draft: MJM HLC.
Writing – review & editing: MJM HLC ESJS.
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Ear Structures of The Naked Mole-Rat, (Rodentia: Bathyergidae)

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Ear Structures of The Naked Mole-Rat, (Rodentia: Bathyergidae)

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RESEARCH ARTICLE

Ear Structures of the Naked Mole-Rat,

1 University of Cambridge, Department of Physiology, Development & Neuroscience, Cambridge, United

Funding: Support was provided by a John Ray

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Materials and Methods

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Table 1. Details of specimens examined in this project, including CT scan information.

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Micro-computed tomography and reconstruction

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The external and middle ears of Heterocephalus

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Table 2. Measurements of middle ear subcavity and bony labyrinth volumes.

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The bony labyrinth in Heterocephalus

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The ear region in very young Heterocephalus (<100 days old)

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The ears of other bathyergid species

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Table 3. Inner ear measurements, made from CT scan data.

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Unusual features of the Heterocephalus ear

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Post-natal ear development in Heterocephalus

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The effect of the middle ear on hearing range

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Subcompartmentalisation of the middle ear is not expected to have a significant impact at

Inner ear structure and function

log10 ðHFLÞ ¼ 2:42 0:994log10 ðBML=CTÞ

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LFL ¼ 1:507 exp½ 0:578ðRbase =Rapex 1Þ

Table 4. Predictions of hearing limits in bathyergid mole-rats.

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Does the ear of Heterocephalus show signs of degeneration?

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LFL ¼ 1:507 exp½ 0:578ðRbase =Rapex 1Þ