Comparative anatomy of the vocal apparatus in bats and implications for the diversity of laryngeal echolocation

Abstract

Most of over 1400 extant bat species produce high-frequency pulses with their larynx for echolocation. However, the debate about the evolutionary origin of laryngeal echolocation in bats remains unresolved. The morphology of the larynx is known to reflect vocal adaptation and thus can potentially help in resolving this controversy. However, the morphological variations of the larynx are poorly known in bats, and a complete anatomical study remains to be conducted. Here, we compare the 3D laryngeal morphology of 23 extant bat species of 11 different families reconstructed by using iodine contrast-enhanced X-ray microtomography techniques. We find that, contrary to previously thought, laryngeal muscle hypertrophy is not a characteristic of all bats and presents differential development. The larynges of Pteropodidae are morphologically similar to those of non-bat mammals. Two morphotypes are described among laryngeal echolocating bats, illustrating morphological differences between Rhinolophoidea and Yangochiroptera, with the main variations being the cricothyroid muscle volume and the shape of the cricoid and thyroid cartilages. For the first time we detail functional specialization for constant frequency echolocation among Rhinolophoidea. Lastly, the nasal-emitting taxa representing a polyphyletic group do not share the same laryngeal form, which raises questions about the potential modular nature of the bat larynx.

Full text

Zoological Journal of the Linnean Society, 2024, XX, 1–20
hps://doi.org/10.1093/zoolinnean/zlad180
Advance access publication 19 January 2024
Original Article
Received 25 June 2023; revised 9 October 2023; accepted 6 November 2023
Original Article
Comparative anatomy of the vocal apparatus in bats and
implications for the diversity of laryngeal echolocation
Nicolas LMBrualla1,, Laura ABWilson2,3,*,, Vuong TanTu4,5,, TaroNojiri6,,
Richard TCarter7,, ongchaiNgamprasertwong8,, anakulWannaprasert8,,
MichaelDoube1,, DaiFukui6,, DaisukeKoyabu1,9,*,
1Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong,
Hong Kong SAR, China
2School of Archaeology and Anthropology, College of Arts and Social Sciences, e Australian National University, Acton, ACT 2601, Australia
3School of Biological, Earth and Environmental Sciences, University of New South Wales, Kensington, NSW 2052, Australia
4Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam
5Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
6Graduate School of Agricultural and Life Sciences, e University of Tokyo, Tokyo, Japan
7Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee, USA
8Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, ailand
9Research and Development Center for Precision Medicine, University of Tsukuba, Tsukuba, Japan
*Corresponding authors. School of Archaeology and Anthropology, College of Arts and Social Sciences, e Australian National University, Acton, ACT 2601, Australia;
School of Biological, Earth and Environmental Sciences, University of New South Wales, Kensington, NSW 2052, Australia. E-mail: laura.wilson@anu.edu.au;
Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong SAR,
China; Research and Development Center for Precision Medicine, University of Tsukuba, Tsukuba, Japan. E-mail: dsk8evoluxion@gmail.com
ABSTRACT
Most of over 1400 extant bat species produce high-frequency pulses with their larynx for echolocation. However, the debate about the evolutionary
origin of laryngeal echolocation in bats remains unresolved. e morphology of the larynx is known to reect vocal adaptation and thus can poten-
tially help in resolving this controversy. However, the morphological variations of the larynx are poorly known in bats, and a complete anatomical
study remains to be conducted. Here, we compare the 3D laryngeal morphology of 23 extant bat species of 11 dierent families reconstructed by
using iodine contrast-enhanced X-ray microtomography techniques. We nd that, contrary to previously thought, laryngeal muscle hypertrophy is
not a characteristic of all bats and presents dierential development. e larynges of Pteropodidae are morphologically similar to those of non-bat
mammals. Two morphotypes are described among laryngeal echolocating bats, illustrating morphological dierences between Rhinolophoidea
and Yangochiroptera, with the main variations being the cricothyroid muscle volume and the shape of the cricoid and thyroid cartilages. For the
rst time we detail functional specialization for constant frequency echolocation among Rhinolophoidea. Lastly, the nasal-emiing taxa repre-
senting a polyphyletic group do not share the same laryngeal form, which raises questions about the potential modular nature of the bat larynx.
Keywords: Chiroptera; cricothyroid muscle; functional adaptation; larynx; mammalian nasopharyngeal morphology; vocal tract; X-ray
microtomography
INTRODUCTION
Bats (order Chiroptera) constitute the second largest mamma-
lian order aer Rodentia in term of species diversity with over
1400 described species (Simmons and Cirranello 2020). ey
are unique mammals that are capable of self-powered ight and
can use echolocation to navigate in dark environments, enabling
them to colonize all continents except Antarctica and to exploit,
and radiate into, unoccupied ecological niches (Grin 1944,
Rayner 1988, Teeling 2009).
Extant bats are classied into two suborders: Yinpteroc-
hiroptera (yinpterochiropterans) uniting the family
Pteropodidae (pteropodids) and the super family Rhinolop-
hoidea (rhinolophoids), which include Rhinolophidae,
Rhinonycteridae, Hipposideridae, Rhinopomatidae,
Craseonycteridae, and Megadermatidae; and Yangochiroptera
(yangochiropterans) comprising all other recognized families
(Simmons and Cirranello 2020). Members of yangochiropterans
and rhinolophoids conduct echolocation by generating
© 2024 e Linnean Society of London.
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2 • Brualla et al.
high-frequency sound produced by the larynx, whereas
pteropodids do not echolocate or employ a primitive form of
echolocation using tongue and/or wing clicks (Yovel et al. 2011,
Boonman et al. 2014, Chaverri et al. 2018). Among laryngeal
echolocating bat families, there are considerable dierences in
echolocation strategies, such as constant frequency (CF), fre-
quency modulated (FM), high-duty cycles (HDC), low-duty
cycles (LDC), nasal or oral emiers (e.g. Fenton et al. 2012).
ese dierences between bat families have raised questions
regarding the origin and evolution of laryngeal echolocation.
Nowadays, two competing hypotheses are debated. e rst ex-
plains a single and common origin of laryngeal echolocation for
all bats with a common ancestor able to laryngeally echolocate,
followed by a loss of the ability in the pteropodids (e.g. Veselka
et al. 2010, Wang et al. 2017). e second hypothesis sup-
ports the idea that laryngeal echolocation appeared independ-
ently multiple times in the dierent bat clades (e.g. Davies et al.
2013, Nojiri et al. 2021). is second hypothesis implies that
pteropodids conserved the same characteristics as the common
ancestor of all bats and that, independently, both rhinolophoids
and yangochiropterans acquired the ability of laryngeal echo-
location by convergence.
Many researchers have examined variation in the ear (e.g.
Davies et al. 2013, Wang et al. 2017, Nojiri et al. 2021), but
very few have undertaken comparative studies of the sound-
producing organ, the larynx (Brualla et al. 2023), probably due
to the dierent preservation of the two organs through time. e
inner ear is located in the petrosal part of the temporal bone,
which is one of the stiest, and therefore best-preserved bones
of the cranium when studying museum specimens of extant spe-
cies and fossils. In contrast, the larynx mainly comprises delicate
so tissues that are not preserved in fossils or dried museum spe-
cimens. Laryngeal so tissues in bats are small but structurally
very complex because most bats are small, making macroscopic
dissections dicult. Today, researchers may study small organ-
isms in a non-destructive way by using a combination of staining
protocols for so tissues and X-ray microtomography scanning
(e.g. Metscher 2009, Gignac et al. 2016, Santana et al. 2019,
Nojiri et al. 2021). ese technologies enable laryngeal anatomy
to be studied in greater detail, potentially yielding critical infor-
mation to further resolve the origin of laryngeal echolocation in
bats.
e larynx is an essential mammalian organ due to its con-
tributions to several vital functions, including vocalization (e.g.
Harrison 1995, Shiba 2010). e morphology of the mam-
malian larynx is described as relatively conserved (Harrison
1995, Saigusa 2011), with only minor morphological changes
having been reported (Brualla et al. 2023). e mammalian
larynx is comprised of four cartilages (one thyroid, one cri-
coid, and a pair of arytenoids) (Fig. 1) that support seven pairs
of intrinsic muscles (cricoarytenoid dorsalis, cricoarytenoid
lateralis, cricothyroid, oblique arytenoid, transverse arytenoid,
thyroarytenoid, and vocalis muscles) (Negus 1949, Harrison
1995, Hoh 2005, 2010, Saigusa 2011, König and Liebich 2020,
Brualla et al. 2023). e muscle contraction on the larynx acts to
incline the thyroid caudally and tilt the arytenoid cartilages lat-
erally, adducting or abducting the vocal folds and puing them
in position to vibrate and produce sound (e.g. Harrison 1995,
Finck and Lejeune 2010, Metzner and Müller 2016, Brown and
Riede 2017). e MyoElastic-AeroDynamic (MEAD) theory
adds that the elasticity of the vocal folds plays a role in the dif-
ferent frequencies emied by mammals (van den Berg 1958,
Brudzynski 2009, Brown and Riede 2017, Švec et al. 2021).
Laryngeal specializations to dierent ecologies and behav-
iours, such as larger laryngeal structures, presence of air sacs,
or of a dorsal tracheal membrane, have been found in several
mammals (Harrison 1995, omas et al. 2004, Reidenberg and
Laitman 2010). Because bats are volant mammals that produce
high-frequency vocalizations, in addition to common morpho-
logical paerns shared with other mammals, their laryngeal
structures are expected to have some unique traits associated
with the adaptative evolution of vocalization (Suthers 2004,
Metzner and Schuller 2010; Metzner and Müller, 2016; Brualla
et al 2023). For instance, bats have been described as having a
similar laryngeal morphology to other mammals, presenting
only minor variations for aspects of the cartilages (shape and
mineralization paerns) and some modications in the muscle
size and activity (e.g. Denny 1976, Harrison 1995, Elemans et
al. 2011, Carter 2020). Hypertrophied intrinsic muscles have
been linked to vocalization needs, especially for laryngeal echo-
location. High-frequency vocalizations observed in bats require
high subgloal pressure, which is possible because of the greatly
developed laryngeal muscles regulating the tension on the vocal
folds (Elemans et al. 2011, Ratclie et al. 2013, Grinnell et al.
2016). Indeed, laryngeal echolocation is produced by the larynx
from the contraction of the dierent intrinsic laryngeal muscles
that twist the arytenoid cartilages and tilt the thyroid cartilage.
is muscular action closes and tenses the vocal folds that pos-
sess thin vocal membranes that vibrate extremely fast, enabling
bats to produce high frequency sounds (e.g. Metzner and Müller,
2016).
Our literature review of pioneer studies of bat laryngeal
morphology (e.g. Elias 1907, Denny 1976), indicated that (i) the
larynges of bats can be classied into two main morphotypes,
one specic to rhinolophoids and the other found only in their
relatives, belonging to the superfamily Vespertilionoidea of
yangochiropterans (vespertilionoids), and that (ii) the dierent
forms of the laryngeal components found in dierent bat taxa
could be correlated with their variations of frequencies, rate of
calls, and amplitudes during sound production (Brualla et al.
2023). As such, the precise descriptions, comparisons and in-
terpretations of the potential dierent laryngeal forms of bats
may be used for elucidating the evolutionary history of laryn-
geal echolocation. However, the anatomy of the larynx and its
relation to ecological diversity remains to be investigated for
most bat species (Brualla et al. 2023). Illustrating the amount
of anatomical variation among bat larynges may challenge the
commonly held idea that mammalian larynges are morpho-
logically uniform and evolutionarily constrained. Formulizing
the paerns of laryngeal forms could also result in beer under-
standing of the evolutionary success of bats, by demonstrating
great modications of the larynx in relation to dierent niches,
ecologies, but also vocal productions.
Here, for the rst time, we describe the paerns and mag-
nitude of variation in the bat larynx (cartilages and muscles),
discussing the diversity of forms and their potential collation
into morphotypes, using iodine contrast-enhanced X-ray
microtomography, and virtual dissection (Fig. 1). In this study,
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Comparative anatomy of the vocal apparatus in bats • 3
we investigated whether dierent laryngeal forms are present
in bats, or whether bat larynges are similar to the general mam-
malian body plan, e.g. those of other laurasiatherians (Harrison
1995, Evans and De Lahunta 2012). As a working hypothesis
based on previous research (reviewed in: Brualla et al. 2023),
we predict that bat larynges display several distinct features
compared to the general mammalian scheme and that discrete
morphotypes are distributed across the phylogeny. We show that
this morphological diversity is unique among mammals, which
underscores the remarkable diversity and ecological success of
Chiroptera.
MATERIALS AND METHODS
X-ray microtomography (XMT) imaging data were collected
from adult specimens, comprising 23 species of bats. One spe-
cies of Eulipotyphla (Suncus murinus Linnaeus 1766) was also
studied as an outgroup. e bat sampling comprises two spe-
cies of non-laryngeal echolocating bats (two Pteropodidae)
and eight species of laryngeal echolocating bats (three
Hipposideridae, one Rhinopomatidae, two Rhinolophidae,
one Megadermatidae, and one Craseonycteridae) among
the yinpterochiropteran suborder and 13 species of la-
ryngeal echolocating bats (three Emballonuridae, three
Phyllostomidae, two Mormoopidae, one Molossidae, and four
Vespertilionidae) among the yangochiropterans (Table 1). e
sample composition was chosen to illustrate the spectrum of
diversity present in bats, by detailing the morphology of the
dierent bat families. We sampled six out of seven families in-
side yinpterochiropterans (86% of the families), and ve of 14
families in the yangochiropterans (36%). us, the majority of
yinpterochiropterans and the families with the most species di-
versity among yangochiropterans are represented in this study.
Phylogenetic relationships between species were produced from
the Timetree (Kumar et al. 2022). Adult body mass within the
sample ranged from 2 g (Craseonycteris thonglongyai Hill 1974)
to 60 g (Eonycteris spelaea Dobson 1871). e dierent laryn-
geal echolocation strategies were also represented in the sample,
with inclusion of constant frequency (CF), frequency modu-
lated (FM), high-duty cycle (HDC), and low-duty cycle (LDC)
laryngeal echolocators. Reference to the bats at a generic level
will be made throughout the paper for brevity, except for mul-
tiple species comparisons within a genus. Specimens originate
from multiple museum collections (Australia, Japan, ailand,
the United States of America, and Vietnam) (Table 1). We gath-
ered specimens stored in 70% ethanol from the collections of
the University Museum of the University of Tokyo, the Vietnam
Academy of Science and Technology, Chulalongkorn University
Museum of Natural History, and the East Tennessee State
University. We also used public datasets from Morphosource.org
provided by the University of Michigan Museum of Zoology and
Stony Brook University.
Figure 1. Detailed visualization of the laryngeal cartilages of Eonycteris spelaea and the specic components found in several bat species
(dashed lines). A, dorsal view; B, cranial view; C,: lateral view. See text for abbreviations.
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4 • Brualla et al.
Image acquisition
As data collection was undertaken from dierent museum col-
lections, other xatives and iodine-staining protocols than the
one cited above are present in the sample, making the data
preparation non-uniform. Iodine staining was undertaken to
enhance the contrast and visualize the so tissues during scan-
ning (Metscher 2009, Gignac et al 2016). e staining protocol
used ethanol with 1% iodine for 14 days (Gignac et al. 2016).
Table 1. Species scanned, their source, collection ID and laryngeal echolocation strategy.
Family Species Source ID (+ DOI/ARK) Laryngeal
echolocation strategy
Eulipotyphla Suncus murinus University Museum of e
University of Tokyo
TS_835A Nil
Pteropodidae Eonycteris spelaea Vietnam Academy of Science
and Technology
VN18-026 Nil
Pteropodidae Macroglossus
sobrinus
Vietnam Academy of Science
and Technology
VN15-017 Nil
Hipposideridae Aselliscus
dongbacanus
Vietnam Academy of Science
and Technology
Vu15-013 CF-HDC
Hipposideridae Coelops ithii Vietnam Academy of Science
and Technology
VN19-196 CF-LDC
Hipposideridae Hipposideros
larvatus
Vietnam Academy of Science
and Technology
VN18-209 CF-HDC
Rhinolophidae Rhinolophus
cornutus
University Museum of e
University of Tokyo
JP21-025 CF-HDC
Rhinolophidae Rhinolophus
macrotis
Vietnam Academy of Science
and Technology
VN11-089 CF-HDC
Megadermatidae Lyroderma lyra Vietnam Academy of Science
and Technology
VN17-535 FM-LDC
Craseonycteridae Craseonycteris
thonglongyai
Chulalongkorn University
Museum of Natural History
CUMZ(M)220213-002 FM/CF-LDC
Rhinopomatidae Rhinopoma
hardwickii
University of Michigan Mu-
seum of Zoology
UMMZ:Mammals:159357 (hps://
doi.org/10.17602/M2/M96954)
FM/CF-LDC
Emballonuridae Saccolaimus mixtus Australian Museum A3257 FM-LDC
Emballonuridae Saccopteryx
bilineata
Stony Brook University L-LD:PE160 (hp://n2t.net/
ark:/87602/m4/393533)
FM-LDC
Emballonuridae Taphozous
melanopogon
Vietnam Academy of Science
and Technology
VN17-0252 FM-LDC
Phyllostomidae Artibeus
jamaicensis
East Tennessee State Uni-
versity
AJ001 FM-LDC
Phyllostomidae Desmodus rotundus University of Michigan Mu-
seum of Zoology
UMMZ:Mammals:112960 (hps://
doi.org/10.17602/M2/M170159)
FM-LDC
Phyllostomidae Rhinophylla
scherae
Stony Brook University L-LD:PE101 (hp://n2t.net/
ark:/87602/m4/394474)
FM-LDC
Mormoopidae Pteronotus
quadridens
Stony Brook University L-LD:DR098 (hp://n2t.net/
ark:/87602/m4/393837)
CF-LDC
Mormoopidae Pteronotus cf. ru-
biginous
University of Michigan Mu-
seum of Zoology
UMMZ:Mammals:74643 (hps://
doi.org/10.17602/M2/M97786)
CF-HDC
Molossidae Molossus molossus Stony Brook University L-LD:PE156 (hp://n2t.net/
ark:/87602/m4/393783)
FM-LDC
Vespertilionidae Kerivoula
hardwickii
Vietnam Academy of Science
and Technology
VN11-0043 FM-LDC
Vespertilionidae Myotis albescens Stony Brook University L-LD:PE008 (hp://n2t.net/
ark:/87602/m4/393605)
FM-LDC
Vespertilionidae Myotis ater Vietnam Academy of Science
and Technology
VN19-016 FM-LDC
Vespertilionidae Myotis siligorensis Vietnam Academy of Science
and Technology
Vu14-018 FM-LDC
CF, constant frequency; FM, frequency modulated; HDC, high-duty cycles; LDC, low-duty cycles.
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Comparative anatomy of the vocal apparatus in bats • 5
All specimens were then XMT scanned with dierent scanners
and protocols (parameters and voxel sizes) depending on the
team acquiring the data. A voxel size between 10 and 30 μm
was required to be able to reconstruct the dierent compo-
nents with sucient resolution: the average thickness of the
laryngeal cartilage was between 50 and 350 μm for most spe-
cies. Manual reconstruction of the three intrinsic cartilages of
the larynx (cricoid, thyroid, and arytenoids) and of the entire
hyoid apparatus was undertaken using the brush tool and inter-
polation function in AMI 5.3.3 soware (ermoFisher). All
the intrinsic muscles were reconstructed with the same method
(cricothyroid, cricoarytenoid dorsalis, cricoarytenoid lateralis,
oblique arytenoid, transverse arytenoid, thyroarytenoid, and
vocalis muscles), to which we added the thyrohyoid muscle for
its connection with the hyoid apparatus and its potential role in
laryngeal echolocation (e.g. Novick and Grin 1961, Griths
1983). e reconstructed 3D surfaces were saved as STL les
and analysed during the current study for anatomical com-
parisons. All 3D surface models are available on the repository
website MorphoMuseum (hps://doi.org/10.18563/journal.
m3.219) or Morphomuseum (Table 1).
We focused on morphological descriptions and comparison
of the size and shape of laryngeal structures. We did not quantify
ossication or degree of mineralization of the cartilage, because
the specimens were acquired by dierent researchers using dif-
ferent protocols, especially regarding staining and parameters
of the XMT scanners. Hence, the images contain dierent con-
trast for potentially the same degree of mineralization from one
specimen to another. For the muscles, we compared the overall
morphology, presence/absence, aachment points/areas, and
size range. Precise volume measurements were not taken as
ethanol preservative and iodine are known to cause so-tissue
shrinkage (Vickerton et al. 2013). We chose to maximize sam-
pling by incorporating specimens that were stained under dif-
ferent protocols, rather than applying strict exclusion criteria.
Lastly, we provide a complete description of all the cartilages
and muscles, followed by detailed comparisons between clades
and laryngeal echolocation strategies.
During reconstruction, certain groups of muscles were classed
as single entities to facilitate identication and reconstruction.
us, the oblique and transverse arytenoid muscles have been
merged as arytenoid muscles. Similarly, the thyroarytenoid and
vocalis muscles have been merged as thyroarytenoid muscles.
e complete list of species with the state of reconstruction of
each component are provided (Supporting Information, Table
S1), illustrating specimens with missing data due to the dif-
ferent acquisition protocols and staining processes. Overall, the
sampling and qualitative descriptions remain robust and the in-
formation extracted from the 3D reconstruction, and detailed
in the result part of this study, are of great value for further in-
vestigations in bat biology. e following abbreviations have
been used for the gures: a, arytenoid muscle; ac, arytenoid
cartilage; bh, basihyal; cad, cricoarytenoid dorsalis muscle; cal,
cricoarytenoid lateralis muscle; cc, cricoid cartilage; ccl, cricoid
cartilage laminae; ch, ceratohyal; cp, corniculate process; ct,
cricothyroid muscle; c, cranial thyroid tubercle; eh, epihyal;
ha, hyoid apparatus; mp, muscular process; mw, muscular wings;
ofr, outwardly ared rim; mc, median crest; sh, stylohyal, ta,
thyroarytenoid muscle; tc, thyroid cartilage; tcac, thyroid caudal
cornua; tch, tracheal chambers; tcrc, thyroid cranial cornua; th,
thyrohyal; thm, thyrohyoid muscle; tl, thyroid laminae; vp, vocal
process.
RESULTS
Cartilages
Cricoid cartilage
e cricoid cartilage displays the greatest variation among
the laryngeal components investigated in this study (Fig. 2).
Within yinpterochiropterans, the cricoid cartilage of the two
species of pteropodids is similar to that observed in the shrew
species, Suncus murinus. e main dierence observed in bats
is the development of a thin median crest on the dorsal part
of the cricoid cartilage. Hipposiderids (Hipposideridae) and
rhinolophids (Rhinolophidae) species have similar cricoid
forms, being narrower than in pteropodids, and with an extreme
development of the median crest and of muscular wings (Fig.
2A). e cricoid cartilage form observed in craseonycterids
(Craseonycteridae) and rhinopomatids (Rhinopomatidae)
species are highly similar. ey have a median crest and mus-
cular wings with less development than the hipposiderids, and
a cranial development of the ventral part of the cartilage (Fig.
2A). e megadermatids (Megadermatidae) species possess
a cricoid cartilage similar to that of craseonycterids, with less
ventral development (Fig. 2A). Yangochiropteran families have
variable cricoid cartilage forms (Fig. 2B). e emballonurids
(Emballonuridae) possess the most caudally elongated, tube-
shaped cartilage. A prominent median crest is present on the
cranial part of the dorsal region of the cricoid, with oval con-
cavities on each side of the crest (Fig. 2B). e phyllostomids
species present interspecic variations and the thinnest cartil-
ages. eir cricoid has a triangular shape instead of being more
oval or rounded as in yinpterochiropterans. Artibeus jamaicensis
(Leach 1821) exhibits an expansion of the cranioventral part
of the cricoid, not seen in the other phyllostomids (Fig. 2B).
All phyllostomids have a reduced median crest and muscular
wings. e cricoid cartilage of mormoorpids is more aened
and elongated ventrally than in phyllostomids and possesses an
outwardly ared rim on the ventral part (Fig. 2B). e muscular
wings and median crest in both Pteronotus (Gray 1838) species
are more developed than most yangochiropterans (except for
the emballonurids). e molossids (Molossidae) have a similar
cricoid cartilage to the rhinopomatids, but with more prominent
muscular wings and a large outwardly ared rim. e cricoid car-
tilage of the vespertilionids is similar, morphologically, to that of
the molossids, with thin cartilage as in phyllostomids and mus-
cular wings such as in rhinolophids. e median crest of both
vespertilionids and molossids is prominent cranially, similarly to
other yangochiropteran species (Fig. 2B).
Tracheal chambers
Tracheal chambers are cartilaginous bullae on the rst tracheal
rings, caudal to the cricoid cartilage (Fig. 3). ey are unique
features found only in the hipposiderids and rhinolophids. e
rostral pair of chambers is large and laterally distributed and the
lower pair is small and dorsally positioned. e rhinolophids
have greater size variation between the upper and lower pairs
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6 • Brualla et al.
Figure 2. Comparison of the dierent cricoid forms. A, outgroup and Yinpterochiroptera species; B, Yangochiroptera. * Rhinolophus cornutus;
** Pteronotus quadridens; *** Myotis albescens. See text for abbreviations.
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Comparative anatomy of the vocal apparatus in bats • 7
than the hipposiderids (Fig. 3). Other families of rhinolophoids,
such as the craseonycterids, megadermatids, and rhinopomatids,
do not possess these tracheal chambers.
yroid cartilage
e pteropodids possess a thyroid cartilage similar in shape
to that observed in the outgroup, with a U shape and the two
cornua oriented craniocaudally (Fig. 4A). Hipposiderids and
rhinolophids species have a more acute angle formed by the
junction of the two laminae making a rounded V shape. Also,
their cranial cornua are thinner than in pteropodids and oriented
more ventrally. eir caudal cornu is extremely reduced and the
cricothyroid joint is larger and more robust than that in other
bats (Fig. 4A). e thyroid cartilage of megadermatids spe-
cies is similar to the rhinolophids. e craseonycterids and
rhinopomatids share a similar morphology, with thinner lam-
inae than the other yinpterochiropterans. eir cornua are also
dierent with the cranial cornu being ventrally oriented, par-
allel to the thyroid laminae, and the caudal cornu more dorsally
oriented than in pteropodids (Fig. 4A). Yangochiropteran spe-
cies possess a distinct thyroid cartilage form with a straighter V
shape and extremely elongated caudal cornua (Fig. 4B). ey
also possess a small cartilaginous development laterally, a cranial
thyroid tubercle. e emballonurids have the most narrowed V
shape and the longest caudal cornua. e phyllostomids have a
wider V-shaped thyroid and their cranial cornua are well devel-
oped, oriented ventrally along the laminae. Rhinophylla scherae
(Carter 1966) possess large thyroid laminae compared to other
species (Fig. 4B). e mormoorpids species have dierent thy-
roid form than the phyllostomids, with a shorter and more ro-
bust caudal cornu, and a straighter V shape. e molossids
thyroids are highly similar to those of the mormoorpids. Lastly,
the vespertilionids have an extremely developed cranial cornu
and cranial thyroid tubercle in Myotis albescens (Georoy
1806). Otherwise, their thyroid is similar to that observed in
phyllostomids (Fig. 4B).
Arytenoid cartilage
e arytenoid cartilages are the most challenging part to recon-
struct due to their low degree of mineralization and their small
size (Fig. 5). In pteropodids, the structures appear dierent to
the outgroup, and all other yinpterochiropteran families illus-
trate similar shape but with variation in size and the degree of
development. e rhinolophids have well-developed arytenoid
cartilages, with a caudally extended muscular process, a prom-
inent corniculate process, and a short, vocal process (Fig. 5A).
e emballonurids have bulky cartilages with extremely devel-
oped corniculate and vocal processes. e phyllostomid species
Figure 3. Comparison of the dierent tracheal chambers in Hipposideros larvatus, Coelops ithii, Aselliscus dongbacanus, and Rhinolophus
cornutus.
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8 • Brualla et al.
Figure 4. Comparison of the dierent thyroid forms. A, outgroup and Yinpterochiroptera species; B, Yangochiroptera. * Rhinolophus cornutus;
** Pteronotus quadridens; *** Myotis albescens. See text for abbreviations.
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Comparative anatomy of the vocal apparatus in bats • 9
Figure 5. Comparison of the dierent arytenoid cartilage forms. A, outgroup and Yinpterochiroptera species; B, Yangochiroptera. *
Rhinolophus cornutus; ** Pteronotus quadridens; *** Myotis albescens. See text for abbreviations.
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10 • Brualla et al.
have arytenoid cartilages developed more ventrally and lat-
erally, with a shorter muscular process, large vocal process, and
an elongated and curved corniculate process (Fig. 5B). In the
mormoorpids, the cartilages are bulky but with short vocal and
corniculate processes. e molossids possess similar arytenoid
cartilages to the phyllostomids and mormoorpids but with a
shorter vocal process. Lastly, the vespertilionids have a more ro-
bust and simple shape than the molossids (Fig. 5B).
Hyoid apparatus
e hyoid apparatus is variable in size and shape among bats
(Fig. 6). e hyoid apparatus in pteropodids is similar to the
outgroup, with only a more curved basal portion. e stylohyal
cartilage is not in direct contact with the tympanic bone in
pteropodids and Suncus (Ehrenberg 1832) (Fig. 6A). In all
other bat species, the paddle-shaped tip of the stylohyal is in
contact or fused with the tympanic bone. e hyoid apparatus
in rhinolophids and hipposiderids are similar to one another,
with a reinforced basihyal ventrally. e craseonycterids possess
a short basal portion that presents a separation between basihyal
and thyrohyals (Fig. 6A). e hyoid apparatus of rhinopomatids
was dicult to reconstruct due to resolution issues and lile can
be inferred from our reconstruction, which may be artefactual.
Saccopteryx bilineata (Temminck 1838) appears to have a longer
greater cornu than all other species, with the development of the
thyrohyals dorsally (Fig. 6B). e form of the hyoid apparatus in
the phyllostomids and mormoorpids is similar to the other bats,
with some internal development of the thyrohyals. e molossid
species have similar shape and size but with the development of
lateral tubercles on the thyrohyals, similar to the cranial thyroid
tubercles (Fig. 6B). e vespertilionids have the most developed
of these tubercles on their thyrohyals, which have a wing shape,
aened laterally. Also, the thyrohyals in vespertilionids species
are separated from the basihyal, as in craseonycterids. Overall,
the basihyal of yangochiropterans is comparatively smaller than
in rhinolophoids.
Muscles
Cricothyroid muscle
Muscle volume is lower in pteropodids than in other bats,
similar to the outgroup (Fig. 7). e hypertrophied muscle in
rhinolophids is similar to that in hipposiderids but does not
cover the ventral part of the cricoid cartilage (Fig. 7C). e
cricothyroid muscle of the megadermatids is well developed
and bulky. In Rhinopoma hardwickii (Gray 1831), the muscle is
thinner than in Lyroderma lyra (Georoy Saint-Hilaire 1810),
and the craseonycterid has a more cranially developed muscle
(Supporting Information, Fig. S1). In all yangochiropterans,
the cricothyroid muscle is extremely well developed and covers
the thyroid cartilage externally but presents some variations
(Fig. 7D; Supporting Information, Fig. S1). In emballonurids,
the ventral part of the larynx is not covered by muscle. e
phyllostomids and mormoorpids have a common shape with
a developed cricothyroid muscle that almost covers the lateral
and ventral portions of the larynx. Finally, the molossids and
vespertilionids possess an extremely well-developed and bulky
cricothyroid muscle, which does not cover the ventral part of the
larynx (Fig. 7D).
Cricoarytenoid dorsalis muscle
e cricoarytenoid dorsalis muscle is relatively voluminous
in bats. In pteropodids and Suncus, the muscle consists of one,
thin, merged layer of muscle (Fig. 7A, B). In the hipposiderids,
rhinolophids, and megadermatids, the muscle is hypertrophied
and separated in two by the median crest (Fig. 7C; Supporting
Information, Fig. S1). In Rhinopoma (Georoy Saint-Hilaire
1818) and Craseonycteris (Hill 1974), the size of the muscle is
reduced. e emballonurids have a thin and extremely elong-
ated muscle due to the tube shape of the cricoid cartilage. eir
cricoarytenoid dorsalis muscle is also not in contact with the
dorsal part of the cricoid but mainly with the elongated caudal
cornua of the thyroid cartilage. e muscle observed in the
phyllostomids and molossids is similar in shape to the one in the
rhinopomatids, but thinner. e cricoarytenoid dorsalis muscle
of Pteronotus is short but bulky and separated by the median crest
(Supporting Information, Fig. S1). Finally, in the vespertilionid
species, the muscle is elongated and relatively thin (Fig. 7D).
Cricoarytenoid lateralis muscle
e cricoarytenoid lateralis muscle varies in size and shape
among the dierent bat families. e muscle is hypertro-
phied in yinpterochiropterans, except for the pteropodids, and
relatively small in yangochiropterans and non-bat mammals
(Fig. 7; Supporting Information, Fig. S1). In pteropodids, the
cricoarytenoid lateralis muscle is shorter than the muscle ob-
served in Suncus. e rhinolophids have an extremely bulky
muscle compared to the other yinpterochiropterans (Fig.
7C). e megadermatids, rhinopomatids, and craseonycterids
possess hypertrophied muscles but of shorter length than in
rhinolophids. e muscle in emballonurids, mormoorpids,
and molossids is highly similar to the small one of pteropodids,
and it is elongated in the phyllostomids species as in Suncus
(Supporting Information, Fig. S1). Lastly, the cricoarytenoid
lateralis muscle of the vespertilionids is similar to that found in
the phyllostomids but slightly curved (Fig. 7D).
yroarytenoid muscle
e pteropodids and phyllostomids have thin thyroaryenoid
muscles. In hipposiderids and rhinolophids, the thyroarytenoid
muscles are elongated and hypertrophied (Fig. 7B, C). e
megadermatids, craseonycterids, and rhinopomatids spe-
cies have more developed thyroarytenoid muscles than the
pteropodids but less hypertrophied than in the hipposiderids.
Similarly, the emballonurids have a well-developed muscle.
e mormoorpids have a ventrally elongated muscle, relatively
similar to the one in pteropodids. e molossid thyroarytenoid
muscles have a similar elongated shape but are bulkier. Finally,
the vespertilionids have thyroarytenoid muscles similar to those
in hipposiderids (Fig. 7D).
Arytenoid muscles
e smallest muscles relative to larynx size are those in the
rhinolophids and non-bats, and the most voluminous aryt-
enoid muscles are observed in the phyllostomids (Fig. 7). e
muscles are bulky in Lyroderma (Peters 1872), Craseonycteris,
and Rhinopoma. e arytenoid muscles are similar in the
emballonurids and Craseonycteris. e phyllostomids possess the
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Comparative anatomy of the vocal apparatus in bats • 11
Figure 6. Comparison of the dierent hyoid forms. A, outgroup and Yinpterochiroptera species; B, Yangochiroptera. * Rhinolophus cornutus; **
Pteronotus quadridens; *** Myotis albescens. See text for abbreviations.
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12 • Brualla et al.
most ventrally and laterally well-developed muscles. Pteronotus,
Molossus molossus (Pallas 1766), and Myotis (Kaup 1829)
have arytenoid muscles of reduced size compared to those of
phyllostomids (Fig. 7D).
yrohyoid muscle
is muscle also illustrates size variations between taxa. For
some, the insertion is on the thyrohyal (Fig. 7); for Suncus it is
on the basihyal. Rhinolophids have small thyrohyoid muscles,
dierent in size from the hypertrophied thyrohyoid muscles
observed in the vespertilionids and mormoorpids (Fig. 7C, D).
e hipposiderids and pteropodids have similar muscle size
and shape. In rhinolophids the muscle is relatively thin. In con-
trast, the thyrohyoid muscle is bulky in Craseonycteris, and also
elongated in Rhinopoma and Lyroderma. e thyrohyoid muscle
is also elongated in the emballonurids species, and the one in
phyllostomid species is similar to the one in megadermatids.
Pteronotus has an extremely craniocaudally elongated muscle
and Molossus (Georoy Saint-Hilaire 1805) has a similar shape
but of shorter length (Supporting Information, Fig. S1). Lastly,
the vespertilionids have an extremely hypertrophied muscle in
contact with the cranial thyroid tubercle and the tubercle on the
thyrohyals. e insertion in Taphozous melanopogon (Temminck
1841), Kerivoula hardwickii (Horseld 1824), and Myotis is on
the basihyal (Fig. 7D).
DISCUSSION
Our observations of the larynx yielded new insights into the
understanding of anatomical diversity among bats and revised
the previous propositions made by dierent studies (and re-
viewed in: Brualla et al. 2023). Dierent potential factors may
impact observed variation in the laryngeal anatomy of bats, such
as phylogenetic relationships, diet, and the capability of laryngeal
echolocation, which for the laer is a highly demanding skill in
terms of vocalization capacities. is study links morphological
changes in bat larynges to dierent laryngeal echolocation strat-
egies through the hypertrophied musculature and the dierent
laryngeal cartilage forms. We noticed that the laryngeal cartil-
ages in vespertilionids and phyllostomids have previously been
described as thinner than in all other bat families (e.g. Elias 1907,
Denny 1976, Carter 2020). Our results conrm this assertion,
and we suggest that the thickness of the cartilage might be correl-
ated with the use of laryngeal echolocation, as the phyllostomids
rely on other senses, rather than only echolocation, to nd their
food, thus, involving less pressure on the laryngeal structures.
Figure 7. Muscle comparison of the dierent bat clades with the outgroup Suncus murinus, considering the main laryngeal echolocation
strategies. A, Suncus murinus; B, Eonycteris spelaea; C, Rhinolophus cornutus; D, Myotis albescens. 1, sagial section of the larynx, at mid-distance
between the median crest and the cricothyroid joint. 2, horizontal section of the larynx, along the main axis of the thyroid cartilage. 3,
horizontal section of the larynx, parallel to section 2, through the cricothyroid joint. * e spectrogram for the FM LDC echolocation strategy
is from Scotophilus kuhlii (Leach 1821), another vespertilionid that has similar echolocation calls to Myotis sp. See text for abbreviations.
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Comparative anatomy of the vocal apparatus in bats • 13
e shape of the cricoid arch in all bats studied herein has
been previously described as a ring shape (e.g. Harrison 1995).
We demonstrate that this is not the case for most of the families
within rhinolophoids, which present an oval and narrowed cri-
coid arch (Fig. 2; Table 2). Also, all yangochiropterans, except
for the emballonurids (that share a similar oval shape with the
rhinolophoids), possess a more triangular shape of their cri-
coid arch (Fig. 2; Table 2). is reduction in size of the cartil-
ages, such as the diameter of the cricoid arch, rst, characterizes
rhinolophoids and yangochiropterans from pteropodids and,
second, opens more space between the cricoid and the thyroid
laminae. It has been suggested that this surface reduction al-
lowed for the muscles to develop, especially for the cricothyroid
muscle involved in laryngeal echolocation (Fig. 7; Håkansson et
al. 2022). To that, we add that the dierential shape modica-
tion of the cricoid arch might be related to air pressure for high-
frequency calls. Rhinolophids and hipposiderids (RH group)
present the most narrowed cricoid arch, which might be correl-
ated with the need for higher air pressure to produce their unique
CF-HDC calls (Table 3; Fenton et al. 2012). Our observation
agrees with the previous statement that the rhinopomatids
and vespertilionids have similar laryngeal shapes (Robin 1881,
Denny 1976). We have shown that similar ventral development
of the cricoid cartilage and the similar shape of the thyroid lam-
inae might have led authors to describe the laryngeal shape of
rhinopomatids as close to that of vespertilionids (Fig. 2A, B).
is ventral development of the cricoid cartilage has previ-
ously been described as exclusive to the megadermatids (Denny
1976), but we nd the same paern in other families, such as
rhinopomatids and craseonycterids for yinpterochiropterans,
and in the phyllostomids, molossids, and vespertilionids for the
yangochiropterans (Figs 8, 9; Table 2). Further work is warranted
to explain this development in these specic bat families. e cri-
coid cartilage of mormoorpids has also been noted as highly dif-
ferent from the one of phyllostomids (Griths 1978, 1983). Our
results suggest a more complex situation. Phyllostomids present
some intrafamilial variations, such as dierential ventral develop-
ment of the cricoid cartilage, and we suggest that morphological
variation in the group may be correlated with its ecological di-
versity. e mormoopids have a cricoid cartilage craniocaudally
aened and the development of larger muscular wings, but
still share an overall similar shape to the phyllostomids, espe-
cially regarding the triangular shape of the cricoid arch. Also, the
mormoopids and molossids share a common development of
an outwardly ared rim, with as yet unknown function. is rim
has a dierent height in the Pteronotus species, as the cricoid car-
tilage of Pteronotus quadridens (Gray 1838) is more ventrally and
cranially developed. In emballonurids, the tube shape of the cri-
coid cartilage is found in all species studied and it contradicts the
previous idea that this shape is exclusive to Taphozous (Georoy
Saint-Hilaire 1818) and Saccolaimus (Temminck 1938) (Fig. 2B;
Robin 1881, Elias 1907, Brualla et al. 2023).
e development of a median crest on the dorsal part of
the cricoid cartilage has previously been described only in
pteropodids (Giannini et al. 2006), hipposiderids (Denny
1976), rhinolophids (e.g. Robin 1881), mormoorpids
[Pteronotus parnellii (Gray 1843); Griths 1983], and vesper-
tilionids (Robin 1881, Elias 1907). Our results show that the
median crest is also developed to some degree on the cricoid
Table 2. Distribution of anatomical features in the dierent bat clades.
Clade Cricoid
Arch Shape
Muscular
Wings
Median
Crest
Cricoid Ventral
Development
Tracheal
Chambers
yroid
Laminae
Shape
Arytenoid
Cartilage
Hyoid
Apparatus
Styohyoid
Shape
Muscle
Hypertrophy
Non-bats and Pteropodidae Rounded - -/ + - - U Average Average Drumstick -
Rhinolophoidea RH Narrowed—
Oval
+++ +++ - ++ Rounded V Developed mus-
cular process
Reinforced
basihyal
Paddle ++ (CAD/
CAL)
MCR Narrowed—
Oval
++ ++ ++ - Rounded V Developed mus-
cular process
Reinforced
basihyal
Paddle ++ (CAD/
CAL)
Yangochiroptera Emballonuridae Narrowed—
Oval
+ ++ ++ - Strict V + cra-
nial tubercle
Developed
vocal process
Shorten
thyrohyals
Elongated and
curved paddle
++ (CT)
Phyllostomidae Narrowed—
Triangle
++ + -/++ - Strict V + cra-
nial tubercle
Developed
vocal process
Shorten
thyrohyals
Elongated and
curved paddle
++ (CT)
Pteronotus sp. Narrowed—
Triangle
+++ +++ ++ (ared rim) - Strict V + cra-
nial tubercle
Developed
vocal process
Reinforced
basihyal
Elongated and
curved paddle
++ (CT)
Others Narrowed—
Triangle
++ ++ -/++ - Strict V + cra-
nial tubercle
Developed
vocal process
Shorten
thyrohyals
Elongated and
curved paddle
++ (CT)
CAD, cricoarytenoid dorsalis muscle; CAL, cricoarytenoid lateralis muscle; CT, cricothyroid muscle; MCR, Megadermatidae, Craseonycteridae and Rhinopomatidae group; RH, Rhinolophidae and Hipposideridae group; -, absent; +, reduced;
++, present; +++, extremely developed; -/++, absent in some species and present in others.
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14 • Brualla et al.
cartilage of all other bat families studied. In contrast to previous
understanding, we postulate that it is a morphological feature
shared among all bats despite dierential development (Fig. 8;
Table 2). e development of the median crest is still a source
of debate concerning its role in laryngeal echolocation, as it
can be visible on the cricoid cartilage of some pteropodids spe-
cies, even being reduced (Fig. 2A; Giannini et al. 2006). Also,
a similar median crest is observed in other mammals of large
body size (e.g. in camel and donkey; Fig. 2; Eshra et al. 2016) to
support the power of the muscle architecture (Harrison 1995),
Table 3. Distribution of anatomical features in the dierent laryngeal echolocation strategies.
Echolocation
Type
Median
Crest
Muscular
Wings
Cricoid Ventral
Development
Tracheal
Chambers
Cricoid
Arch Shape
Stylohyoid
Shape
Muscle
Hypertrophy
None or
Non-LE
-/ + - - - Rounded Drumstick -
CF-HDC +++ +++ -/++ -/++ Narrowed—
Variable
Paddle ++ (CAD/
CAL or CT)
CF-LDC ++ ++ ++ - Narrowed—
Oval
Paddle ++ (CAD/
CAL)
FM-LDC ++ +/ ++ -/ ++ -/ ++* Narrowed—
Triangle
Elongated and
curved paddle
++ (CT)
CAD, cricoarytenoid dorsalis muscle; CAL, cricoarytenoid lateralis muscle; CF, constant frequency; CT, cricothyroid muscle; FM, frequency modulated; HDC, high duty cycles;
LDC, low duty cycles; LE, laryngeal echolocator; -, absent; +, reduced; ++, present; +++, extremely developed; -/++, absent in some species and present in others.
Figure 8. Diversity of laryngeal morphology in bats, with major traits mapped onto the phylogeny (Outgroup and Yinpterochiroptera).
Laryngeal views from le to right: cranial view, ventral view, dorsal view, lateral view. Black lozenges represent the development of specic
morphological traits: 1, development of the median crest; 2, hypertrophied muscles, stylohyoid in paddle shape, development of muscular
wings, rounded V shape of the thyroid cartilage, oval narrowed laryngeal lumen; 3, extremely narrowed laryngeal lumen, extremely developed
median crest and muscular wings, tracheal chambers; 4, ventral development of the cricoid; 5, thyrohyals and basihyal separated. See text for
abbreviations.
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Comparative anatomy of the vocal apparatus in bats • 15
and in smaller mammals such as dogs (Evans and De Lahunta
2012). We suggest that its presence in small mammals, such as
bats and dogs, could be linked to their vocalization and/or to
their requirements for breathing during intense activity such as
running (dog) or ying (bat). en, the extreme development
of the median crest observed in the rhinolophids, hipposiderids,
and Pteronotus species studied here could be a morphological
feature linked to their CF-HDC laryngeal echolocation strategy
(Fig. 7; Tables 2, 3; Supporting Information, Fig. S1). Pteronopus
cf. rubiginous (Wagner 1843) in our dataset is one member of
the Pteronotus cf. parnellii species group, which is the only group
of the yangochiropteran suborder considered to be CF-HDC
emiers (Dávalos 2006, Jones and Teeling 2006, Fenton et al.
2012, De oisy et al. 2014, Pavan and Marroig 2016; López-
Baucells et al. 2017). Pteronotus quadridens has been described to
be a CF-LDC emier (Macías and Mora 2003, Mora and Macías
2011). We found that the cricoid cartilage of P. cf rubiginous is
greatly reduced in height compared to P. quadridens, especially
the cricoid arch, and this could express the dierences between
an HDC and LDC type of CF sound emissions. However, it is
also essential to note that, despite a phylogenetic inertia that po-
tentially constraints the overall laryngeal morphology among
yangochiropterans, P. quadridens has a prominent median crest.
A similar issue is found for muscular wings, previously de-
scribed in the rhinolophids (Harrison 1995). We demonstrate
that muscular wings exist in all the families studied, except
for pteropodids, with dierent degrees of development (Figs
8, 9; Giannini et al. 2006). is trait illustrates, once again,
the distinction between pteropodids and the other bat clades
(Table 2). e function of these muscular wings was theoretic-
ally stated to support the insertion of more voluminous muscles
linked to laryngeal echolocation [e.g. Elias (1907)] and our data
conrmed that enlarged muscular wings support the insertion
of the bulky cricothyroid muscle (e.g. Denny 1976, Harrison
1995). Additionally, it changes the size of the contact surfaces
of the cricothyroid joint (especially for the rhinolophids and
hipposiderids) (Figs 4A, 8, 9). is reinforced connection of
the cricothyroid joint could have an important biomechanical
role, especially in high-frequency sound production (Table 3).
Like the median crest, the extreme development of the muscular
wings in rhinolophids, hipposiderids and Pteronotus species
studied, could be correlated to their unique CF-HDC echoloca-
tion strategy (Figs 8, 9).
Concerning the thyroid cartilage, the narrowed V shape is
shared between all laryngeal echolocators, compared to the
rounded U shape of the cartilage in pteropodids and non-
bats (Giannini et al. 2006). is feature’s variation could be
anatomically compared to the narrowing of the cricoid arch,
which diers among families. A distinction is present between
rhinolophoids and yangochiropterans, with a stricter V-shaped
thyroid cartilage in the yangochiropterans. e rhinolophoids
seem to possess an intermediate morphology of the cartilage,
being between that of pteropodids and of yangochiropterans.
Functionally, the shape modication might be correlated to la-
ryngeal echolocation as pteropodids are the only bats with the
rounded U-shaped cartilage commonly seen in other mammals.
However, no inference regarding specic echolocation strategy
can be made from our results. We noticed that the thyroid
cartilage of vespertilionids was described as unique and div-
ided into two parts, the lateral one being more aened (Robin
1881, Elias 1907). However, our results are not in accordance
with this description as we nd in Myotis a complete lamina of
the thyroid (Fig. 4A). It is possible that extremely low degrees
of mineralization on the thyroid, except on the cricothyroid
joint, the cranial thyroid tubercle parts, as well as the ventral tip
of the laminae, may have led previous researchers to consider
the thyroid cartilage of vespertilionids as two separate mineral-
ized entities. e thyroid cornu in bats have been studied only
by Elias (1907), Denny (1976), and Giannini et al. (2006),
mainly in vespertilionids, rhinopomatids, and pteropodids. Our
results show that the size and orientation of the thyroid cornu
appear dierent in the laryngeal echolocators compared to the
pteropodids (and non-bats) (Giannini et al. 2006). e former
group possesses dorsoventrally tilted cornua and the laer pos-
sesses more craniocaudally orientated cornua. We also describe a
short and large caudal cornu in rhinolophids and hipposiderids,
and an elongated caudal cornu in all yangochiropterans. is dis-
tinguishes rhinolophoids from yangochiropterans and changes
the contact on the cricothyroid joint, potentially enabling spe-
cic laryngeal echolocation strategies (Figs 4A, 8, 9). e aryt-
enoid cartilages have been poorly reconstructed due to their
non-mineralization and their small size, but from our results
we suggest that rhinolophoids and yangochiropterans have dif-
ferent arytenoid development. In rhinolophoids, the muscular
process is greatly developed, potentially to support the bulky
cricoarytenoids involved in CF-HDC laryngeal echolocation
(Fig. 5A). For the yangochiropterans, the vocal process is elong-
ated, and this might change the length of the vocal folds, then
changing the frequencies emied (Fig. 5B).
Our study conrms that the shape of the stylohyal is a main
dierence in the anatomy of the hyoid apparatus between laryn-
geal echolocating bats and non-laryngeal echolocators (Figs 6, 8,
9; Tables 2, 3; Simmons et al. 2008, Veselka et al. 2010). In laryn-
geal echolocators, the paddle-shaped tip of the stylohyal is in con-
tact or fused with the tympanic bone (Nojiri et al. 2021, Snipes
and Carter 2023). Conversely, in non-bats and pteropodids, the
stylohyal is similar to a thin drumstick and disconnected from
the tympanic bone (Simmons et al. 2008, Veselka et al. 2010). In
addition, our results show a suspensory apparatus of the hyoid
positioned more laterally with the basal portion in laryngeal
echolocating bats compared to a more cranially positioned one
in non-laryngeal echolocators. Also, the ceratohyal of all laryn-
geal echolocating bats is diminished in size or absent in some
taxa compared to the pteropodids and non-bat mammals. We
suggest that these dierences could be associated with dierent
needs in sound conduction between laryngeal echolocating bats
and non-laryngeal echolocators (Table 3). Lastly, the basihyal
is reinforced in all rhinolophoids, especially rhinolophids and
hipposiderids, and it contrasts with the reduced basihyal of the
yangochiropterans. We suggest that this reinforcement allows
more strength during vocal production of CF-HDC calls in
these taxa. Overall, most anatomical descriptions of the hyoid
made by Sprague (1943) are veried in this study. However, the
separation of the basihyal and thyrohyal bones in most of the
vespertilionids and in the craseonycterids found in this study
were not described previously (Fig. 6).
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16 • Brualla et al.
e tracheal chambers have been described as potential
Helmholtz resonators that could regulate sound amplitude,
or potentially prevent the reverberation of the sound by tissue
conduction from the lungs to the ears during vocal produc-
tion (Denny 1976, Suthers et al. 1988, Harrison 1995). ey
are reported to be present in hipposiderids, rhinolophids, and
nycterids (Nycteridae) (Robin 1881, Denny 1976, Harrison
1995, Brualla et al. 2023). Our results conrm the presence of
these chambers in hipposiderids and rhinolophids, but we were
not able to include nycterids in this study. No enlargement
has been observed in the rst tracheal rings of rhinopomatids,
emballonurids, mormoorpids, and phyllostomids as com-
pared to previous studies that note this feature (Sprague 1943,
Denny 1976, Griths 1978, 1983, Griths and Smith 1991).
e morphofunction of these features remains unclear and war-
rants further investigation with an expanded taxonomic sample.
More precisely, as no tracheal chambers or enlargement has been
identied in the larynges of Pteropotus species studied, these fea-
tures may not be related to CF-HDC echolocation (Tables 2, 3).
e muscle hypertrophy observed in all laryngeal echolocators
of this study possibly results from the necessity of high-speed call
production. Recently, Håkansson et al. (2022) introduced a new
understanding of bat hypertrophied musculature. In bats, superfast
muscles are somewhat weaker than normal skeletal muscles, due
to the balance between speed and force, the one diminishing if
the other increases. To compensate for the force lost by adapting
for speed, the muscles overgrow to keep their strength, because
force is a function of a muscle’s eective cross-sectional area. It has
been proposed that the cricothyroid muscle is the main muscle in-
volved in laryngeal echolocation and is hypertrophied in laryngeal
echolocators (Figs 7, 8, 9; Griths 1983, Elemans et al. 2011).
Here, we suggest a more complex scenario of muscle involvement.
From our results, rhinolophoids are distinct from other bats by
presenting hypertrophied cricoarytenoid (dorsalis and lateralis)
Figure 9. Diversity of laryngeal morphology in bats, with major traits mapped onto the phylogeny (Yangochiroptera). Laryngeal views from
le to right: cranial view, ventral view, dorsal view, lateral view. Black lozenges represent the development of specic morphological traits: 6,
hypertrophied muscles, stylohyoid in paddle shape, development of muscular wings, narrowed laryngeal lumen, elongated caudal cornu of the
thyroid cartilage, narrow strict V shape of the thyroid cartilage, development of a cranial thyroid tubercle, insertion of the cricothyroid muscle
on the cranial edge of the thyroid cartilage; 7, tube-shaped cricoid cartilage, extreme development of the median crest and the caudal cornu of
the thyroid cartilage; 8, ventral development of the cricoid cartilage, triangular shape of the laryngeal lumen; 9, outwardly ared rim, extreme
development of the muscular wings; 10, outwardly ared rim; 11, extreme development of the cranial thyroid tubercle, thyrohyals laterally
elongated and separated from basihyal. See text for abbreviations.
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Comparative anatomy of the vocal apparatus in bats • 17
and cricothyroid muscles, and the yangochiropterans possess rela-
tively thinner cricoarytenoid muscles but present an extremely
developed cricothyroid muscle that inserts more cranially on the
thyroid laminae, overlapping the thyroid cartilage almost entirely
(Fig. 7; Table 2; Supporting Information, Fig. S1). is distinc-
tion could be linked to the dierent echolocation strategies, CF
calls being potentially produced with a reinforced action of bulky
cricoarytenoid muscles (Table 3). e cricothyroid muscle of
megadermatids is seen as uniquely complex (Griths and Smith
1991), but it appears to be only the two branches of the crico-
thyroid muscle that are well dierentiated from one another com-
pared to the other rhinolophoids. is specicity is also observed
on the distantly related blood-feeding phyllostomid Desmodus
(Wied-Neuwied 1826). We postulate that the cricothyroid muscle
of the vespertilionids illustrates the greatest complexity, having
several large and distinct fascicles (Fig. 7). e importance of a dif-
ferential complexity of development in the cricothyroid muscle,
present in all laryngeal echolocators, requires further clarication.
e thyroarytenoid muscle of the rhinolophids has been de-
scribed as extremely voluminous and both cricoarytenoid (dor-
salis and lateralis) muscles of vespertilionid species have been
considered bulky and hypertrophied (Robin 1881, Elias 1907).
Our observations appear to contradict these descriptions. e
thyroarytenoid muscle in rhinolophids is of normal volume
compared to other bat families of this study, and vespertilionids
species possess some of the less voluminous cricoarytenoid
muscles, especially the cricoarytenoid dorsalis muscle (Figs 7, 8;
Supporting Information, Fig. S1). On the other hand, our results
agree with the previous research describing the cricoarytenoid
muscles of emballonurids as underdeveloped (Elias 1907,
Griths and Smith 1991, Griths et al. 1991). Indeed, we de-
scribe the cricoarytenoid dorsalis muscle as expanded caudally
but relatively thin. e cricoarytenoid lateralis muscle is also
relatively small, potentially due to the massive cricothyroid
muscle of this family developed along the tube-shaped cricoid
cartilage. Finally, the cricothyroid muscle of phyllostomids is de-
scribed as thin and underdeveloped, which does not correspond
to our results (Fig. 9; Griths 1978, 1982). Nonetheless, our
results demonstrate some volume variations of the cricothyroid
muscle in phyllostomids. Desmodus and Rhinophylla (Peters
1865) possess hypertrophied and developed muscles. Only
Artibeus (Leach 1821) present developed but thin cricothyroid
muscles. e cricothyroid muscle of phyllostomids probably il-
lustrates a more complex interconnection between muscle mass,
diet, and echolocation strategy. We nd that the thyrohyoid
muscle originates from the caudal tip of the caudal thyroid cornu
in non-bat mammals and pteropodids but that it originates more
cranially, from the laminae, in most of the laryngeal echolocators.
is dierence correlates with the idea that the thyrohyoid
muscle, not being an intrinsic laryngeal muscle, could play a
role in laryngeal echolocation by pulling the basihyal into the
thyroid for sound conduction through vibration (Novick and
Grin 1961, Griths 1983, Snipes and Carter 2023). However,
the thyrohyoid varies in its insertion areas (basihyal or thyrohyal
bones) in dierent families, so it is dicult to fully understand
its implication in sound production. A similar situation is ob-
served for variation in the size of the thyroarytenoid among bats
(Fig. 7A, D; Supporting Information, Fig. S1).
Overall, we show that the pteropodids (as non-laryngeal
echolocating bats) share similar laryngeal morphology with
other non-laryngeal echolocators, reinforcing the idea that la-
ryngeal echolocation could be responsible for some of the mor-
phological variation in other bat taxa (Figs 7, 8; Table 3). We
observe almost no intrafamilial morphological variation (except
for the phyllostomids). In the rhinolophoids, there are several
main dierences. e RH group possesses tracheal chambers,
extreme development of the muscular wings and median crest,
and extremely narrowed oval shape of the cricoid arch. e
ventral development of the cricoid arch is specic to the MCR
group (megadermatids, craseonycterids, and rhinopomatids)
despite an overall laryngeal form relatively intermediate be-
tween the pteropodids and the RH group (Fig. 8; Table 2). In
addition, among the MCR group, the megadermatids are rela-
tively dierent from the two other families (lesser ventral de-
velopment and muscular wings; Figs 2A, 4A; Table 2), and they
also have a slightly dierent laryngeal echolocation strategy
than the others (FM-LDC laryngeal echolocators with some
short CF calls; Schmidt et al. 2011, Smarsh and Smotherman
2015). e craseonycterids and rhinopomatids have been de-
scribed as CF-LDC (Surlykke et al. 1993, Fenton et al. 1995,
Hiryu et al. 2016), with rhinopomatids species able to produce
FM-LDC calls (Shah and Srinivasulu 2020). e CF calls are
generally considered more behaviourally derived than the FM
calls (Fenton et al. 2012), which could explain why the laryngeal
morphology of the megadermatids appears less derived than the
rhinopomatids and craseonycterids and still modied compared
to the morphology of non-laryngeal echolocators (pteropodids
and non-bats) (Fig. 8). In the same way, the species of the RH
group possess a more derived morphology, which could be a sign
of specialization to the CF-HDC laryngeal echolocation strategy
(e.g. extreme development of median crest and muscular wings).
Among hipposiderids, some dierences are found in the la-
ryngeal form of Coelops ithii (Blyth 1848) compared to other
species, despite an overall similar morphology. Coelops' (Blyth
1848) laryngeal lumen and thyroid laminae are relatively wider
than in other hipposiderids and are features that distinguish the
overall laryngeal form of this species from Hipposideros (Gray
1831) and Aselliscus dongbacanus (Tu et al. 2016). is species is
also distinctive among hipposiderids as it does not use long CF
calls. Instead, it primarily employs FM calls with a short CF com-
ponent at the end (Hughes et al. 2012), which may account for
its morphological divergence from other hipposiderids observed
here. All these dierences express that laryngeal morphology ap-
pears constrained by phylogeny in rhinolophoids and that the
extreme morphological variation found within the RH group is
correlated with the unique laryngeal echolocation strategy used
by these bats. Because yangochiropterans share unique morpho-
logical features among themselves, such as the triangular shape
of the cricoid arch, the V-shaped thyroid cartilage, the cranial
thyroid and hyoid tubercles, and the extreme development of
the cricothyroid muscle, similar conclusions about a phylogen-
etic constraint may be drawn. However, yangochiropterans il-
lustrate some morphological variation with the tube-shaped
cricoid of the emballonurids, the thin diamond-shaped cri-
coid cartilage supporting thin muscles in the phyllostomids,
and the outwardly ared rim on the cricoid cartilage of the
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18 • Brualla et al.
molossids and mormoopids species. is diversity can be ex-
plained by the great diversity of species and ecologies among
yangochiropterans. However, our sample size does not allow us
to draw precise correlations. Despite similar shapes, the develop-
ment of the cricoid and thyroid cartilages of P. quadridens, being
greatly enlarged cranially, contrasts to the relatively thin cartil-
ages of P. cf rubiginous. How these dierences relate to echoloca-
tion strategy appears unclear; indeed, the 3D reconstruction of
P. cf rubiginous has been dicult due to image quality issues, es-
pecially the non-mineralized part of some cartilages (Supporting
Information, Table S1; MorphoMuseum repository 3D models
‘hps://doi.org/10.18563/journal.m3.219’). Still, we can infer
that the strategy of constant frequency echolocation strategy has
more constraints on the overall shape of the cartilages than the
duty-cycle strategies, the rst one being associated with the large
development of the median crest and muscular wings.
Laryngeal echolocation is also characterized by two strat-
egies of sound emission, through the nasal cavities or through
the mouth (e.g. Pedersen 2000, Pedersen and Müller 2013).
e nasal emission is a polyphyletic trait shared convergently
among all rhinolophoids and with the nycterids, phyllostomids
and several species of vespertilionids (Jakobsen et al. 2018).
While bioacoustic research has been conducted to under-
stand the dierences in sound emissions, no specic laryngeal
morphology stands out to dierentiate nasal from oral emiers
(Pedersen and Müller 2013). Similarly, we nd no evidence of
morphological paerns that dierentiate the rhinolophoid and
phyllostomid nasal emiers from the other oral emiers among
yangochiropteran species. Such investigations require further
research, and the qualitative aspect of this article prevents com-
ment on these hypotheses. Furthermore, other parameters such
as diet or ying habits could be inuencing laryngeal anatomy, as
they vary from one family to another. However, the larynx may
be viewed in relation to the concept of modularity (Zelditch and
Goswami 2021), whereby two levels of modularity could be im-
plied to explain nasal sound emission in phylogenetically distant
bat clades, despite the absence of a unifying morphology. e
rst level would consider the laryngeal components (cartilages
and muscles) as modules that function (and potentially evolve)
in interaction with the cartilage developing and functioning to
support the muscle development and function. e second level
of modularity would place the larynx as a module inside the
throat, with the pharynx and the rostrum as other modules. In
this case, the nasal sound emission could still be produced des-
pite an independent evolution of the larynx, by adaptability of
the other modules (pharynx and rostrum).
Perspectives for future studies
Further studies should focus on the collection of quantitative
data to investigate the morphofunction of the laryngeal features
described here. Although this study has a small number of spe-
cies compared to the high number of bat representatives, and
we cannot correlate the morphology for all bat species diver-
sity to a specic functional specialization (e.g. the tube shape of
Taphozous), this study can still illustrate an overall aspect of bats’
laryngeal morphology. Also, our ndings are consistent with the
degrees of mineralization that have already been discussed in pre-
vious studies (e.g. Denny 1976, Carter 2020). Further work on
the larynx should cover the dierent families not yet described
in yangochiropterans, as well as species known to be morpho-
logically unique, such as the hammer-headed bat (Hypsignatus
montrosus Allen 1861), known for its mating vocalizations and
possessing a larynx one-half the length of the spine, and for being
sexually dimorphic (Langevin and Barclay 1990).
CONCLUSIONS
In the present study, we investigated the variation in the laryn-
geal anatomy of bats. We found that several forms were dis-
tributed across bat phylogeny, not specically linked to muscle
hypertrophy. We observed distinctive laryngeal features and
forms between the pteropodids and the rhinolophoids, and be-
tween yangochiropterans and yinpterochiropterans (Figs 8, 9;
Table 2). Rhinolophoids and yangochiropterans possess distinct
laryngeal forms that we consider as two distinct morphotypes
(Figs 8, 9; Table 2). e larynges of pteropodids share with non-
bat mammals several features that are modied in rhinolophoids
and yangochiropterans, such as the modications of the cri-
coid cartilage shape and development of muscular wings, the V
shape of the thyroid cartilage, the hypertrophy of most of the
laryngeal muscles, and the paddle shape of the stylohyoid chain.
e most distinctive aspect of the yangochiropteran laryngeal
morphology is the extreme development of the cricothyroid
muscle, overlapping the entire ventral and lateral surface of the
larynx. Narrowed cricoid cartilage arch, extreme development
of the median crest and muscular wings, enlargement of the
cricothyroid joint, muscular process of the arytenoid cartilage,
and of the basihyal bone may be associated with the CF-HDC
echolocation strategy. No specic form or feature was found to
unite the polyphyletic the nasal emiers. Nasal and oral emiers
inside yangochiropterans share similar forms, distinct from the
rhinolophoids (all nasal emiers), as the most morphological
variations are distributed along the phylogeny.
ese ndings validate several previous statements regarding
the laryngeal anatomy of bats and adjust or improve other ob-
servations thanks to the use of modern protocols (virtual dis-
section of contrast-enhanced X-ray microtomography images).
is study oers new perspectives of research on the laryngeal
morphofunction, its implication in dierent laryngeal echoloca-
tion strategies, and, to some extent, research on the evolutionary
history of bats and their laryngeal echolocation.
SUPPLEMENTARY DATA
Supplementary data are available at Zoological Journal of the
Linnean Society online.
ACKNOWLEDGEMENTS
We thank Cody W. ompson for approving access to the scan data of
the University of Michigan. We thank Laurel Yohe for providing staining
protocol information and sharing her materials on Morphosource. We
thank Kai Ito for his help with scanning.
is study was supported by City University of Hong Kong Start-up
Grant (9610466), JSPS (21H02546, 21K19291, and 22KK0101),
JST (JPMJFR2148) to D.K., and a grant from the Australian Research
Council to L.A.B.W. (FT200100822).
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Comparative anatomy of the vocal apparatus in bats • 19
CREDIT STATEMENT
D.K. and L.A.B.W. conceptualized the project and obtained funding.
D.K., L.A.B.W., V.T.T., T.N., R.T.C., T.N., T.W., and D.F. gathered sam-
ples and performed CT scanning.
D.K., L.A.B.W., and M.D. supervised the study.
N.L.M.B. curated the scan data, processed the 3D reconstructions,
studied the data, and summarized the results.
N.L.M.B., L.A.B.W., and D.K. draed the manuscript; all authors pro-
vided insights, critically revised the text and the gures.
CONFLICT OF INTEREST
e authors declare that they have no conict of interest.
DATA AVAILABILITY
Surface data that support the ndings in the present study are avail-
able from the Morphomuseum repository (hps://doi.org/10.18563/
journal.m3.219) or Morphosource.
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