Comparison of female and male vocalisaon and larynx
morphology in the size dimorphic foot-agging frog species
Doris Preininger1,2, Stephan Handschuh3, Markus Boeckle4, Marc Sztatecsny2 & Walter Hödl2
1Vienna Zoo, Maxingstraße 13b, 1130 Vienna, Austria
2Department of Integrave Zoology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
3VetCore Facility for Research, Imaging Unit, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria
4Department for Psychotherapy and Biopsychosocial Health, Donau-Universität Krems, Doktor-Karl-Dorrek-Straße 30, 3500 Krems, Austria
Herpetological Journal FULL PAPER
Correspondence: Doris Preininger (firstname.lastname@example.org)
Volume 26 (July 2016), 187–197
Published by the Brish
In anurans, males have larger laryngeal structures than females and produce conspicuous species-specic calls in various
social contexts. Knowledge of female vocalisaons is not well established and we start by summarising available spectral and
behavioural informaon on calls in females. We then present novel data on female and male calls in Staurois guatus and ask
how larynx morphology inuences call characteriscs. While there was no dierence in the dominant frequency between the
sexes, sound pressure of female calls was lower than in males suggesng that they could be masked by ambient stream noise
in the natural habitat. In an experimental setup, unrecepve females started calling when approached by a male less than 30
cm away, indicang an agonisc funcon of calling behaviour. In accordance with the overall size dimorphism in S. guatus,
laryngeal muscles as analysed by microCT were larger in females than in males whereas a reverse dimorphism was reported
for most anuran species with silent and vocal females. We argue that in noisy environments such as streams, small male larynx
size associated with high frequency calls is advantageous due to reduced masking and discuss the funconal dierences and
communalies in signalling behaviour between the sexes and in the genus Staurois.
Key words: anuran, female calls, laryngeal structures, noisy environment, visual signal
The communication of anuran amphibians is
characterised by disnct sexual dierences in acousc
signalling behaviour. Males are well known for their
remarkable adversement calls to out-signal competors
and aract females (Wells, 1977). Most males are even
able to display a repertoire of calls depending on the social
context (Duellman & Trueb, 1986): for example courtship
calls are emied when detecng a female or during mang
and disnct territorial signals are used to display a more or
less aggressive defence of resources against rivals (Toledo
et al., 2014). Females on the other hand are generally
considered silent although female vocal behaviour has
been known for over 250 years (Rösel von Rosenhof,
1758). Female calls are produced by over 50 species in
various social contexts (Boistel & Sueur, 2002). The female
repertoire includes release calls when unwillingly clasped
by a male (e.g., Weintraub et al., 1985), reproducve calls
to aract and smulate mates (Schlaepfer & Figeroa-Sandí,
1998) and in some cases even aggressive or territorial
vocalisaons (Capranica, 1968; Wells, 1980; Stewart &
Rand, 1991). Several studies invesgated female defensive
vocalisaons (distress or alarm screams emied when
seized by a predator (Hödl & Gollmann, 1986; Toledo et
al., 2009; Toledo et al., 2011)). Aside from release and
defensive calls, reproducve and aggressive female calling
behaviour is currently described in detail for 21 species
(Table 1) and briey reported for further 12 species (Table
2). The female aggressive call of the common rocket frog
(Colostethus inguinalis) is a so, close range, low-intensity
chirp, given during encounters with either conspecific
sex, predominantly ending with contact and somemes
wrestling with the opponent (Wells, 1980). Vocalisaons
of bullfrog (Lithobates catesbeianus) and common coqui
(Eleutherodactylus coqui) females are similarly given in
defence of territories and if the intruder does not retreat,
a physical attack follows (Capranica, 1968; Stewart &
Rand, 1991). In all reported cases of female aggressive
calls, females exhibit a larger body size, a lower dominant
frequency and shorter call duraon than conspecic males
Anuran call characteriscs are anatomically constrained
by body size, the morphology of the laryngeal structures
and neuronal mechanisms. Male body size, which
corresponds to laryngeal size (McClelland et al., 1996)
D. Preininger et al.
and vocal cord mass (Wilczynski et al., 1993), is inversely
correlated to calling frequency (Ryan & Brenowitz, 1985;
Roelants et al., 2004), generally enabling larger frogs to
produce lower pitched calls (Gerhardt & Huber, 2002).
Temporal characteristics are variable between and
within calls and are considered as dynamic call properes
(Gerhardt & Bee, 2006) mediated by the nervous
system (Walkowiak, 2006). Calls are mainly powered
by contracons of the trunk muscles (Wells, 2001) and
intensity is increased or concentrated to certain frequencies
by the vocal sac (Gridi-Papp, 2008). All females lack vocal
sacs and even in cases of larger body size have smaller
laryngeal structures (McClelland et al., 1997) and trunk
muscles (Gerhardt & Bee, 2006) compared to conspecic
males. Small larynx and trunk muscle size imply shorter
call duraon, higher or similar calling frequency and less
intense calls. Observaons of female call characteriscs,
however, do not always follow predicons derived from
body size and vary across species (Schlaepfer & Figeroa-
The most impressive distinction between body
size and call characteriscs comes from the concaved-
eared torrent frogs (Odorrana tormota) living near noisy
streams in China (Feng et al., 2006). Female body and
larynx size almost doubles that of males (Suthers et al.,
2006) but female reproducve vocalisaons have a higher
fundamental frequency extending into ultrasound (Feng
et al., 2002; Shen et al., 2008). The acousc signals of
males and females of O. tormota might avoid masking and
facilitate communicaon in low-frequency background
noise produced by streams (Narins et al., 2004). Frogs of
the genus Staurois also occur along fast-owing mountain
streams of Borneo and the Philippines. Alternavely and
addionally to high-frequency vocalisaons, males display
foot-agging signals in agonisc male-male encounters
(Grafe & Wanger, 2007; Preininger et al., 2009; Grafe
et al., 2012) to avoid masking in noisy stream habitats
(Boeckle et al., 2009; Grafe et al., 2012). In S. guatus
females also display territorial foot-agging signals in the
presence of signalling conspecic (Grafe & Wanger, 2007)
and heterospecic (e.g., S. latopalmatus; DP pers. obs.)
males. Staurois guatus is the only species of the genus
with reported female vocalisations (Grafe & Wanger,
2007). A few individuals of this diurnal ranid frog species,
endemic to Borneo, constuted the founding generaon
for a conservaon breeding and research program in the
Vienna Zoo (Preininger et al., 2012) and provided the
possibility to invesgate the infrequent calling behaviour
of females rarely observed in the eld.
The aim of this study was to (i) characterise female
calls and compare them to male calls in light of masking
interference of the environmental noise, (ii) compare
conspecific laryngeal structures and (iii) investigate
incidents triggering female calls to beer understand their
funcon and social context.
Study site and acousc recordings
We studied a populaon of Staurois guatus from March
to April 2010 in the Ulu Temburong Naonal Park, Brunei
Darussalam, Borneo (see Grafe et al., 2012 for details on
the study site), where we recorded male and female calls
in the frogs’ natural habitat. From our study populaon
we imported ve males and six females to the Vienna Zoo,
Austria, where all proceeding experiments and recordings
were conducted from April to June 2011 in a bio-secure
container facility (Preininger et al., 2012).
In the eld, we recorded adversement calls of ve male
S. guatus from distances of 1 m using direconal (sound
left) and omni-directional (sound right) microphones
(Sennheiser ME 66, ME 62) and a digital recorder (Zoom
HN4, see Grafe et al. 2012 for details on the recording
methods) at mean temperatures of 25.5°C (±SE 0.08).
Individuals at the Vienna Zoo were separated and
housed in terraria sized 0.6 x 1 m with constantly owing
water and several tree branches with large leaves (the
preferred nightly resting sites) and mean temperature
of 24.5°C (±SE 0.03). Aer an adapon period of three
weeks, all six females were sll unrecepve (no visible
eggs) and consistently perched on the same branches in
their terraria. We recorded female vocalisaons using a
direconal microphone (Sennheiser Me 66), placed 1m
from the focal individual, connected to a digital recorder
(Zoom HN4; sengs: 44.1 kHz, 16-bit resoluon). We also
measured peak sound pressure level (SPL) with a sound
level meter (Voltkraft SL-100, Germany: settings: fast/
max) during each sound recording at a distance of 1 m
to the focal individual. The A-lter frequency weighng
was used because it is approximately at from 1 to 8 kHz,
which comprises the call range of S. guatus. To reduce
reverberaon, the terrarium walls were lined with acousc
foam (egg-box prole, 40 mm deep) and a 5 x 5 cm grid
was drawn on the foam to esmate distances between the
To describe spectral and temporal call parameters, we
used recordings from the directional microphone and
analysed call duraon, note duraon, mean-, minimum-
and maximum frequency. The acousc features of stereo
recordings were extracted and measured using custom
built programs in PRAAT v. 5.2.22 DSP package (Boersma &
Weenik, 2011) that automacally logged these variables in
an output le (Grafe et al., 2012; Preininger et al., 2013a).
To assess the relatedness of female calls recorded in the
eld (n=2) and the zoo (n=6), we randomly selected 20
notes of the mul-note calls in each case. We calculated
the Euclidean distance for each pair of calls entering the
me and frequency parameters note duraon, minimum-,
maximum- and mean frequency together and generated
an acoustic dissimilarity matrix using a transformed
value range between 0 and 1. We generated an expected
dissimilarity matrix, similar to the acoustic matrix, by
dening call pairs from the same locaon as most similar
(similarity=0) and from dierent locaons as most dierent
(similarity=1). We used a Mantel test to determine if the
dissimilarity matrices of observaon-pair distances and
expected-pair distances are correlated (Bonnet & Van de
Peer, 2002). The probability of rejecng the null hypothesis
was based on 10000 randomisaon simulaons.
Female call of Staurois guttatus
We compared spectral and temporal characteriscs of
male and female calls using linear mixed models (LMMs).
The LMMs allow for repeated measurements of the same
individual to be ed in the model as random variable
and controlling for diering number of calls per individual
and note per call. The values of the parameter frequency
and note duraon were entered as dependent variables
in respecve LMMs, with male and female as predictor
variables. The idenes of individual (call) and call (note)
were again entered as nested random variables. The same
comparison was applied for call duraon and note number
with identities of individual (call) entered as nested
Sound pressure (SP) values for comparisons of call
and noise were obtained by analysing omni-direconal
microphone recordings. A period of 1 s aer each male
advertisement call of field recordings was selected to
generate ambient noise files. To obtain SP values of
ambient noise within the frequency range of male and
female calls (ltered ambient noise) we applied a hand
band filter to the spectrum of ambient noise files for
frequencies from 3600–5100 Hz. The extracted relave SP
values for call and noise were transformed into absolute
SP (Pa) by dening the most intensive SP of the complete
sound le (SP absolute=SP relave*SP measured/SP most
intensive). ‘‘SP measured’’ corresponds to the maximum
sound pressure recorded in the eld or Zoo. To test the
hypothesis that S. guatus uses frequencies less masked
by background noise, we compared maximum SP values
of male advertisement calls recorded in the field and
female calls recorded in the Zoo to ambient noise and
ltered ambient noise The SP values of ambient noise,
ltered ambient noise, female and male calls with every
call consisng of 12 or 2 values for every note respecvely,
were entered in the LMM as a dependent variable,
with ambient noise, ltered ambient noise and calls as
predictor variables. The identities of individual (call)
and call (note) were entered in nested terms as random
variable. For post-hoc tests, we used Student’s t stasc
with sequenal Bonferroni correcon for alpha because
of repeated pairwise comparisons. All analyses were
performed using IBM SPSS v. 19.
Three male and female S. guatus specimens originang
from our study populaon were obtained from the Vienna
Natural History Museum. The animals were completely
dehydrated in a graded series of ethanol and subsequently
stained in a soluon of 1% elemental iodine (I2) in absolute
ethanol (Metscher 2009) for seven days. Aer staining,
specimens were rinsed in absolute ethanol for several
hours and mounted in plasc tubes lled with absolute
ethanol for microCT-scanning. Specimens were scanned
using a SCANCO µCT 35 (SCANCO Medical AG, Brüsellen,
Switzerland) equipped with a Hamamatsu microfocus
x-ray source and a 2048*256 pixel digital x-ray detector.
Samples were scanned with 70keV source voltage and
114µA intensity, and projecon images were recorded
with an angular increment of 0.18° over a 180°rotaon.
Depending on specimen size, isotropic voxel size in the
reconstructed volumes varied between 6µm and 10µm.
Reconstructed image stacks were then imported into the
3D soware package Amira (v.5.3.3, Visage Imaging, Berlin,
Germany). In Amira, larynx musculature (for an anatomical
descripon see Trewavas, 1932) was manually segmented
in the Segmentation Editor, and muscle volumes were
extracted based on voxel segmentaon using the Material
Statistics tool. It is, however, important to note that
dehydraon and iodine staining cause some shrinkage of
so ssues, thus the measured muscle volumes do not
exactly resemble muscle volumes in the living animal.
We recorded 34 adversement calls of ve males in the
eld. During data collecon of male calls, we recorded two
coincidental vocalisaons of females on two occasions:
In the first case, a female sitting close to the stream
waterline was approached by the focal male. The female
Fig. 1. Multi-note calls of female (A-B) and male (C)
Staurois guatus. Waveform (±0.5 amplitude relave
20 μPa) and spectrogram of a female territorial call
(A) and a close-up of the two indicated notes (B). A
male advertisement call (C) recorded at the stream.
Spectrogram sengs: FFT method; window length: 0.005
s; number of me steps: 1000 and frequency steps: 1000;
Gaussian window; dynamic range: 40 dB (A-B), 20 dB (C).
D. Preininger et al.
Mean dominant frequency Mean call duraon Mean SVL Reference
[kHz] (± SD) [ms] (± SD) [mm] (± SD)
female 2 1.41 (0.05, n=19) 144 (173, n=13) 34-43aBosch & Márquez, 2001
male 1 1.44 (0.04, n=14) 149.4 (12.4, n=14) 33-39aMarquez & Verrell, 1991
female 2 1.70 (0.16, n=11) 62 (15, n=11) 38b Bush, 1997
male 1 1.80 (0.14, n=28) 102 (17, n=28) 30.6 (2.4, n=28 )
female 2 1.38 (n=1) 119 (n=1) 47 (n=1) Heinzmann, 1970
male 1 1.34 (n=1) 162 (n=1) 45 (n=1)
female 2 3.10 (n=3) 57,7 (n=3) 24.1 (n=1) Schlaepfer & Figeroa-Sandí, 1998
male 1 2.7 (n=2) 43,7 (n=2) 15.9 (n=1)
female 2 0.92 (0.03, n=1) 22100 (5600, n=1) 56.5 (n=1) Boistel & Sueur, 1997
male 1 2.10 (0.10, n=1) 17400 (3400, n=1) 35.7 (n=1)
female 2 0.74 (0.04, n=12)c20 (4, n=12 )cNA Roy et al., 1995
male 1 1.65 (0.04, n=34)c615 (155, n=34)c69 Daniels, 2005
female 2 1.53 (0.20, n=14)c61 (27, n=14)b60dRoy et al., 1995
male 1 2.14 (1.25, n=40)c 503 (101, n=40)b39-43d
female 2 2.03 (0.14, n=1) NA NA Diaz & Estrada, 2000
male 1 2.40 (0.53, n=5) NA NA
female 2 3.12-4.60 (n=14)a48-462 (n=14)a 16.0–25.8a,b Serrano et al.,
male 1 3.17-4.96 (n=82)a 124-763 (n=82)a 16.0–23.5a,b pers. communicaon
female 2 1.01 (0.02, n=32) 19.1 (2.4, n=32) 71.7 (5.8, n=15) da Silva et al., 2008
male 1 1.80 (0.16, n=25) 72 (7.3, n=25) 74.7 (3.2, n=10) da Silva & Giarea, 2009
female 2 0.58 (n=5) 68.8 (n=5) 74.6 (5.7, n=66) Lizana et al., 1994
male 2 0.54 (n=5) 69.0 (n=5) 71.9 (6.0, n=76)
female 2e5.93 (n=8)f NA 58.1 (2.7, n=8) Andreone & Piazza, 1990
male 1 4.47 (n=6)fNA 47.3 (3.5, n=6)
Table 1. Female reproducve and aggressive vocalisaons among anuran species, excluding release and distress
calls. Call types include (1) adversement-, (2) courtship- and (3) territorial calls. Mean dominant frequency, call
duraon, snout-vent length (SVL) and respecve standard deviaon (SD) are presented if not indicated otherwise.
NA=informaon not available, SE=standard error. b esmates retrieved from “amphibiaweb.org”; c n=number of calls,
not number of individuals recorded; d esmates retrieved from “frogsoorneo.org”; e also duet call data available; f
maximum frequency; g esmates for the species; h approximaon from spectrogram; i approximaon of the author.
Female call of Staurois guttatus
female 2 1.20 (n=8) 500 (300, n=8) 110bTobias et al., 1998
male 1 1.80 (SE 0.03, n=33) NA 83bWetzel & Kelley, 1983
female 2 1.30 (n=2) 3195 (777, n=2) 45-50gCui et al., 2010
male 1 0.87 (0.47, n=18) 1740 (500, n=18) 45-50gChen et al., 2011
female 2 0.93 (0.25, n=13) 60 (10, n=13) 59.2 (4.2, n=38) Krishna & Krishna, 2005
male 1 1.22 (0.49, n=22) 1090 (475, n=21) 46.2 (2.3, n=40)
female 2 1.05 (0.11, n=14)c32 (9, n=14)c78bRoy et al., 1995
male 1 2.46 (0.04, n=15)c224 (4, n=15)c48b
female 2 0.72 (n=2) NA 55 (n=2) Given, 1987
male 1 0.46-0.72 (n=2)aNA 52 (n=2)
female 2 7.2 – 9.8a< 150 56 Shen et al., 2008
male 1 5-9 (n=21)
(n=21) 32.5 Feng & Narins, 2008
female 3 1.10-1.50 (n=6)a1050 (SE 120, n=6) 44 (n=25) Stewart & Rand, 1991
male 3 1.40-1.60 (n=4)a1140 (SE 120, n=6) 34 (n=35)
female 3 0.3-0.5h 1400-1800a125b Capranica, 1968
male 3 0.5-0.8 400-600a95-110a,b
female 3 4.24 (0.08, n=6) 3060 (465, n=6) 50.1 (0.7, n=6) current study
male 1 4.67 (0.11, n=7) 301 (29, n=7) 36.1 (1.4, n=5) Grafe & Wanger, 2007
female 3 2.5iNA 27 (n=141) Wells, 1980
male 1 3.20-4.55 (n=6)aNA 25 (n=90)
Mean dominant frequency Mean call duraon Mean SVL Reference
[kHz] (± SD) [ms] (± SD) [mm] (± SD)
started vocalising, crossed the stream and connued to
move away without the male following it. In the second
case, we accidentally disturbed a female at its resng site
and caused it to jump away. The female approached two
nearby males and started calling at a distance of approx.
0.5–1 m from them. Calling connued for 10 min before
it moved away without the males following it.
In the Vienna Zoo we recorded 76 calls of six females.
The females’ predictable behaviour made it possible to
place a male in their terraria and observe the behavioural
response for a period of 30 minutes. The males usually le
their plasc transport boxes within 5 minutes and started
advertising once they discovered the female. Female
calls could be smulated when a male approached the
female to distances less than 30 cm with and without
accompanied vocalisation. Male calls from distances
greater than 30 cm did not evoke calling in females.
Notably, when males actively moved from branch to
branch and gradually approached, females displayed a
series of calls somemes accompanied by foot-agging
behaviour. Staurois guatus females possess no vocal
sac and calls were emied with an open mouth (Video
1, see <hp://www.thebhs.org/pubs_journal_online_
appendices.html>). In response to female vocalisaons,
males either retreated from their posion or remained
moonless at their posion for the rest of the test period.
We never observed any physical contact between tested
Males give a short two note call with narrow
frequency bands, whereas a female call consists of a
Table 1. Connued.
D. Preininger et al.
series of high pitched, frequency-modulated notes with
up to four harmonics (Fig. 1). Males keep their mouths
closed whereas females call with the mouth opened.
Zoo recordings of female calls had an average of 12
notes (range 3–35), and vocalisaons recorded in the
eld consisted of 21 and 25 notes. Euclidean distances
calculated from four acousc note parameters did not
correlate with the expected distances (Mantel test
Pearson correlation: r=-0.008, one-tailed p=0.446)
suggesng high similarity between female vocalisaons
recorded in the eld and in the Zoo.
Female vocalisaons diered in temporal parameters
from male calls, but apart from harmonics no dierences
in spectral call characteriscs could be observed (Table
2). Comparison of SP of male and female calls and noise
produced by the stream revealed signicant dierences
between the sexes (LMM: F3,808=184.670; p<0.001, Fig.
2). Male calls had higher estimated SP values (0.049
Pa±SE 0.003; 68 dB) than female vocalisaons (0.019
Pa±SE 0.002; 60 dB) (LMM: pairwise comparison: ß=0.03;
SE=0.002; t=16.869, p<0.001). Both, female and male
calls, however, had less SP than the noise produced by
the stream (0.064 Pa±SE 0.003; 70 dB, LMM: pairwise
comparison: female: ß=-0.044; SE=0.003; t=-16.657,
p<0.001; male: ß=-0.015; SE=0.003; t=-4.987, p<0.001).
The SP of male adversement calls exceeded the SP of the
stream ltered in the frequency range of the call (0.015
Pa±SE 0.003; 58 dB, LMM: pairwise comparison: ß=0.034;
SE=0.003; t=11.711, p<0.001), but the vocalisaon of
females did not (LMM: pairwise comparison: ß=-0.005;
SE=0.003; t=-1.692, p=0.091).
Female S. guatus were larger (snout-urostyle-length,
SUL±SE: 50.1±0.3 mm, n=6) and heavier (body mass±SE:
9.74±0.2 g, n=6) than males (SUL: 33.6±0.4 mm, n=14,
body mass: 2.69±0.07 g, n=14). The micro-CT scans
Family Species Call type Reference
Bombina variegata courtship Savage, 1932
Ceratobatrachus guentheri courtship Yoshimi et al., 1996
Conraua a. alleni adversement Rödel, 2003
Limnonectes leporinus courtship Emerson, 1992
Limnonectes poilani* courtship Orlov, 1997
Eleutherodactylus angusdigitorum adversement Dixon, 1957
Afrixalus fornasini aggressive Stewart, 1967
Hyperolius marmoratus marginatus aggressive Stewart, 1967
Leptodactylus fallax courtship G. Garcia, M. Goetz & R. Boistel (pers. com.)
Telmatobius culeus courtship G. Garcia & M. Goetz (pers. com)
Pelophylax esculentus aggressive Wahl, 1969
Pelophylax ridibundus adversement Frazer, 1983
Polypedates leucomystax adversement Roy, 1997
Table 2. Female reproducve and aggressive vocalisaons menoned without available data on call characteriscs,
excluding release and distress calls. Call types as described by the authors. *presumably misidened Vietnam samples
Fig. 2. Comparison of sound pressure of female and male
calls of Staurois guttatus and the background noise.
Shown here are estimated means (points), standard
errors (boxes) and 95% condence intervals (whiskers)
of female territorial calls, male advertisement calls,
background noise and noise ltered in the frequency
range of female and male calls. Values without the same
superscript leer (a, b, c) dier signicantly at p<0.001.
Female call of Staurois guttatus
revealed laryngeal muscles of females to have a higher
volume than those of males (Table 3). On average the
dilator and constrictor muscle of females respecvely
had 70% and 66% more volume than in males. We were
unable to measure vocal cord size from the museum
samples due to preservaon eects, however, laryngeal
structures of all three female samples exceeded those of
males (Fig. 3).
Female Staurois guatus emit high pitched calls with an
open mouth that do not dier from male adversement
calls in their dominant frequency but show rich harmonics
and have a signicantly lower SP. The observed dierences
between the sexes most likely originate from the opened
mouth and the lack of a vocal sac in females. The vocal sac
enhances calling ecacy by recycling air and amplifying
the signal (Rand & Dudley, 1993; reviewed in Starnberger
et al., 2014). During calls with open mouth the whole
pulmonary volume is exhaled. These vocalisaons are
generally produced during defensive calls to startle
a predator or interrupt an attack (Hödl & Gollmann,
1986; Toledo et al., 2009). The open mouth likely has
an addional relevance as defensive or agonisc visual
signal. In some species, including S. guttatus, males
perform open mouth displays without vocalisation
during agonisc male-male encounters (Hartmann et al.,
2005; Grafe & Wanger, 2007; Toledo et al., 2011). While
calling an opened mouth causes call energy to spread
over a range of harmonics as demonstrated by arcially
generated calls on euthanised male frogs (Gridi-Papp,
2008). Accordingly, a closed mouth causes the dominant
frequency to be more intense and concentrated in
a narrower frequency range (Gridi-Papp, 2008) as
observed in the male S. guatus call. Gridi-Papp (2008)
and Purgue (1995) suggest that radiang structures and
the frog’s ssue act as lter to narrow the bandwidth of
the call. In addion, laryngeal anatomy contributes to the
heterotypical call characteriscs in anurans. Laryngeal
structures and muscles in male frogs are generally twice
the size of females (McClelland & Wilczynski, 1989;
McClelland et al., 1997). The sexual dimorphism is also
consistent in species with vocalising males and females
(Sassoon & Kelley, 1986; Yager, 1996) corresponding to
less intense and shorter female calls (Emerson & Boyd,
1999). Surprisingly, this common sexual size dimorphism
was reversed in S. guatus with laryngeal muscles being
larger in females. Despite larger muscle size, SPL of female
calls was lower than in males and calls were masked
by noise of the stream measured at a distance of 1 m.
However, females started calling when the distance of an
approaching male was below 30 cm in the experimental
setup. At this inter-individual distance, the reported SPL
of calls would almost triple and improve female acousc
conspicuousness for perceiving males. Male calls need to
be detectable at larger distances to aract females and
detecon and discriminaon of male calls in the genus
Staurois are enhanced by high frequencies (Grafe &
Wanger, 2007; Boeckle et al., 2009; Grafe et al., 2012). As
male body size correlates with vocal cord mass and call
frequency (Gerhardt & Huber, 2002; Roelants et al., 2004;
Narins et al., 2007), sexual selecon might have favoured
smaller males in stream dwelling frogs that produce high
Call parameter Female (n=6) Male (n=5) LMM results
mean frequency [Hz] 4234 (SE 34) 4195 (SE 50) F1/761=0.813; p=0.367
minimum frequency [Hz] 3661 (SE 29) 3699 (SE 45) F1/761=0.884; p=0.347
maximum frequency [Hz] 4807 (SE 41) 4747 (SE 62) F1/761=1.279; p=0.258
call duraon [s] 3.06 (SE 0.19) 0.22 (SE 0.28) F1/106=68.443; p<0.001
note number/call 12.1 (SE 0.7) 1.8 (SE 0.9) F1/106=73.943; p<0.001
note duraon [s] 0.033 (SE 0.001) 0.041 (SE 0.001) F1/765=75.769; p<0.001
Table 3. Comparison of spectral and temporal call characteriscs of male and female Staurois guatus. Values represent
esmated means, standard errors (SE) and p-values of Linear Mixed Models (LMM).
Characteriscs Females Males
1 2 3 mean (±SD) 1 2 3 mean (±SD)
Snout-urostyle-length (mm) 49.1 47 48.2 48.1 (1.1) 32.6 33.4 33.3 33.1 (0.4)
Head width (mm) 15.2 14.2 13.2 14.2 (1.0) 9.5 9.2 9.6 9.4 (0.2)
Body mass (g) 9.91 8.17 7.07 8.38 (1.43) 2.17 2.29 2.56 2.34 (0.20)
Dilator muscle volume (mm3) 2.664 2.601 2.960 2.741 (0.192) 1.794 1.182 1.851 1.609 (0.371)
Constrictor muscle volume (mm3) (mm3) - 1.263 1.549 1.406 (0.202) 0.836 0.848 - 0.842 (0.008)
Table 4. Absolute and mean values of morphological characteriscs of 3 female and 3 male specimens of Staurois
guatus scanned in the micro-CT.
D. Preininger et al.
pitched calls less masked by low-frequency stream noise
(also see Fig. 5 in Boeckle et al., 2009). The only other
report about a larger larynx in females compared to male
frogs comes from the ultrasonic signalling species O.
tormota also living along streams and waterfalls (Suthers
et al., 2006).
The note number of two female calls recorded in the
eld was higher than the average note number recorded
under Zoo sengs. In male and female aggressive calls
of E. coqui shorter vocalisaons are used as low level
warnings and longer calls when an aack is imminent
(Stewart & Rand, 1991). We never observed an aack
or aggressive behaviour in S. guatus; however, males
foot agged in succession to female calls. Foot agging
functions as agonistic signal to defend perching sites
(Preininger et al., 2009; Preininger et al., 2013b) and
most likely evolved from kicking aacks (Preininger et al.,
2013c). We suggest female calls followed by foot-agging
displays are similar to male signals in their behavioural
context and could constitute a stereotyped agonistic
display ritualised from a former costly aggressive
behaviour of direct contact.
Acousc and visual signals of anuran communicaon
as well as the morphological and physiological features
involved in their production are shaped by sexual
selection. Signallers influence receivers via sensory
stimulus, which in turn provides information to the
receiver. Several anuran call characteriscs are related
to physiological and morphological attributes (Ryan,
1988; Gerhardt & Huber, 2002). Males adverse not only
their species identy, locaon and size, but also sexual
recepveness with aracve or aggressive calls (Wells &
Schwartz, 2006). Likewise female signals emied to show
recepveness or unrecepveness (e.g., Ellio & Kelley,
2007) can be used by male receivers to determine the
relevant response. In the present study, males responded
to female calls by stopping adversing and approaching
the female. According to the behavioural context, we
propose that vocalisaons in S. guatus idenfy females
as potenal territorial competors and/or non-recepve
individuals rather than potenal mates.
Staurois guttatus is the only species of the genus
Staurois with reported female calls. Males of sympatric
S. latopalmatus and S. parvus also display agonistic
foot-flagging signals in succession to high frequency
calls and experience similar environmental background
noise (Boeckle et al., 2009; Preininger et al., 2009;
Grafe et al., 2012), but female signalling behaviour
was never observed. The reproductive behaviour in
the three Staurois species seems very similar, but only
male and female S. guatus foot ag during amplexus
when approached by conspecic or heterospecic (S.
latopalmatus) males (DP pers. obs.). Hence, divergent
female signalling behaviour in S. guatus can currently
not be explained by evoluonary responses to diering
environmental factors or reproductive character
displacement, its functional significance in regard to
related species remains unanswered. Grafe and Wanger
(2007) reported an addional so and short call in female
S. guatus, which could not be observed in the present
study. While mate locaon is the most common context
for female reproducve calls (Emerson & Boyd, 1999),
the call repertoire is probably much larger than currently
known. Further investigations of the genus Staurois
would help to expand our understanding of the anuran
communicaon system and evoluonary development
that shapes morphological and physiological features for
signalling in closely related species.
Fig. 3. Cross secons of female and male Staurois guatus retrieved from microCT-scans showing laryngeal structures.
(A) female, (C) male, br brain, c constrictor muscle, d dilator muscle, hy postero-medial process of hyoid plate, ie inner
ear, la larynx, pg pectoral girdle, ph pharynx, sk skull. (For absolute values of dilator and constrictor muscle volume
see Table 4).
Female call of Staurois guttatus
We thank the Universi Brunei Darussalam and the sta
of the Kuala Belalong Field Studies Centre (KBFSC). Special
thanks to T.U. Grafe for his assistance in the eld. We are
grateful for the support of D. Schraer, A. Weissenbacher,
T. Wampula and the sta of the rainforest house from the
Vienna Zoo. We also thank two anonymous reviewers
and F. Toledo for their valuable input. The study was
supported by the Austrian Science Fund (FWF): P22069
and P25612, the Society of Friends of the Vienna Zoo,
and the University Vienna.
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