Syringeal vocal folds do not have a voice in zebra finch vocal development

ABSTRACT Vocal behavior can be dramatically changed by both neural circuit development and postnatal maturation of the body. During song learning in songbirds, both the song system and

syringeal muscles are functionally changing, but it is unknown if maturation of sound generators within the syrinx contributes to vocal development. Here we densely sample the respiratory

pressure control space of the zebra finch syrinx in vitro. We show that the syrinx produces sound very efficiently and that key acoustic parameters, minimal fundamental frequency, entropy

and source level, do not change over development in both sexes. Thus, our data suggest that the observed acoustic changes in vocal development must be attributed to changes in the motor

control pathway, from song system circuitry to muscle force, and not by material property changes in the avian analog of the vocal folds. We propose that in songbirds, muscle use and

training driven by the sexually dimorphic song system are the crucial drivers that lead to sexual dimorphism of the syringeal skeleton and musculature. The size and properties of the

instrument are thus not changing, while its player is. SIMILAR CONTENT BEING VIEWED BY OTHERS FEMALE CALLS PROMOTE SONG LEARNING IN MALE JUVENILE ZEBRA FINCHES Article Open access 16 October

2024 MOLECULAR SPECIALIZATIONS OF DEEP CORTICAL LAYER ANALOGS IN SONGBIRDS Article Open access 30 October 2020 EVOLUTIONARY NOVELTIES UNDERLIE SOUND PRODUCTION IN BALEEN WHALES Article 21

February 2024 INTRODUCTION Many vertebrates change their vocal output over postnatal development1,2,3,4. Especially in species capable of vocal imitation learning, progressive changes in

vocal output are attributed to changes in the underlying brain circuitry. However, the maturation of the vocal organ and tract have profound effect on voice during puberty in humans5, and

can explain transitions from juvenile to adult calls in marmosets6. Thus, the postnatal development of the body can drive changes in vocal behavior, and therefore needs to be included when

studying vocal development. The zebra finch is a widespread animal model to study the mechanisms underlying vocal imitation learning and production7,8. In male zebra finches, vocalizations

change dramatically in structure and variability from the onset of song learning at 25 DPH to adulthood2,3,9. Song develops from variable subsong (~ 25 DPH) to plastic song (50 DPH) to

highly stereotyped crystallized song (100 DPH)9. These changes, including the ones in acoustic features within syllables, such as fundamental frequency (_f_o), amplitude and entropy, are

typically attributed to changes in neural circuitry. However, the vocal organ, the syrinx, is also undergoing significant anatomical and functional changes during song

development10,11,12,13. Coinciding with the transition from the sensory to the sensorimotor phase of learning around 20 DPH, syringeal muscles in male zebra finches increase in mass and

cross-sectional area10. Additionally, the contractile properties of male syringeal muscles increase in speed11, which affects the transformation from neural commands to muscle force13. Thus,

over development functional changes occur in the syrinx that change the transformation from neural signals into force which thereby change motor control of the vocal organ. It is currently

unknown if the syrinx exhibits changes over song development that can affect the complex transformation from muscle forces to sound. Analogous to mammals, birds produce sound when expiratory

airflow from the bronchi induces self-sustained vibrations of vocal fold-like structures, the labia, within the syrinx14,15,16,17 (Fig. 1a) following the myoelastic-aerodynamic (MEAD)

theory for voice production18. Songbirds have two bilateral sets of independently controlled paired folds or labia, one in each bronchus or hemi-syrinx, with the lateral labium on the

lateral side, and the medial vibratory mass (MVM) on the medial side. The MVM is a tissue continuum in which the thicker part is called the medial labium (ML)19,20,21 (Fig. 1b). While in

most songbird species investigated these labia consist of multiple tissue layers, in zebra finches they seem rather homogeneous in the distribution and orientation of collagen and elastin

fibers21,22. According to MEAD, acoustic features, such as source level (SL) and _f_o, or boundary conditions for vibration, such as the phonation threshold pressure, are largely set by the

positioning and tension of, and driving pressures18 on, the vocal folds, as well as their resonance properties and shape. Vocal fold resonance properties in turn are determined by vocal fold

length and structure, such as collagen and elastin fiber composition, the occurrence of tissue layering and layer orientation23. Postnatal changes in size, structure and mechanical

properties of the vocal folds in humans24,25,26, and marmosets6 can cause dramatic changes in vocal output over vocal development. Thus, material property changes of syringeal MVM could also

contribute to changes in vocal output in songbirds. In zebra finches, surprisingly we have no information on either basic morphology, material properties or acoustic output of the MVM as a

function of postnatal development. To our best knowledge syrinx developmental information seems missing for any songbird species. To isolate the contribution of the MVM to the transformation

from neural commands to sound, MVM histology21,22 or mechanical properties, such as tensile tests27, would provide important information, but do not provide the sound ultimately produced.

Sound pressure waveforms or features can be predicted using computational models that require tissue property information. For example, one-dimensional string models can coarsely predict

_f_o or _f_o-ranges from tissue properties27,28, but not an exact _f_o resulting from a specific muscle length or respiratory pressure amplitude, nor other acoustic parameters. Full

complexity 3D fluid–structure-acoustic-interaction models can predict time-resolved pressure waveforms29. A recent model of the (much less complex) pigeon syrinx predicted several acoustic

parameters rather accurately based on geometry and material properties15, but we do not have such a model for the songbird syrinx. However, in either case, models produce mere predictions

that need to be tested against experimental data to validate their accuracy. Thus, we aim at measuring sound output of the vocal organ, and not proxies thereof, as a function of driving

parameters. Furthermore, measuring acoustic features, such as _f_o, SL, harmonic content, or entropy during song over development cannot provide conclusive data on the contribution of a

changing transformation from muscle to sound. First, during trial-and-error learning, individuals are correcting their song for deviations from the targets30,31. Thus, when the acoustic

output of the vocal organ or its control space would be changing, the individual would correct for the resulting error. By looking at in vivo singing behavior we cannot exclude compensatory

correction for changes driven by tissue maturation. Therefore, we need precise experimental control over driving parameters. Second, in many songbird species the left and right hemi-syrinx

contribute differently to the total frequency range32,33,34. Simultaneous air flow through the left and right hemi-syrinx indicates that some syllables are produced by bilateral oscillations

in several songbird species35,36, including zebra finches37,38. When both sides oscillate independently this would give rise to two independent frequencies32,33,34. This phenomenon is one

of the salient features of birdsong. However, other examples show that only one fundamental frequency is produced during simultaneous air flow through the left and right hemi-syrinx, where

flow oscillations can be in or out of phase between sides38. When two mechanical oscillators have similar resonance frequencies and are physically close they can become mechanically coupled

by energy injections39,40. Such injection locking, or injection pulling, can force one oscillator to oscillate at the resonance frequency of the other depending on the coupling strength.

Because the two syrinx sides are physically close, we cannot exclude that the two sides become mechanically coupled by injection locking if the two sides have tissue resonance frequencies

that are sufficiently similar. Thus, despite different resonance frequencies of left and right MVMs19, the two sides can become coupled and oscillate at the same frequency. After bilateral

nerve cuts single _f_o harmonic stacks are produced41,42,43, which supports the idea that the two sides are coupled by injection locking. In summary, we cannot exclude that injection locking

plays a role in zebra finch song in vivo and thereby cannot conclude from in vivo song if the behavior of one side changes over development. Consequently, to isolate the contribution of the

MVM to the transformation from neural commands to sound, we need to start by looking at the contributions of each side individually. Taken together, to measure the complex transformation

from muscle forces to sound, we need to measure the sound produced by each hemi-syrinx, driven by physiologically realistic driving parameters under controlled experimental conditions

without compensatory neural control. Two independent motor systems control the tension and positioning of MVMs in songbirds: the respiratory and the syringeal motor system. The respiratory

system generates the bronchial pressure and flow driving MVM oscillations. The syrinx is suspended in a pressurized air sac, the interclavicular air sac (ICAS), that is essential to achieve

oscillation and sound production14,44,45. Both bronchial and ICAS pressure have been shown to correlate to changes in _f_o during manipulation of _p_icas in vivo45,46 and ex vivo14. However,

we currently lack densely sampled characterization of the pressure control space in the zebra finch syrinx and do not know how both pressures interact to drive acoustic parameters. The

syringeal motor system of songbirds consists of up to 8 pairs of intrinsic and extrinsic syringeal muscles20,47,48 (Fig. 1a), which control the positioning of individual cartilages and bones

within the syringeal skeleton as well as the torque on them. Hereby syringeal muscles control—either directly or indirectly—the adduction level and tension of the labia, which are suspended

within the syringeal skeleton27. Labial adduction level and tension provide fine-control of air flow and _f_o49. The control space of syringeal muscles is vast due to its

multidimensionality and our understanding limited14,48,50. Both changes in the shape and acoustic features of the motor control space could contribute to vocal development, but this is

currently unknown. Here we test if the complex transformation from driving forces to acoustic output of the zebra finch syrinx changes over vocal development. We designed an experimental

paradigm to quantify sound production of the syrinx as a function of physiological driving parameters. In a first step, we did not include the biomechanical effect of syringeal muscles, but

focused on syringeal dynamics controlled by respiratory pressures and densely sampled the control space of bronchial versus air sac pressure. MATERIALS AND METHODS IN VITRO SOUND PRODUCTION

ANIMALS USE AND CARE Zebra finches (_Taeniopygia guttata,_ order Passeriformes) were kept and bred in group aviaries at the University of Southern Denmark, Odense, Denmark on a 12:12 h

light:dark photoperiod and given water and food ad libitum. Adult birds were provided with nesting material, ad libitum_._ Nest boxes were monitored daily, so that birds could be accurately

aged. Breeding began in December 2017 and ended November 2018. We studied vocal output of the freshly dissected syrinx in vitro in four age groups: 25 DPH (N = 5 males, N = 5 females), 50

DPH (N = 4 males, N = 4 females), 75 DPH (N = 6 males, N = 4 females), and 100 DPH (N = 5 males, N = 6 females). Sex was determined by dissection postmortem (25 DPH) or plumage (all other

ages). APPROVAL FOR ANIMAL EXPERIMENTS All experiments were conducted in accordance with the Danish law concerning animal experiments. The protocol was approved by the Danish Animal

Experiments Inspectorate (Ministry of Environment and Food, Glostrup, Denmark, Journal number: 2019-15-0201-00308). The study was carried out in compliance with the ARRIVE guidelines. SYRINX

MOUNTING PROCEDURE Animals were euthanized by Isoflurane (Baxter, Lillerød, Denmark) overdose. The syrinxes were dissected out while being regularly flushed and then submerged in a bath of

oxygenated Ringer’s solution (recipe see51) on ice. Each syrinx was photographed with a Leica DC425 camera mounted on a stereomicroscope (M165-FC, Leica Microsystems, Switzerland) prior to

being placed inside the experimental chamber. Each bronchus was placed over polyethylene tubing (outer diameter 0.97 mm × inner diameter 0.58 mm, Instech Salomon, PA, USA) and secured with

10-0 nylon suture (AROSurgical, Newport Beach, CA, USA). The trachea was placed into a 1 ml pipette which was cut to the length of 15 mm (outer diameter 2.4 mm, inner diameter 1.4 mm). Some

adipose tissue was left on the trachea to ensure an airtight connection. The total length from syrinx to tracheal outlet was 35 mm, similar to the upper vocal tract length of 28–33 mm52. To

control the pressure inside the chamber, it was made airtight with a glass lid, which also allowed visualization of the syrinx through the stereomicroscope. EXPERIMENTAL SETUP We designed an

experimental setup that allows studying the mechanical behavior of the syrinx in vitro or ex vivo (described in detail in14). Both in vitro and ex vivo preparations use fresh, alive

tissues. The ex vivo preparation is also perfused through the original vasculature14. The bronchial pressure (_p_b) and pressure in the experimental chamber (i.e. interclavicular air sac

pressure, _p_icas) can be controlled independently by two dual-valve differential pressure controllers (model PCD, 0–10 kPa, Alicat Scientific, AZ, USA). Bronchial flow through the syrinx

was measured with a MEMS flow sensor (PMF2102V, Posifa Microsystems, San Jose, USA) about 7 cm upstream from the bronchial connection. Sound was recorded with a ½ in. pressure

microphone-pre-amplifier assembly (model 46AD, G.R.A.S, Denmark), amplified and high-pass filtered (0.2 Hz, 3-pole Butterworth filter, model 12AQ, G.R.A.S., Denmark). The sound recording

sensitivity was tested before each experiment using a 1 kHz tone (sound calibrator model 42AB, G.R.A.S., Denmark). The microphone was placed 15 cm from the tracheal connector outlet and

positioned at a 45° angle to avoid the air jet from the tracheal outlet. The sound, pressure and flow signals were low-pass filtered at 10 kHz (model EF120, Thor Labs) and digitized at 50 

kHz (USB 6259, 16 bit, National Instruments, Austin, Texas). EXPERIMENTAL PROTOCOL To explore the acoustic output of the avian syrinx (Fig. 1a), we subjected the syrinxes to _p_b ramps from

0 to 3 kPa (over atmospheric pressure) at 1 kPa/s with _p_icas at a constant pressure. In one run, we applied 13 _p_icas settings at equidistant 0.23 kPa intervals between 0 and 3 kPa in a

randomized order with 2 s pause (_p_b and _p_icas = 0 kPa) between ramps (Fig. 1c). We avoided high flow regimes (_p_t > 2 kPa) to decrease desiccation and avoid potential damage to the

syrinx. When liquid in the bronchi or trachea prevented normal sound production in part of the run, we repeated the entire run. We subjected left and right hemi-syrinx separately in

randomized order to this paradigm by allowing air flow only through one side and kept a 1-min rest between runs. The total duration for the experiment was maximally 20 min per individual.

DATA ANALYSIS Sound was filtered with a 0.1 kHz 3rd order Butterworth high-pass filter, pressure and flow signals were filtered with a 2 kHz 3rd order Butterworth low-pass filter, all with

zero-phase shift implementation (filtfilt function, Matlab). Sound, pressure and flow signals were binned in 2 ms duration bins, with a sliding window of 1 ms, and we calculated several

parameters per bin. We calculated the root mean square (RMS) values for all signals. For the sound signal, we additionally extracted the fundamental frequency (_f_o), source level (SL) and

Wiener entropy (WE). Fundamental frequency was extracted using the yin-algorithm53 by manually optimizing minimal aperiodicity (0.05–0.2) and power (0.25–5.0 mPa). Source level at 1 m

distance of the emitted sound was defined as: $$\mathrm{SL }= 20\mathrm{log}_{10} \frac{P }{P\mathrm{o}} + 20\;\mathrm{ log}_{{10}}\mathrm{r},$$ (1) where _p_ is the RMS of sound pressure

(Pa), _p_o is the reference pressure in air of 20 µPa, and r the distance (0.15 m) from the tracheal outlet to the microphone. To calculate WE, we first computed the amplitude spectrum Px of

each bin using the periodogram method (Fast Fourier transform (FFT) size = 2048, overlap = 1024 samples). WE was defined as: $$WE={\mathrm{log}}_{10}

\left(\frac{geomean(Px)}{mean(Px)}\right).$$ (2) The transmural pressure over the MVM was defined as _p_t = _p_b − _p_icas. Phonation threshold pressures (PTP) were identified for _p_icas,

_p_b, and _p_t as the first pressure value for each _p_b ramp where sound power crossed the set threshold. The minimal _f_o was defined as the average _f_o of the first value for each _p__b_

ramp where sound power crossed the set threshold. Minimal SL was defined as the lowest SL value of all _p__b_ ramps per side. By combining pressure, flow and sound signals we were able to

calculate the mechanical efficiency (ME) as the ratio (in dB) of acoustical power (_P_acoustic) over aerodynamic power (_P_aerodynamic): $$\mathrm{ME }= 10\;\mathrm{ log }_{10} \left(

\frac{P_{{\text{acoustic}}}}{P_{{\text{aerodynamic}}}}\right),$$ (3) The acoustic power (W) was assumed to radiate over half a sphere: \(P_{{\text{acoustic}}}=\frac{4\mathrm{\pi

r}{2}p{2}}{\rho v}\), with air density _ρ_ = 1.2 kg m−3, speed of sound _v_ = 344 m s−1, and _p_ the RMS sound pressure in Pa. The aerodynamic power _P__aerodynamic_ = _p_b_V_ where _p__b_

is the bronchial pressure (Pa) and _V_ is the flow rate (m3 s−1). All data analysis was done in Matlab54 and R version 3.5.155. MVM DIMENSIONS After the experimental protocol, each syrinx

was pinned down in the situ position and kept in 4% (w/v) paraformaldehyde for 24 h at 4 °C. It was then placed in 0.1 M phosphate buffered saline and stored at 4 °C. To ensure that syrinx

geometry remained consistent and resembled the position in vivo, we made photos of the geometry in vivo after opening of the ribcage, during in vitro sound production and after pinning and

fixation. Additionally, the MVM is structurally supported by the tympanum and cartilages B1-B3 that keep it under base tension in a resting position. This position can be mechanically

disturbed, but this would require the bronchi to be pinned in a position or angle clearly outside the in situ range. To quantify the projected size of the MVM of each hemi-syrinx, we

carefully cut the syrinx in half (sagittally) and took pictures of MVMs of each hemi-syrinx using a Leica DC425 camera mounted on a stereomicroscope (M165-FC, Leica Microsystems,

Switzerland) in Leica Application Suite (LAS Ver. 4.7.0). These photos are made perpendicular to the camera plane because images were sharp in the focal plane. We measured the distances

between physiological landmarks in the MVM following19,27: (i) the distance between the _medio-ventral cartilage_ (MVC) and the _lateral dorsal cartilage_ (LDC), and (ii) the distance

between the LDC and the _medio-dorsal cartilage_ (MDC). Distance measurements between cartilages were taken edge-to-edge in the middle of the cartilage. Additionally, we measured the area of

the LDC which is suspended in the MVM. Length and area measurements were taken using ImageJ (Ver. 2.0.0-rc-69/1.52p56). IN VIVO SOUND PRODUCTION To measure the lowest fundamental frequency

produced by an unactuated syrinx in vivo, we performed unilateral tracheosyringeal nerve resections on the right side of the syrinx of juvenile (N = 6) and adult (N = 4) zebra finch males.

TRACHEOSYRINGEAL NERVE LESIONS Unilateral denervation of the tracheosyringeal nerve was performed as previously described57. In brief, birds were anesthetized with isoflurane (induction 3%,

maintenance 1.5–2%) and a 5 mm section of the right tracheosyringeal nerve was removed from the trachea through a small incision of the skin. After that the skin was re-sutured and the birds

were allowed to recover in a heated cage. Adults were subsequently transferred to holding cages. Juveniles received surgery at 30 DPH and were returned to their parents after surgery. After

final song recordings, all animals were sacrificed to confirm the absence of nerve regeneration (adults 20 ± 2 and juveniles 75 ± 3 days post surgery). In all animals we confirmed that the

nerve did not grow back. SOUND RECORDINGS Song was recorded in custom-built, sound attenuated recording boxes (60 × 95 × 57 cm) and vocalizations were recorded continuously using an

omnidirectional microphone (Behringer ECM8000) mounted 12 cm above the cage connected to an amplitude triggered recording software (Sound Analysis Pro58). Sound was digitized at a sampling

rate of 44.1 kHz with 16-bit resolution (Roland octa capture, amplification 40 dB). Adult birds were recorded several days before and 3 weeks after denervation. Juveniles were recorded at 50

and 100 DPH. SONG ANALYSIS Adult birds: For each individual, the most common motif was determined by two experienced observers (IA, CPHE) in recordings acquired before denervation.

Subsequently, the same motif was identified in recordings acquired 3 weeks after denervation (days post-surgery: 20 ± 2). A custom written Matlab script was used to extract motifs from sound

recordings. 134–200 (average 184 ± 26) motifs per bird were selected for analysis. WAV files were high pass filtered at 0.3 kHz using a 3rd order Butterworth filter. Syllables or parts

thereof were assigned to be produced by the left or right hemi-syrinx based on the following procedure. In zebra finch harmonic stacks, expiratory air flow can be positive on both

sides37,38, which requires a careful strategy to assign the contribution each side makes during sound production. The two sides can oscillate independently or with some degree of coupling so

that they open and close in or out of phase. If these two sides oscillate as an uncoupled system, each side would emit an independent _f_o that would be visible as two simultaneous stacks

in the spectrogram. If the _f_os of the oscillators are similar enough, their vibration can become mechanically coupled by energy injections, which would force both sides to oscillate with

the same _f_o (i.e., injection locking) or at an integer ratio. For injection locking to occur the _f_os of both oscillators have to be close, which in zebra finches is around 500 Hz. We

thus focused on analyzing frequency modulated high _f_o syllables (4–6 kHz) that are spaced far from 500 Hz. If these notes were produced by flow through both sides we would expect to see

two independent frequency components after denervating the right side; a frequency modulated high one (left) and an unmodulated low one (right) at the natural resonance of the tissue. If the

syllable was produced by the left side only, we would expect to see no change upon unilateral denervation of the right side. If the syllable was produced by the right side only and we

abolished tension control of the right side by denervation, we would expect to see a frequency drop to the natural resonance of the tissue of the right oscillator without tension control.

Thus, this procedure unambiguously allowed us to assign sides for high notes produced on the right. In line with the literature41,42,43, all high notes in our animals were produced on the

right side. For the left side, we analyzed the syllable with the lowest _f_o trajectory that remained unchanged after denervation. Juvenile birds: Song of juvenile males (N = 6) was analyzed

on recordings from 50 DPH (DPH 52.8 ± 1, days post-surgery: 22.3 ± 0.8). At this age, the syllable structure is not fully stereotyped, but motifs usually can be distinguished. We measured

the lowest fundamental frequency of 50 motifs per bird irrespective of syllable identity. As the juvenile birds were denervated while singing subsong, we couldn’t distinguish which syllable

was produced on which side. Under the assumption that the lowest fundamental is produced by the denervated side, we classified the measured syllables as right produced. In all birds,

frequencies were measured on spectrograms of post denervation recordings (FFT size: 1024, overlap: 75%, Hanning window) in SASlab (Avisoft, Germany). In all juvenile birds the frequency of

the lowest harmonic (i.e. _f_o) was measured. In all other cases, to increase the _f_o resolution, we measured the 3–5th harmonic and _f_o was calculated as: $${f_{\text{o}}} =

{\raise0.7ex\hbox{${{f_n}}$} \!\mathord{\left/ {\vphantom {{{f_n}} n}}\right.\kern-\nulldelimiterspace} \!\lower0.7ex\hbox{$n$}}.$$ (4) where _n_ is the nth harmonic. STATISTICS To assess

which pressure (_p_b, _p_icas, _p_t) drives _f_o and SL in adults, we used a delta Bayesian information criterion (ΔBIC) approach. In a first step, we fit a linear model to the _f_o-pressure

and SL-pressure relationship. We then computed the BIC values for these models and subtracted the BIC of the null model with no pressure variable (_f_o = 1; SL = 1) to calculate the ∆BIC

score. The model with the lowest ∆BIC score represents the model with the best fit. To compare the _f_o–_p_t and SL–_p_b relationship over ages and between side and sex, we fit individual

linear models to the data and extracted the slopes. For _f_o we split the data in two regions: region S1 (_p_t = 0–0.75 kPa) and region S2 (_p_t = 0.75–2 kPa). To test whether age, sex or

side of the syrinx had a significant effect on our response variables, we fit linear mixed effect models (LMMs) to our data using the lmer function of the lmerTest package59 with maximum

likelihood optimization. Sex, side and age were fit as fixed effects and animal was included as a random effect to correct for dependence. Model equations can be found in Tables S1, S2 and

S3. To determine which effects significantly contribute to the model, we performed model selection using a Likelihood Ratio Test (LRT) with the Chi squared distribution using the lme4

package60. The difference between the absolute difference in minimal _f_o between sexes and the difference in minimal _f_o in vivo was assessed with Welch’s t-test. The difference between S1

and S2 slopes for _f_o were assessed using paired t-tests for males and females. All reported values in the text are mean ± SD. All error bars in figures are SD. The outputs of all LMMs are

reported in Supplementary Tables S1, S2, and S3. We chose to present data in figures spilt by sex even when not significant. Statistical significance was accepted at p < 0.05 for all

statistical tests. RESULTS ACOUSTIC OUTPUT OF THE ADULT SYRINX To quantify the functional acoustic output of the adult male and female zebra finch syrinx, we densely sampled the bronchial

(_p_b) and interclavicular (_p_icas) pressure control space in isolated left and right hemi-syrinxes in vitro. For all zebra finch syrinxes tested, sound was produced in a pressure space

enclosed by a minimal _p_b and _p_icas and exclusively in the lower half of the _p_b, _p_icas space (Fig. 1d). Here a differential or transmural pressure (_p_t) exerts force on the MVM that

is positive when directed outwards from bronchus to surrounding air sac. We quantified the phonation threshold pressures (PTP) for the three pressures (Fig. 1g). The PTPb was not

significantly different for sex (LMM, _p_ = 0.418) or side (LMM, _p_ = 0.395) and was 1.01 ± 0.28 kPa (range: 0.64–1.68 kPa, N = 11). The PTPicas was also not significantly different for sex

(LMM, _p_ = 0.638) or side (LMM, _p_ = 0.223) and was 0.45 ± 0.27 kPa (range: 0.22–1.07 kPa, N = 11). The PTPt was also not significantly different for sexes (LMM, _p_ = 0.057) or side

(LMM, _p_ = 0.395) and was 0.15 ± 0.19 kPa (range: 4e−5 to 0.75 kPa, N = 11). Next, we quantified the acoustic output of the adult zebra finch hemi-syrinx within the _p_b-_p_icas control

space for three parameters: fundamental frequency (_f_o), source level (SL) and Wiener entropy (WE). In all animals, fundamental frequency was gradually modulated depending on different

combinations of _p_b and _p_icas (Fig. 1d–f). Frequency jumps were not observed. Of all pressures, _p_t described the _f_o data best (∆BIC = − 528, see “Methods”). The continuous, smooth

increase of _f_o with _p_t can be clearly seen in Fig. 1e,f. In males, the right typically produced higher frequencies than the left as a function of _p_t (Fig. 1e). However, the minimal

_f_o produced was not significantly different and was 511 ± 102 Hz (range: 352–613 Hz, N = 5) and 513 ± 123 Hz (range: 312–625 Hz, N = 5) for left and right hemi-syrinx respectively (Fig. 

1h). To describe _f_o as a function of _p_t, we split the data in to two regions (Fig. 1e,f); region S1 (_p_t = 0–0.75 kPa) and region S2 (_p_t = 0.75–2 kPa) and fit linear models to the

data. The slope of S1 was 189 ± 76 Hz/kPa (range: 67–453 Hz/kPa, N = 5), and was significantly higher than the S2 slope of 55 ± 58 Hz/kPa (range: 25–310 Hz/kPa, N = 5) by 134 ± 18 Hz/kPa

(Fig. 1j) (paired t-test, t = 5.3061, df = 9, _p_ = 5e−4). In females, the minimal _f_o produced was 529 ± 84 Hz (range: 435–678 Hz, N = 6) for the left and 568 ± 57 Hz (range: 518–650 Hz, N

 = 6) for the right hemi-syrinx (Fig. 1f). The slope of the S1 region was 181 ± 108 Hz/kPa (range: 67–453 Hz/kPa, N = 6) and was also significantly higher than the S2 slope of 55 ± 58 Hz/Pa

(range: − 42 to 167 Hz/kPa, N = 6) by 126 ± 51 Hz/kPa (Fig. 1j) (paired t-test, t = 3.31, df = 10, _p_ = 0.0079). Comparing the sexes, the difference in minimal _f_o between left and right

seemed smaller in males than in females (Fig. 1i), but this effect was not significant (Welch’s t-test, t = − 1.08, df = 6.42, p = 0.32). The slopes of both S1 and S2 were not significantly

different between sex and side and were 185 ± 92 Hz/kPa (range: 25–453 Hz/kPa, N = 11) and 55 ± 58 Hz/kPa (range: − 42 to 167, N = 11), for S1 and S2, respectively (Fig. 1j, Supplementary

Table S1). Source level at 1 m distance was best described by _p_b (∆BIC = − 78,996) and increased linearly with pressure in both sexes (Fig. 2a–c). The minimum SL (Fig. 2d) was not

significantly different for sex (LMM, _p_ = 0.149) or side (LMM, _p_ = 0.225) and was 45 ± 4 dB re 20 µPa at 1 m (range: 37–51 dB re 20 µPa at 1 m, N = 11). The slope of the SL-_p_b

relationship (Fig. 2e) did not differ significantly between males and females (LMM, _p_ = 0.704), but was significantly higher on the right side (5 ± 2 dB/kPa, N = 11) than the left side (4 

± 1 dB/kPa, N = 11) (LMM, _p_ = 0.016). We did not show any obvious relation to _p_b_,__ p_icas, or _p_t, and therefore we considered the mean of the entire control space (Fig. 3a). The mean

WE did not differ significantly between sexes (LMM, _p_ = 0.138) or sides (LMM, _p_ = 0.661) and was − 1.8 ± 0.12 dB (range: − 1.6 to − 2.1 dB, N = 11) (Fig. 3b). Our setup allowed us to

calculate the Mechanical Efficiency (ME) of sound production, which estimates how much of the power in the air flow is transformed into sound (see “Methods”, Fig. 4). We found that ME did

not vary systematically with _p_b_,__ p_icas, or _p_t and therefore considered the mean of the entire control space (Fig. 4b). ME was not significantly different between sexes (LMM, _p_ = 

0.118), but was significantly lower (LMM, _p_ = 0.027) on the right side (− 36 ± 1.8 dB, range: − 39 to − 33, N = 10) compared to the left (− 35 ± 2.2 dB, range; − 38 to − 31 dB, N = 10)

(Fig. 4c). ACOUSTIC CONTROL SPACE DOES NOT CHANGE OVER VOCAL DEVELOPMENT Next, we tested if the output of the isolated zebra finch syrinx changed over vocal development from 25 to 100 DPH

(Fig. 5). Like in the adult syrinx, sound was produced exclusively in the lower half of the syringeal _p_b, _p_icas space over vocal development. The PTPb was not significantly affected by

age (LMM, _p_ = 0.257), side (LMM, _p_ = 0.268), or sex (LMM, _p_ = 0.558) and was 0.9 ± 0.24 kPa (range: 0.53–1.68 kPa, N = 32). PTPicas demonstrated a small but significant increase with

age (LMM, _p_ = 0.002), but no significant difference between sexes (LMM, _p_ = 0.762) or sides (LMM, _p_ = 0.274), and went from 0.21 ± 0.8 kPa at 25 DPH (range: 0.006–0.25 kPa, N = 8) to

0.37 ± 0.25 kPa at 100 DPH (range: 0.21–1.1 kPa, N = 11). Consistently, PTPt also demonstrated a small but significant decrease with age from 0.17 ± 0.08 kPa at 25 DPH (range: 0.06–0.31 kPa,

N = 8) to 0.03 ± 0.06 kPa at 100 DPH (range: 4e−5 to 0.16 kPa, N = 11), and marginally, but significantly lower in males (0.0846 ± 0.0981 kPa) compared to females (0.1047 ± 0.0875). We

examined changes in the syringeal _p_b-_p_icas control space for three acoustic parameters: fundamental frequency (_f_o), source level (SL) and Wiener entropy (WE). Over development, _f_o

was also gradually modulated within the _p_b-_p_icas space and did not exhibit frequency jumps (Fig. 5). In males and females, the minimal _f_o produced by each hemi-syrinx in vitro did not

change significantly from 25 to 100 DPH (Fig. 5b), but the left hemi-syrinx produced a significantly lower minimal _f_o than the right (males: L: 487 ± 106 Hz, N = 18, R: 519 ± 104 Hz, N = 

18, females: L: 480 ± 83 Hz, N = 15, R: 590 ± 69 Hz N = 15) (LMM, _p_ < 0.001). Interestingly, in 5 out of 18 males the left–right difference was reversed. The slope of the _f_o-_p_t

relationship in region S1 did not significantly change with age (LMM, _p_ = 0.651), sex (LMM, _p_ = 0.630), or side (LMM, _p_ = 0.270) and was 91 ± 109 Hz/kPa (range: − 204 to 250 Hz/kPa, N 

= 32). The slope in region S2 was significantly different between ages (LMM, _p_ = 0.0273), but not for sex (LMM, _p_ = 0.464), or side (LMM, _p_ = 0.474), and was 88 ± 43 Hz/kPa (range:

31–146 Hz/kPa, N = 8), 62 ± 28 Hz/kPa (range: 18–105 Hz/kPa, N = 7), 70 ± 46 Hz/kPa (range: 5–149 Hz/kPa, N = 7), and 65 ± 50 Hz/kPa (range: − 43 to 135, N = 11) for 25, 50, 75, and 100 DPH

respectively. Source level increased with _p_b for all animals (Fig. 6a,e), but the minimal source level did not change significantly with age (LMM, _p_ = 0.680), sex (LMM, _p_ = 0.374), or

side (LMM, _p_ = 0.271) and was 44 ± 4 dB with reference sound pressure of 20 µPa at 1 m (range: 36–51 dB, N = 32) (Fig. 6b,c). The SL-_p_b slope also did not change with age (LMM, _p_ = 

0.83), or sex (LMM, _p_ = 0.874), but was significantly less steep (LMM, _p_ = 0.037) on the left (4.1 ± 1.0 dB/kPa, range: 1.9–6.2 dB/kPa, N = 32) versus right hemi-syrinx (4.7 ± 1.6 

dB/kPa, range: 0.9–7.4 dB/kPa, N = 32) by 0.6 ± 0.3 dB (N = 32) (Fig. 6d). Mean WE did not change significantly for any of the parameters tested (LMM, age: _p_ = 0.070, sex: _p_ = 0.979,

side: _p_ = 0.084), and was − 1.8 ± 0.1 dB (range: − 2.1 to − 1.6 dB, N = 32; Fig. 7a–b,e–f). Next, we calculated the mechanical efficiency (ME) to test if the ability of the syrinx to

convert mechanical power to sound changed over song development (Fig. 7c,g). ME did not significantly change with sex (LMM, _p_ = 0.091) or side (LMM, _p_ = 0.101), but demonstrated a small

but significant decrease (LMM, _p_ = 0.046) from − 33 ± 1.7 dB (range: − 37 to − 31 dB, N = 8) at 25 DPH to − 35 ± 1.8 dB (range: − 39 to − 33 dB, N = 10) in adults (Fig. 7d,h). Taken

together, we observed that the acoustic output of the syrinx control space did not change considerably over development in males and females in vitro. To test whether these in vitro results

are representative for the mechanical behavior of the syrinx during in vivo sound production, we measured the acoustic output over development in vivo and used unilateral nerve cuts to

exclude the contribution of the syringeal muscles. Because both SL and WE are affected by vocal tract filtering properties, we focused on fundamental frequency. In zebra finches both sides

of the syrinx can contribute to syllable production37,38. To get around this problem, we developed a procedure to assign the produced syllable or frequency component therein (see “Methods”)

to the left or right side. In adults, we observed three types of syllable changes after nerve resection on the right side: (1) The _f_o of high frequency (4–6 kHz) syllables dropped from

4638 ± 725 to 599 ± 77 Hz after denervation on the right side (Fig. 8). We did not observe two simultaneous frequencies in high _f_o notes after nerve cuts. Thus, we could reliably assign

high notes to be produced by the right hemi-syrinx only. (2) In all animals we observed syllables with two simultaneous _f_o trajectories after nerve cuts (Fig. 8a). This suggested that

these syllables were produced by contributions from both sides, and refuted bilateral mechanical injection locking for those syllables (see “Methods”). In these cases, we could also

unambiguously assign side because the unchanged _f_o time trajectory had to be produced by the left side. (3) We also observed syllables whose _f_o trajectories remained unchanged after

right nerve cut, which suggested that these syllables were most likely produced on the left. We measured the lowest fundamental frequency produced by males with unilateral syrinx denervation

at 50 and 100 DPH (see “Methods”). The minimal _f_o produced by the right hemi-syrinx did not change significantly (Welch’s t-test, t = − 0.91, df = 6.01, p = 0.40) with age and was 555 ± 

69 Hz (N = 6) and 599 ± 77 Hz (N = 4) for 50 and 100 DPH, respectively. Furthermore, in adult males the minimal _f_o did not differ between the left (intact) and right (denervated)

hemi-syrinx and was 502 ± 44 Hz (N = 4) and 599 ± 77 Hz (N = 4) for left and right, respectively (paired t-test, t = − 2.10, df = 3, p = 0.13). Thus, the in vivo values were consistent with

the in vitro data (Fig. 5b), strongly suggesting that the output of the syrinx in vitro is representative of the in vivo situation. MORPHOLOGICAL CHANGES OVER DEVELOPMENT We lastly

investigated if the medial vibratory mass (MVM) dimensions change over development (Figs. S1 and S2). The tension in the MVM can be increased by the shortening of two syringeal muscles that

insert on the _medio-ventral cartilage_ (MVC) and _medio-dorsal cartilage_ (MDC)20 (Fig. S1a). A third cartilage, the _lateral dorsal cartilage_ (LDC), is embedded within the MVM. A

thickening of the tissue between the MVC and LDC is called the medial labium. The two syringeal muscles change the tension of the MVM by changing the distance between (1) MVC and LDC and (2)

LDC and MDC (Fig. S1a). The MVC—LDC length did not change with sex (LMM, _p_ = 0.72) or age (LMM, _p_ = 0.446), but was significantly (LMM, _p_ = 0.002) longer on the left (1.13 ± 0.10 mm;

range: 965–1297 μm, N = 29) compared to the right (1.05 ± 0.11 mm, range: 798–1363 μm, N = 29) (Fig. S1b). The LDC–MDC length also did not change with sex (LMM, _p_ = 0.982) or age (LMM, _p_

 = 0.191), but it was significantly (LMM, _p_ = 0.149) longer on the left (0.71 ± 0.16 mm, range: 387–1107 μm, N = 29) compared to right (0.65 ± 0.13 mm, range: 365–858 μm, N = 29) (Fig.

S1c). The area of the LDC was smaller (LMM, _p_ < 0.001) on the left (4.1e4 ± 2.2e4 μm2, range: 1978–9.9e4 μm2, N = 29) compared to the right (6.2e4 ± 3.0e4 μm2, range: 11,867–1.1e5 μm2,

N = 29) hemi-syrinx and increased over development (LMM, _p_ = 0.048; Fig. S1d). Lastly, we approximated overall growth of the syrinx as the distance between both ends of the B4 cartilage in

the bronchus, and found no significant change for side (LMM, _p_ = 0.14), sex (LMM, _p_ = 0.17), or age (LMM, _p_ = 0.8) and was 1.85 ± 0.23 mm (range: 1280–2196 μm, N = 29; Fig. S1e).

DISCUSSION As songbirds learn to sing, both their song system and vocal organ are undergoing postnatal changes that lead to dramatic changes in vocal behavior. Here we show that the acoustic

output of the sound generators within the zebra finch syrinx does not change considerably over vocal development. This strongly suggests that the observed acoustic changes during vocal

development are caused by changes in the motor control pathway, from song system circuitry to muscle force, and not by changes in the avian analog of the vocal folds. In other words, the

properties of the instrument are not changing, but its player is. Our data present a first detailed quantification of the acoustic output of the isolated zebra finch syrinx when driven by

respiratory pressures. We show that both sides of the syrinx, or hemi-syrinxes, produce sound in a well-defined consistent pressure control space. The pressure space is bound by phonation

threshold pressures (PTP) for _p_b, _p_icas and _p_t, which are the same for both sexes and sides. The PTPb values in adults of 1.01 ± 0.28 kPa reported here are consistent with earlier

reported PTPb values of 1.2 kPa in situ61 and in vivo48. The possibility to control the two respiratory pressures (_p_b and _p_icas) independently is different from laryngeal systems where

no air sacs directly apply force on the vocal folds. To what extent birds can actively and independently control the magnitude of _p_t in vivo during song remains unknown. Certainly,

positive pressurization of _p_icas is an essential condition to achieve syringeal vibration in all investigated species in vivo (including zebra finches45), and in vitro14,62,63, and dynamic

_p_t changes up to 1 kPa occur during vocalizations in ringdoves64,65,66. We deliberately covered a larger magnitude pressure range than what the sparse in vivo measurements suggest,

ensuring that the in vivo situation is included and forms a subset of the measured parameter ranges presented here. Our data show that within the investigated control space, source level and

fundamental frequency are modulated by respiratory pressures. SL was driven by bronchial pressure, consistent with laryngeal voiced sound production18. The SL magnitude of our isolated

preparations of 45–60 dB (reference 20 µPa at 1 m) corresponded well to reported SL of zebra finch song in the lab (50–70 dB reference 20 µPa at 1m67). Fundamental frequency (_f_o) was set

predominantly by _p_t, consistent with the idea that _p_t acts as a force on the MVM that increases tension68, which has also been observed in collapsible tubes69. Especially close to the

phonation onset, _f_o modulation driven by _p_t is steep (200 Hz/kPa). If a _p_t difference of 1 kPa occurs in zebra finches, this could thus lead to _f_o modulation of 200 Hz. This range is

of comparable magnitude to full stimulation of the ventral syringeal muscle that changes _f_o by 200 Hz in zebra finches ex vivo14. However, we do not know the magnitude of _p_t during

zebra finch song. In contrast, Wiener Entropy (WE) was not modulated systematically in the pressure space. However, over the course of song learning WE has been shown to change over multiple

time scales, from within a day to weeks and months2,3,70 and increased entropy variance is linked to better learning success3,70. These data suggest that WE is under control of the motor

systems of (i) the syrinx, by modulating collision force and thereby spectral slope of the sound source71, (ii) the upper vocal tract, by changing the airway resonance properties and thereby

frequency content of the radiated sound72,73. Additionally, some degree of mechanical coupling between the tissue resonances and vocal tract may occur. In zebra finches the coupling

strength will be limited because for most syllables the tissue resonances (500–700 Hz) are far away from vocal tract resonances74, and experimental manipulation of tract length does not

affect _f_o75. Mechanical efficiency may have been an important selecting factor during the evolution of the syrinx as a vocal organ75 and our data confirms that the mechanical efficiency of

voiced sound production is indeed high in the avian syrinx compared to the mammalian larynx. Our data shows that about 0.05% (− 33 dB) of the flow energy is converted into sound, while in

mammals, laryngeal ME is only about 1.10–3 to 0.01% (− 50 to − 40 dB) in tigers76 and 3.10–5 to 1.10–3% (− 65 to − 50 dB) in marmosets6). ME in the rooster has been reported to be as high as

1.6% in vivo77. The ME of the zebra finch in vivo is likely to be higher than we measured in vitro, because our preparation does not include a vocal tract, lacks syringeal motor control,

and in vivo both sides can contribute to syllables37,38. The reported SL of the zebra finch song in the lab can be up to 70 dB reference 20 µPa at 1 m67, which is 10 dB louder than the 60 dB

we measured with only respiratory pressure control. A 10 dB SL increase (i.e. root mean square (RMS) is 3 times higher) while maintaining the same aerodynamic power, results in a tenfold

increase in radiated acoustic power and a ME of 0.5% (− 23 dB). This is comparable to the very high efficiency reported in roosters78. Furthermore, we observed slight differences of ME over

development, but these effects were not as pronounced as the changes observed in marmosets, where the adult larynx produces louder and more efficient vocalizations than the infant larynx6.

Thus, the ME of the zebra finch syrinx is very high already at 25 DPH and remains unaffected by vocal development. Our data shows that acoustic output of the syrinx is modulated smoothly

within the pressure boundaries: both SL and _f_o increased continuously with bronchial and air sac pressures. Thus when driven by physiologically relevant pressures, this nonlinear dynamic

system exhibits only one bifurcation from steady (no vibration and no sound) to flow-induced self-sustained vibration, without additional bifurcations, such as period doublings or jumps to

deterministic chaos61,79,80,81,82. Earlier work already showed that in a subset of the pressure control space in situ, additional bifurcations did not occur61. Moreover, the supposed

bifurcations during song in vivo, e.g. frequency jumps, were shown to not be actual bifurcations61, but more likely millisecond scale modulation driven by superfast syringeal muscle83. Here

we also show that in the independently controlled and densely sampled pressure control space of the zebra finch syrinx in vitro, additional bifurcations do not occur. Thus, driven by

physiologically relevant pressures, the acoustic output of the syrinx is continuous, which simplifies sensorimotor control61. Zebra finch song syllables can be produced by MVM oscillations

of one hemi-syrinx or by contributions of both sides37,38, but we did not study mechanical interaction between the two hemi-syrinxes. However, our data shows that the _f_o magnitude of left

and right hemi-syrinx oscillations is about only 50 Hz apart throughout their pressure control space. Because the two sides are also physically close, this raises the possibility that they

can become mechanically coupled in vivo by injection locking or pulling. Indeed, abolishing syringeal motor control by bilateral nerve cuts, shows that single _f_o stacks are

produced41,42,43, which suggest that the two sides are coupled. Thus, next to controlling acoustic output of each hemi-syrinx, we propose that in songbirds the syringeal muscles may also

play an important role in controlling hemi-syrinx coupling strength as a mechanism to control song complexity. Our data show that the above relations between driving pressures and acoustic

output do not change from 25 to 100 DPH over development in zebra finches. The stable acoustic output of the sound generators over development suggests that its mechanical properties are not

changing. The _f_o of sound is determined by positioning and tension of and driving pressures on the vocal folds, as well as their resonance properties19. Because we show that minimum

frequency does not significantly change over development in vitro and in vivo without syringeal control, our data strongly suggest that the effective resonance properties of the syringeal

sound generators when driven by physiological realistic pressures do not change over vocal development. Furthermore, the _f_o-_p_t relationship (slope S1in Fig. 5d) can be seen as a proxy of

MVM stiffness, because _p_t exerts a force on the MVM that causes changes in strain. Since the S1 parameter did not change over development, the change in MVM stiffness as response to

strain is also likely changing very little over development. Taken together, the MVM vibration frequencies and response to external force do not change, which strongly suggest that its

material properties also do not change over development. We think it is unlikely this conclusion can be extended to most other songbird species. The adult zebra finch labia are rather

homogenous in structure21 and we can thus expect a rather isotropic mechanical behavior. However, in the other songbird species investigated, the labia in adults consist of multiple tissue

layers28 with distinct collagen and elastin fiber orientation and likely anisotropic mechanical tissue properties. In humans, the adult vocal folds also consist of several distinct tissue

layers that take over 15 years to developed from a uniform tissue structure at birth to layered after puberty24,25,26,84,85. These layers have different mechanical properties and can

contribute differently to mechanical oscillations in humans85. Over development even small changes in material properties could drive state changes in the vocalizations of marmosets6. Taken

together, we think our study emphasizes our general lack of information on tissue maturation over vocal development in songbirds, but even more so, in birds and mammals in general. We

propose that syringeal sound production on the one hand, and its control on the other, are shaped differentially by genetic and environmental factors in zebra finches. First, we propose that

the development and maintenance of the sound generators is predominantly under genetic control. Second, we propose that the syringeal muscles and skeletal properties are influenced by use

and training, which can ultimately even lead to a sexually dimorphic syrinx. Our data suggest that MVM mechanical properties are set and maintained mostly by genetic factors and less by

usage. First, a salient feature in our dataset is that the adult left hemi-syrinx produces a lower minimal _f_o compared to the right in both sexes, corroborating earlier findings19. In most

songbirds both hemi-syrinxes contribute to the vocal repertoire32,33,34,35,36,37,86,87 and left–right differences in labial morphology21,22 and acoustic output41,88, 89 are common in

songbirds. In all investigated species to date, except for the Bengalese finch14,89 and Australian magpie90, the left hemi-syrinx produces lower frequencies. Here we show for the first time

that differences between left and right sound generators, size and acoustic output, in zebra finches are already established at 25 DPH. Second, our data suggest that the acoustic output of

the MVMs driven by pressure remains stable from 25 DPH. This finding suggests that intrinsic mechanical properties of the bilateral sound generators do not change after 25 DPH, but we would

need detailed structural analysis to establish if the MVM tissues indeed have matured. We believe that this latter feature makes the zebra finch a wonderful model system to study vocal

development compared to mammalian systems where larynx maturation strongly contributes to vocal output6,24,25,26. Third, the MVM mechanical output seems to remain constant even though they

are colliding billions of times over their lifetime and a large variability in use must exist between individuals and sexes. In human vocal folds, strong collisions can lead to several types

of lesions that severely affect vocal output91. Thus, structural repair of vibratory tissues must lead to maintenance of their properties, but what cellular and molecular mechanisms

underlie this maintenance in birds remains unknown. Taken together, these observations support the ideas that the sound generators of the zebra finch syrinx are matured at the onset of vocal

learning and that their composition remains stable, which suggests that their composition and dynamical behavior is set predominantly by genetic factors and less by use. The zebra finch

syrinx is thus a stable instrument whose postnatal development does not add further complexity to song learning. In stark contrast, zebra finch syringeal muscles are changing functionally

over postnatal development during song learning. Until 25 DPH the syrinx of male and female zebra finches is not clearly distinguishable. However, after 25 DPH, the syringeal muscles start

to exhibit sexually dimorphic features, such as increased muscle mass10 and speed in males11, which leads to a changing transformation from motor neuron spikes to force, often referred to as

the neuromuscular transform13. Furthermore, muscle force exerted on bones is known to redirect bone deposition and thus can lead to significant bone remodelling92. Taken together, we

propose the hypothesis that in zebra finches, syringeal muscle activity and resulting forces acting on the syrinx are crucially important drivers to shape the bones and cartilages of the

syringeal skeleton. This hypothesis predicts that some anatomical syrinx differences between sexes in zebra finches, where both sexes produce calls but only the males sing, can be caused by

muscle use and training driven by the sexually dimorphic brain (i.e., song system). This hypothesis remains to be tested experimentally. To what extent this idea can be transferred to other

(song)bird species also remains to be investigated. In several species sexually dimorphic features of the syrinx have been related to differences in song amount and vocal repertoire93,94.

For a correlative study, it would be interesting to investigate the syringeal anatomy of songbird species where both sexes produce calls and sing95. DATA AVAILABILITY The datasets generated

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ancestral in songbirds. _Nat. Commun._ 5, 3379 (2014). Article  ADS  PubMed  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors wish to thank Torben Christensen and Sonja

Jakobsen for technical support, Emil B. Hansen for help with breeding, and John Jackson for assistance with statistical analysis. FUNDING This work was supported by the Danish Research

Council (Grant DFF 5051–00195), the Carlsberg Foundation (Grant CF17-0949) to I.A. and Novo Nordisk Foundation (Grant NNF17OC0028928) to C.P.H.E. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS

* Department of Biology, University of Southern Denmark, 5230, Odense, Denmark Alyssa Maxwell, Iris Adam, Pernille S. Larsen, Peter G. Sørensen & Coen P. H. Elemans Authors * Alyssa

Maxwell View author publications You can also search for this author inPubMed Google Scholar * Iris Adam View author publications You can also search for this author inPubMed Google Scholar

* Pernille S. Larsen View author publications You can also search for this author inPubMed Google Scholar * Peter G. Sørensen View author publications You can also search for this author

inPubMed Google Scholar * Coen P. H. Elemans View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.M., I.A. and C.E. conceived the study. A.M.,

I.A. and C.E. developed technical methods. I.A. and C.E. provided reagents and materials. A.M. and P.L. performed in vitro experiments and collected all data. I.A., P.S. and C.E. performed

in vivo experiments and collected all data. A.M., I.A. & C.E. analyzed the data. A.M. prepared all figures and tables. A.M., I.A. and C.E. wrote the manuscript. All authors read and

approved the final version of the manuscript. CORRESPONDING AUTHOR Correspondence to Coen P. H. Elemans. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

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ARTICLE CITE THIS ARTICLE Maxwell, A., Adam, I., Larsen, P.S. _et al._ Syringeal vocal folds do not have a voice in zebra finch vocal development. _Sci Rep_ 11, 6469 (2021).

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