Peer review of: Vocal state change through laryngeal development (2019)

Reviewers' Comments:  
 
Reviewer #1:  
Remarks to the Author:  
In the present work, Zhang and colleagues investigated the role of biomechanical properties of the 
marmoset larynx on vocal motor production during postnatal development – a largely overseen topic. 
Using a combination of a number of well-controlled, elaborated methodological approaches in a 
sufficient number of animals/larynges they convincingly show that the transition from immature infant 
cries to adult phee calls seems to be mainly caused by the developing larynx and not the underlying 
brain circuits. This transition is mainly due to a sound source switch from vocal fold to vocal 
membrane oscillations.  
 
This is an exciting paper and important work on the largely underestimated role of the developing 
vocal apparatus during vocal development. The results are novel and compelling and the manuscript is 
well-written. This work will be of broad interest and suitable for publication in Nature Communications 
once my concerns below have been sufficiently addressed.  
 
 
Major comments:  
1. Based on their experimental results, the authors explain the loss of cry vocalizations after infancy 
by a change in the biomechanical properties of the marmoset larynx and hypothesize that “the cry 
attractor state is outside the vocal capacity of the adult larynx”. However, recent studies clearly show 
that marmoset monkeys, which experienced limited parental feedback during infancy and adolescence 
do still produce cry vocalizations in certain behavioral contexts at adult age (Gultekin and Hage, Nat 
Commun 2017, Gultekin and Hage, Sci Adv, 2018). However, the vocal performance of these animals 
should not be existent with respect to the model proposed in the present study. It will be important to 
explain how these animals fit into the proposed framework of the present study.  
 
 
Minor comments:  
1. In line 237 the authors state that the larynges were collected from deceased monkeys, in line 256 
they write that the larynges were extracted from euthanized monkeys. Please clarify.  
 
2. Figure 2: The comparison of WE between infant and adult larynges generated with the same 
pressure and vocal fold strain settings show slightly lower yet high WE values also for the adult 
larynges (above -20dB). More specifically, it seems that adult larynges are also able to produce 
sounds with WE values well above -20dB and therefore within the WE range of infant cries (see Fig 
1c). Please clarify.  
 
 
 
Reviewer #2:  
Remarks to the Author:  
This paper provides evidence for the hypothesis that vocal development in marmoset monkeys is 
guided largely by biomechanical changes in the vocal folds, not only by brain development. It is shown 
that the infant marmoset has a capacity for self-sustained oscillation in two modalities, while the adult 
vocalizes in one modality. One modality is low-frequency, low periodicity oscillation of the vocal folds 
proper, which the infant uses in its cries. The other modality is high frequency, high periodicity 
oscillation of a vocal membrane that extends from the superior-medial edge of the vocal folds, which 
the adult uses in its mature “phee” vocalizations. Multiple methodologies are used, including high-
speed imaging of vocal fold motions in excised larynges, stress-strain analysis of vocal fold tissues, 

acoustic analysis, and phonation threshold (PTP) measurement.  
 
While the results add some validity to the hypothesis, they do not propose anything fundamentally 
new. In most fields of motor learning, it has been recognized that the brain develops in conjunction 
with, and often as a result of, changes in the physical plant. In humans, for example, infant cries are 
rough and aperiodic because the alignment of collagen and muscle fibers is slow to develop. The full 
development of the vocal ligament may take several years. Laryngeal musculature is activated to 
enhance this development, and proper control then requires new muscular strategies  
 
The methodology is weakened by the fact that no vocal tract is present when excised larynges are 
used to study self-sustained oscillation. The video images shown here do not clearly reveal whether or 
not there is an alternation of convergent-divergent glottal shapes (upper-lower phase difference) 
when the vocal membrane vibrates. This would appear to be a requirement without vocal tract 
interaction. [The references cited include mathematical treatments of how an acoustically inertive 
vocal tract can interact with a sound source to produce self-sustaine oscillation when there is no 
upper-lower phase difference]. The conclusion drawn by this reviewer is that (1) membrane 
development basically follows ligament development in humans, (2) it is likely that the membrane is 
not vibrating in isolation, but some coupled movement exist between the membrane and the lower 
fold, and (3) the two vibration modalities are similar to chest and falsetto registration found in many 
species.  
 
 
 
Reviewer #3:  
Remarks to the Author:  
In this study, the authors explore the relationship between vocal production and the changing physical 
and material properties of the vocal tract. Combining theory and experiment, the authors present data 
supporting the hypothesis that changing laryngeal dynamics need to be taken into account in order to 
explain vocal development, i.e., vocal development is not a neural control signal change alone. This is 
a fascinating study that builds upon the earlier work of some authors, and will be of great interest to a 
broad range of neuroscientists. However, the paper is not written to be broadly accessible. Control 
experiments are not discussed or described, and a key prediction of the model (phase differences) is 
untested. The discussion, for a topic of such wide interest, is frustratingly brief to non-existent. 
Therefore, while I am positive about this very interesting manuscript, I would like to see the authors 
address some concerns in a major revision.  
 
Major concerns  
 
1) My biggest concern is essentially the lack of a control experiment. While the maturation and 
material changes of vocal tract properties are explored in this study for the cries → phees transition, 
nothing is said about the other sounds that are produced using the same apparatus, yet do not appear 
to acoustically change over development (twitters or trills, as discussed in Takahashi et al 2015). 
These sounds also appear to of high-frequency like the phee, so similar issues would hold. On the flip 
side, it does not seem to be the case that adults are incapable of producing low-frequency sounds – 
for example, Agamaite et al 2015 describe “Egg” and “Ock” sounds that have sub-1KHz f0s. If it were 
the case that the control space for low-frequency production is lost in adults, how are they still able to 
produce these sounds?  
 
2) Which control parameter is responsible for the loudness of the produced sounds? The contact calls 
studied here seem extremely loud, and it appears that if one emphasizes loudness as a critical factor, 
you could take an underlying linear system and over-drive it to produce nonlinear outputs with 

harmonic distortions. Could this be an alternative model to explain why a developmental change is 
seen for phees but not other, quieter, sounds?  
 
3) Related to the authors’ earlier study, how do the authors imagine an instructive signal (such as 
parental feedback) facilitates the transformation from cries → phees? Is it entirely based on the 
synchronization of respiration and muscle tension as their model suggests? But then wouldn’t 
marmosets also need to learn synchronization for the other sounds they produce?  
 
4) In Fig. 2, it seems that the experiments were performed by holding one parameter and ramping the 
other parameter. Given the modeling in Fig. 4, it would be far more interesting to co-vary the 
parameters in-phase or out-of-phase. In an adult vocal tract, maybe it is possible to generate low 
frequency sounds after all by changing this phase difference. This would also go towards addressing 
concern (1) above.  
 
5) Please state the mean lengths of the vocal folds in infants and adults. VF length and VF strain are 
used interchangeably when describing Fig. 2 results, while these are of course related, it would help to 
consistently stick to absolute or relative units. It would be instructive to also provide, as extended 
data figure, the plots in 2e-g, with VF *stress * on the x-axis, as the authors mention in line 125 that 
this could be a critical factor in generating sounds with high f0s.  
 
6) Why are there systematic differences in the sampling of the pressure-strain space between adults 
and infants? Looks like the minimum pressure applied was ~2kPa for infants and ~0.5 kPa for adults. 
The reason for this is not clear from the Methods. What do the blank regions in these plots signify – 
data not acquired, or f0 not quantifiable? Since the f0 of the infant cry is ~500Hz, it would be useful to 
mark this contour in Fig. 2e to g.  
 
7) Are these systematic differences in the VF thickness between infants and adults that could explain 
some of the differences in the stress-strain curves in Fig. 3b for VF? On a related note, I would 
suggest plotting VM and VF on separate plots, it is difficult to compare across adult and infant VF and 
VM, partly because it is scaled for VM. To my eye it seems that the infant stresses at high strains are 
~50% larger than adults, and this could contribute to the steeper slopes seen in Fig. 2 (another 
reason to show pressure-stress axes).  
 
8) The paragraph beginning line 121 could be rewritten much more clearly. If a string model were 
true, adults would be at an advantage for producing lower frequency sounds (longer VF, smaller non-
linearity in stress-strain curves). Yet they don’t, suggesting that the string models are missing an 
important component. Then introduce the VM. A better picture of the larynx showing the VF and VM 
would be helpful.  
 
9) The manuscript is highly technical and hard to read. It is not written with a general audience, such 
as the readership of Nature Communications, in mind. The authors seem to be well within the 
available word limit, and I would highly recommend that they provide more narrative explanations and 
interpretations. The explanation for Fig. 4 is particularly dense and hard to follow.  
 
Additional Comments  
1) Please explain you term the peaks of the parameter distributions in Fig. 1c - f “attractors”. This 
terminology is introduced in the results section without  
 
2) In Fig. 1 it would be helpful to include the spectra of the cry, subharmonic, and phee with f0 
marked as in Ext. Fig. 1b.  
 

3) The red and blue colors for the lines in Fig. 2d should be cued better and defined in the legend. 
Maintain the same color scheme in Ext. Fig. 2 (it is currently flipped).  
 
4) In the supplementary movies, could you mark the orientation of the cranial and caudal sides? The 
difference referred to in line 163 is not at all clear in Movie S1.  
 
5) Line 179 The authors state that the adult VM has increased stiffness, but from Fig. 3b it seems that 
the adult VM slope is actually smaller than the infant VM slope. Is this a typo?  
 
6) line 193 For infants, the two *attractors * ….  

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