Fundamental constraints in synchronous muscle limit superfast motor control in vertebrates

1) It is sometimes unclear for the ZF studies if the dorsal or ventral tracheobronchialis muscle (DTB, VTB) is being studied: The text describing Figure 4B reports results for the VTB, but the figure is labeled DTB. DTB is also the label in Figure 4A but there is no specific reference to DTB or VTB in the text – which one is in Figure 4A? Which one is shown in supplemental figure for Figure 4? Figure 5A refers to DTB – the text just refers to SFM. Figure 6 does not indicate if the results are for the VTB or DTB. The text and figures should be clear as to which muscle is used in each analysis. If the same muscle is not used throughout all of the analyses it should be explained why this is the case.

We apologize for these errors and thank the reviewers for catching them. In our experiments we measured MYH content using qPCR separately for all syringeal muscles in adult males and females. In this manuscript, we decided to only include data on the VTB, for which superfast muscle performance has been established earlier. Unfortunately, we erroneously presented the DTB qPCR data in Figure 4A and B. In our revision we have omitted DTB qPCR data and include only data on VTB. We rephrased the relevant Materials and methods section into:

“We dissected the syringeal muscle tracheobronchialis ventralis (VTB) on ice immediately after isoflurane euthanasia (Elemans et al., 2008).”

Consequently we replaced Figure 4A, B with only VTB data and reran the corresponding statistics presented in Figure 4A and main text. Importantly, none of the statistics changed and as such these changes have no effect on our conclusions.

Furthermore, in Figure 1, Figure 4—figure supplement 1, and Figure 5A we made an error in the labelling and have relabelled these figures. These measurements were all from VTB as mentioned correctly in the relevant methods section.

2) Discussion, first paragraph: The hypoglossal nerve is considered to be homologous to occipital nerve roots in fish (a hypoglossal is not identified in fish) so they are not as distinct as one might be led to think here – recent molecular evidence supports the hypothesis that sonic motor neurons in fish and birds arose from a common progenitor domain distinct from the one that gave rise to laryngeal motor neurons (Albersheim-Carter et al., 2016, Respiratory Physiology and Neurobiology 224: 2-10). This does not mean that the SFMS in birds and fish do not depend upon distinct MYH genes for their function and "descend from different evolutionary events" but the authors might want to be more circumspect in saying that occipital and hypoglossal nerves are "different cranial nerves".

We thank the reviewers for pointing out this very interesting paper. As this new evidence renders our previous statement incorrect, we decided to omit the sentence. We tried adding a brief statement regarding the origin and physiological properties of the vocal/sonic motorneurons to our Discussion both at the end of the first paragraph and also at the last paragraph before the concluding paragraph. However, in both cases this introduced such a significant break of the reading flow and detracted from the main point of the paragraph that we decided to not do so. In the revised manuscript, the first Discussion paragraph ends as follows:

“Since extremely fast crossbridge kinetics are necessary, though not sufficient, for superfast performance (Rome, 2006), and these are set largely by MYH gene expression in vertebrates (Bottinelli, Schiaffino and Reggiani, 1991), the parsimonious conclusion is that the SFM phenotype found in the sound producing organs of bats, songbirds, and toadfish descend from different evolutionary events.”

3) The last paragraph of the Discussion should include a comment on the genomic analysis that supports independent origins for the MYH in each group studied.

We have rephrased the concluding paragraph into:

“To conclude, the non-orthology of the dominant myosin heavy chains expressed in SFMs of birds, mammals, and fish indicates that SFM likely evolved independently in each lineage. […] Motor-driven acoustic modulation rates in complex communication are thus fundamentally limited by muscle architecture.”

4) There are too many abbreviations.

We acknowledge that we used many abbreviations and in our revision aimed to reduce the number of abbreviations as much as possible, especially in the abstract and main text. As such, we omitted six abbreviations in the main text (MY-32, EOM, EDL, VPE, TnC, VTB), but kept the five abbreviations relevant to this paper (ECC, MYH, qPCR, SFM, SR), and some that are widely used (ADP, ATP, Ca²⁺, SERCA -this abbreviation would otherwise be replaced by SR Ca²⁺-ATPase which is not helping readability). In the Discussion we kept essential gene and protein names (MyHC-sf, MyHC-eo, MyHC-2b).

5) The authors should justify their selection of a 100Hz cycle frequency as the boundary for super-fast muscles.

In our revised manuscript we use the definition by Rome (2006) where SFM range from approximately 90 to 250 Hz.

Introduction: “Their ability to repetitively contract and relax fast enough to produce work at cycling rates over around 90 Hz and up to 250 Hz sets them apart from other muscles by almost two orders of magnitude and allows the execution of central motor commands with millisecond temporal precision (Rome, 2006; Rome et al., 1988; Elemans et al., 2004; Elemans et al., 2008; Elemans et al., 2011.”

Figure 1 legend: “SFM (labeled purple) has been defined as synchronous muscle capable of producing work at cycling frequencies in excess of 90 Hz (grey box) (Rome, 2006).”