Peer review of: Comparative analysis of squamate brains unveils multi-level variation in cerebellar (2019)


Reviewers' Comments:  
 
Reviewer #2:  
Remarks to the Author:  
The authors have fully addressed my comments and modified the manuscript accordingly.  
 
 
 
Reviewer #3:  
Remarks to the Author:  
Although Macri et al. have undertaken significant revisions to their manuscript, it still lacks focus and 
some the claims made are not necessarily supported by their analyses or are confirmations of results 
published elsewhere. In particular, the repeated reference to the “accuracy” of their brain 
reconstructions and analyses is an over-statement and I have some concerns over tissue quality based 
on the sources of some of their material.  
 
1. The Introduction still lacks focus and has some inaccuracies. For example, the first paragraph is far 
too general and the statement about studies not describing brain shape or internal neuroanatomy is a 
broad generalization and largely untrue. There many papers that have described brain shape 
accurately and internal anatomy across species. I think the point the authors are trying to make is 
that there few, if any, studies that have integrated multiple approaches to study brain evolution within 
a clade. That statement is entirely correct and what the authors have attempted to execute here.  
 
The choice of cerebellum as a brain region to focus on is not sufficiently justified. There is no evidence 
that is “the most powerful model system for studying brain evolution”. In fact, there is still relatively 
little comparative research on cerebellar anatomy, despite the detailed descriptions of Larsell, who 
first revealed the neuroanatomical diversity of the cerebellum across vertebrates. Further, whether the 
cerebellum can be identified on an endocast or not is irrelevant to the study of cerebellar evolution 
across extant species.  
 
Several other statements were unclear or vague, such as “cellular content”, “multi-level, interspecific 
variations” and the entire sentence on page 3 (lines 10-14). I think what they are trying to say is that 
there is a lot interspecific variation, but the extent to which this is reflected in numbers or sizes of 
neurons, morphology, behaviour or ecology is unclear. This is not, however, how this comes across in 
this paragraph.  
 
In the last paragraph of the Introduction, I will first note that the cerebellum is not a “trait” in this 
context. The authors also do not explain what is meant by “multi-level specializations” and the entire 
sentence does not read like a hypothesis. This paragraph also lacks any predictions or a sense of what 
will be specifically tested in this study, including the methods to be used.  
 
2. One of my main concerns with the analyses presented is the source of the brain material. First, no 
information is provided on fixation and different fixatives can have different effects on brain 
morphology and the amount of shrinkage relative to fresh brains. Second, museum collections 
typically fix specimens in formalin, but then store them long term in 70% ethanol. If the museum 
specimens were stored in ethanol, this can cause major changes in brain morphology and brain region 
sizes as a result of much greater dehydration than keeping brains in formalin. If this was the case 
here, the museum specimens could be biasing the analyses or providing inaccurate reconstructions of 
brain and cerebellar shape. Third, using a variety of different fixatives and storage procedures could 
affect the analyses across species, especially if the majority of a locomotor-group or clade came from 
a single source. The Supplementary Material lacks information on sources of the material, fixatives 

and other details, so it was not possible to evaluate if these potential confounds were affecting their 
data. Fourth, using contrast enhancers can also introduce shrinkage and other artifacts that are not 
discussed and accounted for. If this is layered on top of the fixation/preservation issues I mention 
above, then there might be a range of artifacts affecting cerebellar morphology that could impact the 
results. These issues need to be addressed in order to have confidence in the results as presented.  
 
3. Throughout the Results, there is a heavy emphasis on the Supplementary Material. Although I 
understand that journals such as this have strict page limits, the 20+ references to the Supplementary 
Material indicate that Supplementary Figures 1, 3 and 4 need to actually be embedded in the text of 
the manuscript proper.  
 
There are also some inaccuracies in the text of the Results. For example, the authors claims that they 
“quantified the accurate shape of the brain” for the first time in a squamate. This is not true; Hoops et 
al. (2018) published an MRI brain atlas of a lizard (https://doi.org/10.1002/cne.24480) that is far 
more detailed in its anatomical description than what is provided here. The reference to endocast 
analysis is also irrelevant here.  
I will add that the first part of the Results section was extremely difficult to read because it is 
presented as a single paragraph 3+ pages long with multiple reference to the Supplementary 
Material.  
 
4. One page 7, line 9 there is a reference to “data not shown”. This needs to be shown to demonstrate 
that this statistical assumption was violated and it could easily be included in the Supplementary 
Material. On the same page, line 23 there is a reference to “other parameters”. This needs to be 
spelled out as there are some variables examined in this manuscript and many others that are not 
(e.g., neuron numbers, neuron sizes, neuronal complexity, neuropil volume).  
 
5. In the analysis of Purkinje cell layout, some of the findings were over-stated. For example, the 
“remarkable” variation in Purkinje cell layer organization across squamates has been described before. 
The authors acknowledge that part-way through this section, but the description of this variation 
needs to be tempered to reflect the previous observations. I was also confused by what was means by 
“directly comparing the obtained four groups with similar statistical analysis”. Last, the link between 
locomotor disorders and squamate locomotion seems a bit of a stretch. Yes, some locomotor disorders 
are associated with ectopic Purkinje cells, but this makes it sound as if the evolution of 
burrowing/lateral undulating is the result of cerebellar disorganization when that is unlikely the case. 
The evolution of different locomotor strategies might require different kinds of cerebellar mediated 
coordination, resulting in a different topological organization of the Purkinje cells, which is different to 
abnormal cerebellar development in mutant mice or humans with neurological disorders.  
 
6. On page 9, lines 4-7, I could not determine what the authors were trying to say here and “personal 
observations” are not sufficient for a major journal article to support this statement.  
 
7. Although the gene expression part of this paper is better described in this version, lines 9-12 on 
page 9 are better suited to the Introduction, which lacks any description or mention of 
transcriptomics. I would also say again, that linking some of the findings to mutant mice is a stretch 
and implies here that limbless species are uncoordinated, when they are not. Limbless species have a 
different type of coordination than quadrupedal squamates because the locomotor pattern is different.  
 
8. Like the Introduction, the Conclusions lacks focus and contains inaccurate and/or vague phrases. 
For example, there is no means of determining whether the current analyses are more or less 
accurate than any others. Further, the analyses presented are not as comprehensive as claimed. 
Currently, most studies of this nature quantify neuron numbers and there is a profound lack of 

information on variation in neuron numbers and neuronal morphology across species, for almost every 
vertebrate clade. This is not acknowledged anywhere in the manuscript, but should be discussed here 
and elsewhere. I will also add that the current study does not actually reflect “cerebrotype” analyses, 
which would be dependent on analyses across multiple brain regions or, at the very least, 
quantification within the cerebellum.  
 
9. The methods describes manual segmentation about several brain regions, but there is no actual 
analyses of these presented. Morphometrics can be done without segmenting individual brain regions, 
so an explanation is needed for segmenting non-cerebellar brain regions and an analysis provided 
(which would be a cerebrotype-analysis), or some of this methods section removed.  
 
10. Minor issues.  
a. There were at least two instances of “However, although”, which is grammatically incorrect.  
b. “neuron organization” is not a term  
c. Page 4, lines 29-30. The sentence that begins on line 29 needs to be rephrased.