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

Reviewers' Comments:  
 
Reviewer #1:  
Remarks to the Author:  
In this study the authors performed comparative morphological analyses of extant reptilian brains with 
multiple experimental strategies. The authors particularly focused on the Squamate cerebellum and 
executed 3-D computed tomography, whole mount imaging of Purkinje cell distribution, and 
transcriptomics in adult celrebellum, which conducted substantial associations among specific 
characteristics in these traits and various locomotive behaviors in limbless/limb-reduced or 
quadrupedal characteristics independent to phylogenic relationship. The authors concluded that 
existence of significant correlations among these traits and locomotive behaviors resulted from 
convergent adaptive evolution, and that mosaic patterns of brain evolution can be categorized by 
cerebrotypes based on their definition. Each part of analysis had been performed in detail with enough 
number of samples and data representation were convincing, however, there are conceptual problems 
in the integration of the results and ambiguity in the purpose of the project.  
 
1) The most significant problem is on lack of the causality among morphological traits and specific 
locomotive behaviors across species. It is unsure how unique shape of the cerebellum is functionally 
associated with the pattern of locomotion, Purkinje cell distributions, and transcriptome data 
represented in the manuscript. Without experimental proves or any relevant explanations for the 
functional connections, all the represented results are still fragmental and hard to conduct the 
principle underpinning the brain evolution.  
 
2) Several previous comparative studies of locomotion and relevant neural circuits reveled significant 
associations between the nucleus ruber and reticulo-spinal tracts and quadrupedal behaviors in 
reptiles, while the cerebellum is relatively undeveloped in these animal groups, leading the question 
why the authors focused on the cerebellum but not examined other nuclear structures functionally 
associated with locomotive behaviors.  
 
3) Adult brain morphology must be significantly affected by external physical constraint, i.e., the 
shape of skulls. For example, the length of olfactory tracts is tightly constricted by the morphology of 
the orbit in reptiles. The author have to elaborate how they could eliminate extrinsic influences to the 
brain shapes.  
 
4) The project completely lacks the aspect of developmental biology that is critical to gain mechanistic 
insights into the evolution of species-specific body structures. “Developmental genes” in the adult 
tissue represented in the transcriptome data does not always reflect the functional association with 
embryogenesis, because many developmental genes are pleiotropic in their expressions and functions. 
The author must execute comparative ontogenic analysis of the cerebellum and cellular distributions in 
different species to clarify causal relationships among neuroanatomical architectures, gene expression, 
and unique locomotive behaviors.  
 
 
 
Reviewer #2:  
Remarks to the Author:  
The authors present a unique and extended dataset of Squamata brains which allowed them to 
analyse the shape and size of the different regions. A clear correlation of the cerebellum shape is 
shown with the locomotion type of the species.  
 
The manuscript is well written and illustrated and the information is clear. There are only a few points 

that I believe should be addressed:  
- provide a table with the number of individuals analysed for each type of analyses (3D model, 
histology, light sheet, RNA seq) as well as their age/size. It is important to show and maybe indicate 
in the text that only adult-sized animals were included in the study  
- I am surprised by the small number of one-to-one orhologs, only 630 out of ~9000 unigenes. I 
believe that GO analyses are more meaningful when a larger set of genes is available, especially as 
one gene can belong to several categories. How many genes are shared by nine species and how 
different is the GO distribution of the genes in the three clusters?  
- The brain is known to express a large number of genes where their function is often unknown. In 
cluster 3, the authors detect 12% of the genes (how many genes does this correspond to?) linked to 
locomotory behaviour. I believe it is important to discuss further these findings. Which genes are 
these? Why would their expression in the brain affect the locomotion of the animal? How relevant or 
random is this finding, compared for example to the 'response to inorganic substance' or 'multicellular 
organism reproduction' in clusters 1/2?  
 
 
 
Reviewer #3:  
Remarks to the Author:  
The manuscript submitted by Macri et al. is a very ambitious, inter-disciplinary approach to examining 
brain evolution in a clade that has been the focus of relatively few studies: squamates. An impressive 
suite of approaches were brought to bear on cerebellar anatomy specifically and there are some 
interesting and very novel findings presented. However, the results are presented in a manner that I 
found extremely confusing and it was difficult to distill what conclusions could be made. The over 
emphasis on referring to the supplemental material detracted from the manuscript itself and the 
figures presented within the manuscript were difficult to understand. Adding to the confusion and 
interpretability was a densely written methods section that did not adequately explain why certain 
analyses and tests were done and introduced others that I do not think are appropriate in the context 
of phylogeny-informed analyses. I strongly recommend that the authors consider breaking this 
manuscript up into more than one paper or significantly expanding the current one so that the reader 
can understand what was done and why. I provide more detailed comments below that I hope are 
helpful to the authors.  
 
1. The Introduction emphasizes endocasts repeatedly and the inability to examine the cerebellum 
properly in endocast analyses. This is true, but it has little to do with the paper presented. The 
authors are not doing an endocast analysis, they are using mCT to image the cerebellum itself in fluid 
preserved squamates. I would add that although digital endocasts are a hot and rapidly expanding 
topic at the moment, there is a long history of volumetric and stereological analyses of animal brains, 
including a classic paper by Platel on squamates that was oddly missing from the references section. 
The references to endocasts therefore need to be removed and more emphasis placed on brain 
evolution studies in general, especially those focused on the evolution of size and shape differences of 
brain regions.  
 
2. Related to my previous point, several key references on cerebellar evolution were absent from the 
Introduction and the rest of the manuscript. For example, several key comparative studies on 
cerebellar evolution in birds and mammals published over the past 15 years were noticeably missing. 
More critically, two recent studies from the Wylie lab on lizard and rattlesnake cerebella, including a 
description of the disorganized Purkinje cells in the rattlesnake, were absent as was a reference to the 
lizard brain atlas recently published by Hoops et al. These studies seem crucial to the current 
manuscript for several reasons and need to be cited and discussed.  
 

3. The bulk of the methods section was extremely difficult to read. A long list of R scripts is provided, 
but with little to no explanation regarding why these analyses are being done or what they do. For 
example, I am familiar with many phylogeny-informed statistical packages, but ‘phylolm’ is not one 
that is commonly used and has not been peer-reviewed, in contrast to others that would be suitable 
for this analysis, such as ‘evomap’. The Johnson-Neyman procedure is also unusual and I am unaware 
of papers that have validated or assessed its use within a phylogenetic context. In fact, post-hoc 
comparisons continue to be an issue for most phylogeny-informed statistics. Related to this point, no 
information is provided on what phylogeny was used, how it was constructed or if phylogeny was 
incorporated into the geometric morphometric analyses. I was impressed that the authors went to the 
trouble of brain clearing and light sheet microscopy, but insufficient information was provided on 
quantification. A range of 250-750 Purkinje cells is very broad and without knowing how they were 
selected or quantified, it is not possible to interpret their results. Also lacking was an explanation of 
why the distance between Purkinje cells and the pial surface was measured. I can think of a couple of 
reasons, but none are provided. I also did not follow what analyses could be done of the species or 
locomotor groups when it sounded like n = 1 for each species.  
 
4. I am not an expert in transcriptomics, but the inclusion of this data was justified insufficiently. What 
question is the transcriptomics approach trying to answer? Is it just whether there are differences 
among locomotor groups? If so, how does one know that these are differences due to locomotion and 
not phylogeny?  
 
5. The classification of locomotor groups was another area that required further explanation and 
description. A list of the species and their locomotor classification is provided in the supplementary 
material, but these are not defined anywhere. Anytime you categorize a behaviour or other trait in a 
comparative study, it needs to be defined explicitly so that other authors can employ the same 
categorization (or not as the case may be) in future comparative studies. Without this information, it 
is not possible to determine what the actual behavioural differences are among the different groups.  
 
6. The figures were all of high resolution, but I found them very difficult to follow. For Figures 2-4, the 
reader is expected to constantly refer back to Figure 1 to determine what each of the colours mean. 
This took me 3-4 reads to understand as it was not apparent in Figure 2, what the colours referred to 
and the Results section kept referring to Figure 2 to highlight differences between burrowers and other 
groups. Making it even more problematic was the inclusion of multiple colour schemes within these 
figures. For example, within Figure 2, 2c and 2e are using one rainbow of colours to refer to the 
locomotor groups and another to refer to “consensus shape changes” and relative cerebellar volume 
respectively. This is almost impossible to interpret, especially for readers with subtle colour blindness. 
Note that I put consensus shape changes in quotes because I could not determine from the Results 
section what this meant or what the consensus shape looked like. Interestingly, the figures in the 
supplemental material were far easier to follow and I strongly recommend that the authors make use 
of those and include them in the manuscript proper.  
 
7. I would add that Figure 1 itself is also needlessly complicated. The snakes are monophyletic so I do 
not see a need to have them represented by a different symbol. A bracket labeled “snakes” would be 
sufficient. Also, the colour scheme is not conducive to being interpreted by readers with anomalous 
colour vision. I encourage the authors to seek out resources available online or consult Journal of 
Comparative Neurology guidelines on how best to use colour in figures so that readers with anomalous 
colour vision can still interpret the figures appropriately.  
 
8. The Results section was unfortunately difficult to follow, partially due to the lack of clarity in the 
figures and methods. For example, on page 4, lines 3-4, the authors state “…lizards display an 
inverted tilting causing homologous dorsal regions to project towards opposite directions (Extended 

Data Fig 1b, c).”. I read this several times and I still cannot determine what the authors are trying to 
describe and examining the figure did not help. Similarly, the description of “a gradual morphological 
transition from snakes to quadrupedal lizards, passing through intermediate forms exhibited by 
limbless and limb-reduced lizards” is difficult to follow and not apparent in Figure 2. The placement of 
all of the statistical results in the Extended Data also did not help in reading through the Results as 
this forces the reader to flip back and forth. And the descriptions of cerebellar shape variation were 
extremely difficult to follow. Overall, the Results need better descriptions of the patterns observed, 
details of the statistics embedded within the text itself and more appropriate figures within the 
manuscript so that the differences among the locomotor modes are easier to see and interpret.  
 
9. The morphometric analysis of overall brain shape seemed out of place. The authors introduce the 
cerebellum as the focus of the paper in the Introduction and should stick with the cerebellum. Bringing 
in overall brain shape confuses the reader and whether brain shape as a whole reflects anything about 
behaviour or the sizes of brain regions is unknown.  
 
10. The authors state in other parts of the manuscript that analyzing the size of brain regions is 
insufficient for understanding brain evolution, yet cerebellar size is included in the current analysis. It 
might be more appropriate to tone down the criticism of volumetric studies and perhaps highlight 
more positively that size is only one metric and that other parameters can change independently of 
brain region volume (e.g., neuron sizes, neuron numbers, brain region shape).  
 
11. In their interpretation of the results, the authors comment that squamates have an exceptional 
cerebellar diversity. I do not think this conclusion can be reached based on the data presented. Yes, 
squamates vary in cerebellar size, shape and perhaps Purkinje cell distribution, but not any more so 
than bony fishes or mammals. The key finding here, that locomotor mode is a strong predictor of 
cerebellar size, shape, Purkinje cell distribution and cerebellar transcriptome, is far more important. In 
fact, this suggests that a key change in behaviour and limb morphology is evolutionarily correlated 
with major changes in the cerebellum. Platel and other authors have hinted at something similar 
based on a more limited data set, but I think the authors sell themselves short in relating a major 
behavioural transition with major changes in a brain region.