Convergent mosaic brain evolution is associated with the evolution of novel electrosensory systems in teleost fishes

Article activity feed

1. 

   Version published to 10.7554/elife.74159 on eLife

   Jun 17, 2022
2. 

   eLife

   May 9, 2022

   Author Response

   Reviewer #2 (Public Review):

   Schumacher and Carlson present volumetric data on the brain and main brain areas in several linages of fish that have independently evolved electroreceptors and electrogenesis. The main question is if the evolution of this novel sensory system has led to similar changes in the brain. Previously, the same authors (Sukhum et al 2018) have shown an increase in the relative size of the cerebellum and hindbrain in mormyrid fishes, one group of electrogenic fish. Here they have collected data on South American weakly electric fishes (Gymnotiformes) and weakly electric catfishes (Synodontis spp.) as well as some outgroups. (22 additionally species). I think the question is very interesting, and the inclusion of electrogenic catfishes is particularly interesting as they are a largely understudied …

   Author Response

   Reviewer #2 (Public Review):

   Schumacher and Carlson present volumetric data on the brain and main brain areas in several linages of fish that have independently evolved electroreceptors and electrogenesis. The main question is if the evolution of this novel sensory system has led to similar changes in the brain. Previously, the same authors (Sukhum et al 2018) have shown an increase in the relative size of the cerebellum and hindbrain in mormyrid fishes, one group of electrogenic fish. Here they have collected data on South American weakly electric fishes (Gymnotiformes) and weakly electric catfishes (Synodontis spp.) as well as some outgroups. (22 additionally species). I think the question is very interesting, and the inclusion of electrogenic catfishes is particularly interesting as they are a largely understudied group. I do have some concerns about how the data has been analysed and presented.

   1. A first conclusion is that gymnotiform and siluriform brains are not as enlarged as mormyrid brains, and that this suggests that an increase in brain size is not directly tied to an electrosensory system evolution. I think the story here is more complicated than that. From the data presented, it seems that mormyrids have a different body size-brain volume slope than other groups, but is unclear if this was tested in the PGLS model for brain vs body size, although mormirids show different slopes than other groups in the scaling of the cerebellum to brian volume. This difference in slope for body brain allometry has been confirmed by a manuscript published after the submission of this manuscript (Tsuboi 2021 BBE) with a large data set (~ 850 species, 21 of Osteoglossiformes). This steep slope close to one means that mormyrids with large body size have very large relative brain sizes but smaller mormyrids don't (this can be seen in figure 2). I think this needs to be addressed more carefully. First testing in the PGLS for body size vs brain size if mormyrids have a different slope and then in the discussion. Why mormyrids but not other electrogenic fish have evolved such a unique brain scaling?

   We thank the reviewer for this suggestion. We combined our data with the data from Tsuboi 2021 and assessed how the brain-body allometry has changed across 870 actinopterygians. We identified 3 shifts in lineages with at least 3 descendants and 7 shifts total that were supported by both the OUrjMCMC and PGLS analyses. One of these identified shifts was along the branch leading to osteoglossiforms, with a secondary decrease in one lineage within mormyoids. A second identified shift was along the branch leading to Synodontis multipunctatus. However, we find no shifts along the branches leading to other electrosensory lineages. This suggests that although mormyroids do have a different brain-body allometry compared with other electrogenic fishes, this shift predates the origin of mormyrids as it is found in all osteoglossiforms and thus is unlikely to be related to the evolution of electrosensory systems. These changes are reflected in lines 778-826, 110-153, 513-528, 530-538, 569-575 and figure 3 and associated source data files. See also our detailed response to essential revision 1.

   1. I think the number of outgroups species used are too few and spread among several different linages of teleosts. I think this unfortunately tampers some of the conclusions. Particularly seems to leave unanswered the question if other electrogenic fish have brain larger than non electrosensory or electrogenic fish. A large data set of brain and body size data for teleost has been published (Tsuboi et al 2018; 2021). Adding this data should allow to test for changes in body-brain size relationships in the each electrogenic clades. The addition of the additional data should allow to accurately test for difference in relative brain size between and within electrogenic clades and make it possible to test when exactly in the phylogeny of teleost have grade shits in the body-brain allometry have happened.

   We thank the reviewer for this suggestion. We explicitly addressed this question by fixing shifts along the branches that evolved our three electrosensory phenotypes: evolution of electrogenesis, tuberous electroreceptors, and ampullary electroreceptors. After comparing these models to the unfixed shift model, a model where only osteoglossiforms have a shifted allometry (following the finding of Tsuboi 2021), a model where only intercept can shift, and a model with one shared allometry across all actinopterygians, we found that the unfixed shift model has a better fit than any of the electrosensory phenotype associated models. This further supports the conclusion that a shifted allometry/ large brain size is not necessary to evolve an electrosensory system. These additions are reflected in lines 778-826, 110-153, 513-528, 530-538, 569-575 and figure 3 and associated source data files. See also our detailed response to essential revision 1.

   1. Next, the authors use a principal component analysis and phylogenetic linear models to test how much of brain variation is explained by concerted evolution vs mosaic and where the mosaic change have happened. Here, despite the few non electrogenic/ electrocereptive species, the differences are more clear. I do think that in the case of the linear models, the use brain volume as the independent variable is unnecessary. By regressing the total brain volume, the authors are regressing each structure partially against the same value, and not surprisingly, this generates tight linear correlations. Further, this makes grade shifts (i.e. changes in relative size) less apparent. I think only brain volume -the structure should be used and shown in all figures. This has been the standard in the field when testing for grade shifts.

   We thank the reviewer for this comment. There is much debate in the field regarding whether to use brain volume or brain volume – region of interest as the independent variable, and both are commonly used. Originally, we had looked at both and found qualitatively similar results, but only presented the ‘region x brain volume’ results in the main text for brevity. We have revised this to include the results of statistical analyses for ‘region x brain volume – region’ and the accompanying figures in the main text for both the electrosensory phenotype comparisons and the within electrosensory phenotype comparisons (broadly distributed throughout the results and figure 5—figure supplement 1, figure 5—source data 4-6, figure 7—figure supplement 1, figure 7—source data 2). All of the major findings of relative mosaic shifts between tuberous receptor taxa and non-electric taxa, between electrogenic + ampullary only and non-electric taxa for cerebellum and torus, and no mosaic shifts with electrosensory phenotype in telencephalon hold regardless of the method, and we only find minor differences between the analyses for comparisons that had p values near 0.05. These discrepancies do not change any major conclusions. However, we have kept the reporting of ‘region x total brain volume’ analyses in the main text figures to be consistent with other large comparative studies in the field and our group’s previous work (Yopak et al 2010, Sukhum et al 2018).

   1. Related to the previous point, the authors report significant decreases electrogenic clades in the size of the olfactory bulb, rest of the brain and optic tectum. I think this is and artifact that results from including the cerebellum and other enlarged areas (TS and hindbrain) in the dependent variable. Similarly, the authors state that they found no increase in the size of the telencephalon in electrogenic clades and that non-electric osteoglossiforms have a mosaic increase in telencephalon relative to non-electric otophysans. Again, I think this suffers from the same problem. Figure 4-figure supplement 2 actually provides some insight in this respect. When plotted against the rest of the brain, no apparent differences are found in the size of the optic tectum. In the case of the olfactory bulb only two of the out-group species seem to have larger OB than all other species. Regarding the telencephalon, when plotted against RoB, all osteoglossiform seem to have similar telencephalon size. These conclusions need to be carefully evaluated.

   We thank the reviewer for identifying this miscommunication. We have moved previous figure 4—figure supplement 2 to the main text (now figure 6) and have added the statistical analyses and discussion of this point to both the results and discussion. We have also clarified the distinction between relative and absolute shifts in region sizes throughout but see in particular lines 261-295, 307-317, 330-331, 473-499. See also our detailed response to essential revision 3.

   Reviewer #3 (Public Review):

   The authors use micro-CT scanning and sophisticated statistical techniques to compare the sizes of various major brain regions across a sample of 32 fish species, including lineages that have independently evolved passive electroreception and, in a smaller subset, the ability to generate and sense weakly electric fields. They found that most of the variation in brain region sizes is linked to variation in total brain size, indicating concerted evolution. However, the analysis also reveals that the electrogenic lineages/species have selectively enlarged the cerebellum, the midbrain torus semicircularis, and the hindbrain. These findings are interesting and usefully extend the last author's prior work on a subset of these species.

   A significant strength of the work is that it includes a relatively large number of species, makes a good attempt to understand how these species are related to one another (though the authors admit that the phylogeny is tentative), and that the analytical methods are quantitative and relatively sophisticated. It is also true that other researchers have long argued about the relative frequency and importance of concerted versus mosaic evolution. The present study is a valiant attempt to address this issue.

   However, some key results must be viewed cautiously. Most important is that the dramatic increase in the cerebellum (and torus semicircularis and hindbrain), relative to the rest of the brain, must necessarily lead to some other brain regions appearing to have decreased in size. Therefore, their absolute size may well have stayed the same or even increased in evolution; it's just that the enlarged brain regions decrease the proportions of at least some other regions. The authors mentioned this caveat in their previous paper on mormyroids (Sukhum et al., 2018), but not in the present manuscript. As a result of the problem, it is difficult to interpret the documented variation in olfactory bulb, optic tectum, or telencephalon size; is that variation "real" or just artifacts of major changes in the size of other brain regions (mainly cerebellum, torus, and hindbrain). The best way to address this problem would have been to repeat the analysis using a "reference" brain region that is thought not to vary dramatically in size across the species of interest (e.g., "rest of brain"). However, I acknowledge that this approach also has limitations. Still, the problem should be addressed somehow.

   We thank the reviewer for identifying this miscommunication. We have moved previous figure 4—figure supplement 2 to the main text (now figure 6) and have added the statistical analyses and discussion of this point to both the results and discussion. We have also clarified the distinction between relative and absolute shifts in region sizes throughout but see in particular lines 261-295, 307-317, 330-331, 473-499. See also our detailed response to essential revision 3.

   One strength of the manuscript is that it provides information about y-intercepts and slopes. Many other studies simply note increases or decreases in average volume (before or after correcting for absolute brain size). I like knowing which changes in relative brain region size are grade shifts (changes in intercept) versus changes in slope. However, the authors don't really do anything with those results. What do they mean? Are there different kinds of evo-devo mechanisms that underlie the two types of changes (slope versus intercept)?

   We thank the reviewer for this suggestion. We have added some discussion on potential mechanisms for evolutionary changes in intercept and slope (lines 543-559). Unfortunately, this topic is not well studied in fishes, which have extensive adult neurogenesis.

   On a related note, do the major brain regions vary in allometric slope within a given lineage? The realization that such differences do exist (at least in mammals and cartilaginous fishes) contributed much to the excitement around the concept of concerted evolution, since it means that evolutionary changes in absolute brain size can lead to major shifts in brain region proportions, but the authors seemingly ignore this point.

   We thank the reviewer for this suggestion. We do find variability in slope for different regions of each lineage. We reported these values (figure 5—source data 1, figure 7—source data 1) and add discussion of this point (lines 539-542).

   Finally, I must confess that some of the study's findings didn't surprise me. It is well known among fish neurobiologists that mormyrids have a dramatically enlarged cerebellum and that all electrogenic gymnotoids and mormyroids have a very large torus semicircularis and dorsal/alar hindbrain. One didn't need the fancy analytical techniques to confirm this. To be fair, however, it had not been clear whether the cerebellum is enlarged in gymnotoid electric fish and their non-electrogenic relatives (the authors report that it is). Nor was it known that the weakly electric catfishes have a larger cerebellum (not so much for the torus) than their non-electric relatives. This is new information that raises interesting questions about how the electric catfishes are using their electrosensory system (I would have liked to see some discussion of this).

   We thank the reviewer for this comment. We too agree that electric catfishes warrant further study into which species are electrogenic, whether their discharges are sporadic versus continuous, and how they are using their electrosensory systems. We have added further discussion on electric catfishes (lines 411-416, 425-437).

   On balance, I appreciate that the authors have provided a large and useful data set , which they used to address an interesting set of questions about how brain evolution "works." I'm just disappointed that, for me, there are relatively few significant, novel insights. For example, the notion that "selection can impact structural brain composition to favor specific regions involved in novel behaviors" (last sentence of the abstract) is one that I've accepted for a long time. Maybe the conclusion can be made more interesting by focusing more explicitly on changes in the size of major brain regions versus smaller cell groups (where mosaic evolution is widely accepted).

   We thank the reviewer for this suggestion. We agree that mosaic evolution is more readily detected in smaller subregions/ nuclei/ circuits and is found less so at the scale of major brain regions. We have adjusted the text throughout to further highlight this distinction, but see in particular lines 42-48, 500-528.

   Reviewer #4 (Public Review):

   The authors present a detailed and thorough comparative analysis of brain composition across 3 different lineages of weakly electric fish, and several non-electric fishes. The goal of this comparison was to determine whether the evolution of electrosensory systems is associated with common changes in brain composition across the three lineages. Several aspects of this research are highly novel, such as the use of m-CT imaging and phylogeny-informed multivariate statistics. Overall, the authors show that cerebellar enlargement is key to the evolution of electrosensory systems of all three groups and the enlargement of the hindbrain and torus semicircularis varies depending on the types of electroreceptors and electrical signals produced. This is one of very few examples in evolutionary neuroscience of convergent evolution of brain anatomy and behaviour and sets the stage for future research on other sensory specialists and clades.

   Strengths

   The comprehensive analysis provided by Schumacher and Carlson has several strengths. First, the use of m-CT scans to derive neuroanatomical measurements in fish is relatively novel and the detailed descriptions of brain region borders were greatly appreciated. Few papers that focus on comparative neuroanatomy put this degree of effort into describing how regions were differentiated and defined, but the level of detail provided here will allow other researchers to acquire data in an identical method and is therefore an important resource.

   Second, the statistical analysis is phylogeny-informed and uses an array of approaches. Too many neurobiology papers either avoid phylogeny-informed statistics or execute them poorly. This paper is neither of those and should serve as a template for future studies in the field.

   Third, the inclusion of some recording data for Synodontis is an important contribution. I am not an expert on weakly electric fish, but I do know that the catfish are understudied compared with gymnotiforms and mormyroids. Hopefully, this will result in some well-deserved attention to the diversity of catfishes.

   Fourth, I found the manuscript as a whole well written and presented. In particular, the authors provided a novel way of incorporating additional statistical information into Figures 3 and 4.

   Last, the supplemental video was great addition to the data presented.

   Weaknesses

   First, the Introduction was a bit brief for readers unfamiliar with weakly electric fishes. It would be helpful to provide a bit more information to a general audience. Including a figure depicting the phylogenetic relationships among some (not all) bony fish clade to illustrate the independent evolution of electrosensory systems across the three clades would be particularly helpful in this regard.

   We thank the reviewer for this comment. We have included more background on the evolution of electrosensory systems in actinopterygians and included a figure showing this (lines 76-83, figure 1).

   Second, I think it is important to determine if the principal component analysis changes if the volumetric data is scaled. One issue that can affect multivariate analyses is including variables that differ greatly in scale. For example, if one brain region varies between 0.5-1.2 mm3, but another varies from 10-50 mm3 across species, that difference in scale can sometimes affect the PCA. I suggest checking that the analyses are broadly the same if the volumetric data is scaled (e.g., converting to z-scores).

   We thank the reviewer for this suggestion. We z-score normalized the regions and repeated the pPCA and found nearly identical results (lines 175-177, figure 4—figure supplement 1).

   Third is there any information regarding malapteurid catfish? Are they similar enough to Synodontis or could they exhibit yet another brain type from that discussed in this study? The reason I ask is that the authors raise the issue of Torpedo, but do not discuss other strongly electric fish like Malapteurus (which is a siluriform related to Synodontis).

   We thank the reviewer for this comment. We too agree that they would be worthwhile species to add. Unfortunately, there is no data available on malapteurid catfish, and we were unable to sample any. We have added discussion of this point to lines 411-416.

   Last, some of the graphs in the supplemental material are too small with datapoints too crowded to effectively read them. Larger graphs would enable a more effective evaluation of how the various clades differ from one another.

   We thank the reviewer for this comment. We enlarged the region x region plots and plotted species means instead to make it easier to visualize these data (Figure 6, figure 7—figure supplement 2-4).

   Read the original source
3. 

   eLife

   May 9, 2022

   Evaluation Summary:

   This ms examines changes in brain region size in several groups of weakly electric fishes, the Mormyroidea, and the Gymnotiformes and weakly electric catfishes (Synodontis spp.), which evolved electroreception independently of mormyrids. These are an interesting group for examination of mosaic growth. Many analyses are thoughtful and well executed, but there is some concern about whether the observed volumetric decreases are a consequence of the method.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   May 9, 2022

   Reviewer #1 (Public Review):

   The authors examined changes in brain region size in several groups of weakly electric fishes (Mormyroidea), the Gymnotiformes and weakly electric catfishes (Synodontis spp.), which evolved electroreception independently of mormyroids.

   The major strengths and weaknesses of the methods and results are as follows.

   Major strengths are the careful examination of many weakly electric fish, which are an interesting group for examination of mosaic growth. The analyses are thoughtful and well done. The use of phylogenetically informed linear models is an additional strength. One concern is whether the significant decreases in other brain areas are real.

   The authors achieved their aims, and in general the results support their conclusions, except for the above mentioned concern about whether there are significant …

   Reviewer #1 (Public Review):

   The authors examined changes in brain region size in several groups of weakly electric fishes (Mormyroidea), the Gymnotiformes and weakly electric catfishes (Synodontis spp.), which evolved electroreception independently of mormyroids.

   The major strengths and weaknesses of the methods and results are as follows.

   Major strengths are the careful examination of many weakly electric fish, which are an interesting group for examination of mosaic growth. The analyses are thoughtful and well done. The use of phylogenetically informed linear models is an additional strength. One concern is whether the significant decreases in other brain areas are real.

   The authors achieved their aims, and in general the results support their conclusions, except for the above mentioned concern about whether there are significant decreases in other brain areas.

   Read the original source
5. 

   eLife

   May 9, 2022

   Reviewer #2 (Public Review):

   Schumacher and Carlson present volumetric data on the brain and main brain areas in several linages of fish that have independently evolved electroreceptors and electrogenesis. The main question is if the evolution of this novel sensory system has led to similar changes in the brain. Previously, the same authors (Sukhum et al 2018) have shown an increase in the relative size of the cerebellum and hindbrain in mormyrid fishes, one group of electrogenic fish. Here they have collected data on South American weakly electric fishes (Gymnotiformes) and weakly electric catfishes (Synodontis spp.) as well as some outgroups. (22 additionally species). I think the question is very interesting, and the inclusion of electrogenic catfishes is particularly interesting as they are a largely understudied group. I do have …

   Reviewer #2 (Public Review):

   Schumacher and Carlson present volumetric data on the brain and main brain areas in several linages of fish that have independently evolved electroreceptors and electrogenesis. The main question is if the evolution of this novel sensory system has led to similar changes in the brain. Previously, the same authors (Sukhum et al 2018) have shown an increase in the relative size of the cerebellum and hindbrain in mormyrid fishes, one group of electrogenic fish. Here they have collected data on South American weakly electric fishes (Gymnotiformes) and weakly electric catfishes (Synodontis spp.) as well as some outgroups. (22 additionally species). I think the question is very interesting, and the inclusion of electrogenic catfishes is particularly interesting as they are a largely understudied group. I do have some concerns about how the data has been analysed and presented.

   1. A first conclusion is that gymnotiform and siluriform brains are not as enlarged as mormyrid brains, and that this suggests that an increase in brain size is not directly tied to an electrosensory system evolution. I think the story here is more complicated than that. From the data presented, it seems that mormyrids have a different body size-brain volume slope than other groups, but is unclear if this was tested in the PGLS model for brain vs body size, although mormirids show different slopes than other groups in the scaling of the cerebellum to brian volume. This difference in slope for body brain allometry has been confirmed by a manuscript published after the submission of this manuscript (Tsuboi 2021 BBE) with a large data set (~ 850 species, 21 of Osteoglossiformes). This steep slope close to one means that mormyrids with large body size have very large relative brain sizes but smaller mormyrids don't (this can be seen in figure 2). I think this needs to be addressed more carefully. First testing in the PGLS for body size vs brain size if mormyrids have a different slope and then in the discussion. Why mormyrids but not other electrogenic fish have evolved such a unique brain scaling?

   2. I think the number of outgroups species used are too few and spread among several different linages of teleosts. I think this unfortunately tampers some of the conclusions. Particularly seems to leave unanswered the question if other electrogenic fish have brain larger than non electrosensory or electrogenic fish. A large data set of brain and body size data for teleost has been published (Tsuboi et al 2018; 2021). Adding this data should allow to test for changes in body-brain size relationships in the each electrogenic clades. The addition of the additional data should allow to accurately test for difference in relative brain size between and within electrogenic clades and make it possible to test when exactly in the phylogeny of teleost have grade shits in the body-brain allometry have happened.

   1. Next, the authors use a principal component analysis and phylogenetic linear models to test how much of brain variation is explained by concerted evolution vs mosaic and where the mosaic change have happened. Here, despite the few non electrogenic/electrocereptive species, the differences are more clear. I do think that in the case of the linear models, the use brain volume as the independent variable is unnecessary. By regressing the total brain volume, the authors are regressing each structure partially against the same value, and not surprisingly, this generates tight linear correlations. Further, this makes grade shifts (i.e. changes in relative size) less apparent. I think only brain volume -the structure should be used and shown in all figures. This has been the standard in the field when testing for grade shifts.

   2. Related to the previous point, the authors report significant decreases electrogenic clades in the size of the olfactory bulb, rest of the brain and optic tectum. I think this is and artifact that results from including the cerebellum and other enlarged areas (TS and hindbrain) in the dependent variable. Similarly, the authors state that they found no increase in the size of the telencephalon in electrogenic clades and that non-electric osteoglossiforms have a mosaic increase in telencephalon relative to non-electric otophysans. Again, I think this suffers from the same problem. Figure 4-figure supplement 2 actually provides some insight in this respect. When plotted against the rest of the brain, no apparent differences are found in the size of the optic tectum. In the case of the olfactory bulb only two of the out-group species seem to have larger OB than all other species. Regarding the telencephalon, when plotted against RoB, all osteoglossiform seem to have similar telencephalon size. These conclusions need to be carefully evaluated.

   Read the original source
6. 

   eLife

   May 9, 2022

   Reviewer #3 (Public Review):

   The authors use micro-CT scanning and sophisticated statistical techniques to compare the sizes of various major brain regions across a sample of 32 fish species, including lineages that have independently evolved passive electroreception and, in a smaller subset, the ability to generate and sense weakly electric fields. They found that most of the variation in brain region sizes is linked to variation in total brain size, indicating concerted evolution. However, the analysis also reveals that the electrogenic lineages/species have selectively enlarged the cerebellum, the midbrain torus semicircularis, and the hindbrain. These findings are interesting and usefully extend the last author's prior work on a subset of these species.

   A significant strength of the work is that it includes a relatively large number …

   Reviewer #3 (Public Review):

   The authors use micro-CT scanning and sophisticated statistical techniques to compare the sizes of various major brain regions across a sample of 32 fish species, including lineages that have independently evolved passive electroreception and, in a smaller subset, the ability to generate and sense weakly electric fields. They found that most of the variation in brain region sizes is linked to variation in total brain size, indicating concerted evolution. However, the analysis also reveals that the electrogenic lineages/species have selectively enlarged the cerebellum, the midbrain torus semicircularis, and the hindbrain. These findings are interesting and usefully extend the last author's prior work on a subset of these species.

   A significant strength of the work is that it includes a relatively large number of species, makes a good attempt to understand how these species are related to one another (though the authors admit that the phylogeny is tentative), and that the analytical methods are quantitative and relatively sophisticated. It is also true that other researchers have long argued about the relative frequency and importance of concerted versus mosaic evolution. The present study is a valiant attempt to address this issue.

   However, some key results must be viewed cautiously. Most important is that the dramatic increase in the cerebellum (and torus semicircularis and hindbrain), relative to the rest of the brain, must necessarily lead to some other brain regions appearing to have decreased in size. Therefore, their absolute size may well have stayed the same or even increased in evolution; it's just that the enlarged brain regions decrease the proportions of at least some other regions. The authors mentioned this caveat in their previous paper on mormyroids (Sukhum et al., 2018), but not in the present manuscript. As a result of the problem, it is difficult to interpret the documented variation in olfactory bulb, optic tectum, or telencephalon size; is that variation "real" or just artifacts of major changes in the size of other brain regions (mainly cerebellum, torus, and hindbrain). The best way to address this problem would have been to repeat the analysis using a "reference" brain region that is thought not to vary dramatically in size across the species of interest (e.g., "rest of brain"). However, I acknowledge that this approach also has limitations. Still, the problem should be addressed somehow.

   One strength of the manuscript is that it provides information about y-intercepts and slopes. Many other studies simply note increases or decreases in average volume (before or after correcting for absolute brain size). I like knowing which changes in relative brain region size are grade shifts (changes in intercept) versus changes in slope. However, the authors don't really do anything with those results. What do they mean? Are there different kinds of evo-devo mechanisms that underlie the two types of changes (slope versus intercept)?

   On a related note, do the major brain regions vary in allometric slope within a given lineage? The realization that such differences do exist (at least in mammals and cartilaginous fishes) contributed much to the excitement around the concept of concerted evolution, since it means that evolutionary changes in absolute brain size can lead to major shifts in brain region proportions, but the authors seemingly ignore this point.

   Finally, I must confess that some of the study's findings didn't surprise me. It is well known among fish neurobiologists that mormyrids have a dramatically enlarged cerebellum and that all electrogenic gymnotoids and mormyroids have a very large torus semicircularis and dorsal/alar hindbrain. One didn't need the fancy analytical techniques to confirm this. To be fair, however, it had not been clear whether the cerebellum is enlarged in gymnotoid electric fish and their non-electrogenic relatives (the authors report that it is). Nor was it known that the weakly electric catfishes have a larger cerebellum (not so much for the torus) than their non-electric relatives. This is new information that raises interesting questions about how the electric catfishes are using their electrosensory system (I would have liked to see some discussion of this).

   On balance, I appreciate that the authors have provided a large and useful data set , which they used to address an interesting set of questions about how brain evolution "works." I'm just disappointed that, for me, there are relatively few significant, novel insights. For example, the notion that "selection can impact structural brain composition to favor specific regions involved in novel behaviors" (last sentence of the abstract) is one that I've accepted for a long time. Maybe the conclusion can be made more interesting by focusing more explicitly on changes in the size of major brain regions versus smaller cell groups (where mosaic evolution is widely accepted).

   Read the original source
7. 

   eLife

   May 9, 2022

   Reviewer #4 (Public Review):

   The authors present a detailed and thorough comparative analysis of brain composition across 3 different lineages of weakly electric fish, and several non-electric fishes. The goal of this comparison was to determine whether the evolution of electrosensory systems is associated with common changes in brain composition across the three lineages. Several aspects of this research are highly novel, such as the use of m-CT imaging and phylogeny-informed multivariate statistics. Overall, the authors show that cerebellar enlargement is key to the evolution of electrosensory systems of all three groups and the enlargement of the hindbrain and torus semicircularis varies depending on the types of electroreceptors and electrical signals produced. This is one of very few examples in evolutionary neuroscience of …

   Reviewer #4 (Public Review):

   The authors present a detailed and thorough comparative analysis of brain composition across 3 different lineages of weakly electric fish, and several non-electric fishes. The goal of this comparison was to determine whether the evolution of electrosensory systems is associated with common changes in brain composition across the three lineages. Several aspects of this research are highly novel, such as the use of m-CT imaging and phylogeny-informed multivariate statistics. Overall, the authors show that cerebellar enlargement is key to the evolution of electrosensory systems of all three groups and the enlargement of the hindbrain and torus semicircularis varies depending on the types of electroreceptors and electrical signals produced. This is one of very few examples in evolutionary neuroscience of convergent evolution of brain anatomy and behaviour and sets the stage for future research on other sensory specialists and clades.

   Strengths

   The comprehensive analysis provided by Schumacher and Carlson has several strengths. First, the use of m-CT scans to derive neuroanatomical measurements in fish is relatively novel and the detailed descriptions of brain region borders were greatly appreciated. Few papers that focus on comparative neuroanatomy put this degree of effort into describing how regions were differentiated and defined, but the level of detail provided here will allow other researchers to acquire data in an identical method and is therefore an important resource.

   Second, the statistical analysis is phylogeny-informed and uses an array of approaches. Too many neurobiology papers either avoid phylogeny-informed statistics or execute them poorly. This paper is neither of those and should serve as a template for future studies in the field.

   Third, the inclusion of some recording data for Synodontis is an important contribution. I am not an expert on weakly electric fish, but I do know that the catfish are understudied compared with gymnotiforms and mormyroids. Hopefully, this will result in some well-deserved attention to the diversity of catfishes.

   Fourth, I found the manuscript as a whole well written and presented. In particular, the authors provided a novel way of incorporating additional statistical information into Figures 3 and 4.

   Last, the supplemental video was great addition to the data presented.

   Weaknesses

   First, the Introduction was a bit brief for readers unfamiliar with weakly electric fishes. It would be helpful to provide a bit more information to a general audience. Including a figure depicting the phylogenetic relationships among some (not all) bony fish clade to illustrate the independent evolution of electrosensory systems across the three clades would be particularly helpful in this regard.

   Second, I think it is important to determine if the principal component analysis changes if the volumetric data is scaled. One issue that can affect multivariate analyses is including variables that differ greatly in scale. For example, if one brain region varies between 0.5-1.2 mm3, but another varies from 10-50 mm3 across species, that difference in scale can sometimes affect the PCA. I suggest checking that the analyses are broadly the same if the volumetric data is scaled (e.g., converting to z-scores).

   Third is there any information regarding malapteurid catfish? Are they similar enough to Synodontis or could they exhibit yet another brain type from that discussed in this study? The reason I ask is that the authors raise the issue of Torpedo, but do not discuss other strongly electric fish like Malapteurus (which is a siluriform related to Synodontis).

   Last, some of the graphs in the supplemental material are too small with datapoints too crowded to effectively read them. Larger graphs would enable a more effective evaluation of how the various clades differ from one another.

   Read the original source
8. 

   Version published to 10.1101/2021.09.29.462233v1 on bioRxiv

   Oct 1, 2021