μCT of the brains and skulls were performed at P0. Images were obtained using a Bruker SKYSCAN 1173 μCT scanner. To obtain skull images, neonates’ heads were scanned following these parameters: isotropic voxel size of 8.19 μm, 30 kV, and 200 μA. For brains, we rescanned heads, but they were previously soaked for 24 hours in Lugol’s iodine as a contrast agent following the work by Metscher (46). Scanning parameters for brain images with contrast were an isotropic voxel size of 8.19 μm, 50 kV, and 160 μA.

We used Amira software to build three-dimensional reconstructions and to register the position of anatomical points (landmarks). For skulls, a set of 42 bilateral landmarks was registered for each specimen, as described by Gonzalez et al. (fig. S13) (22). For brains, we followed the protocol proposed by Gonzalez et al. (22) to obtain the position of 57 landmarks and semilandmarks. Data collection was performed by one of the authors (J.B.-A.), who had previously applied the same protocol and measured the intraobserver error for this kind of data (22). Landmark and semilandmark coordinates were superimposed (Procrustes Analysis), and comprehensive measures of skull and brain size (centroid size) were obtained. Briefly, centroid size is defined as the square root of the summed squared distances of all digitized landmarks to the centroid of the configuration. Total brain size was estimated following this formula and using all the digitized points. In addition, since each point was digitized in an identifiable brain structure, for further analysis, we examined those landmarks and semilandmarks describing only the cortex. Figure S12 shows a transversal view of the cortex described by points 7 to 16, while in the sagittal, the cortex is represented by 16 to 23. Then, this restricted set of landmarks and semilandmarks describing the cortex was used to calculate cortex centroid size. A ratio between cortex and total brain centroid size was obtained for each experimental group as a variable to compare the proportion of the cortex in relation to the whole brain. Larger ratios correspond to brains in which cortex is relatively larger, while smaller ratios indicate that the cortex is relatively reduced.

While we did not digitize semilandmarks in the skull, in the brain, its structure demanded the use of semilandmarks (fig. S12). In this case, semilandmaks were sliced previously to superimposition using the function procSym of the Morpho R package.

The relationship between body weight and brain size was examined to understand whether the registered brain sizes correspond to the expected values for their body weights. For this purpose, a linear model was carried out for the brain centroid size on body weight and the resulting residuals, which represent the deviations of the actual brain size of each case from the predicted value according to the body weight, as described by Gonzalez et al. (22). Positive values for the residuals indicate that brain size is larger than expected for certain body weight, while negative values suggest that brain size is smaller for body weight.

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