DOI: 10.5061/dryad.3n5tb2rs2
title: Endocranial development in non-avian dinosaurs reveals an ontogenetic brain trajectory distinct from extant archosaurs

description: Modern birds possess highly encephalized brains that evolved from
 non-avian dinosaurs. Evolutionary shifts in developmental timing, namely
 juvenilization of adult phenotypes, have been proposed as a driver of head
 evolution along the dinosaur-bird transition, including brain morphology.
 Testing this hypothesis requires a sufficient developmental sampling of
 brain morphology in non-avian dinosaurs. In this study, we harness, for
 the first time, brain endocasts of a postnatal growth series of the
 ornithischian dinosaur Psittacosaurus and several other
 immature and mature non-avian dinosaurs to investigate how evolutionary
 changes to brain development are implicated in the origin of the avian
 brain. Using three-dimensional characterization of neuroanatomical shape
 across archosaur reptiles, we demonstrate that (i) the brain of non-avian
 dinosaurs underwent a distinct developmental trajectory compared to
 alligators and crown birds; (ii) ornithischian and non-avialan theropod
 dinosaurs shared a similar developmental trajectory, suggesting that their
 derived trajectory evolved in their common ancestor; and (iii) the
 evolutionary shift in developmental trajectories is partly consistent with
 paedomorphosis underlying overall brain shape evolution along the
 dinosaur-bird transition; however, the heterochronic signal is not uniform
 across time and neuroanatomical region suggesting a highly mosaic
 acquisition of the avian brain form.
description: Taken from manuscript's Methods section (in-text citations
 refer to references of manuscript):
 <em>Specimens</em> We amassed 85
 previously published endocranial reconstructions that spans 52 species of
 non-avian and avian dinosaurs in addition to American alligators (Table
 1). The data include interspecific sampling of 36 crown birds, a juvenile
 <em>Archaeopteryx</em><sup>32</sup>, and 19
 non-avialan dinosaurs<sup>1,3,17–20,33</sup>, plus ontogenetic
 series of the domestic chicken (<em>Gallus</em>
 <em>gallus; n</em> = 14), the American alligator
 (<em>Alligator mississippiensis</em>; <em>n</em> =
 14), and the ornithischian <em>Psittacosaurus</em>
 <em>lujiatunensis</em><sup>3,17,34</sup>
 (<em>n</em> = 4). Among non-avialan dinosaurs, several
 isolated immature specimens of both ornithischian and non-avian theropod
 dinosaurs, including <em>Lambeosaurus </em>(ROM 758) and
 troodontids <em>Zanabazar</em> and an unnamed specimen IGM
 100/1126<sup>19–21,35–38</sup> were sampled for both
 ontogenetic and phylogenetic diversity. The taxonomically ambiguous
 tyrannosaurid specimen, CMNH 7541, has been considered a relatively
 immature specimen within Tyrannosauridae<sup>39–43</sup>. The
 major results and conclusions of this study are not dependent on the
 taxonomic assignment of this specimen, but rather the ontogenetic stage of
 CMNH 7541. Considering that no organism attributable to a tyrannosaur
 similar to, and including, CMNH 7541 has preserved an external fundamental
 system that indicates the cessation of rapid growth and the onset of
 skeletal maturity<sup>39,45</sup>, we treat CMNH 7541 as an
 immature individual.  The specimen and data acquisition protocols for
 <em>Alligator</em> and <em>Gallus</em> specimens
 were approved by Institutional Animal Care and Use Committee at respective
 institutions (Stony Brook IACUC Protocol #236370-1; Oklahoma State
 University Center for Health Sciences IACUC Protocol #2015-1; American
 Museum of Natural History 2014). Please refer to the references listed in
 Table 1 for additional details about those
 specimens.<em> </em> <em>Imaging and
 Endocranial Reconstructions</em> Endocranial
 reconstructions were generated from computed tomography (CT) imaging of
 cranial specimens. Considering the incomplete nature of the PKUP 1053 and
 PKUP 1054 endocasts<sup>18</sup>, they were reconstructed
 again for this study. We found that the original specimens and
 reconstructed models were of sufficient quality for morphometric analysis.
 These reconstructions were paired with two other
 <em>Psittacosaurus</em> specimens, IVPP V15451 and IVPP
 V12617, to reconstruct an ontogenetic series for the species <em>P.
 lujiatunensis</em>. The endocast of the tyrannosaurid
 <em>Alioramus altai</em> (IGM 100/1844) was retrodeformed
 using a local symmetrization algorithm<sup>46</sup>
 implemented in the “Morpho” R package<sup>47</sup> due to its
 sheared appearance. Because the combined data comprise multiple sources,
 the scanner, scanning parameters, and segmentation protocol naturally
 differ across datasets (Table 1). Nevertheless, we considered variation in
 resulting shape data due to differences in these practices to be
 negligible compared to the large interspecific variation observed across
 archosaurs<sup>48</sup>. However, to adopt some
 standardization across endocranial reconstructions, we used the
 “QuickSmooth” function in Geomagic Wrap v2021 (Artec 3D, Senningerberg,
 Luxembourg) to remove cranial nerve and vascular projections from the main
 body of all the endocasts at the base.
 <em>Morphometric Data</em> We
 adopted the same high-density 3D landmark scheme from previous studies to
 characterize the overall shape of endocasts and their major regions,
 including the cerebrum, optic lobe, cerebellum, and
 brainstem<sup>3,33,34</sup> (Fig. 1c; Table S1). The ‘patch’
 tool in Landmark Editor v3.6<sup>49</sup> was used to place
 discrete landmarks that are anatomically defined, curve semi-landmarks
 that delimit regions, and surface semi-landmarks that capture the surface
 morphology within regions on both left and right sides of the endocast. We
 did not collect coordinate data from the olfactory tract and bulbs due to
 their incomplete preservation in the fossils, as well as their diminutive
 size in most crown birds. We acknowledge that endocasts are generally
 accurate representations of actual brain morphology among
 archosaurs<sup>34,50</sup>, but their correspondence is
 relatively low in the hindbrain due to greater presence of tissues
 surrounding the brain. This correspondence is lower in
 crocodilians<sup>27</sup> and presumably in non-avialan
 dinosaurs, especially the presence of dorsal eminence in some
 endocasts<sup>19,20,51</sup>. We have noted in the manuscript
 where the lower brain-endocast correspondence in the hindbrain,
 particularly the ‘cerebellum’ region, may influence the interpretation of
 results. The coordinate data were subjected to
 generalized Procrustes alignment<sup>52,53</sup> minimizing
 total bending energy, allowing semi-landmarks to slide on the mesh
 surface<sup>54,55</sup> using the ‘slider3d’ and ‘gpagen’
 functions in the “Morpho”<sup>56</sup> and
 “geomorph”<sup>57</sup> R packages. After alignment,
 (semi-)landmarks on the right side were removed to remove redundant shape
 information while minimizing artifacts caused by alignment of one-sided
 coordinate data of bilaterally symmetric
 structures<sup>58,59</sup>. The resulting shape data to be
 analyzed are comprised of 14 discrete landmarks, 49 curve semi-landmarks,
 and 56 surface semi-landmarks. For region-specific analyses, the size and
 shape of each region was extracted by calculating centroid size and
 performing localized alignment on a subset of landmarks that characterize
 each region. <em>Time-Calibrated
 Phylogeny</em> To perform phylogenetic
 comparative analysis, we constructed a time-calibrated phylogeny that
 includes all the sampled species. Because the crown group sampling is
 equivalent to a previous study<sup>3</sup>, fossil taxa were
 grafted to the existing tree file (additional details on the base tree
 construction<sup>3</sup>). These additional taxa included
 <em>Corythosaurus</em>, <em>Hypacrosaurus</em>,
 <em>Psittacosaurus</em>, <em>Gorgosaurus</em>, and
 <em>Tyrannosaurus</em>, which were incorporated into the
 existing phylogeny based on the mid-point of range of first occurrence age
 recorded on the Paleobiology Database (paleobiodb.org). When the maximum
 age of a species was identical to that of its clade, the age of the
 internal node was set to equally bisect the parent and descendent branches
 (‘Equal Branching Method’)<sup>60</sup>.
 <em>Analysis</em> All statistical
 analyses for this project were performed in R
 v4.2.1<sup>61</sup>. Patterns of endocast shape variation were
 visualized as morphospaces for the cerebrum, cerebellum, optic lobe, and
 brainstem regions of the endocasts and were constructed using scores from
 PCAs made from shape data. The functions physignal, procD.lm, and
 procD.pgls of the geomorph package were used to assess the level of
 phylogenetic signal, allometry, and evolutionary allometry, with 1000
 pseudoreplications. The multivariate statistical tests outlined above were
 chosen due to their robusticity against type I errors and loss of power
 that often accompanies differences in specimen and landmark
 sampling<sup>62–64</sup>. Allometric trends were visualized by
 plotting the PC1 of residuals from the overall shape-to-size relationship
 against scores along this allometric
 relationship<sup>65</sup>. We used Morpho package’s “CAC”
 function to extract CAC scores and residuals from shape data. Statistical
 differences between endocranial shapes and allometric trajectories between
 clades were tested with the procD.lm function, where differences in clades
 were tested on data accounting for log-transformed centroid size. In
 addition, we used the “trajectory.analysis” function in the ‘RRPP” R
 package<sup>66</sup> to test for differences between vectors
 of evolutionary and developmental shape change in total shape
 space.
description: # Endocranial development in non-avian dinosaurs reveals an ontogenetic
 brain trajectory distinct from extant archosaurs
 [https://doi.org/10.5061/dryad.3n5tb2rs2](https://doi.org/10.5061/dryad.3n5tb2rs2) The contents comprise: 1\. King_etal_2024_Analysis.R (R code for performing analyses presented in the associated article) 2\. SuppData1.csv (Procrustes-aligned endocranial shape data) 3\. SuppData2.csv (centroid size of endocasts) 4\. SuppData3.tre (phylogenetic tree file used in comparative analyses) ## Description of the data and file structure SuppData1.csv and SuppData2.csv are CSV files where each row represents a specimen and columns are morphometric variables. SuppData3.tre is a plain text file containing time-calibrated phylogenetic tree in Newick format. ## Sharing/Access information These files can also be found on the author's GitHub page: [https://github.com/akiopteryx/analyses/](https://github.com/akiopteryx/analyses/) ## Code/Software King_etal_2024_Analysis.R is written in R.