Flightless Birds Are Not Neuroanatomical Analogs of Non-Avian Dinosaurs Maria Eugenia Leone Gold1,2,3* and Akinobu Watanabe3,4,5

Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 https://doi.org/10.1186/s12862-018-1312-0

RESEARCHARTICLE Open Access Flightless birds are not neuroanatomical analogs of non-avian dinosaurs Maria Eugenia Leone Gold1,2,3* and Akinobu Watanabe3,4,5

Abstract Background: In comparative neurobiology, major transitions in behavior are thought to be associated with proportional size changes in brain regions. Bird-line theropod dinosaurs underwent a drastic locomotory shift from terrestrial to volant forms, accompanied by a suite of well-documented postcranial adaptations. To elucidate the potential impact of this locomotor shift on neuroanatomy, we first tested for a correlation between loss of flight in extant birds and whether the brain morphology of these birds resembles that of their flightless, non-avian dinosaurian ancestors. We constructed virtual endocasts of the braincase for 80 individuals of non-avian and avian theropods, including 25 flying and 19 flightless species of crown group birds. The endocasts were analyzed using a three-dimensional (3-D) geometric morphometric approach to assess changes in brain shape along the dinosaur-bird transition and secondary losses of flight in crown-group birds (Aves). Results: While non-avian dinosaurs and crown-group birds are clearly distinct in endocranial shape, volant and flightless birds overlap considerably in brain morphology. Phylogenetically informed analyses show that locomotory mode does not significantly account for neuroanatomical variation in crown-group birds. Linear discriminant analysis (LDA) also indicates poor predictive power of neuroanatomical shape for inferring locomotory mode. Given current sampling, Archaeopteryx, typically considered the oldest known bird, is inferred to be terrestrial based on its endocranial morphology. Conclusion: The results demonstrate that loss of flight does not correlate with an appreciable amount of neuroanatomical changes across Aves, but rather is partially constrained due to phylogenetic inertia, evident from sister taxa having similarly shaped endocasts. Although the present study does not explicitly test whether endocranial changes along the dinosaur-bird transition are due to the acquisition of powered flight, the prominent relative expansion of the cerebrum, in areas associated with flight-related cognitive capacity, suggests that the acquisition of flight may have been an important initial driver of brain shape evolution in theropods. Keywords: 3-D geometric morphometrics, Endocasts, Neuroanatomy, Theropoda, Aves, Powered flight, Locomotion

Background clades, birds provide arguably the best system within Major behavioral transitions often correlate with neuro- which to study the evolution of flight and associated anatomical changes because novel sensory inputs and morphological adaptations because they have a long motor control pathways can form new or more robust stem lineage that is represented by a rich fossil record connections, increasing the volume and density of asso- [7, 8]. This record has elucidated the origins of many of ciated regions of the brain [1–5]. One such evolutionary the postcranial ‘adaptations’ traditionally considered transition, from non-volancy to powered flight, has been directly related to flight and has shown that these acquired independently by three different vertebrate characters, such as the furcula and hollow long bones, groups—pterosaurs, bats, and birds [6]. Among these arose not at the origin of this locomotory mode—near the appearance of Archaeopteryx lithographica—but – * Correspondence: [email protected] earlier among non-avian theropods [9 11]. 1Biology Department, Suffolk University, Boston, MA 02108, USA The early history of neuroanatomical adaptions related 2 Department of Anatomical Sciences, Stony Brook University, Stony Brook, to this transition, however, is more obscure. Previous NY 11779, USA Full list of author information is available at the end of the article studies of avian neuroanatomical evolution indicated

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 2 of 11

that the avian brain is characterized by an enlarged the Microscopy and Imaging Facility at the AMNH or cerebrum and dorsoventrally flexed lateral profile. These the high-resolution source at the University of Texas at studies divided endocasts into distinct volumes based on Austin High-Resolution X-ray Computed Tomography major brain regions (e.g. cerebrum, cerebellum, optic Facility (Additional file 1: Tables S1 and S2). Raphus lobe). The fossil record shows that these ‘avian-like’ cere- cucullatus (NHMUK PV A9040) [23] was scanned at the bral volumes evolved at least as far back as the origin of NHMUK Imaging Facility. Scan data were imported into Maniraptora, ~ 160 Mya [11, 12]. This indicates that the VGStudio MAX v2.2 (Volume Graphics GmbH, size of the brain follows the more general trend of other Heidelberg, Germany) to construct a 3-D model of the ‘avian’ characteristics, evolving well before the origin of endocranial cavity (endocast) following the protocol set powered flight. Purely volumetric analyses, however, are forth by Balanoff et al. [24]. Because the brain fills nearly limited in their capacity to characterize morphology. the entire cranial cavity in birds [25, 26], its morphology Neural pathways important in creating and regulating is accurately reflected by use of an endocast [24, 27]. locomotor behavior often are distributed differentially The 3-D models were smoothed in VGStudio MAX and among multiple regions of the brain [5], thus under- exported as PLY files for the software Landmark Editor standing the regional shape changes has the potential to v3.6 to virtually place landmarks [28]. inform the evolutionary tempo and mode of fight capacity along the theropod lineage. Time-calibrated phylogenetic tree Here, we use the loss of flight in crown group birds as A time-calibrated tree was used to perform comparative a potential reverse analogue to the acquisition of flight phylogenetic analyses on endocranial shape. For extant in theropods. To determine if there is a link between birds, we constructed a topology using the data provided neuroanatomy and loss of flight, we examined the online by Jetz et al. [29](birdtree.org), pruned to include endocasts of multiple independent pairs of flightless only species sampled in the current study. The Jetz et al. birds and their closest volant relatives to test for modifi- [29] trees were chosen for their dense species sampling. cations in brain shape. We then compared the endocasts Our dataset has, in some cases, multiple species that are to the plesiomorphic condition in flightless, non-avian very closely related and the increased tip sampling of theropod dinosaurs. We used high-resolution X-ray that tree allowed us to retain all of our species. computed tomographic (CT) imaging to construct TreeAnnotator v1.8.1 [30] was used to construct a three-dimensional (3-D) digital endocasts of the cranial maximum clade credibility tree from 1000 posterior cavity of a broad sample of modern flying and flightless trees based on the Hackett et al. [31] tree backbone [29]. members of Aves (sensu [13]) and extinct non-avian the- Because some of the sampled taxa are not included in ropods. Shape data from these specimens were collected the Jetz et al. [29] dataset, we replaced these tip labels using a high-dimensional geometric morphometric with the closest relatives to each of these species in our (GM) approach [14–16] and they were subsequently analysis. Thus, Alle alle, Chunga burmeisteri, and Goura subjected to a suite of multivariate analyses [17, 18]. cristata, and Rollandia rolland were replaced by Pingui- With this approach, we evaluated whether (1) the loss of nus impennis, Pelecyornis australis, Raphus cucullatus, flight incurs predictable changes to neuroanatomical and Podilymbus gigas, respectively. We considered the shape, such as a reduction in cerebral areas known to above listed extinct crown group avians to exist in the function in flight [5]; and (2) the endocast of modern Recent for the phylogeny (i.e., 0 Ma) in the context of flightless birds returns to a shape more similar to that of deep time. The dataset included Struthio molybdophanes non-avian theropods than to their flying relatives. and S. camelus, however, only the latter was present in the Jetz et al. [29] dataset. Both species were included Methods for the non-phylogenetically informed analyses (e.g. PCA CT imaging and digital endocast reconstruction and LDA), but only S. camelus was used for the con- Taxonomic sampling included the skulls of 80 specimens struction of the time-calibrated tree and phylogenetically representing 51 species: 25 flying and 19 flightless informed analyses. This tree was used for the avians, and 7 non-avian dinosaurs: Alioramus altai [19] crown-group Aves dataset (Fig. 1). For the Coelurosaur (IGM 100/1844), Khaan mckennai [20] (IGM 100/973), dataset, we used Mesquite v3.02 [32] to incorporate Citipati osmolskae [20] (IGM 100/978), Incisivosaurus non-avian dinosaurs into the time-calibrated avian phyl- gauthieri [21] (IVPP V 13326), Zanabazar junior [22] ogeny based on the phylogenetic relationships proposed (IGM 100/1), an unnamed troodontid (IGM 100/1126 by Brusatte et al. [8]. Branch lengths were calculated for [13]) and Archaeopteryx lithographica [11] (NMNH PV non-avian coelurosaurs based on oldest fossil ages from OR 37001). We CT scanned each specimen using a Gen- the Paleobiology Database (http://www.paleobiodb.org) eral Electric v|tome Phoenix Computed Tomography in sister subclades for each internal node (e.g., the spe- (CT) scanner (General Electric, Heidelberg, Germany) at cies range for Incisivosaurus is 122.46–125.45 Ma and Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 3 of 11 Coelurosauria Alioramus altai Incisivosaurus gauthieri Citipati osmolskae Non-avian Khaan mckennai Coelurosaurs Zanabazar junior IGM100/1126 Archaeopteryx lithographica Archaeopterygidae Paleognathae Crypturellus tataupa Aves Struthio camelus X Maniraptora Rhea americana X Apteryx australis X Dromaius novaehollandiae X Casuarius casuarius X Casuarius unappendiculatus X Neognathae Gallus gallus Galloanseriformes Chauna chavaria Anas platyrhynchos ves A Tachyeres patachonicus Tachyeres leucocephalus X Tachyeres pteneres X Tachyeres brachypterus X Chordeiles minor Caprimulgiformes Caloenas nicobarica Raphus cucullatus X Ptilinopus melanospilus Columbea Podilymbus podiceps Podilymbus gigas X Gavia immer Diomedea immutabilis Eudyptes chrysocome Aequornithes Fregata magnificens Phalacrocorax penicillatus Phalacrocorax harrisi X Rallus philippensis Gallirallus rovianae X Gruiformes Gallirallus australis X Grus canadensis Phaethon rubricauda Phaethoniformes Alca torda Pinguinus impennis X Bucorvus abyssinicus Afroaves Melanerpes aurifrons Coragyps atratus Haliaeetus leucocephalus Cariama cristata Pelecyornis australis X Brotogeris chrysopteris Australaves Nestor meridionalis Strigops habroptilus X Sturnus vulgaris

175.0 150.0 125.0 100.0 75.0 50.0 25.0 0.0 Million Years Ago Fig. 1 Time-calibrated phylogeny of taxa sampled in the study. Maximum clade credibility tree based on Jetz et al. [26] with a Hackett et al. [28] backbone. Non-avian dinosaurs and Archaeopteryx were incorporated into the phylogeny based on fossil occurrence data from Paleobiology Database (paleobiodb.org). Colors correspond to major clades and are consistent across figures. Red crosses next to taxonomic names indicate a flightless taxon for Oviraptorosauria is 130.0 Ma, based on the first fossil the optic lobe, cerebrum, and cerebellum). These land- occurrence of Caudipteryx zoui; subtracting the first marks were used to define patches to place sliding semi- occurrence age from the latter gives 4.55 Myr, which landmarks on each brain section. With this approach, is the branch length for Incisivosaurus). This combined each patch comprises discrete landmarks anchoring the extant and fossil tree was used for the Coelurosaur dataset patch boundary, 3-D semilandmarks on lines between (Fig. 1). discrete landmarks along the surface of the endocast model, and 3-D surface semilandmarks within the patch. Landmark data Patches of 6 × 6 sliding semilandmarks were used for each To collect landmark data, each 3-D endocranial recon- half of the cerebrum (Fig. 2,Additionalfile1: Tables S3 struction was imported into Landmark Editor [28] for and S4). 4 × 4 patches were used for each optic lobe. A digital placement of 3-D Cartesian coordinate points. single patch of 4 × 5 semilandmarks was used for the cere- The relatively featureless surface of an endocast presents bellum. In places where the edges of neighboring patches an issue for landmark placement due to the lack of clear overlapped, we manually deleted the duplicated landmarks surficial unions of tissues or other large features that in R, leaving a total of 109 landmarks. We chose the num- help define discrete landmarks [17]. We placed evenly ber of semilandmarks for each patch so as to capture the spaced 3-D semilandmarks on five sections of the endo- shape of each lobe without oversampling it, which we con- cast (i.e., left and right cerebrum, left and right optic lobes, firmed using the function LaSEC in the LaMBDA R pack- and cerebellum). To do this, we first placed discrete Type age [35]. The raw landmark files are available on Dryad. I or Type II landmarks (sensu [33], also see [34]) along The landmark data were imported into R v3.1.1 [36], and major endocranial features (e.g. the triple point between aligned using “geomorph” v3.0.3 [37] with generalized Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 4 of 11

Fig. 2 Landmark scheme on the endocast of Crypturellus tataupa (AMNH 604) in a) dorsal, b) ventral, c) caudal, and d) left lateral views. Cerebrum (red), cerebellum (yellow), optic lobes (blue), landmarks on the division between cerebrum and cerebellum (orange), triple point between optic lobe, cerebellum, and cerebrum (brown)

Procrustes superimposition and with sliding semiland- (LDA) using the “MASS” R package v7.3–45 [40]. For marks minimizing total bending energy [15, 16]. The the Coelurosaur dataset, we used the first 17 PC axes aligned coordinates were imported into MorphoJ that encompass 95% of the total shape variation. This v1.06a [38]astwodatasets:1)fullsamplewith analysis looks to maximally separate the a priori non-avian and avian dinosaurs (N =80; ‘Coelurosaur locomotor groups (e.g. terrestrial, volant, secondarily dataset’) and 2) subsampled dataset with the crown-group flightless). If the LDA is able to adequately separate the birds (N =73; ‘Aves dataset’). An analysis using the Coel- shape data into a priori groups, then it suggests that urosaur dataset indicated that landmark placement on locomotor mode induces certain neuroanatomical Alioramus may have been incorrect, probably due to de- shapes across taxonomic groups. If not, then those formation and lack of easily identifiable landmarks. There- groups are not supported. Group membership was fore, a third dataset was created excluding Alioramus, cross-validated to see if the LD axes are able to correctly which is discussed below as the ‘Coelurosaur dataset.’ Due predict group assignment. Two locomotor categories to bilateral symmetry, the symmetrical component of the (‘protoflying’ and ‘swimming’) had single members and shape data was used for further analyses. Species with therefore those groups were not included in the dataset multiple specimens were aligned with the pooled data and for the analysis. The data point for these members were the mean shape was calculated for each species. subsequently projected onto the morphospace based on LDA using the ‘predict’ function in R. We also con- Analyses ducted cross-validation analysis to assess the ability to We subjected each shape dataset to a principal compo- correctly assign locomotory mode based on endocranial nents analysis (PCA) in MorphoJ. Plotting the scores shape. associated with the first three PC axes creates a morpho- These analyses were run twice. The first analysis was space of the overall shape differences among specimens run with crown-group avians (N = 73 specimens repre- and identifies the morphological changes occurring senting 44 species) and their locomotory mode (e.g. along each axis. In addition to endocast images, ‘lolli- volant or flightless). 'Flightlessness' among extant birds pop’ diagrams were used to visualize major shape was defined by a complete loss of lift generation, i.e., a changes occurring in morphospace along PC axes, total inability to create sufficient lift to raise the body off where the vectors indicate the direction and magnitude the ground for any amount of time, such as the ostrich of change from the mean shape. Using the “geomorph” (Struthio camelus), dodo (Raphus cucullatus), and ka- R package [37], we also created a phylomorphospace kapo (Strigops habroptilus) and was labeled ‘flightless’ in based on the first two PC axes. The plot shows the cor- these analyses. A penguin, Eudyptes chrysocome,is respondence of phylogenetic relatedness to morpho- present in the dataset. Penguins use subaqueous flight to logical resemblance and can illustrate morphological propel through the water [41] and therefore it was innovation through the amount of morphospace ex- placed in its own locomotion category (‘swimming’). As plored by taxa [39]. This analysis visualizes unequal it was the only specimen for the ‘swimming’ locomotor magnitude of change per clade or branch and unequal category, it was removed from the dataset for LDA. morphological innovation by the geometry and relative Therefore, the first LDA was performed with two a length of the branches [39]. priori groups: ‘volant’ (N = 37 specimens, representing A dataset containing the PC axes encompassing 95% 25 species), and ‘flightless’ (N = 35 specimens, represent- of the total shape variation was exported from MorphoJ ing 18 species), for a total of 72 specimens and and imported into R to run a linear discriminant analysis 43 species. Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 5 of 11

The second LDA contained the original set of location relative to the cerebrum, and a posterior shift crown-group birds and six non-avian dinosaurs (three and dorsoventral expansion of the optic lobes (Add- oviraptorosaurs, two troodontids, and Archaeopteryx). itional file 1: Figure S2B). PC2 correlates with lateral and The non-avian dinosaurs (excluding Archaeopteryx) anterior expansion of the anterior cerebrum, posteroven- were categorized as ‘terrestrial’ because their primary tral reduction of the cerebellum, and a poteromedial locomotory mode was not inherited from flying ances- shift of the optic lobes (Additional file 1: Figure S2C). tors. The distinction here between ‘secondarily flightless’ Importantly, the plot of PC2 versus PC1 results in no and ‘terrestrial’ is important because ‘secondarily flight- clear visual separation between flying and flightless less’ indicates that the species evolved from birds, but rather data points tend to cluster by clade flight-capable ancestors, whereas ‘terrestrial’ indicates (Fig. 3, Additional file 1: Figure S1). There is variable dir- their ancestors were never capable of volant activity. Al- ectionality in shape change within pairs of though a penguin (Eudyptes chrysocome) is present in volant-flightless sister taxa. For example, there is a nega- the dataset, it was again removed for this analysis. Simi- tive shift along PC1 in Columbiformes (from Caloenas larly, because of continuing debate concerning the level nicobarica to Raphus cucullatus), but a positive shift of flying ability of Archaeopteryx [12, 42, 43], it was de- along PC1 in Psittaciformes (from Nestor meridionalis to fined as a ‘proto-flyer’ and was the only member of that Strigops habroptilus), and a positive shift in PC2 in Cariami- locomotor category. As such, Archaeopteryx was also re- formes (from Cariama cristata to Pelecyornis australis) moved from the dataset for the LDA. Therefore, this (Additional file 1:FigureS1).Insomegroups,flight- LDA was performed with three a priori groups: ‘terres- less members radiate in multiple directions away from trial’ (N = 5 specimens, each representing a single spe- volant members (e.g. Gruiformes and Anseriformes). cies), ‘volant’ (N = 37 specimens, representing 25 Within the Coelurosaur dataset the first 17 axes account species), and ‘flightless’ (N = 35 specimens, representing for 95% of the symmetric component of shape variation, 18 species), for a total of 77 specimens and 48 species. with the first three axes associated with 66.4% of the vari- Once the analysis was complete, the ‘predict’ function ation (38.7, 17.5, and 10.4%, respectively) (Fig. 3). The was used to place Archaeopteryx and Eudyptes into the shape changes corresponding to PC1 and PC2 axes are LD morphospace. equivalent to those in the Aves dataset. PC1 correlates To test explicitly whether locomotory mode and other with a relative anterodorsal shift of the anterior margin of factors, such as allometry and phylogenetic inertia, drive the cerebrum, anteroventral shift of the anterior margin of predictable changes to endocranial shape, we performed the cerebellum and relative decrease in dorsoventral several regression analyses. First, regressions of the height of the cerebrum, and a posteroventral shift of the symmetric component of the shape data against posterior margin of the cerebellum (Fig. 3 inset endocra- log-transformed centroid size were run for each dataset nia, Additional file 1: Figure S3B). Along PC2, more posi- in MorphoJ using 10,000 replicates to assess the allometric tive numbers indicate an anteroventral shift in the effect. Second, phylogenetically-informed least-squares anterior cerebrum, a lateral reduction of the optic lobes, analyses on locomotory mode and shape data were and a posterior shift and expansion of the cerebellum (Fig. performed using the ‘procD.pgls’ function in the “ geo- 3 inset endocrania, Additional file 1: Figure S3C). The cu- morph” R package. In addition, we evaluated the effect of mulative effect of these shifts is an anteroposteriorly lon- phylogenetic signal using the ‘physignal’ function in the ger and mediolaterally narrower endocast towards positive “geomorph” R package and allometry based on PC1 and a shorter, rounder endocast towards negative log-transformed centroid sizes exported from MorphoJ PC1. The PC morphospace of the Coelurosaur dataset that were used as a proxy for endocranial size [44, 45]. shows no clear visual distinction between modern flying and flightless birds, however non-avian dinosaurs, as well Results as Archaeopteryx, occupy a unique region of morphospace Principal components analyses (Fig. 3). Plots of PC3 versus PC1 show some separation be- The PCA of the Aves dataset generated 17 PC axes ac- tween flying and flightless taxa in both the Aves (Add- counting for 95% of the symmetric component of shape itional file 1: Figure S4) and Coelurosaur datasets (Fig. 4), variation, with the first three axes associated with 66.6% with non-avian dinosaurs again occupying their own mor- of the variation (36.4, 16.2, and 13.8%, respectively) phospace. In both analyses, changes along PC3 are driven (Additional file 1: Figure S1; overall distribution of data primarily by the lateral expansion of the cerebrum, a ven- points resemble Coelurosaur dataset, Fig. 3). The shape tral shift of the anterior margin of the cerebrum, and a of the posterior aspects of the endocast drive neuroana- dorsolateral shift in the optic lobe (Fig. 4 inset endocrania, tomical changes along PC1, where positive scores Additional file 1:FigureS5). indicate an anteroventral shift in the posterior margin of In phylomorphospace, the early history of the coeluro- the cerebrum, a posteroventral shift in cerebellar saurian lineage begins on the positive end of PC1 and Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 6 of 11

Nestor meridionalis

Anas platyrhynchos PC 2 (17.5%)

Volant Flightless −0.1Terrestrial 0.0 0.1 Protoflying Swimming Non-Avians Archaeopterygidae Paleognathae Galloanseriformes Caprimulgiformes Columbea −0.2 Aequornithes Citipati osmolskae Gruiformes Phaethoniformes Chordeiles minor Afroaves Australaves

−0.3 −0.2 −0.1 0.0 0.1 0.2 PC 1 (38.6%) Fig. 3 Endocranial shape variation and phylomorphospace for the Coelurosaur dataset. Morphospace constructed from PC1 and PC2 of symmetric component of shape. Symbols by locomotor mode and color-coded taxonomically. Bold, black branches indicate stem Neornithes. Branches leading to clades are color-coded with the same colors, and their hypothetical ancestral shapes are noted with a small circle of the same color 0.2

Chordeiles minor 0.1 0.0 PC3 (10.4%) Volant Flightless Terrestrial Protoflying Swimming Non-Avians −0.1 Archaeopterygidae Paleognathae Galloanseriformes Caprimulgiformes Columbea Aequornithes Gruiformes Phaethoniformes Afroaves Phalacrocorax penicillatus Australaves −0.2

−0.2 −0.1 0.0 0.1 0.2 PC1 (38.6%) Fig. 4 Endocranial shape variation in the Coelurosaur dataset. Morphospace constructed from PC1 and PC3 of symmetric shape component in Coelurosaur dataset, color-coded taxonomically Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 7 of 11

− shifts negatively (Fig. 3). Along PC1, Archaeopteryx oc- and 0.252e 29, respectively). The Rockhopper penguin, cupies a position that lies between non-avian dinosaurs Eudyptes chrysocome, falls among the volant birds with a and crown-group birds (Fig. 3). Many internal nodes posterior probability of 0.853, versus 0.147 for flightless − along the avian stem, as estimated by ancestral state re- and 2.66e 16 for running. A cross-validation analysis constructions, appear in a small section of morphospace, indicated that some taxa were misclassified (Additional which may be due to poor sampling at the base of Aves. file 1: Table S5). Rallus philippensis (two of three speci- Radiations from this small section of morphospace con- mens), Gallus gallus, Nestor meridionalis, Diomedea tain several subclades and their hypothetical ancestors immutabilis, Phalacrocorax penicillatus (all three speci- that correspond to the origin of Neoaves. The test for mens), Coragyps atratus, Tachyeres patachonicus, Anas phylogenetic signal indicates that phylogenetic signal is platyrhynchos,andPodilymbus podiceps are all volant very low, but significant, in both the Aves dataset birds that were classified as secondarily flightless. (Blomberg’s K = 0.04; P < 0.0001) and the Coelurosaur Phalacrocorax harrisi (one of two specimens), Rhea dataset (K = 0.05;P< 0.0001). americana (one of three specimens), Pelecyornis australis, Gallirallus rovianae (two of three specimens), Raphus Linear discriminant analysis cucullatus, Tachyeres pteneres (one of three specimens), Based on PC axes that account for 95% of symmetric Strigops habroptilus,andGallirallus australis (both speci- component of shape, the morphospace constructed from mens) are flightless birds that were classified as volant. LD axes and the Coelurosaur dataset shows crown Lastly, IGM 100/1126, a troodontid, which is terrestrial, group birds in a distinct cluster from non-avian was classified as flying. The other 53 specimens were cor- dinosaurs and is able to visually distinguish between rectly classified by this analysis. The LDA for the Aves locomotory modes (Fig. 5). LD1 distinguishes non-avian dataset (Additional file 1: Figure S6), shows separation dinosaurs and crown-group birds. However, volant and along the single LD axis between volant and flightless flightless crown-group birds are separated along LD2, birds with small overlap in value between the two locomo- orthogonal to major neuroanatomical changes along the tory modes. dinosaur-bird transition. Shape changes towards negative LD2 are anteroposterior contraction of the cerebrum, Covariates posterolateral expansion of the cerebrum, narrowing of The lack of parallel neuroanatomical changes associated the cerebellum both in terms of lateral and anteroposter- with the dinosaur-bird transition and volant-flightless ior extent, and an anterior and ventral shift of the optic birds implies that shifts between these locomotory lobes. Interestingly, the cross-validation technique based modes incur contrasting changes to brain shape or these on LDA classifies Archaeopteryx as terrestrial with a shifts have not contributed substantially to neuroana- posterior probability of ~1 (neither flying nor secondarily tomical variation. To explicitly assess the impact of − flightless, which had posterior probabilities of 3.89 e 30 locomotory mode on endocranial shape, we tested for

Dromaius novaehollandiae

Citipati osmolskae LD2

Flightless Protoflying * Volant −3 −2 −1 0 1 2 Terrestrial Bucorvus abyssinicus Swimming

−2 0 2 4 6 8 10 LD1 Fig. 5 Morphospace constructed from LD1 and LD2 of endocranial shape variation in the Coelurosaur dataset, color-coded by locomotion. Endocasts of representative taxa are shown in dorsal (left) and lateral (right) views Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 8 of 11

the effect of locomotory mode on endocranial shape flightless birds, relative to their volant counterparts, gen- before and after correcting for phylogenetic structure erally exhibit anteroposterior expansion of the cerebrum, and allometric signal. The ahistorical analyses indicate decreased dorsal convexity of the cerebrum, narrowing that size has a statistically significant effect in both the of the cerebellum, and inward contraction of the optic Aves dataset (R2 = 0.169; P < 0.0001) and the Coelurosaur lobes. dataset: (R2 =0.106; P< 0.0001). However, when ex- Meanwhile, cross-validation analysis misclassifies the amined in a phylogenetic context, both size and loco- locomotory mode of approximately 30% of the speci- motion independently and together failed to reject mens based on both the Aves and Coelurosaur dataset, thenullhypothesisofnoeffectontheshapedatafor indicating that neuroanatomical shape is not as reliable the Aves dataset (P >0.44; Table 1). The Coelurosaur a neuroanatomical correlate for locomotor categories as dataset returned significant results for the effect of the LD morphospace would make it seem. Among size (R2 = 0.397; P = 0.049) and locomotion (R2 = non-avian dinosaurs, IGM 100/1126 is misclassified as 0.007; P = 0.004) separately, but not the combined flying (Additional file 1: Table S5). IGM 100/1126 falls effects of size and locomotion (R2 = 0.295; P = 0.496; closer to the PC morphospace area of crown group birds Table 1). than the other non-avian dinosaurs and Archaeopteryx (Fig. 3), even though it falls within the non-avian mor- Discussion phospace in the LDA (Fig. 5). However, the small num- ber of non-avian coelurosaurs sampled for this study (N Our study presents several notable results regarding the = 5) may simply have contributed greater degree of un- relationship between flight capacity and brain shape evo- certainty in estimating locomotory mode. Sampling add- lution. These results, visually shown by the PC morphos- itional non-avian coelurosaurs through additional paces, are not congruent with the hypothesis that discoveries of well-preserved skulls may enhance the flightless birds revert to a plesiomorphic brain shape that capacity of endocranial shape to distinguish between ter- is similar to their terrestrial, non-avian relatives. In fact, restrial and secondarily flightless modes. Importantly, there is comparatively little shape difference between none of the flightless birds were misclassified as terres- flightless birds and their closest flying relatives (Fig. 3), trial and based on Aves dataset, the locomotory mode of indicating that flightless birds largely retain brain shapes birds is misclassified for 25 of 72 taxa. This result cor- more similar to their closest living relatives. Moreover, roborates the observation from PC morphospace that non-volant birds do not collectively approach the flightless and volant birds overlap in their neuroanatom- endocranial shape of non-avian theropods. Therefore, ical shape, but are distinct from non-avian dinosaurs. based on visual inspection of the PC morphospace, the Taken together, the results from PC morphospace and neuroanatomy of flightless birds is clearly not a reverse LDA imply that although some neuroanatomical analogue of flightless non-avian dinosaurs. differences may be associated with secondary loss of The LD morphospace, based on axes that maximize flight, these changes account for very small proportion separation between group means, distinguishes between of shape variation to be a meaningful predictor for non-avian and avian groups, as expected from PCA. locomotory mode. Importantly, it shows an overall separation between Furthermore, whereas ahistorical regression analyses volant and flightless birds along LD2 with some overlap on both the Aves and Coelurosaur datasets indicate a (Fig. 5). This shows that some aspects of endocranial significant effect of allometric signal on brain shape, shape can distinguish volant and flightless birds. The phylogenetically-informed regression analyses indicate shape variation associated with LD2 implies that that locomotory mode is not a significant predictor of endocranial shape with or excluding allometric signal for Table 1 Results from the phylogenetically informed regression the Aves dataset (Table 1). The phylogenetically-informed of symmetric component of shape onto locomotory mode and analysis of variance of the Coelurosaur dataset returned centroid size significant results for the independent effect of locomo- Analysis Variable Results tory mode and size on the shape of the endocast but insig- 2 R FP nificant results when tested together (Table 1). As such, Aves Locomotion 0.423 14.69 0.439 there is no evidence that locomotory mode is a strong pre- Log Centroid Size 0.497 40.45 0.570 dictor of endocranial shape when corrected for size. Taken Locomotion and Log Centroid Size 0.006 2.61 0.678 together, major shifts in locomotory mode have not driven Coelurosaur Locomotion 0.007 0.03 <0.001 uniform neuroanatomical changes within crown-group birds. Another possibility explaining the lack of locomotor Log Centroid Size 0.397 30.90 0.049 signal is that the divergence of more recently evolved fly- Locomotion and Log Centroid Size 0.295 4.26 0.496 ing and flightless avian sister taxa occurred on a much Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 9 of 11

shorter time-scale than the dinosaur-bird transition. current sampling classifies Archaeopteryx as terrestrial, Consequently, many flightless lineages may not have and not volant. This is an intriguing result given the yet had the time to evolve clear neuroanatomical ongoing debate about its flight capabilities [42, 43]. The changes that would be associated with flightlessness. endocast of Archaeopteryx seems to more closely resem- The latter scenario, however, is unlikely for paleog- ble its non-avian relatives rather than crown group birds, naths, which have been flightless since the early meaning it may not have been capable of extensive vo- Cenozoic [46]. lant behaviors. Increased taxonomic sampling that fills It is worth noting that the shape changes in this study in the gap between Archaeopteryx and the modern radi- only reflect surficial differences in neuroanatomy ation of birds may strengthen our ability to classify the because we employed endocasts. As such, shifts in locomotory mode of the earliest members of Aves. The locomotory mode may have an impact on internal LDA classifies Eudyptes as a volant bird. Early studies neuroanatomy that cannot be detected through the use note that the penguin uses subaqueous flight for of endocasts. Nevertheless, some surficial anatomy may locomoting underwater [41], and our study shows that reflect changes occurring internally. Although it cannot its neuroanatomical shape corroborates the notion that be confirmed with the currently available data, the sep- subaqueous flight is equivalent to aerial flight. This aration of non-avian dinosaurs and crown-group birds in outcome may be due to the fact that the evolution of PC morphospaces indicate a clear trend towards relative wing-propelled diving (i.e. the acquisition of a new expansion of the cerebrum along the dinosaur-bird tran- behavior) may have played a larger role in shaping the sition (Figs. 3 and 4), which may be related to increased brain than the initial loss of aerial flight (i.e. the loss of stimulus processing ability [47]. A recent study suggests an established behavior) [48]. that the Wulst (a visual and somatosensory integration If not locomotory mode, then what are the major and processing center) and the entopallium (a visual drivers of brain shape evolution in birds? Previous GM interpretation area) are important in fast-paced visual studies on avian endocasts have shown that phylogenetic processing during flight [5]. Although the landmark history, allometry, and orbit shape account for statisti- scheme used here did not explicitly test for shape differ- cally significant (P < 0.001), but small proportions of ences in the Wulst, the expansion of the posterodorsal endocranial shape variation [17, 49]. With different taxo- region of the cerebrum in avian evolution (Fig. 3, nomic sampling and use of high-dimensional shape Additional file 1: Figure S2), where these flight-related characterization, our results corroborate these find- nuclei lie, may correspond to an increased trend in ings, indicating that there is allometric signal and a volant behaviors [5]. Previous studies based on pro- very weak, albeit statistically significant, phylogenetic portional volumes have shown that the cerebral ex- signal present in the endocranial shape datasets. pansion did not appear abruptly at the origin of While these factors have variably contributed to Avialae, but rather, occurred more gradually through- evolutionary changes in bird brains, they collectively out coelurosaurian evolutionary history [11, 12]. In- account for a small proportion (~ 10%) of the total flated, ‘avian-like’ brain volumes first appear at the variation [e.g., 49], suggesting that additional drivers base of Maniraptora [11] and significant volumetric are yet to be identified [49]. expansion of the cerebrum does not occur until well In addition, increased taxonomic sampling is still within the crown group [12]. However, the shape data needed between the base of Avialae and the origin of liv- here demonstrate that the neuroanatomical shape ing birds. Unfortunately, most of the fossils from this differences between non-avian dinosaurs and interval are fragmentary [50] or crushed [51] making crown-group birds are primarily driven by the cere- endocranial construction extremely difficult. This brum. Collectively, these results suggest that cerebral paucity of specimens contributes to the apparent gap shape may have changed in response to the increased between Archaeopteryx and modern birds in the use of flight behaviors between non-avian dinosaurs phylomorphospace, preventing a more reliable model of and the modern radiation of birds even as propor- neuroanatomical evolution along this lineage. Early avia- tional volume remained relatively constant [12]. Intri- lans undertook a variety of locomotor strategies, from guingly, LD2, that separates volant and flightless the fully volant Enantiornithines [52] to the swimming birds, is associated with slight reduction in the con- Hesperornithiformes [53]. Sampling this region of the vexity of the cerebrum, including the Wulst, suggest- tree, perhaps from recently discovered specimens [54], ing that its relative expansion could reflect flight might bridge the gap from the non-volant theropods and capacity across coelurosaurs. Such investigation re- at least partially volant Archaeopteryx to the fully volant quires explicit characterization of Wulst morphology. crown group and provide examples of transitional brain Although the overall predictive power of endocranial morphologies not represented by living or extinct shape for locomotory mode is weak, the LDA based on theropods. Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 10 of 11

Conclusions Acknowledgments The osteological changes associated with the evolution We thank A. Balanoff for sharing endocast data. We thank A. Balanoff and M. Norell for their comments on early drafts. We thank the Microscopy and of flight in theropod dinosaurs is well documented in Imaging Facility at the American Museum of Natural History, especially M. the fossil record, but the accompanying neuroanatomical Hill and H. Towbin, and the University of Texas High Resolution CT changes are less known. Here, we used 3-D GM tech- Laboratory, especially J. Maisano and M. Colbert for imaging the specimens. E. Stanley and M. Colbert taught MELG the process of making endocasts. A. niques on endocasts of extinct and modern dinosaurs Perrard, J. Foox, M. Siddall, A. Laing, A. West, and K.J. Soda assisted with (birds) to quantify and evaluate neuroanatomical phylogenetic and statistical analyses. E. Wilberg provided helpful feedback changes related to this major locomotory change. In on figures. We thank four anonymous reviewers for their constructive feedback. particular, we analyzed closely-related sets of volant and flightless birds to use as a potential reverse analogue to Funding the initial acquisition of flight. The results demonstrate MELG was supported by the Richard Gilder Graduate School, American Museum of Natural History, NSF DDIG (1311790). MELG and AW were that loss of flight capacity does not incur predictive supported by NSF DEB (1801224). The funding agencies had no role in the changes to endocranial shape in extant birds. In design of the study, in the collection, analysis, or interpretation of the data, addition, the brain morphology of flightless birds do not nor in writing the manuscript. converge to that of non-avian dinosaurs. While add- Availability of data and materials itional sampling may close the gap between non-avian The landmark data sets and tree file supporting the results of this article are dinosaurs and Neornithes, brain evolution from available in the Dryad repository, https://doi.org/10.5061/dryad.n1k111m. non-avian dinosaurs to birds appears to have been a uni- “ ” Authors’ contributions directional phenomenon or point of no return. Among MELG and AW conceived of the study. MELG procured specimens. MELG crown-group birds, returning to an ancestral locomotory collected the data. MELG performed the analyses. MELG wrote the mode (i.e., flightlessness) does not correlate with a rever- manuscript, with the help of AW. MELG and AW edited the manuscript. MELG and AW read and approved the manuscript. sion to the more pleisiomorphic brain shape found in terrestrial non-avian dinosaurs. Modern flightless birds, Ethics approval and consent to participate therefore, are not reverse neuroanatomical analogues of Not applicable. non-avian dinosaurs. Consent for publication Not applicable.

Additional file Competing interests The authors declare that they have no competing interests. Additional file 1: Table S1. CT scanning parameters used for each specimen scanned for this project. Table S2. CT scanning parameters Publisher’sNote used for additional specimens used for this project from Balanoff et al. Springer Nature remains neutral with regard to jurisdictional claims in [11]. Table S3. Discrete landmarks and anatomical descriptions. published maps and institutional affiliations. Table S4. Sliding semilandmarks used and anatomical descriptions. Table S5. Posterior probabilities from the cross-validation analysis of the Author details LDA of the Coelurosaur dataset. Figure S1. Endocranial shape variation 1Biology Department, Suffolk University, Boston, MA 02108, USA. in the Aves dataset, illustrated in a morphospace constructed from PC1 2Department of Anatomical Sciences, Stony Brook University, Stony Brook, and PC2. Figure S2. Shape changes along positive PC1 and PC2 for the NY 11779, USA. 3Division of Paleontology, American Museum of Natural Aves dataset shown as lollipop diagrams in dorsal and right lateral views. History, New York, NY 10024, USA. 4Department of Anatomy, New York Figure S3. Shape changes along positive PC1 and PC2 for the Coelurosaur Institute of Technology College of Osteopathic Medicine, Old Westbury, NY dataset shown as lollipop diagrams in dorsal and right lateral views. 11568, USA. 5Life Sciences Department Vertebrates Division, Natural History Figure S4. Endocranial shape variation in the Aves dataset, illustrated in a Museum, London SW7 5BD, UK. morphospace constructed from PC1 and PC3. Figure S5. Shape changes along positive PC3 for the Aves and Coelurosaur datasets shown as lollipop Received: 13 September 2017 Accepted: 28 November 2018 diagrams in dorsal and right lateral views. Figure S6. Bar plot of the single linear discriminant axis constructed from the LDA of endocranial shape variation in the Aves dataset. (DOCX 1520 kb) References 1. Andrews TJ, Halpern SD, Purves D. Correlated size variations in human visual cortex, lateral geniculate nucleus, and optic tract. J Neurosci. 1997;17(8):2859–68. Abbreviations 2. Herculano-Houzal S, Manger PM, Kaas JH. Brain scaling in mammalian AMNH (FARB): American Museum of Natural History, New York, NY, USA evolution as a consequence of concerted and mosaic changes in numbers (Fossil Amphibian, Reptile, and Bird Collection); FMNH: Field Museum of of neurons and average neuronal cell size. Front Neuroanat. 2014;8(77):1–28. Natural History, Chicago, IL, USA; IGM: Mongolian Institute of Geology, 3. Robinson CD, Patton MS, Andre BM, Johnson MA. Convergent evolution of Ulaanbaatar, Mongolia; IVPP: Institute of Vertebrate Paleontology and brain morphology and communication modalities in lizards. Curr Zool. Paleoanthropology Beijing, People’s Republic of China; KU: University of 2015;61(2):281–91. Kansas Natural History Museum, Lawrence, KS, USA; NHMUK (PV OR, 4. Olkowicz S, Kocourek M, Lučan RK, Porteš M, Fitch WT, Herculano-Houzel S, A): Natural History Museum, London, United Kingdom (Paleontology Němec P. Birds have primate-like numbers of neurons in the forebrain. Vertebrates and Old Register, Aves); NMNH: Smithsonian Institution National P Natl Acad Sci. 2016;113(26):7255–60. Museum of Natural History; TCWC: Texas Cooperative Wildlife Collection, 5. Gold MEL, Schulz D, Budassi M, Gignac PM, Vaska P, Norell MA. Flying College Station, Texas, USA; TMM: University of Texas Memorial Museum, starlings, PET and the evolution of volant dinosaurs. Curr Biol. Austin, Texas, USA 2016;26:R265–7. Gold and Watanabe BMC Evolutionary Biology (2018) 18:190 Page 11 of 11

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