Paul Gignac

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• Balanoff, A., Ferrer, E., Saleh, L., Gignac, P. M., Gold, M. E., Marugán-Lobón, J., Norell, M., Ouellette, D., Salerno, M., Watanabe, A., Wei, S., Bever, G., & Vaska, P. (2024). Quantitative functional imaging of the pigeon brain: implications for the evolution of avian powered flight. Proceedings of the Royal Society B: Biological Sciences, 291(2015). doi:10.1098/rspb.2023.2172
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  The evolution of flight is a rare event in vertebrate history, and one that demands functional integration across multiple anatomical/physiological systems. The neuroanatomical basis for such integration and the role that brain evolution assumes in behavioural transformations remain poorly understood. We make progress by (i) generating a positron emission tomography (PET)-based map of brain activity for pigeons during rest and flight, (ii) using these maps in a functional analysis of the brain during flight, and (iii) interpreting these data within a macroevolutionary context shaped by non-avian dinosaurs. Although neural activity is generally conserved from rest to flight, we found significant increases in the cerebellum as a whole and optic flow pathways. Conserved activity suggests processing of self-movement and image stabilization are critical when a bird takes to the air, while increased visual and cerebellar activity reflects the importance of integrating multimodal sensory information for flight-related movements. A derived cerebellar capability likely arose at the base of maniraptoran dinosaurs, where volumetric expansion and possible folding directly preceded paravian flight. These data represent an important step toward establishing how the brain of modern birds supports their unique behavioural repertoire and provide novel insights into the neurobiology of the bird-like dinosaurs that first achieved powered flight.
• Gignac, P. M., Valdez, D., Morhardt, A. C., & Lynch, L. M. (2024). Buffered Lugol's Iodine Preserves DNA Fragment Lengths. Integrative organismal biology (Oxford, England), 6(1), obae017.
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  Museum collections play a pivotal role in the advancement of biological science by preserving phenotypic and genotypic history and variation. Recently, contrast-enhanced X-ray computed tomography (CT) has aided these advances by allowing improved visualization of internal soft tissues. However, vouchered specimens could be at risk if staining techniques are destructive. For instance, the pH of unbuffered Lugol's iodine (IKI) may be low enough to damage deoxyribonucleic acid (DNA). The extent of this risk is unknown due to a lack of rigorous evaluation of DNA quality between control and experimental samples. Here, we used formalin-fixed mice to document DNA concentrations and fragment lengths in nonstained, ethanol-preserved controls and 3 iodine-based staining preparations: (1) 1.25% weight-by-volume (wt/vol.) alcoholic iodine (IE); (2) 3.75% wt/vol. IKI; and (3) 3.75% wt/vol. buffered IKI. We tested a null hypothesis of no significant difference in DNA concentrations and fragment lengths between control and treatment samples. We found that DNA concentration decreases because of staining-potentially an effect of measuring intact double-stranded DNA only. Fragment lengths, however, were significantly higher for buffered IKI and control samples, which were not, themselves, significantly different. Our results implicate buffered IKI as the appropriate choice for contrast-enhanced CT imaging of museum wet collections to safely maximize their potential for understanding genetic and phenotypic diversity.
• Gignac, P., Aceves, V., Baker, S., Bell, J., Barnes, J., Boyer, D., Cunningham, D., De Carlo, F., Chase, M., Coen, K., Colbert, M., Creed, T., Daza, J., Dickinson, E., Leonardo, V., Dougan, L., Duffy, F., Dunham, C., Early, C., , Edey, D., et al. (2024). The Role of Networks to Overcome Large-scale Challenges in Tomography: The Non-Clinical Tomography Users Research Network. Tomography of Materials and Structures, 5, 100031. doi:10.1016/j.tmater.2024.100031
• Gignac, P., Balanoff, A., Ferrer, E., Saleh, L., Gold, M., Marugán-Lobón, J., Norell, M., Ouellette, D., Salerno, M., Watanabe, A., Wei, S., Bever, G., & Vaska, P. (2024). Quantitative functional imaging of the pigeon brain: implications for the evolution of avian powered flight. Proceedings of the Royal Society B: Biological Sciences, 202327172. doi:10.1098/rspb.2023.2172
• Gignac, P., Valdez, D., Morhardt, A., & Lynch, L. (2024). Buffered Lugol's Iodine Preserves DNA Fragment Lengths. Integrative Organismal Biology, 6(1). doi:10.1093/iob/obae017
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  Synopsis Museum collections play a pivotal role in the advancement of biological science by preserving phenotypic and genotypic history and variation. Recently, contrast-enhanced X-ray computed tomography (CT) has aided these advances by allowing improved visualization of internal soft tissues. However, vouchered specimens could be at risk if staining techniques are destructive. For instance, the pH of unbuffered Lugol's iodine (I2 KI) may be low enough to damage deoxyribonucleic acid (DNA). The extent of this risk is unknown due to a lack of rigorous evaluation of DNA quality between control and exper- imental samples. Here, we used formalin-fixed mice to document DNA concentrations and fragment lengths in nonstained, ethanol-preserved controls and 3 iodine-based staining preparations: (1) 1.25% weight-by-volume (wt/vol.) alcoholic iodine (I2 E); (2) 3.75% wt/vol. I2 KI; and (3) 3.75% wt/vol. buffered I2 KI. We tested a null hypothesis of no significant difference in DNA concentrations and fragment lengths between control and treatment samples. We found that DNA concentration de- creases because of staining-potentially an effect of measuring intact double-stranded DNA only. Fragment lengths, however, were significantly higher for buffered I2 KI and control samples, which were not, themselves, significantly different. Our re- sults implicate buffered I2 KI as the appropriate choice for contrast-enhanced CT imaging of museum wet collections to safely maximize their potential for understanding genetic and phenotypic diversity.
• Gignac, P., Vasquez, T., Brewer, P., & Gold, M. (2024). Tumour or Hemmorrhage?: Differential Diagnosis of an Unknown Mass within the Brain of a Budgerigar using a Novel Imaging Pipeline. Veterinary Record Case Reports, e899. doi:10.1002/vrc2.899
• Gray, J. A., Gignac, P. M., & Stanley, E. L. (2024). The first full body diffusible iodine-based contrast-enhanced computed tomography dataset and teaching materials for a member of the Testudines. Anatomical record (Hoboken, N.J. : 2007), 307(3), 535-548.
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  Diffusible iodine-based contrast-enhanced Computed Tomography (diceCT) is now a widely used technique for imaging metazoan soft anatomy. Turtles present a particular challenge for anatomists; gross dissection is inherently destructive and irreversible, whereas their near complete shell of bony plates, covered with keratinous scutes, presents a barrier for iodine diffusion and significantly increases contrast-enhanced CT preparation time. Consequently, a complete dataset visualizing the internal soft anatomy of turtles at high resolution and in three dimensions has not yet been successfully achieved. Here we outline a novel method that augments traditional diceCT preparation with an iodine injection technique to acquire the first full body contrast-enhanced dataset for the Testudines. We show this approach to be an effective method of staining the soft tissues inside the shell. The resulting datasets were processed to produce anatomical 3D models that can be used in teaching and research. As diceCT becomes a widely employed method for nondestructively documenting the internal soft anatomy of alcohol preserved museum specimens, we hope that methods applicable to the more challenging of these, such as turtles, will contribute toward the growing stock of digital anatomy in online repositories.
• Plateau, O., Green, T., Gignac, P., & Foth, C. (2024). Comparative digital reconstruction of Pica pica and Struthio camelus and their cranial suture ontogenies. Anatomical Record, 307(1). doi:10.1002/ar.25275
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  To date, several studies describe post-hatching ontogenetic variation in birds; however, none of these studies document and compare ontogenetic variation of the entire skull in multiple avian species. Therefore, we studied ontogenetic skull variation of two bird species with very different ecologies, Pica pica, and Struthio camelus, using μCT based 3D reconstructions. For each specimen, we performed bone-by-bone segmentation in order to visualize and describe the morphological variation of each bone during ontogeny and estimated the average sutural closure of the skulls to identify different ontogenetic stages. Although bone fusion of P. pica occurs more rapidly than that of S. camelus the general sequence of bone fusion follows a similar trend from posterior to anterior, but a more detailed analysis reveals some interspecific variation in the fusion patterns. Although growth persists over a longer period in S. camelus than in P. pica and adults of the former species are significantly larger, the skull of the most mature S. camelus is still less fused than that of P. pica. Different growth and fusion patterns of the two species indicate that the interspecific ontogenetic variation could be related to heterochronic developments. Nevertheless, this hypothesis needs to be tested in a broader phylogenetic framework in order to detect the evolutionary direction of the potential heterochronic transformations.
• Straight, P. J., Gignac, P. M., & Kuenzel, W. J. (2024). A histological and diceCT-derived 3D reconstruction of the avian visual thalamofugal pathway. Scientific reports, 14(1), 8447.
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  Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.
• Straight, P. J., Gignac, P. M., & Kuenzel, W. J. (2024). Mapping the avian visual tectofugal pathway using 3D reconstruction. The Journal of comparative neurology, 532(2), e25558.
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  Image processing in amniotes is usually accomplished by the thalamofugal and/or tectofugal visual systems. In laterally eyed birds, the tectofugal system dominates with functions such as color and motion processing, spatial orientation, stimulus identification, and localization. This makes it a critical system for complex avian behavior. Here, the brains of chicks, Gallus gallus, were used to produce serial brain sections in either coronal, sagittal, or horizontal planes and stained with either Nissl and Gallyas silver myelin or Luxol fast blue stain and cresyl echt violet (CEV). The emerging techniques of diffusible iodine-based contrast-enhanced computed tomography (diceCT) coupled with serial histochemistry in three planes were used to generate a comprehensive three-dimensional (3D) model of the avian tectofugal visual system. This enabled the 3D reconstruction of tectofugal circuits, including the three primary neuronal projections. Specifically, major components of the system included four regions of the retina, layers of the optic tectum, subdivisions of the nucleus rotundus in the thalamus, the entopallium in the forebrain, and supplementary components connecting into or out of this major avian visual sensory system. The resulting 3D model enabled a better understanding of the structural components and connectivity of this complex system by providing a complete spatial organization that occupied several distinct brain regions. We demonstrate how pairing diceCT with traditional histochemistry is an effective means to improve the understanding of, and thereby should generate insights into, anatomical and functional properties of complicated neural pathways, and we recommend this approach to clarify enigmatic properties of these pathways.
• Straight, P., Gignac, P., & Kuenzel, W. (2024). A histological and diceCT-derived 3D reconstruction of the avian visual thalamofugal pathway. Scientific Reports, 14(1). doi:10.1038/s41598-024-58788-z
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  Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.
• Vasquez, T., Gignac, P., Brewer, P., & Gold, M. (2024). Tumour or haemorrhage?: Differential diagnosis of an unknown mass within the brain of a budgerigar (Melopsittacus undulatus) using a novel imaging pipeline. Veterinary Record Case Reports, 12(3). doi:10.1002/vrc2.899
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  We report on a previously healthy zoo specimen of an adult budgerigar (Melopsittacus undulatus, obtained with permission from Southwick's Zoo) found deceased in its enclosure. To assess cause of death and ensure the absence of an infectious neoplasia, we used an integrated multiscale brain-imaging workflow, previously only used on mammals. The specimen was imaged with microcomputed tomography before and after enhancing soft-tissue contrast with diffusible iodine-based contrast-enhanced microcomputed tomography. Scans revealed an orbital blowout fracture and an unidentified large mass across majority of the diencephalon, striatum and midbrain caudal to the right orbit. After destaining, neural pathohistology confirmed the mass as a brain haemorrhage with no evidence of neoplasia or inflammation. We conclude that this specimen died of head trauma, likely from a head-on collision within its enclosure. This multiscale imaging workflow (diffusible iodine-based contrast-enhanced microcomputed tomography followed by destaining and pathohistology) can improve our evaluation of differential diagnoses in avian specimens.
• Gray, J. A., Gignac, P. M., & Stanley, E. L. (2023). The first full body diffusible iodine‐based contrast‐enhanced computed tomography dataset and teaching materials for a member of the Testudines. The Anatomical Record, 307(3), 535-548. doi:10.1002/ar.25282
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  Diffusible iodine-based contrast-enhanced Computed Tomography (diceCT) is now a widely used technique for imaging metazoan soft anatomy. Turtles present a particular challenge for anatomists; gross dissection is inherently destructive and irreversible, whereas their near complete shell of bony plates, covered with keratinous scutes, presents a barrier for iodine diffusion and significantly increases contrast-enhanced CT preparation time. Consequently, a complete dataset visualizing the internal soft anatomy of turtles at high resolution and in three dimensions has not yet been successfully achieved. Here we outline a novel method that augments traditional diceCT preparation with an iodine injection technique to acquire the first full body contrast-enhanced dataset for the Testudines. We show this approach to be an effective method of staining the soft tissues inside the shell. The resulting datasets were processed to produce anatomical 3D models that can be used in teaching and research. As diceCT becomes a widely employed method for nondestructively documenting the internal soft anatomy of alcohol preserved museum specimens, we hope that methods applicable to the more challenging of these, such as turtles, will contribute toward the growing stock of digital anatomy in online repositories.
• Green, T. L., & Gignac, P. M. (2023). Osteological comparison of casque ontogeny in palaeognathous and neognathous birds: insights for selecting modern analogues in the study of cranial ornaments from extinct archosaurs. Zoological Journal of the Linnean Society, 199(1), 10-25. doi:10.1093/zoolinnean/zlad016
• Straight, P. J., Gignac, P. M., & Kuenzel, W. J. (2023). Mapping the avian visual tectofugal pathway using 3D reconstruction. Journal of Comparative Neurology, 532(2). doi:10.1002/cne.25558
  More info

  Image processing in amniotes is usually accomplished by the thalamofugal and/or tectofugal visual systems. In laterally eyed birds, the tectofugal system dominates with functions such as color and motion processing, spatial orientation, stimulus identification, and localization. This makes it a critical system for complex avian behavior. Here, the brains of chicks, Gallus gallus, were used to produce serial brain sections in either coronal, sagittal, or horizontal planes and stained with either Nissl and Gallyas silver myelin or Luxol fast blue stain and cresyl echt violet (CEV). The emerging techniques of diffusible iodine-based contrast-enhanced computed tomography (diceCT) coupled with serial histochemistry in three planes were used to generate a comprehensive three-dimensional (3D) model of the avian tectofugal visual system. This enabled the 3D reconstruction of tectofugal circuits, including the three primary neuronal projections. Specifically, major components of the system included four regions of the retina, layers of the optic tectum, subdivisions of the nucleus rotundus in the thalamus, the entopallium in the forebrain, and supplementary components connecting into or out of this major avian visual sensory system. The resulting 3D model enabled a better understanding of the structural components and connectivity of this complex system by providing a complete spatial organization that occupied several distinct brain regions. We demonstrate how pairing diceCT with traditional histochemistry is an effective means to improve the understanding of, and thereby should generate insights into, anatomical and functional properties of complicated neural pathways, and we recommend this approach to clarify enigmatic properties of these pathways.
• Watanabe, A., Marshall, S. S., & Gignac, P. M. (2023). Dumbbell‐shaped brains of Polish crested chickens as a model system for the evolution of novel brain morphologies. Journal of Anatomy, 243(3), 421-430. doi:10.1111/joa.13883
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  The evolutionary history of vertebrates is replete with emergence of novel brain morphologies, including the origin of the human brain. Existing model organisms and toolkits for investigating drivers of neuroanatomical innovations have largely proceeded on mammals. As such, a compelling non-mammalian model system would facilitate our understanding of how unique brain morphologies evolve across vertebrates. Here, we present the domestic chicken breed, white crested Polish chickens, as an avian model for investigating how novel brain morphologies originate. Most notably, these crested chickens exhibit cerebral herniation from anterodorsal displacement of the telencephalon, which results in a prominent protuberance on the dorsal aspect of the skull. We use a high-density geometric morphometric approach on cephalic endocasts to characterize their brain morphology. Compared with standard white Leghorn chickens (WLCs) and modern avian diversity, the results demonstrate that crested chickens possess a highly variable and unique overall brain configuration. Proportional sizes of neuroanatomical regions are within the observed range of extant birds sampled in this study, but Polish chickens differ from WLCs in possessing a relatively larger cerebrum and smaller cerebellum and medulla. Given their accessibility, phylogenetic proximity, and unique neuroanatomy, we propose that crested breeds, combined with standard chickens, form a promising comparative system for investigating the emergence of novel brain morphologies.
• Feldman, K., O'Keefe, Y., Gignac, P., & O'Brien, H. (2022). Highest resolution microCT scan of the human brainstem reveals putative anatomical basis for infrequency of medial medullary syndrome. NeuroImage: Clinical, 36(Issue). doi:10.1016/j.nicl.2022.103272
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  Ischemic strokes affecting the medial medulla are exceedingly rare. The anatomical basis for the relative infrequency of this stroke syndrome has been largely uninvestigated due to historically coarse MRI and CT scan resolution. We capture and digitally dissect the highest-ever-resolution diffusible iodine-based contrast-enhanced CT (diceCT) scanned images of a cadaveric brainstem to map arterial territories implicated in medial medullary infarctions. 3D reconstructions show that within the anterior spinal artery territory previously implicated in medial medullary syndrome (MMS), there are numerous, small sulcal artery branches perforating the medulla within the anterior median fissure. These branches proceed in parallel through the anteroposterior depth of the medulla as expected; however, we also identify a network of intraparenchymal, rostrocaudal anastomoses between these sulcal perforating branches. This network of intraparenchymal sulcal artery anastomoses has never been described and may provide a significant collateral supply of oxygenated blood flow throughout the medial medulla. By ramifying deeper tissues, these anastomoses can help explain the infrequency of MMS.
• Gignac, P., Smaers, J., & O'Brien, H. (2022). Unexpected bite-force conservatism as a stable performance foundation across mesoeucrocodylian historical diversity. Anatomical Record, 305(10). doi:10.1002/ar.24768
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  Effective interpretation of historical selective regimes requires comprehensive in vivo performance evaluations and well-constrained ecomorphological proxies. The feeding apparatus is a frequent target of such evolutionary studies due to a direct relationship between feeding and survivorship, and the durability of craniodental elements in the fossil record. Among vertebrates, behaviors such as bite force have been central to evaluation of clade dynamics; yet, in the absence of detailed performance studies, such evaluations can misidentify potential selective factors and their roles. Here, we combine the results of a total-clade performance study with fossil-inclusive, phylogenetically informed methods to assess bite-force proxies throughout mesoeucrocodylian evolution. Although bite-force shifts were previously thought to respond to changing rostrodental selective regimes, we find body-size dependent conservation of performance proxies throughout the history of the clade, indicating stabilizing selection for bite-force potential. Such stasis reveals that mesoeucrocodylians with dietary ecologies as disparate as herbivory and hypercarnivory maintain similar bite-force-to-body-size relationships, a pattern which contrasts the precept that vertebrate bite forces should vary most strongly by diet. Furthermore, it may signal that bite-force conservation supported mesoeucrocodylian craniodental disparity by providing a stable performance foundation for the exploration of novel ecomorphospace.
• Steppan, S., Meyer, A., Barrow, L., Alhajeri, B., Al-Zaidan, A., Gignac, P., & Erickson, G. (2022). Phylogenetics and the evolution of terrestriality in mudskippers (Gobiidae: Oxudercinae). Molecular Phylogenetics and Evolution, 169(Issue). doi:10.1016/j.ympev.2022.107416
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  The initial vertebrate conquest of land by stegocephalians (Sarcopterygia) allowed access to new resources and exploitation of untapped niches precipitating a major phylogenetic diversification. However, a paucity of fossils has left considerable uncertainties about phylogenetic relationships and the eco-morphological stages in this key transition in Earth history. Among extant actinopterygians, three genera of mudskippers (Gobiidae: Oxudercinae), Boleophthalmus, Periophthalmus and Periophthalmodon are the most terrestrialized, with vertebral, appendicular, locomotory, respiratory, and epithelial specializations enabling overland excursions up to 14 h. Unlike early stegocephalians, the ecologies and morphologies of the 45 species of oxudercines are well known, making them viable analogs for the initial vertebrate conquest of land. Nevertheless, they have received little phylogenetic attention. We compiled the largest molecular dataset to date, with 29 oxudercine species, and 5 nuclear and mitochondrial loci. Phylogenetic and comparative analyses revealed strong support for two independent terrestrial transitions, and a complex suit of ecomorphological forms in estuarine environments. Furthermore, neither Oxudercinae nor their presumed sister-group the eel gobies (Amblyopinae, a group of elongated gobies) were monophyletic with respect to each other, requiring a merging of these two subfamilies and revealing an expansion of phenotypic variation within the “mudskipper” clade. We did not find support for the expected linear model of ecomorphological and locomotory transition from fully aquatic, to mudswimming, to pectoral-aided mudswimming, to lobe-finned terrestrial locomotion proposed by earlier morphological studies. This high degree of convergent or parallel transitions to terrestriality, and apparent divergent directions of estuarine adaptation, promises even greater potential for this clade to illuminate the conquest of land. Future work should focus on these less-studied species with “transitional” and other mud-habitat specializations to fully resolve the dynamics of this diversification.
• Gignac, P., Green, T., Oehler, D., Malatos, J., Hollinger, C., & Paré, J. (2021). Diffusible iodine-based contrast-enhanced computed tomography as a necropsy aid: A case report evaluating respiratory disease in macrocephalon maleo. Journal of Zoo and Wildlife Medicine, 52(1). doi:10.1638/2020-0086
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  Abstract: This study describes the novel use of diffusible iodine-based contrast-enhanced computed tomography (diceCT) as a digital necropsy aid. DiceCT was used postmortem to evaluate the cause of progressive respiratory disease in a juvenile maleo (Macrocephalon maleo). The technique facilitated soft-Tissue contrast and a three-dimensional investigation of sinus and choanal anatomy as a means to identify normal and pathologic morphologies. Results showed right-sided narial occlusion by mucoid debris, along with left-sided choanal stenosis caused by osteomyelitis and reactive bone formation. The high spatial resolution afforded by diceCT enabled targeted histology and quantification of the clinical impact of pathologies, which contributed to an effective 60% loss in nasal airway aperture for this individual. This study demonstrates how adding diceCT to traditional necropsy can proffer additional understanding of an individual's pathology, and the resulting data can enhance research programs in vertebrate anatomy, evolution, and health.
• Gignac, P., Sanchez, J., Vazquez-Sanroman, D., & O’Brien, H. (2021). Multiscale imaging of the rat brain using an integrated diceCT and histology workflow. Brain Structure and Function, 226(7). doi:10.1007/s00429-021-02316-6
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  Advancements in tissue visualization techniques have spurred significant gains in the biomedical sciences by enabling researchers to integrate their datasets across anatomical scales. Of particular import are techniques that enable the interpolation of multiple hierarchical scales in samples taken from the same individuals. In this study, we demonstrate that two-dimensional histology techniques can be employed on neural tissues following three-dimensional diffusible iodine-based contrast-enhanced computed tomography (diceCT) without causing tissue degradation. This represents the first step toward a multiscale pipeline for brain visualization. We studied brains from adolescent male Sprague–Dawley rats, comparing experimental (diceCT-stained then de-stained) to control (without diceCT) brains to examine neural tissues for immunolabeling integrity, compare somata sizes, and distinguish neurons from glial cells within the telencephalon and diencephalon. We hypothesized that if experimental and control samples do not differ significantly in morphological cell analysis, then brain tissues are robust to the chemical, temperature, and radiation environments required for these multiple, successive imaging protocols. Visualizations for experimental brains were first captured via micro-computed tomography scanning of isolated, iodine-infused specimens. Samples were then cleared of iodine, serially sectioned, and prepared again using immunofluorescent, fluorescent, and cresyl violet labeling, followed by imaging with confocal and light microscopy, respectively. Our results show that many neural targets are resilient to diceCT imaging and compatible with downstream histological staining as part of a low-cost, multiscale brain imaging pipeline.
• Green, T., & Gignac, P. (2021). Osteological description of casque ontogeny in the southern cassowary (Casuarius casuarius) using micro-CT imaging. Anatomical Record, 304(3). doi:10.1002/ar.24477
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  Extant cassowaries (Casuarius) are unique flightless birds found in the tropics of Indo-Australia. They have garnered substantial attention from anatomists with focus centered on the bony makeup and function of their conspicuous cranial casques, located dorsally above the orbits and neurocranium. The osteological patterning of the casque has been formally described previously; however, there are differing interpretations between authors. These variable descriptions suggest that an anatomical understanding of casque anatomy and its constituent elements may be enhanced by developmental studies aimed at further elucidating this bizarre structure. In the present study, we clarify casque osteology of the southern cassowary (C. casuarius) by detailing casque anatomy across an extensive growth series for the first time. We used micro-computed tomography (μCT) imaging to visualize embryonic development and post-hatching ontogeny through adulthood. We also sampled closely related emus (Dromaius novaehollandiae) and ostriches (Struthio camelus) to provide valuable comparative context. We found that southern cassowary casques are comprised of three paired (i.e., nasals, lacrimals, frontals) and two unpaired elements (i.e., mesethmoid, median casque element). Although lacrimals have rarely been considered as casque elements, the contribution to the casque structure was evident in μCT images. The median casque element has often been cited as a portion of the mesethmoid. However, through comparisons between immature C. casuarius and D. novaehollandiae, we document the median casque element as a distinct unit from the mesethmoid.
• To, K., O'brien, H., Stocker, M., & Gignac, P. (2021). Cranial Musculoskeletal Description of Black-Throated Finch (Aves: Passeriformes: Estrildidae) with DiceCT. Integrative Organismal Biology, 3(1). doi:10.1093/iob/obab007
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  Dietary requirements and acquisition strategies change throughout ontogeny across various clades of tetrapods, including birds. For example, birds hatch with combinations of various behavioral, physiological, and morphological factors that place them on an altricial-precocial spectrum. Passeriformes (=songbirds) in particular, a family constituting approximately more than half of known bird species, displays the most drastic difference between hatchling and adults in each of these aspects of their feeding biology. How the shift in dietary resource acquisition is managed during ontogeny alongside its relationship to the morphology of the feeding apparatus has been largely understudied within birds. Such efforts have been hampered partly due to the small size of many birds and the diminutive jaw musculature they employ. In this study, we used standard and diffusible iodine-based contrast-enhanced computed tomography in conjunction with digital dissection to quantify and describe the cranial musculature of the Black-throated Finch (Poephila cincta) at fledgling and adult stages. Our results reveal that in both the fledgling and the adult, cranial musculature shows clear and complex partitioning in the Musculus adductor mandibulae externus that is consistent with other families within Passeriformes. We quantified jaw-muscle sizes and found that the adult showed a decrease in muscle mass in comparison to the fledgling individual. We propose that this could be the result of low sample size or a physiological effect of parental care in Passeriformes. Our study shows that high-resolution visualization techniques are informative at revealing morphological discrepancies for studies that involve small specimens such as Passeriformes especially with careful specimen selection criteria.
• Watanabe, A., Balanoff, A., Gignac, P., Gold, M., & Norell, M. (2021). Novel neuroanatomical integration and scaling define avian brain shape evolution and development. eLife, 10(Issue). doi:10.7554/elife.68809
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  How do large and unique brains evolve? Historically, comparative neuroanatomical studies have attributed the evolutionary genesis of highly encephalized brains to deviations along, as well as from, conserved scaling relationships among brain regions. However, the relative contributions of these concerted (integrated) and mosaic (modular) processes as drivers of brain evolution remain unclear, especially in non-mammalian groups. While proportional brain sizes have been the predominant metric used to characterize brain morphology to date, we perform a high-density geometric morphometric analysis on the encephalized brains of crown birds (Neornithes or Aves) compared to their stem taxa—the non-avialan coelurosaurian dinosaurs and Archaeopteryx. When analyzed together with developmental neuroanatomical data of model archosaurs (Gallus, Alligator), crown birds exhibit a distinct allometric relationship that dictates their brain evolution and development. Furthermore, analyses by neuroanatomical regions reveal that the acquisition of this derived shape-to-size scaling relationship occurred in a mosaic pattern, where the avian-grade optic lobe and cerebellum evolved first among non-avialan dinosaurs, followed by major changes to the evolutionary and developmental dynamics of cerebrum shape after the origin of Avialae. Notably, the brain of crown birds is a more integrated structure than non-avialan archosaurs, implying that diversification of brain morphologies within Neornithes proceeded in a more coordinated manner, perhaps due to spatial constraints and abbreviated growth period. Collectively, these patterns demonstrate a plurality in evolutionary processes that generate encephalized brains in archosaurs and across vertebrates.
• Ksepka, D., Balanoff, A., Smith, N., Bever, G., Bhullar, B., Bourdon, E., Braun, E., Burleigh, J., Clarke, J., Colbert, M., Corfield, J., Degrange, F., De Pietri, V., Early, C., Field, D., Gignac, P., Gold, M., Kimball, R., Kawabe, S., , Lefebvre, L., et al. (2020). Tempo and Pattern of Avian Brain Size Evolution. Current Biology, 30(11). doi:10.1016/j.cub.2020.03.060
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  Relative brain sizes in birds can rival those of primates, but large-scale patterns and drivers of avian brain evolution remain elusive. Here, we explore the evolution of the fundamental brain-body scaling relationship across the origin and evolution of birds. Using a comprehensive dataset sampling> 2,000 modern birds, fossil birds, and theropod dinosaurs, we infer patterns of brain-body co-variation in deep time. Our study confirms that no significant increase in relative brain size accompanied the trend toward miniaturization or evolution of flight during the theropod-bird transition. Critically, however, theropods and basal birds show weaker integration between brain size and body size, allowing for rapid changes in the brain-body relationship that set the stage for dramatic shifts in early crown birds. We infer that major shifts occurred rapidly in the aftermath of the Cretaceous-Paleogene mass extinction within Neoaves, in which multiple clades achieved higher relative brain sizes because of a reduction in body size. Parrots and corvids achieved the largest brains observed in birds via markedly different patterns. Parrots primarily reduced their body size, whereas corvids increased body and brain size simultaneously (with rates of brain size evolution outpacing rates of body size evolution). Collectively, these patterns suggest that an early adaptive radiation in brain size laid the foundation for subsequent selection and stabilization. Ksepka et al. reconstruct brain-body scaling in a study of >2,000 birds and non-avian dinosaurs. Their results show that avian brain size evolution was profoundly impacted by the K-Pg mass extinction, in the aftermath of which many clades achieved larger relative brain sizes via body size reduction.
• Schwab, J., Young, M., Neenan, J., Walsh, S., Witmer, L., Herrera, Y., Allain, R., Brochu, C., Choiniere, J., Clark, J., Dollman, K., Etches, S., Fritsch, G., Gignac, P., Ruebenstahl, A., Sachs, S., Turner, A., Vignaud, P., Wilberg, E., , Xu, X., et al. (2020). Inner ear sensory system changes as extinct crocodylomorphs transitioned from land to water. Proceedings of the National Academy of Sciences of the United States of America, 117(19). doi:10.1073/pnas.2002146117
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  Major evolutionary transitions, in which animals develop new body plans and adapt to dramatically new habitats and lifestyles, have punctuated the history of life. The origin of cetaceans from land-living mammals is among the most famous of these events. Much earlier, during the Mesozoic Era, many reptile groups also moved from land to water, but these transitions are more poorly understood. We use computed tomography to study changes in the inner ear vestibular system, involved in sensing balance and equilibrium, as one of these groups, extinct crocodile relatives called thalattosuchians, transitioned from terrestrial ancestors into pelagic (open ocean) swimmers. We find that the morphology of the vestibular system corresponds to habitat, with pelagic thalattosuchians exhibiting a more compact labyrinth with wider semicircular canal diameters and an enlarged vestibule, reminiscent of modified and miniaturized labyrinths of other marine reptiles and cetaceans. Pelagic thalattosuchians with modified inner ears were the culmination of an evolutionary trend with a long semiaquatic phase, and their pelagic vestibular systems appeared after the first changes to the postcranial skeleton that enhanced their ability to swim. This is strikingly different from cetaceans, which miniaturized their labyrinths soon after entering the water, without a prolonged semiaquatic stage. Thus, thalattosuchians and cetaceans became secondarily aquatic in different ways and at different paces, showing that there are different routes for the same type of transition.
• O'Brien, H., Lynch, L., Vliet, K., Brueggen, J., Erickson, G., & Gignac, P. (2019). Crocodylian head width allometry and phylogenetic prediction of body size in extinct crocodyliforms. Integrative Organismal Biology, 1(1). doi:10.1093/iob/obz006
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  Body size and body-size shifts broadly impact life-history parameters of all animals, which has made accurate body-size estimates for extinct taxa an important component of understanding their paleobiology. Among extinct crocodylians and their precursors (e.g., suchians), several methods have been developed to predict body size from suites of hard-tissue proxies. Nevertheless, many have limited applications due to the disparity of some major suchian groups and biases in the fossil record. Here, we test the utility of head width (HW) as a broadly applicable body-size estimator in living and fossil suchians. We use a dataset of sexually mature male and female individuals (n=76) from a comprehensive sample of extant suchian species encompassing nearly all known taxa (n=22) to develop a Bayesian phylogenetic model for predicting three conventional metrics for size: body mass, snout-vent length, and total length. We then use the model to estimate size parameters for a select series of extinct suchians with known phylogenetic affinity (Montsechosuchus, Diplocynodon, and Sarcosuchus). We then compare our results to sizes reported in the literature to exemplify the utility of our approach for a broad array of fossil suchians. Our results show that HW is highly correlated with all other metrics (all R2≥0.85) and is commensurate with femoral dimensions for its reliably as a body-size predictor. We provide the R code in order to enable other researchers to employ the model in their own research.
• Watanabe, A., Gignac, P., Balanoff, A., Green, T., Kley, N., & Norell, M. (2019). Are endocasts good proxies for brain size and shape in archosaurs throughout ontogeny?. Journal of Anatomy, 234(3). doi:10.1111/joa.12918
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  Cranial endocasts, or the internal molds of the braincase, are a crucial correlate for investigating the neuroanatomy of extinct vertebrates and tracking brain evolution through deep time. Nevertheless, the validity of such studies pivots on the reliability of endocasts as a proxy for brain morphology. Here, we employ micro-computed tomography imaging, including diffusible iodine-based contrast-enhanced CT, and a three-dimensional geometric morphometric framework to examine both size and shape differences between brains and endocasts of two exemplar archosaur taxa – the American alligator (Alligator mississippiensis) and the domestic chicken (Gallus gallus). With ontogenetic sampling, we quantitatively evaluate how endocasts differ from brains and whether this deviation changes during development. We find strong size and shape correlations between brains and endocasts, divergent ontogenetic trends in the brain-to-endocast correspondence between alligators and chickens, and a comparable magnitude between brain–endocast shape differences and intraspecific neuroanatomical variation. The results have important implications for paleoneurological studies in archosaurs. Notably, we demonstrate that the pattern of endocranial shape variation closely reflects brain shape variation. Therefore, analyses of endocranial morphology are unlikely to generate spurious conclusions about large-scale trends in brain size and shape. To mitigate any artifacts, however, paleoneurological studies should consider the lower brain–endocast correspondence in the hindbrain relative to the forebrain; higher size and shape correspondences in chickens than alligators throughout postnatal ontogeny; artificially ‘pedomorphic’ shape of endocasts relative to their corresponding brains; and potential biases in both size and shape data due to the lack of control for ontogenetic stages in endocranial sampling.
• Gignac, P., & Erickson, G. (2017). The Biomechanics behind Extreme Osteophagy in Tyrannosaurus rex. Scientific Reports, 7(1). doi:10.1038/s41598-017-02161-w
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  Most carnivorous mammals can pulverize skeletal elements by generating tooth pressures between occluding teeth that exceed cortical bone shear strength, thereby permitting access to marrow and phosphatic salts. Conversely, carnivorous reptiles have non-occluding dentitions that engender negligible bone damage during feeding. As a result, most reptilian predators can only consume bones in their entirety. Nevertheless, North American tyrannosaurids, including the giant (13 metres [m]) theropod dinosaur Tyrannosaurus rex stand out for habitually biting deeply into bones, pulverizing and digesting them. How this mammal-like capacity was possible, absent dental occlusion, is unknown. Here we analyzed T. rex feeding behaviour from trace evidence, estimated bite forces and tooth pressures, and studied tooth-bone contacts to provide the answer. We show that bone pulverization was made possible through a combination of: (1) prodigious bite forces (8,526-34,522 newtons [N]) and tooth pressures (718-2,974 megapascals [MPa]) promoting crack propagation in bones, (2) tooth form and dental arcade configurations that concentrated shear stresses, and (3) repetitive, localized biting. Collectively, these capacities and behaviors allowed T. rex to finely fragment bones and more fully exploit large dinosaur carcasses for sustenance relative to competing carnivores.
• Balanoff, A., Bever, G., Colbert, M., Clarke, J., Field, D., Gignac, P., Ksepka, D., Ridgely, R., Smith, N., Torres, C., Walsh, S., & Witmer, L. (2016). Best practices for digitally constructing endocranial casts: examples from birds and their dinosaurian relatives. Journal of Anatomy, 229(2). doi:10.1111/joa.12378
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  The rapidly expanding interest in, and availability of, digital tomography data to visualize casts of the vertebrate endocranial cavity housing the brain (endocasts) presents new opportunities and challenges to the field of comparative neuroanatomy. The opportunities are many, ranging from the relatively rapid acquisition of data to the unprecedented ability to integrate critically important fossil taxa. The challenges consist of navigating the logistical barriers that often separate a researcher from high-quality data and minimizing the amount of non-biological variation expressed in endocasts – variation that may confound meaningful and synthetic results. Our purpose here is to outline preferred approaches for acquiring digital tomographic data, converting those data to an endocast, and making those endocasts as meaningful as possible when considered in a comparative context. This review is intended to benefit those just getting started in the field but also serves to initiate further discussion between active endocast researchers regarding the best practices for advancing the discipline. Congruent with the theme of this volume, we draw our examples from birds and the highly encephalized non-avian dinosaurs that comprise closely related outgroups along their phylogenetic stem lineage.
• Gignac, P., & Erickson, G. (2016). Ontogenetic bite-force modeling of Alligator mississippiensis: implications for dietary transitions in a large-bodied vertebrate and the evolution of crocodylian feeding. Journal of Zoology, 299(4). doi:10.1111/jzo.12349
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  Crocodylians undergo substantial increases in size during ontogeny. The American alligator, Alligator mississippiensis, in particular traverses nearly four orders of body mass between hatching and senescence. Accompanying such changes are modifications in rostrodental morphology and feeding capabilities that facilitate major shifts in diet. How such anatomical changes relate to ecological niche occupation across sizes is not well understood. In this study, we focused on the effects of ontogenetic changes on the force-generating mechanisms for jaw closure to assess the impacts of scaling on feeding biomechanics. We developed dissection-based, musculoskeletal models of maximum bite-force generation throughout ontogeny and compared and tested their veracity with data from an A. mississippiensis developmental series, for which bite forces were directly measured. Through examinations of the scaling patterns within the parameters of our models, we discuss how muscle pennation and positive allometry in the American alligator jaw adductor system facilitate capture strategies and oral processing of prey, and contribute to developmental niche shifts in this large-bodied taxon. On the basis of conservation of the crocodylian jaw adductor system, we argue that our findings are broadly applicable to crown Crocodylia and reflect an important, but often overlooked, aspect of the crocodylian feeding ecomorphology: littoral, sit-and-wait predation is enhanced by posteroventrally displaced, exceptionally large, and forceful ventral pterygoideus muscles, in particular. Future studies on the ontogeny and evolution of feeding in crocodylians should not neglect the functional and ecological implications of these muscles' contributions to diet.
• Hughes, D., Walker, E., Gignac, P., Martinez, A., Negishi, K., Lieb, C., Greenbaum, E., & Khan, A. (2016). Rescuing perishable neuroanatomical information from a threatened biodiversity hotspot: Remote field methods for brain tissue preservation validated by cytoarchitectonic analysis, immunohistochemistry, and x-ray microcomputed tomography. PLoS ONE, 11(5). doi:10.1371/journal.pone.0155824
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  Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue. Third, immersion-fixation of lizard brains, prepared under identical environmental conditions, was readily compatible with subsequent iodine-enhanced X-ray microcomputed tomography, which facilitated the non-destructive imaging of the intact brain within its skull. In summary, we have validated multiple approaches to preserving intact lizard brains in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.
• Leone Gold, M., Schulz, D., Budassi, M., Gignac, P., Vaska, P., & Norell, M. (2016). Flying starlings, PET and the evolution of volant dinosaurs. Current Biology, 26(7). doi:10.1016/j.cub.2016.02.025
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  Birds have evolved behavioral and morphological adaptations for powered flight. Many aspects of this transition are unknown, including the neuroanatomical changes that made flight possible [1]. To understand how the avian brain drives this complex behavior, we utilized positron emission tomography (PET) scanning and the tracer 18F-fluorodeoxyglucose (FDG) to document regional metabolic activity in the brain associated with a variety of locomotor behaviors. FDG studies are typically employed in rats [2] though the technology has been applied to birds [3]. We examined whole-brain function in European Starlings (Sturnus vulgaris), trained to fly in a wind tunnel while metabolizing the tracer. Drawing on predictions from early anatomical studies [4], we hypothesized increased metabolic activity in the Wulst and functionally related visual brain regions during flight. We found that flight behaviors correlated positively with entopallia and Wulst activity, but negatively with thalamic activity.
• O'Brien, H. D., Gignac, P. M., Hieronymus, T. L., & Witmer, L. M. (2016). A comparison of postnatal arterial patterns in a growth series of giraffe (Artiodactyla: Giraffa camelopardalis). PeerJ, 2016(Issue 2). doi:10.7717/peerj.1696
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  Nearly all living artiodactyls (even-toed ungulates) possess a derived cranial arterial pattern that is highly distinctive from most other mammals. Foremost among a suite of atypical arterial configurations is the functional and anatomical replacement of the internal carotid artery with an extensive, subdural arterial meshwork called the carotid rete. This interdigitating network branches from the maxillary artery and is housed within the cavernous venous sinus. As the cavernous sinus receives cooled blood draining from the nasal mucosa, heat rapidly dissipates across the high surface area of the rete to be carried away from the brain by the venous system. This combination yields one of the most effective mechanisms of selective brain cooling. Although arterial development begins from the same embryonic scaffolding typical of mammals, possession of a rete is typically accompanied by obliteration of the internal carotid artery. Among taxa with available ontogenetic data, the point at which the internal carotid obliterates is variable throughout development. In small-bodied artiodactyls, the internal carotid typically obliterates prior to parturition, but in larger species, the vessel may remain patent for several years. In this study, we use digital anatomical data collection methods to describe the cranial arterial patterns for a growth series of giraffe (Giraffa camelopardalis), from parturition to senescence. Giraffes, in particular, have unique cardiovascular demands and adaptations owing to their exceptional body form and may not adhere to previously documented stages of cranial arterial development. We find the carotid arterial system to be conserved between developmental stages and that obliteration of the giraffe internal carotid artery occurs prior to parturition.
• Gignac, P., & Erickson, G. (2015). Ontogenetic changes in dental form and tooth pressures facilitate developmental niche shifts in American alligators. Journal of Zoology, 295(2). doi:10.1111/jzo.12187
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  Between hatching and late adulthood American alligators Alligator mississippiensis show up to 7000-fold increases in body mass. Concurrent with such changes in body size are absolute and relative modifications in rostral proportions, dental form, feeding capacities and dietary preferences. How these major anatomical changes accommodate prey-resource shifts is poorly understood. In this study, we focus on the effects of ontogenetic changes in bite-force capacities and dental form to address how these factors relate to tooth-pressure generation and diet. We derive absolute values of tooth pressure along the crowns of the most prominent teeth (the first documentation of tooth pressures throughout ontogeny and after initial tooth contact for any animal) and show that these pressures increase with positive allometry during ontogeny. In addition, we discuss how American alligator tooth-pressure values explain their capacities for seizure and oral processing of typical prey, and how tooth-pressure changes facilitate developmental niche shifts in this large-bodied taxon.
• Erickson, G., Gignac, P., Lappin, A., Vliet, K., Brueggen, J., & Webb, G. (2014). A comparative analysis of ontogenetic bite-force scaling among Crocodylia. Journal of Zoology, 292(1). doi:10.1111/jzo.12081
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  Interspecific adult bite forces for all extant crocodylian species are now known. However, how bite forces scale during ontogeny across the clade has yet to be studied. Here we test the hypotheses that extant crocodylians share positively allometric and statistically comparable developmental scaling coefficients for maximal bite-force capacity relative to body size. To do this, we measured bite forces in the Australian freshwater crocodile Crocodylus johnsoni and the Saltwater crocodile C.porosus, and determined how performance changed during ontogeny. We statistically compared these results with those for the American alligator Alligator mississippiensis using 95% prediction intervals and interpreted our findings in a phylogenetic context. We found no observable taxon-specific shifts in the intraspecific scaling of biomechanical performance. Instead, all bite-force values in our crocodylid dataset fell within the bounds of the A.mississippiensis 95% prediction intervals, suggesting similar bite-force capacity when same-sized individuals are compared. This holds true regardless of differences in developmental stage, potential adult body size, rostro-dental form, bone mineralization, cranial suturing, dietary differences or phylogenetic relatedness. These findings suggest that intraspecific bite-force scaling for crocodylians with feeding ecologies comparable with those of extant forms has likely remained evolutionarily static during their diversification. copy; 2013 The Zoological Society of London.
• Gignac, P., & Kley, N. (2014). Iodine-enhanced micro-CT imaging: Methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 322(3). doi:10.1002/jez.b.22561
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  The now widespread use of non-destructive X-ray computed tomography (CT) and micro-CT (μCT) has greatly augmented our ability to comprehensively detail and quantify the internal hard-tissue anatomy of vertebrates. However, the utility of X-ray imaging for gaining similar insights into vertebrate soft-tissue anatomy has yet to be fully realized due to the naturally low X-ray absorption of non-mineralized tissues. In this study, we show how a wide diversity of soft-tissue structures within the vertebrate head-including muscles, glands, fat deposits, perichondria, dural venous sinuses, white and gray matter of the brain, as well as cranial nerves and associated ganglia-can be rapidly visualized in their natural relationships with extraordinary levels of detail using iodine-enhanced (i-e) μCT imaging. To date, Lugol's iodine solution (I2KI) has been used as a contrast agent for μCT imaging of small invertebrates, vertebrate embryos, and certain isolated parts of larger, post-embryonic vertebrates. These previous studies have all yielded promising results, but visualization of soft tissues in smaller invertebrate and embryonic vertebrate specimens has generally been more complete than that for larger, post-embryonic vertebrates. Our research builds on these previous studies by using high-energy μCT together with more highly concentrated I2KI solutions and longer staining times to optimize the imaging and differentiation of soft tissues within the heads of post-embryonic archosaurs (Alligator mississippiensis and Dromaius novaehollandiae). We systematically quantify the intensities of tissue staining, demonstrate the range of anatomical structures that can be visualized, and generate a partial three-dimensional reconstruction of alligator cephalic soft-tissue anatomy. © 2014 Wiley Periodicals, Inc.
• Erickson, G., Gignac, P., Steppan, S., Lappin, A., Vliet, K., Brueggen, J., Inouye, B., Kledzik, D., & Webb, G. (2012). Insights into the ecology and evolutionary success of crocodilians revealed through bite-force and tooth-pressure experimentation. PLoS ONE, 7(3). doi:10.1371/journal.pone.0031781
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  Background: Crocodilians have dominated predatory niches at the water-land interface for over 85 million years. Like their ancestors, living species show substantial variation in their jaw proportions, dental form and body size. These differences are often assumed to reflect anatomical specialization related to feeding and niche occupation, but quantified data are scant. How these factors relate to biomechanical performance during feeding and their relevance to crocodilian evolutionary success are not known. Methodology/Principal Findings: We measured adult bite forces and tooth pressures in all 23 extant crocodilian species and analyzed the results in ecological and phylogenetic contexts. We demonstrate that these reptiles generate the highest bite forces and tooth pressures known for any living animals. Bite forces strongly correlate with body size, and size changes are a major mechanism of feeding evolution in this group. Jaw shape demonstrates surprisingly little correlation to bite force and pressures. Bite forces can now be predicted in fossil crocodilians using the regression equations generated in this research. Conclusions/Significance: Critical to crocodilian long-term success was the evolution of a high bite-force generating musculo-skeletal architecture. Once achieved, the relative force capacities of this system went essentially unmodified throughout subsequent diversification. Rampant changes in body size and concurrent changes in bite force served as a mechanism to allow access to differing prey types and sizes. Further access to the diversity of near-shore prey was gained primarily through changes in tooth pressure via the evolution of dental form and distributions of the teeth within the jaws. Rostral proportions changed substantially throughout crocodilian evolution, but not in correspondence with bite forces. The biomechanical and ecological ramifications of such changes need further examination. © 2012 Erickson et al.
• Pfaller, J., Gignac, P., & Erickson, G. (2011). Erratum: Ontogenetic changes in jaw-muscle architecture facilitate durophagy in the turtle Sternotherus minor (The Journal of Experimental Biology 214 (1655-1667)). Journal of Experimental Biology, 214(11). doi:10.1242/jeb059493
• Pfaller, J., Gignac, P., & Erickson, G. (2011). Ontogenetic changes in jaw-muscle architecture facilitate durophagy in the turtle Sternotherus minor. Journal of Experimental Biology, 214(10). doi:10.1242/jeb.048090
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  Differential scaling of musculoskeletal traits leads to differences in performance across ontogeny and ultimately determines patterns of resource use during development. Because musculoskeletal growth of the feeding system facilitates high bite-force generation necessary to overcome the physical constraints of consuming more durable prey, durophagous taxa are well suited for investigations of the scaling relationships between musculoskeletal growth, bite-force generation and dietary ontogeny. To elucidate which biomechanical factors are responsible for allometric changes in bite force and durophagy, we developed and experimentally tested a static model of bite-force generation throughout development in the durophagous turtle Sternotherus minor. Moreover, we quantified the fracture properties of snails found in the diet to evaluate the relationship between bite force and the forces required to process durable prey. We found that (1) the static bite-force model accurately predicts the ontogenetic scaling of bite forces, (2) biteforce positive allometry is accomplished by augmenting muscle size and muscle pennation, and (3) the rupture forces of snails found in the diet show a similar scaling pattern to bite force across ontogeny. These results indicate the importance of muscle pennation for generating high bite forces while maintaining muscle size and provide empirical evidence that the allometric patterns of musculoskeletal growth in S. minor are strongly linked to the structural properties of their primary prey. © 2011.
• Gignac, P., Makovicky, P., Erickson, G., & Walsh, R. (2010). A description of Deinonychus antirrhopus bite marks and estimates of bite force using tooth indentation simulations. Journal of Vertebrate Paleontology, 30(4). doi:10.1080/02724634.2010.483535
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  We report the discovery of a specimen of Tenontosaurus tilletti from the Cloverly Formation that bears lesions we interpret as bite marks of Deinonychus antirrhopus. Some of the bite marks are in the form of exceptionally deep punctures through the long bone cortices. These provide a rare opportunity to estimate the bite-force capacities of this taxon through tooth indentation simulations. These experiments showed that approximately 4100 N of bite force were required to generate one of the bite marks, and 8200 N would have been generated simultaneously at a distal-most tooth position. These values are higher than those reported for large carnivoran mammals but similar to values recorded for comparably sized crocodilians. Although current evidence does not indicate how D. antirrhopus actually used its claws and teeth to acquire prey resources, it is clear that large individuals were capable of generating forces great enough to bite through bone. © 2010 by the Society of Vertebrate Paleontology.
• Pfaller, J., Herrera, N., Gignac, P., & Erickson, G. (2010). Ontogenetic scaling of cranial morphology and bite-force generation in the loggerhead musk turtle. Journal of Zoology, 280(3). doi:10.1111/j.1469-7998.2009.00660.x
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  The feeding systems of durophagous vertebrates are well suited for studying how the performance of feeding structures is affected by growth. For these animals, feeding structures that deviate from isometric growth (i.e. allometry) may be biologically meaningful in terms of disproportionate increases in bite-force generation across ontogeny. We measured body size, cranial morphology and bite-force generation in an ontogenetic series of loggerhead musk turtles Sternotherus minor and compared the scaling coefficients with predictions based on isometry. We found that morphological growth in S. minor is characterized by positive allometry in the dimensions of the head and beak (rhamphotheca) relative to body and head size. Because negative allometry or isometry in head size relative to body size is a nearly universal trait among vertebrates, S. minor appears to be unique in this regard. In addition, we found that bite forces scaled with positive allometry relative to nearly all morphological measurements. These results suggest that modified lever mechanics, and/or increased physiological cross-sectional area through changes in muscle architecture (i.e. fiber lengths, degree of pennation) of the jaw adductor musculature may have more explanatory power for bite-force generation than external head measures in this taxon. Lastly, we found that bite force scaled with negative allometry relative to lower beak depth and symphyseal length, indicating that the development of high bite forces requires a disproportionately more robust mandible. These results indicate how deviations from isometric growth may make it possible for durophagous vertebrates to generate, transfer and dissipate mechanical forces during growth. © 2009 The Authors. Journal compilation © 2009 The Zoological Society of London.
• Hoekman, D., Terhorst, C., Bauer, A., Braun, S., Gignac, P., Hopkins, R., Joshi, S., Laskis, K., Sanscrainte, N., Travis, J., & Miller, T. (2007). Oviposition decreased in response to enriched water: A field study of the pitcher-plant mosquito, Wyeomyia smithii. Ecological Entomology, 32(1). doi:10.1111/j.1365-2311.2006.00840.x
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  1. Environmental cues are known to influence oviposition behaviour in mosquitoes, with important consequences for larval survival and insect population dynamics. Enriched microhabitats have been shown to be preferred oviposition sites. 2. In a field experiment designed to determine whether ovipositing mosquitoes are sensitive to different levels of nutrient enrichment, new pitcher-plant (Sarracenia purpurea) leaves were opened and enriched with 0, 2, or 20 dead ants, and the number of pitcher-plant mosquito (Wyeomyia smithii) larvae resulting from subsequent oviposition were measured. 3. Oviposition rates were higher in leaves with low levels of enrichment (0 and 2 ants per leaf), although larval development was enhanced at the highest enrichment level. 4. Results suggest that, although these mosquito larvae are nutrient limited, ovipositing females preferentially avoid highly enriched leaves. This counterintuitive result may be due to low oxygen concentrations or a masked cue in enriched leaves, and contrasts with other oviposition studies. © 2007 The Royal Entomological Society.
• Prieto-Marquez, A., Gignac, P., & Joshi, S. (2007). Neontological evaluation of pelvic skeletal attributes purported to reflect sex in extinct non-avian archosaurs. Journal of Vertebrate Paleontology, 27(3). doi:10.1671/0272-4634(2007)27[603:NEOPSA]2.0.CO;2
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  Sex in non-avian archosaurs has been inferred using a variety of osteological attributes. However, little quantitative data have been presented showing that these phenotypes truly exist. In this study, testing for the presence of pelvic osteological correlates of sex in extant archosaurs was conducted, using skeletons of wild-caught A. mississippiensis as a neontological model. For outgroup comparison, the squamate Iguana iguana is included. A sample of 16 females and 19 males of A. mississippiensis, and 18 females and 10 males of I. iguana were examined. Measurements included pelvic canal area, dorsoventral depth, and mediolateral width of the pelvic canal, mediolateral width between the dorsal edge of each ilium, and ischium orientation. These data were analyzed using analyses of covariance, a t-test, and a recently developed geodesic distance shape analysis. Results indicate that there is sexual dimorphism in the proportions of the pelvic canal in A. mississippiensis, with females typically having deeper pelvic canals than males. This dimorphism might be synapomorphic for Archosauria. No dimorphism was found in I. iguana. The detection of dimorphism in A. mississipiensis required large sample sizes owing to substantial overlap between sexes. Thus, sexing isolated specimens using this metric is tenuous at best. Assuming similar variance in the relative pelvic depth versus width in other non-avian archosaurs, this criterion would also produce imprecise determinations of sex for these taxa. © 2007 by the Society of Vertebrate Paleontology.