Augmenting Trait-Dependent Diversification Estimations with Fossil Evidence: A Case Study Using Osmoregulatory Neurovasculature.

When comparative neuromorphological studies are extended into evolutionary contexts, traits of interest are often linked to diversification patterns. Features demonstrably associated with increases in diversification rates and the infiltration or occupation of novel niche spaces are often termed "key innovations." Within the past decade, phylogenetically informed methods have been developed to test key innovation hypotheses and evaluate the influence these traits have had in shaping modern faunas. This is primarily accomplished by estimating state-dependent speciation and extinction rates. These methods have important caveats and guidelines related to both calculation and interpretation, which are necessary to understand in cases of discrete (qualitative) character analysis, as can be common when studying the evolution of neuromorphology. In such studies, inclusion of additional characters, acknowledgement of character codistribution, and addition of sister clade comparison should be explored to ensure model accuracy. Even so, phylogenies provide a survivor-only examination of character evolution, and paleontological contexts may be necessary to replicate and confirm results. Here, I review these issues in the context of selective brain cooling - a neurovascular-mediated osmoregulatory physiology that dampens hypothalamic responses to heat stress and reduces evaporative water loss in large-bodied mammals. This binary character provides an example of the interplay between sample size, evenness, and character codistribution. Moreover, it allows for an opportunity to compare phylogenetically constrained results with paleontological data, augmenting survivor-only analyses with observable extinction patterns. This trait- dependent diversification example indicates that selective brain cooling is significantly associated with the generation of modern large-mammal faunas. Importantly, paleontological data validate phylogenetic patterns and demonstrate how suites of characters worked in concert to establish the large-mammal communities of today.

Physical and computational fluid dynamics models for the hemodynamics of the artiodactyl carotid rete.

In the mammalian order Artiodactyla, the majority of arterial blood entering the intracranial cavity is supplied by a large arterial meshwork called the carotid rete. This vascular structure functionally replaces the internal carotid artery. Extensive experimentation has demonstrated that the artiodactyl carotid rete drives one of the most effective selective brain cooling mechanisms among terrestrial vertebrates. Less well understood is the impact that the unique morphology of the carotid rete may have on the hemodynamics of blood flow to the cerebrum. It has been hypothesized that, relative to the tubular internal carotid arteries of most other vertebrates, the highly convoluted morphology of the carotid rete may increase resistance to flow during extreme changes in cerebral blood pressure, essentially protecting the brain by acting as a resistor. We test this hypothesis by employing simple and complex physical models to a 3D surface rendering of the carotid rete of the domestic goat, Capra hircus. First, we modeled the potential for increased resistance across the carotid rete using an electrical circuit analog. The extensive branching of the rete equates to a parallel circuit that is bound in series by single tubular arteries, both upstream and downstream. This method calculated a near-zero increase in resistance across the rete. Because basic equations do not incorporate drag, shear-stress, and turbulence, we used computational fluid dynamics to simulate the impact of these computationally intensive factors on resistance. Ultimately, both simple and complex models demonstrated negligible changes in resistance and blood pressure across the arterial meshwork. We further tested the resistive potential of the carotid rete by simulating blood pressures known to occur in giraffes. Based on these models, we found resistance (and blood pressure mitigation as a whole) to be an unlikely function for the artiodactyl carotid rete.

A foundational description of Antilocapra americana pronghorn core osteohistology.

Cranial bony projections ("headgear") have diverse forms and functions, such as defense, species recognition, mate selection, and thermoregulation. Most commonly, they are associated with the artiodactyl infraorder, Pecora. All pecoran headgear-antlers, horns, ossicones, and pronghorns-are osseous protrusions of the frontal or parietal bone with an integumentary covering, although there is taxonomic, developmental, and compositional variation. However, compared with other pecorans, there is a dearth of literature addressing extant antilocaprids-Antilocapra americana. This study provides a foundational osteohistological description of A. americana pronghorn cores in order to start building a framework to better understand the complex interplay among microanatomy, development, behavior, environment, and phylogenetic history of pronghorn headgear. Osteohistological analysis of adult A. americana pronghorn cores reveal the inner medullary region is composed of trabecular bone. Based on similar studies in bovids, we propose that these trabeculae may function to reduce the effects of repeated loading incurred by intraspecific combat. The deep aspect of the outer region was found to be composed of compacted coarse cancellous bone and primary bone remodeled to dense Haversian bone, in both male and female specimens, respectively, and superficially composed of highly vascularized fibrolamellar bone. The presence of fibrolamellar bone may indicate that the bone is fast-growing, and its presence at the periosteal surface suggests protracted growth of the pronghorn core beyond sexual maturity.

Unexpected bite-force conservatism as a stable performance foundation across mesoeucrocodylian historical diversity.

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.

Highest resolution microCT scan of the human brainstem reveals putative anatomical basis for infrequency of medial medullary syndrome.

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.

The earliest Pleistocene record of a large-bodied hominin from the Levant supports two out-of-Africa dispersal events.

The paucity of early Pleistocene hominin fossils in Eurasia hinders an in-depth discussion on their paleobiology and paleoecology. Here we report on the earliest large-bodied hominin remains from the Levantine corridor: a juvenile vertebra (UB 10749) from the early Pleistocene site of 'Ubeidiya, Israel, discovered during a reanalysis of the faunal remains. UB 10749 is a complete lower lumbar vertebral body, with morphological characteristics consistent with Homo sp. Our analysis indicates that UB-10749 was a 6- to 12-year-old child at death, displaying delayed ossification pattern compared with modern humans. Its predicted adult size is comparable to other early Pleistocene large-bodied hominins from Africa. Paleobiological differences between UB 10749 and other early Eurasian hominins supports at least two distinct out-of-Africa dispersal events. This observation corresponds with variants of lithic traditions (Oldowan; Acheulian) as well as various ecological niches across early Pleistocene sites in Eurasia.

Appendicular skeletal morphology of North American Martes reflect independent modes of evolution in conjunction with Pleistocene glacial cycles.

Pleistocene glacial cycles are thought to have driven ecological niche shifts, including novel niche formation. North American pine martens, Martes americana and M. caurina, are exemplar taxa thought to have diverged molecularly and morphologically during Pleistocene glaciation. Previous research found correlations between Martes limb morphology with biome and climate, suggesting that appendicular evolution may have occurred via adaptation to selective pressures imposed by novel and shifting habitats. Such variation can also be achieved through non-adaptive means such as genetic drift. Here, we evaluate whether regional genetic differences reflect limb morphology differences among populations of M. americana and M. caurina by analyzing evolutionary tempo and mode of six limb elements. Our comparative phylogenetic models indicate that genetic structure predicts limb shape better than size. Marten limb size has low phylogenetic signal, and the best supported model of evolution is punctuational (kappa), with morphological and genetic divergence occurring simultaneously. Disparity through time analysis suggests that the tempo of limb evolution in Martes tracks Pleistocene glacial cycles, such that limb size may be responding to shifting climates rather than population genetic structure. Contrarily, we find that limb shape is strongly tied to genetic relationships, with high phylogenetic signal and a lambda mode of evolution. Overall, this pattern of limb size and shape variation may be the result of geographic isolation during Pleistocene glacial advance, while declines in disparity suggest hybridization during interglacial periods. Future inclusion of extinct populations of Martes, which were more morphologically and ecologically diverse, may further clarify Martes phenotypic evolution.

Multiscale imaging of the rat brain using an integrated diceCT and histology workflow.

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.

From Anomalous Arteries to Selective Brain Cooling: Parallel Evolution of the Artiodactyl Carotid Rete.

Terrestrial artiodactyls (even-toed ungulates) inhabit some of the world's most extreme environments, including arid deserts and high elevations. As medium-to-large-bodied mammals, artiodactyls have a suite of specialized physiologies to facilitate occupation of regions unavailable to other large mammals. One such physiology is selective brain cooling, wherein reduction of brain temperature below core body temperature has been demonstrated to reduce evaporative water loss. This physiology is enabled by an arterial heat-exchanger called the carotid rete. The ubiquity of the carotid rete throughout the clade, as well as its evolutionary history, is currently uninvestigated. Here, I use osteological correlates to survey clade-wide presence and morphology of the carotid rete, prior to conducting a preliminary evolutionary analysis. Nearly all living artiodactyls possess a carotid rete and are capable of selective brain cooling; however, major arteries supplying the rete are derived from different embryonic aortic arches on a suborder-specific basis. Ancestral character estimation infers this pattern of variation to be the result of independent evolutionary processes, suggesting carotid rete homoplasy arising via parallelism. This is a surprising finding given the role this structure plays in driving a physiology that has been implicated in mitigating artiodactylan responses to extreme environmental conditions. Future studies should incorporate extinct species represented in the fossil record to better parse between parallel and convergent mechanisms, as well as to better understand the relationship between the carotid rete, selective brain cooling, and survivorship of climate perturbation. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 303:308-317, 2020. © 2018 American Association for Anatomy.

Crocodylian Head Width Allometry and Phylogenetic Prediction of Body Size in Extinct Crocodyliforms.

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 R 2≥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.

Alometría del Ancho de la Cabeza de Cocodrilo y Predicción Filogenética del Tamaño Corporal en Cocodrilos Extintos (Crocodylian Head Width Allometry and Phylogenetic Prediction of Body Size in Extinct Crocodyliforms)El tamaño corporal y los cambios de tamaño corporal afectan ampliamente los parámetros de la historia de vida de todos los animales, lo que ha hecho que las estimaciones precisas del tamaño corporal de los taxones extintos sean un componente importante para comprender su paleobiología. Entre los crocodilianos extintos y sus precursores (por ejemplo, los suquios), se han desarrollado varios métodos para predecir el tamaño corporal a partir de conjuntos de indicadores de tejido duro. Sin embargo, muchos tienen aplicaciones limitadas debido a la disparidad de algunos grupos importantes de crocodiliformes y sesgos en el registro fósil. Aquí, probamos la utilidad del ancho de la cabeza como un estimador de tamaño corporal ampliamente aplicable en crocodiliformes vivos y fósiles. Utilizamos un conjunto de datos de individuos machos y hembras sexualmente maduros (n=76) de una muestra exhaustiva de especies existentes de cocodrilos que abarcan casi todos los taxones conocidos (n=22) para desarrollar un modelo filogenético bayesiano y predecir tres métricas convencionales para el tamaño: masa corporal, longitud del orificio de ventilación y longitud total. Luego usamos el modelo para estimar los parámetros de tamaño para una serie selecta de crocodiliformes extintos con afinidad filogenética conocida (Montsechosuchus, Diplocynodon, y Sarcosuchus). Luego comparamos nuestros resultados con los tamaños reportados en la literatura para demostrar la utilidad de nuestro enfoque en una gama amplia de tales fósiles. Nuestros resultados muestran que el ancho de la cabeza está altamente correlacionado con todas las otras métricas (todo R 2≥0.85) y es conmensurable a las dimensiones femorales debido a su confiabilidad como predictor del tamaño corporal. Proporcionamos el código R para permitir que otros investigadores empleen el modelo en su propia investigación.Translated to Spanish by C.A. Alfonso (calfonsoc@vt.edu).

Cranial arterial patterns of the alpaca (Camelidae: Vicugna pacos).

Artiodactyl cranial arterial patterns deviate significantly from the standard mammalian pattern, most notably in the possession of a structure called the carotid rete (CR)-a subdural arterial meshwork that is housed within the cavernous venous sinus, replacing the internal carotid artery (ICA). This relationship between the CR and the cavernous sinus facilitates a suite of unique physiologies, including selective brain cooling. The CR has been studied in a number of artiodactyls; however, to my knowledge, only a single study to date documents a subset of the cranial arteries of New World camelids (llamas, alpacas, vicugñas and guanacoes). This study is the first complete description of the cranial arteries of a New World camelid species, the alpaca (Vicugna pacos), and the first description of near-parturition cranial arterial morphology within New World camelids. This study finds that the carotid arterial system is conserved between developmental stages in the alpaca, and differs significantly from the pattern emphasized in other long-necked ruminant artiodactyls in that a patent, homologous ICA persists through the animal's life.

Body water conservation through selective brain cooling by the carotid rete: a physiological feature for surviving climate change?

Some mammals have the ability to lower their hypothalamic temperature below that of carotid arterial blood temperature, a process termed selective brain cooling. Although the requisite anatomical structure that facilitates this physiological process, the carotid rete, is present in members of the Cetartiodactyla, Felidae and Canidae, the carotid rete is particularly well developed in the artiodactyls, e.g. antelopes, cattle, sheep and goats. First described in the domestic cat, the seemingly obvious function initially attributed to selective brain cooling was that of protecting the brain from thermal damage. However, hyperthermia is not a prerequisite for selective brain cooling, and selective brain cooling can be exhibited at all times of the day, even when carotid arterial blood temperature is relatively low. More recently, it has been shown that selective brain cooling functions primarily as a water-conservation mechanism, allowing artiodactyls to save more than half of their daily water requirements. Here, we argue that the evolutionary success of the artiodactyls may, in part, be attributed to the evolution of the carotid rete and the resulting ability to conserve body water during past environmental conditions, and we suggest that this group of mammals may therefore have a selective advantage in the hotter and drier conditions associated with current anthropogenic climate change. A better understanding of how selective brain cooling provides physiological plasticity to mammals in changing environments will improve our ability to predict their responses and to implement appropriate conservation measures.

A comparison of postnatal arterial patterns in a growth series of giraffe (Artiodactyla: Giraffa camelopardalis).

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.

Unexpected Convergent Evolution of Nasal Domes between Pleistocene Bovids and Cretaceous Hadrosaur Dinosaurs.

The fossil record provides tangible, historical evidence for the mode and operation of evolution across deep time. Striking patterns of convergence are some of the strongest examples of these operations, whereby, over time, similar environmental and/or behavioral pressures precipitate similarity in form and function between disparately related taxa. Here we present fossil evidence for an unexpected convergence between gregarious plant-eating mammals and dinosaurs. Recent excavations of Late Pleistocene deposits on Rusinga Island, Kenya, have uncovered a catastrophic assemblage of the wildebeest-like bovid Rusingoryx atopocranion. Previously known from fragmentary material, these new specimens reveal large, hollow, osseous nasal crests: a craniofacial novelty for mammals that is remarkably comparable to the nasal crests of lambeosaurine hadrosaur dinosaurs. Using adult and juvenile material from this assemblage, as well as computed tomographic imaging, we investigate this convergence from morphological, developmental, functional, and paleoenvironmental perspectives. Our detailed analyses reveal broad parallels between R. atopocranion and basal Lambeosaurinae, suggesting that osseous nasal crests may require a highly specific combination of ontogeny, evolution, and environmental pressures in order to develop.

Cranial arterial pattern of the Sri Lankan spotted chevrotain, Moschiola memmina, and comparative basicranial osteology of the Tragulidae.

The cranial arterial pattern of artiodactyls deviates significantly from the typical mammalian pattern. One of the most striking atypical features is the rete mirabile epidurale: a subdural arterial meshwork that functionally and anatomically replaces the arteria carotis interna. This meshwork facilitates an exceptional ability to cool the brain, and was thought to be present in all artiodactyls. Recent research, however, has found that species of mouse deer (Artiodactyla: Tragulidae) endemic to the Malay Archipelago possess a complete a. carotis interna instead of a rete mirabile epidurale. As tragulids are the sister group to pecoran ruminants, the lack of a rete mirabile epidurale in these species raises intriguing evolutionary questions about the origin and nature of artiodactyl thermoregulatory cranial vasculature. In this study, cranial arterial patterns are documented for the remaining species within the Tragulidae. Radiopaque latex vascular injection, computed tomography (CT-scanning), and digital 3-dimensional anatomical reconstruction are used to image the cranial arteries of a Sri Lankan spotted chevrotain, Moschiola meminna. Sites of hard and soft tissue interaction were identified, and these osteological correlates were then sought in nine skulls representative of the remaining tragulid species. Both hard and soft tissue surveys confirm that the presence of an a. carotis interna is the common condition for tragulids. Moreover, the use of a 3-D, radiographic anatomical imaging technique enabled identification of a carotico-maxillary anastomosis that may have implications for the evolution of the artiodactyl rete mirabile epidurale.

Using biplanar fluoroscopy to guide radiopaque vascular injections: a new method for vascular imaging.

Studying vascular anatomy, especially in the context of relationships with hard tissues, is of great interest to biologists. Vascular studies have provided significant insight into physiology, function, phylogenetic relationships, and evolutionary patterns. Injection of resin or latex into the vascular system has been a standard technique for decades. There has been a recent surge in popularity of more modern methods, especially radiopaque latex vascular injection followed by CT scanning and digital "dissection." This technique best displays both blood vessels and bone, and allows injections to be performed on cadaveric specimens. Vascular injection is risky, however, because it is not a standardizable technique, as each specimen is variable with regard to injection pressure and timing. Moreover, it is not possible to view the perfusion of injection medium throughout the vascular system of interest. Both data and rare specimens can therefore be lost due to poor or excessive perfusion. Here, we use biplanar video fluoroscopy as a technique to guide craniovascular radiopaque latex injection. Cadaveric domestic pigs (Sus scrofa domestica) and white-tailed deer (Odocoileus virginianus) were injected with radiopaque latex under guidance of fluoroscopy. This method was found to enable adjustments, in real-time, to the rate, location, and pressure at which latex is injected in order to avoid data and specimen loss. In addition to visualizing the injection process, this technique can be used to determine flow patterns, and has facilitated the development of consistent markers for complete perfusion.