New frontiers in imaging, anatomy, and mechanics of crocodylian jaw muscles.

New imaging and biomechanical approaches have heralded a renaissance in our understanding of crocodylian anatomy. Here, we review a series of approaches in the preparation, imaging, and functional analysis of the jaw muscles of crocodylians. Iodine-contrast microCT approaches are enabling new insights into the anatomy of muscles, nerves, and other soft tissues of embryonic as well as adult specimens of alligators. These imaging data and other muscle modeling methods offer increased accuracy of muscle sizes and attachments without destructive methods like dissection. 3D modeling approaches and imaging data together now enable us to see and reconstruct 3D muscle architecture which then allows us to estimate 3D muscle resultants, but also measurements of pennation in ways not seen before. These methods have already revealed new information on the ontogeny, diversity, and function of jaw muscles and the heads of alligators and other crocodylians. Such approaches will lead to enhanced and accurate analyses of form, function, and evolution of crocodylians, their fossil ancestors and vertebrates in general.

Clarifying relationships between cranial form and function in tapirs, with implications for the dietary ecology of early hominins.

Paleontologists and paleoanthropologists have long debated relationships between cranial morphology and diet in a broad diversity of organisms. While the presence of larger temporalis muscle attachment area (via the presence of sagittal crests) in carnivorans is correlated with durophagy (i.e. hard-object feeding), many primates with similar morphologies consume an array of tough and hard foods-complicating dietary inferences of early hominins. We posit that tapirs, large herbivorous mammals showing variable sagittal crest development across species, are ideal models for examining correlations between textural properties of food and sagittal crest morphology. Here, we integrate dietary data, dental microwear texture analysis, and finite element analysis to clarify the functional significance of the sagittal crest in tapirs. Most notably, pronounced sagittal crests are negatively correlated with hard-object feeding in extant, and several extinct, tapirs and can actually increase stress and strain energy. Collectively, these data suggest that musculature associated with pronounced sagittal crests-and accompanied increases in muscle volume-assists with the processing of tough food items in tapirs and may yield similar benefits in other mammals including early hominins.

Tempo and Pattern of Avian Brain Size Evolution.

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.

Reply to: Charlier et al. 2018. Mudslide and/or animal attack are more plausible causes and circumstances of death for AL 288 ('Lucy'): a forensic anthropology analysis. Medico-Legal Journal 86(3) 139-142, 2018.

The Pliocene hominin fossil 'Lucy' (A.L. 288-1, Australopithecus afarensis) was discovered in the Afar region of Ethiopia in 1974 and dates to 3.18 million years in age. In Kappelman et al.,1 we presented the results of a detailed investigation of the skeleton that for the first time identified and described unusual bone-into-bone compressive fractures at several of the major long bone joints. Using multiple criteria, we concluded that these fractures are more likely to be perimortem than postmortem in nature. We next evaluated a number of possible mechanisms that could have produced these fractures and, on the basis of all of the evidence, hypothesised that a fall from considerable height, likely out of a tree, with its resulting vertical deceleration event, most closely matched the pattern of fractures preserved in the skeleton and was also the probable cause of death. Charlier et al. disagree with our approach and hypothesis, and instead present what they consider to be better evidence supporting two of the other possible mechanisms for breakage that we also investigated, a mudslide/flood, or an animal attack. We here show that the evidence presented by Charlier et al. is incorrectly interpreted, and that these two alternative hypotheses are less likely to be responsible for the fractures.

Perimortem fractures in Lucy suggest mortality from fall out of tall tree.

The Pliocene fossil 'Lucy' (Australopithecus afarensis) was discovered in the Afar region of Ethiopia in 1974 and is among the oldest and most complete fossil hominin skeletons discovered. Here we propose, on the basis of close study of her skeleton, that her cause of death was a vertical deceleration event or impact following a fall from considerable height that produced compressive and hinge (greenstick) fractures in multiple skeletal elements. Impacts that are so severe as to cause concomitant fractures usually also damage internal organs; together, these injuries are hypothesized to have caused her death. Lucy has been at the centre of a vigorous debate about the role, if any, of arboreal locomotion in early human evolution. It is therefore ironic that her death can be attributed to injuries resulting from a fall, probably out of a tall tree, thus offering unusual evidence for the presence of arborealism in this species.

X-ray computed tomography datasets for forensic analysis of vertebrate fossils.

We describe X-ray computed tomography (CT) datasets from three specimens recovered from Early Cretaceous lakebeds of China that illustrate the forensic interpretation of CT imagery for paleontology. Fossil vertebrates from thinly bedded sediments often shatter upon discovery and are commonly repaired as amalgamated mosaics grouted to a solid backing slab of rock or plaster. Such methods are prone to inadvertent error and willful forgery, and once required potentially destructive methods to identify mistakes in reconstruction. CT is an efficient, nondestructive alternative that can disclose many clues about how a specimen was handled and repaired. These annotated datasets illustrate the power of CT in documenting specimen integrity and are intended as a reference in applying CT more broadly to evaluating the authenticity of comparable fossils.

Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.

Morphologists have historically had to rely on destructive procedures to visualize the three-dimensional (3-D) anatomy of animals. More recently, however, non-destructive techniques have come to the forefront. These include X-ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard-tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT-based research is the use of chemical agents to render visible, and differentiate between, soft-tissue structures in X-ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine-based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine-based, contrast-enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting-edge applications of diffusible iodine-based contrast-enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.

Best practices for digitally constructing endocranial casts: examples from birds and their dinosaurian relatives.

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.

An investigation of the efficacy and mechanism of contrast-enhanced X-ray computed tomography utilizing iodine for large specimens through experimental and simulation approaches.

BACKGROUND: Iodine-based solutions have long been known to be effective in aiding the differentiation among soft tissues in both fundamental anatomical research and for clinical diagnoses. Recently the combination of this particular contrasting agent with micro-computed tomography (micro-CT) has resulted in an array of high-quality image data, in which anatomical structures not visible in conventional CT can be identified and quantified. However, there has been only limited data available to inform detailed protocols for staining large specimens. Further, modeling of the staining mechanism has focused on simple diffusion processes. RESULTS: A low concentration of iodine-based buffered formalin solution with a long staining period was used to visualize soft-tissue structures in a large goose head. The staining effect was analyzed by serially measuring the micro-CT profiles across coronal sections throughout the staining period. Regular replacement of the staining solution combined with a longer staining period significantly improved contrast within tissues. A simplified one-dimensional Diffusion-Sorption model with a three-zone domain was used to simulate the diffusion process by calculating the concentration profile of iodine across the adductor region, which fits well with the experiment data. Observations of changes in the concentration of the staining agent and simulation results suggest that the sorption of iodine by tissues significantly affects the effective diffusion coefficient for the contrasting agent. CONCLUSIONS: The Diffusion-Sorption model better explains previously reported difficulties in staining large samples comprised of tissues with high partition coefficients (K d ). Differences in partition coefficient (K d ), bulk density (ρ b ), and porosity (θ) could further explain the observed variation in staining rate and maximal staining effect among different tissues. Recommended protocols for staining large specimens are detailed.

A new fossil species supports an early origin for toothed whale echolocation.

Odontocetes (toothed whales, dolphins and porpoises) hunt and navigate through dark and turbid aquatic environments using echolocation; a key adaptation that relies on the same principles as sonar. Among echolocating vertebrates, odontocetes are unique in producing high-frequency vocalizations at the phonic lips, a constriction in the nasal passages just beneath the blowhole, and then using air sinuses and the melon to modulate their transmission. All extant odontocetes seem to echolocate; however, exactly when and how this complex behaviour--and its underlying anatomy--evolved is largely unknown. Here we report an odontocete fossil, Oligocene in age (approximately 28 Myr ago), from South Carolina (Cotylocara macei, gen. et sp. nov.) that has several features suggestive of echolocation: a dense, thick and downturned rostrum; air sac fossae; cranial asymmetry; and exceptionally broad maxillae. Our phylogenetic analysis places Cotylocara in a basal clade of odontocetes, leading us to infer that a rudimentary form of echolocation evolved in the early Oligocene, shortly after odontocetes diverged from the ancestors of filter-feeding whales (mysticetes). This was followed by enlargement of the facial muscles that modulate echolocation calls, which in turn led to marked, convergent changes in skull shape in the ancestors of Cotylocara, and in the lineage leading to extant odontocetes.

Morphology and variation in porpoise (Cetacea: Phocoenidae) cranial endocasts.

Evolution of endocranial anatomy in cetaceans is important from the perspective of echolocation ability, intelligence, social structure, and alternate pathways for circulation to the brain. Apart from the importance of studying brain shape and asymmetries as they relate to aspects of behavior and intelligence, cranial endocasts can show a close correspondence to the hydrostatic shape of the brain in life, and canals and grooves can preserve features of the circulatory system. Multiple samples are rarely available for studies of individual variation, especially in fossils, thus a first step in quantifying variation and making comparisons with fossils is made possible with CT scans of osteological specimens. This study presents a series of high-resolution X-ray CT-derived cranial endocasts of six extant species of Phocoenidae, a clade including some of the smallest and one of the rarest cetaceans. Degree of gyrification varies interspecifically and intraspecifically, possibly resulting from variation in preservation of the ossified meninges. Computed tomographic data show that visually assessed asymmetry in the cranial endocasts is not correlated with volumetric measurements, but nonetheless may reflect torsion in the skull's shape such that the right cerebral and cerebellar hemispheres extend rostrally and laterally more than the left. Vasculature and canals are similar to other described cetacean species, but the hypophyseal casts are unusual. Similarities between brain shape and volume measurements in the different species can be attributed to paedomorphism and concomitant variation in ecological preferences. This may explain similarities Neophocaena phocaenoides and Phocoena sinus share with the juvenile Phocoena phocoena specimen studied.

Auditory morphology and hearing sensitivity in fossil New World monkeys.

In recent years it has become possible to investigate the hearing capabilities in fossils by analogy with studies in living taxa that correlate the bony morphology of the auditory system with hearing sensitivity. In this analysis, we used a jack-knife procedure to test the accuracy of one such study that examined the functional morphology of the primate auditory system and we found that low-frequency hearing (sound pressure level at 250 Hz) can be predicted with relatively high confidence (±3-8 dB depending on the structure). Based on these functional relationships, we then used high-resolution computed tomography to examine the auditory region of three fossil New World monkeys (Homunculus, Dolicocebus, and Tremacebus) and compared their morphology and predicted low-frequency sensitivity with a phylogenetically diverse sample of extant primates. These comparisons reveal that these extinct taxa shared many auditory characteristics with living platyrrhines. However, the fossil with the best preserved auditory region (Homunculus) also displayed a few unique features such as the relative size of the tympanic membrane and stapedial footplate and the degree of trabeculation of the anterior accessory cavity. Still, the majority of evidence suggests that these fossil species likely had similar low-frequency sensitivity to extant South American monkeys. This research adds to the small but growing body of evidence on the evolution of hearing abilities in extinct taxa and lays the groundwork for predicting hearing sensitivity in additional fossil primate specimens.

Correlations between auditory structures and hearing sensitivity in non-human primates.

Primates show distinctions in hearing sensitivity and auditory morphology that generally follow phylogenetic patterns. However, few previous studies have attempted to investigate how differences in primate hearing are directly related to differences in ear morphology. This research helps fill this void by exploring the form-to-function relationships of the auditory system in a phylogenetically broad sample of non-human primates. Numerous structures from the outer, middle, and inner ears were measured in taxa with known hearing capabilities. The structures investigated include the overall size and shape of the pinna, the areas of the tympanic membrane and stapedial footplate, the masses and lever arm lengths of the ossicles, the volumes of the middle ear cavities, and the length of the cochlea. The results demonstrate that a variety of auditory structures show significant correlations with certain aspects of hearing (particularly low-frequency sensitivity). Although the majority of these relationships agree with expectations from auditory theory, some traditional (and possibly outdated) ideas were not supported. For example, the common misconception that higher middle ear transformer ratios (e.g., impedance transformer ratio) result in increased hearing sensitivity was not supported. Although simple correlations between form and function do not necessarily imply causality, the relationships defined in this study not only increase our understanding of auditory patterns in extant taxa but also lay the foundation to begin investigating the hearing in fossil primates.

Polymorphism in developmental timing: intraspecific heterochrony in a Lake Victoria cichlid.

Biologists measure developmental time by dividing development into arbitrary time blocks called "stages." This is a reasonable approach, provided that developmental timing is precisely controlled within a species. However, the degree of this precision is unknown. This is unfortunate because precision in developmental timing at the population level is a central issue to the whole research program of heterochrony. To examine this issue, we apply Ontogenetic Sequence Analysis to 261 embryos of the Lake Victoria cichlid Haplochromis piceatus. The result of our analysis can be mapped as a complex web of 26,880 equally parsimonious developmental sequences. This topology reflects timing polymorphism (intraspecific heterochrony) among embryos of this species. Because of this timing polymorphism, it is not possible to define discrete "stages" in this cichlid (although there is sufficient sequence signal to assess the maturity of embryos). More generally, we show that sequence polymorphism creates uncertainty about how a given embryo will develop implying that the mechanisms controlling developmental timing in embryos lack precision. For this reason, it is imperative to consider patterns of embryonic variability when measuring developmental time.

Ontogenetic Sequence Analysis: using parsimony to characterize developmental sequences and sequence polymorphism.

Ontogenetic sequences are a pervasive aspect of development and are used extensively by biologists for intra- and interspecific comparisons. A tacit assumption behind most such analyses is that sequence is largely invariant within a species. However, recent embryological and experimental work emphasizes that ontogenetic sequences can be variable and that sequence polymorphism may be far more prevalent than is generally realized. We present a method that uses parsimony algorithms to map hierarchic developmental patterns that capture variability within a sample. This technique for discovering and formalizing sequences is called the "Ontogenetic Sequence Analysis" (OSA). Results of OSA include formalized diagrams of reticulating networks, describe all most parsimonious sequences, and can be used to develop statistics and metrics for comparison of both intraspecific and interspecific sequence variation. The method is tested with examples of human postnatal skeletal ossification, comprising a time-calibrated data set of human hand and wrist epiphyseal unions, and a longitudinal data set of human wrist ossification. Results illustrate the validity of the method for discovering sequence patterns and for predicting morphologies not represented in analytic samples. OSA demonstrates the potential and challenges of incorporating ontogenetic sequences of morphological information into evolutionary analyses.

Olfactory fossa of Tremacebus harringtoni (platyrrhini, early Miocene, Sacanana, Argentina): implications for activity pattern.

CT imaging was undertaken on the skull of approximately 20-Myr-old Miocene Tremacebus harringtoni. Here we report our observations on the relative size of the olfactory fossa and its implications for the behavior of Tremacebus. The endocranial surface of Tremacebus is incomplete, making precise estimate of brain size and olfactory fossa size imprecise. However, olfactory fossa breadth and maximum endocranial breadth measured from CT images of one catarrhine species and eight platyrrhine species for which volumes of the olfactory bulb and brain are known show that the osteological proxies give a reasonably accurate indication of relative olfactory bulb size. Nocturnal Aotus has the largest relative olfactory fossa breadth and the largest olfactory bulb volume compared to brain volume among extant anthropoids. Tremacebus had a much smaller olfactory fossa breadth and, by inference, bulb volume--within the range of our sample of diurnal anthropoids. Variations in the relative size of the olfactory bulbs in platyrrhines appear to relate to the importance of olfaction in daily behaviors. Aotus has the largest olfactory bulbs among platyrrhines and relies more on olfactory cues when foraging than Cebus, Callicebus, or Saguinus. As in other examples of nocturnal versus diurnal primates, nocturnality may have been the environmental factor that selected for this difference in Aotus, although communication and other behaviors are also likely to select for olfactory variation in diurnal anthropoids. Considering the olfactory fossa size of Tremacebus, olfactory ability of this Miocene monkey was probably not as sensitive as in Aotus and counts against the hypothesis that Tremacebus was nocturnal. This finding accords well with previous observations that the orbits of Tremacebus are not as large as nocturnal Aotus.

Three-dimensional analysis of plant structure using high-resolution X-ray computed tomography.

High-resolution X-ray computed tomography (HRCT) is a non-invasive approach to 3D visualization and quantification of biological structure. The data, based on differential X-ray attenuation, are analogous to those otherwise obtainable only by serial sectioning. Requiring no fixing, sectioning or staining, HRCT produces a 3D digital map of the specimen that allows measurements and visualizations, including arbitrarily oriented sections. In spite of its application throughout the natural sciences, HRCT has yet to be applied in extant plant structural research.