Patterns of early embryogenesis and growth in the olfactory system of chick (Gallus gallus domesticus) based on iodine-enhanced micro-computed tomography.

BACKGROUND: The vertebrate olfactory system entails a complex set of neural/support structures that bridge morphogenetic regions. The developmental mechanisms coordinating this bridge remain unclear, even for model organisms such as chick, Gallus gallus. Here, we combine previous growth data on the chick olfactory apparatus with new samples targeting its early embryogenesis. The purpose is to illuminate how early developmental dynamics integrate with scaling relationships to produce adult form and, potentially, evolutionary patterns. Olfactory structures, including epithelium, turbinate, nerve, and olfactory bulb, are considered in the context of neighboring nasal and brain structures. RESULTS: Axonal outgrowth from the olfactory epithelium, which eventually connects receptor neurons with the brain, begins earlier than previously established. This dynamic marks the beginning of a complex pattern of early differential growth wherein the olfactory bulbs scale with positive allometry relative to both brain volume and turbinate area, which in turn scale isometrically with one another. CONCLUSIONS: The mechanisms driving observed patterns of organogenesis and growth remain unclear awaiting experimental evidence. We discuss competing hypotheses, including the possibility that broad-based isometry of olfactory components reflects constraints imposed by high levels of functional/structural integration. Such integration would include the frontonasal prominence having a strong influence on telencephalic patterning.

Exploring contrast-enhancing staining agents for studying adipose tissue through contrast-enhanced computed tomography.

Contrast-enhanced computed tomography offers a nondestructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using contrast-enhanced computed tomography but fail to provide chemical information. This study introduces isotonic Lugol's iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae and bovine muscle tissue strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte gray values post-IL staining were associated with chemical distinctions between bovine muscle tissue and murine caudal vertebrae. More specific, a difference in the degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of gray value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.

Structural analysis of the female reptile reproductive system by micro-computed tomography and optical coherence tomography†.

Volumetric data provide unprecedented structural insight to the reproductive tract and add vital anatomical context to the relationships between organs. The morphology of the female reproductive tract in non-avian reptiles varies between species, corresponding to a broad range of reproductive modes and providing valuable insight to comparative investigations of reproductive anatomy. However, reproductive studies in reptilian models, such as the brown anole studied here, have historically relied on histological methods to understand the anatomy. While these methods are highly effective for characterizing the cell types present in each organ, histological methods lose the 3D relationships between images and leave the architecture of the organ system poorly understood. We present the first comprehensive volumetric analyses of the female brown anole reproductive tract using two non-invasive, non-destructive imaging modalities: micro-computed tomography (microCT) and optical coherence tomography (OCT). Both are specialized imaging technologies that facilitate high-throughput imaging and preserve three-dimensional information. This study represents the first time that microCT has been used to study all reproductive organs in this species and the very first time that OCT has been applied to this species. We show how the non-destructive volumetric imaging provided by each modality reveals anatomical context including orientation and relationships between reproductive organs of the anole lizard. In addition to broad patterns of morphology, both imaging modalities provide the high resolution necessary to capture details and key anatomical features of each organ. We demonstrate that classic histological features can be appreciated within whole-organ architecture in volumetric imaging using microCT and OCT, providing the complementary information necessary to understand the relationships between tissues and organs in the reproductive system. This side-by-side imaging analysis using microCT and OCT allows us to evaluate the specific advantages and limitations of these two methods for the female reptile reproductive system.

Comparative muscle anatomy of the anuran pelvis and hindlimb in relation to locomotor mode.

Frogs have a highly conserved body plan, yet they employ a diverse array of locomotor modes, making them ideal organisms for investigating the relationships between morphology and locomotor function, in particular whether anatomical complexity is a prerequisite for functional complexity. We use diffusible iodine contrast-enhanced microCT (diceCT) imaging to digitally dissect the gross muscle anatomy of the pelvis and hindlimbs for 30 species of frogs representing five primary locomotor modes, including the first known detailed dissection for some of the world's smallest frogs, forming the largest digital comparative analysis of musculoskeletal structure in any vertebrate clade to date. By linking musculoskeletal dissections and phylogenetic comparative methods, we then quantify and compare relationships between anatomy and function across over 160 million years of anuran evolution. In summary, we have found that bone lengths and pelvic crest sizes are generally not reliable predictors of muscle sizes, which highlights important implications for future palaeontological studies. Our investigation also presents previously unreported differences in muscle anatomy between frogs specialising in different locomotor modes, including several of the smallest frog hindlimb muscles, which are extremely difficult to extract and measure using traditional approaches. Furthermore, we find evidence of many-to-one and one-to-many mapping of form to function across the phylogeny. Additionally, we perform the first quantitative analysis of how the degree of muscle separation can differ between frogs. We find evidence that phylogenetic history is the key contributing factor to muscle separation in the pelvis and thigh, while the separation of shank muscles is influenced more strongly by locomotor mode. Finally, our anatomical 3D reconstructions are published alongside this manuscript to contribute towards future research and serve as educational materials.

Ostrich (Struthio camelus) syrinx morphology and vocal repertoire across postnatal ontogeny and sex: Implications for understanding vocal evolution in birds.

Vocal production in birds has been the target of considerable research that mostly has focused on phylogenetically well-nested songbirds. Anatomical descriptions and recordings of many non-songbirds have often only focused on a single ontogenetic stage or sex. While basic morphology of the vocal organ (syrinx) of ostrich (Palaeognathae, Struthio camelus) has been known since the 1800s, descriptions of its vocal repertoire and syrinx anatomy since then have been incomplete or inconsistent. New toolkits now enable detailed qualitative description of internal anatomy and meristic data and allow it to be compared to vocal production. Here we describe the anatomy of the syrinx in Struthio camelus for three post-hatching ontogenetic stages and both an adult male and female utilizing dissection and contrast enhanced X-ray computed tomography (diceCT). We find changes in ring geometry and spacing through ontogeny as well as lateral labia thickness. We document a small unpaired, midline, cartilaginous structure, a "pessuliform process" at the tracheobronchial juncture present throughout ontogeny and in both males and females. Investigation of the vocal repertoire of ostriches across ontogeny using a new dataset of 77 recordings led to identification of four vocalizations not previously reported in the literature, including the simultaneous production of a hiss and tonal. We find syrinx morphology largely consistent across ontogeny and in male and female adults. Both are capable of producing long duration tonal calls, but these may be more frequent in male birds. Closed-mouth boom calls remain unique to males. A detailed understanding of diversity in parts of early diverging clades is pivotal in attempting to estimate features of the ancestral syrinx in birds and how avian vocalization evolved.

Differential performance of aqueous- and ethylic-Lugol's iodine stain to visualize anatomy in µCT-scanned vertebrates.

Museum specimens are an increasingly important tool for studying global biodiversity. With the advent of diffusible iodine-based contrast-enhanced computed tomography (diceCT), researchers can now visualize an organism's internal soft tissue anatomy without the need for physical dissection or other highly destructive sampling methods. However, there are many considerations when deciding which method of staining to use for diceCT to produce the best gray-scale contrast for facilitating downstream anatomical analyses. The general lack of direct comparisons among staining methodologies can make it difficult for researchers to determine which approaches are most appropriate for their study. Here, we compare the performance of ethylic-Lugol's iodine solution with aqueous-Lugol's staining solution across several vertebrate orders to assess differential imaging outcomes. We found that ethylic-Lugol's is better for visualizing muscle attachment to bone but provides overall lower contrast between soft tissue types. Comparatively, aqueous-based Lugol's provides high-contrast imaging among soft tissue types, although bone is more difficult to discern. We conclude that the choice of staining methodology largely depends on the type of anatomical data the researcher wishes to collect, and we provide a decision-based framework for assessing which staining methodology (ethylic or aqueous) is most appropriate for desired imaging results.

Comparative morphology of oral glands in snakes of the family Homalopsidae reveals substantial variation and additional independent origins of salt glands within Serpentes.

Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we examined the morphology of the oral glands of 12 species of the family Homalopsidae. Snakes of this family exhibit substantial interspecific morphological variation in their oral glands. Particular variables are the venom glands, ranging from large (e.g., Subsessor bocourti) to small (e.g., Erpeton tentaculatum). The supra- and infralabial glands are more uniform in morphology, being the second most developed in almost all the sampled species. Premaxillary glands distinct from the supralabial glands were observed in five species (Myron richardsonii, Bitia hydroides, Cantoria violacea, Fordonia leucobalia, and Gerarda prevostiana), in addition to Cerberus rynchops, the only species in which this condition was previously documented associated with the excretion of salt. In the three species of the saltwater group of homalopsids (C. violacea, F. leucobalia, and G. prevostiana), the premaxillary glands also extend posteriorly, occupying a large area above the supralabial gland, a condition not observed in any other species of snake studied thus far. Character evolution analyses indicate that premaxillary glands differentiated from the supralabial gland and evolved independently three or four times in the family, always in lineages that invaded marine habitats. Our results suggest that the differentiated premaxillary glands are likely salt glands, as is the case in C. rynchops. If corroborated, this increases to six or seven the number of independent evolutionary origins of salt glands in snakes that have undergone an evolutionary transition to marine life.

3D atlas of tinamou (Neornithes: Tinamidae) pectoral morphology: Implications for reconstructing the ancestral neornithine flight apparatus.

Palaeognathae, the extant avian clade comprising the flightless ratites and flight-capable tinamous (Tinamidae), is the sister group to all other living birds, and recent phylogenetic studies illustrate that tinamous are phylogenetically nested within a paraphyletic assemblage of ratites. As the only extant palaeognaths that have retained the ability to fly, tinamous may provide key information on the nature of the flight apparatus of ancestral crown palaeognaths-and, in turn, crown birds-as well as insight into convergent modifications to the wing apparatus among extant ratite lineages. To reveal new information about the musculoskeletal anatomy of tinamous and facilitate development of computational biomechanical models of tinamou wing function, we generated a three-dimensional musculoskeletal model of the flight apparatus of the extant Andean tinamou (Nothoprocta pentlandii) using diffusible iodine-based contrast-enhanced computed tomography (diceCT). Origins and insertions of the pectoral flight musculature of N. pentlandii are generally consistent with those of other extant volant birds specialized for burst flight, and the entire suite of presumed ancestral neornithine flight muscles are present in N. pentlandii with the exception of the biceps slip. The pectoralis and supracoracoideus muscles are robust, similar to the condition in other extant burst-flying birds such as many extant Galliformes. Contrary to the condition in most extant Neognathae (the sister clade to Palaeognathae), the insertion of the pronator superficialis has a greater distal extent than the pronator profundus, although most other anatomical observations are broadly consistent with the conditions observed in extant neognaths. This work will help form a basis for future comparative studies of the avian musculoskeletal system, with implications for reconstructing the flight apparatus of ancestral crown birds and clarifying musculoskeletal modifications underlying the convergent origins of ratite flightlessness.

First evidence of hemiclitores in snakes.

Female genitalia are conspicuously overlooked in comparison to their male counterparts, limiting our understanding of sexual reproduction across vertebrate lineages. This study is the first complete description of the clitoris (hemiclitores) in female snakes. We describe morphological variation in size and shape (n = 9 species, 4 families) that is potentially comparable to the male intromittent organs in squamate reptiles (hemipenes). Dissection, diffusible iodine contrast-enhanced micro-CT and histology revealed that, unlike lizard hemiclitores, the snake hemiclitores are non-eversible structures. The two individual hemiclitores are separated medially by connective tissue, forming a triangular structure that extends posteriorly. Histology of the hemiclitores in Australian death adders (Acanthophis antarcticus) showed erectile tissue and strands/bundles of nerves, but no spines (as is found in male hemipenes). These histological features suggest the snake hemiclitores have functional significance in mating and definitively show that the hemiclitores are not underdeveloped hemipenes or scent glands, which have been erroneously indicated in other studies. Our discovery supports that hemiclitores have been retained across squamates and provides preliminary evidence of differences in this structure among snake species, which can be used to further understand systematics, reproductive evolution and ecology across squamate reptiles.

Hind foot drumming: Volumetric micro-computed tomography investigation of the hind limb musculature of three African mole-rat species (Bathyergidae).

Several species of African mole-rats use seismic signalling by means of hind foot drumming for communication. The present study aimed to create three-dimensional reconstructions and compare volumetric measurements of 27 muscles of the hind limb of two drumming (Georychus capensis and Bathyergus suillus) and one non-drumming (Cryptomys hottentotus natalensis) species of African mole-rats. Diffusible iodine contrast-enhanced micro-computed tomography (diceCT) scans were performed on six specimens per species. Manual segmentation of the scans using VGMAX Studio imaging software allowed for individual muscles to be separated while automatically determining the volume of each muscle. The volume of the individual muscles was expressed as a percentage of the total hind limb volume and statistically compared between species. Subsequently, three-dimensional reconstructions of these muscles were created. Musculus gracilis anticus had a significantly larger percentage of the total hind limb muscle volume in both drumming species compared to the non-drumming C. h. natalensis. Furthermore, several hip and knee extensors, namely mm. gluteus superficialis, semimembranosus, gluteofemoralis, rectus femoris and vastus lateralis, had significantly larger muscle volume percentages in the two drumming species (G. capensis and B. suillus) compared to the non-drumming species. While not statistically significant, G. capensis had larger muscle volume percentages in several key hip and knee extensors compared to B. suillus. Additionally, G capensis had the largest summed percentage of the total hind limb volume in the hip flexor, hip extensor, knee extensor and ankle plantar flexor muscle groups in all the three species. This could be indicative of whole muscle hypertrophy in these muscles due to fast eccentric contractions that occur during hind foot drumming. However, significantly larger muscle volume percentages were observed in the scratch digging B. suillus compared to the other two chisel tooth digging species. Moreover, while not statistically significant, B. suillus had larger muscle volume percentages in several hip extensor and knee flexor muscles compared to G. capensis (except for m. vastus lateralis). These differences could be due to the large relative size of this species but could also be influenced by the scratch digging strategy employed by B. suillus. Therefore, while the action of hind foot drumming seems to influence certain key muscle volumes, digging strategy and body size may also play a role.

Sharp and fully loaded: 3D tissue reconstruction reveals how snake fangs stay deadly during fang replacement.

Snake venom is produced, transported and delivered by the sophisticated venom delivery system (VDS). When snakes bite, the venom travels from the venom gland through the venom duct into needle-like fangs that inject it into their prey. To counteract breakages, fangs are continuously replaced throughout life. Currently, the anatomy of the connection between the duct and the fang has not been described, and the mechanism by which the duct is reconnected to the replacement fang has not been identified. We examined the VDS in 3D in representative species from two families and one subfamily (Elapidae, Viperidae, Atractaspidinae) using contrast-enhanced microCT (diceCT), followed by dissection and histology. We observed that the venom duct bifurcates immediately anterior to the fangs so that both the original and replacement fangs are separately connected and functional in delivering venom. When a fang is absent, the canal leading to the empty position is temporarily closed. We found that elapid snakes have a crescent-shaped venom reservoir where venom likely pools before it enters the fang. These findings form the final piece of the puzzle of VDS anatomy in front-fanged venomous snakes. Additionally, they provide further evidence for independent evolution of the VDS in these three snake taxa.

DiceCT applied to fossilized hard tissues: A preliminary case study using a miocene bird.

Iodine-based contrasting agents for computed tomography (CT) have been used for decades in medicine. Agents like Lugol's iodine enhance the contrasts between soft tissues and mineralized (skeletal) tissues. Because a recent study on extant avian heads showed that iodine-ethanol (I2 E) is a better contrast enhancer overall than the standard Lugol's iodine, here, we tested if I2 E could also enhance the CT contrasts of two fossilized skeletal tissues: bone and calcified cartilage. For this, we used a partial ankle joint from an extinct pheasant from the Late Miocene of Northwest China (Linxia Basin). The pre-staining CT scans showed no microstructural details of the sample. After being immersed into a solution of 1% I2 E for 8 days and scanned a second time, the contrasts were drastically enhanced between the mineralized tissues (bony trabeculae and calcified cartilage) and the sediments and minerals inside vascular spaces. After three other staining-scanning cycles in 2%, 3%, and 6% I2 E solutions, the best contrasts were obtained after immersion in 6% I2 E for 7 days. Energy Dispersive Spectroscopy showed that iodine was preferentially absorbed by the mineralized tissues and the minerals in the vascular spaces, but not by the sediments. This method not only effectively increased the contrasts of two different fossilized skeletal tissues, it was also non-destructive and reversible because part of the fossil was successfully de-stained after a few days in pure ethanol. These preliminary results indicate that iodine-ethanol has the potential to be used widely in vertebrate paleontology to improve CT imaging of fossilized tissues.

Enhancing CT imaging: A safe protocol to stain and de-stain rare fetal museum specimens using diffusible iodine-based staining (diceCT).

Computed tomography (CT) scanning is being increasingly employed in the study of natural history, particularly to investigate the internal anatomy of unique specimens in museum collections. Different techniques to enhance the contrast between tissues have been developed to improve the quality of the scans while preserving the integrity of these rare specimens. Diffusible iodine-based contrast enhanced computed tomography (diceCT) was found to be particularly effective and reversible for staining tissues in formalin preserved specimens. While it can also be effectively employed to stain ethanol-preserved specimens of small size, the reversibility of this process and the applicability to large-bodied animals has never been thoroughly tested. Here, we describe a novel diceCT protocol developed to stain and de-stain ethanol-preserved prenatal specimens of baleen whales (Mysticeti, Cetacea). These large (10-90 cm in length only considering early fetal stages) specimens present unique challenges as they are rare in collections and irreplaceable, therefore it is imperative to not damage them with the staining process. Before trying this protocol on baleen whales' specimens, we conducted a pilot study on commercially available fetal pigs using the same parameters. This allowed us to optimize the staining time to obtain the best results in CT scanning and to test first-hand the effect of de-staining on ethanol-based specimens. External coloration of the specimens is also a concern with iodine-staining, as stained specimens assume a bright red color that needs to be removed from both internal and external tissues before they can be stored. To test the reversibility of the stain in ethanol-preserved specimens with fur, we also conducted a small experiment using commercially acquired domestic mice. After these trials were successful, we applied the staining and de-staining protocol to multiple fetal specimens of mysticetes up to 90 cm in length, both ethanol- and formalin-preserved. Specimens were soaked in a solution of 1% pure iodine in 70% ethanol for 1-28 days, according to their size. After scanning, specimens are soaked in a solution of 3% sodium thiosulfate in 70% ethanol that is able to completely wash out the iodine from the tissues in a shorter time frame, between a few hours and 14 days. The same concentrations were used for formalin-preserved specimens, but DI water was used as solvent instead of ethanol. The staining technique proved particularly useful to enhance the contrast difference between cartilage, mineralized bone, teeth, and the surrounding soft tissues even when the specimens where scanned in medical-grade CT scans. The specimens did not suffer any visible damage or shrinkage due to the procedure, and in both the fetal samples and in the mice the color of the stain was completely removed by the de-staining process. We conclude therefore that this protocol can be safely applied to a variety of ethanol-preserved museum specimens to enhance the quality of the CT scanning and highlight internal morphological features without recurring to dissection or other irreversible procedures. We also provide tips to best apply this protocol, from how to mix the solutions to how to minimize the staining time.

Anatomical and physiological variation of the hyoid musculature during swallowing in infant pigs.

The function of a muscle is impacted by its line of action, activity timing and contractile characteristics when active, all of which have the potential to vary within a behavior. One function of the hyoid musculature is to move the hyoid bone during swallowing, yet we have little insight into how their lines of action and contractile characteristics might change during a swallow. We used an infant pig model to quantify the contractile characteristics of four hyoid muscles during a swallow using synchronized electromyography, fluoromicrometry and high-speed biplanar videofluoroscopy. We also estimated muscle line of action during a swallow using contrast-enhanced CT-scanned muscles animated to move with the hyoid bone and found that as the hyoid elevated, the line of action of the muscles attached to it became greater in depression. We also found that muscles acted eccentrically and concentrically, which was correlated with hyoid movement. This work contributes to our understanding of how the musculature powering feeding functions during swallowing.

Pectoral myology of limb-reduced worm lizards (Squamata, Amphisbaenia) suggests decoupling of the musculoskeletal system during the evolution of body elongation.

BACKGROUND: The evolution of elongated body forms in tetrapods has a strong influence on the musculoskeletal system, including the reduction of pelvic and pectoral girdles, as well as the limbs. However, despite extensive research in this area it still remains unknown how muscles within and around bony girdles are affected by these reductions. Here we investigate this issue using fossorial amphisbaenian reptiles, or worm lizards, as a model system, which show substantial variation in the degree of reductions of girdles and limbs. Using iodine-based contrast-enhanced computed tomography (diceCT), we analyze the composition of the shoulder muscles of the main clades of Amphisbaenia and their outgroups relative to the pectoral skeleton. RESULTS: All investigated amphisbaenian taxa retain the full set of 17 shoulder muscles, independent of the degree of limb and girdle reductions, whereas in some cases muscles are fused to complexes or changed in morphology relative to the ancestral condition. Bipes is the only taxon that retains forelimbs and an almost complete pectoral girdle. All other amphisbaenian families show more variation concerning the completeness of the pectoral girdle having reduced or absent girdle elements. Rhineura, which undergoes the most severe bone reductions, differs from all other taxa in possessing elongated muscle strands instead of discrete shoulder muscles. In all investigated amphisbaenians, the shoulder muscle agglomerate is shortened and shifted anteriorly relative to the ancestral position as seen in the outgroups. CONCLUSIONS: Our results show that pectoral muscle anatomy does not necessarily correspond to the loss or reduction of bones, indicating a decoupling of the musculoskeletal system. Muscle attachment sites change from bones to non-skeletal areas, such as surrounding muscles, skin or connective tissue, whereas muscle origins themselves remain in the same region where the pectoral bones were ancestrally located. Our findings indicate a high degree of developmental autonomy within the musculoskeletal system, we predict that the observed evolutionary rearrangements of amphisbaenian shoulder muscles were driven by functional demands rather than by developmental constraints. Nevertheless, worm lizards display a spatial offset of both pectoral bones and muscles relative to the ancestral position, indicating severe developmental modifications of the amphisbaenian body axis.

Prenatal developmental sequence of the skull of minke whales and its implications for the evolution of mysticetes and the teeth-to-baleen transition.

Baleen whales (Mysticeti) have an extraordinary fossil record documenting the transition from toothed raptorial taxa to modern species that bear baleen plates, keratinous bristles employed in filter-feeding. Remnants of their toothed ancestry can be found in their ontogeny, as they still develop tooth germs in utero. Understanding the developmental transition from teeth to baleen and the associated skull modifications in prenatal specimens of extant species can enhance our understanding of the evolutionary history of this lineage by using ontogeny as a relative proxy of the evolutionary changes observed in the fossil record. Although at present very little information is available on prenatal development of baleen whales, especially regarding tooth resorption and baleen formation, due to a lack of specimens. Here I present the first detailed description of prenatal specimens of minke whales (Balaenoptera acutorostrata and Balaenoptera bonaerensis), focusing on the skull anatomy and tooth germ development, resorption, and baleen growth. The ontogenetic sequence described consists of 10 specimens of both minke whale species, from the earliest fetal stages to full term. The internal skull anatomy of the specimens was visualized using traditional and iodine-enhanced computed tomography scanning. These high-quality data allow detailed description of skull development both qualitatively and quantitatively using three-dimensional landmark analysis. I report distinctive external anatomical changes and the presence of a denser tissue medial to the tooth germs in specimens from the final portion of gestation, which can be interpreted as the first signs of baleen formation (baleen rudiments). Tooth germs are only completely resorbed just before the eruption of the baleen from the gums, and they are still present for a brief period with baleen rudiments. Skull shape development is characterized by progressive elongation of the rostrum relative to the braincase and by the relative anterior movement of the supraoccipital shield, contributing to a defining feature of cetaceans, telescoping. These data aid the interpretation of fossil morphologies, especially of those extinct taxa where there is no direct evidence of presence of baleen, even if caution is needed when comparing prenatal extant specimens with adult fossils. The ontogeny of other mysticete species needs to be analyzed before drawing definitive conclusions about the influence of development on the evolution of this group. Nonetheless, this work is the first step towards a deeper understanding of the most distinctive patterns in prenatal skull development of baleen whales, and of the anatomical changes that accompany the transition from tooth germs to baleen. It also presents comprehensive hypotheses to explain the influence of developmental processes on the evolution of skull morphology and feeding adaptations of mysticetes.

The Utility of DiceCT Imaging for High-Throughput Comparative Neuroanatomical Studies.

Advancements in imaging techniques have drastically improved our ability to visualize, study, and digitally share complex, often minute, anatomical relationships. The recent adoption of soft-tissue X-ray imaging techniques, such as diffusible iodine-based contrast-enhanced computed tomography (diceCT), is beginning to offer previously unattainable insights into the detailed configurations of soft- tissue complexes across Metazoa. As a contrast agent, dissolved iodine diffuses deeply throughout preserved specimens to bind fats and carbohydrates that are natural ly present within metazoan soft tissues, increasing the radiodensities of these tissues in predictable ways. Like the current "gold standard" of magnetic resonance imaging, diceCT does not require physical dissection and can differentiate between the lipid content of myelinated versus nonmyelinated tissues, thereby offering great potential for neuroanatomical studies. Within the brain, for example, diceCT distinguishes myelinated fiber tracts from unmyelinated cortices, nuclei, and ganglia and allows three-dimensional visualization of their anatomical interrelationships at previously unrealized spatial scales. In this study, we illustrate the utility of diceCT for the rapid visualization of both external and internal brain anatomy in vertebrates - alongside the intact bones of the skull and the complete, undisturbed pathways of peripheral nerves, up to and including the target organs that they innervate. We demonstrate the transformative potential of this technique for developing high-resolution neuroanatomical datasets and describe best practices for imaging large numbers of specimens for broad evolutionary studies across vertebrates.

Assessing Soft-Tissue Shrinkage Estimates in Museum Specimens Imaged With Diffusible Iodine-Based Contrast-Enhanced Computed Tomography (diceCT).

The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six "fresh" bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

Genital interactions during simulated copulation among marine mammals.

Genitalia are morphologically variable across many taxa and in physical contact during intromission, but little is known about how variation in form correlates with function during copulation. Marine mammals offer important insights into the evolutionary forces that act on genital morphology because they have diverse genitalia and are adapted to aquatic living and mating. Cetaceans have a fibroelastic penis and muscular vaginal folds, while pinnipeds have a baculum and lack vaginal folds. We examined copulatory fit in naturally deceased marine mammals to identify anatomical landmarks in contact during copulation and the potential depth of penile penetration into the vagina. Excised penises were artificially inflated to erection with pressurized saline and compared with silicone vaginal endocasts and within excised vaginas in simulated copulation using high-resolution, diffusible iodine-based, contrast-enhanced computed tomography. We found evidence suggestive of both congruent and antagonistic genital coevolution, depending on the species. We suggest that sexual selection influences morphological shape. This study improves our understanding of how mechanical interactions during copulation influence the shape of genitalia and affect fertility, and has broad applications to other taxa and species conservation.

Application of diceCT to Study the Development of the Zika Virus-Infected Mouse Brain.

Zika virus (ZIKV) impacts the developing brain. Here, a technique was applied to define, in 3D, developmental changes in the brains of ZIKV-infected mice. Postnatal day 1 mice were uninfected or ZIKV-infected, then analysed by iodine staining and micro-CT scanning (diffusible iodine contrast-enhanced micro-CT; diceCT) at 3-, 6-, and 10-days post-infection (dpi). Multiple brain regions were visualised using diceCT: the olfactory bulb, cerebrum, hippocampus, midbrain, interbrain, and cerebellum, along with the lens and retina of the eye. Brain regions were computationally segmented and quantitated, with increased brain volumes and developmental time in uninfected mice. Conversely, in ZIKV-infected mice, no quantitative differences were seen at 3 or 6 dpi when there were no clinical signs, but qualitatively, diverse visual defects were identified at 6-10 dpi. By 10 dpi, ZIKV-infected mice had significantly lower body weight and reduced volume of brain regions compared to 10 dpi-uninfected or 6 dpi ZIKV-infected mice. Nissl and immunofluorescent Iba1 staining on post-diceCT tissue were successful, but RNA extraction was not. Thus, diceCT shows utility for detecting both 3D qualitative and quantitative changes in the developing brain of ZIKV-infected mice, with the benefit, post-diceCT, of retaining the ability to apply traditional histology and immunofluorescent analysis to tissue.